Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo.

BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.

Peri-pubertal exposure to testicular hormones organizes response to novel environments and social behaviour in adult male rats.

Previous research has shown that exposure to testicular hormones during the peri-pubertal period of life has long-term, organizational effects on adult sexual behaviour and underlying neural mechanisms in laboratory rodents. However, the organizational effects of peri-pubertal testicular hormones on other aspects of behaviour and brain function are less well understood. Here, we investigated the effects of manipulating peri-pubertal testicular hormone exposure on later behavioural responses to novel environments and on hormone receptors in various brain regions that are involved in response to novelty. Male rodents generally spend less time in the exposed areas of novel environments than females, and this sex difference emerges during the peri-pubertal period. Male Lister-hooded rats (Rattus norvegicus) were castrated either before puberty or after puberty, then tested in three novel environments (elevated plus-maze, light-dark box, open field) and in an object/social novelty task in adulthood. Androgen receptor (AR), oestrogen receptor (ER1) and corticotropin-releasing factor receptor (CRF-R2) mRNA expression were quantified in the hypothalamus, hippocampus and medial amygdala. The results showed that pre-pubertally castrated males spent more time in the exposed areas of the elevated-plus maze and light-dark box than post-pubertally castrated males, and also confirmed that peri-pubertal hormone exposure influences later response to an opposite-sex conspecific. Hormone receptor gene expression levels did not differ between pre-pubertally and post-pubertally castrated males in any of the brain regions examined. This study therefore demonstrates that testicular hormone exposure during the peri-pubertal period masculinizes later response to novel environments, although the neural mechanisms remain to be fully elucidated.

Ginkgo biloba supplement abates lead-induced endothelial and testicular dysfunction in Wistar rats via up-regulation of Bcl-2 protein expression, pituitary-testicular hormones and down-regulation of oxido-inflammatory reactions.

BACKGROUND: Apoptotic and oxido-inflammatory pathways have been found to be up-regulated in lead acetate poisoning which has been associated to endothelial and testicular dysfunctions. It is yet uncertain, nevertheless, if treatment with Ginkgo biloba supplements (GBS), a flavonoid-rich natural product can lessen the adverse effects of lead on endothelial and testicular functions. This study investigated the impact of Ginkgo biloba supplementation on lead-induced endothelial and testicular dysfunctions. METHODS: The animals were treated with GBS (50 mg/kg and 100 mg/kg orally) for 14 days following oral exposure to lead acetate (25 mg/kg) for 14 days. After euthanasia, blood samples, epididymal sperm, testes, and aorta were collected. The quantities of the hormones (testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH), as well as the anti-apoptotic, oxidative, nitrergic, inflammatory markers, were then determined using immunohistochemistry, ELISA, and conventional biochemical methods. RESULTS: GBS reduced lead-induced oxidative stress by increasing the levels of the antioxidant enzymes catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD), while lowering malondialdehyde (MDA) in endothelium and testicular cells. Normal testicular weight was restored by GBS which also decreased endothelial endothelin-I and increased nitrite levels. TNF-α and IL-6 were decreased while Bcl-2 protein expression was enhanced. Lead-induced alterations in reproductive hormones (FSH, LH, and testosterone) were also restored to normal. CONCLUSION: According to our result, using Ginkgo biloba supplement prevented lead from causing endothelial and testicular dysfunction by raising pituitary-testicular hormone levels, boosting Bcl-2 protein expression and lowering oxidative and inflammatory stress in the endothelium and testes.

Members of the murine Pate family are predominantly expressed in the epididymis in a segment-specific fashion and regulated by androgens and other testicular factors.

BACKGROUND: Spermatozoa leaving the testis are not able to fertilize the egg in vivo. They must undergo further maturation in the epididymis. Proteins secreted to the epididymal lumen by the epithelial cells interact with the spermatozoa and enable these maturational changes, and are responsible for proper storage conditions before ejaculation. The present study was carried out in order to characterize the expression of a novel Pate (prostate and testis expression) gene family, coding for secreted cysteine-rich proteins, in the epididymis. METHODS: Murine genome databases were searched and sequence comparisons were performed to identify members of the Pate gene family, and their expression profiles in several mouse tissues were characterized by RT-PCR. Alternate transcripts were identified by RT-PCR, sequencing and Northern hybridization. Also, to study the regulation of expression of Pate family genes by the testis, quantitative (q) RT-PCR analyses were performed to compare gene expression levels in the epididymides of intact mice, gonadectomized mice, and gonadectomized mice under testosterone replacement treatment. RESULTS: A revised family tree of Pate genes is presented, including a previously uncharacterized Pate gene named Pate-X, and the data revealed that Acrv1 and Sslp1 should also be considered as members of the Pate family. Alternate splicing was observed for Pate-X, Pate-C and Pate-M. All the Pate genes studied are predominantly expressed in the epididymis, whereas expression in the testis and prostate is notably lower. Loss of androgens and/or testicular luminal factors was observed to affect the epididymal expression of several Pate genes. CONCLUSIONS: We have characterized a gene cluster consisting of at least 14 expressed Pate gene members, including Acrv1, Sslp1 and a previously uncharacterized gene which we named Pate-X. The genes code for putatively secreted, cysteine-rich proteins with a TFP/Ly-6/uPAR domain. Members of the Pate gene cluster characterized are predominantly expressed in the murine epididymis, not in the testis or prostate, and are regulated by testicular factors. Similar proteins are present in venoms of several reptiles, and they are thought to mediate their effects by regulating certain ion channels, and are thus expected to have a clinical relevance in sperm maturation and epididymal infections.

Testicular hormones mediate robust sex differences in impulsive choice in rats.

Impairments in choosing optimally between immediate and delayed rewards are associated with numerous psychiatric disorders. Such 'intertemporal' choice is influenced by genetic and experiential factors; however, the contributions of biological sex are understudied and data to date are largely inconclusive. Rats were used to determine how sex and gonadal hormones influence choices between small, immediate and large, delayed rewards. Females showed markedly greater preference than males for small, immediate over large, delayed rewards (greater impulsive choice). This difference was neither due to differences in food motivation or reward magnitude perception, nor was it affected by estrous cycle. Ovariectomies did not affect choice in females, whereas orchiectomies increased impulsive choice in males. These data show that male rats exhibit less impulsive choice than females and that this difference is at least partly maintained by testicular hormones. These differences in impulsive choice could be linked to gender differences across multiple neuropsychiatric conditions.

Mullerian-inhibiting substance induces Gro-beta expression in breast cancer cells through a nuclear factor-kappaB-dependent and Smad1-dependent mechanism.

Mullerian-inhibiting substance (MIS), a transforming growth factor-beta family member, activates the nuclear factor-kappaB (NF-kappaB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-beta. Inhibiting NF-kappaB activation with a phosphorylation-deficient IkappaBalpha mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-beta promoter-driven reporter expression and Gro-beta mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-beta mRNA, suggesting that regulation of Gro-beta expression by MIS was dependent on activation of NF-kappaB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of kappaB-binding sequences within Gro-beta, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-kappaB complex bound to the kappaB site of the Gro-beta promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-beta promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-kappaB signaling in which enhancement of NF-kappaB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the kappaB consensus site within the promoter.

Recombinant human mullerian inhibiting substance inhibits human ocular melanoma cell lines in vitro and in vivo.

Since Mullerian Inhibiting Substance (MIS) causes regression of the Mullerian duct, the anlagen of the uterus, vagina, and fallopian tube, we expected and have previously observed that purified recombinant human MIS causes regression of gynecological tumors. However, recent experiments indicating that neural crest derivatives might be responsive to MIS prompted study of a group of human ocular melanoma cell lines in 4 in vitro inhibition assays, and a subrenal capsule assay in vivo. Ocular melanoma cell lines that grew well in a respective assay were studied with MIS to determine whether this biological modifier could inhibit growth. Three human ocular melanomas, OM431 (P less than 0.01), OM467 (P less than 0.02), and OM482 (P less than 0.03), were growth-inhibited by highly purified human recombinant MIS in soft agarose. A dose-dependent tumor inhibition was noted when OM431 cells were incubated with MIS in a liquid colony inhibition assay (P less than 0.05). In addition, OM467 was inhibited (P less than 0.05) by MIS in a multicellular tumor spheroid assay. Cell cycle analysis indicated that OM431 cells were inhibited in monolayer by MIS while in G1. At 100-fold lower serum concentrations than required in the media of in vitro assays, MIS delivered via i.p. osmotic pumps inhibited (P less than 0.05) in vivo the growth of OM431 implanted beneath the renal capsule of nude and CD-1 irradiated mice when compared to mice given implants of pumps containing no MIS. The responsiveness of ocular melanoma to MIS broadens the spectrum of tumors that might be treated with MIS and suggests further investigation of other neural crest tumors.

Minimal antiproliferative effect of recombinant müllerian inhibiting substance on gynecological tumor cell lines and tumor explants.

Múllerian Inhibiting Substance (MIS) is a testicular hormone that promotes involution of the Múllerian duct during embryogenesis. The Múllerian duct gives rise to adult female reproductive ducts including the fallopian tubes, uterus, and upper vagina. Thus, testicular MIS ensures the regression of female sex organ primordia. Partially purified bovine MIS was reported to inhibit proliferation of tumor cells derived from human gynecological cancers. These observations suggest that MIS might be an effective anticancer agent for some human tumors. Recombinant human MIS (rHu-MIS) has recently become available. To assess the antiproliferative activity of rHu-MIS, we examined its effects on 11 ovarian, six endometrial, and two nongynecological human tumor cell lines. rHu-MIS had no effect on proliferation of these cell lines in five independent assays. Forty-three primary human tumor explants were also examined in human tumor colony forming assays, gel-supported primary culture assays, and subrenal capsule assays. rHu-MIS significantly inhibited the growth of five of these tumors including four ovarian and one small cell lung cancer explant. The four ovarian cancer responses include three of 13 (23%) explants tested in human tumor colony-forming assays and one of eight (12.5%) explants tested in gel-supported primary culture assays. We conclude that rHu-MIS may have antiproliferative activity against some human ovarian cancers.

Human müllerian inhibiting substance inhibits tumor growth in vitro and in vivo.

Müllerian inhibiting substance (MIS) causes regression of the müllerian duct in the male fetus. Bovine MIS has been reported to inhibit the growth of some gynecological tumors. Recombinant human MIS (rhMIS) produced in transfected Chinese hamster ovary cells has been highly purified by immunoaffinity chromatography. The introduction of a salt wash prior to elution of MIS from the affinity column removes a growth-stimulating factor(s) derived from Chinese hamster ovary cells. This immunopurified rhMIS caused significant inhibition (34-59% survival) of A431 (a vulvar epidermoid carcinoma), HT-3 (a cervical carcinoma), HEC-1-A (an endometrial adenocarcinoma), NIH:OVCAR-3 (an ovarian adenocarcinoma), and OM431 (an ocular melanoma) human cell lines in colony inhibition assays. Two cell lines, Hep 3B (a hepatocellular carcinoma) and RT4 (a bladder transitional cell papilloma), were unresponsive to immunopurified rhMIS. Using an in vivo subrenal capsule assay in irradiated CD-1 mice, the growth of A431 and OM431 cells was inhibited by immunopurified rhMIS. We conclude that rhMIS inhibits the growth of certain tumor cell lines in vitro and in vivo.

Anti-Müllerian hormone: its role in follicular growth initiation and survival and as an ovarian reserve marker.

In this paper the role in the ovary of anti-Müllerian hormone (AMH), a member of the transforming growth factor-beta family of growth and differentiation factors, is reviewed. AMH has an inhibitory effect on primordial follicle recruitment and may also inhibit follicle-stimulating homone-dependent selection of follicles for dominance. In addition to its functional role in the ovary, AMH in serum is an excellent candidate marker as an indication of the ovarian reserve, not only in infertility clinic patients but also in women during and after cancer treatment.

Anti-Müllerian hormone initiates growth of human primordial follicles in vitro.

Survival and growth of follicles in human ovarian tissue is presently only performed with limited success. We evaluated the effect of anti-Müllerian hormone (AMH) and/or testosterone on follicular growth during a 4-week culture period using ovarian cortical tissue from six women in their reproductive years. The cortex of each biopsy was isolated and immediately cryopreserved upon collection and stored in liquid nitrogen. After thawing the tissue was placed in culture. After the culture period all follicles were counted on histological sections and classified for viability and stage of development. Based on evaluation of 6603 follicles it was found that the number of growing follicles significantly increased during the culture period as compared to the uncultured control, irrespective of the composition of the culture medium. Furthermore, significantly more follicles advanced to the primary and secondary stage (p<0.05) in tissue cultured with AMH (54%) as compared to tissue cultured in control medium (41%). The mean diameter of follicles classified as primary follicles was significantly enhanced in tissue cultured in the presence of AMH (p=0.002) and AMH plus testosterone (p<0.001) as compared to that observed in tissue cultured with control medium and medium containing testosterone alone. In contrast the mean diameter of the oocyte and its nucleus remained similar irrespective of culture medium. In conclusion, AMH seems to affect early stages of human follicular development by enhancing recruitment, survival and/or growth during a 4-week culture period.

Jost's hormone inhibitrice comes of age.

The First International Workshop on Anti-Müllerian hormone/Müllerian inhibiting substance was held in Aix-en-Provence, France, from 7-8 October 2002.

Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.

Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.

Regulation of Sertoli cell differentiation by the testicular paracrine factor PModS: potential role of immediate-early genes.

Testicular peritubular cells produce a paracrine factor, PModS, under androgen control that modulates Sertoli cell functions that are essential for the process of spermatogenesis. PModS has a more dramatic effect on Sertoli cell differentiated functions in vitro than any regulatory agent previously shown to influence the cell, including FSH. Investigation of the actions of PModS on a molecular level have used transferrin expression as a marker of Sertoli cell differentiation. PModS was found to stimulate transferrin gene expression while having no effect on transferrin mRNA stability. The ability of PModS to elevate transferrin mRNA levels was inhibited by cycloheximide. Therefore, the actions of PModS require ongoing protein synthesis and appear to be indirectly mediated through trans-acting early event genes. PModS was found to dramatically increase mRNA levels for c-fos, but had no effect on c-jun mRNA levels. The c-fos mRNA levels increased transiently within a few minutes to a maximal level of stimulation at 1 h and returned to basal levels within 6 h. The rise in c-fos mRNA preceded the elevation in transferrin mRNA, which started to increase at 2 h to a maximum level between 6-12 h that was maintained at high levels for several days in cell culture. Treatment of Sertoli cells with an antisense c-fos oligonucleotide was found to inhibit the actions of PModS on transferrin expression. Combined results support the hypothesis that PModS acts indirectly through transcription factors (e.g. c-fos) to induce Sertoli cell differentiated functions (e.g. transferrin expression). Therefore, PModS appears to act as a differentiation-type factor to promote and maintain optimal Sertoli cell function.

Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.

A model system for studying inhibin.

We report here a sensitive model for studying inhibin-like activity in crude ovine testicular extracts (CoTE). The administration of CoTE in 25-100 mg amounts to 35-day-old immature male rats, orchidectomized just prior to use, resulted in the prevention of a rise in plasma FSH levels, seen 10 h post-treatment. In a second model, CoTE was injected at 1000 h of day 35 to a group of rats that was castrated 12-24 h prior to injection, and the animals were sacrificed 6 h later; plasma FSH levels were found to be significantly suppressed. CoTE, administered subcutaneously, both in multiple doses and as a single injection, was found to be equally effective. A single injection suppressed FSH levels within 3-6 h while LH levels were unaffected. The suppressive effect was dose-dependent, reaching a maximum value at doses of 100 mg CoTE and above. FSH levels could not be suppressed below the tonic level, either by the administration of a single large dose of CoTE, or by repetitive injections of maximal doses of CoTE at 3 h intervals. The suppression in FSH levels caused by a single injection of CoTE waned with time and totally disappeared by 36h. CoTE was prepared by the heat treatment (55 C for 30 min) of an aqueous extract of ovine testis, followed by centrifugation and ether extraction of the supernatant. The aqueous layer was then dialyzed and lyophilized. The lyophilized material was found to be free of testosterone, as measured by a specific radioimmunoassay, indicating that the active factor is a heat-stable, lyophilizable, nondialyzable material, free of contamination by testosterone and probably by other steroids.

Evidence for a hypothalamic site of action of inhibin to suppress FSH release.

To determine if inhibin has a hypothalamic site of action to suppress FSH release, highly purified inhibin preparations from the rete testis fluid (RTF) of rams were injected into adult male rats which had been orchidectomized (ORDX) 24 hours earlier. Third ventricular (3rd V) injection of a potent inhibin fraction (RTF 38-I) significantly depressed plasma FSH concentrations, without influencing LH, 4-10 h after treatment. A less active preparation of inhibin (RTF 38-II) at the same dose had no effect. A higher dose of another less potent fraction (RTF 1A) significantly reduced FSH 2-6 h following 3rd V administration, accompanied by slight but significant decrements in LH at 2 and 4 only. To determine responsiveness of the pituitary, luteinizing hormone-releasing hormone (LHRH) was injected intravenously at 6 h. It induced similar elevations of FSH and LH in inhibin- and saline-treated groups. Systemic administration of RTF 38-II at a dose 2.5-fold higher than the dose effective centrally failed to modify either FSH or LH levels up to 6 h. These results provide evidence that inhibin from the male can preferentially suppress FSH release by a CNS site of action in addition to its well-known pituitary site of action.

Inhibition of de novo synthesis of FSH by the Sertoli cell factor (SCF).

The effects of SCF, a substance produced by isolated rat Sertoli cells in culture, on the synthesis of FSH and LH were studied in vitro. Anterior pituitaries from adult male rats were organ-cultured 24-48h in the presence and absence of SCF in the culture medium. The incorporation of 3H-leucine into FSH and LH was determined following 6h incubation with this labelled precursor and immunoprecipitation with specific FSH or LH antibodies. Total FSH and LH were measured by radioimmunoassay. The results indicate that SCF can selectively reduce the incorporation of 3H-leucine into immunoprecipitable FSH without decreasing the incorporation into LH.

A simple and rapid in vitro bioassay for inhibin.

This report describes a quantitative bioassay for inhibin which can be used to monitor purification and establish the physiological role of this hormone in spermatogenesis and folliculogenesis. Anterior pituitary cells from adult male Sprague-Dawley rats were dispersed and precultured for 18 h before the addition of inhibin-containing materials to the culture medium. After a further 72 h in culture, the medium was removed by aaspiration, and the cells were lysed releasing their intracellular hormone into RIA buffer. Cell content of FSH was reduced in inhibin-containing preparations, but without changes in LH, GH, and PRL concentrations. The inhibin dose-response curve, based on the inhibition of FSH in 15 experiments using an ovine testicular lymph preparation as a standard, had indices of precision between -0.032 and -0.098, and Finney's g ranged from 0.003--0.025. The interassay variability ranged from 15.0--16.9%. The assay had a practical capacity of 300--400 wells, which permitted the measurement of dose-response curves of 15 or more unknowns with quadruplicate wells per dose. The potency of unknown preparations was calculated with reference to the inhibin standard, which had a designated potency of 1 U/mg. Preparations showing nonparallelism were excluded. This assay presented advantages over those described previously, since it showed specificity of response to FSH and accommodated a large number of samples without loss of precision or reproducibility. It is therefore suitable for measurement of the inhibin-like activity in some biological fluids.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.