Testosterone Synthesis

Androgens are synthesized from cholesterol in testicular Leydig cells, the adrenal cortex, and ovarian thecal cells (see Fig. 38-1). In the adult gonads the principal regulator of testosterone synthesis and secretion is luteinizing hormone (LH), which is produced by the anterior pituitary gland (Chapter 38). The precursor cholesterol is synthesized in the Leydig cells from acetate and stored as cholesterol esters in lipid droplets. A cholesterol ester hydrolase mobilizes free cholesterol from the lipid droplets, which in turn is transferred to the inner mitochondrial membrane. Stimulation of this transfer represents a major action of LH and is mediated by the steroidogenic acute regulatory (StAR) protein. Leydig cells can convert a small fraction of testosterone to estradiol (see Chapter 40 and Fig. 38-1). LH increases the level of enzymes in this synthetic pathway.

Unlike the peptide hormones, the intracellular storage of steroid hormones, which can be mobilized and secreted, is minimal. The amount of testosterone in the human testis is approximately 300 ng/g of wet tissue. Assuming a normal, adult testis in humans weighs 15 g, total testicular testosterone is approximately 9 µg, or 0.1% of its daily production (5 to7 mg).

During human development, testosterone synthesis begins during the first trimester of pregnancy and is regulated by placental chorionic gonadotropin (hCG). At this stage of fetal development, male sexual differentiation is dependent on placental hCG until fetal pituitary gonadotrophs become functional at the end of the first trimester. During the second trimester, gonadotrophs become able to provide adequate gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin formation. Gonadotropin secretion and sex steroid production decline late in fetal life, followed by a prominent postnatal surge lasting 2 to 3 months; by 3 or 4 months of age, testosterone secretion is significantly reduced. At puberty, gonadotropin secretion increases and again stimulates the Leydig cell to produce testosterone. The neurotransmitters gamma aminobutyric acid, neuropeptide Y, and kisspeptins (ligands of the orphan G-protein-coupled receptor GPR54) have each been proposed to influence puberty by regulating GnRH.

Gonadotropin secretion in early puberty follows a diurnal rhythm, with elevated concentrations of LH and testosterone at night. In adult men the diurnal rhythm for LH is less demonstrable. Specifically, testosterone levels in the early morning are approximately 25% higher than in the late afternoon. LH secretion fluctuates every 1 to 2 hours, resulting from intermittent stimulation of gonadotrophs by GnRH. GnRH secretory episodes in turn are coupled to excitatory discharges of a neural oscillator system. Intermittent GnRH secretion is required for the pituitary to function normally, and testosterone is released into the circulation in pulses in response to the pulsatile stimulation of Leydig cells by LH.

Androgen Production by the Adrenal Glands

Although glucocorticoids and mineralocorticoids are the principal products of the adult adrenal gland, dihydroepiandrosterone (DHEA), androstenedione, and testosterone, as well as some DHEA sulfate and estrone, can be also secreted (see Fig. 38-1). The concentrations of DHEA, DHEA sulfate, and androstenedione in the circulation increase between 7 to 10 years of age. This process has been termed adrenarche, to distinguish it from gonadarche, which is the onset of adult gonadal function at puberty. Adrenal androgen secretion declines in the elderly and during severe illness.

Regulation of Testosterone Synthesis and Secretion

The major site of testosterone synthesis and secretion is the Leydig cell, which has cell-surface LH receptors that associate with the Gs subunit of adenylyl cyclase. Steroidogenesis mediated by LH requires mobilization of intracellular Ca⁺⁺ and the Ca⁺⁺-binding protein calmodulin, and activation of phospholipase C (see Chapter 1). The effects of LH involve rapid stimulation of testosterone production (within minutes), which is mediated by the StAR. Other hormones that influence testosterone synthesis include prolactin, cortisol, insulin, insulin-like growth factors, estradiol, activin, and inhibin. There is a growing appreciation of the multiple factors involved in testosterone synthesis that are produced within the seminiferous tubules by germ cells and Sertoli cells or peritubular myoid cells. These factors maintain the serum concentration of testosterone in adult men at 0.3 to 1.0 mg/dL (10 to 30 nM). During illness, LH production declines, and cytokines suppress testosterone production.

Sertoli cells are somatic cells within the seminiferous tubules. Tight junctions between these cells at the base of seminiferous tubules form a blood-testis barrier, which prevents circulating proteins from entering the tubular compartment. Sertoli cells secrete many types of proteins, some of which enter the tubular lumen and are important in spermatogenesis. Others are secreted through the basal end of the cell and enter the circulation. Among these proteins are the androgen-binding protein, transferrin, and inhibin-B. Follicle-stimulating hormone (FSH) is the major regulator of Sertoli cell function. The FSH receptor is also membrane-bound and acts through both cyclic adenosine monophosphate and Ca⁺⁺. Insulin and insulin-like growth factors, testosterone, vitamin A, and β-endorphins also influence Sertoli cell function.

The hormones of the hypothalamus, pituitary, and testes form an internally regulated unit (Fig. 41-1). Not only are the testes stimulated by pituitary gonadotropins, but the testes also regulate LH and FSH secretion through negative-feedback mechanisms. Testosterone suppresses gonadotropin secretion by slowing the pulsatile release of GnRH. Estradiol, which is synthesized from testosterone in the ovary, testes, adipose tissue, liver, and brain, inhibits gonadotropin release through effects on both the hypothalamus and pituitary. Inhibin-B selectively reduces FSH synthesis and secretion.

FIGURE 41–1 Hormonal control of testicular function.

Normally, women produce approximately 0.25 mg/day of testosterone compared with the 5 to 7 mg/day for adult men. Most testosterone circulating in women is derived from the peripheral conversion of androstenedione secreted by the ovaries and adrenals (see Chapter 40). Benign and malignant tumors of the adrenal and ovary, congenital steroidogenic enzyme defects, and disturbances of gonadotropin secretion can be associated with increased androgen production in women.

Androgen Action

Endogenous testosterone or exogenous testosterone derivatives are transported to their target tissues through the blood. The primary mode of circulation of testosterone is a high-affinity interaction with the hepatic glycoprotein, sex hormone binding-globulin (SHBG). Approximately 1% to 3% of circulating testosterone is unbound and available for entering target tissues and altering gene activity.

There is evidence that SHBG binds androgen target cells and may play a role in the action of testosterone. SHBG formation is increased by estrogens and thyroxine and decreased by androgens, growth hormone, and insulin. The higher levels of estrogen in women promote twofold to threefold higher levels of SHBG than in men. Also, patients with hyperthyroidism exhibit higher SHBG levels. Obesity is associated with low concentrations of SHBG, perhaps because of hyperinsulinemia and insulin resistance.

Depending on the tissue, intracellular testosterone or a more active metabolite DHT can interact directly with ARs (Fig. 41-2). When testosterone enters the prostate gland or any tissue with significant 5α-reductase activity, nearly 90% of it is metabolized to DHT. There are two isoenzymes of 5α-reductase, encoded by two different genes. Type I 5α-reductase is found in liver, skin, sebaceous glands, most hair follicles, and prostate, whereas 5α-reductase type II predominates in genital skin, beard and scalp hair follicles, and prostate. The presence of ambiguous genitalia in patients with inactivating mutations of the 5α-reductase type II gene is indicative of the importance of this enzyme in normal development of male external genitalia. The distribution of the isozymes has been exploited to develop tissue-specific inhibitors of 5α-reductase activity.

FIGURE 41–2 Androgen action at target cells.

Androgen binding to ARs and the events that follow are similar to those of other steroid hormones (see Chapter 1). The AR is encoded by a gene on the X chromosome and is expressed in most tissues. When a ligand binds to the AR, the conformation of the receptor is altered, it binds to DNA response elements, multiple coactivator proteins are recruited, and the transcription of messenger RNAs for tissue-specific proteins ensues. Although most actions of androgens are mediated by transcriptional activity of the receptor, others are mediated through second messengers, such as the mitogen-activated protein kinase pathway.

Androgen regulation of target tissues may be positive, as in the stimulation of androgen-dependent proteins within the prostate, or negative, as in the inhibition of pituitary gonadotropin α-subunit gene expression and GnRH release by the hypothalamus. Negative regulation is less well understood but in certain cases has been explained by AR binding to, and interfering with, the action of stimulatory transcription factors.

Antiandrogens and Androgen Receptor Antagonists

There are two basic mechanisms to block the activity of androgen. These include inhibition of androgen formation or antagonism of androgen-AR interactions. Strategies for suppression of androgen formation include blockade of gonadotropin formation using GnRH analogs and the use of spironolactone and ketoconazole to competitively inhibit the activities of steroid 17α-hydroxylase (CYP17) and cholesterol side-chain cleavage enzyme (CYP11A), respectively (see Fig. 38-1). Another agent, finasteride, is a competitive inhibitor of the 5α-reductase isozymes and antagonizes the formation DHT, which has potent androgenic action. All these drugs are substrate analogs and compete with the natural substrates for active sites on the enzymes.

In addition to inhibiting androgen synthesis, antagonism of the effects of androgen may be achieved with AR antagonists. The compounds compete with testosterone or DHT for binding to the AR and include flutamide, nilutamide, bicalutamide, cyproterone, and spironolactone.

Testosterone Metabolism

The nonhepatic metabolism of testosterone is summarized in Figure 38-1; the hepatic metabolism is shown in Figure 41-3. Nearly half of hepatic testosterone is metabolized to the 17-ketosteroids, 5α-androsterone and 5β-etiocholanolone, both of which are excreted in the urine. These compounds, however, constitute only a small fraction of the 17-ketosteroids in urine. Most urinary 17-ketosteroids are metabolites of androstenedione and DHEA produced by adrenals. In addition, testosterone is also conjugated and excreted as glucuronides and sulfates.

FIGURE 41–3 Primary hepatic metabolism of testosterone. 17β-HSD, 17β-Hydroxysteroid dehydrogenase.

DHT and estradiol are also formed from testosterone metabolism. Although these compounds represent minor (5%) metabolites, they are highly biologically active. Circulating levels of DHT are approximately 10% that of testosterone, and estradiol levels in men approximate those in women in the early follicular phase of the menstrual cycle. Estradiol plays a role in bone matrix regulation in males and females and contributes to negative feedback control of GnRH and gonadotropins.

Androgens

The pharmacokinetic properties for the androgens available for clinical use are listed in Table 41-1. Testosterone administered orally is rapidly cleared by the liver by first-pass metabolism and is ineffective for clinical use.

Testosterone esterified at the 17β-hydroxyl position (e.g., as the propionate, cypionate, or enanthate) and contained in an oil suspension is administered by intramuscular (IM) injection. Esterification increases lipid solubility and decreases hepatic metabolism, prolonging the duration of action. The esters are converted to free testosterone in the circulation. The high levels of testosterone and estradiol in the days after injection may produce acne, polyhemia, and gynecomastia and are associated with mood swings in some patients.

The transdermal delivery of testosterone was developed to produce stable physiological drug concentrations by avoiding first-pass hepatic metabolism. Recent developments include gels and a buccal tablet that forms a gel after it is placed on the surface of the gums.

Testosterone implants in pellet form are inserted subcutaneously using a trocar cannula to lie on top of the rectus sheath in subcutaneous fat. Although popular in some countries, the need for surgical implantation and the tendency for the pellets to extrude through the skin have limited their use in the United States. Another method of delivery being tested is the IM injection of biodegradable microspheres containing testosterone.

The synthetic alkylated androgens, methyltestosterone and fluoxymesterone, are less extensively metabolized by the liver than testosterone and are available for sublingual or oral use. Methyltestosterone and fluoxymesterone have relatively short durations of action. These drugs are used only after sexual development, because they lack the potency to facilitate sexual development.

Danazol is only weakly androgenic and interacts with progesterone and androgen receptors. It inhibits the pulsatile release of gonadotrophin, with a subsequent decline in serum concentrations of estradiol and estrone in women. Danazol undergoes extensive hepatic metabolism, and peak concentrations occur within 2 hours after oral administration, with a half-life of 4.5 hours. The metabolic and excretion kinetics of the anabolic steroid, nandrolone, varies among individuals.

Anabolic Steroids in Normal Men

Anabolic steroids are used therapeutically in children to promote growth and can be combined with exercise to increase muscle mass and improve physical performance. These drugs are believed to be more anabolic than androgenic. Whether the mechanisms by which anabolic steroids act differ from androgens is controversial, because ARs in skeletal muscle are not known to differ from those in seminal vesicles and prostate. However, prostate contains 5α-reductase, whereas skeletal muscle does not. Although this enzyme catalyzes the formation DHT that has a much stronger affinity for ARs, it does not influence the potency of most testosterone derivatives and may reduce the potency of 19-nortestosterone. Thus tissue-specific metabolism may influence the potency of various androgens differently. Coactivators recruited to ARs may be ligand-dependent and also contribute to tissue-specific responses. Drugs commonly used for their anabolic activity include nandrolone, oxandrolone, oxymetholone, and stanozolol.

Other Uses of Androgens

Patients of both sexes with wasting resulting from chronic disease or malnutrition are often androgen deficient. For example, plasma testosterone concentrations are often reduced in men with AIDS, among whom testosterone replacement increases muscle mass and strength, although there is no evidence that androgens prolong survival.

The erythropoietic effect of androgens is well established, as shown by the fact that the hemoglobin concentration is 1 to 2 g/dL higher in men than in women or children, and mild anemia is common in hypogonadal men. Polycythemia may occur as an unwanted effect of androgen therapy.

Androgens have been shown to stimulate erythropoiesis by increasing renal erythropoietin production (see Chapter 26). Androgens also have a direct effect on erythrocyte maturation. Because 5β-androgens (which bind weakly to ARs) are more effective than 5α-androgens, this may constitute a novel mechanism explaining the direct effect of androgens on bone marrow cells. Androgens may be used to treat patients with aplastic anemia, although responses vary. Danazol is an androgen derivative that has been used for the treatment of endometriosis, fibrocystic disease of the breast, and premenstrual tension syndrome. Danazol is used in women, rather than testosterone, because it is weakly androgenic. Danazol is also used to prevent attacks of hereditary angioneurotic edema, a disorder characterized by recurrent edema of the skin and mucosa. These patients lack the function of the inhibitor of the activated first component of complement, and androgens increase serum concentrations of this protein.

Androgens have also been used for treatment of inoperable breast cancer, postpartum breast pain, and engorgement. The mechanisms by which androgens affect the normal breast and modify growth of breast cancer cells are uncertain. The rate of positive responses in women with breast cancer, which average 30%, is less than that seen for other hormonal therapies.

Antiandrogens and Androgen Receptor Antagonists

Androgen synthesis inhibitors and receptor antagonists are used for treatment of female hirsutism, alopecia, acne, precocious puberty in males, benign prostate hyperplasia, and other diseases. In addition, several gonadotropin suppressants that inhibit testosterone production, including leuprolide, buserelin, nafarelin, and goserelin, have been approved by the United Stated Food and Drug Administration to treat prostate cancer. Although the role of androgens in the pathogenesis of benign and malignant prostate disease remains uncertain, patients with disseminated prostate cancer are treated by decreasing testosterone production with long-acting GnRH analogs and impeding androgen action with AR antagonists, because the symptoms of bone pain are lessened and patient survival is prolonged.

Finasteride is a competitive inhibitor of 5α-reductase type II that blocks conversion of testosterone to 5α-DHT in tissues containing this enzyme but has little activity against 5α-reductase type I. Finasteride reduces prostate DHT content by 80%, decreasing prostate size, and is used to treat benign prostatic hyperplasia. Another therapy that can be used to improve urinary flow is monotherapy with α₁-adrenergic receptor antagonists (see Chapter 11) or combination therapy with finasteride, as symptoms progress. Finasteride has little effect in treating established prostate cancer. In a large multicenter primary prevention trial, finasteride prevented or delayed the appearance of prostate cancer (6.3% vs. 8.7% of men followed developed cancer), but unexpectedly, the risk of high-grade prostate cancer increased. Consequently, it is not recommended for prevention of prostate cancer. Finasteride at a reduced dose of 1 mg/day is also approved to treat male-pattern baldness. After 12 months of therapy, visible improvement occurs in approximately 50% of treated men.

Dutasteride, a competitive inhibitor of both types of 5α-reductase, was developed for treatment of men with moderate to severe lower urinary tract symptoms caused by benign prostatic hyperplasia.

Spironolactone is a synthetic steroid that is used primarily as an aldosterone antagonist in the treatment of primary and secondary hyperaldosteronism. It can be used to treat hypertension and as a treatment for heart failure (see Chapters 20 and 23). In addition to effects at aldosterone receptors, spironolactone interacts with ARs. Further, spironolactone inhibits testosterone synthesis. Progesterone concentration increases, because its further metabolism is inhibited. However, a decrease in serum androgen concentration in men produces an increase in gonadotropin secretion, which may return serum testosterone concentrations to normal. Because of its ability to block testosterone synthesis and impede androgen action, spironolactone is used in treatment of hirsute women.

Ketoconazole is a broad-spectrum antimycotic agent used in treatment of systemic fungal infections (see Chapter 50). It inhibits the synthesis of ergosterol in fungi, resulting in altered membrane permeability. It also inhibits the synthesis of cholesterol and interferes with the action of cytochrome P450 enzymes in several mammalian cell types, including Leydig cells. The result is a dose-dependent decline in circulating testosterone concentrations in adult men and a rise in serum 17α-hydroxyprogesterone concentrations. Serum LH and FSH concentrations rise because of the decline in testosterone negative feedback. This action of ketoconazole has prompted its investigational use in treatment of prostate cancer and gonadotropin-independent precocious puberty in boys. However, the extent to which it suppresses testosterone synthesis in men is highly variable. Ketoconazole also inhibits cortisol biosynthesis and is used as an adjunct therapy in patients with Cushing’s syndrome. Gynecomastia may develop in ketoconazole-treated men.

Megestrol acetate is a progestin with antiandrogenic activity. It is used as an appetite stimulant in patients with cancer anorexia/cachexia and as a treatment for metastatic breast cancer in postmenopausal women. Cyproterone acetate, a synthetic steroid derived from 17α-hydroxyprogesterone, is a steroidal antiandrogen. Both megestrol and cyproterone activate the glucocorticoid receptor and suppress the hypothalamic pituitary axis. In combination with estrogen, these drugs suppress gonadotropin secretion, inhibit ovulation, and reduce circulating testosterone concentrations.

Flutamide, nilutamide, and bicalutamide are nonsteroidal androgen receptor antagonists approved for immediate and adjuvant treatment of prostate cancer. These drugs can be used in combination with GnRH analogs to offset their initial stimulatory effect on LH secretion and thereby testosterone production. These drugs are also used to treat hirsutism in women, but in healthy women use is limited by potential hepatotoxicity. Flutamide is rapidly and extensively metabolized, with a hydroxylated derivative responsible for mediating its antiandrogenic effects.

The histamine receptor antagonist cimetidine, used to decrease gastric acid secretion in treatment of peptic ulcer disease and esophagitis (see Chapter 14), also acts as an antiandrogen. Thus it has been reported to produce gynecomastia when given in large doses, such as those used in the treatment of patients with Zollinger-Ellison syndrome. Gynecomastia occurs in less than 1% of patients treated with the doses used in peptic ulcer disease. Cimetidine interacts with ARs approximately 0.01% as effectively as testosterone and has been used with limited effectiveness to treat hirsutism in women.