Activation of protein kinase C induces cortical granule exocytosis in a Ca²⁺‐independent manner, but not the resumption of cell cycle in porcine eggs

Abstract

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca(2+)-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.

The role of nitric oxide in parthenogenetic activation of pig oocytes: A review

“…The exocytosis of cortical granules does not always accompany oocyte activation by artificial activating stimuli, and this was the case, for example, when activation had been induced by the inhibition of protein kinases (Mayes et al, 1995). These observations are in agreement with those of Sun et al (1997), who proved that the activation of oocytes and the exocytosis of cortical granules were independent processes. Quite a long exposure to an NO-donor (10 hours) is necessary for the activation of pig oocytes.…”

“…Fan et al (2002b) demonstrated a change in the localization of PKC -α, -β I , -γ in maturing porcine oocytes and after their activation. On the other hand, Sun et al (1997) did not observe metaphase II transition and meiosis resumption in mature porcine oocytes after PKC stimulation, which indicates that PKC does not trigger these processes. Green et al (1999) reported that PKC inhibition together with protein kinase A and myosin light chain kinase inhibition resulted in the activation of the porcine oocyte.…”

Effects of protein kinase C on parthenogenetic activation of pig oocytes using calcium ionophore or nitric oxide-donor

“…Fan et al (2002b) demonstrated a change in the localization of PKC-α, -β I , -γ in maturing porcine oocytes after their activation. On the other hand, Sun et al (1997) did not observe any metaphase II transition and meiosis resumption in mature porcine oocytes after PKC stimulation, which indicates that PKC does not trigger these processes. Green et al (1999) reported that PKC inhibition, together with protein kinase A and myosin light chain kinase inhibition, results in the activation of porcine oocyte.…”

“…The PKC activity itself is insufficient for the activation of porcine oocytes (Sun et al, 1997;Fan et al, 2002b). However, its activity is necessary for complete oocyte activation.…”

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

“…Finally, the PKC-z, -l, -t isotypes belong to the atypical PKCs (aPKCs) and are independent of both Ca 21 and DAG (Liu and Heckman, 1998). The role of PKC activation in the exit from metaphase II of porcine oocytes has been studied using PKC-activators such as phorbol-12-myristate-13-acetate (PMA: an activator of all isotypes of PKC; Sun et al, 1997;Ito et al, 2003) and 1-oleoyl-2-acetylglycerol (OAG: an activator of calcium-dependent PKC isotypes; Sun et al, 1997). After oocyte treatment and their cultivation for an additional 20 h, the female pronucleus was not observed either with PMA or with OAG .…”