Regulation Of Testicular Steroidogenesis Research Articles

Role of sox30 in regulating testicular steroidogenesis of common carp

Expression of transcription factors is crucial for the regulation of steroidogenesis and gonadal development in fish. SRY-related box (SOX) proteins regulate gene expression of various events related to vertebrate reproduction. This study reports the role of sox30 and its influence on sox9a/b in regulating testicular steroidogenesis of the common carp, Cyprinus carpio. Tissue distribution showed predominant expression of sox30 in gonads, while gonadal ontogeny indicated significant dimorphic expression of sox30 from 120 days post hatch. Higher sox30 transcripts during the spawning season, an elevation of sox30 after human chorionic gonadotropin induction, and 11-ketotestosterone (11-KT) treatment authenticate gonadotropin dependency. Treatment of 17α–methyl-di-hydroxy-testosterone to juvenile common carp for mono-sex induction, vis-à-vis elevated sox30 expression. Sox30 protein was detected abundantly in spermatocytes and spermatid/sperm of carp testis. Transient silencing of sox30 using small interfering RNAs decreased sox9a/b expression, lead to downregulation of certain molecule/factor, transcription factor, germ/stem cell marker, and steroidogenesis-related enzyme genes. Serum testosterone and 11-KT decreased significantly upon transient silencing of sox30, in vivo. Concomitantly, a reduction in testicular microsomal 11-β hydroxysteroid dehydrogenase activity was observed. These results demonstrate the influence of sox30 as well as sox9a/b in the regulation of testicular steroidogenesis in common carp.

Developmental, behavioral and endocrine alterations in male rats at early and late postnatal life following in utero exposure to low dose di-n-butylphthalate.

Environmental chemical pollutants that interfere with hormonal homeostasis or hormone signaling are the relevant agents inducing congenital or postnatally developed reproductive abnormalities in human beings, wild and domestic animals. In this study, we are examining reproductive effects of prenatal exposure of male rats to a low dose di-n-butylphthalate (DBP). Wistar female rats were given intragastrically DBP at a daily dose of 100mg/kg b.w. during 15th-21st days of pregnancy. Anogenital distance(AGD) in male offspring decreased on postnatal day (PND) 2 followed by its normalization on PND 7 and 10. There were no other visible teratogenic lesions in the newborns. The testicle descent into scrotum of control males occurred on PND 38.5 ± 0.1, while in DBP group it accelerated by 5.3days on the average. At the age of 6months, DBP-exposed animals exhibited double increase of blood plasma testosterone level as compared to controls, and hyperactive male sexual behavior in the presence of receptive female. The duration of latent periods of the first mount and the first intromission, as well as post-ejaculatory refractory period, have been shortened; the number of mounts with intromission and the number of ejaculations increased significantly. Histological examination of the testes indicated activation of Leydig cells. The female-type sexual behavior as evaluated by appearance of lordosis of orchidectomized and primed with estradiol and progesterone 10-month-old males in response to mount or approach of sexually active normal male was enhanced in DBP-group. Both 10-month-old and aging males (18months), castrated and hormone-primed, displayed homosexual type of behavior. Prenatal low dose DBP caused in 18-month-old males premature atrophy of the testes and accessory sexual glands, increased number of Leydig cell adenomas, a twice decrease of plasma testosterone level and exhausting of sexual potency. We concluded that prenatal exposition of male rats to low dose DBP determines epigenetic alterations of programming of sex brain differentiation and regulation of testicular steroidogenesis that leads to reproductive disorders and accelerated aging of reproductive system.

Ранние стероидогенные эффекты гонадотропина и низкомолекулярного агониста рецептора лютеинизирующего гормона при введении их субмаксимальных доз самцам крыс

The receptors of luteinizing hormone (LH) play a key role in the regulation of testicular steroidogenesis. They can be activated by gonadotropins, LH and human chorionic gonadotropin (hCG), and by the low-molecular-weight agonists, including the derivatives of thieno[2,3-d]pyrimidines, which bind to an allosteric site located in the transmembrane channel of LH receptor. The differences in the steroidogenic effects of gonadotropins and thieno[2,3-d]pyrimidines in the in vitro experiments and in a long-term administration to male rats were previously identified, but their early steroidogenic effects in the testes when administered at submaximal doses, have not been studied . The aim of the work was to study the effect of a single administration of hCG and TP03, a thieno[2,3-d]pyrimidine derivative, in submaximal doses on testosterone (T) plasma levels and on the expression of steroidogenic protein genes of, as well as the pattern of steroid hormones, the precursors of T, in the testes for 5 hours after the treatment of male rats. We demonstrate that TP03 (15 mg/kg) and hCG (50 IU/rat) 30 minutes after their administration to rats cause an increase in the intratesticular levels of T and its precursors, such as 17-hydroxyprogesterone and androstenedione, and also reduce the level of pregnenolone, which indicates its expenditure for progesterone synthesis. The gene expression of steroidogenic proteins significantly changed 3 hours after drug administration. The administration of TP03 and hCG led to an increase in the expression of the Star and Cyp17a1 genes encoding the transport protein StAR and the cytochrome P450-17α, the key components of the steroidogenesis system. At the same time, the difference between the expressions of the Hsd17b gene encoding the dehydrogenase HSD17β, which were higher in the hCG-treated group as compared to the TP03-treated group, is shown. An increase in the plasma T levels one and three hours after treatment of rats with hCG was higher than in the TP03-treated group, but after 5 hours the T levels in both groups of rats were comparable, which was due to the weakening of the steroidogenic effect of hCG while maintaining the corresponding effect of TP03. Thus, despite the similarity of the early steroidogenic effects of TP03 and hCG used at the submaximal doses, even in the first hours after their administration to rats, the peculiarities of their effect on testicular steroidogenesis begin to appear, which, after a long-term administration, lead to significant differences in their steroidogenic activity.

Anethum graveolens as a possible modulator of testicular steroidogenesis

The aim of this study was to evaluate the possible in vitro effects of Anethum graveolens L. (dill) extract on the steroidogenesis in testicular tissue. Specifically, we focused to determine the dominant chemical components of the dill extract followed by a subsequent assessment of its in vitro effects on the production of cholesterol, dehydroepiandrosterone, androstenedione and testosterone by rat testicular fragments. Dill extract was subjected to high performance liquid chromatography (HPLC) which determined cyranosid, carvone, chlorogenic acid, p-hydroxybenzoic acid and salicylic acid among the predominant biomolecules. For the in vitro experiments, testicular fragments from 12 sexually mature Wistar rats were incubated in the absence (control) or presence of 10, 100 or 1000 μg/mL extract for 24 h. Cholesterol levels were quantified via photometry and the hormones were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). Our data showed that the release of cholesterol and androstenedione (but not dehydroepiandrosterone and testosterone) by the testicular fragments obtained from seminiferous tubules was significantly affected by the dill extract in a dose-dependent manner. Exposure to the extract led to a significant decrease of both cholesterol as well as androstenedione (P<0.01 in case of 10 μg/mL; P<0.001 with respect to 100 and 1000 ug/mL extract). Our results suggest that the bioactive molecules present in Anethum graveolens L. could have dose-dependent impact on the secretion of selected male reproductive hormones, playing a role in the regulation of testicular steroidogenesis. Nevertheless, the exact effect of individual biomolecules on the endocrine testicular function has yet to be investigated.

The neuropeptide orexin A - search for its possible role in regulation of steroidogenesis in adult mice testes.

Orexin A, a hypothalamic neuropeptide, regulates food intake and sleep-wake cycle by binding to orexin receptor 1. Besides brain, orexin A and orexin receptor 1 are also present in peripheral organs. In our earlier studies, localization and expression of orexin A and orexin receptor 1 have been shown in adult mouse testis, and further their role in testicular development in neonatal mouse was also demonstrated. In this study, role of orexin A and orexin receptor 1 in the testis of adult mouse by blocking binding of orexin A to orexin receptor 1 using an orexin receptor 1 antagonist, SB-334867, was investigated under invivo and exvivo conditions. Mice were given a single bilateral intratesticular injection of the antagonist at doses of 4 and 12μg/mouse and were sacrificed 24h post-injection. The antagonist treatment caused degenerative changes in the seminiferous tubules in the testis and also caused alterations in steroidogenesis, with a concomitant decrease in the level of testosterone (T) and an increase in the level of 17β-estradiol (E2 ) in serum and in testis. Further, expressions of SF1, StAR, P450scc and 17β-HSD were downregulated, while the expressions of 3β-HSD and P450arom were upregulated in antagonist-treated mice compared with controls. Also, the oxidative stress in the testis was increased in treated mice. In exvivo study, antagonist treatment to the testis caused a marked decrease in the level of T and an increase in the level of E2 in the media, accompanied by downregulated expression of SF1, StAR, P450scc and 17β-HSD and an upregulation in the expression of 3β-HSD and P450arom, indicating a direct role of orexin A in regulation of testicular steroidogenesis. The results of exvivo study supported the findings of invivo study. In conclusion, the results suggest potential involvement of orexin A and orexin receptor 1 in regulation of steroidogenesis and spermatogenesis in the testis of adult Parkes mice.

Regulation of testicular steroidogenesis by Foxa3 via transcriptional modulation of ERα signaling in type 2 diabetes mellitus (T2DM)

Although both insulin and estrogen receptor α (ERα) are known to exert inhibitory effects on testicular steroidogenesis, it remains unknown whether these pathways regulate testosterone (T) production under certain pathological conditions [e.g., type 2 diabetes mellitus (T2DM)] in a coordinated manner. Here, we found that the expression of forkhead box protein A3 (Foxa3), an essential transcriptional regulator engaged in adipogenesis and energy metabolism, was significantly down-regulated in the Leydig cells (LCs) from T-deficient T2DM mice. Functionally, upon hCG stimulation, Foxa3 recruits to the Esr1 promoter and suppresses the transactivation of Esr1 gene. Disruption of this recruitment by T2DM-elicited hyperinsulinemia led to abnormal activation of ERα pathway, inhibited steroidogenic enzyme genes expression, and thus caused inadequate T production. Therapeutically, insulin-impaired and Foxa3 ablation-compromised steroidogenesis were effectively rescued by a pharmacological inhibitor of the ERα pathway. These findings reveal an obligatory coregulatory role of Foxa3 in the regulation of ERα expression and of the Foxa3/ERα cascade, at least in part, in the pathogenesis of androgen deficiency caused by T2DM.

Effect of neonatal hypothyroidism on prepubertal mouse testis in relation to thyroid hormone receptor alpha 1 (THRα1)

Thyroid hormones (THs) are important for growth and development of many tissues, and altered thyroid status affects various organs and systems. Testis also is considered as a thyroid hormone responsive organ. Though THs play an important role in regulation of testicular steroidogenesis and spermatogenesis, the exact mechanism of this regulation remains poorly understood. The present study, therefore, is designed to examine the effect of neonatal hypothyroidism on prepubertal Parkes (P) strain mice testis in relation to thyroid hormone receptor alpha 1 (THRα1). Hypothyroidism was induced by administration of 6-propyl-2-thiouracil (PTU) in mother’s drinking water from birth to day 28; on postnatal day (PND) 21 only pups, and on PND 28, both pups and lactating dams were euthanized. Serum T3 and T4 were markedly reduced in pups at PND 28 and in lactating mothers, while serum and intra-testicular testosterone levels were considerably decreased in pups of both age groups. Further, serum and intra-testicular levels of estrogen were significantly increased in hypothyroid mice at PND 28 with concomitant increase in CYP19 expression. Histologically, marked changes were noticed in testes of PTU-treated mice; immunohistochemical and western blot analyses of testes in treated mice also revealed marked decrease in the expression of THRα1 at both age groups. Semiquantitative RT-PCR and western blot analyses also showed reductions in both testicular mRNA and protein levels of SF-1, StAR, CYP11A1 and 3β-HSD in these mice. In conclusion, our results suggest that neonatal hypothyroidism alters localization and expression of THRα1 and impairs testicular steroidogenesis by down-regulating the expression SF-1, thereby affecting spermatogenesis in prepubertal mice.

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura)

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder’s organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17–20 lyase (CypP450c17) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3β-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis.

Role of leptin in seasonal adiposity associated changes in testicular activity of vespertilionid bat, Scotophilus heathi

The aim of this study was to investigate changes in circulating level of leptin in natural population of male bat, Scotophilus heathi in relation to seasonal changes in body mass, adipose tissue mass and testicular activity. The peak serum leptin level coincided with the period of maximum body mass due to heavy accumulation of adipose tissue. The in vivo study showed both inhibitory and stimulatory effects on testicular activity depending on circulating level of leptin. Moderate increase in circulating concentration of leptin coincided with the stimulatory effect on testicular activity in October, whereas peak circulating concentration of leptin coincided with suppressed testicular activity in December. Immunohistochemical and Western blot analysis confirmed expression of leptin receptors (Ob-R) in the testis, particularly in Leydig cells during different reproductive stages suggesting the direct action of leptin in regulation of testicular steroidogenesis. The in vitro study confirmed the in vivo observations of inhibitory effect of leptin on testosterone production through dose-dependent StAR expression in the testis. This study further showed that the leptin affects testicular activity through STAT3 mediated regulation of StAR expression. Thus, increased adiposity associated rise in circulating leptin level during winter dormancy impairs testicular activity by suppressing StAR mediated testosterone production in S. heathi. These results are suggestive of a link between the adipocyte derived leptin and testicular function.

Nitric oxide: An autocrine regulator of Leydig cell steroidogenesis in the Asian catfish, Clarias batrachus

Nitric oxide has been recognized as an important inter- and intra-cellular modulator of testicular steroidogenesis in higher vertebrates with conflicting results. Moreover, its role in regulation of testicular steroidogenesis in ectothermic vertebrates is non-existent. The present study was, therefore, undertaken to examine whether Leydig cells of a freshwater catfish, Clarias batrachus produce nitric oxide (NO), if so, does it regulate its steroidogenic activity? The purified Leydig cells were stained histochemically for NADPH-diaphorase (NADPH-d) activity, and immunocytochemically for neuronal and inducible nitric oxide synthase (nNOS and iNOS) like molecules. Leydig cells were also incubated with NOS inhibitor, N-nitro- l-arginine methyl ester ( l-NAME), and NO donor, sodium nitroprusside (SNP). NO and testosterone released in incubation medium were analyzed. A distinct positive NAPDH-d staining was observed in Leydig cells. These cells also exhibited immunoprecipitation of variable intensity with nNOS and iNOS antibodies. Further, l-NAME treatment caused significant suppression in NO production and elevation in testosterone secretion by Leydig cells. On the contrary, exposure of Leydig cells to SNP resulted in increased NO production with concomitant decline in testosterone level. Thus, the present study reports NO production by Leydig cells in fish for the first time, which appears to inhibit its own androgen production.

Galanin stimulates steroidogenesis in rat leydig cells

The present study was designed to evaluate whether galanin could play a role in the regulation of testicular steroidogenesis. To this purpose, using purified rat Leydig cells, we examined the effects of galanin on basal and hCG- or LHRH-induced testosterone production and the interference of a specific galanin receptor antagonist, galantide, on galanin activity. Moreover, since it has been shown that galanin-induced stimulation of LHRH secretion appears to involve the release of prostaglandin E 2 (PGE 2) as intracellular mediator, we evaluated also the effect of galanin on Leydig cells PGE 2 output and the interference of indomethacin, a cycloxygenase blocker, on its activity. Furthermore, the effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, was also examined. Data obtained indicate that galanin amplified testosterone response to hCG or LHRH whilst galantide prevented its potentiating activity. Moreover, galanin stimulated PGE 2 output though this fatty acid is not involved in galanin activity on Leydig cells as indomethacin failed to affect its amplification of testosterone production. The possible involvement of leukotrienes should also be excluded as NDGA did not modify galanin action. In summary, the present study indicates that galanin potentiates acute gonadotropin or LHRH steroidogenic action on Leydig cells and that this activity is specific and receptor-mediated as it is prevented by a specific receptor antagonist.

Vasoactive intestinal peptide stimulates testosterone production by cultured fetal rat testicular cells

As a part of a series of studies on the regulation of testicular steroidogenesis in the fetal rat, we found that VIP stimulated fetal testicular cAMP production at a dose of 10 −9 mol/l, while a dose as low as 10 −12 mol/l stimulated testosterone production. RT-PCR analysis could not reveal either VIP mRNA in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, while VIP2 receptor mRNA was detected in fetal testes as early as E15.5. The testicular VIP content was unmeasurable by our radioimmunoassay method (<1 fmol/testis), while the circulating levels of VIP were 82.9±1.1 pmol/l at E17.5 and decreased with advanced fetal ages. In conclusion, our results suggest that VIP, from and extratesticular source, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5.

Isolation, purification and culture of Sertoli cells from immature piglet testes

Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.

Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists

Clinical and experimental studies are described on the effects of a gonadotropin-releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions.

Cytoplasmic estradiol binding sites in testis of fertile and infertile men.

Free cytoplasmic estradiol receptor was determined in testicular biopsies from six adults showing various primary testicular disorders, and in testicular tissue obtained at autopsy from 18 males of various ages. Estradiol receptors were found in all cases examined. Receptor concentration varied during testicular maturation and from one patient to another. The presence of the receptor at an early stage of development and the changes observed in its concentration during testicular maturation and dysfunction support the idea that estradiol may play, through its receptor interaction, an important role in the regulation of testicular steroidogenesis during postnatal human development.

Comparable testosterone responses are produced by constant infusion of LH or GnRH in normal men.

Luteinizing hormone (LH) was infused continuously at a rate of 1.3 IU/min to 4 normal adult men. A 4 to 5-fold increase in serum LH was noted by 8 hours. Serum FSH declined steadily throughout the infusion period in the face of rising concentrations of gonadal steroids. Basal plasma testosterone of 4.7 +/- 0.4 ng/ml rose progressively to a peak of 11.1 +/- 0.9 ng/ml at hour 56 (p less than 0.005). A similar pattern was demonstrated by plasma androstenedione. Plasma 17 alpha-hydroxyprogesterone rose from a basal concentration of 0.81 +/- 0.14 ng/ml to a peak concentration of 2.6 +/- 0.3 ng/ml at hour 36 of the infusion and subsequently declined. A similar course was followed by serum estradiol-17 beta, which achieved a maximal concentration of 70.0 +/- 10.4 pg/ml at hour 36. Results are compared to those obtained with continuous infusion of GnRH in normal adult men. Testosterone responses were similar, whereas elevations in 17 alpha-hydroxyprogesterone and estradiol were higher following GnRH infusion. This difference may be consequent upon a direct gonadal effect of GnRH, or may be secondary to local regulation of testicular steroidogenesis by estradiol-17 beta.

HCG‐STIMULATION OF TESTICULAR STEROIDOGENESIS DURING INDUCED HYPER—AND HYPOPROLACTINAEMIA IN MAN

In order to elucidate the role of prolactin in the regulation of testicular steroidogenesis in man, hyper- and hypoprolactinaemia were induced by sulpiride (group 2) and bromocriptine (group 3) administration in eight and nine young adults, respectively, and the responses of testosterone and six other steroids to a single dose of 5000 iu hCG were compared with those obtained under basal conditions (group 1) in eight men. During hyperprolactinaemia (group 2) the responses of pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5 alpha-dihydrotestosterone tended to be greater than in the control and hypoprolactinaemia groups, but these changes were not statistically significant. In contrast to the behaviour of these steroids, oestradiol showed diminished peak values at 24-36 hCG in the sulpiride-treated group and the response was significantly smaller (P less than 0 . 01) than during hypoprolactinaemia. These findings suggest that testicular aromatization is modulated by prolactin and thus the partial inhibition of oestradiol production, observed during short-term hyperprolactinaemia, may result in a better androgen response to hCG.

Effect of clomiphene treatment on the human testicular response to a single dose of hCG.

The effect of antioestrogen treatment on the human testicular response to hCG was investigated in 17 adult men to further clarify the role of endogenous oestradiol in the regulation of testicular steroidogenesis. Clomiphene citrate was administered in 2 different modes. Group 1 (n = 8) was treated for 6 days (100 mg of the antioestrogen once a day) and a single dose of hCG (5000 IU im) was given at the beginning of the experiment. In group 2 (n = 9), the treatment was started 7 days prior to the hCG injection and was continued for additional 6 days. In both groups peripheral blood samples were collected up to 6 days after hCG, and the sera were analysed for FSH, prolactin and 8 steroids. In group 1, the steroidogenic response was identical to that found previously in untreated men. In group 2, the 7-day treatment with clomiphene citrate led to elevated serum concentrations of LH, FSH, pregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and oestradiol. When compared with these elevated values, the response of serum 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione and testosterone to hCG were diminished. The ratios of the steroid concentrations support previous reports that hCG-induced inhibition of 17-hydroxylase, 17--20 desmolase and 3 beta-hydroxysteroid dehydrogenase-delta 4-5 isomerase is decreased during antioestrogen administration. This further substantiates the idea of a central role for endogenous testicular oestradiol in the mediation of steroidogenic lesions following acute large doses of hCG.