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

Abstract

Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 mM) and L-a-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 mM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP-DPAM, SNAP-OAG and SNAP-PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 mM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 6 10.0%, OAG 63.3 6 1.0% and PMA 45.0 6 16.5%) as well as the NO-donor alone (48.7 6 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP-OAG 0, SNAP-DPAM 17.4 6 2.5% and SNAP-PMA 38.4 6 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 6 1.7%, whereas CGs were partially released after Ca 21 ionophore treatment (13.0 6 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 6 2.5, 6.7 6 1.9 and 0.7 6 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 6 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP-DPAM 20.7 6 2.6%, SNAP-OAG 16.7 6 2.9% or SNAP-PMA 20.0 6 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.Keywords: protein kinase C, nitric oxide, pig oocyte, activation, cortical granules ImplicationsParthenogenetic activation of mammalian oocytes is commonly used in reproductive biotechnologies, for example, cloning using nuclear transfer, and its success depends on an adequate artificial stimulus. Nitric oxide (NO) has been described as an oocyte activator but it does not induce a cortical reaction. Protein kinases C (PKC) play an important role in the cortical reaction but the PKC cascade is not triggered after oocyte activation by an NO-donor. There is a possibilit...

Gasotransmitters in Gametogenesis and Early Development: Holy Trinity for Assisted Reproductive Technology—A Review

“…In accordance with this variable effect, cortical granules exocytosis has been reported in Xenopus oocytes [44] but not in porcine oocytes [46]. The interspecies differences of NO action during fertilization are obvious and NO seems to be even nonessential during mammalian fertilization (Figure 2).…”

PKCβ1 regulates meiotic cell cycle in mouse oocyte

“…PKCβ1 is an important member of the family of conventional protein kinase C that play a crucial role in regulating the mitotic cell cycle [21,45]. Until now, the involvement of PKC in the regulation of meiosis has been studied by utilizing PKC activators or inhibitors, which are unable to discriminate between the different types of PKC [30,46]. In this study, we investigated the expression, localization and possible roles of PKCβ1 during mouse oocyte meiotic maturation.…”