Medical Physiology

628 PART IX ENDOCRINE PHYSIOLOGY Glycogen Protein Glycogen Glycogen synthesis synthase phosphorylase Glucokinase Amino Amino hexokinase acids acids Protein Glucose Glucose 6-phosphate Glucose-6- phosphatase Protein degradation Fructose-1,6-diphosphatase Phosphofructokinase Phosphoenolpyruvate Pyruvate kinase carboxykinase LIVER CELL, MUSCLE CELL, ADIPOCYTE Pyruvate dehydrogenase Pyruvate carboxylase FIGURE 35.6 Effects of insulin on protein synthesis and protein degradation. Insulin promotes the ac- cumulation of protein by stimulating (heavy arrows) amino acid Citric acid cycle uptake and protein synthesis and by inhibiting (light arrows) pro- tein degradation in liver, skeletal muscle, and adipose tissue. Insulin stimulation of glycogen synthesis FIGURE 35.4 and glucose metabolism. Insulin promotes glucose uptake into target tissues, stimulates glycogen synthesis, synthesis. Insulin also increases the amount of protein syn- and inhibits glycogenolysis. In addition it promotes glycolysis in thesis machinery in cells by promoting ribosome synthesis. its target tissues. Heavy arrows indicate processes stimulated by Third, insulin inhibits protein degradation by reducing lyso- insulin; light arrows indicate processes inhibited by insulin. some activity and possibly other mechanisms as well. Glucagon Primarily Affects the Liver Metabolism Effects of Insulin on Protein Synthesis and Protein Degra- dation. Insulin promotes protein accumulation in its pri- of Carbohydrates, Lipids, and Proteins mary target tissues—liver, adipose tissue, and muscle—in The primary physiological actions of glucagon are exerted three specific ways (Fig. 35.6). First, it stimulates amino acid in the liver. Numerous effects of glucagon have been docu- uptake. Second, it increases the activity of several factors in- mented in other tissues, primarily adipose tissue, when the volved in protein synthesis. For example, it increases the ac- hormone has been added at high, nonphysiological con- tivity of protein synthesis initiation factors, promoting the centrations in experimental situations. While these effects start of translation and increasing the efficiency of protein may play a role in certain abnormal situations, the normal daily effects of glucagon occur primarily in the liver. Adipocyte Mechanism of Glucagon Action. Glucagon initiates its biological effects by interacting with one or more types of Endothelial cell cell membrane receptors. Glucagon receptors are coupled Cytoplasm Stored lipid to G proteins and promote increased intracellular cAMP, Capillary via the activation of adenylyl cyclase, or elevated cytosolic blood calcium as a result of phospholipid breakdown to form IP 3. Glucose Glucose Glucagon and Glycogenolysis. Glucagon is an impor- Glucose tant regulator of hepatic glycogen metabolism. It produces a net effect of glycogen breakdown by increasing intracel- lular cAMP levels, initiating a cascade of phosphorylation Acetyl-CoA α-Glycerol Lipoprotein phosphate events that ultimately results in the phosphorylation of phosphorylase b and its activation by conversion into Lipoprotein phosphorylase a. Similarly, glucagon promotes the net lipase Fatty acids breakdown of glycogen by promoting the inactivation of glycogen synthase (Fig. 35.7). Fatty acids Triglyceride Glucagon and Gluconeogenesis. In addition to promot- ing hepatic glucose production by stimulating glycogenol- Effects of insulin on lipid metabolism in FIGURE 35.5 ysis, glucagon stimulates hepatic gluconeogenesis (Fig. adipocytes. Insulin promotes the accumulation of lipid (triglycerides) in adipocytes by stimulating the processes 35.8). It does this principally by increasing the transcrip- shown by the heavy arrows and inhibiting the processes shown tion of mRNA coding for the enzyme phosphoenolpyru- by the light arrows. Similar stimulatory and inhibitory effects oc- vate carboxykinase (PEPCK), a key rate-limiting enzyme in cur in liver cells. gluconeogenesis. Glucagon also stimulates amino acid

CHAPTER 35 The Endocrine Pancreas 629 Glucose Glycogen α-Glycerol phosphate Glycogen Glycogen synthase phosphorylase Triglycerides Glucose 6- Glucose Glucose phosphate Acetyl CoA Fatty Hormone- acids sensitive Ketogenesis lipase Ketones Glycerol LIVER CELL The role of glucagon in glycogenolysis and FIGURE 35.7 glucose production in liver cells. Heavy ar- rows indicate processes stimulated by glucagon; light arrows indi- cate processes inhibited by glucagon. Ketones Fatty transport into liver cells and the degradation of hepatic acids proteins, helping provide substrates for gluconeogenesis. LIVER CELL Glucagon and Ureagenesis. The glucagon-enhanced The role of glucagon in lipolysis and keto- conversion of amino acids into glucose leads to increased FIGURE 35.9 genesis in liver cells. Heavy arrows indicate formation of ammonia. Glucagon assists in the disposal of processes stimulated by glucagon; light arrows indicate processes ammonia by increasing the activity of the urea cycle en- inhibited by glucagon. zymes in liver cells (see Fig. 35.8). Glucagon and Lipolysis. Glucagon promotes lipolysis in Glucagon and Ketogenesis. Glucagon promotes ketogen- liver cells (Fig. 35.9), although the quantity of lipids stored esis, the production of ketones, by lowering the levels of mal- in liver is small compared to that in adipose tissue. onyl CoA, relieving an inhibition of palmitoyl transferase and allowing fatty acids to enter the mitochondria for oxida- tion to ketones (see Fig 35.9). Ketones are an important Amino source of fuel for muscle cells and heart cells during times of acids starvation, sparing blood glucose for other tissues that are obligate glucose users, such as the central nervous system. During prolonged starvation, the brain adapts its metabolism to use ketones as a fuel source, lessening the overall need for hepatic glucose production (see Chapter 34). Protein synthesis Gluconeo- The Insulin-Glucagon Ratio Determines genesis Metabolic Status Glucose Amino Protein acids In most instances, insulin and glucagon produce opposing effects. Therefore, the net physiological response is deter- Ammonia Protein mined by the relative levels of both hormones in the blood degradation plasma, the insulin-glucagon ratio (I/G ratio). Urea synthesis I/G Ratio in the Fed and Fasting States. The I/G ratio Urea may vary 100-fold or more because the plasma concentra- tion of each hormone can vary considerably in different nu- tritional states. In the fed state, the molar I/G ratio is ap- proximately 30. After an overnight fast, it may fall to about 2, and with prolonged fasting, it may fall to as low as 0.5. LIVER CELL The role of glucagon in gluconeogenesis Inappropriate I/G Ratios in Diabetes. A good example of FIGURE 35.8 and ureagenesis in liver cells. Heavy arrows the profound influence of the I/G ratio on metabolic status indicate processes stimulated by glucagon; light arrow indicates is in insulin-deficient diabetes. Insulin levels are low, so processes inhibited by glucagon. pathways that insulin stimulates operate at a reduced level.

630 PART IX ENDOCRINE PHYSIOLOGY However, insulin is also necessary for alpha cells to sense 50% or less chance that the second will develop the dis- blood glucose appropriately; in the absence of insulin, the ease. The specific environmental factors have not been secretion of glucagon is inappropriately elevated. The re- identified, although much evidence implicates viruses. sult is an imbalance in the I/G ratio and an accentuation of Therefore, it appears that a combination of genetics and glucagon effects well above what would be seen in normal environment are strong contributing factors to the devel- states of low insulin, such as in fasting. opment of type 1 diabetes. Because the primary defect in type 1 diabetes is the in- ability of beta cells to secrete adequate amounts of insulin, these patients must be treated with injections of insulin. In DIABETES MELLITUS an attempt to match insulin concentrations in the blood Diabetes mellitus is a disease of metabolic dysregulation— with the metabolic requirements of the individual, various most notably a dysregulation of glucose metabolism—ac- formulations of insulin with different durations of action companied by long-term vascular and neurological complica- have been developed. Patients inject an appropriate tions. Diabetes has several clinical forms, each of which has a amount of these different insulin forms to match their di- distinct etiology, clinical presentation, and course. Insights etary and lifestyle requirements. into diabetes and its complications have been gleaned from The long-term control of type 1 diabetes depends on extensive metabolic studies, the use of radioimmunoassays for maintaining a balance between three factors: insulin, diet, insulin and glucagon, and the application of molecular biol- and exercise. To strictly control their blood glucose, pa- ogy strategies. Diabetes is the most common endocrine dis- tients are advised to monitor their diet and level of physical order. Some 16 million people may have the disease in the activity, as well as their insulin dosage. Exercise per se, United States; the exact number is not known because many much like insulin, increases glucose uptake by muscle. Dia- people have a borderline, subclinical form of the disorder. betic patients must take this into account and make appro- Many deaths attributed to cardiovascular disease are in fact priate adjustments in diet or insulin whenever general exer- the result of complications from diabetes. cise levels change dramatically. Diagnosing diabetes mellitus is not difficult to do. Symptoms usually include frequent urination, increased thirst, increased food consumption, and weight loss. The Type 2 Diabetes Mellitus Primarily Originates standard criterion for a diagnosis of diabetes is an elevated in the Target Tissue plasma glucose level after an overnight fast on at least two Type 2 diabetes mellitus results primarily from impaired separate occasions. A glucose value above 126 mg/dL (7.0 ability of target tissues to respond to insulin. There are mmol/L) is often used as the diagnostic value. multiple forms of the disease, each with a different etiol- Diabetes mellitus is a heterogeneous disorder. The ogy. In some cases, it is a permanent, lifelong disorder; in causes, symptoms, and general medical outcomes are vari- others, it results from the secretion of counterregulatory able. Generally, the disease takes one of two forms, type 1 hormones in a normal (e.g., pregnant) or pathophysio- diabetes or type 2 diabetes. Other forms of diabetes, such logical (e.g., Cushing’s disease) state. Gestational dia- as gestational diabetes, are also well known. betes occurs in 2 to 5% of all pregnancies but usually dis- appears after delivery. Women who have had gestational diabetes have an increased risk of developing type 2 dia- Most Forms of Type 1 Diabetes Mellitus betes later in life. Involve an Autoimmune Disorder Type 1 diabetes is characterized by the inability of beta Insulin Resistance in Type 2 Diabetes. In most cases of cells to produce physiologically appropriate amounts of in- type 2 diabetes, normal or higher-than-normal amounts of sulin. In some instances, this may result from a mutation in insulin are present in the circulation. Therefore, there is no the preproinsulin gene. However, the most common form impairment in the secretory capacity of pancreatic beta of type 1 diabetes results from destruction of the pancreatic cells but only in the ability of target cells to respond to in- beta cells by the immune system. The initial pathological sulin. In some instances, it has been demonstrated that the event is insulitis, involving a lymphocytic attack on beta fundamental defect is in the insulin receptor. In most cases, cells. Antibodies to beta cell cell-surface antigens have also however, receptor function appears normal, and the im- been found in the circulation of many persons with type 1 pairment in insulin action is ascribed to a postreceptor de- diabetes, but this is not a primary causative factor and prob- fect. Since the exact mechanism of insulin action has not ably results from the initial cellular damage. been determined, it is difficult to explore the causes of in- Studies of identical twins have provided important in- sulin resistance in much greater depth. formation regarding the genetic basis of type 1 diabetes. If one twin develops type 1 diabetes, the odds that the second Genetics, Environment, and Type 2 Diabetes. As with will develop the disease are much higher than for any ran- type 1 diabetes, key information on the influence of genet- dom individual in the population, even when the twins are ics and environmental factors in type 2 diabetes comes raised apart under different socioeconomic conditions. In from studies of identical twins. These studies indicate that addition, individuals with certain cell-surface HLA antigens there is a strong genetic component to the development of bear a higher risk for the disease than others. type 2 diabetes and that environmental factors, including Environmental factors are involved as well because the diet, play a considerably lesser role. If one identical twin development of type 1 diabetes in one twin predicts only a develops type 2 diabetes, chances are nearly 100% that the

CHAPTER 35 The Endocrine Pancreas 631 second will as well, even if they are raised apart under en- tant electrolytes. Excessive ketone production in type 1 dia- tirely different conditions. betes results in acidosis, a loss of cations, and a loss of fluids. Many persons with type 2 diabetes are overweight, and Emergency department procedures are directed toward im- often the severity of their disease can be lessened simply by mediate correction of these acute problems and usually in- weight loss. However, no strict cause-and-effect relation- volve the administration of base, fluids, and insulin. ship between these two conditions has been established. The complex sequence of events that can result from un- Clearly, not all persons with type 2 diabetes are obese, and controlled type 1 diabetes is shown in Figure 35.10. If left not all obese individuals develop diabetes. unchecked, many of these complications can have an addi- tive effect to further the severity of the disease state. Treatment Options for Type 2 Diabetes. In milder forms Persons with type 2 diabetes are generally not ketotic of type 2 diabetes, dietary restriction leading to weight loss and do not develop acidosis or the electrolyte imbalances may be the only treatment necessary. Commonly, however, characteristic of type 1 diabetes. Hyperglycemia leads to dietary restriction is supplemented by treatment with one of fluid loss and dehydration. Severe cases may result in hy- several orally active agents, most often of the sulfonylurea perosmolar coma as a result of excessive fluid loss. The ini- class. These drugs appear to act in two ways. First, they pro- tial objective of treatment in these individuals is the ad- mote insulin action in target cells, lessening insulin resistance ministration of fluids to restore fluid volumes to normal and in tissues. Second, they correct or reverse a somewhat slug- eliminate the hyperosmolar state. gish response of pancreatic beta cells often seen in type 2 di- abetes, normalizing insulin secretory responses to glucose. Chronic Secondary Complications of Diabetes. With The exact mechanisms of these effects are unknown. In some good control of their disease, most persons with diabetes cases, persons with type 2 diabetes may also be treated with can avoid the acute complications described above; how- insulin, although in the most of cases a regimen of oral agents ever, it is rare that they will not suffer from some of the and dietary manipulation is sufficient. chronic secondary complications of the disease. In most in- stances, such complications will ultimately lead to reduced life expectancy. Diabetes Mellitus Complications Present Most lesions occur in the circulatory system, although Major Health Problems the nervous system is also often affected. Large vessels of- If left untreated or if glycemic control is poor, diabetes ten show changes similar to those in atherosclerosis, with leads to acute complications that may prove fatal. How- the deposition of large fatty plaques in arteries. However, ever, even with reasonably good control of blood glucose, most of the circulatory complications in diabetes occur in over a period of years, most diabetics develop secondary microvessels. The common finding in affected vessels is a complications of the disease that result in tissue damage, primarily involving the cardiovascular and nervous systems. Acute Complications of Diabetes. The nature of acute complications that develop in type 1 and type 2 diabetics differs. Persons with poorly controlled type 1 diabetes of- ten exhibit hyperglycemia, glucosuria, dehydration, and di- abetic ketoacidosis. As blood glucose becomes elevated above the renal plasma threshold, glucose appears in the urine. As a result of osmotic effects, water follows glucose, leading to polyuria, excessive loss of fluid from the body, and dehydration. With fluid loss, the circulating blood vol- ume is reduced, compromising cardiovascular function, which may lead to circulatory failure. Excessive ketone formation leads to acidosis and elec- trolyte imbalances in persons with type 1 diabetes. If uncon- trolled, ketones may be elevated in the blood to such an ex- tent that the odor of acetone (one of the ketones) is noticeable on the breath. Production of the primary ketones, -hydroxybutyric acid and acetoacetic acid, results in the generation of excess hydrogen ions and a metabolic acidosis. Ketones may accumulate in the blood to such a degree that they exceed renal transport capacities and appear in the urine. As a result of osmotic effects, water is also lost in the urine. In addition, the pK of ketones is such that, even with the most acidic urine, a normal kidney can produce about half of the excreted ketones in the salt (or base) form. To en- FIGURE 35.10 Events resulting from acute deficiency in sure electrical neutrality, these must be accompanied by a type 1 diabetes mellitus. If left untreated, in- cation, usually either sodium or potassium. The loss of ke- sulin deficiency may lead to several complications, which may have additive or confounding effects that may ultimately result in death. tones in the urine, therefore, also results in a loss of impor-

632 PART IX ENDOCRINE PHYSIOLOGY CLINICAL FOCUS BOX 35.1 The Diabetic Foot traumatic amputations in the United States each year are Despite efforts to control their disease and maintain a nor- due to diabetes. Breakdown of the foot in persons who mal glycemic state, most persons with diabetes eventually are diabetic is commonly due to a combination of neu- develop one or more secondary complications of the dis- ropathy, vascular impairment, and infection. In a typical ease. These complications may be somewhat subtle in on- scenario, small lesions on the foot result from dryness of set and slow in progression; however, they account for the the skin due to a combination of neural and vascular com- high rates of morbidity and mortality. While the specific plications. Impairments in sensory nerve function may re- mechanisms involved remain areas of debate and research sult in these small lesions going unnoticed by the patient activity, most secondary complications are vascular or until a severe infection or gangrene has become well es- neural in nature. tablished. Vascular complications may involve atherosclerotic-like Loss of the affected foot or limb often can be avoided lesions in the large blood vessels or impaired function in with patient and physician education. The focus in manag- the microcirculation. Damage to the basement membrane ing patients with diabetes is the maintenance of normal of capillaries in the eye (diabetic retinopathy) or kidney blood glucose levels; avoiding primary complications, (diabetic nephropathy) is commonly seen. Although such as diabetic ketoacidosis or hyperosmolar coma; and there is no satisfactory direct treatment for diabetic vascu- initial secondary complications, such as diabetic retinopa- lar disease, its progression is often monitored closely as an thy. There is an increasing awareness of the importance of indirect indicator of the overall diabetic state. assessing the feet of a diabetic patient at each visit. The re- Diabetic neuropathy typically involves symmetric sen- sults of one study show that the likelihood of amputation sory loss in the distal lower extremities or autonomic is reduced by half if patients with diabetes simply remove neuropathy, leading to impotence, GI dysfunction, or an- their shoes for foot inspection during every outpatient hidrosis (lack of sweating) in the lower extremities. The clinic visit. Therefore, while the underlying physiological diabetic foot is an example of several complicating fac- mechanisms of the problem may be complex, the problem tors exacerbating one another. About 50 to 70% of non- can be relatively easily avoided. thickening of the basement membrane. This condition Diabetic peripheral neuropathy is also a common com- leads to impaired delivery of nutrients and hormones to the plication of long-standing diabetes. This disorder usually tissues and inadequate removal of waste products, resulting involves sensory nerves and those of the autonomic nerv- in irreparable tissue damage. ous system. Many persons with diabetes experience dimin- Some of the more disabling consequences of diabetic ished sensation in the extremities, especially in the feet and circulatory impairment are deterioration of blood flow to legs, which compounds the problem of diminished blood the retina of the eye, causing retinopathy and blindness; flow to these areas (see Clinical Focus Box 35.1). Often, deterioration of blood flow to the extremities, causing, in impaired sensory nerve function results in lack of awareness some cases, the need for foot or leg amputation; and dete- of severe ulcerations of the feet caused by reduced blood rioration of glomerular filtration in the kidneys, leading to flow. Men may develop impotence, and both men and renal failure. women may have impaired bladder and bowel function. REVIEW QUESTIONS DIRECTIONS: Each of the numbered (B) Stimulation of glucose uptake into would be most characteristic of his items or incomplete statements in this all tissues in the body form of the disease? section is followed by answers or (C) Inhibition of protein degradation (A) Insulin resistance completions of the statement. Select the in skeletal muscle (B) Treatment with exogenous insulin ONE lettered answer or completion that is (D) Stimulation of hormone-sensitive (C) Sulfonylurea treatment BEST in each case. lipase in adipose tissue (D) Virtual absence of secondary 3. The effects of glucagon include complications 1. Which of the following stimulate the (A) Inhibition of insulin secretion by 5. Type 2 diabetes secretion of both insulin and glucagon pancreatic beta cells (A) Has a strong genetic component to from the pancreas? (B) Primary actions in adipose tissue the development of the disease (A) Epinephrine (C) Promotion of gluconeogenesis and (B) Is characterized by low or (B) Amino acids urea synthesis in liver cells negligible circulating insulin (C) Acetylcholine (D) Indirect stimulation of (C) Occurs only in obese individuals (D) Both amino acids and ketogenesis in liver cells by the (D) Is treated in the same manner as acetylcholine inhibition of pancreatic somatostatin type 1 diabetes 2. The effects of insulin include secretion 6. Which of the following would you (A) Inhibition of amino acid uptake 4. A 55-year-old man was diagnosed with least likely see in a person with long- into skeletal muscle type 1 diabetes at the age of 8. Which standing type 2 diabetes? (continued)

CHAPTER 35 The Endocrine Pancreas 633 (A) Neuropathy (B) Immediately after a high-protein Rifkin’s Diabetes Mellitus. 5th Ed. Stam- (B) Nephropathy meal ford, CT: Appleton & Lange, 1997. (C) Retinopathy (C) After an overnight fast Saltiel AR, Kahn CR. Insulin signaling and (D) Ketoacidosis (D) After a 3-day fast the regulation of glucose and lipid me- 7. Delta cells of the islets of Langerhans tabolism. Nature 2001;414:799–806. produce which hormone? SUGGESTED READING Virkamaki A, Ueki K, Kahn CR. Protein- (A) Insulin American Diabetes Association Web site. protein interaction in insulin signaling (B) Glucagon Available at: http://www.diabetes.org. and the molecular mechanisms of in- (C) Acetylcholine Elmendorf JS, Pessin JE. Insulin signaling sulin resistance. J Clin Invest (D) Somatostatin regulating the trafficking and plasma 1999;103:931–943. 8. The insulin-glucagon ratio would be membrane fusion of GLUT4-contain- Wilson JD, Foster DW, Kronenberg HM, expected to be lowest ing intracellular vesicles. Exp Cell Res Larsen PR, eds. Williams Textbook of (A) Immediately after a high- 1999:253:55–62. Endocrinology. 9th Ed. Philadelphia: carbohydrate meal Porte D Jr, Sherwin RS, eds. Ellenberg & WB Saunders, 1998.

Endocrine Regulation of CHAPTER 36 36 Calcium, Phosphate, and Bone Metabolism Daniel E. Peavy, Ph.D. CHAPTER OUTLINE ■ AN OVERVIEW OF CALCIUM AND PHOSPHORUS IN ■ REGULATION OF PLASMA CALCIUM AND THE BODY PHOSPHATE CONCENTRATIONS ■ MECHANISMS OF CALCIUM AND PHOSPHATE ■ ABNORMALITIES OF BONE MINERAL HOMEOSTASIS METABOLISM KEY CONCEPTS 1. When plasma calcium levels fall below normal, sponta- vital role in calcium and phosphate homeostasis, and acts neous action potentials can be generated in nerves, lead- on bones, kidneys, and intestine to raise the plasma cal- ing to tetany of muscles, which, if severe, can result in cium concentration and lower the plasma phosphate con- death. centration. 2. About half of the circulating calcium is in the free or ion- 5. Vitamin D is converted to the active hormone 1,25-dihy- ized form, about 10% is bound to small anions, and about droxycholecalciferol by sequential hydroxylation reactions 40% is bound to plasma proteins. Most of the phosphorus in the liver and kidneys. This hormone stimulates intestinal circulates free as orthophosphate. calcium absorption and, thereby, raises the plasma cal- 3. The majority of ingested calcium is not absorbed by the GI cium concentration. tract and leaves the body via the feces; by contrast, phos- 6. Calcitonin, a polypeptide hormone produced by the thyroid phate is almost completely absorbed by the GI tract and glands, tends to lower the plasma calcium concentration, leaves the body mostly via the urine. but its physiological importance in humans has been ques- 4. Secretion of parathyroid hormone (PTH), a polypeptide tioned. hormone produced by the parathyroid glands, is stimu- 7. Osteoporosis, osteomalacia and rickets, and Paget’s disease lated by a decrease in plasma-ionized calcium. PTH plays a are the most common forms of metabolic bone disease. he plasma calcium concentration is among the most lead to soft tissue calcification and formation of stones. Tclosely regulated of all physiological parameters in the Phosphorus also plays important roles in the body. body. Typically, it varies by only 1 to 2% daily or even weekly. Such stringent regulation in a biological system usually implies that the parameter plays an important role Calcium Plays Key Roles in Nerve and Muscle in one or more critical processes. Excitation, Muscle Contraction, Enzyme Function, Phosphate also plays a variety of important roles in the and Bone Mineral Balance body, although its concentration is not as tightly regulated Calcium affects nerve and muscle excitability, neurotrans- as that of calcium. Many of the factors involved in regulat- mitter release from axon terminals, and excitation-contrac- ing calcium also affect phosphate. tion coupling in muscle cells. It serves as a second or third messenger in several intracellular signal transduction path- ways. Some enzymes use calcium as a cofactor, including some in the blood-clotting cascade. Finally, calcium is a AN OVERVIEW OF CALCIUM AND major constituent of bone. PHOSPHORUS IN THE BODY Of all these roles, the one that demands the most care- Calcium plays a key role in many physiologically important ful regulation of plasma calcium is the effect of calcium on processes. A significant decrease in plasma calcium can rap- nerve excitability. Calcium affects the sodium permeability idly lead to death. A chronic increase in plasma calcium can of nerve membranes, which influences the ease with which 634

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 635 action potentials are triggered. Low plasma calcium can lead to the generation of spontaneous action potentials in TABLE 36.2 Inorganic Constituents of Bone nerves. When motor neurons are affected, tetany of the muscles of the motor unit may occur; this condition is Percentage of Total Body Content called hypocalcemic tetany. Latent tetany may be revealed Constituent Present in Bone in certain diagnostically important signs. Trousseau’s sign Calcium 99 is a characteristic spasm of the muscles of the forearm that Phosphate 86 causes flexion of the wrist and thumb and extension of the Carbonate 80 fingers. It may occur spontaneously or be elicited by infla- Magnesium 50 tion of a blood pressure cuff placed on the upper arm. Sodium 35 Chvostek’s sign is a unilateral spasm of the facial muscles Water 9 that can be elicited by tapping the facial nerve at the point where it crosses the angle of the jaw. Calcium and Phosphorus Are Present Phosphate Participates in pH Buffering and Is a Major Constituent of Macromolecules and Bones in the Plasma in Several Forms In humans, the normal plasma calcium concentration is 9.0 Phosphorus (usually as phosphate) also participates in many important metabolic processes. Phosphate serves as an im- to 10.5 mg/dL. Plasma calcium exists in three forms: ion- portant component of intracellular pH buffering and various ized or free calcium (50% of the total), protein-bound cal- metabolic intermediates. DNA, RNA, and phosphoproteins cium (40%), and calcium bound to small diffusible anions, all contain phosphate as an integral part of their structure. such as citrate, phosphate, and bicarbonate (10%). The as- Phosphate is also a major component of bones. sociation of calcium with plasma proteins is pH-dependent. At an alkaline pH, more calcium is bound; the opposite is true at an acidic pH. The Distributions of Calcium and Plasma phosphorus concentrations may fluctuate signif- Phosphorus Differ icantly during the course of a day, from 50 to 150% of the average value for any particular individual. In adults, the Table 36.1 shows the relative distributions of calcium and normal range of plasma concentrations is 3.0 to 4.5 mg/dL phosphate in a healthy adult. The average adult body con- (expressed in terms of milligrams of phosphorus). tains approximately 1 to 2 kg of calcium, roughly 99% of it Phosphorus circulates in the plasma primarily as inor- in bones. Despite its critical role in excitation-contraction ganic orthophosphate (PO 4 ). At a normal blood pH of 7.4, coupling, only about 0.3% of total body calcium is located in 80% of the phosphate is in the HPO 4 form and 20% is in 2– muscle. About 0.1% of total calcium is in extracellular fluid. the H 2 PO 4 form. Nearly all plasma inorganic phosphate Of the roughly 600 g of phosphorus in the body, most is ultrafilterable. In addition to free orthophosphate, phos- is in bones (86%). Compared with calcium, a much larger phate is present in small amounts in the plasma in organic percentage of phosphorus is located in cells (14%). The form, such as in hexose or lipid phosphates. amount of phosphorus in extracellular fluid is rather low (0.08% of body content). Bones also contain a relatively high percentage of the to- The Homeostatic Pathways for Calcium tal body content of several other inorganic substances and Phosphorus Differ Quantitatively (Table 36.2). About 80% of the total carbonate in the body is located in bones. This carbonate can be mobilized into Both calcium and phosphate are obtained from the diet. The the blood to combat acidosis; thus, bone participates in pH ultimate fate of each substance is determined primarily by buffering in the body. Long-standing uncorrected acidosis the gastrointestinal (GI) tract, the kidneys, and the bones. can result in considerable loss of bone mineral. Significant percentages of the body’s magnesium and sodium and Calcium Handling by the GI Tract, Kidneys, and Bones. nearly 10% of its total water content are in bones. The approximate tissue distribution and average daily flux of calcium among tissues in a healthy adult are shown in Figure 36.1. Dietary intakes may vary widely, but an “aver- age” diet contains approximately 1,000 mg/day of calcium. Intakes up to twice that amount are usually well tolerated, Body Content and Tissue Distribution of TABLE 36.1 Calcium and Phosphorus in a Healthy but excessive calcium intake can result in soft tissue calcifi- Adult cation or kidney stones. Only about one third of ingested calcium is actually absorbed from the GI tract; the remain- Calcium Phosphorus der is excreted in the feces. The efficiency of calcium up- Total Body Content 1,300 g 600 g take from the GI tract varies with the individual’s physio- Relative Tissue Distribution logical status. The percentage uptake of calcium may be (% of total body content) increased in young growing children and pregnant or nurs- Bones and teeth 99% 86% ing women; often it is reduced in older adults. Extracellular fluid 0.1% 0.08% Figure 36.1 also indicates that approximately 150 Intracellular fluid 1.0% 14% mg/day of calcium actually enter the GI tract from the

636 PART IX ENDOCRINE PHYSIOLOGY Cells mg/day of calcium excreted in the urine represent only 11,000 mg about 1% of the calcium initially filtered by the kidneys; the remaining 99% is reabsorbed and returned to the blood. Therefore, small changes in the amount of calcium reab- sorbed by the kidneys can have a dramatic impact on cal- Calcium in diet cium homeostasis. 1,000 mg/day Bone 1,000 g Phosphate Handling by the GI Tract, Kidneys, and Bones. Figure 36.2 shows the overall daily flux of phosphate in the body. A typical adult ingests approximately 1,400 mg/day of phosphorus. In marked contrast to calcium, most (1,300 Absorption Deposition mg/day) of this phosphorus is absorbed from the GI tract, 300 mg/day 500 mg/day typically as inorganic phosphate. There is an obligatory Extracellular fluid contribution of phosphorus to the contents of the GI tract Secretion 900 mg Resorption (about 200 mg/day), much like that for calcium, resulting in 150 mg/day 500 mg/day a net uptake of phosphorus of 1,100 mg/day and excretion of 300 mg/day via the feces. Thus, the majority of ingested phosphate is absorbed from the GI tract and little passes Glomerular through to the feces. filtrate Reabsorbed 10,000 mg/day 9,850 mg/day Cells Fecal excretion 84 g 850 mg/day Kidney Phosphorus in diet 1,400 mg/day Bone 500 g Absorption Deposition Urinary excretion 1,300 mg/day 200 mg/day 150 mg/day Extracellular Typical daily exchanges of calcium between fluid FIGURE 36.1 different tissue compartments in a healthy Secretion 900 mg Resorption adult. Fluxes of calcium (mg/day) are shown in color. Total cal- 200 mg/day 200 mg/day cium content in each compartment is shown in black. Note that the majority of ingested calcium is eliminated from the body via the feces. Glomerular filtrate Reabsorbed 6,000 mg/day 4,900 mg/day body. This component of the calcium flux partly results from sloughing of mucosal cells that line the GI tract and Fecal excretion also from calcium that accompanies various secretions into 300 mg/day Kidney the GI tract. This component of calcium metabolism is rel- atively constant, so the primary determinant of net calcium uptake from the GI tract is calcium absorption. Intestinal absorption is important in regulating calcium homeostasis. Bone in an average individual contains approximately 1,000 g of calcium. Bone mineral is constantly resorbed and deposited in the remodeling process. As much as 500 mg/day of calcium may flow in and out of the bones (see Fig. 36.1). Since bone calcium serves as a reservoir, both Urinary excretion bone resorption and bone formation are important in regu- 1,100 mg/day lating plasma calcium concentration. Typical daily exchanges of phosphorus be- In overall calcium balance, the net uptake of calcium FIGURE 36.2 tween different tissue compartments in a from the GI tract presents a daily load of calcium that will healthy adult. Fluxes of phosphorus (mg/day) are shown in eventually require elimination. The primary route of elimi- color. Total phosphorus content in each compartment is shown nation is via the urine, and therefore, the kidneys play an in black. Note that the majority of ingested phosphorus is ab- important role in regulating calcium homeostasis. The 150 sorbed and eventually eliminated from the body via the urine.

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 637 Because most ingested phosphate is absorbed, phos- bound to small diffusible anions. The remaining 40% of the phate homeostasis is greatly influenced by renal excretory total calcium is bound to plasma proteins and is not filter- mechanisms. Since the majority of circulating phosphate is able by the glomeruli. Ordinarily, 99% of the filtered cal- readily filtered in the kidneys, tubular phosphate reabsorp- cium is reabsorbed by the kidney tubules and returned to tion is a major process regulating phosphate homeostasis. the plasma. Reabsorption occurs both in the proximal and distal tubules and in the loop of Henle. Approximately 60% of filtered calcium is reabsorbed in the proximal tubule, MECHANISMS IN CALCIUM AND 30% in the loop of Henle, and 9% in the distal tubule; the PHOSPHATE HOMEOSTASIS remaining 1% is excreted in the urine. Renal calcium excre- tion is controlled primarily in the late distal tubule; As indicated above, the GI tract, kidneys, and bone each parathyroid hormone stimulates calcium reabsorption here, play a role in the regulation of calcium and phosphate promoting calcium retention and lowering urinary calcium. homeostasis. Parathyroid hormone is an important regulator of plasma calcium concentration. Calcium and Phosphate Are Absorbed Renal Handling of Phosphate. Most ingested phosphate is Primarily by the Small Intestine absorbed from the GI tract, and the primary route of excre- Calcium absorption in the small intestine occurs by both tion of this phosphate is via the urine. Therefore, the kidneys active transport and diffusion. The relative contribution of play a key role in regulating phosphate homeostasis. Ordi- each process varies with the region and with total calcium narily about 85% of filtered phosphate is reabsorbed and intake. Uptake of calcium by active transport predominates 15% is excreted in the urine. Phosphate reabsorption occurs in the duodenum and jejunum; in the ileum, simple diffu- via active transport, mainly in the proximal tubule where 65 sion predominates. The relative importance of active trans- to 80% of filtered phosphate is reabsorbed. Parathyroid hor- port in the duodenum and jejunum versus passive diffusion mone inhibits phosphate reabsorption in the proximal tubule in the ileum depends on several factors. At very high levels and has a major regulatory effect on phosphate homeostasis. of calcium intake, active transport processes are saturated It increases urinary phosphate excretion, leading to the con- and most of the uptake occurs in the ileum, partly because dition of phosphaturia, with an accompanying decrease in of its greater length, compared with other intestinal seg- the plasma phosphate concentration. ments. With moderate or low calcium intake, however, ac- tive transport predominates because the gradient for diffu- Substantial Amounts of Calcium and Phosphate sion is low. Active transport is the regulated variable in controlling Enter and Leave Bone Each Day calcium uptake from the small intestine. Metabolites of vi- Although bone may be considered as being a relatively inert tamin D provide a regulatory signal to increase intestinal material, it is active metabolically. Considerable amounts of calcium absorption. Under the influence of 1,25-dihydrox- calcium and phosphate both enter and exit bone each day, ycholecalciferol, calcium-binding proteins in intestinal mu- and these processes are hormonally controlled. cosal cells increase in number, enhancing the capacity of these cells to transport calcium actively (see Chapter 27). Composition of Bone. Mature bone can be simply de- The small intestine is also a primary site for phosphate ab- scribed as inorganic mineral deposited on an organic frame- sorption. Uptake occurs by active transport and passive diffu- work. The mineral portion of bone is composed largely of sion, but active transport is the primary mechanism. As indi- calcium phosphate in the form of hydroxyapatite crystals, cated in Figure 36.2, phosphate is efficiently absorbed from the small intestine; typically, 80% or more of ingested phos- which have the general chemical formula Ca 10 (PO 4 ) 6 (OH) 2 . The mineral portion of bone typically com- phate is absorbed. However, phosphate absorption from the prises about 25% of its volume, but because of its high den- small intestine is regulated very little. To a minor extent, ac- sity, the mineral fraction is responsible for approximately tive transport of phosphate is coupled to calcium transport. half the weight of bone. Bone contains considerable Therefore, when active transport of calcium is low, as with vi- amounts of the body’s content of carbonate, magnesium, tamin D deficiency, phosphate absorption is also low. and sodium in addition to calcium and phosphate (see Table 36.2). The Kidneys Play an Important Role in Regulating The organic matrix of bone on which the bone mineral Plasma Concentrations of Calcium and Phosphate is deposited is called osteoid. Type I collagen is the pri- mary constituent of osteoid, comprising 95% or more. Col- As a result of regulating the urinary excretion of calcium lagen in bone is similar to that of skin and tendons, but and phosphate, the kidneys are in a key position to regulate bone collagen exhibits some biochemical differences that the total body balance of these two ions. Hormones are an impart increased mechanical strength. The remaining non- important signal to the kidneys to direct the excretion or collagen portion (5%) of organic matter is referred to as retention of calcium and phosphate. ground substance. Ground substance consists of a mixture of various proteoglycans, high-molecular-weight com- Renal Handling of Calcium. As discussed in Chapter 24, pounds consisting of different types of polysaccharides filterable calcium comprises about 60% of the total calcium linked to a polypeptide backbone. Typically, they are 95% in the plasma. It consists of free calcium ions and calcium or more carbohydrate.

638 PART IX ENDOCRINE PHYSIOLOGY Electron microscopic study of bone reveals needle-like As osteoblasts are progressively incorporated into min- hydroxyapatite crystals lying alongside collagen fibers. This eralized bone, they lose much of their bone-forming ability orderly association of hydroxyapatite crystals with the colla- and become quiescent. At this point they are called osteo- gen fibers is responsible for the strength and hardness char- cytes. Many of the cytoplasmic connections in the os- acteristic of bone. A loss of either bone mineral or organic teoblast stage are maintained into the osteocyte stage. matrix greatly affects the mechanical properties of bone. These connections become visible channels or canaliculi Complete demineralization of bone leaves a flexible collagen that provide direct contact for osteocytes deep in bone framework, and the complete removal of organic matrix with other osteocytes and with the bone surface. It is gen- leaves a bone with its original shape, but extremely brittle. erally believed that these canaliculi provide a mechanism for the transfer of nutrients, hormones, and waste products Cell Types Involved in Bone Formation and Bone Re- between the bone surface and its interior. sorption. The three principal cell types involved in bone Osteoclasts are cells responsible for bone resorption. formation and bone resorption are osteoblasts, osteocytes, They are large, multinucleated cells located on bone sur- and osteoclasts (Fig. 36.3). faces. Osteoclasts promote bone resorption by secreting Osteoblasts are located on the bone surface and are re- acid and proteolytic enzymes into the space adjacent to sponsible for osteoid synthesis. Like many cells that ac- the bone surface. Surfaces of osteoclasts facing bone are tively synthesize proteins for export, osteoblasts have an ruffled to increase their surface area and promote bone abundant rough ER and Golgi apparatus. Cells actively en- resorption. Bone resorption is a two-step process. First, gaged in osteoid synthesis are cuboidal, while those less ac- osteoclasts create a local acidic environment that in- tive are more flattened. Numerous cytoplasmic processes creases the solubility of surface bone mineral. Second, connect adjacent osteoblasts on the bone surface and con- proteolytic enzymes secreted by osteoclasts degrade the nect osteoblasts with osteocytes deeper in the bone. Os- organic matrix of bone. teoid produced by osteoblasts is secreted into the space ad- jacent to the bone. Eventually, new osteoid becomes Bone Formation and Bone Remodeling. Early in fetal mineralized, and in the process, osteoblasts become sur- development, the skeleton consists of little more than a car- rounded by mineralized bone. tilaginous model of what will later form the bony skeleton. The process of replacing this cartilaginous model with ma- ture, mineralized bone begins in the center of the cartilage and progresses toward the two ends of what will later form the bone. As mineralization progresses, the bone increases in thickness and in length. The epiphyseal plate is a region of growing bone of par- ticular interest because it is here that the elongation and growth of bones occurs after birth. Histologically, the epi- physeal plate shows considerable differences between its leading and trailing edges. The leading edge consists pri- marily of chondrocytes, which are actively engaged in the synthesis of cartilage of the epiphyseal plate. These cells gradually become engulfed in their own cartilage and are replaced by new cells on the cartilage surface, allowing the process to continue. The cartilage gradually becomes calci- fied, and the embedded chondrocytes die. The calcified cartilage begins to erode, and osteoblasts migrate into the area. Osteoblasts secrete osteoid, which eventually be- comes mineralized, and new mature bone is formed. In the epiphyseal plate, therefore, the continuing processes of cartilage synthesis, calcification, erosion, and osteoblast in- vasion result in a zone of active bone formation that moves away from the middle or center of the bone toward its end. Chondrocytes of epiphyseal plates are controlled by hormones. Insulin-like growth factor I (IGF-I), primarily produced by the liver in response to growth hormone, serves as a primary stimulator of chondrocyte activity and, ultimately, of bone growth. Insulin and thyroid hormones provide an additional stimulus for chondrocyte activity. Beginning a few years after puberty, the epiphyseal plates in long bones (as in the legs and arms) gradually become less responsive to hormonal stimuli and, eventu- ally, are totally unresponsive. This phenomenon is re- The location and relationship of the three FIGURE 36.3 primary cell types involved in bone me- ferred to as closure of the epiphyses. In most individuals, tabolism. epiphyseal closure is complete by about age 20; adult

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 639 height is reached at this point, since further linear growth Hormonal Mechanisms Provide High-Capacity, is impossible. Not all bones undergo closure. For exam- Long-Term Regulation of Plasma Calcium and ple, those in the fingers, feet, skull, and jaw remain re- Phosphate Concentrations sponsive, which accounts for the skeletal changes seen in acromegaly, the condition of growth hormone overpro- The hormonal mechanisms described here have a large ca- duction (see Chapter 32). pacity and the ability to make long-term adjustments in cal- The flux of calcium and phosphate into and out of bone cium and phosphate fluxes, but they do not respond in- each day reflects a turnover of bone mineral and changes in stantaneously. It may take several minutes or hours for the bone structure generally referred to as remodeling. Bone re- response to occur and adjustments to be made. However, modeling occurs along most of the outer surface of the bone, these are the principal mechanisms that regulate plasma making it either thinner or thicker, as required. In long calcium and phosphate concentrations. bones, remodeling can also occur along the inner surface of the bone shaft, next to the marrow cavity. Remodeling is an The Chemistry of Parathyroid Hormone, Calcitonin, and adaptive process that allows bone to be reshaped to meet 1,25-Dihydroxycholecalciferol and the Regulation of changing mechanical demands placed on the skeleton. It also Their Production. One of the primary regulators of allows the body to store or mobilize calcium rapidly. plasma calcium concentrations is parathyroid hormone (PTH). PTH is an 84-amino acid polypeptide produced by the parathyroid glands. Synthetic peptides containing the first 34 amino terminal residues appear to be as active as the REGULATION OF PLASMA CALCIUM native hormone. AND PHOSPHATE CONCENTRATIONS There are two pairs of parathyroid glands, located on Regulatory mechanisms for calcium include rapid nonhor- the dorsal surface of the left and right lobes of the thyroid monal mechanisms with limited capacity and somewhat gland. Because of this close proximity, damage to the slower hormonally regulated mechanisms with much parathyroid glands or to their blood supply may occur dur- greater capacity. There are also similar mechanisms in- ing surgical removal of the thyroid gland. volved in regulating plasma phosphate concentrations. The primary physiological stimulus for PTH secretion is a decrease in plasma calcium. Figure 36.4 shows the relation- ship between serum parathyroid hormone concentration Nonhormonal Mechanisms Can Rapidly and total plasma calcium concentration. It is actually a de- Buffer Small Changes in Plasma Concentrations crease in the ionized calcium concentration that triggers an of Free Calcium increase in PTH secretion. The net effect of PTH is to in- The calcium bound to plasma proteins and a small fraction of that in bone mineral can help prevent a rapid decrease in the plasma calcium concentration. 4,000 Protein-Bound Calcium. The association of calcium with proteins is a simple, reversible, chemical equilibrium process. 700 Protein-bound calcium, therefore, has the capacity to serve as a buffer of free plasma calcium concentrations. This effect 3,000 600 is rapid and does not require complex signaling pathways; however, the capacity is limited, and the mechanism cannot 500 serve a long-term role in calcium homeostasis. Serum PTH (pg/mL) PTH CT A Readily Exchangeable Pool of Calcium in Bones. 2,000 400 Serum CT (pg/mL) Recall that approximately 99% of total body calcium is 300 present in bones, and a healthy adult body has about 1 to 2 kg of calcium. Most of the calcium in bones exists as 1,000 200 mature, hardened bone mineral that is not readily ex- changeable but can be moved into the plasma via hor- monal mechanisms (described below). However, approx- Undetectable imately 1% (or 10 g) of the calcium in bones is in a simple Undetectable chemical equilibrium with plasma calcium. This readily exchangeable calcium source is primarily located on the 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 surface of newly formed bones. Any change in free cal- Serum calcium (mg/dL) cium in the plasma or extracellular fluid results in a shift of calcium either into or out of the bone mineral until a FIGURE 36.4 Effect of changes in plasma calcium on parathyroid hormone (PTH) and calcitonin new equilibrium is reached. Although this mechanism, (CT) secretion. (Modified from Arnaud, CD, Littledike T, Tsao like that described above, provides for a rapid defense HS. Simultaneous measurements of calcitonin and parathyroid against changes in free calcium concentrations, it is lim- hormone in the pig. In: Taylor S, Foster GV, eds. Proceedings of ited in capacity and can provide for only short-term ad- the Symposium on Calcitonin and C Cells. London: Heinemann, justments in calcium homeostasis. 1969, p. 99).

640 PART IX ENDOCRINE PHYSIOLOGY crease the flow of calcium into plasma, and return the volved, it is difficult to specify a minimum exposure time. plasma calcium concentration toward normal. However, exposure to moderately bright sunlight for 30 to Calcitonin (CT) is a 32-amino acid polypeptide. Also 120 min/day usually provides enough vitamin D to satisfy known as thyrocalcitonin, CT is produced by parafollicu- the body’s needs without any dietary supplementation. lar cells of the thyroid gland (see Fig. 33.1). Unlike PTH, Vitamins D 3 and D 2 are by themselves relatively inactive. for which only the initial amino terminal segment is re- However, they undergo a series of transformations in the quired, the full polypeptide is required for CT activity. liver and kidneys that convert them into powerful calcium- Salmon calcitonin differs from human calcitonin in 9 of 32 regulatory hormones (see Fig. 36.6). The first step occurs in amino acid residues and is 10 times more potent than hu- the liver and involves addition of a hydroxyl group to carbon man CT in its hypocalcemic effect. The higher potency 25, to form 25-hydroxycholecalciferol (25-OH D 3 ). This re- may be due to a greater affinity for receptors and slower action is largely unregulated, although certain drugs and liver degradation by peripheral tissues. CT is often used clini- diseases may affect this step. Next, 25-hydroxycholecalcif- cally as a synthetic peptide matching the sequence of erol is released into the blood, and it undergoes a second hy- salmon calcitonin. droxylation reaction on carbon 1 in the kidney. The product In contrast to PTH, CT secretion is stimulated by an in- is 1,25-dihydroxycholecalciferol, also known as 1,25-dihy- crease in plasma calcium (see Fig. 36.4). Hormones of the droxyvitamin D 3 or calcitriol, the principal hormonally ac- GI tract, especially gastrin, also promote CT secretion. Be- tive form of the vitamin. The biological activity of 1,25-di- cause the net effect of CT is to promote calcium deposition hydroxycholecalciferol is approximately 100 to 500 times in bone, the stimulation of CT secretion by GI hormones greater than that of 25-hydroxycholecalciferol. The reaction provides an additional mechanism for facilitating the up- in the kidney is catalyzed by the enzyme 1-hydroxylase, take of calcium into bone after the ingestion of a meal. which is located in tubule cells. The third key hormone involved in regulating plasma The final step in 1,25-dihydroxycholecalciferol forma- calcium is vitamin D 3 (cholecalciferol). More precisely, a tion is highly regulated. The activity of 1-hydroxylase is metabolite of vitamin D 3 serves as a hormone in calcium regulated primarily by PTH, which stimulates its activity. homeostasis. The D vitamins, a group of lipid-soluble com- Therefore, if plasma calcium levels fall, PTH secretion in- pounds derived from cholesterol, have long been known to creases; in turn, PTH promotes the formation of 1,25-di- be effective in the prevention of rickets. Research during hydroxycholecalciferol. In addition, enzyme activity in- the past 30 years indicates that vitamin D exerts it effects creases in response to a decrease in plasma phosphate. This through a hormonal mechanism. does not appear to involve any intermediate hormonal sig- Figure 36.5 shows the structure of vitamin D 3 and the re- nals but apparently involves direct activation of either the lated compound vitamin D 2 (ergocalciferol). Ergocalciferol enzyme or cells in which the enzyme is located. Both a de- is the form principally found in plants and yeasts and is crease in plasma calcium, which triggers PTH secretion, commonly used to supplement human foods because of its and a decrease in circulating phosphate result in the activa- relative availability and low cost. Although it is less potent tion of 1-hydroxylase and an increase in 1,25-dihydroxy- on a mole-per-mole basis, vitamin D 2 undergoes the same cholecalciferol synthesis. metabolic conversion steps and, ultimately, produces the same biological effects as vitamin D 3 . The physiological ac- tions of vitamin D 3 also apply to vitamin D 2 . The Actions of Parathyroid Hormone, Calcitonin, and Most hormones gener- Vitamin D 3 can be provided by the diet or formed in the 1,25-Dihydroxycholecalciferol. ally improve the quality of life and the chance for survival skin by the action of ultraviolet light on a precursor, 7-de- when an animal is placed in a physiologically challenging hydrocholesterol, derived from cholesterol (Fig. 36.6). In situation. However, PTH is essential for life. The complete many countries where food is not systematically supple- absence of PTH causes death from hypocalcemic tetany mented with vitamin D, this pathway provides the major within just a few days. The condition can be avoided with source of vitamin D. Because of the number of variables in- hormone replacement therapy. The net effects of PTH on plasma calcium and phos- phate and its sites of action are shown in Figure 36.7. PTH causes an increase in plasma calcium concentration while decreasing plasma phosphate. This decrease in phosphate concentration is important with regard to calcium home- ostasis. At normal plasma concentrations, calcium and phosphate are at or near chemical saturation levels. If PTH were to increase both calcium and phosphate levels, they would simply crystallize in bone or soft tissues as calcium phosphate, and the necessary increase in plasma calcium concentration would not occur. Thus, the effect of PTH to lower plasma phosphate is an important aspect of its role in regulating plasma calcium. Parathyroid hormone has several important actions in the The structures of vitamin D 3 and vitamin kidneys (see Fig. 36.7). It stimulates calcium reabsorption in FIGURE 36.5 D 2 . Note that they differ only by a double bond the thick ascending limb and late distal tubule, decreasing between carbons 22 and 23 and a methyl group at position 24. calcium loss in the urine and increasing plasma concentra-

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 641 The conver- FIGURE 36.6 sion pathway of vitamin D 3 into 1,25-dihy- droxycholecalciferol [1,25- (OH) 2 D 3 ]. tions. It also inhibits phosphate reabsorption in the proximal lar cells) does not lead to overt clinical abnormalities of cal- tubule, leading to increased urinary phosphate excretion and cium homeostasis. Second, CT hypersecretion, such as a decrease in plasma phosphate. Another important effect of from thyroid tumors involving parafollicular cells, does not PTH is to increase the activity of kidney 1-hydroxylase, cause any overt problems. On a daily basis, calcitonin prob- which is involved in forming active vitamin D. ably only fine-tunes the calcium regulatory system. In bone, PTH activates osteoclasts to increase bone re- The overall action of calcitonin is to decrease both cal- sorption and the delivery of calcium from bone into plasma cium and phosphate concentrations in plasma (Fig. 36.8). (see Fig. 36.7). In addition to stimulating active osteoclasts, The primary target of CT is bone, although some lesser ef- PTH stimulates the maturation of immature osteoclasts into fects also occur in the kidneys. In the kidneys, CT de- mature, active osteoclasts. PTH also inhibits collagen syn- creases the tubular reabsorption of calcium and phosphate. thesis by osteoblasts, resulting in decreased bone matrix This leads to an increase in urinary excretion of both cal- formation and decreased flow of calcium from plasma into cium and phosphate and, ultimately, to decreased levels of bone mineral. The actions of PTH to promote bone re- both ions in the plasma. In bones, CT opposes the action of sorption are augmented by 1,25-dihydroxycholecalciferol. PTH on osteoclasts by inhibiting their activity. This leads PTH does not appear to have any major direct effects on to decreased bone resorption and an overall net transfer of the GI tract. However, because it increases active vitamin calcium from plasma into bone. Calcitonin has little or no D formation, it ultimately increases the absorption of both direct effect on the GI tract. calcium and phosphate from the GI tract (see Fig. 36.7). The net effect of 1,25-dihydroxycholecalciferol is to in- Calcitonin is important in several lower vertebrates, but crease both calcium and phosphate concentrations in despite its many demonstrated biological effects in humans, plasma (Fig. 36.9). The activated form of vitamin D prima- it appears to play only a minor role in calcium homeostasis. rily influences the GI tract, although it has actions in the This conclusion mostly stems from two lines of evidence. kidneys and bones as well. First, CT loss following surgical removal of the thyroid In the kidneys, 1,25-dihydroxycholecalciferol increases gland (and, therefore, removal of CT-secreting parafollicu- the tubular reabsorption of calcium and phosphate, pro-

642 PART IX ENDOCRINE PHYSIOLOGY Plasma calcium Parathyroid glands PTH secretion Plasma PTH Kidneys Phosphate 1,25-(OH) 2 D 3 Bone reabsorption formation resorption Calcium reabsorption Urinary excretion Plasma of phosphate 1,25-(OH) 2 D 3 Urinary excretion Release of calcium of calcium into plasma Intestine Calcium absorption Effects of parathyroid hormone FIGURE 36.7 (PTH) on calcium and phosphate Plasma phosphate Plasma calcium metabolism. Plasma calcium Parafollicular cells CT secretion Plasma CT Kidneys Phosphate Calcium Bone reabsorption reabsorption resorption Urinary excretion Urinary excretion Calcium of phosphate of calcium release Effects of calcitonin (CT) on calcium Plasma phosphate Plasma calcium FIGURE 36.8 and phosphate metabolism.

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 643 Plasma calcium Plasma PTH Renal 1α-hydroxylase activity 1,25-(OH) 2 D 3 formation Plasma 1,25-(OH) 2 D 3 Kidneys Bone Phosphate Calcium promotes PTH reabsorption reabsorption action Intestine Urinary excretion Phosphate Calcium of phosphate absorption absorption Urinary excretion of calcium FIGURE 36.9 Effects of 1,25-dihydroxycholecalciferol [1,25-(OH) 2 D 3 ] on calcium and phos- Plasma phosphate Plasma calcium phate metabolism. moting the retention of both ions in the body. However, cus Box 36.1). Osteoporosis involves a reduction in total this is a weak and probably only minor effect of the hor- bone mass with an equal loss of both bone mineral and or- mone. In bones, the hormone promotes actions of PTH on ganic matrix. Several factors are known to contribute di- osteoclasts, increasing bone resorption (see Fig. 36.9). rectly to osteoporosis. Long-term dietary calcium defi- In the gastrointestinal tract, 1,25-dihydroxycholecalcif- ciency can lead to osteoporosis because bone mineral is erol stimulates calcium and phosphate absorption by the mobilized to maintain plasma calcium levels. Vitamin C de- small intestine, increasing plasma concentrations of both ficiency also can result in a net loss of bone because vitamin ions. This effect is mediated by increased production of cal- C is required for normal collagen synthesis to occur. A de- cium transport proteins resulting from gene transcription fect in matrix production and the inability to produce new events and usually requires several hours to appear. bone eventually result in a net loss of bones. For reasons that are not entirely understood, a reduction in the me- chanical stress placed on bone can lead to bone loss. Im- ABNORMALITIES OF BONE mobilization or disuse of a limb, such as with a cast or paral- MINERAL METABOLISM ysis, can result in localized osteoporosis of the affected limb. Space flight can produce a type of disuse osteoporo- There are several metabolic bone diseases, all typified by sis resulting from the condition of weightlessness. ongoing disruption of the normal processes of either bone Most commonly, osteoporosis is associated with ad- formation or bone resorption. The conditions most fre- vancing age in both men and women, and it cannot be as- quently encountered clinically are osteoporosis, osteomala- signed to any specific definable cause. For several rea- cia, and Paget’s disease. sons, women are more prone to develop the disease than men. Figure 36.10 shows the average bone mineral con- tent (as grams of calcium) for men and women versus age. Osteoporosis Is a Reduction in Bone Mass Until about the time of puberty, males and females have Osteoporosis is a major health problem, particularly be- similar bone mineral content. However, at puberty, males cause older adults are more prone to this disorder and the begin to acquire bone mineral at a greater rate; peak bone average age of the population is increasing (see Clinical Fo- mass may be approximately 20% greater than that of

644 PART IX ENDOCRINE PHYSIOLOGY CLINICAL FOCUS BOX 36.1 The Toll of Osteoporosis What causes osteoporosis, and what can be done to Osteoporosis is often called the “silent disease” because prevent or treat the disease? While it is known that a diet bone loss initially occurs without symptoms. People may low in calcium or vitamin D, certain medications such as not know that they have significant bone loss until their glucocorticoids and anticonvulsants, and excessive inges- bones become so weak that a sudden strain, bump, or fall tion of aluminum-containing antacids can cause osteo- causes a fracture. Osteoporosis is a major public health porosis, in most cases, the exact cause is unknown. How- threat in the United States because it affects some 28 mil- ever, several identified risk factors associated with the lion Americans. Some 10 million individuals have been di- disease are being a woman (especially a postmenopausal agnosed with the disease and another 18 million have low woman); being Caucasian or Asian; being of advanced bone mass, placing them at increased risk for osteoporo- age; having a family history of the disease; having low sis. Approximately 80% of those affected by osteoporosis testosterone levels (in men); having an inactive lifestyle; are women. While osteoporosis is often thought of as an cigarette smoking; and an excessive use of alcohol. older person’s disease, it can strike at any age. The seeds A comprehensive program to help prevent osteoporo- of osteoporosis are sown in childhood, and it takes a life- sis includes a balanced diet rich in calcium and vitamin D, time of effort to prevent the disease. A frightening number regular weight-bearing exercise, a healthy lifestyle with no of children do not get sufficient exercise, vitamin D, and smoking or excessive alcohol use, and bone density test- calcium to ensure protection from developing the disease ing and medication when appropriate. Although at present later in life. there is no cure for osteoporosis, there are five FDA-ap- Osteoporosis is responsible for more than 1.5 million proved medications to either prevent or treat the disease in fractures annually, including 300,000 hip fractures, 700,000 women: estrogens; alendronate and risedronate (both bis- vertebral fractures, 250,00 wrist fractures, and 300,000 phosphonates); calcitonin; and raloxifene, a selective es- fractures at other sites. It is estimated that osteoporosis trogen receptor modulator. Although 20% of all osteo- costs some 10 to 15 billion dollars a year for hospitalization porosis cases occur in men, only alendronate and and nursing home care. Additional losses in wages and risedronate are currently FDA approved for use in men and productivity send these numbers far higher. Nearly one only for cases of corticosteroid-induced osteoporosis. third of people who have hip fractures end up in nursing Testosterone replacement therapy is often helpful in a man homes within a year; nearly 20% die within a year. with a low testosterone level. women. Maximum bone mass is attained between 30 and Osteomalacia and Rickets Result From 40 years of age and then tends to decrease in both sexes. Inadequate Bone Mineralization Initially this occurs at an approximately equivalent rate, but women begin to experience a more rapid bone min- Osteomalacia and rickets are characterized by the inade- eral loss at the time of menopause (about age 45 to 50). quate mineralization of new bone matrix, such that the ra- This loss appears to result from the decline in estrogen tio of bone mineral to matrix is reduced. As a result, bones secretion that occurs at menopause. Low-dose estrogen may have reduced strength and are subject to distortion in supplementation of postmenopausal women is usually ef- response to mechanical loads. When the disease occurs in fective in retarding bone loss without causing adverse ef- adults, it is called osteomalacia; when it occurs in children, fects. This condition of increased bone loss in women af- it is called rickets. In children, the condition often produces ter menopause is called postmenopausal osteoporosis a bowing of the long bones in the legs. In adults, it is often (see Clinical Focus Box 36.2). associated with severe bone pain. TABLE 36.3 Causes of Osteomalacia and Rickets Inadequate availability of Dietary deficiency or lack of exposure vitamin D to sunlight Fat-soluble vitamin malabsorption Defects in metabolic 25-Hydroxylation (liver) activation of vitamin D Liver disease Certain anticonvulsants, such as phenobarbital 1-Hydroxylation (kidney) Renal failure Hypoparathyroidism Impaired action of 1,25- Certain anticonvulsants Changes in bone calcium content as a func- dihydroxycholecalciferol 1,25-Dihydroxycholecalciferol FIGURE 36.10 tion of age in males and females. These on target tissues receptor defects changes can be roughly extrapolated into changes in bone mass Uremia and bone strength

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 645 CLINICAL FOCUS BOX 36.2 Cytokines, Estrogens, and Osteoporosis in bone remodeling by suppressing the formation of these It is well established that a decline in circulating levels of cytokines. As a result of its ability to interact with bone 17-estradiol is a major contributing factor in the develop- cells and their precursors to regulate local paracrine sig- ment of osteoporosis in postmenopausal women. Until re- naling mechanisms, estradiol produces anti-osteoporotic cently, specific mechanisms by which estradiol might in- effects in bone. fluence bone metabolism were largely unknown. Recent When estradiol is present, as in a premenopausal state, studies suggest that estradiol influences the production it acts as a governor to reduce cytokine production and and/or modulates the activity of several cytokines involved limit osteoclast activity. When estradiol levels are reduced, in regulating bone remodeling. the governor is lost, secretion of these cytokines increases, Normal bone remodeling involves a regulated balance and osteoclast formation and activity increase, resulting in between the processes of bone formation and bone re- increased bone resorption. sorption. Osteoclast-mediated bone resorption involves Current research efforts attempt to define more two processes: the activation of mature, functional osteo- clearly the specific source(s) and roles of the cytokines clasts and the recruitment and differentiation of osteoclast involved. The elucidation of these factors might allow precursors. In addition to PTH, the cytokines interleukin-1 the development of diagnostic tools, such as the assess- (IL-1) and tumor necrosis factor (TNF) are involved in the ment of cytokine levels, to monitor osteoporosis. In ad- activation of mature osteoclasts to cause bone resorption. dition, such knowledge should facilitate the develop- For maturation of osteoclast precursors, the cytokines ment of drugs that might interfere with cytokine action macrophage-colony stimulating factor (M-CSF) and inter- and potentially be of value in the treatment of osteo- leukin-6 (IL-6) appear to be involved. Estradiol plays a role porosis. The primary cause of osteomalacia and rickets is a defi- Paget’s Disease Leads to ciency in vitamin D activity. Vitamin D may be deficient in Disordered Bone Formation the diet; it may not be adequately absorbed by the small in- testine; it may not be converted into its hormonally active Paget’s disease affects about 3% of people older than 40. It form; or target tissues may not adequately respond to the is typified by disordered bone formation and resorption (re- active hormone (Table 36.3). Dietary deficiency is gener- modeling) and may occur at a single local site or at multiple ally not a problem in the United States, where vitamin D is sites in the body. Radiographs of affected bone often exhibit added to many foods; however it is a major health problem increased density, but the abnormal structure makes the in other parts of the world. Because the liver and kidneys bone weaker than normal. Often those with Paget’s disease are involved in converting vitamin D 3 into its hormonally experience considerable pain, and in severe cases, crippling active form, primary disease of either of these organs may deformities may lead to serious neurological complications. result in vitamin D deficiency. Impaired vitamin D actions The cause of the disease is not well understood. Both ge- are somewhat rare but can be produced by certain drugs. In netic and environmental factors (probably viral) appear to be particular, some anticonvulsants used in the treatment of important. Several therapies are available for treating the dis- epilepsy may produce osteomalacia or rickets with pro- ease, including treatment with CT, but these typically offer longed treatment. only temporary relief from pain and complications. REVIEW QUESTIONS DIRECTIONS: Each of the numbered (B) 50% (D) Bile items or incomplete statements in this (C) 60% 4. 1,25-Dihydroxycholecalciferol can be section is followed by answers or by (D) 100% formed in the body by metabolism of completions of the statement. Select the 2. A healthy individual consumed 1,000 g cholesterol. Which of the following is ONE lettered answer or completion that is of calcium during a 24-hour period. not either directly or indirectly BEST in each case. What is the major route of calcium involved in formation of 1,25- excretion from the body? dihydroxycholecalciferol? 1. As part of a routine physical exam, a (A) Urine (A) Bone patient’s serum electrolyte levels were (B) Sweat (B) Skin measured. Among the measurements, it (C) Feces (C) Kidney was determined that total plasma (D) Bile (D) Liver calcium concentration was 10.2 mg/dL. 3. The major route by which ingested 5. A 42-year-old woman develops an What percentage of total plasma phosphate leaves the body is via the autoimmune disease that damages her calcium is normally present as the free (A) Urine kidneys. Of the following conversions, Ca 2 ion? (B) Sweat which is most likely to be impaired in (A) 1% (C) Feces this person? (continued)

646 PART IX ENDOCRINE PHYSIOLOGY (A) Cholecalciferol to 7- bone, kidney, and indirectly the GI 9th Ed. Philadelphia: WB Saunders, dehydrocholesterol tract. The net result of PTH actions is 1998;1397–1496. that it tends to Bilezikian JP, Kurland ES, Rosen CJ. Male (B) Vitamin D 3 to vitamin D 2 (C) 25-Hydroxycholecalciferol to Raise plasma calcium and phosphate skeletal health and osteoporosis. 1,25-dihydroxycholecalciferol (A) Lower plasma calcium and Trends Endocrinol Metab (D) Calcium to hydroxyapatite phosphate 1999;10:244–250. 6. A 62-year-old woman stumbles on a (B) Lower plasma calcium and raise Griffin JE, Ojeda SR, eds. Textbook of En- crack in the sidewalk, falls, and breaks plasma phosphate docrine Physiology. 4th Ed. Oxford: her right hip. She suffers from a form (C) Raise plasma calcium and lower Oxford University Press, 2000. of metabolic bone disease in which plasma phosphate Henry HL. The 25-hydroxyvitamin D 1- there is an equivalent loss of bone hydroxylase. In: Feldman D, Glorieux mineral and organic matrix. What is FH, Pike JW, eds. Vitamin D. San this disease? SUGGESTED READING Diego: Academic Press, 1997. (A) Paget’s disease Aurbach GD, Marx SJ, Spiegel AM. National Osteoporosis Foundation Web (B) Rickets Parathyroid hormone, calcitonin, and site. Available at: http:/www.nof.org (C) Osteoporosis the calciferols. In: Wilson JD, Foster Norman AW, Litwack G. Hormones. (D) Osteomalacia DW, Kronenberg HM, Larsen PR, eds. 2nd Ed. San Diego: Academic Press, 7. Parathyroid hormone has effects on Williams Textbook of Endocrinology. 1997. CASE STUDIES FOR PART IX • • • CASE STUDY FOR CHAPTER 31 CASE STUDY FOR CHAPTER 32 Diabetes Mellitus Growth Hormone Charlie was diagnosed with type 1 diabetes mellitus A 6-year-old boy was brought to the clinic to be evalu- during the summer of his eighth year. Charlie’s mother ated for GH deficiency. The boy’s height is between 2 suspected he was drinking excessive amounts of fluids and 3 standard deviations below the average height for that summer; however, he was in and out of the house his age. Initial physical examination rules out head and visiting friends, and she couldn’t be certain. Dur- trauma, chronic illness, and malnutrition. The patient’s ing an afternoon at a friend’s birthday party, Charlie family history does not suggest similar short stature in drank nearly 3 quarts of fruit juice; his mother became immediate relatives. Thyroid hormones are normal. alerted to a possible problem and took him to their Questions family doctor. 1. Why would the doctor order a blood test for levels of Charlie’s tests are normal, except that he tests positive IGFBP3 and IGF-I? for glucose in the urine (dipstick test) and his fasting 2. The levels of IGFBP3 and IGF-I are below the normal range blood sugar is elevated (620 mg/dL). Plasma insulin (5 in this patient. What does this finding suggest? U/mL) and C-peptide (0.6 ng/mL) are reduced. Charlie is 3. What is GH resistance, and what measurements would sup- placed on a regimen of daily insulin injections, along port the presence of this problem? with monitoring of blood and urine glucose concentra- 4. Why is it important to treat GH deficiency and short stature tions. His mother is instructed about changes in Charlie’s prior to the onset of puberty? diet. During the next year, Charlie returns to the doctor 5. Why is resistance to insulin action a potential adverse effect for several follow-up visits and to adjust his insulin of giving extremely high pharmacological doses of GH for a dosage. Data from his 1-year visit are as follows: fasting long time? blood glucose, 120 mg/dL; C-peptide, 0.1 ng/mL. Answers to Case Study Questions for Chapter 32 Questions 1. GH is released in a pulsatile manner; between pulses of GH 1. What might be the reason for the decrease in Charlie’s C- secretion the blood concentration may be undetectable in peptide after one year? normal individuals. GH induces the synthesis and secretion 2. Why weren’t plasma insulin values measured after one of IGF-I and IGFBP3, both of which are easily detectable in year? the serum. Low IGF-I and IGFBP3 levels would indicate in- sufficient GH secretion. Answers to Case Study Questions for Chapter 31 2. In most cases, low levels of IGF-I and IGFBP3 in the blood 1. The decrease in C-peptide reflects further destruction of would indicate insufficient GH release. However, low levels Charlie’s insulin-producing pancreatic beta cells and indi- of IGF-I and IGFBP3 could also be due to a defect in the GH cates a further impairment in his own insulin production ca- receptor, resulting in GH resistance. pacity. 3. GH resistance is characterized by impaired growth as a re- 2. Because Charlie is taking insulin injections, measurement of sult of low levels of IGF-I and IGFBP3 in the blood. However, circulating insulin levels would not have provided any infor- the blood concentration of GH is high. Defects in the GH re- mation about insulin secretion. This information is inferred ceptor, which prevent GH from stimulating the production from the C-peptide data. of IGF-I and IGFBP3, are a common cause of GH resistance.

CHAPTER 36 Endocrine Regulation of Calcium, Phosphate, and Bone Metabolism 647 Measurement of a GH in the blood should detect the ex- 5. A thionamide compound should first be used to inhibit thy- tremely high levels of the hormone to confirm diagnosis. roid hormone synthesis. This treatment will relieve the 4. GH and IGF-I stimulate the epiphyseal growth plate of the symptoms of hyperthyroidism and may result in a reduction long bones to grow. The epiphyseal plate fuses several in immune response. The drug may be withdrawn after sev- years after puberty, at which time GH and IGF-I can no eral months of treatment to determine whether the disease longer stimulate the growth of the bone. Therefore, the ear- is in remission. If thyroid hormone levels increase with ces- lier GH therapy is initiated, the greater will be the chance of sation of the drug, ablation of the thyroid gland with 131 I (or achieving normal adult height before long bone growth less commonly with surgery) would be indicated. stops. 5. GH has diabetogenic actions, which oppose the actions of CASE STUDY FOR CHAPTER 34 insulin. Thus, chronic, high doses of GH can impair the ac- tions of insulin. Insulin resistance is a condition in which tis- Congenital Adrenal Hyperplasia sues in the body do not respond very well to insulin (see The pediatric endocrinologist is called in to consult on Chapter 35). the case of a 1-week-old girl. The baby was born at home Reference and is now in the emergency department because she Grumbach MM, Bin-Abbas BS, Kaplan SL. The growth hor- appeared listless and has not nursed during the past 24 mone cascade: progress and hours. On physical examination, the baby exhibits signs long-term results of growth hormone treatment in growth hor- of virilization (growth of pubic hair) and volume deple- mone deficiency. Horm Res 1998;49(Suppl 2):41–57. tion, and laboratory results indicate hyponatremia and hyperkalemia. CASE STUDY FOR CHAPTER 33 1. Based on the history, physical examination, and laboratory findings, what would be a reasonable initial hypothesis? Thyroiditis 2. What are the two most likely congenital defects in adrenal steroidogenic enzymes that could explain the findings in A 35-year-old woman is seen in the Endocrine Clinic for this child? evaluation of thyroid disease. The patient complains of 3. From a blood sample, what hormones/metabolites should weight loss, irritability, and restlessness. Physical exami- the laboratory measure, and what would be the likely re- nation reveals enlargement of the thyroid gland, weak- sults? ness in maintaining the leg in an extended position, 4. From the hormone/metabolite analysis, how would the two warm moist skin, and tachycardia. Family history indi- most likely causes for this case of congenital adrenal hyper- cates that the patient’s mother had hypothyroidism after plasia be distinguished? the birth of the patient’s brother and an aunt had 5. A genetic screen utilizing DNA from the baby’s white cells Hashimoto’s disease. identifies an inactivating mutation in the gene (CYP21A2) Questions for 21-hydroxylase. What would be appropriate hormone re- 1. Based on the history and physical examination, what would placement for this patient? be a reasonable initial diagnosis? 2. From a blood sample, what hormone concentrations should Answers to Case Study Questions for Chapter 34 1. A reasonable initial hypothesis is that the baby has a form the laboratory measure, and what would be the likely re- of congenital adrenal hyperplasia. The virilization (appear- sults? 3. What antibody titers should the laboratory determine? ance of pubic hair) suggests the presence of excess andro- gen production by the adrenal gland. The hyponatremia, hy- Which antibody titer is the most useful in the diagnosis of perkalemia, and volume depletion suggest a “salt wasting” Hashimoto’s disease? syndrome. 4. Which antibody titer would be most useful in the diagnosis 2. Mutations in CYP21A2 , which encodes 21-hydroxylase, ac- of Graves’ disease? 5. The antibody titers indicate that the patient has Graves’ dis- count for more than 90% of all cases of adrenal hyperplasia associated with excess androgen production. Mutations in ease. What treatment would be appropriate for this patient? CYP11B1, which encodes 11-hydroxylase, would also re- Answers to Case Study Questions for Chapter 33 sult in excess adrenal androgen production. 1. The physical findings, including the presence of goiter, sug- 3. Adrenal androgens would be significantly elevated in pa- gest that the patient may be hyperthyroid. However, goiter tients with virilizing forms of congenital adrenal hyperpla- can also occur in hypothyroidism. Since autoimmune thy- sia. Adrenal hyperplasia is usually due to defects in cortisol roid disease runs in families, the family history suggests production. Therefore, the serum concentrations of precur- that the thyroiditis might be due to an autoimmune re- sors of cortisol biosythesis such as progesterone, 17-hy- sponse. droxyprogesterone, and 11-deoxycortisol could be elevated. 2. The laboratory should determine the blood levels of thyroid In addition, serum ACTH would be elevated as a result of hormones (T 4 and T 3 ) and TSH. Thyroid hormones should the lack of negative feedback from the absent cortisol. be increased. TSH may be increased if it is early in the pro- 4. Genetic defects in the gene for 11-hydroxylase, resulting in gression of Hashimoto’s disease or decreased if the patient a reduction in the activity of this enzyme, would result in in- has Graves’ disease. creased 11-deoxycortisol. Defects in the gene for 21-hydrox- 3. The laboratory should measure antibodies to TSH receptor, ylase, which impair the activity of the enzyme, would not thyroid peroxidase, and thyroglobulin. Antibodies to thyroid lead to the production of 11-deoxycortisol. Since 11-deoxy- peroxidase are elevated to the greatest extent in cortisol has significant mineralocorticoid activity, excess Hashimoto’s disease. production of this steroid is usually associated with hyper- 4. Antibodies to TSH receptor, thyroid peroxidase, and thy- tension, rather than the volume depletion and hypotension roglobulin can all be elevated in Graves’ disease. However, observed in this patient. the presence of TSH receptor antibodies is diagnostic. 5. Treatment would be directed toward replacement of gluco-

648 PART IX ENDOCRINE PHYSIOLOGY corticoids and mineralocorticoids. Glucocorticoids would re- 3. Exercise not only helps to control weight, it stimulates glu- place the missing cortisol and also suppress ACTH secre- cose uptake in skeletal muscle, lessening the requirements tion. With less ACTH stimulation of steroid production from for injected insulin. the adrenal gland, the hyperandrogenemia should subside. Mineralocorticoids are given to treat the “salt wasting” that CASE STUDY FOR CHAPTER 36 occurs in the absence of aldosterone. Bone Fractures CASE STUDY FOR CHAPTER 35 A 38-year-old Caucasian man recently came to the atten- tion of his physician when he suffered the second of two Type 2 Diabetes bone fractures in the past year and a half. He previously A 65-year-old semi-retired college professor was diag- was in relatively good health, was not a smoker, and used nosed with type 2 diabetes about 4 years ago during a alcohol only moderately. However, his only form of exer- routine physical examination at his family doctor’s of- cise was cutting the lawn on weekends during the sum- fice. Treatment for the diabetes initially consisted of one mer months. He has not required any major surgeries tablet daily of an oral antidiabetic drug of the sulfony- during his lifetime, and had only minor bouts of the typi- lurea class and two daily injections of insulin. The pa- cal childhood illnesses. However, at age eight he was di- tient’s doctor also recommended modest weight loss agnosed with asthma after he suffered severe respiratory and a regular exercise program. With diligence to the problems during a baseball game on a hot summer day. treatment program, the patient was able to control his He has been treated ever since with a daily tablet of a syn- blood sugar levels adequately. thetic glucocorticoid and the occasional use of an inhaler About 2 years ago, the patient developed gallstones, when needed to relieve acute symptoms of the disease. which required surgery to remove the gallbladder. For The fractures that the patient experienced were to the about one week after the surgery, the patient had to in- left wrist and the right forearm. In both cases, the trauma crease his insulin dosage to maintain normal blood glu- that caused the fracture was relatively minor. Suspecting cose levels. He gradually returned to his presurgery in- that there may be an underlying problem, his physician sulin dose. orders a series of bone density scans. Results of these Because of the surgery, the patient vows to take better studies show that the patient has a considerable reduc- care of himself. He increases his physical activity and be- tion in bone mass compared with other men of the same gins a diet that results in loss of 7 kg in 3 months. The age. weight loss and exercise result in the cessation of the pa- Questions tient’s need for insulin injections, although he still takes 1. What is the most probable diagnosis? his daily oral medication. 2. What is the most probable underlying cause for the pa- Questions tient’s problem? 1. Why might the gallbladder disease and resulting surgery 3. What risk factors are present (or absent) in this case? have increased the patient’s need for insulin? Answers to Case Study Questions for Chapter 36 2. What might be the consequences if the patient were to re- 1. Osteoporosis and, perhaps, glucocorticoid-induced osteo- gain the weight he lost after surgery? porosis. 3. Why is exercise an important part of the treatment regimen 2. Because the patient is young and has a relatively healthy for type 2 diabetes? lifestyle, the most probable cause of his osteoporosis is his Answers to Case Study Questions for Chapter 35 30-year history of treatment with glucocorticoids for 1. Stress, such as surgery, results in increased production of asthma. Glucocorticoids increase bone loss by inhibiting os- epinephrine and norepinephrine, both of which inhibit in- teoblasts, stimulating bone resorption, impairing intestinal sulin secretion. The patient’s pancreas will produce less in- calcium absorption, increasing urinary calcium loss, inhibit- sulin, and thus, more exogenous insulin will need to be pro- ing secretion of sex hormones, and other effects. vided. 3. The patient lacks the risk factors of smoking, excessive alco- 2. If the patient were to regain weight, he would most likely hol intake, and being female. He does appear, however, to have to go back to taking insulin injections. have the risk factor of a somewhat sedentary lifestyle.

PART X Reproductive Physiology CHAPTER The Male 37 37 Reproductive System Paul F. Terranova, Ph.D. CHAPTER OUTLINE ■ AN OVERVIEW OF THE MALE REPRODUCTIVE ■ SPERMATOGENESIS SYSTEM ■ TESTICULAR STEROIDOGENESIS ■ REGULATION OF TESTICULAR FUNCTION ■ THE ACTIONS OF ANDROGENS ■ THE MALE REPRODUCTIVE ORGANS ■ REPRODUCTIVE DYSFUNCTIONS KEY CONCEPTS 1. In the testes, luteinizing hormone (LH) controls the synthe- 5. LH and FSH secretion by the anterior pituitary are con- sis of testosterone by Leydig cells, and follicle-stimulating trolled by gonadotropin-releasing hormone (GnRH). hormone (FSH) increases the production of 6. Testosterone mainly reduces LH secretion, whereas inhibin 2. androgen-binding protein, inhibin, and estrogen by Sertoli reduces the secretion of FSH. The testicular hormones cells. complete a negative-feedback loop with the hypothalamic- 3. Spermatozoa are produced within the seminiferous pituitary axis. tubules of both testes. Sperm develop from spermatogonia 7. Androgens have several target organs and have roles in through a series of developmental stages that include regulating the development of secondary sex characteris- spermatocytes and spermatids. tics, the libido, and sexual behavior. 4. The sperm mature and are stored in the epididymis. At the 8. The most potent natural androgen is dihydrotestosterone, time of ejaculation, sperm are moved by muscular contrac- which is produced from the precursor, testosterone, by the tions of the epididymis and vas deferens through the ejacu- action of the enzyme 5
-reductase. latory ducts into the prostatic urethra. The sperm are finally 9. Male reproductive dysfunction is often due to a lack of LH moved out of the body through the urethra in the penis. and FSH secretion or abnormal testicular morphology. he testes have two primary functions, spermatogene- and include the typical male hair pattern, deep voice, and Tsis, the process of producing mature sperm, and large muscle and bone masses. steroidogenesis, the synthesis of testosterone. Both processes are regulated by the pituitary gonadotropins LH and FSH. Testosterone is the primary sex hormone in AN OVERVIEW OF THE MALE the male and is responsible for primary and secondary sex REPRODUCTIVE SYSTEM characteristics. The primary sex characteristics include those structures responsible for promoting the develop- A diagram of reproduction regulation in the male is pre- ment, preservation, and delivery of sperm. The second- sented in Figure 37.1. The system is divided into factors af- ary sex characteristics are those structures and behavioral fecting male function: brain centers, which control pitu- features that make men externally different from women itary release of hormones and sexual behavior; gonadal 649

650 PART X REPRODUCTIVE PHYSIOLOGY Environment ported via the urethra through the penis and are ultimately expelled by ejaculation. The accessory structures of the Age Drugs male reproductive tract include the prostate gland, seminal vesicles, and bulbourethral glands. These glands contribute Brain centers several constituents to the seminal fluid that are necessary for maintaining functional sperm. Hypothalamus REGULATION OF TESTICULAR FUNCTION GnRH Testicular function is regulated by LH and FSH. LH regu- lates the secretion of testosterone by the Leydig cells and Anterior pituitary  FSH, in synergy with testosterone, regulates the produc- tion of spermatozoa. FSH

LH Inhibin Follistatin Hypothalamic Neurons Produce Testes
Gonadotropin-Releasing Hormone Activin Hypothalamic neurons produce gonadotropin-releasing Testosterone hormone (GnRH), a decapeptide, which regulates the se-


cretion of luteinizing hormone (LH) and follicle-stimulat- ing hormone (FSH). Although neurons that produce Accessory Secondary sex reproductive Behavior GnRH can be located in various areas of the brain, their characteristics tissues highest concentration is in the medial basal hypothalamus, in the region of the infundibulum and arcuate nucleus. Regulation of reproduction in the male. FIGURE 37.1 GnRH enters the hypothalamic-pituitary portal system and The main reproductive hormones are shown in boxes. Positive and negative regulations are depicted by plus and binds to receptors on the plasma membranes of pituitary minus signs, respectively. cells, resulting in the synthesis and release of LH and FSH. A variety of external cues and internal signals influence the secretion of GnRH, LH, and FSH. For example, the structures, which produce sperm and hormones; a ductal amount of GnRH, FSH, and LH secreted changes with age, system, which stores and transports sperm; and accessory stress levels, and hormonal state. In addition, various dis- glands, which support sperm viability. ease states lead to hyposecretion of GnRH. Little, if any, The endocrine glands of the male reproductive system secretion of hypothalamic GnRH occurs in patients with include the hypothalamus, anterior pituitary, and testes. prepubertal hypopituitarism, resulting in a failure of the de- The hypothalamus processes information obtained from velopment of the testes, primarily a result of a lack of LH, the external and internal environment using neurotransmit- FSH, and testosterone. ters that regulate the secretion of gonadotropin-releasing Male patients with Kallmann’s syndrome are hypogo- hormone (GnRH). GnRH moves down the hypothalamic- nadal from a deficiency in LH and FSH secretion because pituitary portal system and stimulates the secretion of LH of a failure of GnRH neurons to migrate from the olfactory and FSH by the gonadotrophs of the anterior pituitary. LH bulbs, their embryological site of origin. These patients do binds to receptors on the Leydig cells and FSH binds to re- not have a sufficient hypothalamic source of GnRH to ceptors on the Sertoli cells. Leydig cells reside in the inter- maintain secretion of LH and FSH, and the testes fail to un- stitium of the testes, between seminiferous tubules, and dergo significant development. produce testosterone. Sertoli cells are located within the GnRH originates from a large precursor molecule called seminiferous tubules, support spermatogenesis, contain preproGnRH (Fig. 37.2). PreproGnRH consists of a signal FSH and testosterone receptors, and produce estradiol, al- peptide, native GnRH, and a GnRH-associated peptide beit at low levels. (GAP). The signal peptide (or leader sequence) allows the Testosterone belongs to a class of steroid hormones, the protein to cross the membrane of the rough ER. However, androgens, which promote “maleness.” It carries out multiple both the signal peptide and GAP are enzymatically cleaved functions, including feedback on the hypothalamus and ante- at the rough ER prior to GnRH secretion. rior pituitary; the support of spermatogenesis; the regulation of behavior, including sexual behavior; and the development Distinct Gonadotrophs Produce LH and FSH and maintenance of secondary sex characteristics. Sertoli cells also produce glycoprotein hormones— nhibin, activin, and Three distinct pituitary LH- and FSH-secreting cells have follistatin—that regulate the secretion of FSH. been identified. Gonadotrophs contain either LH or FSH, The duct system that transports sperm from the testis to and some cells contain both LH and FSH. GnRH can in- the outside through the penis includes the epididymis, vas duce the secretion of both hormones simultaneously be- deferens, and urethra. The sperm acquire motility and the cause GnRH receptors are present on all of these cell types. capability to fertilize in the epididymis; they are stored in LH and FSH each contain two polypeptide subunits, re- the epididymis and in the vas deferens. They are trans- ferred to as alpha and beta chains, that are about 15 kDa in

CHAPTER 37 The Male Reproductive System 651 Processing sites 23 AA 10 AA 56 AA Signal peptide GnRH GnRH-associated peptide (GAP) N GnRH C terminus terminus FIGURE 37.2 The precursor molecule, pre- proGnRH, that contains GnRH. The amino acid sequence of GnRH, a decapeptide is pyroGLU-HIS-TRP-SER-TYR-GLY-LEU-ARG-PRO-GLY-NH 2 indicated at the bottom. size. Both hormones contain the same
subunit but differ- rectly indicate that GnRH pulses have occurred. Numerous ent  subunits. Each hormone is glycosylated prior to re- human studies measuring pulsatile secretion of LH and FSH lease into the general circulation. Glycosylation regulates in peripheral blood at various times have provided much of the half-life, protein folding for receptor recognition, and the information regarding the role of LH and FSH in regu- biological activity of the hormone. lating testicular development and function. However, the LH and FSH bind membrane receptors on Leydig and exact relationship between endogenous GnRH pulses and Sertoli cells, respectively. The activation of LH and FSH LH and FSH secretion in humans is unknown. receptors on these cells increases the intracellular second Hypogonadal eunuchoid men exhibit low levels of LH messenger cAMP. The two gonadotropin receptors are in serum and do not exhibit pulsatile secretion of LH. Pul- linked to G proteins and adenylyl cyclase for the produc- satile injections of GnRH restore LH and FSH secretion tion of cAMP from ATP. For the most part, cAMP can ac- and increase sperm counts. FSH pulses tend to be smaller in count for all of the actions of LH and FSH on testicular amplitude than LH pulses, mostly because FSH has a longer cells. cAMP binds to protein kinase A, which activates tran- half-life than LH in the circulation. scription factors such as steroidogenic factor-1 (SF-1) and Although the exact identity of the cells responsible for cAMP response element binding protein (CREB). These generating GnRH pulsatility is unknown, the presence of a factors activate the promoter region of the genes of pulse generator in the hypothalamus has been postulated. steroidogenic enzymes that control testosterone produc- The putative pulse generator resides in the medial basal hy- tion by Leydig cells. Similar signal-transducing events oc- pothalamus and is responsible for the synchronized and cur in Sertoli cells that regulate the production of estradiol. rhythmic firing of a population of neurons. The activity of The testis converts testosterone and some other androgens the pulse generator is modified by several factors. For ex- to estradiol by the process of aromatization, although estra- ample, castration causes a large increase in basal LH levels diol production is low in males. in serum, as evidenced by an increase in frequency and am- Another major function of the testis is the production of plitude of LH pulses. Therefore, the pulse generator may be mature sperm, inhibin (a protein produced by Sertoli cells tonically inhibited by testosterone. However, GnRH neu- that suppresses FSH secretion), and androgen-binding pro- rons lack receptors for gonadal steroids, suggesting that tein. Activin and follistatin production by testicular cells in humans is currently being investigated. GnRH Is Secreted in a Pulsatile Manner GnRH GnRH in the hypothalamus is secreted in a pulsatile man- ner into the hypothalamic-hypophyseal portal blood. GnRH pulsatility is ultimately necessary for proper func- tioning of the testes because it regulates the secretion of Portal GnRH conc (pg/mL) FSH and LH, which are also released in a pulsatile fashion (Fig. 37.3). Continuous exposure of gonadotrophs to GnRH results in desensitization of GnRH receptors, lead- ing to a decrease in LH and FSH release. Therefore, the pulsatile pattern of GnRH release serves an important Peripheral LH conc (ng/mL) physiological function. The administration of GnRH at an improper frequency results in a decrease in circulating con- centrations of LH and FSH. LH Most evidence for GnRH pulses has come from animal studies because GnRH must be measured in hypothalamic- 19 2468753 hypophyseal portal blood, an extremely difficult area to Time (hr) obtain blood samples in humans. Since discrete pulses of GnRH are followed by distinct pulses of FSH and LH, A diagram of the pulsatile release of GnRH FIGURE 37.3 measurements of the pulses of LH and FSH in serum indi- in portal blood and LH in peripheral blood.

652 PART X REPRODUCTIVE PHYSIOLOGY steroidal effects are mediated by other neurons whose neu- The Testis Is the Site of Sperm Formation ropeptides, neurohormones, or vasoactive agents regulate the activity of the GnRH-producing neurons. During embryonic stages of development, the testes lie at- tached to the posterior abdominal wall. As the embryo elon- gates, the testes move to the inguinal ring. Between the sev- Steroids and Polypeptides From the enth month of pregnancy and birth, the testes descend Testis Regulate LH and FSH Secretion through the inguinal canal into the scrotum. The location of the testes in the scrotum is important for sperm production, Testosterone, estradiol, inhibin, activin, and follistatin are which is optimal at 2 to 3C lower than core body tempera- major testicular hormones that regulate the release of the ture. Two systems help maintain the testes at a cooler tem- gonadotropins LH and FSH. Generally, testosterone, estra- perature. One is the pampiniform plexus of blood vessels, diol, and inhibin reduce the secretion of LH and FSH in the which serves as a countercurrent heat exchanger between male. Activin stimulates the secretion of FSH, whereas fol- warm arterial blood reaching the testes and cooler venous listatin inhibits FSH secretion. blood leaving the testes. The second is the cremasteric mus- Testosterone inhibits LH release by decreasing the se- cle, which responds to changes in temperature by moving cretion of GnRH and, to a lesser extent, by reducing go- the testes closer or farther away from the body. Prolonged nadotroph sensitivity to GnRH. Estradiol formed from exposure of the testes to elevated temperature, fever, or testosterone by aromatase also has an inhibitory effect on thermoregulatory dysfunction can lead to temporary or per- GnRH secretion. Acute testosterone treatment does not al- manent sterility as a result of a failure of spermatogenesis, ter pituitary responsiveness to GnRH, but prolonged expo- whereas steroidogenesis is unaltered. sure significantly reduces the secretory response to GnRH. The testes are encapsulated by a thick fibrous connec- Removal of the testes results in increased circulating lev- tive tissue layer, the tunica albuginea. Each human testis els of LH and FSH. Replacement therapy with physiologi- contains hundreds of tightly packed seminiferous tubules, cal doses of testosterone restores LH to precastration levels ranging from 150 to 250 m in diameter and from 30 to 70 but does not completely correct FSH levels. This observa- cm long. The tubules are arranged in lobules, separated by tion led to a search for a gonadal factor that specifically in- extensions of the tunica albuginea, and open on both ends hibits FSH release. The polypeptide hormone inhibin was into the rete testis. Examination of a cross section of a testis eventually isolated from seminal fluid. Inhibin is produced reveals distinct morphological compartmentalization. by Sertoli cells, and has a molecular weight of 32 to 120 Sperm production is carried out in the avascular seminifer- kDa, the 32-kDa form being the most prominent. Inhibin is ous tubules, whereas testosterone is produced by the Ley- composed of two dissimilar subunits,
and , which are dig cells, which are scattered in a vascular, loose connective held together by disulfide bonds. There are two  subunit tissue between the seminiferous tubules in the interstitial forms, called A and B. Inhibin B consists of the
subunit compartment. bound by a disulfide bridge to the B subunit and is the Each seminiferous tubule is composed of two somatic physiologically important form of inhibin in the human cell types (myoid cells and Sertoli cells) and germ cells. The male. Inhibin acts directly on the anterior pituitary and in- seminiferous tubule is surrounded by a basement membrane hibits the secretion of FSH but not LH. (basal lamina) with myoid cells on its perimeter, which de- Activin is produced by Sertoli cells, stimulates the se- fine its outer limit. On the inside of the basement mem- cretion of FSH, has an approximate molecular weight of 30 brane are large, irregularly shaped Sertoli cells, which ex- kDa and has multiple forms based on the A and B sub- tend from the basement membrane to the lumen (Fig. 37.4). Sertoli cells are attached to one another near their base by units of inhibin. The multiple forms of activin are called ac- tight junctions (Fig. 37.5). The tight junctions divide each tivin A (two A subunits linked by a disulfide bridge), ac- tubule into a basal compartment, whose constituents are tivin B (two B subunits), and activin AB (one A and one exposed to circulating agents, and an adluminal compart- B subunit). The major form of activin in the male is cur- ment, which is isolated from bloodborne elements. The rently unknown although both Sertoli and Leydig cells tight junctions limit the transport of fluid and macromole- have been implicated in its secretion. cules from the interstitial space into the tubular lumen, Follistatin is a 31 to 45 kDa single-chain protein hor- forming the blood-testis barrier. mone, with several isoforms, that binds and deactivates ac- Located between the nonproliferating Sertoli cells are tivin. Thus, the deactivation of activin by binding to follis- germ cells at various stages of division and differentiation. tatin reduces FSH secretion. Follistatin is apparently Mitosis of the spermatogonia (diploid progenitors of sper- produced by Sertoli cells and acts as a paracrine factor on matozoa) occurs in the basal compartment of the seminifer- the developing spermatogenic cells. ous tubule (see Fig. 37.5). The early meiotic cells (primary spermatocytes) move across the junctional complexes into the adluminal compartment, where they mature into sper- matozoa or gametes after meiosis. The adluminal compart- THE MALE REPRODUCTIVE ORGANS ment is an immunologically privileged site. Spermatozoa The testes produce spermatozoa and transport them that develop in the adluminal compartment are not recog- through a series of ducts in preparation for fertilization. nized as “self” by the immune system. Consequently, males The testes also produce testosterone that regulates devel- can develop antibodies against their own sperm, resulting in opment of the male gametes, male sex characteristics, and infertility. Sperm antibodies neutralize the ability of sperm male behavior. to function. Sperm antibodies are often present after vasec-

CHAPTER 37 The Male Reproductive System 653 Spermatogonium Spermatozoon m Lumen 300 Sertoli cell Basement membrane Leydig cell surrounding the seminiferous tubule The testis. This cross-sectional FIGURE 37.4 view shows the anatomic relation- ship of the Leydig cells, basement membrane, semi- niferous tubules, Sertoli cells, spermatogonia, and spermatozoa. (Modified from Alberts B, Bray D, Lewis M, et al. Molecular Biology of the Cell. 3rd Ed. New York: Garland, 1994.) tomy or testicular injury and in some autoimmune diseases and testosterone increases. Receptors for FSH, present only where the adluminal compartment is ruptured, allowing on the plasma membranes of Sertoli cells, are glycoproteins sperm to mingle with immune cells from the circulation. linked to adenylyl cyclase via G proteins. FSH exerts mul- tiple effects on the Sertoli cell, most of which are mediated by cAMP and protein kinase A (Fig. 37.6). FSH stimulates Sertoli Cells Have Multiple Functions the production of androgen-binding protein and plasmino- Sertoli cells are critical to germ cell development, as indi- gen activator, increases secretion of inhibin, and induces cated by their close contact. As many as 6 to 12 spermatids aromatase activity for the conversion of androgens to es- may be attached to a Sertoli cell. Sertoli cells phagocytose trogens. The testosterone receptor is within the nucleus of residual bodies (excess cytoplasm resulting from the trans- the Sertoli cell. formation of spermatids to spermatozoa) and damaged Androgen-binding protein (ABP) is a 90-kDa protein, germ cells, provide structural support and nutrition for made of a heavy and a light chain, that has a high binding germ cells, secrete fluids, and assist in spermiation, the fi- affinity for dihydrotestosterone and testosterone. It is sim- nal detachment of mature spermatozoa from the Sertoli cell ilar in function, with some homology in structure, to an- into the lumen. Spermiation may involve plasminogen ac- other binding protein, sex hormone-binding globulin tivator, which converts plasminogen to plasmin, a prote- (SHBG), synthesized in the liver. ABP is found at high con- olytic enzyme that assists in the release of the mature sperm centrations in the human testes and epididymis. It serves as into the lumen. Sertoli cells also synthesize large amounts a carrier of testosterone in Sertoli cells, as a storage protein of transferrin, an iron-transport protein important for for androgens in the seminiferous tubules, and as a carrier sperm development. of testosterone from the testes to the epididymis. During the fetal period, Sertoli cells and gonocytes form Other products of the Sertoli cell are inhibin, follistatin, the seminiferous tubules as Sertoli cells undergo numerous and activin. Inhibin suppresses FSH release from the pitu- rounds of cell divisions. Shortly after birth, Sertoli cells itary gonadotrophs. The pituitary gonadotrophs and testic- cease proliferating, and throughout life, the number of ular Sertoli cells form a classical negative-feedback loop in sperm produced is directly related to the number of Sertoli which FSH stimulates inhibin secretion and inhibin sup- cells. At puberty, the capacity of Sertoli cells to bind FSH presses FSH release. Inhibin also functions as a paracrine

654 PART X REPRODUCTIVE PHYSIOLOGY agent in the testes. Activin stimulates the release of FSH. Follistatin, an activin-binding protein, reduces FSH secre- Leydig cell tion induced by activin. Basement membrane surrounding seminiferous tubule Leydig Cells Produce Testosterone Leydig cells are large polyhedral cells that are often found Spermatogonium in clusters near blood vessels in the interstitium between Basal compartment seminiferous tubules. They are equipped to produce Tight junction steroids because they have numerous mitochondria, a Adluminal compartment prominent smooth ER, and conspicuous lipid droplets. Leydig cells undergo significant changes in quantity and Spermatocyte activity throughout life. This mechanism may depend on a nuclear transcription factor, steroidogenic factor-1 (SF-1), that recognizes a sequence in the promoter of all genes en- Spermatid Nucleus coding CYP enzymes. In the human fetus, the period from weeks 8 to 18 is marked by active steroidogenesis, which is Nucleus obligatory for differentiation of the male genital ducts. Ley- dig cells at this time are prominent and very active, reach- Intercellular space ing their maximal steroidogenic activity at about 14 weeks, when they constitute more than 50% of the testicular vol- Spermatozoon ume. Because the fetal hypothalamic-pituitary axis is still underdeveloped, steroidogenesis is controlled by human chorionic gonadotropin (hCG) from the placenta, rather Sertoli cell Sertoli cell than by LH from the fetal pituitary (see Chapter 39); LH and hCG bind the same receptor. After this period, Leydig cells slowly regress. At about 2 to 3 months of postnatal life, male infants have a significant rise in testosterone pro- Lumen duction (infantile testosterone surge), the regulation and Sertoli cells. Sertoli cells are connected by FIGURE 37.5 function of which are unknown. Leydig cells remain quies- tight junctions, which divide the intercellular cent throughout childhood but increase in number and ac- space into a basal compartment and an adluminal compart- tivity at the onset of puberty. ment. Spermatogonia are located in the basal compartment and maturing sperm in the adluminal compartment. Spermatocytes are Leydig cells do not have FSH receptors, but FSH can in- formed from the spermatogonia and cross the tight junctions into crease the number of developing Leydig cells by stimulat- the adluminal compartment, where they mature into spermatozoa. ing the production of growth stimulators from Sertoli cells (Modified from Alberts B, Bray D, Lewis M, et al. Molecular Biol- that subsequently enhance the growth of the Leydig cells. ogy of the Cell. 3rd Ed. New York: Garland, 1994.) In addition, androgens stimulate the proliferation of devel- oping Leydig cells. Estrogen receptors are present on Ley- dig cells, and they reduce the proliferation and activity of these cells. Leydig cell Sertoli cell Lumen of seminiferous Cholesterol tubule ABP LH Receptor cAMP Capillary ATP Basement membrane T-ABP Pregnenolone T T Testosterone (T)  ATP E cAMP Receptor FSH T Estradiol Regulation, (E) FIGURE 37.6 R hormonal E products, and interactions R between Leydig and Sertoli cells. ABP, androgen-binding protein; E, estradiol; T, testos- Proteins Proteins terone; R, receptor.

CHAPTER 37 The Male Reproductive System 655 Leydig cells have LH receptors, and the major effect of vasodilation of the arterioles and corpora cavernosa. The LH is to stimulate androgen secretion via a cAMP-depend- smooth muscles in those structures relax, and the blood ent mechanism (see Fig. 37.6). The main product of Leydig vessels dilate and begin to engorge with blood. The thin- cells is testosterone, but two other androgens of less bio- walled veins become compressed by the swelling of the logical activity, dehydroepiandrosterone (DHEA) and an- blood-filled arterioles and cavernosa, restricting blood drostenedione, are also produced. flow. The result is a reduction in the outflow of blood from There are bidirectional interactions between Sertoli and the penis, and blood is trapped in the surrounding erectile Leydig cells (see Fig. 37.6). The Sertoli cell is incapable of tissue, leading to engorgement, rigidity, and elongation of producing testosterone but contains testosterone receptors the penis in an erect position. as well as FSH-dependent aromatase. The Leydig cell does Semen, consisting of sperm and the associated fluids, not produce estradiol but contains receptors for it, and is expelled by a neuromuscular reflex that is divided into estradiol can suppress the response of the Leydig cell to two sequential phases: emission and ejaculation. Emis- LH. Testosterone diffuses from the Leydig cells, crosses the sion moves sperm and associated fluids from the cauda basement membrane, enters the Sertoli cell, and binds to epididymis and vas deferens into the urethra. The latter ABP. As a result, androgen levels can reach high local con- process involves efferent stimuli originating in the lum- centrations in the seminiferous tubules. Testosterone is bar areas (L1 and L2) of the spinal cord and is mediated obligatory for spermatogenesis and the proper functioning by adrenergic sympathetic (hypogastric) nerves that in- of Sertoli cells. In Sertoli cells, testosterone also serves as a duce contraction of smooth muscles of the epididymis precursor for estradiol production. The daily role of estra- and vas deferens. This action propels sperm through the diol in the functioning of Leydig cells is unclear, but it may ejaculatory ducts and into the urethra. Sympathetic dis- modulate responses to LH. charge also closes the internal urethral sphincter, which prevents retrograde ejaculation into the urinary bladder. Ejaculation is the expulsion of the semen from the penile The Duct System Functions in Sperm Maturation, urethra; it is initiated after emission. The filling of the Storage, and Transport urethra with sperm initiates sensory signals via the pu- dendal nerves that travel to the sacrospinal region of the After formation in the seminiferous tubules, spermatozoa are transported to the rete testes and from there through cord. A spinal reflex mechanism that induces rhythmic the efferent ductules to the epididymis. This movement of contractions of the striated bulbospongiosus muscles sur- sperm is accomplished by ciliary movement in the efferent rounding the penile urethra results in propelling the se- ductules, by muscle contraction, and by the flow of fluid. men out of the tip of the penis. The epididymis is a single, tightly coiled duct, 4 to 5 m The secretions of the accessory glands promote long. It is composed of a head (caput), a body (corpus), and sperm survival and fertility. The accessory glands that a tail (cauda) (Fig. 37.7). The functions of the epididymis contribute to the secretions are the seminal vesicles, are storage, protection, transport, and maturation of sperm prostate gland, and bulbourethral glands. The semen cells. Maturation at this point includes a change in func- contains only 10% sperm by volume, with the remainder tional capacity as sperm make their way through the epi- consisting of the combined secretions of the accessory didymis. The sperm become capable of forward mobility glands. The normal volume of semen is 3 mL with 20 to during migration through the body of the epididymis. A 50 million sperm per milliliter; normal is considered significant portion of sperm maturation is carried out in the more than 20 million sperm per milliliter. The seminal caput, whereas sperm are stored in the cauda. vesicles contribute about 75% of the semen volume. Frequent ejaculation results in reduced sperm numbers Their secretion contains fructose (the principal substrate and increased numbers of immotile sperm in the ejaculate. for glycolysis of ejaculated sperm), ascorbic acid, and The cauda connects to the vas deferens, which forms a di- prostaglandins. In fact, prostaglandin concentrations are lated tube, the ampulla, prior to entering the prostate. high and were first discovered in semen but were mis- The ampulla also serves as a storage site for sperm. Cut- takenly considered the product of the prostate. Seminal ting and ligation of the vas deferens or vasectomy is an ef- vesicle secretions are also responsible for coagulation of fective method of male contraception. Because sperm are the semen seconds after ejaculation. Prostate gland se- stored in the ampulla, men remain fertile for 4 to 5 weeks cretions (
0.5 mL) include fibrinolysin, which is re- after vasectomy. sponsible for liquefaction of the coagulated semen 15 to 30 minutes after ejaculation, releasing sperm. Erection and Ejaculation Are Neurally Regulated Erection is associated with sexual arousal emanating from SPERMATOGENESIS sexually related psychic and/or physical stimuli. During sexual arousal, impulses from the genitalia, together with Spermatogenesis is a continual process involving mitosis of nerve signals originating in the limbic system, elicit motor the male germ cells that undergo extensive morphological impulses in the spinal cord. These neuronal impulses are changes in cell shape and, ultimately, meiosis to produce carried by the parasympathetic nerves in the sacral region the haploid spermatozoa. Sperm are produced throughout of the spinal cord via the cavernous nerve branches of the life beginning with puberty. Sperm production declines in prostatic plexus that enter the penis. Those signals cause the elderly.

656 PART X REPRODUCTIVE PHYSIOLOGY Urinary bladder Ampulla Prostatic Seminal urethra vesicle Vas deferens Ejaculatory Urethra duct Prostate gland Corpus cavernosum Bulbourethral Epididymis gland A Tunica albuginea Vas deferens Epididymis Caput (head) Seminiferous Corpus (body) tubules Cauda (tail) Rete testis The male repro- FIGURE 37.7 ductive organs. The top drawing is a general side view. The bottom enlargement shows B a sagittal section of the testis, epi- didymis, and vas deferens. Spermatogenesis Is an Ongoing Process The time required to produce mature spermatozoa from From Puberty to Senescence the earliest stage of spermatogonia is 65 to 70 days. Because several developmental stages of spermatogenic cells occur Spermatogenesis is the process of transformation of male during this time frame, the stages are collectively known as germ cells into spermatozoa. This process can be divided the spermatogenic cycle. There is synchronized develop- into three distinct phases. The phases include cellular pro- ment of spermatozoa within the seminiferous tubules, and liferation by mitosis, two reduction divisions by meiosis to each stage is morphologically distinct. A spermatogonium produce haploid spermatids, and cell differentiation by a becomes a mature spermatozoon after going through sev- process called spermiogenesis, in which the spermatids dif- eral rounds of mitotic divisions, a couple of meiotic divi- ferentiate into spermatozoa (Fig. 37.8). Spermatogenesis sions, and a few weeks of differentiation. Hormones can al- begins at puberty, so the seminiferous tubules are quiescent ter the number of spermatozoa, but they generally do not throughout childhood. Spermatogenesis is initiated shortly affect the duration of the cycle. Spermatogenesis occurs before puberty, under the influence of the rising levels of along the length of each seminiferous tubule in successive gonadotropins and testosterone, and continues throughout cycles. New cycles are initiated at regular time intervals life, with a slight decline during old age. (every 2 to 3 weeks) before the previous ones are com-

CHAPTER 37 The Male Reproductive System 657 Primordial germ cell Enters adluminal portion of seminiferous tubule Spermatogonium Mitosis Diploid spermatogonia divide by mitosis inside seminiferous tubule Primary spermatocyte Meiotic division I Secondary spermatocyte Meiosis Meiotic division II Spermatids XX Y Y Differentiation Spermiogenesis Mature XXY Y spermatozoa The process of spermatogene- FIGURE 37.8 sis, showing successive cell di- visions and remodeling leading to the formation of haploid spermatozoa. (Modified from Alberts B, Bray D, Lewis M, et al. Molecular Biology of the Cell. 3rd Ed. New York: Garland, 1994.) pleted. Consequently, cells at different stages of develop- normally detected and destroyed by the immune system, ment are spaced along each tubule in a “spermatogenic the blood-testis barrier isolates advanced germ cells from wave.” Such a succession ensures the continuous produc- immune surveillance. tion of fresh spermatozoa. Approximately 200 million sper- If the blood-testis barrier is ruptured by physical injury or matozoa are produced daily in the adult human testes, infection and sperm cells within the barrier are exposed to cir- which is about the same number of sperm present in a nor- culating immune cells, it is possible that antibodies will de- mal ejaculate. velop to the sperm cells. In the past, it was thought that the Since sperm cells are rapidly dividing and undergoing development of antisperm antibodies could lead to male in- meiosis, they are sensitive to external agents that alter cell fertility. It appears that men with high levels of antisperm an- division. Chemical carcinogens, chemotherapeutic agents, tibodies may exhibit some infertility problems. However, certain drugs, environmental toxins, irradiation, and ex- studies of men who have developed low or moderate levels of treme temperatures are factors that can reduce the number antisperm antibodies after vasectomy and who have had their of replicating germ cells or cause chromosomal abnormali- vasa deferens reconnected have normal fertility if the vasec- ties in individual cells. While defective somatic cells are tomy was for a relatively short time. Vasectomy does not ap-

658 PART X REPRODUCTIVE PHYSIOLOGY pear to change hormone or sperm production by the testes. hence, it must fulfill several prerequisites: It should pos- Nevertheless, in some cases, a high level of antisperm anti- sess an energy supply and means of locomotion, it should bodies in men and women leads to infertility. be able to withstand a foreign and even hostile environ- ment, it should be able to recognize and penetrate an egg, and it must carry all the genetic information necessary to Spermatogonia Undergo Mitotic and Meiotic create a new individual. Divisions and Become Spermatids The mature spermatozoon exhibits a remarkable degree Spermatogonia undergo several rounds of mitotic division of structural and functional specialization well adapted to prior to entering the meiotic phase (see Fig. 37.8). The carry out these functions. The cell is small, compact, and spermatogonia remain in contact with the Sertoli cells, mi- streamlined; it is about 1 to 2 m in diameter and can ex- grate away from the basal compartment near the walls of ceed 50 m in length in humans. It is packed with special- the seminiferous tubules and cross into the adluminal com- ized organelles and long axial fibers but contains only a few partment of the tubule (see Fig. 37.5). After crossing into of the normal cytoplasmic constituents, such as ribosomes, the adluminal compartment, the cells differentiate into ER, and Golgi apparatus. It has a very prominent nucleus, a spermatocytes prior to undergoing meiosis I. The first mei- flexible tail, numerous mitochondria, and an assortment of otic division of primary spermatocytes gives rise to diploid proteolytic enzymes. (2n chromosomes) secondary spermatocytes. The spermatozoon consists of three main parts: a head, a The second meiotic division produces haploid (1 set of middle piece, and a tail. The two major components in the chromosomes) cells called spermatids. Of every four head are the condensed chromatin and the acrosome. The spermatids emanating from a primary spermatocyte, two haploid chromatin is transcriptionally inactive throughout contain X chromosomes and two have Y chromosomes the life of the sperm until fertilization, when the nucleus de- (see Fig. 37.8). Because of the numerous mitotic divisions condenses and becomes a pronucleus. The acrosome con- and two rounds of meiosis, each spermatogonium com- tains proteolytic enzymes, such as hyaluronidase, acrosin, mitted to meiosis should have yielded 256 spermatids, if neuraminidase, phospholipase A, and esterases. They are in- all cells survive. active until the acrosome reaction occurs upon contact of There are numerous developmental disorders of sper- the sperm head with the egg (see Chapter 39). Their prote- matogenesis. The most frequent is Klinefelter’s syndrome, olytic action enables sperm to penetrate through the egg which causes hypogonadism and infertility in men. Patients membranes. The middle piece contains spiral sheaths of mi- with this disorder have an accessory X chromosome caused tochondria that supply energy for sperm metabolism and lo- by meiotic nondisjunction. The typical karyotype is 47 comotion. The tail is composed of a 9  2 arrangement of XXY, but there are other chromosomal mosaics. Testicular microtubules, which is typical of cilia and flagella, and is sur- volume is reduced more than 75% and ejaculates contain rounded by a fibrous sheath that provides some rigidity. The few, if any, spermatozoa. Spermatogonic cell differentia- tail propels the sperm by a twisting motion, involving inter- tion beyond the primary spermatocyte stage is rare. actions between tubulin fibers and dynein side arms and re- quiring ATP and magnesium. The Formation of a Mature Spermatozoon Requires Extensive Cell Remodeling Testosterone Is Essential for Sperm Production and Maturation Spermatids are small, round, and nondistinctive cells. Dur- ing the second half of the spermatogenic cycle they un- Spermatogenesis requires high intratesticular levels of dergo considerable restructuring to form mature spermato- testosterone, secreted from the LH-stimulated Leydig cells. zoa. Notable changes include alterations in the nucleus, the The testosterone diffuses across the basement membrane of formation of a tail, and a massive loss of cytoplasm. The nu- the seminiferous tubule, crosses the blood-testis barrier, cleus becomes eccentric and decreases in size, and the and complexes with ABP. Sertoli cells, but not spermato- chromatin becomes condensed. The acrosome, a lyso- genic cells, contain receptors for testosterone. Sertoli cells some-like structure unique to spermatozoa, buds from the also contain FSH receptors. However, recent studies using Golgi apparatus, flattens, and covers most of the nucleus. mice, in which the  subunit of FSH has been mutated to The centrioles, located near the Golgi apparatus, migrate an inactive form, reveal that the testes are small but do pro- to the caudal pole and form a long axial filament made of duce sperm. The absolute requirement for FSH in sperm nine peripheral doublet microtubules surrounding a central production remains unknown. From these data, it appears pair (9  2 arrangement). This becomes the axoneme or that testosterone may be sufficient for spermatogenesis. major portion of the tail. Throughout this reshaping The actions of FSH and testosterone at each point of process, the cytoplasmic content is redistributed and dis- sperm cell production are unknown. Upon entering meio- carded. During spermiation, most of the remaining cyto- sis, spermatogenesis appears to depend on the availability plasm is shed in the form of residual bodies. of FSH and testosterone. In human males, FSH is thought The reasons for this lengthy and metabolically costly to be required for the initiation of spermatogenesis before process become apparent when the unique functions of puberty. When adequate sperm production has been this cell are considered. Unlike other cells, the spermato- achieved, LH alone (through stimulation of testosterone zoon serves no apparent purpose in the organism. Its only production) or testosterone alone is sufficient to maintain function is to reach, recognize, and fertilize an egg; spermatogenesis.

CHAPTER 37 The Male Reproductive System 659 TESTICULAR STEROIDOGENESIS hydrogenase), which substitutes the keto group in posi- tion 17 with a hydroxyl group. Unlike all the preceding Following spermatogenesis, the second primary function of enzymatic reactions, this is a reversible step but tends to the testes is steroidogenesis. Steroidogenesis is the pro- favor testosterone. duction of the steroid hormones, mainly testosterone. Although estrogens are only minor products of testicu- Testosterone is then converted to dihydrotesterone lar steroidogenesis, they are normally found in low con- (DHT), the most biologically active androgen, and to centrations in men. Androgens (C19) are converted to es- estradiol, the most biologically estrogen. trogens (C18) by the action of the enzyme complex aromatase (CYP19). Aromatization involves the removal of Testosterone Production Requires Two the methyl group in position 19 and the rearrangement of ring A into an unsaturated aromatic ring. The products of Intracellular Compartments and Several Enzymes aromatization of testosterone and androstenedione are Steroid hormones are produced from cholesterol by the ad- estradiol and estrone, respectively (see Fig. 37.9). In the renal cortex, ovaries, testes, and placenta. Cholesterol, a testis, the Sertoli cell is the main site of aromatization, 27-carbon (C27) steroid, can be obtained from the diet or which is stimulated by FSH; however, aromatization may synthesized within the body from acetate. Each organ uses also occur in peripheral tissues that lack FSH receptors a similar steroid biosynthetic pathway, but the relative (e.g., adipose tissue). amount of the final products depends on the particular sub- set of enzymes expressed in that tissue and the trophic hor- mones (LH, FSH, ACTH) stimulating specific cells within The Effects of LH on Leydig Cells Are the organ. The major steroid produced by the testis is Primarily Mediated by cAMP testosterone, but other androgens, such as androstenediol, androstenedione, and dehydroepiandrosterone (DHEA), as The action of LH on Leydig cells is mediated through spe- well as a small amount of estradiol, are also produced. cific LH receptors on the plasma membrane. A Leydig cell Cholesterol from low-density lipoprotein (LDL) and has about 15,000 LH receptors, and occupancy of less than high-density lipoprotein (HDL) is released in the Leydig 5% of these is sufficient for maximal steroidogenesis. This cell and transported from the outer mitochondrial mem- is an example of “spare receptors” (see Chapter 31). Excess brane to the inner mitochondrial membrane, a process reg- receptors increase target cell sensitivity to low circulating ulated by steroidogenic acute regulatory protein (StAR). levels of hormones by increasing the probability that suffi- Under the influence of LH, with cAMP as a second mes- cient receptors will be occupied to induce a response. After senger, cholesterol is converted to pregnenolone (C21) by exposure to a high LH concentration, the number of LH re- cholesterol side-chain cleavage enzyme (CYP11A1), which ceptors and testosterone production decrease. However, in removes 6 carbons attached to the 21 position. Preg- response to the initial high concentration of LH, testos- nenolone is a key intermediate for all steroid hormones in terone production will increase and then decrease. There- various steroidogenic organs (Fig. 37.9; see also Fig. 34.5). after, subsequent challenges with LH lead to no response or Pregnenolone is transported out of mitochondria by spe- decreased responses. This so-called desensitization in- cific transport proteins. The pregnenolone then moves by volves a loss of surface LH receptors as a result of internal- diffusion to the smooth ER, where the remainder of sex ization and receptor modification by phosphorylation. hormone biosynthesis takes place. The LH receptor is a single 93-kDa glycoprotein com- Pregnenolone can be converted to testosterone via two posed of three functional domains: a glycosylated extracel- pathways, the delta 5 pathway and the delta 4 pathway. In lular hormone-binding domain, a transmembrane spanning the delta 5 pathway, the double bond is in ring B; in the domain that contains seven noncontiguous segments, and delta 4 pathway the double bond is in ring A (see Fig. 37.9). an intracellular domain. The receptor is coupled to a stim- The delta 5 intermediates include 17
-hydroxypreg- ulatory G protein (G s ) via a loop of one of the LH receptor nenolone, DHEA, and androstenediol, while the delta 4 in- transmembrane segments. The activation of G s results in in- termediates are progesterone, 17
-hydroxyprogesterone, creased adenylyl cyclase activity, the production of cAMP, and androstenedione. and the activation of protein kinase A (Fig. 37.10). The conversion of C21 steroids (the progestins) to an- Low doses of LH can stimulate testosterone production drogens (C19 steroids) proceeds in two steps: first, 17
- without detectable changes in total cell cAMP concentra- hydroxylation of pregnenolone (to form 17
-hydrox- tion. However, the amount of cAMP bound to the regula- ypregnenolone) and second, C17,20 cleavage; thus, two tory subunit of protein kinase A (PKA) increases in response carbons are removed to form DHEA. This hydroxylation to such low doses of LH. This response emphasizes the im- and cleavage is accomplished by a single enzyme, 17
- portance of compartmentalization for both enzymes and hydroxylase or 17,20-lyase (CYP17). DHEA is converted substrates in mediating hormonal action. Other intracellular to androstenedione by another two-step enzymatic reac- mediators, such as the phosphatidylinositol system or cal- tion: dehydrogenation in position 3 (catalyzed by 3-hy- cium, have roles in regulating Leydig cell steroidogenesis, droxysteroid dehydrogenase [3-HSD]) and shifting of but it appears that the PKA pathway may predominate. the double bond from ring B to ring A (catalyzed by delta The proteins phosphorylated by PKA are specific for 4,5-ketosteroid isomerase); these two may be the same each cell type. Some of these, such as cAMP response ele- enzyme. The final reaction yielding testosterone is carried ment binding protein (CREB), which functions as a DNA- out by 17-ketosteroid reductase (17-hydroxysteroid de- binding protein, regulate the transcription of cholesterol

660 PART X REPRODUCTIVE PHYSIOLOGY CH 3 17 CH CD 3 Leydig AB cell HO 3 5 4 6 Cholesterol Cholesterol side- chain cleavage CH 3 CH 3 enzyme CO CO (CYPllAl) CH 3 CH 3 CH 3β-HSD CH 3 3 HO O Progesterone Pregnenolone 17α-Hydroxylase 17α-Hydroxylase CH 3 (CYP17) CH 3 (CYP17) CO CO CH CH 3 OH 3 OH CH 3β-HSD CH 3 3 HO O 17α-Hydroxypregnenolone 17α-Hydroxyprogesterone 17,20-Lyase 17,20-Lyase (CYP17) O (CYP17) O O CH CH CH 3 3 3 3β-HSD Aromatase CH CH 3 3 HO O HO Dehydroepiandrosterone Androstenedione Estrone 17β-OH steroid 17 Ketosteroid 17-Ketosteroid dehydrogenase OH reductase OH reductase OH CH CH CH 3 3 3 3β-HSD Aromatase CH CH 3 3 HO O HO Androstenediol Testosterone 17β-Estradiol 5α-Reductase OH CH 3 Steroidogen- FIGURE 37.9 CH 3 esis in Leydig Target Cells cells and further modifications O of androgens in target cells. H Solid arrows represent the delta Dihydrotestosterone 5 pathway. Dashed arrows repre- (DHT) sent the delta 4 pathway. side-chain cleavage enzyme (CYP11A1), the rate-limiting which releases cholesterol from its intracellular stores. The enzyme in the conversion of cholesterol to pregnenolone. other is the activation of CYP11A1. cAMP is inactivated by phosphodiesterase to AMP. This Leydig cells also contain receptors for prolactin enzyme plays a major role in regulating LH (and, possibly, (PRL). Hyperprolactinemia in men with pituitary tumors, FSH) responses because phosphodiesterase is activated by usually microadenomas, is associated with decreased gonadotropin stimulation. The increase in phosphodi- testosterone levels. This condition is due to a direct ef- esterase reduces the response to LH (and FSH). Certain fect of elevated circulating levels of PRL on Leydig cells, drugs can inhibit phosphodiesterase; gonadotropin hor- reducing the number of LH receptors or inhibiting down- mone responses will increase dramatically in the presence stream signaling events. In addition, hyperprolactinemia of those drugs. Numerous isoforms of phosphodiesterase may decrease LH secretion by reducing the pulsatile na- and adenylyl cyclase exist; specific types of each in the ture of its release. Under nonpathological conditions, testis have not yet been revealed. however, PRL may synergize with LH to stimulate testos- LH stimulates steroidogenesis by two principal activa- terone production by increasing the number of LH re- tions. One is the phosphorylation of cholesterol esterase, ceptors.

CHAPTER 37 The Male Reproductive System 661 5'-AMP LH Adenylyl cyclase Receptor Phosphodiesterase G protein ATP cAMP Nucleus ATP Protein Cholesterol ester PKA HDL Cholesterol esterase Protein-PO 4 ADP Cholesterol Protein-PO 4 LDL Acetate StAR Pregnenolone Cholesterol Pregnenolone Testosterone CYPllAl FIGURE 37.10 A proposed intracellular Mitochondrion Estradiol mechanism by which LH Smooth ER stimulates testosterone syn- thesis. THE ACTIONS OF ANDROGENS the circulation much more slowly if bound to a protein. Any type of liver damage or disease will generally reduce DHT enhances development of the male reproductive SHBG production. The latter can upset the hormonal bal- tract, accompanying accessory ducts and glands, and male ance between LH and testosterone. For example, if SHBG sex characteristics, including behavior. A lack of androgen declines acutely, then free testosterone may increase secretion or action causes feminization. while the total amount of circulating testosterone would decrease. In response to the increase in free testosterone, LH levels would decline in a homeostatic attempt to re- Peripheral Tissues Process and duce testosterone production. Metabolize Testosterone Once testosterone is released into the circulation, its Testosterone is not stored in Leydig cells but diffuses into fate is variable. In most target tissues, testosterone func- the blood immediately after being synthesized. An adult tions as a prohormone and is converted to the biologically man produces 6 to 7 mg testosterone per day. This amount active derivatives DHT by 5
-reductase or estradiol by slowly declines after age 50 and reaches about 4 mg/day in aromatase (Fig. 37.11). Skin, hair follicles, and most of the the seventh decade of life. Therefore, men do not undergo male reproductive tract contain an active 5
-reductase. a sudden cessation of sex steroid production upon aging, as The enzyme irreversibly catalyzes the reduction of the women do during their postmenopausal period, when the double bond in ring A and generates DHT (see Fig. 37.9). ova are completely depleted. DHT has a high binding affinity for the androgen receptor Testosterone circulates bound to plasma proteins, with and is 2 to 3 times more potent than testosterone. only 2 to 3% present as the free hormone. About 30 to Congenital deficiency of 5
-reductase in males results 40% is bound to albumin and the remainder to sex hor- in ambiguous genitalia containing female and male char- mone-binding globulin (SHBG), a 94-kDa glycoprotein acteristics because DHT is critical for directing the nor- produced by the liver. SHBG binds both estradiol and mal development of male external genitalia during embry- testosterone, with a higher binding affinity for testos- onic life (see Chapter 39). Without DHT, the female terone. Because its production is increased by estrogens pathway may predominate, even though the genetic sex is and decreased by androgens, plasma SHBG concentration male and small, undescended testes are present in the in- is higher in women than in men. SHBG serves as a reser- guinal region. DHT is nonaromatizable and cannot be voir for testosterone, and therefore, a sudden decline in converted to estrogens. newly formed testosterone may not be evident because of Drugs that inhibit 5
-reductase are currently used to re- the large pool bound to proteins. SHBG, in effect, deacti- duce prostatic hypertrophy because DHT induces hyperpla- vates testosterone because only the unbound hormone sia of prostatic epithelial cells. In addition, analogs of GnRH, can enter the cell. SHBG also prolongs the half-life of cir- as either agonists or antagonists, can be given to patients to culating testosterone because testosterone is cleared from reduce the secretion of androgen in androgen-dependent

662 PART X REPRODUCTIVE PHYSIOLOGY Plasma Testosterone testes, pituitary, muscle testosterone Conjugating enzymes Aromatase Estradiol Conjugates fat, liver, CNS, skin, hair liver, kidney 17β-Dehydrogenase 5α-Reductase Dihydrotestosterone prostate, scrotum, 17-Ketosteroids penis, bone liver, kidney Biologically active Excretory metabolites Conversion of testosterone to different products in extratesticular sites. FIGURE 37.11 neoplasia or cancer (see Clinical Focus Box 37.1). In the case most every tissue, including alteration of the primary sex of the GnRH antagonist, this analog blocks the secretion of structures (i.e., the testes and genital tract) and stimulation LH. In contrast, GnRH agonists given in large quantities ini- of the secondary sex structures (i.e., accessory glands) and tially induce the secretion of LH (and androgen). However, development of secondary sex characteristics responsible this response is followed by down-regulation of GnRH re- for masculine phenotypic expression. Androgens also affect ceptors on the pituitary gonadotrophs and, ultimately, a dra- both sexual and nonsexual behavior. The relative potency matic decline in circulating LH and androgen. ranking of androgens is DHT  testosterone  an- Aromatization of some androgens to estrogens occurs in drostenedione  DHEA. The action of sex steroid hor- fat, liver, skin, and brain cells. Circulating levels of total es- mones on somatic tissue, such as muscle, is referred to as trogens (estradiol plus estrone) in men can approach those “anabolic” because the end result is increased muscle size. of women in their early follicular phase. Men are protected This action is mediated by the same molecular mechanisms from feminization as long as production of and tissue re- that result in virilization. sponsiveness to androgens are normal. The treatment of Between 8 and 18 weeks of fetal life, androgens medi- hypogonadal male patients with high doses of aromatizable ate differentiation of the male genitalia. The organogene- testosterone analogs (or testosterone), the use of anabolic sis of the wolffian (mesonephric) ducts into the epi- steroids by athletes, abnormal reductions in testosterone didymis, vas deferens, and seminal vesicles is directly secretion, estrogen-producing testicular tumors, and tissue influenced by testosterone, which reaches these target tis- insensitivity to androgens can lead to gynecomastia or sues by diffusion rather than by a systemic route. The dif- breast enlargement. All of these conditions are character- ferentiation of the urogenital sinus and the genital tuber- ized by a decrease in the testosterone-to-estradiol ratio. cle into the penis, scrotum, and prostate gland depends on Androgens are metabolized in the liver to biologically testosterone being converted to DHT. Toward the end of inactive water-soluble derivatives suitable for excretion fetal life, the descent of the testes into the scrotum is pro- by the kidneys. The major products of testosterone me- moted by testosterone and insulin-like hormones from tabolism are two 17-ketosteroids, androsterone and etio- Leydig cells (see Chapter 39). cholanolone. These, as well as native testosterone, are The onset of puberty is marked by enhanced androgenic conjugated in position 3 to form sulfates and glu- activity. Androgens promote the growth of the penis and curonides, which are water-soluble and excreted into the scrotum, stimulate the growth and secretory activity of the urine (see Fig. 37.11). epididymis and accessory glands, and increase the pigmen- tation of the genitalia. Enlargement of the testes occurs un- der the influence of the gonadotropins (LH and FSH). Androgens Have Effects on Reproductive Spermatogenesis, which is initiated during puberty, de- pends on adequate amounts of testosterone. Throughout and Nonreproductive Tissues adulthood, androgens are responsible for maintaining the An androgen is a substance that stimulates the growth of structural and functional integrity of all reproductive tis- the male reproductive tract and the development of sec- sues. Castration of adult men results in regression of the re- ondary sex characteristics. Androgens have effects on al- productive tract and involution of the accessory glands.

CHAPTER 37 The Male Reproductive System 663 CLINICAL FOCUS BOX 37.1 Prostate Cancer dogenous GnRH from binding to those receptors, and sub- Some prostate cancers are highly dependent upon andro- sequently reduce LH and FSH secretion. Shortly after treat- gens for cellular proliferation; therefore, physicians at- ment, testicular concentrations of androgens decline be- tempt to totally ablate the secretion of androgens by the cause of the low levels of circulating LH and FSH. The testes. Generally, two options for those patients are surgi- expectation is that androgen-dependent cancer cells will cal castration and chemical castration. Surgical castration cease or slow proliferation and, ultimately, die. is irreversible and requires the removal of the testes, while GnRH agonists (leuprolide acetate [trade name chemical castration is reversible. Lupron]) are usually used in combination with other drugs One option for chemical treatment of these patients is in order to block most effectively androgenic activity. For the use of analogs of GnRH, the hormone that regulates the example, one of the androgen-blocking drugs includes 5
- secretion of LH and FSH. Long-acting GnRH agonists or an- reductase inhibitors that prevent the conversion of testos- tagonists reduce LH and FSH secretion by different mecha- terone to the highly active androgen dihydrotestosterone nisms. GnRH agonists reduce gonadotropin secretion by (DHT). In addition, desensitization of the pituitary gonadotrophs to GnRH, antiandrogens, such as flutamide, bind to the androgen leading to a reduction of LH and FSH secretion. GnRH ago- receptor and prevent binding of endogenous androgen. nists initially stimulate GnRH receptors on pituitary cells Some prostate cancers are androgen-independent, and and ultimately reduce their numbers. GnRH antagonists the treatment requires nonhormonal therapies, including bind to GnRH receptors on the pituitary cells, prevent en- chemotherapy and radiation. Androgens Are Responsible for Secondary Sex drogens have multiple effects on skeletal and cardiac mus- Characteristics and the Masculine Phenotype cle. Because 5
-reductase activity in muscle cells is low, the androgenic action is due to testosterone. Testosterone Androgens effect changes in hair distribution, skin texture, stimulates muscle hypertrophy, increasing muscle mass; pitch of the voice, bone growth, and muscle development. however, it has minimal or no effect on muscle hyperplasia. Hair is classified by its sensitivity to androgens into non- Testosterone, in synergy with GH, causes a net increase in sexual (eyebrows and extremities); ambisexual (axilla), muscle protein. which is responsive to low levels of androgens; and sexual Other nonreproductive organs and systems are affected, (face, chest, upper pubic triangle), which is responsive only directly or indirectly, by androgens, including the liver, to high androgen levels. Hair follicles metabolize testos- kidneys, adipose tissue, and hematopoietic and immune terone to DHT or androstenedione. Androgens stimulate systems. The kidneys are larger in males, and some renal the growth of facial, chest, and axillary hair; however, enzymes (e.g., -glucuronidase and ornithine decarboxy- along with genetic factors, they also promote temporal hair lase) are induced by androgens. HDL levels are lower and recession and loss. Normal axillary and pubic hair growth triglyceride concentrations higher in men, compared to in women is also under androgenic control, whereas excess premenopausal women, a fact that may explain the higher androgen production in women causes the excessive prevalence of atherosclerosis in men. Androgens increase growth of sexual hair (hirsutism). red blood cell mass (and, hence, hemoglobin levels) by The growth and secretory activity of the sebaceous stimulating erythropoietin production and by increasing glands on the face, upper back, and chest are stimulated by stem cell proliferation in the bone marrow. androgens, primarily DHT, and inhibited by estrogens. In- creased sensitivity of target cells to androgenic action, es- pecially during puberty, is the cause of acne vulgaris in The Brain Is a Target Site for Androgen Action both males and females. Skin derived from the urogenital ridge (e.g., the prepuce, scrotum, clitoris, and labia majora) Many sites in the brain contain androgen receptors, with remains sensitive to androgens throughout life and contains the highest density in the hypothalamus, preoptic area, an active 5
-reductase. Growth of the larynx and thicken- septum, and amygdala. Most of those areas also contain ing of the vocal cords are also androgen-dependent. Eu- aromatase and many of the androgenic actions in the brain nuchs maintain the high-pitched voice typical of prepuber- result from the aromatization of androgens to estrogens. tal boys because they were castrated prior to puberty. The pituitary also has abundant androgen receptors, but no The growth spurt of adolescent males is influenced by a aromatase. The enzyme 5
-reductase is widely distributed complex interplay between androgens, growth hormone in the brain, but its activity is generally higher during the (GH), nutrition, and genetic factors. The growth spurt in- prenatal period than in adults. Sexual dimorphism in the cludes growth of the vertebrae, long bones, and shoulders. size, number, and arborization of neurons in the preoptic The mechanism by which androgens (likely DHT) alter area, amygdala, and superior cervical ganglia has been re- bone metabolism is unclear. Androgens accelerate closure cently recognized in humans. of the epiphyses in the long bones, eventually limiting fur- Unlike most species, which mate only to produce off- ther growth. Because of the latter, precocious puberty is as- spring, in humans, sexual activity and procreation are not sociated with a final short adult stature, whereas delayed tightly linked. Superimposed on the basic reproductive puberty or eunuchoidism usually results in tall stature. An- mechanisms dictated by hormones are numerous psycho-

664 PART X REPRODUCTIVE PHYSIOLOGY logical and societal factors. In normal men, no correlation is To establish the cause(s) of reproductive dysfunction, found between circulating testosterone levels and sexual physical examination and medical history, semen analysis, drive, frequency of intercourse, or sexual fantasies. Simi- hormone determinations, hormone stimulation tests, and larly, there is no correlation between testosterone levels and genetic analysis are performed. Physical examination impotence or homosexuality. Castration of adult men re- should establish whether eunuchoidal features (i.e., infan- sults in a slow decline in, but not a complete elimination of, tile appearance of external genitalia and poor or absent de- sexual interest and activity. See Clinical Focus Box 37.2 for velopment of secondary sex characteristics) are present. In a discussion of the effects of testosterone administration. men with adult-onset reproductive dysfunction, physical examination can uncover problems such as cryptorchidism (nondescendent testes), testicular injury, varicocele (an ab- REPRODUCTIVE DYSFUNCTIONS normality of the spermatic vasculature), testicular tumors, prostatic inflammation, or gynecomastia. Medical and fam- Male reproductive dysfunctions may by caused by en- ily history help determine delayed puberty, anosmia (an in- docrine disruption, morphological alterations in the repro- ability to smell, often associated with GnRH dysfunction), ductive tract, neuropathology, and genetic mutations. Sev- previous fertility, changes in sexual performance, ejacula- eral medical tests, including serum hormone levels, tory disturbances, or impotence (an inability to achieve or physical examination of the reproductive organs, and maintain erection). sperm count are important in ascertaining causes of repro- One step in the evaluation of fertility is semen analysis. ductive dysfunctions. Semen are analyzed on specimens collected after 3 to 5 days of sexual abstinence, as the number of sperm ejaculated re- mains low for a couple of days after ejaculation. Initial ex- Hypogonadism Can Result From amination includes determination of viscosity, liquefaction, Defects at Several Levels and semen volume. The sperm are then counted and the Male hypogonadism may result from defects in spermato- percentage of sperm showing forward motility is scored. genesis, steroidogenesis, or both. It may be a primary de- The spermatozoa are evaluated morphologically, with at- fect in the testes or secondary to hypothalamic-pituitary tention to abnormal head configuration and defective tails. dysfunction, and determining whether the onset of gonadal Chemical analysis can provide information on the secretory failure occurred before or after puberty is important in es- activity of the accessory glands, which is considered abnor- tablishing the cause. However, several factors must be con- mal if semen volume is too low or sperm motility is im- sidered. First, normal spermatogenesis almost never occurs paired. Fructose and prostaglandin levels are determined to with defective steroidogenesis, but normal steroidogenesis assess the function of the seminal vesicles and levels of zinc, can be present with defective spermatogenesis. Second, magnesium, and acid phosphatase to evaluate the prostate. primary testicular failure removes feedback inhibition from Terms used in evaluating fertility include aspermia (no se- the hypothalamic-pituitary axis, resulting in elevated men), hypospermia and hyperspermia (too small or too plasma gonadotropins. In contrast, hypothalamic and/or large semen volume), azoospermia (no spermatozoa), and pituitary failure is almost always accompanied by decreased oligozoospermia (reduced number of spermatozoa). gonadotropin and steroid levels and reduced testicular size. Serum testosterone, estradiol, LH, and FSH analyses are Third, gonadal failure before puberty results in the absence performed using radioimmunoassays. Free and total testos- of secondary sex characteristics, creating a distinctive clin- terone levels should be measured; because of the pulsatile ical presentation called eunuchoidism. In contrast, men nature of LH release, several consecutive blood samples are with a postpubertal testicular failure retain masculine fea- needed. Dynamic hormone stimulation tests are most valu- tures but exhibit low sperm counts or a reduced ability to able for establishing the site of abnormality. A failure to in- produce functional sperm. crease LH release upon treatment with clomiphene, an CLINICAL FOCUS BOX 37.2 Effects of Testosterone Administration to a suppression of testosterone production by the Ley- Although testosterone has a role in stimulating spermato- dig cells and a further decrease in testicular testosterone genesis, infertile men with a low sperm count do not ben- concentrations. Ultimately, because LH levels decrease efit from testosterone treatment. Unless given at supra- when exogenous testosterone is administered, testicular physiological doses, exogenous testosterone cannot size decreases, as has been reported for men who abuse achieve the required local high concentration in the testis. androgens. One function of androgen-binding protein in the testis is to High levels of androgens have an anabolic effect on sequester testosterone, which significantly increases its lo- muscle tissue, leading to increased muscle mass, strength, cal concentration. and performance, a desired result for body builders and Exogenous testosterone given to men would normally athletes. Androgen abuse has been associated with abnor- inhibit endogenous LH release through a negative-feed- mally aggressive behavior and the potential for increased back effect on the hypothalamic-pituitary axis, and lead incidence of liver and brain tumors.

CHAPTER 37 The Male Reproductive System 665 antiestrogen, likely indicates a hypothalamic abnormality. lactinemia, whether from hypothalamic disturbance or pi- Clomiphene blocks the inhibitory effects of estrogen and tuitary adenoma, often results in decreased GnRH pro- testosterone on endogenous GnRH release. An absence of duction, hypogonadotropic state, impotence, and de- or blunted testosterone rise after hCG injection suggests a creased libido. It can be treated with dopaminergic primary testicular defect. Genetic analysis is used when agonists (e.g., bromocryptine), which suppress PRL re- congenital defects are suspected. The presence of the Y lease (see Chapter 38). Excess androgens can also result in chromosome can be revealed by karyotyping of cultured suppression of the hypothalamic-pituitary axis, resulting peripheral lymphocytes or direct detection of specific Y in lower LH levels and impaired testicular function. This antigens on cell surfaces. condition often results from congenital adrenal hyperpla- sia and increased adrenal androgen production from 21- hydroxylase (CYP21A2) deficiency (see Chapter 34). Reproductive Disorders Are Associated With Hypergonadotropic hypogonadism usually results from Hypogonadotropic or Hypergonadotropic States impaired testosterone production, which can be congenital or acquired. The most common disorder is Klinefelter’s Endocrine factors are responsible for approximately 50% of hypogonadal or infertility cases. The remainder is of un- syndrome discussed earlier. known etiology or the result of injury, deformities, and en- vironmental factors. Endocrine-related hypogonadism can Male Pseudohermaphroditism Often Results be classified as hypothalamic-pituitary defects (hypogo- From Resistance to Androgens nadotropic because of the lack of LH and/or FSH), primary gonadal defects (hypergonadotropic because go- A pseudohermaphrodite is an individual with the gonads nadotropins are high as a result of a lack of negative feed- of one sex and the genitalia of the other. One of the most back from the testes), and defective androgen action (usu- interesting causes of male reproductive abnormalities is an ally the result of absence of androgen receptor or end organ insensitivity to androgens. The best character- 5
-reductase). Each of these is further subdivided into sev- ized syndrome is testicular feminization, an X-linked re- eral categories, but only a few examples are discussed here. cessive disorder caused by a defect in the testosterone re- Hypogonadotropic hypogonadism can be congenital, ceptor. In the classical form, patients are male idiopathic, or acquired. The most common congenital form pseudohermaphrodites with a female phenotype and an XY is Kallmann’s syndrome, which results from decreased or male genotype. They have abdominal testes that secrete absent GnRH secretion, as mentioned earlier. It is often as- testosterone but no other internal genitalia of either sex sociated with anosmia or hyposmia and is transmitted as an (see Chapter 39). They commonly have female external autosomal dominant trait. Patients do not undergo pubertal genitalia, but with a short vagina ending in a blind pouch. development and have eunuchoidal features. Plasma LH, Breast development is typical of a female (as a result of pe- FSH, and testosterone levels are low, and the testes are im- ripheral aromatization of testosterone), but axillary and pu- mature and have no sperm. There is no response to bic hair, which are androgen-dependent, are scarce or ab- clomiphene, but intermittent treatment with GnRH can sent. Testosterone levels are normal or elevated, estradiol produce sexual maturation and full spermatogenesis. levels are above the normal male range, and circulating go- Another category of hypogonadotropic hypogo- nadotropin levels are high. The inguinally located testes nadism, panhypopituitarism or pituitary failure, can oc- usually have to be removed because of an increased risk of cur before or after puberty and is usually accompanied by cancer. After orchiectomy, patients are treated with estra- a deficiency of other pituitary hormones. Hyperpro- diol to maintain a female phenotype. REVIEW QUESTIONS DIRECTIONS: Each of the numbered (D) Failure of the hypothalamus to (A) Storage and transport of mature sperm items or incomplete statements in this respond to testosterone (B) Initiating the development of section is followed by answers or by (E) Increased number of FSH receptors spermatozoa completions of the statement. Select the in the testis (C) Secretion of estrogens ONE lettered answer or completion that is 2. The major function of follistatin is (D) Production of inhibin BEST in each case. (A) Bind FSH and increase FSH (E) Secretion of fluids that contribute secretion to semen 1. A major causal factor in some cases of (B) Inhibit the production of seminal 4. The production of mature spermatozoa hypogonadism is fluid from spermatogonia (A) Reduced secretion of (C) Reduce testosterone secretion by (A) Takes 32 days gonadotropin-releasing hormone Leydig cells (B) Takes 70 days (GnRH) (D) Stimulate the production of (C) Takes 150 days (B) Hypersecretion of pituitary LH and spermatogonia (D) Is unaffected by Kallmann’s FSH as the result of increased GnRH (E) Bind activin and thus decrease FSH syndrome (C) Excess secretion of testicular secretion (E) Is independent of testicular activin by Sertoli cells 3. A major function of the epididymis is temperature (continued)

666 PART X REPRODUCTIVE PHYSIOLOGY 5. The first enzymatic reaction, which is (C) Decreases the half-life of SUGGESTED READING the rate-limiting step, in the testosterone Burger H, DeKretser D. The Testis. New production of testosterone (D) Stimulates the secretion of inhibin York: Raven Press, 1989. (A) Occurs in the mitochondria (E) Blocks the synthesis of androgen- Fawcett DW. A Textbook of Histology. (B) Occurs in the ribosomes binding protein 12th Ed. New York: Chapman & Hall, (C) Involves aromatization 8. The production of estradiol by the 1994;796–850. (D) Generates progesterone as the testes requires Griswold MD, Russell LD. Sertoli cells, immediate derivative (A) Sertoli cell follistatin function. In: Knobil E, Neill JD, eds. (E) Is stimulated by FSH (B) LH and Leydig cells The Encyclopedia of Reproduction. 6. Testosterone is (C) Activin but not LH New York: Academic Press, (A) Bound to high-density lipoprotein (D) Leydig cell, Sertoli cells, LH, and 1999;371–380. (HDL) FSH Johnson L, McGowen TA, Keillor GE. (B) Bound to activin (E) Leydig cells and FSH Testis, overview. In: Knobil E, Neill (C) Converted to dihydrotestosterone 9. Eunuchs are tall because JD, eds. The Encyclopedia of Repro- in the prostate (A) Estrogens stimulate the growth of duction. New York: Academic Press, (D) Converted to 17- long bones 1999;769–784. hydroxyprogesterone in the liver (B) Excess LH delays epiphyseal Payne A, Hardy M, Russell L. The Leydig (E) Metabolized by cholesterol side- closure of the long bones Cell. Vienna, IL: Cache River Press, chain cleavage enzyme (C) Reduced androgen and estrogen 1996. 7. Sex hormone-binding globulin (SHBG) delays epiphyseal closure in long Redman JF. Male reproductive system, (A) Binds testosterone with a higher bones human. In: Knobil E, Neill JD, eds. affinity than estradiol (D) The lack of testes stimulates The Encyclopedia of Reproduction. (B) Reduces the total amount of closure of the epiphyses New York: Academic Press, circulating testosterone (E) They secrete excess androgen 1999;30–41.

The Female CHAPTER 38 38 Reproductive System Paul F. Terranova, Ph.D. CHAPTER OUTLINE ■ AN OVERVIEW OF THE FEMALE REPRODUCTIVE ■ FORMATION OF THE CORPUS LUTEUM FROM THE SYSTEM POSTOVULATORY FOLLICLE ■ THE HYPOTHALAMIC-PITUITARY AXIS ■ THE MENSTRUAL CYCLE ■ THE FEMALE REPRODUCTIVE ORGANS ■ ESTROGEN, PROGESTIN, AND ANDROGEN: ■ FOLLICULOGENESIS, STEROIDOGENESIS, ATRESIA, TRANSPORT AND METABOLISM AND MEIOSIS ■ PUBERTY ■ FOLLICLE SELECTION AND OVULATION ■ MENOPAUSE ■ INFERTILITY KEY CONCEPTS 1. Pulses of hypothalamic GnRH regulate the secretion of LH 7. The formation of a functional corpus luteum requires the and FSH, which enhance follicular development, steroido- presence of an LH surge, adequate numbers of LH recep- genesis, ovulation, and formation of the corpus luteum. tors, sufficient granulosa cells, and significant proges- 2. LH and FSH, in coordination with ovarian theca and granu- terone secretion. losa cells, regulate the secretion of follicular estradiol. 8. The uterine cycle is regulated by estradiol and proges- 3. Ovulation occurs as the result of a positive feedback of fol- terone, such that estradiol induces proliferation of the uter- licular estradiol on the hypothalamic-pituitary axis that in- ine endometrium, whereas progesterone induces differen- duces LH and FSH surges. tiation of the uterine endometrium and the secretion of 4. Follicular development occurs in distinct steps: primordial, distinct products. primary, secondary, tertiary, and graafian follicle stages. 9. During puberty, the hypothalamus begins to secrete in- 5. Follicular rupture (ovulation) requires the coordination of creasing quantities of GnRH, which increases LH and FSH appropriately timed LH and FSH surges that induce in- secretion, enhances ovarian function, and leads to the first flammatory reactions in the graafian follicle, leading to ovulation. dissolution at midcycle of the follicular wall by several 10. Menopause ensues from the loss of numerous oocytes in ovarian enzymes. the ovary and the subsequent failure of follicular develop- 6. Follicular atresia results from the withdrawal of go- ment and estradiol secretion. LH and FSH levels rise from nadotropin support. the lack of negative feedback by estradiol. he fertility of the mature human female is cyclic. The tive-feedback effects on the hypothalamus and on pituitary Trelease from the ovary of a mature female germ cell or gonadotrophs, generating the cyclic pattern of LH and ovum occurs at a distinct phase of the menstrual cycle. The FSH release characteristic of the female reproductive sys- secretion of ovarian steroid hormones, estradiol and prog- tem. Since the hormonal events during the menstrual cycle esterone, and the subsequent release of an ovum during the are delicately synchronized, the menstrual cycle can be menstrual cycle are controlled by cyclic changes in LH and readily affected by stress and by environmental, psycho- FSH from the pituitary gland, and estradiol and proges- logical, and social factors. terone from the ovaries. The cyclic changes in steroid hor- The female cycle is characterized by monthly bleeding, mone secretion cause significant changes in the structure resulting from the withdrawal of ovarian steroid hormone and function of the uterus in preparing it for the reception support of the uterus, which causes shedding of the super- of a fertilized ovum. At different stages of the menstrual cy- ficial layers of the uterine lining at the end of each cycle. cle, progesterone and estradiol exert negative- and posi- The first menstrual cycle occurs during puberty. Menstrual 667

668 PART X REPRODUCTIVE PHYSIOLOGY cycles are interrupted during pregnancy and lactation and lation. Both LH and FSH regulate follicular steroidogenesis cease at menopause. Menstruation signifies a failure to con- and androgen and estradiol secretion, and LH regulates the ceive and results from regression of the corpus luteum and secretion of progesterone from the corpus luteum. Ovarian subsequent withdrawal of luteal steroid support of the su- steroids inhibit the secretion of LH and FSH with one ex- perficial endometrial layer of the uterus. ception: Just prior to ovulation (at midcycle), estradiol has a positive-feedback effect on the hypothalamic-pituitary axis and induces significant increases in the secretion of AN OVERVIEW OF THE FEMALE GnRH, LH, and FSH. The ovary also produces three REPRODUCTIVE SYSTEM polypeptide hormones. Inhibin suppresses the secretion of FSH. Activin (an inhibin-binding protein) increases the se- An overview of the interactions of hormonal factors in fe- cretion of FSH, and follistatin (an activin-binding protein) male reproduction is shown in Figure 38.1. The female reduces the secretion of FSH. hormonal system consists of the brain, pituitary, ovaries, Shortly after fertilization, the embryo begins to develop and reproductive tract (oviduct, uterus, cervix, and vagina). placenta cells, which attach to the uterine lining and unite In the brain, the hypothalamus produces gonadotropin-re- with the maternal placental cells. The placenta produces leasing hormone (GnRH), which controls the secretion of several pituitary-like and ovarian steroid-like hormones. luteinizing hormone (LH) and follicle-stimulating hor- These hormones support placental and fetal development mone (FSH). throughout pregnancy and have a role in parturition. The The mature ovary has two major functions: the matura- mammary glands are also under the control of pituitary tion of germ cells and steroidogenesis. Each germ cell is ul- hormones and ovarian steroids, and provide the baby with timately enclosed within a follicle, a major source of steroid immunological protection and nutritional support through hormones during the menstrual cycle. At ovulation, the lactation. Lactation is hormonally controlled by prolactin ovum or egg is released and the ruptured follicle is trans- (PRL) from the anterior pituitary, which regulates milk formed into a corpus luteum, which secretes progesterone production, and oxytocin from the posterior pituitary, as its main product. FSH is primarily involved in stimulat- which induces milk ejection from the breasts. ing the growth of ovarian follicles, while LH induces ovu- THE HYPOTHALAMIC-PITUITARY AXIS Environment Age Drugs The hypothalamic-pituitary axis has an important role in regulating the menstrual cycle. GnRH, a decapeptide pro- duced in the hypothalamus and released in a pulsatile man- Brain 
ner, controls the secretion of LH and FSH through a portal Centers vascular system (see Chapter 32). Blockade of the portal system reduces the secretion of LH and FSH and leads to ovarian atrophy and a reduction in ovarian hormone secre- Hypothalamus
tion. The secretion of GnRH by the hypothalamus is regu- lated by neurons from other brain regions. Neurotransmit- ters, such as epinephrine and norepinephrine, stimulate the GnRH Dopamine secretion of GnRH, whereas dopamine and serotonin in- hibit secretion of GnRH. In addition, ovarian steroids and peptides and hypothalamic neuropeptides can regulate the Anterior pituitary
secretion of GnRH. GnRH stimulates the pituitary go- nadotrophs to secrete LH and FSH. GnRH binds to high- affinity receptors on the gonadotrophs and stimulates the FSH/LH 
PRL secretion of LH and FSH through a phosphoinositide-pro- tein kinase C-mediated pathway (see Chapter 1). Inhibin
, activin  , Ovary  A graph of LH release throughout the female life span is follistatin
shown in Figure 38.2. During the neonatal period, LH is re- leased at low and steady rates without pulsatility; this pe- riod coincides with lack of development of mature ovarian Estradiol, follicles and very low to no ovarian estradiol secretion. Pul- progesterone, androgen satile release begins with the onset of puberty and for sev-   eral years is expressed only during sleep; this period coin- cides with increased but asynchronous follicular Reproductive Secondary sex development and with increased secretion of ovarian estra- tract characteristics diol. Upon the establishment of regular functional men- Regulation of the reproductive tract in the strual cycles associated with regular ovulation, LH pulsatil- FIGURE 38.1 female. The main reproductive hormones are ity prevails throughout the 24-hour period, changing in a shown in boxes. Positive and negative regulations are depicted by monthly cyclic manner. In postmenopausal women whose plus and minus signs. ovaries lack sustained follicular development and exhibit

CHAPTER 38 The Female Reproductive System 669 Day Night Day Night Day Night Day Night Day Night Plasma LH conc. FIGURE 38.2 Relative levels of LH release in hu- fied from Yen SSC, et al. In: Ferin M, et Neonatal Pubertal Tonic Midcycle Postmenopausal man females throughout life. (Modi- al., eds. Biorhythms and Human Repro- Reproductive duction. New York: Wiley, 1974.) low ovarian estradiol secretion, mean circulating LH levels tures change in a cyclic manner under the influence of the are high and pulses occur at a high frequency. reproductive hormones. The ovaries are in the pelvic portion of the abdominal cav- ity on both sides of the uterus and are anchored by ligaments (Fig. 38.3). An adult ovary weighs 8 to 12 g and consists of an THE FEMALE REPRODUCTIVE ORGANS outer cortex and an inner medulla, without a sharp demarca- The female reproductive tract has two major components: tion. The cortex is surrounded by a fibrous tissue, the tunica the ovaries, which produce the mature ovum and secrete albuginea, covered by a single layer of surface epithelium progestins, androgens, and estrogens; and the ductal sys- continuous with the mesothelium covering the other organs tem, which transports ovum, is the place of the union of the in the abdominal cavity. The cortex contains oocytes en- sperm and egg, and maintains the developing conceptus closed in follicles of various sizes, corpora lutea, corpora al- until delivery. The morphology and function of these struc- bicantia, and stromal cells. The medulla contains connective Isthmus Fundus Ampulla Corpus Broad ligament Uterus Oviduct Myometrium Fimbria Endometrium Ovary Infundibulum Primordial follicle Cervix Primary follicle Vagina Ovarian ligament Atretic follicle Ovarian vessels Early antrum formation Corpus albicans Mature corpus luteum Ovary Graafian follicle Early corpus luteum Stroma Germinal epithelium Ovulation The female reproductive organs. (Modified from Patton BM. Human Embryology. New FIGURE 38.3 York: McGraw-Hill, 1976.)

670 PART X REPRODUCTIVE PHYSIOLOGY and interstitial tissues. Blood vessels, lymphatics, and nerves cation (keratinization) of the vaginal epithelium, whereas enter the medulla of the ovary through the hilus. progesterone opposes those actions and induces the influx On the side that ovulates, the oviduct (fallopian tube) of polymorphonuclear leukocytes into the vaginal fluids. receives the ovum immediately after ovulation. The Estradiol also activates vaginal glands that produce lubri- oviducts are the site of fertilization and provide an envi- cating fluid during coitus. ronment for development of the early embryo. The oviducts are 10 to 15 cm long and composed of sequential regions called the infundibulum, ampulla, and isthmus. FOLLICULOGENESIS, STEROIDOGENESIS, The infundibulum is adjacent to the ovary and opens to the ATRESIA, AND MEIOSIS peritoneal cavity. It is trumpet-shaped with finger-like pro- jections called fimbria along its outer border that grasp the Most follicles in the ovary will undergo atresia. However, ovum at the time of follicular rupture. Its thin walls are cov- some will develop into mature follicles, produce steroids, ered with densely ciliated projections, which facilitate and ovulate. As follicles mature, oocytes will also mature by ovum uptake and movement through this region. The am- entering meiosis, which produces the proper number of pulla is the site of fertilization. It has a thin musculature and chromosomes in preparation for fertilization. well-developed mucosal surface. The isthmus is located at the uterotubal junction and has a narrow lumen surrounded The Primordial Follicle Contains an by smooth muscle. It has sphincter-like properties and can Oocyte Arrested in Meiosis serve as a barrier to the passage of germ cells. The oviducts transport the germ cells in two directions: sperm ascend to- Female germ cells develop in the embryonic yolk sac and ward the ampulla and the zygote descends toward the migrate to the genital ridge where they participate in the uterus. This requires coordination between smooth muscle development of the ovary (Table 38.1). Without germ contraction, ciliary movement, and fluid secretion, all of cells, the ovary does not develop. The germs cells, called which are under hormonal and neuronal control. oogonia, actively divide by mitosis. Oogonia undergo mi- The uterus is situated between the urinary bladder and tosis only during the prenatal period. By birth, the ovaries rectum. On each upper side, an oviduct opens into the uter- contain a finite number of oocytes, estimated to be about 1 ine lumen, and on the lower side, the uterus connects to the million. Most of them will die by a process called atresia. By vagina. The uterus is composed of two types of tissue. The puberty, only 200,000 oocytes remain; by age 30, only outer part is the myometrium, composed of multiple layers 26,000 remain; and by the time of menopause, the ovaries of smooth muscle. The inner part, lining the lumen of the are essentially devoid of oocytes. uterus, is the endometrium, which contains a deep stromal When oogonia cease the process of mitosis, they are called layer next to the myometrium and a superficial epithelial oocytes. At that time they enter the meiotic cycle (or meio- layer. The stroma is permeated by spiral arteries and con- sis, to prepare for the production of a haploid ovum), become tains much connective tissue. The epithelial layer is inter- arrested in prophase of the first meiotic division, and remain rupted by uterine glands, which also penetrate the stromal arrested in that phase until they either die or grow into ma- layer and are lined by columnar secretory cells. The uterus ture oocytes at the time of ovulation. The primordial follicle provides an environment for the developing fetus, and (Fig. 38.4) is 20 m in diameter and contains an oocyte, eventually, the myometrium will generate rhythmic con- which may or may not be surrounded by a single layer of flat- tractions that assist in expelling the fetus at delivery. tened (squamous) pregranulosa cells. When pregranulosa The cervix (neck) is a narrow muscular canal that con- cells surround the oocyte, a basement membrane develops, nects the vagina and the body (corpus) of the uterus. It separating the granulosa from the ovarian stroma. must dilate in response to hormones to allow the expulsion of the fetus. The cervix has numerous glands with a colum- A Graafian Follicle Is the Final Stage of nar epithelium that produces mucus under the control of estradiol. As more and more estradiol is produced during Follicle Development the follicular phase of the cycle, the cervical mucus changes Folliculogenesis (also called follicular development) is the from a scanty viscous material to a profuse watery and process by which follicles develop and mature (see Fig. highly elastic substance called spinnbarkeit. The viscosity 38.3). Follicles are in one of the following physiological of the spinnbarkeit can be tested by touching it with a piece states: resting, growing, degenerating, or ready to ovulate. of paper and lifting vertically. The mucus can form a thread During each menstrual cycle, the ovaries produce a group up to 6 cm under the influence of elevated estradiol. If a of growing follicles of which most will fail to grow to ma- drop of the cervical mucus is placed on a slide and allowed turity and will undergo follicular atresia (death) at some to dry, it will form a typical ferning pattern when under the stage of development. However, one dominant follicle influence of estradiol. generally emerges from the cohort of developing follicles The vagina is well innervated, and has a rich blood sup- and it will ovulate, releasing a mature haploid ovum. ply. It is lined by several layers of epithelium that change Primordial follicles are generally considered the non- histologically during the menstrual cycle. When estradiol growing resting pool of follicles, which gets progressively levels are low, as during the prepubertal or post- depleted throughout life; by the time of menopause, the menopausal periods, the vaginal epithelium is thin and the ovaries are essentially devoid of all follicles. Primordial fol- secretions are scanty, resulting in a dry and infection-sus- licles are located in the ovarian cortex (peripheral regions ceptible area. Estradiol induces proliferation and cornifi- of the ovary) beneath the tunica albuginea.

CHAPTER 38 The Female Reproductive System 671 TABLE 38.1 Different Stages in the Development of an Ovum and Follicle Stage Process Ovum Follicle Fetal life Migration Primordial germ cells Mitosis Oogonia Primordial follicle First meiotic division begins Primary oocyte Primary follicle Birth Arrest in prophase Growth of oocyte and follicle Puberty Follicular maturation Secondary follicle Cycle Antral follicle Ovulation Resumption of meiosis Secondary oocyte Graafian follicle Emission of first polar body Arrest in metaphase Corpus luteum Fertilization Second meiotic division complete Zygote Emission of second polar body Implantation Mitotic divisions Embryo Blastocyst Parturition Body Patterning Fetus Corpus albicans Progression from primordial to the next stage of follicu- acquire receptors for FSH and start producing small lar development, the primary stage, occurs at a relatively amounts of estrogen. The theca externa remains fibroblastic constant rate throughout fetal, juvenile, prepubertal, and and provides structural support to the developing follicle. adult life. Once primary follicles leave the resting pool, Development beyond the primary follicle is go- they are committed to further development or atresia. Most nadotropin-dependent, begins at puberty, and continues in become atretic, and typically only one fully developed fol- a cyclic manner throughout the reproductive years. As the licle will ovulate. The conversion from primordial to pri- follicle continues to grow, theca layers expand, and fluid- mary follicles is believed to be independent of pituitary go- filled spaces or antra begin to develop around the granulosa nadotropins. The exact signal that recruits a follicle from a cells. This early antral stage of follicle development is re- resting to a growing pool is unknown; it could be pro- ferred to as the tertiary follicular stage (see Fig. 38.4). The grammed by the cell genome or influenced by local ovarian critical hormone responsible for progression from the pre- growth regulators. antral to the antral stage is FSH. Mitosis of the granulosa The first sign that a primordial follicle is entering the cells is stimulated by FSH. As the number of granulosa cells growth phase is a morphological change of the flattened increases, the production of estrogens, the binding capac- pregranulosa cells into cuboidal granulosa cells. The ity for FSH, the size of the follicle, and the volume of the cuboidal granulosa cells proliferate to form a single contin- follicular fluid all increase significantly. uous layer of cells surrounding the oocyte, which has en- As the antra increase in size, a single, large, coalesced larged from 20 m in the primordial stage to 140 m in di- antrum develops, pushing the oocyte to the periphery of ameter. At this stage, a glassy membrane, the zona the follicle and forming a large 2- to 2.5-cm-diameter pellucida, surrounds the oocyte and serves as means of at- graafian follicle (preovulatory follicle; see Fig. 38.4). Three tachment through which the granulosa cells communicate distinct granulosa cell compartments are evident in the with the oocyte. This is the primary follicular stage of de- graafian follicle. Granulosa cells surrounding the oocyte are velopment, consisting of one layer of cuboidal granulosa cumulus granulosa cells (collectively called cumulus cells and a basement membrane. oophorus). Those cells lining the antral cavity are called The follicle continues to grow, mainly through prolifer- antral granulosa cells and those attached to the basement ation of its granulosa cells, so that several layers of granu- membrane are called mural granulosa cells. Mural and losa cells exist in the secondary follicular stage of develop- antral granulosa cells are more steroidogenically active ment (see Fig. 38.4). As the secondary follicle grows deeper than cumulus cells. into the cortex, stromal cells, near the basement membrane, In addition to bloodborne hormones, antral follicles have begin to differentiate into cell layers called theca interna a unique microenvironment in which the follicular fluid con- and theca externa, and a blood supply with lymphatics and tains different concentrations of pituitary hormones, nerves forms within the thecal component. The granulosa steroids, peptides, and growth factors. Some are present in layer remains avascular. the follicular fluid at a concentration 100 to 1,000 times The theca interna cells become flattened, epithelioid, higher than in the circulation. Table 38.2 lists some parame- and steroidogenic. The granulosa cells of secondary follicles ters of human follicles at successive stages of development in

672 PART X REPRODUCTIVE PHYSIOLOGY Primordial Basement membrane follicle Oocyte Granulosa cells Primary Basement membrane follicle Granulosa cells Fully grown oocyte Zona pellucida Secondary Basement membrane follicle Granulosa cells Zona pellucida Fully grown oocyte Presumptive theca Theca externa Basement membrane Fully grown oocyte Early tertiary Multiple layers of follicle granulosa cells Zona pellucida Antrum Theca interna Graafian follicle Theca interna Cumulus oophorus Zona pellucida The developing follicle, Antrum (follicular fluid) FIGURE 38.4 Corona radiata from primordial through graafian. (Modified from Erickson GF. In: Basement membrane Sciarra JJ, Speroff L, eds. Reproductive En- Granulosa cells docrinology, Infertility, and Genetics. New Theca externa York: Harper & Row, 1981.) the follicular phase. There is a 5-fold increase in follicular di- The follicular fluid contains other substances, including ameter and a 25-fold rise in the number of granulosa cells. As inhibin, activin, GnRH-like peptide, growth factors, opioid the follicle matures, the intrafollicular concentration of FSH peptides, oxytocin, and plasminogen activator. Inhibin and does not change much, whereas that of LH increases and activin inhibit and stimulate, respectively, the release of that of PRL declines. Of the steroids, the concentrations of FSH from the anterior pituitary. Inhibin is secreted by estradiol and progesterone increase 20-fold, while androgen granulosa cells. In addition to its effect on FSH secretion, levels remain unchanged. inhibin also has a local effect on ovarian cells. TABLE 38.2 Different Parameters of Follicles During the First Half of the Menstrual Cycle Granulosa Cycle Diameter Volume Cells FSH 6 (day) (mm) (mL) (10 ) ng/mL LH PRL A E2 P4 1 4 0.05 2 2.5 — 60 800 100 — 4 7 0.15 5 2.5 — 40 800 500 100 7 12 0.50 15 3.6 2.8 20 800 1,000 300 12 20 0.50 50 3.6 2.8 5 800 2,000 2,000 FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; A, androstenedione; E2, estradiol; P4, progesterone. (Modi- fied from Erickson GF. An analysis of follicle development and ovum maturation. Semin Reprod Endocrinol 1986;4:233–254.)

CHAPTER 38 The Female Reproductive System 673 Granulosa and Theca Cells Both Participate is subsequently converted to androstenedione by 3-hy- in Steroidogenesis droxysteroid dehydrogenase. The androgens contain 19 carbons. Testosterone and androstenedione diffuse from The main physiologically active steroid produced by the the thecal compartment, cross the basement membrane, follicle is estradiol, a steroid with 18 carbons. Steroidoge- and enter the granulosa cells. nesis, the process of steroid hormone production, depends In the granulosa cell, under the influence of FSH, with on the availability of cholesterol, which originates from cAMP as a second messenger, testosterone and androstene- several sources and serves as the main precursor for all of dione are then converted to estradiol and estrone, respec- steroidogenesis. Ovarian cholesterol can come from plasma tively, by the enzyme aromatase, which aromatizes the A lipoproteins, de novo synthesis in ovarian cells, and choles- ring of the steroid and removes one carbon (see Fig. 38.5; terol esters within lipid droplets in ovarian cells. For ovar- see Fig. 37.9). Estrogens typically have 18 carbons. Estrone ian steroidogenesis, the primary source of cholesterol is can then be converted to estradiol by 17-hydroxysteroid low-density lipoprotein (LDL). dehydrogenase in granulosa cells. The conversion of cholesterol to pregnenolone by cho- In summary, estradiol secretion by the follicle requires lesterol side-chain cleavage enzyme is a rate-limiting step cooperation between granulosa and theca cells and coordi- regulated by LH using the second messenger cAMP nation between FSH and LH. An understanding of this (Fig. 38.5). LH binds to specific membrane receptors on two-cell, two-gonadotropin hypothesis requires recogni- theca cells, activates adenylyl cyclase through a G protein, tion that the actions of FSH are restricted to granulosa cells and increases the production of cAMP. cAMP increases because all other ovarian cell types lack FSH receptors. LH LDL receptor mRNA, the uptake of LDL cholesterol, and actions, on the other hand, are exerted on theca, granulosa, cholesterol ester synthesis. cAMP also increases the trans- and stromal (interstitial) cells and the corpus luteum. The port of cholesterol from the outer to the inner mitochondr- expression of LH receptors is time-dependent because ial membrane, the site of pregnenolone synthesis, using a theca cells acquire LH receptors at a relatively early stage, unique protein called steroidogenic acute regulatory pro- whereas LH receptors on granulosa cells are induced by tein (StAR). Pregnenolone, a 21-carbon steroid of the FSH in the later stages of the maturing follicle. progestin family, diffuses out of the mitochondria and en- The biosynthetic enzymes are differentially expressed ters the ER, the site of subsequent steroidogenesis. in the two cells. Aromatase is expressed only in granulosa Two steroidogenic pathways may be used for subse- cells, and its activation and induction are regulated by quent steroidogenesis (see Fig. 37.9). In theca cells, the FSH. Granulosa cells are deficient in 17-hydroxylase delta 5 pathway is predominant; in granulosa cells and the and cannot proceed beyond the C-21 progestins to gen- corpus luteum, the delta 4 pathway is predominant. Preg- erate C-19 androgenic compounds (see Fig. 38.5). Conse- nenolone gets converted to either progesterone by 3-hy- quently, estrogen production by granulosa cells depends droxysteroid dehydrogenase in the delta 4 pathway or to on an adequate supply of exogenous aromatizable andro- 17-hydroxypregnenolone by 17-hydroxylase in the gens, provided by theca cells. Under LH regulation, theca delta 5 pathway. In the delta 4 pathway, progesterone gets cells produce androgenic substrates, primarily an- converted to 17-hydroxyprogesterone (by 17-hydroxy- drostenedione and testosterone, which reach the granu- lase), which is subsequently converted to androstenedione losa cells by diffusion. The androgens are then converted and testosterone by 17,20-lyase and 17-hydroxysteroid to estrogens by aromatization. dehydrogenase (17-ketosteroid reductase), respectively. In In follicles, theca and granulosa cells are exposed to dif- the delta 5 pathway, 17-hydroxypregnenolone gets con- ferent microenvironments. Vascularization is restricted to verted to dehydroepiandrosterone (by 17,20-lyase), which the theca layer because blood vessels do not penetrate the Theca cell Granulosa cell Cholesterol Cholesterol cAMP  Receptor LH FIGURE 38.5 The two- LH Receptor cAMP  Pregnenolone ATP cell, two- ATP gonadotropin hypothesis. Capillary Pregnenolone Basement membrane The follicular theca cells, un- Progesterone der control of LH, produce 17OH pregnenolone androgens that diffuse to the follicular granulosa cells, Androstenedione where they are converted to Dehydroepiandrosterone cAMP estrogens via an FSH-sup-  ported aromatization reac- Testosterone tion. The dashed line indi- Androstenedione  ATP Receptor FSH cates that granulosa cells cAMP cannot convert progesterone Estradiol Estrone to androstenedione because Testosterone of the lack of the enzyme 17-hydroxylase.

674 PART X REPRODUCTIVE PHYSIOLOGY basement membrane. Theca cells, therefore, have better ac- hypertrophy and may remain in the ovary for extended pe- cess to circulating cholesterol, which enters the cells via riods of time. LDL receptors. Granulosa cells, on the other hand, prima- rily produce cholesterol from acetate, a less efficient process than uptake. In addition, granulosa cells are bathed Meiosis Resumes During the Periovulatory Period in follicular fluid and exposed to autocrine, paracrine, and All healthy oocytes in the ovary remain arrested in prophase juxtacrine control by locally produced peptides and growth of the first meiosis. When a graafian follicle is subjected to a factors. “Juxtacrine” describes the interaction of a mem- surge of gonadotropins (LH and/or FSH), the oocyte within brane-bound growth factor on one cell with its membrane- undergoes the final stages of meiosis, resulting in the pro- bound receptor on an adjacent cell. duction of a mature gamete. This maturation is accomplished FSH acts on granulosa cells by a cAMP-dependent by two successive cell divisions in which the number of chro- mechanism and produces a broad range of activities, in- mosomes is reduced, producing haploid gametes. At fertil- cluding increased mitosis and cell proliferation, the stimu- ization, the diploid state is restored. lation of progesterone synthesis, the induction of aro- Primary oocytes arrested in meiotic prophase 1 (of the matase, and increased inhibin synthesis. As the follicle first meiosis) have duplicated their centrioles and DNA matures, the number of receptors for both gonadotropins (4n DNA) so that each chromosome has two identical increases. FSH stimulates the formation of its own recep- chromatids. Crossing over and chromatid exchange occur tors and induces the appearance of LH receptors. The com- during this phase, producing genetic diversity. The re- bined activity of the two gonadotropins greatly amplifies sumption of meiosis, ending the first meiotic prophase estrogen production. and beginning of meiotic metaphase 1, is characterized by Androgens are produced by theca and stromal cells. disappearance of the nuclear membrane, condensation of They serve as precursors for estrogen synthesis and also the chromosomes, nuclear dissolution (germinal vesicle have a distinct local action. At low concentrations, andro- breakdown), and alignment of the chromosomes on the gens enhance aromatase activity, promoting estrogen pro- equator of the spindle. At meiotic anaphase 1, the homol- duction. At high concentrations, androgens are converted ogous chromosomes move in opposite directions under by 5-reductase to a more potent androgen, such as dihy- the influence of the retracting meiotic spindle at the cel- drotestosterone (DHT). When follicles are overwhelmed lular periphery. At meiotic telophase 1, an unequal divi- by androgens, the intrafollicular androgenic environment sion of the cell cytoplasm yields a large secondary oocyte antagonizes granulosa cell proliferation and leads to apop- (2n DNA) and a small, nonfunctional cell, the first polar tosis of the granulosa cells and subsequent follicular atresia. body (2n DNA). Each cell contains half the original 4n number of chromosomes (only one member of each ho- Follicular Atresia Probably Results From a mologous pair is present, but each chromosome consists Lack of Gonadotropin Support of two unique chromatids). The secondary oocyte is formed several hours after the Follicular atresia, the degeneration of follicles in the ovary, initiation of the LH surge but before ovulation. It rapidly is characterized by the destruction of the oocyte and gran- begins the second meiotic division and proceeds through a ulosa cells. Atresia is a continuous process and can occur at short prophase to become arrested in metaphase. At this any stage of follicular development. During a woman’s life- stage, the secondary oocyte is expelled from the graafian time approximately 400 to 500 follicles will ovulate; those follicle. The second arrest period is relatively short. In re- are the only follicles that escape atresia, and they represent sponse to penetration by a spermatozoon during fertiliza- a small percentage of the 1 to 2 million follicles present at tion, meiosis 2 resumes and is rapidly completed. A second birth. The cause of follicular atresia is likely due to lack of unequal cell division soon follows, producing a small sec- gonadotropin support of the growing follicle. For example, ond polar body (1n DNA) and a large fertilized egg, the at the beginning of the menstrual cycle, several follicles are zygote (2n DNA, 1n from the mother and 1n from the fa- selected for growth but only one follicle, the dominant fol- ther). The first and second polar bodies either degenerate licle, will go on to ovulate. Because the dominant follicle or divide, yielding small nonfunctional cells. If fertilization has a preferential blood supply, it gets the most FSH (and does not occur, the secondary oocyte begins to degenerate LH). Other reasons for the lack of gonadotropin support of within 24 to 48 hours. nondominant follicles could be a lack of FSH and LH re- ceptors or the inability of granulosa cells to transduce the gonadotropin signals. FOLLICLE SELECTION AND OVULATION During atresia, granulosa cell nuclei become pyknotic (referring to an apoptotic process characterized by DNA The number of ovulating eggs is species-specific and is in- laddering), and/or the oocyte undergoes pseudomatura- fluenced by genetic, nutritional, and environmental factors. tion, characteristic of meiosis. During the early stages of In humans, normally only one follicle will ovulate, but mul- oocyte death, the nuclear membrane disintegrates, the tiple ovulations in a single cycle (superovulation) can be chromatin condenses, and the chromosomes form a induced by the timed administration of gonadotropins or metaphase plate with a spindle; the term pseudomaturation is antiestrogens. The mechanism by which one follicle is se- appropriate because these oocytes are not capable of suc- lected from a cohort of growing follicles is poorly under- cessful fertilization. During atresia of follicles containing stood. It occurs during the first few days of the cycle, im- theca cells, the theca layer may undergo hyperplasia and mediately after the onset of menstruation. Once selected,

CHAPTER 38 The Female Reproductive System 675 the follicle begins to grow and differentiate at an exponen- is also an increased production of follicular fluid, disaggrega- tial rate and becomes the dominant follicle. tion of granulosa cells, and detachment of the oocyte-cumu- In parallel with the growth of the dominant follicle, the lus complex from the follicular wall, moving it to the central rest of the preantral follicles undergo atresia. Two main fac- portion of the follicle. The basement membrane separating tors contribute to atresia in the nonselected follicles. One is theca cells from granulosa cells begins to disintegrate, gran- the suppression of plasma FSH in response to increased estra- ulosa cells begin to undergo luteinization, and blood vessels diol secretion by the dominant follicle. The decline in FSH begin to penetrate the granulosa cell compartment. support decreases aromatase activity and estradiol produc- Just prior to follicular rupture, the follicular wall thins by tion and interrupts granulosa cell proliferation in those non- cellular deterioration and bulges at a specific site called the dominant follicles. The dominant follicle is protected from a stigma, the point on the follicle that actually ruptures. As fall in circulating FSH levels because it has a healthy blood ovulation approaches, the follicle enlarges and protrudes supply, FSH accumulated in the follicular fluid, and an in- from the surface of the ovary at the stigma. In response to the creased density of FSH receptors on its granulosa cells. An- LH surge, plasminogen activator is produced by theca and other factor in selection is the accumulation of atretogenic granulosa cells of the dominant follicle and converts plas- androgens, such as DHT, in the nonselected follicles. The minogen to plasmin. Plasmin is a proteolytic enzyme that increase in DHT changes the intrafollicular ratio of estrogen acts directly on the follicular wall and stimulates the produc- to androgen and antagonizes the actions of FSH. tion of collagenase, an enzyme that digests the connective As the dominant follicle grows, vascularization of the tissue matrix. The thinning and increased distensibility of the theca layer increases. On day 9 or 10 of the cycle, the vascu- wall facilitates the rupture of the follicle. The extrusion of the larity of the dominant follicle is twice that of the other antral oocyte-cumulus complex is aided by smooth muscle con- follicles, permitting a more efficient delivery of cholesterol traction. At the time of rupture, the oocyte-cumulus complex to theca cells and better exposure to circulating go- and follicular fluid are ejected from the follicle. nadotropins. At this time, the main source of circulating The LH surge triggers the resumption of the first meiosis. estradiol is the dominant follicle. Since estradiol is the pri- Up to this point, the primary oocyte has been protected by mary regulator of LH and FSH secretion by positive and neg- unknown factors within the follicle from premature cell divi- ative feedback, the dominant follicle ultimately determines sion. The LH surge also causes transient changes in plasma its own fate. estradiol and a prolonged increase in plasma progesterone The midcycle LH surge occurs as a result of rising levels concentrations. Within a couple of hours after the initiation of circulating estradiol, and it causes multiple changes in the of the LH surge, the production of progesterone, androgens, dominant follicle, which occur within a relatively short time. and estrogens begins to increase. Progesterone, acting These include the resumption of meiosis in the oocyte (as al- through the progesterone receptor on granulosa cells, pro- ready discussed); granulosa cell differentiation and transfor- motes ovulation by releasing mediators that increase the dis- mation into luteal cells; the activation of proteolytic en- tensibility of the follicular wall and enhance the activity of zymes that degrade the follicle wall and surrounding tissues; proteolytic enzymes. As LH levels reach their peak, plasma increased production of prostaglandins, histamine, and other estradiol levels plunge because of down-regulation by LH of local factors that cause localized hyperemia; and an increase FSH receptors on granulosa cells and the inhibition of gran- in progesterone secretion. Within 30 to 36 hours after the ulosa cell aromatase. Eventually, LH receptors on luteinizing onset of the LH surge, this coordinated series of biochemical granulosa cells escape the down-regulation, and proges- and morphological events culminates in follicular rupture terone production increases. and ovulation. The midcycle FSH surge is not essential for ovulation because an injection of either LH or human chori- onic gonadotropin (hCG) before the endogenous go- FORMATION OF THE CORPUS LUTEUM FROM nadotropin surge can induce normal ovulation. However, THE POSTOVULATORY FOLLICLE only follicles that have been adequately primed with FSH will ovulate because they contain sufficient numbers of LH In response to the LH and FSH surges and after ovulation, receptors for ovulation and subsequent luteinization. the wall of the graafian follicle collapses and becomes con- Four ovarian proteins are essential for ovulation: the prog- voluted, blood vessels course through the luteinizing gran- esterone receptor, the cyclooxygenase enzyme (which con- ulosa and theca cell layers, and the antral cavity fills with verts arachidonic acid to prostaglandins), cyclin D2 (a cell blood. The granulosa cells begin to cease their proliferation cycle regulator), and a transcription factor called C/EBP and begin to undergo hypertrophy and produce proges- (CCAAT/enhancer binding protein). The mechanisms by terone as their main secretory product. The ruptured follicle which these proteins interact to regulate follicular rupture are develops a rich blood supply and forms a solid structure largely unknown. However, mice with specific disruption of called the corpus luteum (yellow body). The mature corpus genes for any of these proteins fail to ovulate, and these pro- luteum develops as the result of numerous biochemical and teins are likely to have a functional role in human ovulation. morphological changes, collectively referred to as luteiniza- The earliest responses of the ovary to the midcycle LH tion. The granulosa cells and theca cells in the corpus lu- surge are the release of vasodilatory substances, such as his- teum are called granulosa-lutein cells and theca-lutein tamine, bradykinin, and prostaglandins, which mediate in- cells, respectively. creased ovarian and follicular blood flow. The highly vascu- Continued stimulation by LH is needed to ensure mor- larized dominant follicle becomes hyperemic and edematous phological integrity (healthy luteal cells) and functionality and swells to a size of at least 20 to 25 mm in diameter. There (progesterone secretion). If pregnancy does not occur, the

676 PART X REPRODUCTIVE PHYSIOLOGY corpus luteum regresses, a process called luteolysis or luteal LH; therefore, LH is referred to as a luteotropic hormone. regression. Luteolysis occurs as a result of apoptosis and Lack of LH can lead to luteal insufficiency (see Clinical Fo- necrosis of the luteal cells. After degeneration, the cus Box 38.1). luteinized cells are replaced by fibrous tissue, creating a Regression of the corpus luteum at the end of the cycle is nonfunctional structure, the corpus albicans. Therefore, the not understood. Luteal regression is thought to be induced corpus luteum is a transient endocrine structure formed from by locally produced luteolytic agents that inhibit LH action. the postovulatory follicle. It serves as the main source of cir- Several ovarian hormones, such as estrogen, oxytocin, culating steroids during the luteal (postovulatory) phase of prostaglandins, and GnRH, have been proposed, but their the cycle and is essential for maintaining pregnancy during role as luteolysins is controversial. The corpus luteum is res- the first trimester (see Case Study) as well as maintaining cued from degeneration in the late luteal phase by the action menstrual cycles of normal length. of human chorionic gonadotropin (hCG), an LH-like hor- The process of luteinization begins before ovulation. Af- mone that is produced by the embryonic trophoblast during ter acquiring a high concentration of LH receptors, granu- the implantation phase (see Chapter 39). This hormone losa cells respond to the LH surge by undergoing morpho- binds the LH receptor and increases cAMP and proges- logical and biochemical transformation. This change terone secretion. involves cell enlargement (hypertrophy) and the develop- ment of smooth ER and lipid inclusions, typical of steroid- secreting cells. Unlike the nonvascular granulosa cells in the THE MENSTRUAL CYCLE follicle, luteal cells have a rich blood supply. Invasion by capillaries starts immediately after the LH surge and is facil- Under normal conditions, ovulation occurs at timed inter- itated by the dissolution of the basement membrane be- vals. Sexual intercourse may occur at any time during the cy- tween theca and granulosa cells. Peak vascularization is cle, but fertilization occurs only during the postovulatory reached 7 to 8 days after ovulation. period. Once pregnancy occurs, ovulation ceases, and after Differentiated theca and stroma cells, as well as granulosa parturition, lactation also inhibits ovulation. The first men- cells, are incorporated into the corpus luteum, and all three strual cycle occurs in adolescence, usually around age 12. classes of steroids—androgens, estrogens, and progestins— The initial period of bleeding is called the menarche. The are synthesized. Although some progesterone is secreted first few cycles are usually irregular and anovulatory, as the before ovulation, peak progesterone production is reached 6 result of delayed maturation of the positive feedback by to 8 days after the LH surge. The life span of the corpus lu- estradiol on a hypothalamus that fails to secrete significant teum is limited. Unless pregnancy occurs, it degenerates GnRH. During puberty, LH secretion occurs more during within about 13 days after ovulation. During the menstrual periods of sleep than during periods of being awake, result- cycle, the function of the corpus luteum is maintained by ing in a diurnal cycle. CLINICAL FOCUS BOX 38.1 Luteal Insufficiency ceptors mediate the action of LH, which stimulates prog- Occasionally, the corpus luteum will not produce sufficient esterone secretion. An insufficient number of LH receptors progesterone to maintain pregnancy during its very early could be due to insufficient priming of the developing fol- stages. Initial signs of early spontaneous pregnancy termi- licle with FSH. It is well known that FSH increases the num- nation include pelvic cramping and the detection of blood, ber of LH receptors in the follicle. Third, the LH surge could similar to indications of menstruation. If the corpus luteum have been inadequate in inducing full luteinization of the is truly deficient, then fertilization may occur around the ide- corpus luteum, yet there was sufficient LH to induce ovu- alized day 14 (ovulation), pregnancy will terminate during lation. It has been estimated that only 10% of the LH surge the deficient luteal phase, and menses will start on sched- is required for ovulation, but the amount required for full ule. Without measuring levels of hCG, the pregnancy detec- luteinization and adequate progesterone secretion to tion hormone, the woman would not know that she is preg- maintain pregnancy is not known. nant because of the continuation of regular menstrual If progesterone values are low in consecutive cycles at cycles. Luteal insufficiency is a common cause of infertil- the midluteal phase and do not match endometrial biop- ity. Women are advised to see their physician if pregnancy sies, exogenous progesterone may be administered in does not result after 6 months of unprotected intercourse. order to prevent early pregnancy termination during a Analysis of the regulation of progesterone secretion by fertile cycle. Other options include the induction of follic- the corpus luteum provides insights into this clinical prob- ular development and ovulation with clomiphene and lem. There are several reasons for luteal insufficiency. hCG. This treatment would likely produce a large, First, the number of luteinized granulosa cells in the corpus healthy, estrogen-secreting graafian follicle with suffi- luteum may be insufficient because of the ovulation of a cient LH receptors for luteinization. The exogenous hCG small follicle or the premature ovulation of a follicle that is given to supplement the endogenous LH surge and to was not fully developed. Second, the number of LH recep- ensure full stimulation of the graafian follicle, ovulation, tors on the luteinized granulosa cells in the graafian follicle adequate progesterone, and luteinization of the develop- and developing corpus luteum may be insufficient. LH re- ing corpus luteum.

CHAPTER 38 The Female Reproductive System 677 LH peak 50 50 40 (mIU/mL) 30 FSH 30 20 20 40 (mIU/mL) 10 10 LH 0 0 20 300 Progesterone (ng/mL) 1 Estradiol (pg/mL) 200 P E 2 β 17-OH P 2 (ng/mL) 17-Hydroxyprogesterone 10 1 100 0 Follicle diameter (mm) 20 regression 20 diameter (mm) Corpus luteum Luteal 10 10 Day: 0246 8 10 12 14 16 18 20 22 24 26 28 Menses Ovulation Phase: Menstrual Follicular Ovulatory Luteal Day of menstrual cycle Hormonal and ovarian events during the menstrual cycle. P, progesterone; E 2 , estra- FIGURE 38.6 diol; 17-OH P, 17-hydroxyprogesterone. The average menstrual cycle length in adult women is 28 ception and lactation and is subjected to modulation by days, with a range of 25 to 35 days. The interval from ovu- physiological, psychological, and social factors. lation to the onset of menstruation is relatively constant, av- eraging 14 days in most women and is dictated by the fixed life span of the corpus luteum. In contrast, the interval from The Menstrual Cycle Requires Synchrony the onset of menses to ovulation (the follicular phase) is Among the Ovary, Brain, and Pituitary more variable and accounts for differences in cycle lengths among ovulating women. The menstrual cycle requires several coordinated elements: The menstrual cycle is divided into four phases hypothalamic control of pituitary function, ovarian follicu- (Fig. 38.6). The menstrual phase, also called menses or lar and luteal changes, and positive and negative feedback menstruation, is the bleeding phase and lasts about 5 days. of ovarian hormones at the hypothalamic-pituitary axis. The ovarian follicular phase lasts about 10 to 16 days; folli- We have discussed separately the mechanisms that regulate cle development occurs, estradiol secretion increases, and the synthesis and release of the reproductive hormones; the uterine endometrium undergoes proliferation in re- now we put them together in terms of sequence and inter- sponse to rising estrogen levels. The ovulatory phase lasts action. For this purpose, we use a hypothetical cycle of 28 24 to 48 hours, and the luteal phase lasts 14 days. In the days (see Fig. 38.6), divided into four phases as follows: luteal phase, progesterone is produced, and the en- menstrual (days 0 to 5), follicular (days 0 to 13), ovulatory dometrium secretes numerous proteins in preparation for (days 13 to 14), and luteal (days 14 to 28). implantation of an embryo. During menstruation, estrogen, progesterone, and in- The cycles become irregular as menopause approaches hibin levels are very low as a result of the luteal regression around age 50, and cycles cease thereafter. During the re- that has just occurred and the low estrogen synthesis by im- productive years, menstrual cycling is interrupted by con- mature follicles. The plasma FSH levels are high while LH