Increased Testosterone Production Research Articles

Hormonal and cellular brain mechanisms regulating the amplexus of male and female water frog (Rana esculenta).

The role of nitric oxide (NO) synthase, prostaglandin E2-9-ketoreductase, and aromatase brain activities in regulating frog amplexus was assessed in the water frog (Rana esculenta). Plasma concentrations of testosterone were higher, and concentrations of 17beta-oestradiol lower, in amplexing males than in unamplexing males; while concentrations of testosterone and PGE2 were lower, and those of 17beta-oestradiol and PGF2alpha higher, in amplexing females compared to unamplexing females. Hormone release rescued from frog brains in vitro mirrored plasma hormone measures. Brain aromatase activity was lower in amplexing males; NO synthase was lower and PGE2-9-ketoreductase and aromatase were higher in amplexing females. In male brains, PGE2-9-ketoreductase inhibitor decreased PGF2alpha release and increased that of PGE2; aromatase inhibitor decreased 17beta-oestradiol and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor increased testosterone and PGE2 release and decreased that of 17beta-oestradiol and PGF2alpha; NO synthase inhibitor decreased testosterone release and PGE2 and increased 17beta-oestradiol and PGF2alpha release; PGF2alpha decreased testosterone release and increased 17beta-oestradiol release; aromatase inhibitor decreased 17beta-oestradiol release and increased testosterone release. In female brains, NO donor and PGE2-9-ketoreductase inhibitor decreased PGE2-9-ketoreductase and aromatase activities; PGF2alpha increased aromatase activity; NO synthase inhibitor increased PGE2-9-ketoreductase and aromatase activity. The data suggest that, in amplexing female brains, external and/or internal stimuli inhibit NO synthase, decreasing NO and activating PGE2-9-ketoreductase; in turn, PGF2alpha increases aromatase activity and 17beta-oestradiol release; while, in amplexing male brains, stimuli inhibit aromatase activity, thereby increasing testosterone production.

Stimulation of steroidogenic acute regulatory protein (StAR) gene expression by d-aspartate in rat Leydig cells

d-aspartate and human chorionic gonadotropin act synergistically to increase testosterone production in purified rat Leydig cells, and d-aspartate stimulates testosterone synthesis even in the absence of human chorionic gonadotropin stimulation. In addition, d-aspartate enhances steady-state cellular mRNA and protein levels of steroidogenic acute regulatory protein, which is a key regulatory factor in gonadal and adrenal steroidogenesis, d-aspartate therefore appears to increase testosterone production in rat Leydig cells by stimulating steroidogenic acute regulatory protein gene expression. To our knowledge, this is the first report demonstrating a direct effect of d-aspartate on gene expression in mammalian cells.

Isolation method of Leydig cells from mature male Djungarian hamsters (Phodopus sungorus) and their steroidogenic activityin vitro

For studies addressing the functions of Leydig cells, isolated cells are often better suited than intact animals. Here, the isolation procedure of Leydig cells from adult male Djungarian hamsters (Phodopus sungorus) is described. Cells were isolated using a procedure involving enzymatic dissociation and Percoll-gradient centrifugation. For each experiment, approximately 4.4 x 10(6) Leydig cells from six animals were obtained. The cells showed high steroidogenic responsiveness to physiological (ovine luteinizing hormone (oLH) and human chorionic gonadotropin (hCG)) and nonphysiological (forskolin) stimuli in vitro. Approximately 98% of cells were viable as assessed by trypan blue exclusion, and the purity varied from 80 to 95% as tested by 3 beta-hydroxysteroid dehydrogenase activity. Leydig cells were also identified by a bright yellow halo under phase-contrast microscopy. They contained numerous lipid droplets and showed round nuclei and prominent nucleoli. The cells responded to oLH, hCG and forskolin with an increased testosterone production in a dose-dependent manner. Dose-response curves in these studies suggest that Leydig cells of Djungarian hamsters undergo desensitization, probably due to down regulation of their LH/CG receptors.

Molecular Mechanisms of Thyroid Hormone-stimulated Steroidogenesis in Mouse Leydig Tumor Cells: INVOLVEMENT OF THE STEROIDOGENIC ACUTE REGULATORY (StAR) PROTEIN

Using a mouse Leydig tumor cell line, we explored the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis. Triiodothyronine (T3) induced a approximately 3.6-fold increase in the steady-state level of StAR mRNA which paralleled with those of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production was effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displayed the highest affinity of [125I]iodo-T3 binding and was most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increased testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression were similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) showed marginal elevation of StAR expression, but coordinately increased T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1 markedly diminished the SF-1 mRNA expression, and concomitantly abolished T3-mediated responses. Noteworthy, T3 augmented the SF-1 mRNA expression while inhibition of the latter by DAX-1 strongly impaired T3 action. Northern hybridization analysis revealed four StAR transcripts which increased 3-6-fold following T3 stimulation. These observations clearly identified a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis.

Recombinant Human Follicle-Stimulating Hormone Administration Increases Testosterone Production in Men, Possibly by a Sertoli Cell-Secreted Nonsteroid Factor

Recombinant Human Follicle-Stimulating Hormone Administration Increases Testosterone Production in Men, Possibly by a Sertoli Cell-Secreted Nonsteroid Factor

Does age-associated reduced Leydig cell testosterone production in Brown Norway rats result from under-stimulation by luteinizing hormone?

Previous studies have shown that reductions in Leydig cell testosterone production occur with aging in the Brown Norway rat. The recent observation that changes in luteinizing hormone (LH) pulse interval and amplitude also occur with aging suggests the possibility that age-related reduced Leydig cell steroidogenesis might be related to changes in LH. We reasoned that, if this is the case, exogenously administered LH should restore testosterone production by aged rat Leydig cells to the higher levels produced by Leydig cells of young rats. To test this hypothesis, young (4-month-old) and aged (21-month-old) rats received testosterone- and estradiol-containing Silastic implants designed to suppress LH and, thus, endogenous Leydig cell testosterone production. At the same time, the rats received miniosmotic pumps programmed to deliver pulsatile ovine LH at a predetermined daily dose. In some experiments, treatment effects were determined by measuring testosterone production by testes perfused in vitro with maximally stimulating ovine LH. In others, Leydig cells were isolated by centrifugal elutriation and Percoll density gradient centrifugation, and their in vitro ability to produce testosterone in response to maximally stimulating LH was determined. Testes or isolated Leydig cells from untreated young rats produced about twice as much testosterone as that produced by Leydig cells from aged rats. The administration of testosterone- and estradiol-filled implants for 5 days reduced testosterone production significantly at both ages. In young rats administered 24 microg LH/day for 5 days, along with the implants, testosterone production was maintained at the high level of the young controls. Comparable treatment of aged rats resulted in testosterone production only at the low level of the aged controls. Indeed, even with higher LH doses (36 microg/day), testosterone production by the aged rat Leydig cells did not rise above the aged-control level. The inability of exogenously administered LH to increase testosterone production by testes and Leydig cells of aged rats suggests that Leydig cell steroidogenic deficits in the aged Brown Norway rat are unlikely to be the result of age-related changes in LH.

Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats.

We examined the temporal relationship between protein synthesis and testosterone production by rat Leydig cells in primary culture. Leydig cells were isolated from adult control Sprague-Dawley rats and from rats that had received LH-suppressive testosterone and estradiol (TE) implants in vivo for 10 days. The cells were incubated for 1-4 h with [35S]methionine in the presence or absence of maximally stimulating ovine LH, and newly synthesized proteins were examined by two-dimensional PAGE autoradiography. Approximately 800-900 newly synthesized polypeptides were readily visible on all autoradiograms, most of which did not differ in the cells from intact control and TE-treated rats. Incubation of cells from the control and treated rats with maximally stimulating LH for 4 h in both cases resulted in significant increases in testosterone production and in three newly synthesized polypeptides. These polypeptides, along with two others that changed little in response to LH, were similar in apparent molecular mass, 30 kDa, but differed in isoelectric point. Time-course studies revealed a temporal relationship between stimulation of the three 30-kDa proteins and of testosterone production. Western blot analysis identified the 30-kDa proteins as steroidogenic acute regulatory protein (StAR). The results of these studies, for the first time utilizing primary cultures of highly purified, testosterone-producing Leydig cells, provide further correlative evidence of a role for StAR protein in the acute regulation of Leydig cell testosterone biosynthesis by LH.

Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages.

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.

Effects of thyroid hormone (thyroxine) and testosterone on hepatic 11beta-hydroxysteroid dehydrogenase mRNA and activity in pubertal hypothyroid male rats.

To investigate the effects of thyroid hormone and testosterone on 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), we measured changes in hepatic 11beta-dehydrogenase activity and its mRNA levels in pubertal methimazole (MMI)-induced hypothyroid male rats following treatment with thyroxine ([T4] 50 microg/kg/d) or testosterone (250 microg/d) for 14 days. Hypothyroidism in male rats markedly reduced hepatic 11beta-HSD1 mRNA levels and serum testosterone concentrations (P < .01). Subcutaneous injection of T4 in the hypothyroid rats significantly (P < .01) increased hepatic 11beta-HSD1 mRNA to approximately normal levels and simultaneously increased serum testosterone levels. However, the same daily dose of T4 administered to castrated male hypothyroid rats for 14 days did not elevate hepatic 11beta-HSD1 activity. Treatment with testosterone for 14 days in castrated hypothyroid male rats and rats without gonadectomy significantly (P < .01) increased the enzyme activity without administration of T4. Variations in hepatic 11beta-HSD1 activity were demonstrated to be accompanied by changes in serum testosterone levels in the rats following alteration of the thyroid hormone state. These results suggest that the effect of T4 in increasing the subnormal 11beta-HSD1 gene expression in hypothyroid male rats is mediated by its ability to increase testosterone production in these rats, because in castrated hypothyroid rats, T4 does not elevate 11beta-HSD1 gene expression.

Increased testosterone production in vitro by Leydig cells from rats with severe autoimmune orchitis.

We have previously observed (M. O. Suescun et al., 1994, Journal of Andrology, 15, 442-448) that rats with autoimmune orchitis (EAO) exhibit increased testosterone production in vitro by isolated testes. The aim of the present study was to determine whether the increase in testosterone production correlated with an enhanced number of Leydig cells and/or enhanced steroidogenic capacity per Leydig cell. For this purpose, EAO was induced in adult Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. At 80 days after the primary immunization, 60% of rats presented with severe testicular damage characterized by sloughing of the seminiferous epithelium, seminiferous tubule atrophy and interstitial mononuclear cell infiltration. At 160 days after the first immunization, testicular lesions were more severe. A morphometric study, by light microscopy, showed an increase in the number of Leydig cells in rats with EAO (45% increase at 80 days and 50% at 160 days). By electronmicroscopy, testicular sections of rats with EAO revealed the presence of numerous Leydig cells closely associated with macrophages. Most Leydig cells exhibited ultrastructural features of active steroid secreting cells. The steroidogenic capacity of Percoll-purified Leydig cells from tests of rats with EAO, killed at 80 and 160 days, was evaluated. Leydig cells from rats with EAO exhibited an enhanced steroidogenic response to hCG in vitro at 80 days (38%) and an increase in basal (77%) and post-hCG testosterone production (115%) at 160 days compared to controls. However, these cells were less sensitive to hCG. In conclusion, the results indicate that the enhancement of in-vitro testosterone production observed in rats with EAO is accounted for both by the increased number of Leydig cells and by the increased testosterone production of each Leydig cell.

Local Effect of PACAP and VIP on Testicular Function in Immature and Adult Rats

Csaba, Zs., V. Csernus, and I. Gerendai. Local effect of PACAP and VIP on testicular function in immature and adult rats. Peptides 18(10) 1561–1567, 1997.—PACAP, VIP, anti-PACAP and anti-VIP antisera were injected intratesticularly. In 9-day-old hemicastrated rats PACAP or VIP decreased basal testosterone secretion. In 22-day-old hemicastrates VIP but not PACAP reduced compensatory testicular hypertrophy, however, neither PACAP nor VIP altered steroidogenesis. Anti-VIP antiserum to this age group increased testosterone production and enhanced compensatory testicular hypertrophy. In adult hemicastrates neither the peptides nor the antisera influenced steroidogenesis. Neither in immatures nor in adults treatment of both testes with PACAP or VIP had any effect. Data indicate that both PACAP and VIP might exert a local action on testicular steroidogenesis, on compensatory testicular hypertrophy, and these effects are age-dependent.

Purification and characterization of a lipophilic factor from testicular macrophages that stimulates testosterone production by Leydig cells.

Testicular macrophages have been shown to secrete a factor that stimulates testosterone production by Leydig cells. The purpose of this investigation was to purify and characterize this factor. Medium was collected from 24- to 48-hour cultures of testicular macrophages isolated from adult rats. This medium induced a sevenfold increase in testosterone production by cultured Leydig cells. When the medium was extracted with ether, all biological activity was found in the organic phase, indicating that the factor was lipophilic. The ether extract was then fractionated on a C18 reversed-phase high-performance liquid chromatography (HPLC) column, using a gradient of acidified methanol as the mobile phase. Leydig cell-stimulating activity eluted at approximately 11 minutes. Standards of testosterone, dihydroepiandrosterone (DHEA), pregnenolone, progesterone, dihydrotestosterone (DHT), and prostaglandin E2 (PGE2) all had elution times of between 5 and 6 minutes, under identical column conditions. The biological activity of the HPLC-purified fraction was partly resistant to boiling but was completely abolished by Dextran-coated charcoal treatment. Biological activity of testicular macrophage-conditioned medium was not abolished following chymotrypsin treatment, indicating that this molecule was not a hydrophilic peptide. It was found that the factor obtained by reversed-phase HPLC could be further purified by normal-phase HPLC. The results of this investigation demonstrate that the testicular macrophage-derived factor that stimulates testosterone production by Leydig cells can be purified by organic extraction and HPLC, and that it is a highly potent chymotrypsin-resistant heat-stable lipophilic factor.

Evidence for Germ Cell Control of Sertoli Cell Function in Three Models of Germ Cell Depletion in Adult Rat

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight [TW]: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.

Effects of Ammonium Perfluorooctanoate on Leydig-Cell Function: In Vitro, in Vivo, and ex Vivo Studies

Ammonium perfluorooctanoate (C8) produced a dose-dependent increase in Leydig cell adenomas in Crl:CD BR (CD) rats fed 0, 30, or 300 ppm for 2 years. Administration of C8 to adult male CD rats, by gavage for 14 days, produced decreased serum and testicular interstitial fluid testosterone levels and increased serum estradiol levels. The C8-mediated decrease in the serum testosterone levels appeared to be due to a lesion at the level of the testis. These endocrine changes may play a role in the C8 induction of Leydig cell tumors. In the present work, C8 was examined for its ability to (1) directly affect Leydig cells in vitro using isolated Leydig cells from untreated rats and ex vivo using Leydig cells isolated from C8-treated rats, (2) affect testicular interstitial fluid hormone levels, and (3) induce aromatase activity. These studies were conducted to investigate the hypothesis that C8 produces an increase in estradiol by inducing cytochrome P450 XIX (aromatase), which converts testosterone to estradiol, and that the elevated estradiol levels ultimately produce Leydig cell hyperplasia and adenoma formation by acting as a mitogen or enhancing growth factor secretion. In the in vivo and ex vivo studies, adult male CD rats were gavaged with either 0 or 25 mg/kg/day C8 for 14 days. In addition to the ad libitum control, a second control group was pair-fed to the 25 mg/kg/day C8 group. In the in vitro and ex vivo studies, Leydig cells were isolated from testes of adult males by collagenase digestion followed by enrichment over Percoll gradients. A dose-dependent decrease in testosterone levels was observed in hCG-stimulated Leydig cells treated in vitro with C8 for 5 hr (IC50 approximately 200 μM). In contrast, ex vivo studies demonstrated an increase in testosterone production in hCG-stimulated Leydig cells from C8-treated rats when compared to Leydig cells isolated from either the ad libitum or pair-fed control rats. The in vitro data demonstrate that C8 directly inhibits testosterone release from Leydig cells, while the ex vivo data demonstrate that this inhibition is reversible. In the in vivo study, serum and testicular interstitial fluid estradiol concentrations were significantly higher in the C8-treated rats when compared to the pair-fed control group. In addition, testicular interstitial fluid transforming growth factor α (TGF α) levels were also increased, consistent with the hypothesis that estradiol may modulate growth factor expression within the testis. In a follow-up in vivo study, C8 produced a 4.5-fold increase in hepatic aromatase when compared to the pair-fed control, but did not increase aromatase activity in the testis, muscle, or fat. Collectively, these data suggest that the increase in serum estradiol levels from C8-treated rats is primarily due to aromatase induction in the liver.

A novel mutation of the luteinizing hormone receptor gene causing male gonadotropin-independent precocious puberty.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant gonadotropin-independent disorder. Affected males generally develop signs of precocious puberty in early childhood. They typically show Leydig cell hyperplasia and increased testosterone production typical for their age, whereas circulating LH concentrations remain prepubertal. Several dominant point mutations of the LH receptor gene were identified in pedigrees with familial male-limited precocious puberty and were shown to cosegregate with the disease. Here we report a novel heterozygote point mutation in the LH receptor gene of a Brazilian boy with gonadotropin-independent precocious puberty. This mutation substitutes alanine 568 with valine at the carboxyterminus of the third cytosolic loop of the LH receptor. The unoccupied mutant receptors confer constitutive activation of adenyl cyclase activity when expressed in COS-7 cells, resulting in 4-fold higher cAMP concentrations over baseline compared with cells expressing an equivalent number of wild-type receptors. The affinity of the mutant receptors to 125I-labeled human LH was not altered compared with the wild type. Mutations of the homologue alanine residue in the alpha 1-adrenergic (in vitro), FSH (in vitro), and TSH (naturally occurring) receptors also result in constitutive adenyl cyclase activation, suggesting that this alanine residue is crucial for signal transduction and a potential site for upregulatory/oncogenic mutations in G-protein coupled receptors.

Effect of Oxytocin on Testosterone Production by Isolated Rat Leydig Cells is Mediated Via a Specific Oxytocin Receptor1

The effect of the neurohypophysial hormones oxytocin and arginine vasopressin (AVP) on testicular steroidogenesis was reevaluated by use of short-term (< 10 h) cultures of isolated adult rat Leydig cells. Oxytocin at 10(-9), 10(-7), and 10(-5) M concentrations significantly increased basal testosterone production in a dose-dependent manner but had no effect on LH-stimulated testosterone production. The specificity of the effect was determined by use of the specific oxytocin receptor antagonist (OTA). OTA from 10(-9) to 10(-5) M concentrations inhibited the oxytocin-stimulated increase in testosterone production. Furthermore, the oxytocin agonist Thr4 Gly7 oxytocin also induced a dose-dependent increase in basal testosterone production. In contrast, AVP from 10(-9) to 10(-5) M concentrations did not consistently affect basal testosterone production by isolated Leydig cells, but significantly decreased LH-stimulated testosterone production. Inclusion of 10(-7) and 10(-5) M OTA with 10(-7) M AVP did not alter the inhibitory effect of the AVP. These data show that oxytocin and AVP have different effects on testosterone production by Leydig cells in vitro and support the hypothesis that oxytocin acts in the testis through a specific oxytocin receptor.

HORMONAL PROFILES IN WOMEN WITH BREAST CANCER

The literature findings on endogenous hormonal profiles in women with breast cancer are reviewed in detail. It is concluded that four sets of findings are valid: (1) diminished adrenal androgen production, probably genetic, in women with premenopausal breast cancer; (2) ovarian dysfunction (luteal inadequacy plus increased testosterone production) in breast cancer at all ages; (3) increased 16 alpha-hydroxylation of estradiol in breast cancer at all ages; and (4) evidence that prolactin is a permissive risk factor for breast cancer, and that the pregnancy-induced decrease in prolactin levels may account for the protective effect of early pregnancy against breast cancer.

The effects of fetal serum and gonadotropins on testosterone production by cultured chick testicular cells

This report describes the influences of fetal serum and gonadotropins on the development and function of chick testicular cells in a culture system that allows long term control of culture conditions. Testis of new born male chicks were dissociated and cultured in different conditions and the production of testosterone measured by RIA. 1. 1.|To investigate the effect of serum the cells were wither cultured in a defined medium composed by Dulbecco's modified medium (DMM) plus 0.1% albumin or media containing DMM plus 10% fetal bovine serum (FBS). The presence of serum increased protein content of the cultures, but no differences were found in basal testosterone production. When hCG (2IU/ml) was added to both culture media, the increment in testosterone production hence produced, was greater when cells were cultured in defined medium. 2. 2.|Cells were precultured for 24 hr in either defined serum media with or without hCG. The media were then replaced with DMM plus BSA and 1-methyl 3-isobutyl xanthine (IBMX) in the presence or absence of hCG and cells further incubated for 2 hr. Addition of hCG increased testosterone production by cells cultured in defined medium and enhanced its subsequent response to hCG stimulation. Cells cultured in serum medium maintained their ability to respond to hCG, but the addition of this hormone to the medium, abolished the response to further hCG-stimulation. 3. 3.|hCG caused a dose-related increased in testosterone production by cells cultured in defined and serum containing media, but concentrations of hCG above 2 IU/ml depressed testosterone production in the latter group. 4. 4.|Cells cultured in defined medium were incubated for 6 hr either with or without FSH of different purity grade. Both preparations stimulated the production of testosterone, the pure one being less effective than the LH-contaminated one. 5. 5.|Cells cultured in defined medium were incubated for 6 hr either with or without FSH and further stimulated with hCG (2 IU/ml). A significant increment in the response to hCG was observed when cells had been exposed to FSH. Present results show that FBS influences the function and development of chick testis cells in culture. Since both FSH and hCG modified testosterone secretion, a role for gonadotropins in the functional capacity of neonatal Leydig cells is also suggested.

Stimulatory and inhibitory factors of Leydig cell steroidogenesis are secreted simultaneously by the rat seminiferous tubules and do not affect Leydig cell inhibin production in vitro.

The effect of conditioned medium from rat seminiferous tubules (at Stages VII-VIII and Stages IX-VI) cultured with or without follicle-stimulating hormone (FSH) on the production of testosterone and immunoactive inhibin by Leydig cells was examined. Low doses of conditioned medium from unstimulated tubules at Stages VII-VIII significantly (P < 0.05) increased the mean testosterone production to greater than 31 +/- 11% over that achieved with luteinizing hormone (LH) alone. At the highest dose, the conditioned medium significantly inhibited (P < 0.05) LH-stimulated testosterone production by 13 +/- 7%. Low doses of conditioned medium from unstimulated tubules at Stages IX-VI increased the mean testosterone production to 22 +/- 10%, whereas at higher doses, a significant reversal in the stimulation occurred although not to the same extent as that found with medium from tubules at Stages VII-VIII. Conditioned medium from FSH-stimulated tubules at Stages VII-VIII and Stages IX-VI, significantly increased testosterone production to 39 +/- 7% and 31 +/- 13% respectively. Immunoactive inhibin production by the Leydig cells remained unaffected by exposure to conditioned medium from FSH stimulated and unstimulated tubules at Stages VII-VIII and Stages IX-VI. The data demonstrate that tubule culture medium contains FSH-modulated activities which can specifically stimulate and inhibit testosterone synthesis by adult rat Leydig cells in vitro and therefore explains the contradictory reports in the literature.

Differential regulation of steroidogenic enzymes during differentiation optimizes testosterone production by adult rat Leydig cells

The postnatal differentiation of rat Leydig cells may be subdivided into three stages based on morphology and steroid production. The purpose of this study was to clarify the developmental mechanisms underlying increased testosterone production by measuring steady state levels of the mRNAs for three steroidogenic enzymes in isolated Leydig cells at each stage of differentiation. These include Leydig cell progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90. The steroidogenic enzymes were 1) cholesterol side-chain cleavage enzyme (CSCC), 2) 17 alpha-hydroxylase (P450-17 alpha), and 3) 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD). We report that levels of CSCC and P450-17 alpha mRNAs increase, whereas 3 alpha HSD mRNA levels decline during the course of Leydig cell differentiation. The levels of 3 alpha HSD mRNA were high in progenitor Leydig cells that appeared to contain little smooth endoplasmic reticulum and decreased in cells as smooth endoplasmic reticulum developed and other enzyme mRNAs increased. These observations suggest that the factors that regulate 3 alpha HSD mRNA levels are startlingly different from those that regulate the mRNA levels of CSCC and P450-17 alpha. We conclude that the progressive increase in the capacity of differentiating Leydig cells to produce testosterone can be explained in part by an increase in the activity of enzymes that synthesize testosterone (CSCC and P450-17 alpha) and a decrease in the activity of an enzyme that metabolizes testosterone and its precursors (3 alpha HSD).