Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, FSAA, Université Laval, Québec, Québec, Canada.
Biol Reprod. 2012 Aug 30;87(2):46. doi: 10.1095/biolreprod.112.099754. Print 2012 Aug.
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.
细胞间缝隙连接通讯（GJC）在卵巢细胞生理学中发挥着重要作用。GJC 的闭合被认为参与了卵母细胞的成熟，特别是在体内和体外，通过控制减数分裂抑制剂，如 cAMP 和 cGMP 的流动来控制减数分裂的恢复。了解 GJC 动力学在体外成熟（IVM）期间是如何调节的，可能为控制体外减数分裂恢复和卵母细胞成熟提供有力工具。由于人们对卵丘细胞之间 GJC 动态调节知之甚少，我们开发了一种基于钙黄绿素荧光在光漂白卵丘细胞中恢复的测定法，即间隙 FRAP 测定法。在猪 IVM 的最初几个小时内，我们对 GJC 特征进行了描述。我们表明，马绒毛膜促性腺激素（eCG）和表皮生长因子（EGF）下调了卵丘细胞之间的 GJC 有效性。然而，人绒毛膜促性腺激素并没有下调 GJC 有效性。我们还表明，在此期间 GJC 网络扩大，而这种效应不受促性腺激素的调节。成熟培养基中的猪卵泡液也对 GJC 调节有影响，增加了 GJC 网络的建立和钙黄绿素在卵丘细胞之间转移速率的有效性。这些结果表明，eCG 和 EGF 都在 IVM 后 4.5 小时下调 GJC 有效性，而网络扩展则与促性腺激素无关。因此，卵丘细胞之间的 GJC 调节将在体外 IVM 期间受到特异性调节。
