Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

Abstract

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 µM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 µM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 µM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

“…The failure of high concentrations of LY29004 to block AREG-induced meiotic resumption observed in our study can be explained by a damage of the surrounding cumulus cells that are undoubtedly involved in that inhibitory mechanism. The damage of cumulus cells was displayed in our experiments by a lack to undergo expansion after washing and it was also documented by high incidence of apoptotic cells in cumuli treated by high doses of LY294002 (Shimada et al 2003).…”

Effects of PI3K and FSH on steroidogenesis, viability and embryo development of the cumulus–oocyte complex after in vitro culture

“…PI3K is one of the most relevant proteins that controls steroidogenesis (Moore et al, 2001;Shimada et al, 2003;Yu et al, 2005;Su et al, 2006;Ebeling et al, 2011) and has defined actions in the activation and survival of primordial follicles, and in the recruitment of follicles (Zheng et al, 2012). PI3K controls steroidogenesis, an effect that has been observed by using 100 μM of its inhibitor, LY294002, a dose used by Hoshino et al (2004).…”

Qualité ovocytaire et embryonnaire : les marqueurs apoptotiques ont-ils leur place dans le potentiel préimplantatoire ?

“…Puis, une augmentation d'activité d'AKT favorise, d'une part, la production de progestérone par les cellules du cumulus, ce qui favoriserait la reprise méiotique et, d'autre part, la signalisation de survie. D'ailleurs, l'utilisation d'un inhibiteur pharmacologique de la voie PI(3)K bloquant l'activation d'AKT, augmente l'activité de type caspase 3 et la proportion de cellules apoptotiques dans les cellules du cumulus oophorus et affecte la reprise méiotique stimulée des ovocytes porcins [104]. Or l'utilisation de milieu basique pour la culture des complexes cumulo-ovocytaires ne permet pas le maintien de l'activité d'AKT qui chute de façon drastique en quelques heures après la mise en culture [104].…”

“…D'ailleurs, l'utilisation d'un inhibiteur pharmacologique de la voie PI(3)K bloquant l'activation d'AKT, augmente l'activité de type caspase 3 et la proportion de cellules apoptotiques dans les cellules du cumulus oophorus et affecte la reprise méiotique stimulée des ovocytes porcins [104]. Or l'utilisation de milieu basique pour la culture des complexes cumulo-ovocytaires ne permet pas le maintien de l'activité d'AKT qui chute de façon drastique en quelques heures après la mise en culture [104]. Il est probable que la perte de cette signalisation de survie dans les complexes cumulo-ovocytaires provoque l'apoptose des cellules du cumulus oophorus.…”