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Gametogenetin (Ggn) is a germ cell-specific gene with multiple splicing variants giving rise to three predicted protein products, gametogenetin protein 1 (GGN1), gametogenetin protein 2 (GGN2), and gametogenetin protein 3 (GGN3). GGN1 and GGN3 were reported to interact with Fanconi anemia complementation group L (FANCL) per proliferation of germ cells (POG), a ubiquitin E3 ligase involved in germ-cell-deficient (gcd) mutation. While GGN2, another protein from Ggn by alternative splicing did not interact with FANCL/POG since it lacked the domain mediating the interaction. Little is known about the expression and function of GGN2. Here through Northern blotting experiment we showed that Ggn was mainly expressed in the testis but hardly detectable in the ovary or the somatic tissues. By preparing GGN2-specific antibody we showed that GGN2 was detectable and only detectable in the testis. By comparing the expression of Ggn mRNA and GGN2 protein in developing mouse testis, we showed that there was no evident delay of the translation of Ggn mRNA after their transcription. Both the subcellular localization study and the germ cell membrane protein fractionation implied that GGN2 associated with the intracellular membrane system. Co-fractionation on Superdex and yeast two-hybrids suggested that like GGN1, GGN2 was also a potential interaction partner of gametogenetin binding protein 1 (GGNBP1). Our data suggested that gametogenetin proteins were mainly involved in male germ cell development and GGN2 was also a possible interaction partner of GGNBP1. Like GGN1, GGN2 was also possibly involved in cell trafficking. The possible involvement of GGN2 in acrosome biogenesis was proposed.

In mammals, the Y-linked Sry gene is normally responsible for testis induction. However, testes develop in the absence of Sry in human patients and animal models with Sry-negative XX sex reversal. The mechanism of testis induction in this disorder is presently unknown. Characterization of gene expression in normal embryos contributes to the framework within which the canine Sry-negative XX sex reversal model can be evaluated. The objective of this study was to add two molecular milestones to the canine sex determination pathway by determining the temporal and spatial expression patterns of Sf1 and Mis in normal urogenital ridges (UGR) at various gestational stages. The onset of Sf1 expression signifies the start of the sex determination period, whereas initial Mis expression identifies the end of the testis induction period. Sf1 expression in UGR was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and whole mount in situ hybridization (WMISH) at Carnegie stages (CS) 15 to 20. Canine sex determination begins at CS 15 with Sf1 expression in the emerging indifferent gonad. Gonadal Sf1 expression was detected in both sexes at all ages, and in the presumptive adrenal primordium at CS 15 and 17. At stages > or = CS 17, Sf1 expression was pronounced in male and female gonads. Mis expression, assayed by WMISH at CS 13.5-20, was observed only in male gonads > or = CS 18, indicating that the testis induction period ends at CS 18. The expression patterns of both genes are similar to those observed in humans and domestic animals.

Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is one of the earliest and best-known markers of Sertoli cell differentiation and is expressed until around puberty. The present study is aimed at the better understanding of the molecular pathways involved in testicular development and establishment of adult functions with regards to AMH regulation. We found, within the mouse AMH promoter, putative GATA motifs (A/T)GATA(A/G), known to be specifically bound by members of the GATA transcription factor family. We then carried out RNase protection assays and immunohistochemical techniques aimed at comparing precisely the chronological expression patterns of AMH and GATA-1, this latter being expressed in the testis after birth. Using both approaches we found an inverse and close relationship between AMH and GATA-1 mRNA and protein expression during the pre-pubertal period. These results allowed us to define a transitory 4-5-day period, starting from 3 dpp when both proteins are heterogeneously expressed in Sertoli cells and showed that the appearance of GATA-1 is associated with the decrease of AMH expression in these cells. Furthermore DNA-protein interaction in in vitro studies showed first that GATA-1 binds with various affinities on sites found in the AMH promoter and second that the proximity of the two strongest affinity sites leads to a synergistic binding effect. Altogether, the present study suggests that GATA-1 participates in AMH gene repression during the pre-pubertal period.

Sertoli cell secretion of Mullerian-inhibiting substance (MIS) begins shortly after testis differentiation. Mullerian ducts regress following MIS exposure during an embryonic critical period. In dogs with XX sex reversal, Mullerian ducts persist in the presence of testicular tissue. This study was conducted to determine whether MIS is present in ovotestes of XX sex-reversed embryos during the period for Mullerian duct regression in normal males. XX sex-reversed embryos and normal littermates were identified by a combination of karyotype and gonadal histology. The degree of regression in the adjacent Mullerian duct was scored. Immunohistochemical staining was used to detect MIS in the contralateral gonad. Testicular differentiation and MIS secretion were identified in XY embryos at all ages studied (35–46 days). Seminiferous tubules were not observed in gonads of embryos at risk of XX sex reversal between 35–38 days (n = 15), but were observed at 40 and 46 days (n = 3). Although positive staining for MIS was observed in ovotestes, adjacent Mullerian ducts persisted. The degree of seminiferous tubule development was reduced and MIS secretion was delayed in ovotestes, compared to normal testes. Mullerian duct persistence in this model is apparently due to an abnormality in the quantity and timing of MIS secretion during embryonic development.© 1994 Wiley-Liss, Inc.

The epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region-specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol-binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alternations in the composition of protein factors within the tubule lumen.

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.

Anti-Mullerian hormone (Amh) is expressed in the granulosa cells of growing and preovulatory follicles in the mouse ovary where it acts to decrease responsiveness to follicle stimulating hormone (FSH) and plays a role in inhibiting primordial follicle recruitment. Recently, co-culture of isolated oocytes and granulosa cells has demonstrated that Amh expression is up-regulated in the presence of oocytes and, at preantral stages, this effect is dependent upon close contact. In Sertoli cells, Amh expression is regulated by several transcription factors including SOX9, SF1, GATA4, WT1, and DAX1, which, with the exception of SOX9, are also expressed in granulosa cells where GATA4 is known to up-regulate Amh expression antagonised by FOG2. Here, we demonstrate that Sox8, which is closely related to Sox9 and encodes the protein SOX8 which can transactivate Amh, is expressed in the postnatal mouse ovarian follicle but is not co-expressed with Amh in the granulosa cells of preantral follicles. Sox8 expression was found only in the oocytes of preantral follicles and in the oocytes, cumulus cells, and mural granulosa cells of preovulatory follicles. Also, increased expression of Amh in granulosa cells co-cultured with oocytes was not associated with increased mRNA levels of the transcription factors Sf1, Gata4, Wt1, Dax1, or Fog2. These findings reveal Sox8 expression in the ovarian follicle and show that oocyte regulation of Amh expression is not due to oocyte regulation of Sf1, Gata4, Wt1, Dax1, or Fog2 expression in granulosa cells.

A novel gene Ggnbp1 was identified during yeast two-hybrid screening of gametogenetin protein 1 (GGN1)-interacting proteins. Ggnbp1 gene was found in mouse, rat, and human genomes but not in sequenced yeast, worms, fly, or fish genomes. Northern blotting analysis revealed that the gene was specifically expressed in the testis but not expressed in the other tissues. In situ hybridization showed that it was testicular germ cell-specific and was specifically expressed in later primary spermatocytes, meiotic cells, and early round spermatids. Western blotting analysis detected a protein of expected size in and only in the testis. By making membrane and cytosolic fractions of germ cells, we were able to show that GGNBP1 associated with the membrane. The identification and characterization of a novel germ cell-specific gene Ggnbp1 is the first step toward the defining of the functions of Ggnbp1 in spermatogenesis.

Anti-Müllerian hormone (AMH) is a member of the TGF-beta family which elicits its main action during male sex differentiation. This hormone is probably the most convenient marker of Sertoli cell differentiation and maturation throughout testicular development. Studying AMH gene regulation may thus be one way of identifying effectors of Sertoli cell differentiation. To this end we first tried to locate and then to characterise DNA elements responsible for in vivo transcriptional control of AMH expression. We obtained transgenic mice expressing a reporter gene (LacZ), under control of various putative AMH regulatory sequences. Analysis of transgenic animals revealed that activation of the AMH gene probably requires a two-step regulatory process. The first step corresponds to the initial activation of the AMH gene occurring at around 12.0 dpc. It requires the presence of regulatory DNA encompassed within a maximum of 370 bp upstream of the translation start site of the gene, delimited by the presence of an upstream housekeeping gene (SAP-62). Following this initial transient phase, a second phase seems to account for the persistence of AMH gene expression until the onset of puberty. As the 370 bp regulatory region is not sufficient on its own to allow the triggering of this second phase, it seems possible that additional control elements are required for normal AMH expression throughout testicular development. The complete array of regulatory elements remains to be located. Mol. Reprod. Dev. 59:256-264, 2001.

Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.

The present study was conducted to reveal effects of in ovo injection of nonsteroidal aromatase inhibitor (Fadrozole) or estradiol at day 3 of incubation on mRNA levels of P45017ahydroxylase (P450c17), P450 aromatase (P450arom) and anti- Mullerian hormone (AMH) in the chicken gonads. The mRNA levels in the gonads at days 4-8 of incubation were assessed by in situ hybridization analysis using digoxigenin labeling method. The in situ hybridization data were analyzed by relative expression of specific hybridizable signals of each mRNA corrected by the non-specific background by employing an image ana- lyzer. P450c17 mRNA expression increased rapidly at day 6 of incubation in the male but decreased thereaf- ter. In contrast to the transient expression in the male, the expression was gradually increased in the female. P450arom mRNA was not expressed in the male but was detectable in the female as early as day 6 and increased subsequently with days of incubation. AMH mRNA was expressed as early as day 5 of incubation followed by a sharp increase on day 6, which was maintained in the male thereafter. In contrast, the female showed very little expression. The injection of Fadrozole caused no effect on P450c17 mRNA expres- sion, while it suppressed P450arom mRNA expression but increased AMH mRNA expression in the female. In contrast, the injection of estradiol induced P450arom mRNA expression significantly but suppressed AMH mRNA expression in the male. These results indicate that expression of P450arom and AMH is sexually dimorphic and is reciprocally regulated during early ontogenic life in chicken gonads. Mol. Reprod. Dev. 55:20-30, 2000. r 2000 Wiley-Liss, Inc.

We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs.

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of 3H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less 3H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 microM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 microM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP--TCP-11 interactions modulate AC/cAMP.

This report presents data that suggest that the tissue-restricted polyoma enhancer activator protein (PEA3) of the Ets oncogene family of DNA-binding proteins is a putative modulator of the epididymis-specific glutathione peroxidase 5 gene gpx5. Northern and polymerase chain reactions on reverse-transcribed epididymal RNAs were used to show that the PEA3 factor is spatially and temporally expressed within the mouse epididymis in a manner consistent with gpx5 characteristics of expression. Then, using contransfection experiments carried out in heterologous tissue-culture cells with various deletions of the gpx5 promoter driving a CAT reporter gene, we have shown that the transcriptional activity of the gpx5 promoter is modulated by the presence of the PEA3 protein. Subsequently, we have shown using gel-shift assays that DNA sequences located within the 5' flanking region of the gpx5 gene have the ability to bind specifically to the PEA3 protein. Finally, using Northern assays we present data that suggest that PEA3 mRNA accumulation in the mouse caput epididymidis is controlled by androgens and testicular factors. Altogether, these results strongly suggest that the PEA3 factor might participate in the transcriptional control of the murine epididymis caput-specific gpx5 gene.

Using immunohistochemistry and Western blotting analyses, we present a detailed study of the distribution of the glutathione peroxidase protein (GPX5) within the mouse epididymis. We have shown that the expression of the epididymis-specific protein is restricted to the caput and essentially localized to the apical cell border of the caput epithelium. Secretion of the protein was detected as early as the proximal segment of the caput and GPX5 was subsequently found in the lumen of corpus and cauda epididymis duct. Within the caput, Western blot analyses have shown that equivalent quantities of GPX5 protein were found in segments I, II, and III. During ontogenesis, GPX5 appeared at 20 days postnatal, before the completion of the morphological differentiation of the caput and concomitantly with the appearance of spermatozoa within the epididymis, in agreement with what was reported earlier regarding the transcription of its corresponding gene during epididymal ontogenesis (Faure et al., 1991). Hormonal privation by castration abolished the accumulation of the GPX5 protein confirming previous data obtained on GPX5 mRNA levels. Treatments such as testosterone replacement or hemicastration led to the restriction of the protein to the caput epithelium, suggesting that protein secretion partly depends both on the presence of testicular factors and on spermatozoa. Using electron microscopy, we have shown that the secreted protein binds to spermatozoa and is found predominantly on the sperm acrosomic region. Finally, we report here that the GPX5 protein can be detected in fluids recovered from the uterine horns of freshly mated female mice. These results suggest that GPX5 might play an important role in sperm maturation from the early events up to the onset of fertilization and therefore could potentially be used as a tool to monitor sperm quality.

MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis-secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis-secreted protein and a protein of the sperm plasma membrane.

Sry is the Y-chromosomal gene responsible for initiating the pathway of male development in mammals, presumably by regulating downstream target genes. As a basis for examining the role played by Sry and other candidate sex-determining genes, we have used reverse-transcriptase PCR to produce a precise temporal profile of expression of Sry in the developing gonad. Sry expression first occurs at 10.5 days postcoitum (dpc) and is upregulated to reach a maximum level from 11.25 to 12 dpc. Expression is then steadily downregulated from 12.25 to 13.25 dpc and is completely extinguished by 13.5 dpc. Two genes implicated in sexual development are Amh, encoding anti-müllerian hormone (AMH), and Fra1, encoding Fos-related antigen-1. Expression profiles of Amh and Sry in fetal gonads indicate a possible role for AMH in the downregulation of Sry expression. We show, however, that the timing of Sry downregulation is unaffected in Amh-deficient mice, excluding a role for AMH as a negative regulator of Sry. Further, we have examined the possibility that SRY may activate Fra1 during testis determination by analysing the expression of Fra1 in mouse fetal gonads. Fra1 is not expressed at any stage at or around the critical period of sex determination when Sry transcripts are present, thus excluding a role for Fra1 in sex determination and differentiation.

Epididymis-secreted proteins D and E have been purified to homogeneity and partially characterized, and it is shown that monoclonal antibody (MAb) 4E9 (raised against a detergent extract of rat caudal epididymal sperm [Moore et al., 1994: Mol Reprod Dev 37(2):181-194]) recognizes protein E, but not protein D. The molecular weight of protein D (approximately 30 kD) is approximately 2 kD lower than protein E (approximately 32 kD). The NH2-terminus of each protein is blocked; however, microsequencing of internal peptides confirms earlier reports of significant sequence identity between the two proteins. High performance liquid chromatography tryptic peptide mapping showed peak differences between the two proteins, but it was not possible to obtain amino acid sequence in the peaks that were different. The epitope for MAb 4E9 was localized in the blocked NH2-terminus-CNBr peptide derived from protein E. The epitope was destroyed by protease treatment of protein E. Removal of N-linked oligosaccharides did not destroy the epitope for MAb 4E9 and did not affect the molecular weight difference between the proteins.

We have examined the transcription of Müllerian inhibiting substance (MIS) in testis by the sensitive technique of reverse transcription polymerase chain reaction (RTPCR). A developmental study of testis by this nonquantitative technique showed expression at all postnatal stages, including adults while liver and kidney provided negative controls. Cell separation studies indicated that highly purified interstitial cells, as well as less homogeneous Sertoli cell-enriched and germ cell-enriched fractions, contained RNA for MIS. The transcription of MIS in an interstitial cell type was confirmed by finding MIS mRNA in purified Leydig cells. Inasmuch as the germ cell-enriched fraction contains some Sertoli cells, and XX,Sxra and XX,Sxrb which have germ cell-depleted testes, contain MIS mRNA, a Sertoli cell source remains likely for the seminiferous tubule compartment.

The present discussion surveys some of the recently published studies utilizing transgenic strategies to address questions in reproductive endocrinology. Beginning with a brief introduction of the transgenic method itself, the following areas are covered: 1. Sexual development and Mullerian-inhibiting substance; 2. Hypogonadal mice and hypothalamic GnRH; 3. The GnRH neuron: generation of immortalized rare cell types; 4. Glycoprotein hormones: immortalized cells, development and evolution; 5. Growth hormone and reproduction; and, 6. Gestation and the insulin-like growth factors. In each section, the discussion attempts to be integrative with respect to the significance of the results to physiological, cellular and molecular biology. We believe this approach is appropriate, as transgenic science itself is necessarily an integration of all of these levels of investigation and participation from those working at all levels is needed.

During embryogenesis normal male phenotypic development requires the action of Müllerian Inhibiting Substance (MIS) which is secreted by Sertoli cells of the fetal testis. As testes differentiate in genetic (XY) males, they produce MIS which causes regression of the Müllerian ducts, the anlagen of the female reproductive tract. Soon thereafter, testicular androgens stimulate the Wolffian ducts. In females, on the other hand, MIS is not produced by grandulosa cells until after birth, before which, estrogens induce Müllerian duct development, while the Wolffian ducts passively atrophy in the absence of androgenic stimulation. High serum MIS levels in males are maintained until puberty, whereupon they fall to baseline levels. In females MIS is undetectable in serum until the peripubertal period when values approach the baseline levels of males. This distinct pattern of sexual and ontogenic expression presupposes and requires tight regulation. MIS may play a role in gonadal function and development. Our laboratory has shown that an important role for ovarian MIS is to inhibit oocyte meiosis, perhaps providing maximal oocyte maturation prior to selection for ovulation and subsequent fertilization. Furthermore, Vigier et al. (Development 100:43-55) have recently obtained evidence that MIS may influence testicular differentiation, coincident with inhibition of aromatase activity. Current structure-function studies demonstrate that MIS, like other growth regulators in its protein family, requires proteolytic cleavage to exhibit full biological activity. MIS can be inhibited by epidermal growth factor. This antagonism, which is common to all MIS functions so far investigated, is associated with inhibition of EGF receptor autophosphorylation. We have provided evidence that bovine MIS can inhibit female reproductive tract tumors arising in adults.(ABSTRACT TRUNCATED AT 250 WORDS)