Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery.

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The 'nest' chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample 'cradles' that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Rationally Designed Probe for Reversible Sensing of Zinc and Application in Cells.

Biologically compatible fluorescent ion sensors, particularly those that are reversible, represent a key tool for answering a range of fundamental biological questions. We report a rationally designed probe with a 6'-fluoro spiropyran scaffold (5) for the reversible sensing of zinc (Zn2+) in cells. The 6'-fluoro substituent overcomes several limitations normally associated with spiropyran-based sensors to provide an improved signal-to-background ratio and faster photoswitching times in aqueous solution. In vitro studies were performed with 5 and the 6'-nitro analogues (6) in HEK 293 and endothelial cells. The new spiropyran (5) can detect exogenous Zn2+ inside both cell types and without affecting the proliferation of endothelial cells. Studies were also performed on dying HEK 293 cells, with results demonstrating the ability of the key compound to detect endogenous Zn2+ efflux from cells undergoing apoptosis. Biocompatibility and photoswitching of 5 were demonstrated within endothelial cells but not with 6, suggesting the future applicability of sensor 5 to study intracellular Zn2+ efflux in these systems.

Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult individual; indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc.

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.

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.

Oocyte induction of EGF responsiveness in somatic cells is associated with the acquisition of porcine oocyte developmental competence.

Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

Modifications of human growth differentiation factor 9 to improve the generation of embryos from low competence oocytes.

Growth differentiation factor 9 (GDF9) is an oocyte-derived growth factor that plays a critical role in ovarian folliculogenesis and oocyte developmental competence and belongs to the TGF-ß family of proteins. Recombinant human GDF9 (hGDF9) is secreted in a latent form, which in the case of the fully processed protein, has the proregion noncovalently associated with the mature region. In this study, we investigated a number of amino acid residues in the mature region of hGDF9 that are different from the corresponding residues in the mouse protein, which is not latent. We designed, expressed, and purified 4 forms of chimeric hGDF9 (M1-M4) that we found to be active in a granulosa cell bioassay. Using a porcine in vitro maturation model with inherent low developmental competence (yielding 10%-20% blastocysts), we tested the ability of the chimeric hGDF9 proteins to improve oocyte maturation and developmental competence. Interestingly, one of the chimeric proteins, M3, was able to significantly increase the level of embryo production using such low competence oocytes. Our molecular modeling studies suggest that in the case of hGDF9 the Gly(391)Arg mutation probably increases receptor binding affinity, thereby creating an active protein for granulosa cells in vitro. However, for an improvement in oocyte developmental competence, a second mutation (Ser(412)Pro), which potentially decreases the affinity of the mature region for the proregion, is also required.

Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period.

Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare's serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus-oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15:Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

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.

Aberrant GDF9 expression and activation are associated with common human ovarian disorders.

CONTEXT: Growth differentiation factor 9 (GDF9) is a central regulator of folliculogenesis and ovulation rate. Fourteen mutations in human (h) GDF9 have been reported in women with premature ovarian failure or polycystic ovarian syndrome as well as in mothers of dizygotic twins, implicating GDF9 in the etiology of these conditions. We sought to determine how these mutations alter the biological activity of hGDF9. OBJECTIVE: The objective of the study was to determine whether aberrant GDF9 expression or activation is associated with common ovarian disorders. DESIGN: Homology modeling was used to predict the location of individual mutations within structurally important regions of the pro domains and mature domains of hGDF9. Each hGDF9 variant was generated by site-directed mutagenesis, expressed from human embryonic kidney 293T cells and assessed as to whether it resulted in defective production or the enhanced activation of mature hGDF9 in an in vitro granulosa cell proliferation bioassay. RESULTS: Mutations observed in mothers of dizygotic twins (P103S and P374L) completely abrogated GDF9 expression, suggesting that women heterozygous for these mutations would have a 50% reduction in GDF9 levels. Comparable declines in GDF9 in ewes result in a 2-fold increase in ovulation rate and fecundity. Remarkably, three prodomain mutations associated with premature ovarian failure (S186Y, V216M, and T238A) all resulted in the activation of hGDF9. Mechanistically, these mutations reduced the affinity of the prodomain for mature hGDF9, allowing the growth factor to more readily access its signaling receptors. CONCLUSIONS: Together these findings indicate that alterations to hGDF9 synthesis and activity can contribute to the most common ovarian pathologies.

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence.

STUDY QUESTION: Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? SUMMARY ANSWER: Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. WHAT IS KNOWN ALREADY: In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. STUDY DESIGN, SIZE, DURATION: An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on individual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence; however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. LIMITATION, REASONS FOR CAUTION: Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. WIDER IMPLICATIONS OF THE FINDINGS: This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

Activation of latent human GDF9 by a single residue change (Gly 391 Arg) in the mature domain.

Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-ß superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly(391)) confers latency to hGDF9. Gly(391) forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly(391)Arg) had similar activity to mGDF9 (EC(50) 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly(391)Arg) was also a potent stimulator of murine granulosa cell proliferation (EC(50) 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals.

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.

Differences in the participation of TGFB superfamily signalling pathways mediating porcine and murine cumulus cell expansion.

It is widely held that mammalian cumulus cell (CC) expansion requires oocyte-paracrine signalling, however in three of the four species studied to date, CC expansion occurs in the absence of the oocyte. This study was conducted to examine the paracrine and SMAD/MAPK intracellular signalling mechanism mediating porcine CC expansion, and to compare these to the mouse. Cumulus-oocyte complexes (COCs) and oocyte-free complexes (OOXs) from pigs and eCG-primed mice were treated in vitro with FSH and a broad range of TGFB superfamily antagonists. Expansion of porcine COCs and OOXs was unaffected by neutralisation of growth differentiation factor 9, TGFB, activin A, activin B and a broad spectrum bone morphogenetic protein antagonist. A SMAD-responsive luciferase reporter assay confirmed that porcine oocytes secreted factors that activate SMAD3 and SMAD1/5/8 in granulosa cells, but murine oocytes activated SMAD3 only. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) partially inhibited porcine CC expansion and expression of TNFAIP6, but ablated murine CC expansion. SB431542 was equally effective at attenuating porcine CC expansion in the presence or absence of the oocyte. By contrast, a SMAD1/5/8 phosphorylation inhibitor (dorsomorphin) had no effect on porcine or murine CC function. Inhibition of ERK1/2 and p38 MAPK signalling pathways prevented porcine COC expansion and expression of most matrix genes examined. The activation of CC SMAD signalling by oocytes, and the requirement of SMAD2/3 signalling for expansion, is notably contrasted in pigs and mice. Nonetheless, porcine CC SMAD2/3 signalling is likely to be needed for optimal matrix formation, possibly by facilitating essential MAPK signals.

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.

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion.

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion.

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex, which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II and is currently the primary candidate molecule for the cumulus-expansion enabling factor. This study was conducted to determine whether GDF9 is the mouse cumulus-expansion enabling factor. Cumulus-oocyte complexes were collected from mice, and the oocyte was microsurgically removed to generate an oocytectomized (OOX) complex. OOX complexes treated with FSH alone or recombinant mouse GDF9 alone failed to expand, whereas expansion was induced in the presence of FSH by GDF9, TGFbeta1, or coculture with oocytes. A specific GDF9-neutralizing antibody, mAb-GDF9-53, neutralized the expansion of OOX complexes in response to GDF9 but not the expansion of OOX complexes cocultured with oocytes. Using real-time RT-PCR, hyaluronan synthase 2 (HAS2) mRNA expression by OOXs was up-regulated 4- to 6-fold by oocytes and GDF9. Monoclonal neutralizing antibody-GDF9-53 attenuated GDF9-induced OOX HAS2 expression but not oocyte-induced HAS2 expression. A TGFbeta antagonist neutralized TGFbeta-induced, but not oocyte-induced, expansion of OOX complexes, and when combined with monoclonal neutralizing antibody-GDF9-53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the bone morphogenetic protein receptor II extracellular domain, which is a known GDF9 antagonist, completely antagonized GDF9-induced expansion but only partially neutralized oocyte-induced expansion. This study provides further evidence that like TGFbeta, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFbeta alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion.