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.

Gap junctional communication (GJC) plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx), proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.

It is generally accepted that mammalian females are born with a finite pool of oocytes and that this is the sole source of ovules throughout the reproductive life of the adult. This dogma was shaken in 2003 when researchers showed that the oocyte stock might be renewable in adult mammals. It has been proposed that hematopoietic stem cells might be a source of new oocytes. These discoveries have puzzled many researchers and remain controversial. In our study, we attempted to determine if transplanted bone marrow cells could provide new oocytes in PU.1 mice and in severe combined immunideficiency (SCID) mice after treatment with chemotherapeutic agents. We also examined the possibility that grafted bovine embryonic ovarian cortex might provide an environment favoring such a response. We found no evidence that transplanted bone marrow cells provide new fertilizable oocytes in PU.1 mice, in SCID mice treated with chemotherapeutic agents, or with bovine embryonic ovarian tissue grafts. However, transplanted bone marrow cells have improved the fertility of SCID mice previously treated with chemotherapeutic agents. These data suggest that bone marrow cells cannot provide new oocytes but can positively influence ovarian physiology to improve the fertility of mice previously treated with chemotherapeutic agents.

This study was supported by the Colorado Center for Reproductive Medicine (CCRM, Lone Tree, Colorado USA). We declare no conflict of interest.

C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.