Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

PURPOSE: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFß superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. METHODS: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. RESULTS: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. CONCLUSION: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes.

Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-ß Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.

Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone.

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs-namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes' ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.

Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply.

This study assessed the participation of amphiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus-oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle-stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG-treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD(++), were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autofluorescence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting that oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems.

TGF-ß mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-ß1, TGF-ß2, and TGF-ß3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-ß3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-ß isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-ß induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-ß neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-ß isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-ß in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Metabolic differences in bovine cumulus-oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15.

Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% ± 7.4%; with FSH and rhBMP15, 51.5% ± 5.4%; P < 0.05). Glucose uptake and lactate production was significantly increased by greater than 2-fold with FSH (P < 0.05), whereas rhBM15 supplementation did not increase these levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P < 0.05). Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD(++)) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD(++):NAD(P)H). The significant increase in FAD(++) and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.

Signalling pathways mediating specific synergistic interactions between GDF9 and BMP15.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are two proteins selectively expressed in the oocyte which are essential for normal fertility. Both of these proteins are members of the transforming growth factor beta (TGF-ß) superfamily and as such are produced as pre-proproteins, existing after proteolytic processing as a complex of the respective pro and mature regions. Previous work has shown that these two proteins interact both at the genetic and cellular signalling levels. In this study, our aim was to determine if the purified mature regions of GDF9 and BMP15 exhibit synergistic interactions on granulosa cells and to determine if such interactions are specific to these two proteins. We have used primary cultures of murine granulosa cells and [(3)H]-thymidine incorporation or transcriptional reporter assays as our readouts. We observed clear synergistic interactions between the mature regions of GDF9 and BMP15 when either DNA synthesis or SMAD3 signalling were examined. GDF9/BMP15 synergistic interactions were specific such that neither factor could be replaced by an analogous TGF-ß superfamily member. The GDF9/BMP15 synergistic signalling response was inhibited by the SMAD2/3 phosphorylation inhibitor SB431542, as well as inhibition of the mitogen-activated protein kinase or rous sarcoma oncogene (SRC) signalling pathways, but not the nuclear factor kappa B pathway. In this study, we show that purified mature regions of GDF9 and BMP15 synergistically interact in a specific manner which is not dependent on the presence of a pro-region. This synergistic interaction is targeted at the SMAD3 pathway, and is dependent on ERK1/2 and SRC kinase signalling.

Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation.

OBJECTIVE: In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM). MATERIALS AND METHODS: In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV) oocytes (as F and V) were In vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of In vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos. RESULTS: These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression of the studied genes. CONCLUSION: Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent IVF outcomes.

Activin B and Activin C Have Opposing Effects on Prostate Cancer Progression and Cell Growth.

Current prognostic and diagnostic tests for prostate cancer are not able to accurately distinguish between aggressive and latent cancer. Members of the transforming growth factor-ß (TGFB) family are known to be important in regulating prostate cell growth and some have been shown to be dysregulated in prostate cancer. Therefore, the aims of this study were to examine expression of TGFB family members in primary prostate tumour tissue and the phenotypic effect of activins on prostate cell growth. Tissue cores of prostate adenocarcinoma and normal prostate were immuno-stained and protein expression was compared between samples with different Gleason grades. The effect of exogenous treatment with, or overexpression of, activins on prostate cell line growth and migration was examined. Activin B expression was increased in cores containing higher Gleason patterns and overexpression of activin B inhibited growth of PNT1A cells but increased growth and migration of the metastatic PC3 cells compared to empty vector controls. In contrast, activin C expression decreased in higher Gleason grades and overexpression increased growth of PNT1A cells and decreased growth of PC3 cells. In conclusion, increased activin B and decreased activin C expression is associated with increasing prostate tumor grade and therefore have potential as prognostic markers of aggressive prostate cancer.

Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain-Fc fusion protein (BMPR2ecd-Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd-Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd-Fc during the postnatal days 4-12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd-Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4-28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd-Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

Oocyte peptides as paracrine tools for ovarian stimulation and oocyte maturation.

Recent studies report the production and isolation of a stable bioactive recombinant human bone morphogenetic protein 15 (rhBMP15) that is appropriately processed in HEK-293 cells and activates the SMAD 1/5/8 pathway in mouse granulosa cell cultures. Further, the purified rhBMP15 induces the expression of genes associated with cumulus expansion. Thanks to recent research, we have a greater understanding of the importance of the dialogue that occurs between the oocyte and the granulosa cell layer with regard to regulating folliculogenesis and the acquisition of oocyte developmental competence and maturation. BMP15 is one of the critical components of these intra-follicular communication pathways. The production of recombinant human BMP15 is important for understanding the biochemistry of this specific pathway and for also fully understanding its functional contributions to mediating oocyte development. The production of a stable recombinant human BMP15 is also important for use in experiments aimed at optimizing ovarian stimulation protocols and in vitro oocyte maturation methods. This is required to improve oocyte and embryonic developmental competence and increase our ability to effectively use in vitro methods for animal production and the treatment of human infertility.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells.

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

Smad signalling in the ovary.

It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.

Surface expression of GluR-D AMPA receptor is dependent on an interaction between its C-terminal domain and a 4.1 protein.

Dynamic regulation of the number and activity of AMPA receptors is believed to underlie many forms of synaptic plasticity and is presumably mediated by specific protein-protein interactions involving the C-terminal domain of the receptor. Several proteins interacting with the C-terminal tails of the glutamate receptor (GluR)-A and GluR-B subunits have been identified and implicated in the regulation of endocytosis and exocytosis, clustering, and anchoring of AMPA receptors to the cytoskeleton. In contrast, little is known of the molecular interactions of the GluR-D subunit, or of the mechanisms regulating the traffic of GluR-D-containing AMPA receptors. We analyzed the subcellular localization of homomeric GluR-D receptors carrying C-terminal deletions in transfected human embryonic kidney (HEK) 293 cells and in primary neurons by immunofluorescence microscopy and ELISA. A minimal requirement for a 14-residue cytoplasmic segment for the surface expression of homomeric GluR-D receptors was identified. Previously, a similar region in the GluR-A subunit was implicated in an interaction with 4.1 family proteins. Coimmunoprecipitation demonstrated that GluR-D associated with 4.1 protein(s) in both HEK293 cells and rat brain. Moreover, glutathione S-transferase pull-down experiments showed that the same 14-residue segment is critical for 4.1 binding to GluR-A and GluR-D. Point mutations within this segment dramatically decreased the surface expression of GluR-D in HEK293 cells, with a concomitant loss of the 4.1 interaction. Our findings demonstrate a novel molecular interaction for the GluR-D subunit and suggest that the association with the 4.1 family protein(s) plays an essential role in the transport to and stabilization of GluR-D-containing AMPA receptors at the cell surface.

Adenoviral gene transfer allows Smad-responsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa luteal cells.

In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-beta, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-beta signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-beta superfamily.