Topic No. & Title: Topic 4 Biosynthesis and secretion of adrenal, ovarian and testicular hormones-factors influencing secretion

[Academic Script] Biosynthesis and secretion of adrenal, ovarian and testicular hormones-factors influencing secretion Subject: Zoology Course: B.Sc. 2 nd Year Paper No. & Title: Z-203B Vertebrate Endocrinology & Reproductive Biology Topic No. & Title: Topic 4 Biosynthesis and secretion of adrenal, ovarian and testicular hormones-factors influencing secretion Lecture Title: Biosynthesis and secretion of adrenal, ovarian and testicular hormones-factors influencing secretion

Academic Script 1. Introduction The first steroidal precursor for biosynthesis of steroid hormones in the adrenals, ovaries, and testes is cholesterol. In these endocrine glands, cholesterol can be synthesized de novo from acetate by a complex series of reactions. Cholesterol is converted to a variety of steroid hormones in the endocrine glands through the action of specific enzymes, encoded by different genes. The first and rate-limiting reaction in the formation of steroid hormones is the conversion of cholesterol to pregnenolone, which is stimulated by adrenocorticotropic hormone (ACTH) in the adrenals and by Luteinizing hormone (LH) in the ovaries and testes to synthesize C21,C19 and C18 compounds respectively. This reaction is complex and occurs in the mitochondria. It is catalyzed by the enzyme C20-22-desmolase, which is encoded by the CYP11A gene. A key step in the reaction is the transport of cholesterol from extracellular sources to the inner mitochondrial membrane, and subsequent loading of the precursor into the active site of the enzyme in these steroid genic cells. Both the delivery of cholesterol to the enzyme and the enzyme level are primarily under the control of tropic hormones (LH or ACTH) using cyclic AMP or calcium as the intracellular messenger. Once pregnenolone is formed, it can then be converted to progesterone, androgens, estrogens, and corticosteroids. For this reason, pregnenolone is sometimes referred to as the mother steroid. 2. Steroid Hormone Biosynthesis In The Adrenal Cortex The adult adrenal cortex produces three classes of steroid hormones: glucocorticoids, mineralocorticoids, and adrenal androgens. The

specialized fetal adrenal, in conjunction with the fetal liver, the placenta, and some maternal organs, produces the steroid hormones of pregnancy. Cortisol, the major glucocorticoid in humans, is rapidly synthesized and secreted in response to ACTH; this is part of a response to stress and increases circulating levels of energyproviding compounds: glucose, free fatty acids and free amino acids. Aldosterone, the major human mineralocorticoid, is synthesized and secreted in response to angiotensin II; it helps prevent extracellular fluid depletion by promoting sodium reabsorption and fluid retention. The physiological roles of adrenal androgens are poorly understood. Because the actions of these steroid hormones result from effects on gene expression, the effects generally have a slow onset and are long-lived. The adrenal is surrounded by the capsule, a tough layer of connective tissue that protects the organ, and helps to maintain its structure and shape. Within the capsule are three layers, or zones, of cells that comprise the steroid genic structures of the adrenal: the zona glomerulosa, the zona fasciculata and the zona reticularis. The zona glomerulus, the outermost zone, is the site of aldosterone production. The zona fasciculate is the largest zone, and is the major site of cortisol synthesis; the zona fasciculate also produces the adrenal androgens dehydroepiandrosterone ( DHEA) and dehydroepiandrosterone sulphate (DHEAS). The innermost steroid genic zone is the zona reticularis, which also produces cortisol, DHEA, and DHEAS. The major synthetic pathways for the adrenal steroid hormones begin with cholesterol and goes to the adrenal products aldosterone, cortisol, and DHEA(S), and to the non-adrenal steroid hormones

progesterone, testosterone, and estradiol. The steroids within the grey box are not produced by the adrenal; these steroids are either produced from cholesterol in gonadal tissues or are produced from circulating DHEA and androstenedione in peripheral tissues. Note that while progesterone is synthesized in the adrenal, adrenal progesterone is generally not released in significant quantities; instead it merely serves as a precursor for some of the adrenal hormones. In normal individuals, hormonally relevant levels of progesterone are produced only by the ovary or by the placenta. The steps appears complex but is logically organized. In each case the difference between one row and the next lower row is the reaction catalyzed by a single enzyme. Each of the reactions linked by a box is catalyzed by the same enzyme. Thus, the first row (with the steroids pregnenolone, 17α hydroxy- pregnenolone, and DHEA contains 3β -hydroxy- Δ5 steroids, where Δ5 refers to the presence of a double bond between the 5 and 6 postions. The second row (progesterone, 17α-hydroxyprogesterone, and androstenedione) contains 3- keto- Δ4 steroids. All are produced from first row steroids by the enzyme 3β -hydroxysteroid dehydrogenase/ Δ 5- Δ 4- isomerase. The third row contains 21-hydroxysteroids (deoxycorticosterone and deoxycortisol). These are produced from the corresponding second row steroids by the enzyme 21- hydroxylase. The fourth row contains 11β -hydroxysteroids (corticosterone and cortisol), products of the enzyme 11β - hydroxylase (cortisol, and some corticosterone), or 18-hydroxylase (corticosterone); in the case of corticosterone production, the same reaction can be catalyzed by two different enzymes. The fifth row contains a single steroid, aldosterone, the final product of the 18- hydroxylase.

The columns are similarly organized; the first column contains 21- carbon steroids, the second column contains 21-carbon, 17α - hydroxysteroids, and the third column contains 19-carbon steroids. Note that P450-17 α catalyzes two separate reactions: 17α - hydroxylation of pregnenolone and progesterone, and the cleavage of the short side chain to produce the 19-carbon steroids. The enzyme can release the 21-carbon 17α - hydroxysteroids, allowing the production of (eventually) cortisol, or can catalyze the second step, and release the 19-carbon final product. The thick arrows in the steroid biosynthesis pathway chart are cytochrome P450 enzymes. Three of the enzymes, all cytochromes P450 (P450scc, P450-11β, P450-18) are located within the mitochondria; all of the other steroidogenic enzymes are found in the smooth endoplasmic reticulum. The first enzyme in the pathway is cytochrome P450scc, also known as cholesterol side chain cleavage enzyme. P450scc catalyzes the first, slowest, and therefore, rate limiting step for hormone synthesis. As you might expect from the observation that P450scc catalyzes the rate-limiting step, P450scc is also the major site of physiological regulation. In each steroid synthetic tissue, the control hormone for that endocrine gland increases the activity of P450scc. Pregnenolone, the product of the P450scc reaction, is converted to the final hormone product by sequential steps along the pathway depending on the enzymes that are present in that tissue. Thus, the zona glomerulosa makes aldosterone, because it contains 3β -HSD, P450-21, and P450-18, and lacks P450-17α. Gonadal tissues lack P450-21 and therefore can only make the sex steroids progesterone, testosterone and estradiol. P450-18 is closely related (about 95% sequence identity) to P450-11β, and has both 11β-hydroxylase activity and the ability to catalyze

the additional reactions that produce aldosterone. The majority of human corticosterone is produced by the action of 11β -hydroxylase on deoxycorticosterone present in the zona fasciculata; normally, 18- hydroxylase carries out all of the reactions required to convert deoxycorticosterone to aldosterone without releasing the intermediates. This chart is important in understanding the normal biosynthetic processes involved in steroid production. In cases of genetic enzyme deficiencies, however, the patient will make products determined by enzymes that are still present. For example, a patient lacking P450-21 can only make androgens. A patient lacking P450-17α has an entire adrenal that acts like the zona glomerulosa, producing large amounts of deoxycorticosterone, corticosterone, and aldosterone, but nothing else. Regulation of Adrenal Steroid Synthesis Adrenocorticotropic hormone (ACTH), of the hypophysis, regulates the hormone production of the zona fasciculata and zona reticularis. ACTH receptors in the plasma membrane of the cells of these tissues activate adenylate cyclase with production of the second messenger, camp. The effect of ACTH on the production of cortisol is particularly important, with the result that a classic feedback loop is prominent in regulating the circulating levels of corticotropin releasing hormone (CRH), ACTH, and cortisol. Mineralocorticoid secretion from the zona glomerulosa is stimulated by an entirely different mechanism. Angiotensins II and III, derived from the action of the kidney protease renin on liver-derived angiotensinogen, stimulate zona glomerulosa cells by binding a plasma membrane receptor coupled to phospholipase C. Thus, angiotensin II and III binding to their receptor leads to the activation

of PKC and elevated intracellular Ca 2+ levels. These events lead to increased P450ssc activity and increased production of aldosterone. In the kidney, aldosterone regulates sodium retention by stimulating gene expression of mrna for the Na + /K + ATPase responsible for the reaccumulation of sodium from the urine. The interplay between renin from the kidney and plasma angiotensinogen is important in regulating plasma aldosterone levels, sodium and potassium levels, and ultimately blood pressure. 3. Hormone Biosynthesis In The Adrenal Medulla Cells in the adrenal medulla synthesize secrete epinephrine and norepinephrine. Following release into blood, these hormones bind adrenergic receptors on target cells, where they induce essentially the same effects as direct sympathetic nervous stimulation. Synthesis of catecholamines begins with the amino acid tyrosine, which is taken up by chromaffin cells in the medulla and converted to norepinephrine and epinephrine through the following steps: Hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA) is the rate-determining reaction and is catalyzed by the enzyme tyrosine hydroxylase. Activity of this enzyme is inhibited by catecholamines (product inhibition) and stimulated by phosphorylation. In this way, regulatory adjustments are made rapidly and are closely tied to bursts of secretion. A protracted increase in secretory activity induces synthesis of additional enzyme after a lag time of about 12 hours.

Tyrosine hydroxylase and DOPA decarboxylase are cytosolic enzymes, but the enzyme that catalyzes the β-hydroxylation of dopamine to form norepinephrine resides within the secretory granule.dopamine is pumped into the granule by an energy dependent, stereospeciftc process. For sympathetic nerve endings and those adnenomedullary cells that produce norepinephrine, synthesis is complete with the formation of norepinephrine, and the hormone remains in the granule until it is secreted. Synthesis of epinephrine, however, requires that norepinephrine reenter the cytosol for the final methylation reaction. The enzyme required for this reaction, phenylethanolamine-n-methyltransferase (PNMT), is at least partly inducible hy glucocorticoids. Induction requires concentrations of Cortisol that are considerably higher than those found in peripheral blood. The vascular arrangement in the adrenals is such that interstitial fluid surrounding cells of the medulla can equilibrate with venous blood that drains the cortex and therefore has a much higher content of glucocorticoids than arterial blood. Glucocorticoids may thus determine the ratio of epinephrine to norepinephrine production. Once methylated, epinephrine is pumped back into the storage granule, whose membrane protects stored catecholamines from oxidation by cytosolic enzymes. 4. Steroid Hormone Biosynthesis In The Ovaries And Testes Although many steroids are produced by the testes and the ovaries, the most important are testosterone, progesterone and estrogen. These compounds are under tight biosynthetic control, with short and long negative feedback loops that regulate the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) by the

pituitary and gonadotropin-releasing hormone (GnRH) by the hypothalamus. Low levels of circulating sex hormone reduce feedback inhibition on GnRH synthesis (the long loop), leading to elevated FSH and LH. The latter peptide hormones bind to gonadal tissue and stimulate P450ssc activity, resulting in sex hormone production via camp and PKA mediated pathways. The roles of camp and PKA in gonadal tissue are the same as that described for glucocorticoid production in the adrenals, but in this case adenylate cyclase activation is coupled to the binding of LH to plasma membrane receptors. The biosynthetic pathway to sex hormones in male and female gonadal tissue includes the production of the androgens, androstenedione and DHEA. Testes and ovaries contain an additional enzyme, a 17β-hydroxysteroid dehydrogenase that enables androgens to be converted to testosterone. In males, LH binds to Leydig cells, stimulating production of the principal Leydig cell hormone, testosterone. Testosterone is secreted to the plasma and also carried to Sertoli cells by androgen binding protein (ABP). In Sertoli cells the Δ4 double bond of testosterone is reduced, producing dihydrotestosterone (DHT). Testosterone and (DHT) are carried in the plasma, and delivered to target tissue, by a specific gonadal-steroid binding globulin (GBG). In a number of target tissues, testosterone can be converted to DHT. DHT is the most potent of the male steroid hormones, with an activity that is 10 times that of testosterone. Because of its relatively lower potency, testosterone is sometimes considered to be a prohormone.

Testosterone is also produced by Sertoli cells but in these cells it is regulated by FSH, again acting through a camp- and PKA-regulatory pathway. In addition, FSH stimulates Sertoli cells to secrete androgen-binding protein (ABP), which transports testosterone and DHT from Leydig cells to sites of spermatogenesis. There, testosterone acts to stimulate protein synthesis and sperm development. In females, LH binds to thecal cells of the ovary, where it stimulates the synthesis of androstenedione and testosterone by the usual camp- and PKA-regulated pathway. An additional enzyme complex known as aromatase is responsible for the final conversion of the latter 2 molecules into the estrogens. Aromatase is a complex endoplasmic reticulum enzyme found in the ovary and in numerous other tissues in both males and females. Its action involves hydroxylations and dehydrations that results in aromatization of the A ring of the androgens. Aromatase activity is also found in granulosa cells, but in these cells the activity is stimulated by FSH. Normally, thecal cell androgens produced in response to LH diffuse to granulosa cells, where granulosa cell aromatase converts these androgens to estrogens. As granulosa cells mature they develop competent large numbers of LH receptors in the plasma membrane and become increasingly responsive to LH, increasing the quantity of estrogen produced from these cells. Granulosa cell estrogens are largely, if not all, secreted into follicular fluid. Thecal cell estrogens are secreted largely into the circulation, where they are delivered to target tissue by the same globulin (GBG) used to transport testosterone.

Cholesterol undergoes double oxidation to produce 20,22- dihydroxycholesterol. This vicinal diol is then further oxidized with loss of the side chain starting at position C-22 to produce pregnenolone. This reaction is catalyzed by cytochrome P450scc. The conversion of pregnenolone to progesterone takes place in two steps. First, the 3-hydroxyl group is oxidized to a keto group and second; the double bond is moved to C-4, from C-5 through a keto/enol tautomerization reaction. This reaction is catalyzed by 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(4) isomerase. The Steroid hormones are released into the blood circulation as soon as they are formed, travel to various parts of the body, and act on specific cells to bring about specific responses. All the Steroid hormones exert their action by passing through the plasma membrane and binding to intracellular receptors. The steroid hormone-receptor complexes exert their action by binding to specific nucleotide squences in the DNA of responsive genes. These DNA sequences are identified as HREs (Hormone Response Elements). The interaction of Steroid-Receptor complexes with DNA can induce or repress the transcription of their associated genes. A number of endocrine disorders are also attributed to defects in steroid biosynthesis due to specific enzyme defects. Inability to secrete normal levels of adrenal steroids may result in CAH (Congenital Adrenal Hyperplasia). In the majority of cases, this syndrome is due to CYP21A2 deficiency, and is associated with increased adrenal androgen secretion and partial virilization in girls. Defects in testicular Androgen synthesis (CYP17 or HSD17B deficiency) can lead to male Pseudo hermaphrodites. And other related diseases in the male dueto its altered levels.

5. Summary All steroid hormones are produced by steroidogenic tissues like adrenal (cortex) and gonads as well as catecholamines by adrenal medulla. Steroid hormones are C18, C19 and C21 compounds derived from a precursor, cholesterol in these tissue as these have specific enzymes for synthesis in them followed by the stimulation of respective pituitary hormones. All these hormones have common steroid nucleus having four structured rings. The adrenocortical hormones are involved in fight-or-flight response. Gonadal hormones are important for reproductive functions. All these steroid molecules bring about their effects through their receptor complex activating specific genes of the nucleus in their target sites. Nerves and feed back system control their production, storage and release.