Regulation of gap-junctional communication between cumulus cells during in vitro maturation in swine, a gap-FRAP study

Intercellular gap-junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors, such as cAMP and cGMP. Understanding how GJC dynamics are regulated during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and oocyte maturation in vitro. Since little is known about the GJC dynamic regulation between cumulus cells, we have developed an assay based on recovery of calcein fluorescence in photobleached cumulus cells, a gap-FRAP assay. The GJC profile has been characterized during the first hours of porcine IVM. We showed that equine chorionic gonadotropin (eCG) and epidermal growth factor (EGF) down-regulated GJC effectiveness between cumulus cells. However, human chorionic gonadotropin was not down-regulating GJC effectiveness. We also showed that the GJC network expanded during this period and that this effect was not regulated by gonadotropins. Porcine follicular fluid present in the maturation medium also had an impact on GJC regulation, increasing GJC network establishment and the effectiveness of calcein transfer rate between cumulus cells. These results show that both eCG and EGF are regulating the decrease in GJC effectiveness after 4.5 h of IVM, while the network extension is gonadotropin independent. Regulation of GJC between cumulus cells would then be specifically regulated during in vitro IVM.     

Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) belong to the TGF-beta superfamily and are involved in the regulation of folliculogenesis. Though there are many reports concerning the expression and regulation of GDF9 in the process of oocyte maturation, expression of BMP15 during oocyte maturation is still not clearly understood. It has been reported that BMP15 and GDF9 expression is important in folliculogeneiss and that the regulation of these two proteins is complex and species-specific. In this report, we investigated the expression of BMP15 and GDF9 genes during in vitro maturation (IVM) at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h for porcine oocytes. Porcine GDF9 gene was found to be highly expressed in immature oocytes and declined slowly during the oocyte maturation process. BMP15mRNA and its encoded protein were expressed at low levels in immature oocytes and increased to the highest level at 18 h of IVM, which coincides with the time of cumulus cell expansion. Thus, these two genes were differentially expressed during the oocyte maturation process and BMP15 is specifically expressed during cumulus cell expansion in porcine oocytes.     

Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

Developmental competence of porcine oocytes after in vitro maturation and in vitro culture under different oxygen concentrations

In this study, we investigated the effect of two oxygen concentrations (5 and 20%) during in vitro maturation (IVM) and during in vitro culture (IVC) on porcine embryo development and analysed differences in gene expression between cumulus-oocyte complexes matured under 5 or 20% oxygen and the resulting blastocysts cultured under 5% or 20% oxygen following parthenogenetic activation. There was no significant difference in oocyte maturation rate. However, the numbers of resulting blastocysts were significantly increased in the 5% IVC group compared with the 20% IVC group. Moreover, the M20C5 treatment group (23.01%) supported greater blastocyst development compared with the M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. However, total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. GLUT1, G6PD and LDHA were up-regulated in cumulus cells that had been matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas cyclin B1 (CCNB) and MnSOD (Mn-superoxide dismutase) were upregulated in cumulus cells that had been matured in high oxygen, which suggests a higher activity of mitosis-promoting factor and antioxidant response. In spite of these differential effects on cumulus cells, oocytes could mature normally regardless of different oxygen concentrations. Therefore, it can be concluded that high oxygen concentration during in vitro maturation and low oxygen during in vitro culture may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development (p < 0.05) but not blastocyst quality.     

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.     

Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis

In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.     

Expression, regulation, and function of progesterone receptors in bovine cumulus oocyte complexes during in vitro maturation

Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence.     

PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Identification of hyaluronic acid-binding proteins and their expressions in porcine cumulus-oocyte complexes during in vitro maturation

Hyaluronic acid-binding proteins (HABPs) are necessary for expansion of the cumulus-oocyte complex (COC) during oocyte maturation. In this study, to obtain the detailed information of HABPs during cumulus expansion, we examined the expression of HABPs in porcine COCs during in vitro maturation (IVM). After maturation culture, proteins were extracted from porcine COCs and separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After transfer, the membranes were subjected to ligand blotting with biotinylated hyaluronic acid (bHA) or fluorescein isothiocyanate-labeled hyaluronic acid (FITC-HA). Furthermore, the extracted proteins were subjected to immunoprecipitation, Western blotting, and immunofluorescence analysis to dissect the HABPs. Ligand blotting with FITC-HA could detect HABPs. Using this ligand-blotting method, 13 and 14 bands of HABPs were detected in porcine COCs after 0 and 48 h in culture, respectively. Of these, the level of expression of 85-kDa HABP increased with cumulus expansion during IVM and was newly detected after culture. Immunoprecipitation, Western blotting, and immunofluorescent analysis confirmed that the 85-kDa HABP corresponded to CD44 and that it existed on/in the membrane of cumulus cells. The present results indicated that HABP expressed in porcine COCs during IVM, particularly CD44, may form a network of the matrices in the extracellular space of the oocyte with cumulus expansion during IVM.