Production of testosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and androsterone by dispersed testicular interstitial cells and whole testes in vitro.

The production of 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), androsterone and testosterone by whole rat testes and testicular interstitial cells dispersed with collagenase was studied in vitro. Luteinizing hormone stimulated the production of each of the androgens by cells prepared from 31- to 34-day-old rats. Half maximum stimulation of the production of each androgen occurred with approximately 3.5 ng NIH-LH-B9/ml medium. Androstanediol was the predominant product then androsterone and then testosterone. Luteinizing hormone stimulated the production of testerone, but not androstanediol or androsterone by dispersed interstitial cells from 200-day-old rats. The time-course of production and the effect of the concentration of cells on the production of these androgens suggested that in dispersed testicular interstitial cells from immature animals androstanediol and androsterone are formed, at least partially, by the metabolism of testosterone. In these experiments LH-stimulated testosterone production increased during incubation for 15--60 min and then remained constant up to 180 min. The concentrations of androstanediol and androsterone increased in a linear manner during incubation for 60--180 min. Varying the number of cells incubated yielded a positive correlation between cell concentration and the ratio 5 alpha-reduced androgen : testosterone produced. Luteinizing hormone stimulated production of each androgen by whole tests obtained from rats at 30--175 days of age. The serum concentration of testosterone in these rats increased abruptly at 50 days of age. Significant changes in androgen production in vitro also observed at this age included: (1) increased production of the three steroids when incubated in either the presence or absence of LH and (2) testosterone production, either in the presence or absence of LH, which represented a greater percentage of the total production of the three androgens.     

Inhibitory effect of gonadotropin releasing hormone upon cultured testicular cells

The direct inhibitory of gonadotropin-releasing hormone (GnRH) upon testicular androgen production was studied in cultured testicular cells. Enzyme-dispersed testicular cells from immature hypophysectomized rats were cultured for 6 days in a serum-free medium with or without various hormones. Culture media were changed every 2 days and media concentration of a androgens was measured by radioimmunoassay. The testis cultures from immature rats secrete predominately 5 alpha-androstane-3 alpha, 17 beta-diol (A-diol) and androsterone but negligible amounts of androstenedione, testosterone and dihydrotestosterone. Incubation of testicular cells with hCG and FSH increased A-diol and androsterone production as compared to control cultures. In contrast, treatment with GnRH or a GnRH agonist (des-Gly10, D-Leu6 (N alpha Me)Leu7, Pro9NHEt-GnRH) inhibited the gonadotropin effect in a dose-dependent manner; 10(-8) M GnRH inhibited A-diol production by approximately 20% on day 4 and 6 of culture whereas 10(-6) M GnRH inhibited A-diol production by greater than 80%. The observed effect of GnRH upon testicular cells in primary culture demonstrates that the hypothalamic peptide directly inhibits testicular steroidogenesis.     

5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.     

Maturational changes in steroidogenic enzyme activities metabolizing testosterone and dihydrotestosterone in two populations of testicular interstitial cells

The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21-73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21-35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21-50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5 alpha-androstane-3 beta,17 beta-diol in Band 2 cells, while 5 alpha-androstane-3 beta,17 beta-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.     

Steroid 5alpha-reductase 1 promotes 5alpha-androstane-3alpha,17beta-diol synthesis in immature mouse testes by two pathways

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.     

Steroid production in vitro by rat ovaries during sexual maturation

To determine changes in steroidogenesis by rat ovaries during sexual maturation, ovaries obtained at various ages (days 10-35) and at the first pro-oestrus were incubated in the absence or presence of LH and the accumulation of steroids in the medium was measured. Basal and LH-stimulated oestradiol-17 beta and testosterone release into the medium, expressed in pmol/4 h per mg ovary, was high at day 10 of age and at first pro-oestrus. Between days 20 and 35 basal oestradiol and testosterone release was low and could not be stimulated by LH. Addition of testosterone to the culture medium increased oestradiol production at all ages studied. Release of progesterone occurred at all ages even in LH-free medium. Incubation in the presence of LH resulted in a dose-dependent increase in progesterone with a maximal response at pro-oestrus. Androsterone and 5 alpha-androstane-3 alpha,17 beta-diol production in the absence or presence of LH was high during the entire prepuberal period. Production of 5 alpha-reduced androgens in response to LH increased from days 10 to 20 but decreased thereafter. Similarly, 5 alpha-reductase activity, measured in ovarian homogenates, increased from days 10 to 20 but was decreased again by first pro-oestrus. A further decrease in basal and LH-stimulated 5 alpha-reduced androgen production occurred after first ovulation. These results demonstrated age-related changes in steroid release after in-vitro incubation. At day 10 progesterone can be converted to aromatizable androgens allowing production of oestrogens, while after day 10 progesterone is converted to 5 alpha-reduced C19 steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine regulation of sex-dependent hydroxysteroid dehydrogenase activities in rat kidney: NADP-dependent microsomal 3alpha- and 20beta-hydroxysteroid dehydrogenase.

The NADP-dependent microsomal kidney enzymes, 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSDH), which exhibit considerable sex differences in their activities (male:female activity ratios, 16:1 and 30:1 respectively), were investigated after interference with the pituitary-gonad and pituitary-adrenal systems. Prepubertal gonadectomy as well as hypophysectomy of mature male rats led to a decline in HSDH activity to almost that found in the normal female rat, whereas activities in female rats were unaffected. Testosterone induced typical male 3alpha-HSDH activity in both gonadectomized and hypophysectomized rats of either sex. Administration of 5alpha-dihydrotestosterone (5alpha-DHT) or 5alpha-androstane-3alpha, 17beta-diol to hypophysectomized male rats was equally effective in restoring full 3alpha- and 20beta-HSDH activities whereas 5alpha-androstane-3beta, 17beta-diol was less effective and dehydroepiandrosterone was ineffective. Simultaneous administration of cyproterone acetate did not block the inductive action of 5alpha-DHT. Administration of chorionic gonadotrophin, pregnant mare serum gonadotrophin or a combination of luteinizing hormone and follicle-stimulating hormone to hypophysectomized male rats all led to parallel increases in the weight of the seminal vesicles and in both renal enzyme activities; administration of growth hormone, prolactin or thyroid-stimulating hormone was ineffective. Adrenalectomy of gonadectomized, but not of hypophysectomized male rats, caused a further drop in activity to the normal female level. Adrenalectomy of otherwise intact rats did not affect either enzyme activity. The hypophysis was involved in the regulation of the two NADP-dependent renal HSDH activities through its gonadotrophic function in male rats; adrenal secretions were of little physiological significance.     

Unsolved problems in male physiology: studies in a marsupial

Testicular androgens induce formation of the male urogenital tract in all mammals. In marsupials male development occurs after birth and over a prolonged period. For example, in the tammar wallaby virilization of the Wolffian ducts begins by day 20, prostate formation begins about day 25, and phallic development starts after day 80 of pouch life. Between days 20 and 40 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in tammar testes and secreted into plasma. Administration of 5alpha-adiol to pouch young females induces urogenital sinus virilization by day 40 and formation of a mature male prostate and phallus by day 150. 5alpha-Adiol is synthesized in pouch young testes by two pathways, one involving testosterone and dihydrotestosterone and the other 5alpha-pregnane-3alpha,17alpha-diol-20-one and androsterone as intermediates, both utilizing steroid 5alpha-reductase. In target tissues 5alpha-adiol acts via the androgen receptor after conversion to dihydrotestosterone but may have other actions as well. Whether 5alpha-adiol plays a role in male development in placental mammals is uncertain.     

Plasma concentrations of ovarian steroids in the freshwater European silver eel (Anguilla anguilla L.): effects of hypophysectomy and transfer to sea water

Intact and hypophysectomized freshwater (FW) silver eels were transferred to tanks of FW or artificial sea water (SW; salinity = 0.60 osmol/l) which were simultaneously renewed twice a week. Fish were killed 2 months after transfer and plasma was assayed for ovarian steroids. In all fish, 5 alpha-androstane-3 beta,17 beta-diol was present, while 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol were undetectable. In intact FW eels, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were approximately 0.15 nmol/l. In intact SW eels, no change in plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol was found, whereas the concentration of oestradiol-17 beta was increased significantly (P less than 0.01), indicating stimulation of aromatase activity. In hypophysectomized compared with intact FW fish, plasma levels of testosterone and 5 alpha-androstane-3 beta,17 beta-diol were decreased (P less than 0.05) and there was a slight but significant (P less than 0.01) augmentation of the plasma concentration of oestradiol-17 beta which may have involved the removal of pituitary-dependent inhibition of aromatase activity, possibly by 5 alpha-reduced compounds. In hypophysectomized compared with intact SW fish, plasma levels of testosterone, 5 alpha-androstane-3 beta,17 beta-diol and oestradiol-17 beta were decreased (P less than 0.05); in the case of oestradiol-17 beta, this may have reflected the diminished ovarian synthesis of testosterone, its precursor. The plasma level of oestradiol-17 beta was, however, higher in SW than in FW fish, even in hypophysectomized eels. This suggests that extra-pituitary mechanisms mediate, at least partly, the effects of transfer to SW on aromatase activity.     

Metabolism of C19 steroids in vitro by the submaxillary salivary gland of mature and immature domestic pigs

Homogenates of the submaxillary glands of immature and mature pigs were incubated with 3H-labelled C19 steroids which have been shown previously to be metabolized in vitro by the submaxillary gland of mature boars. Dehydroepiandrosterone was metabolized largely to androstenedione, 5 alpha-androstane-3,17-dione and androsterone, and to small amounts of testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstanediols. Testosterone yielded predominantly 5 alpha-androstane-3 alpha, 17 beta-diol with smaller amounts of other 5 alpha-reduced products, i.e. 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione and androsterone; 5 alpha-dihydrotestosterone and the two epimeric 5 alpha-androstane-3 alpha/3 beta, 17 beta-diols were interconverted. These and earlier results show that the porcine submaxillary gland has the capacity in vitro to metabolize selected C19 steroids in a way which is not related to either sexual maturity or sex of the animal; in this respect the findings support certain aspects of previous histochemical studies.     

Tissue androgen concentrations in golden hamsters with benign prostatic tumours

Using gas chromatography-high resolution mass spectrometry with selected ion detection, the concentrations of testosterone, 5alpha-dihydrotestosterone, androsterone, 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17alpha-diol and 5alpha-androstane-3beta,17beta-diol were measured in the dorsal and ventral prostates of two strains of golden hamster (Mesocricetus auratus) at various ages from 60 to 250 days. The tissue concentrations from a control strain were compared with those of the BIO 87.2 strain of hamster, the latter developing benign hyperplasia of the prostate between 90 and 120 days of age. Marked increases in the concentration of total 5alpha-androstanediols in both prostatic lobes of the BIO 87.2 strain were detected, with the highest level of over 345 nmol/g protein being found at 200 days of age. In comparison, the control strain showed a less pronounced increase in concentration at 150 days. Increases in total 5alpha-androstanediols were mainly associated with increases in the concentration of 5alpha-androstane-3alpha,17beta-diol. Concentrations of testosterone in prostatic tissue were also found to increase in the BIO 87.2 animals with peak values being seen at 200 days. Increases in concentration of androsterone were observed by 150 days and these levels were maintained up to 250 days of age. Control animals showed no comparable increases in testosterone and androsterone during the time-course studied. Surprisingly, no significant variation in 5alpha-dihydrotestosterone concentration was detected in either strain from 60 to 250 days although levels were slightly but consistently higher in the BIO 87.2 strain. There were no significant fluctuations in any of the androgens assayed either before or during tumour development. The increases in 5alpha-androstane-3alpha,17beta-diol, testosterone and androsterone were detected only after tumours had become established.     

Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats

The direct effect of gonadotropin-releasing hormone (GnRH) and its agonist on testicular LH receptor and steroidogenesis was studied in hypophysectomized immature and adult rats. Hypophysectomized rats were treated daily with varying doses of GnRH or [des-Gly10,D-Leu6(N alpha Me)Leu7, Pro9-NHEt]GnRH(a potent agonist). Some animals were also treated concomitantly with FSH, PRL, GH and/or LH to prevent the hypophysectomy-induced loss of testicular LH receptor and steroidogenic capacity. At the end of 5 days of treatment, testicular LH/hCG receptor concentration was measured by a [125I]-hCG-binding assay and steroidogenic responsiveness was determinded by in vitro incubations. GnRH and the GnRH agonist reduced testicular LH receptor in control and FSH-treated hypophysectomized immature rats. As little as 0.5 microgram agonist/day induced a greater than 40% decrease in the LH receptor content, whereas GnRH was less potent, with 50 micrograms/day inducing about a 50% decrease. The inhibitory effect of GnRH was shown to be the result of decreases in the concentration of LH receptor rather than changes in the receptor affinity (Kd = 1.1 X 10(-10)M). GnRH did not interfere with the [125I]hCG receptor assay. Treatment with PRL, GH, and FSH, alone or in various combinations, increased the testicular LH receptor content. The stimulatory effect of these pituitary hormones was depressed by concomitant treatment with the GnRH agonist. Similar inhibitory effects of GnRH and the agonist on testicular LH receptor were demonstrated in adult hypophysectomized rats. In vitro studies demonstrated that treatment with the GnRH agonist in vivo inhibited both basal and hCG-stimulated androgen production in FSH-primed immature hypophysectomized rats. Associated with decreases in androgens (testosterone and androstenedione) and reduced androgens (dihydrotestosterone, androstanediol, and androsterone), there was marked suppression of 17 alpha-hydroxylated precursors and C-21 steroid intermediates in animals treated with the GnRH agonist, thus suggesting that the inhibitory effect of the GnRH agonist was associated with possible defects in 17 alpha-hydroxylase and side-chain cleavage enzymes. Likewise, treatment with the GnRH agonist inhibited in vitro testicular steroidogenic responses in adult hypopysectomized rats. These results demonstrate the extrapituitary inhibitory effect of GnRH on testicular LH receptor content and Leydig cell steroidogenesis in immature and adult hypophysectomized rats.     

In-vitro testosterone metabolism in the mouse preputial gland: intercellular co-operation and changes with cell maturation

In-vitro [14C]testosterone metabolism was investigated in isolated cells of adult male mouse preputial sebaceous glands. Labelled steroids were extracted and chromatographed after a 2-h incubation, and were identified as 5 alpha-dihydrotestosterone, androstenedione, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol, androsterone and 3-epiandrosterone. In cells separated according to state of maturity (lipid content) by isopycnic centrifugation in a metrizamide gradient, maximal testosterone metabolism occurred in large, nearly mature cells. In this population, mean hydroxysteroid 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities were 3.8 and 2.3 nmol/10(6) cells per 2h respectively, more than 100-fold greater than in the densest population, comprised of undifferentiated and early differentiating cells. It was also found that the profile of testosterone metabolites was dependent on the proportion of the label metabolized. The metabolite index (MI), i.e. the average number of enzymatic steps undergone per molecule of metabolite, increased with increasing substrate utilization. Metrizamide showed reversible, non-specific inhibition of testosterone metabolism and reduction of the MI. Thus, it was postulated that testosterone is metabolized sequentially by different cells, with metrizamide inhibiting cellular uptake and intercellular substrate transport. This suggested that most of the metabolites would be found in the medium, rather than in the cellular compartment. Further, in incubations run without cell disaggregation, efficient substrate cycling among cells should result in a high MI, independent of metrizamide concentration and substrate utilization. These predictions were all confirmed, providing strong evidence that testosterone metabolism is a co-operative effort among several cells in this tissue.     

Direct inhibitory effect of glucocorticoids upon testicular luteinizing hormone receptor and steroidogenesis in vivo and in vitro

The direct effects of glucocorticoids on testicular LH receptor content and steroidogenesis were studied in vivo and in vitro. Immature hypophysectomized rats were treated with varying doses of dexamethasone, corticosterone, or a synthetic progestin, 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020). Some animals were also treated concomitantly with FSH to prevent the hypophysectomy-induced decrease in testis functions. At the end of 5 days of treatment, testicular LH/hCG receptor content was measured by [125I]hCG binding assay while steroidogenic responsiveness was measured by in vitro incubation of testes. Dexamethasone decreased testicular LH receptor in control and FSH-treated hypophysectomized rats in doses as low as 10 microgram/day, whereas corticosterone (10 microgram/day) decreased testicular LH receptor in the FSH-treated rats but had no effect in rats not treated with FSH. In contrast, R5020 had no effect on testicular LH receptor content. In vivo treatment of hypophysectomized rats with FSH increased both basal and hCG-stimulated production of androstanediol in vitro. In contrast, concomitant treatment with dexamethasone, but not R5020, decreased both basal and hCG-stimulated testicular androstanediol production. The direct effect of glucocorticoids on testicular steroidogenic potentials was also studied in primary culture of testicular cells obtained from adult hypophysectomized rats. Treatment of cultured testicular cells wtih hCG increased testosterone production. The addition of various natural and synthetic glucocorticoids, but not R5020, to hCG-treated cells decreased testosterone production in a dose- and time-related manner (triamcinolone greater than or equal to dexamethasone greater than cortisol greater than or equal to corticosterone). A 40% decrease in testosterone production was apparent at 6 h after addition of 10(-7) M dexamethasone to hCG-treated cells. These results demonstrate the direct inhibitory effect of glucocorticoids on testicular LH receptor content and steroidogenesis, suggesting the adrenal glucocorticoids may regulate testis functions.     

Relationship between the structures and steroidogenic functions of the testes of the urohaze-goby (Glossogobius olivaceus)

The testis of the brackishwater goby (Glossogobius olivaceus, the urohaze-goby in this text) consists of two main components, the glandular and the seminiferous tissue. After manual separation of the two tissues, in vitro steroidogenesis in each tissue was examined using testes from mature males in the breeding season. Cell-free homogenates (800g supernatant fluid) of each tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, or 5 alpha-pregnane-3,20-dione in the presence of NAD+ or NADPH. (1) Glandular tissue: Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively, in the presence of NAD+. In the presence of NADPH, the following metabolism of steroids was established. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product), 17 alpha-hydroxyprogesterone, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, and androstenedione. 17 alpha-Hydroxyprogesterone was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione (main product), 3 beta, 17 alpha-dihydroxy-5 alpha-pregnan-20-one, androstenedione, and 5 alpha-androstane-3,17-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone (main product), 5 alpha-androstane-3 beta, 17 beta-diol, epiandrosterone, and 5 alpha-androstane-3,17-dione. 5 alpha-Pregnane-3,20-dione was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, 5 alpha-androstane-3,17-dione, epiandrosterone, and 5 alpha-dihydrotestosterone. (2) Seminiferous tissue: Almost all of the above metabolites were obtained, but the yield was much smaller, especially for 5 alpha-reduced metabolites, compared with that for glandular tissue. From these results, it is concluded that steroidogenesis in the testis of G. olivaceus is characterized by the predominant activity of 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase and that these are localized mainly in glandular tissue, together with delta 5-3 beta-hydroxysteroid dehydrogenase + delta 5-delta 4 isomerase, 17 alpha-hydroxylase, and C-17-C-20 lyase.     

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.     

Feminization of hepatic steroid metabolism in male rats following electrothermic lesion of the hypothalamus.

The metabolism of [4-14C]androst-4-ene-3,17-dione, [4-14C]5alpha-androstane-3alpha,17beta-diol and [1,2-3H]5alpha-androstane-3alpha,17beta-diol, 3,17-disulfate in the 105,000 X g supernatant and microsomal fractions of liver was studied in male and female rats after electrothermic lesion of the hypothalamus including the median eminence. Following electrothermic lesion, hepatic steroid metabolism in male rats was generally "feminized" (increased 5alpha-reduction and decreased 6beta- and 16alpha-hydroxylation of 4-androstene-3,17-dione, decreased 2alpha-, 2beta-, 18- and 7beta-hydroxylation of 5alpha-androstane-3alpha, 17beta-diol and induced 15beta-hydroxylation of 5alpha-androstane-3alpha,17beta-diol,3,17-disulfate), whereas hepatic metabolism in female rats remained essentially unchanged. Previous investigations have pointed to the occurrence of a sex-specific secretion of "feminizing factor" from the female pituitary that is responsible for the "feminization" of the basically "masculine" type of metabolism characterizing the rat liver. Taken together with these findings, the present results indicate that the release of the pituitary "feminizing factor" is controlled by means of a release-inhibiting factor from the hypothalamus. This factor is not secreted in female rats; it is suggested that its secretion in male rats is turned on as a result of neonatal imprinting by testicular androgens.     

Testosterone metabolites do not participate in the control of hypothalamic LH-releasing hormone

To determine whether the ability of testosterone to increase intrahypothalamic LH-releasing hormone (LHRH) in orchidectomized rats might be explained by the conversion of the hormone into either its 5 alpha-reduced or oestrogenic metabolites, testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) (2 mg/rat per day for 6 days) and oestradiol (0.1, 0.5, 1.0 and 5.0 micrograms/rat per day for 6 days) were injected into castrated male rats. After 6 days the rats were killed and serum LH levels and intrahypothalamic LHRH stores measured using specific radioimmunoassay procedures. Testosterone and its 5 alpha-reduced metabolites were used in either the free alcohol or the propionate form (dipropionates in the case of the diols); oestradiol was used as oestradiol-17 beta or in the benzoate form. Treatment with testosterone, DHT, 3 alpha-diol and 3 beta-diol resulted in a significant decrease in serum LH levels; all the 5 alpha-reduced testosterone derivatives were more effective than testosterone in this respect. Testosterone and DHT propionates suppressed LH release following orchidectomy totally; 3 alpha-diol and 3 beta-diol dipropionates were less effective. Testosterone increased intrahypothalamic LHRH stores, this effect being much higher after testosterone propionate, i.e. when intrahypothalamic LHRH stores were restored to pre-castration levels. None of the 5 alpha-reduced steroids was capable of modifying the low intrahypothalamic levels of LHRH found following orchidectomy; only 3 alpha-diol dipropionate exhibited some activity, but this was much lower than that of testosterone propionate. Oestradiol-17 beta was totally ineffective in decreasing serum LH in orchidectomized animals; in contrast, oestradiol benzoate progressively decreased serum LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The backdoor pathway to dihydrotestosterone.

Dihydrotestosterone (DHT) is the androgen responsible for formation of the male external genitalia during embryogenesis and for most androgen-mediated events at male puberty. In most circumstances, testosterone (T) derived from the testis is converted to DHT by 5alpha-reductase type 2 in genital skin and prostate. By contrast, the testes of pouch young of the tammar wallaby and immature postnatal testes of several species synthesize 5alpha-androstane-3alpha,17beta-diol, which is the proximal precursor of DHT in androgen-target tissues. Human steroidogenic enzymes efficiently catalyze all the required steps in a route to DHT that does not involve the T intermediate, called the 'backdoor pathway'. This alternative pathway of DHT production appears to explain how potent androgens are produced in some normal and pathological conditions when the conventional androgen-biosynthetic pathways fail to account completely for the of patterns androgen synthesis that are observed.     

Evidence that luteinizing hormone-stimulated differentiation of purified ovarian thecal-interstitial cells is mediated by both type I and type II adenosine 3',5'-monophosphate-dependent protein kinases

LH has been shown to be the principal hormone regulating ovarian thecal-interstitial cell (TIC) differentiation. It has been well documented that LH stimulates cAMP production and that cAMP analogs mimick the stimulatory actions of LH, but the mechanisms by which LH and cAMP stimulate TIC differentiation are unknown. The purpose of these studies was to characterize LH-stimulated differentiation of isolated TIC in serum-free medium and examine the role of cAMP-dependent protein kinase (PKA) isoenzymes in TIC differentiation. Highly purified (greater than 90%) TIC which were free from granulosa cell contamination were isolated from collagenase-dispersed ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When the purified TIC (20,000 viable cells/well) were cultured (2 days) in serum-free medium (0.2 ml in 96-well plates), low levels of steroids were produced. LH stimulated a dose-related (ED50 = 2.6 +/- 0.4 ng/ml) increase (50-fold) in androsterone, the principal androgen produced. LH stimulated an immediate dose-related increase in cAMP production, but there was a 20-h lag before LH stimulated an increase in androsterone production, which reached maximum levels at 30 h. LH-stimulated progesterone production increased immediately to a maximum at 10 h, then progesterone levels decreased as androsterone production increased. To determine the role of PKA in stimulating androsterone and progesterone production, TIC were cultured (2 days) with 8-aminohexylamino-cAMP (100 microM) plus N6-benzoyl-cAMP (30 microM) or 8-thiomethyl-cAMP (30 microM) plus N6-benzoyl-cAMP (30 microM) to directly and selectively activate type I or type II PKA, respectively. Selective activation of either isoenzyme increased androsterone and progesterone production by TIC. Immunoblots revealed that either type I or type II PKA increased the contents of P450scc and P45017 alpha in TIC. This is the first demonstration that direct activation of either type I or type II PKA stimulates TIC differentiation. These results indicate that LH stimulates TIC differentiation by a mechanism mediated by activation of one or both PKA isoenzymes.