Regulation of Gap-Junctional Communication Between Cumulus Cells During In Vitro Maturation in Swine, a Gap-FRAP Study1

Abstract

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.

Myosin VI facilitates connexin 43 gap junction accretion

“…In accordance with our micropipette loading assay, calcein AM fluorescence recovery as calculated by linear regression analysis (Santiquet et al, 2012) was reduced when myosin VI was lost (Fig. 5A).…”

The Dynamics of Connexin Expression, Degradation and Localisation Are Regulated by Gonadotropins during the Early Stages of In Vitro Maturation of Swine Oocytes

“…We therefore hypothesized that modulation of GJC can be explained in terms of changes in the total number of junctions, junction localization, opening frequency or gap junction pore size [13], [46], [47], [48]. The present study was undertaken to determine whether changes in connexin expression, degradation, and localization during the first 8.5 h of IVM are correlated with the previously observed dynamics of GJC [45].…”

“…Groups of 50 COCs were cultured in Nunclon Δ four-well dishes in 500 µl of standard porcine IVM culture medium: BSA-free NCSU 23 medium [50] containing 25 µM of 2-mercaptoethanol, 0.1 mg/ml cysteine and 10% (vol/vol) of filtered porcine follicular fluid as already described [45]. Depending on the experiment, the medium also contained 5 IU/well of human chorionic gonadotropin (hCG) and/or equine chorionic gonadotropin (PMSG/eCG, Intervet, Whitby, Ontario, Canada).…”

“…The Gap-FRAP assay was performed as described previously [45]. Briefly, this assay consisted of measuring the transfer of the fluorescent dye calcein from cells to a cell that had been laser-bleached to eliminate calcein fluorescence.…”

“…We have demonstrated recently that in swine, gonadotropins can regulate the effectiveness of GJC between cumulus cells during the first several hours of IVM [45]. We therefore hypothesized that modulation of GJC can be explained in terms of changes in the total number of junctions, junction localization, opening frequency or gap junction pore size [13], [46], [47], [48].…”