The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative.In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.

We have developed a sensitive and specific assay for AMH. Serum AMH in men with male factor infertility is lower than in normal men. Levels of AMH in pregnancy are higher than normal menstrual cycle levels suggesting a foeto-placental contribution.

The TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGF beta and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.

Activins are cytokines of the transforming growth factor β family, which plays a central role in the determination of cell fate and the regulation of tissue balance. Family members are composed of two subunits and this dimerization is critical for liganding their cognate receptors and execution of proper functions. In the current study we focused on the localization of activin β A , β B , β C and β E subunits in the adult rat and analyzed the composition of putative activin β dimers. By dissecting tissue distribution of various activins, we found that the liver, in particular the hepatocytes, is the major source for activin β C and β E transcripts, since other tissues almost failed to express these isoforms. In sharp contrast, the emergence of activin β A and β B appeared ubiquitous. Using a highly selective proteome approach, we were able to identify homo-as well as heterodimers of individual activin subunits, indicating a high redundancy of ligand composition. Certainly, this broad potential to homo-and heterodimerize has to be considered in future studies on activin function.

Activins are formed by dimerization of beta-subunits and, as members of the TGF-beta superfamily, have diverse roles as potent growth and differentiation factors. As the biological function of the activin C homodimer (betaC-betaC) is unknown, we sought to compare activin A (betaA-betaA), B (betaB-betaB), and C homodimer bioactivities and to investigate the consequences of activin betaC-subunit overexpression in prostate tumor cells. Exogenous activin A and B homodimers inhibited cell growth and activated activin-responsive promoters. In contrast, the activin C homodimer was unable to elicit these responses. We previously showed that the activin betaC-subunit heterodimerized with activin betaA in vitro to form activin AC. Therefore, we hypothesize that the activin betaC-subunit regulates the levels of bioactive activin A by the formation of activin AC heterodimers. To test this hypothesis, we measured activin AC heterodimer production using a novel specific two-site ELISA that we developed for this purpose. In the PC3 human prostate tumor cell line, activin betaC-subunit overexpression increased activin AC heterodimer levels, concomitantly reduced activin A levels, and decreased activin signaling. Overall, these data are consistent with a role for the activin betaC-subunit as a regulatory mechanism to reduce activin A secretion via intracellular heterodimerization.

The mRNA expression of two activin growth factor subunits ( A-and C-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of A-activin and C-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague-Dawley rats, 12-240 h (n=3-5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. A-and C-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. A-and C-activin mRNA dropped to,50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when A-activin expression increased to three times sham control values and C-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24-48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of A-activin mRNA. C-activin immunoreactive homo-and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained C-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for A-activin. We conclude that A-and C-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.