Evidence that protein kinase C can be activated at fertilization trough a Ca2+- indipendent pathway

Introduction: Fertilization initiates a series of rapid changes that remodels the oocyte into the zygote and triggers embryonic development. These early changes involve cytoplasmic signalling pathways activated by the sperm-induced Ca2 rise, a signal essential for both cortical granule exocytosis and meiosis resumption. Numerous studies have proposed a role for protein kinase C (PKC) as one of the Ca2 effectors implicated in the control of mammalian oocyte activation. However in mouse oocytes, in addition to Ca2-dependent isotypes, the presence of PKC isotypes that do not have a requirement for Ca2 has been reported. Therefore, at fertilization PKC could be activated not only in response to the Ca2 signal but also as a result of activation of Ca2-independent signalling pathways. To test this hypothesis, in the present work we have monitored PKC activity in fertilized mouse oocytes where the sperminduced Ca2 increase was prevented by a specific intracellular Ca2 chelator. In addition, we investigated whether changes in PKC activity were accompanied by intracellular redistribution of a Ca2-independent PKC isoform detected in mouse oocytes. Materials and methods: Ovulated oocytes from CD1 mice were deprived of zona pellucida and then incubated in the presence of 200 000 sperm/ml (control) or exposed to the Ca2 chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N,N-tetra-acetic acid acetoxymethyl ester (BAPTA-AM; 10 μmol/l) for 30 min prior to insemination. Experiments where, at 10 min of insemination, sperm–oocyte fusion rate monitored by the Hoechsttransfer technique was 90% were discarded. In a first trial, at different times of insemination, oocytes were processed for biochemical analysis of PKC activity by using MARKS as a specific PKC substrate. In a second trial oocytes were immunostained by using a primary antibody against δ-PKC and a secondary antibody conjugated with the fluorochrome Cy-3 and analysed by a laser scanning confocal microscope. Negative control samples were evaluated in which the primary antibody was omitted. Results: Data from the biochemical assay revealed a significant increase in PKC activity as early as 10 min of insemination in fertilized oocytes where the level of Ca2 was clamped low with the Ca2 chelator. However, this level of activity was lower than that observed in the control group that was in the presence of the Ca2 signal. Immunocytochemical analysis showed that at fertilization, at the same time PKC activity increased, the δ-PKC gradually changed its cytoplasmic distribution and enriched at the site of the meiotic spindle during the metaphase–anaphase transition. In fertilized oocytes where the Ca2 signal was prevented, δ-PKC staining on the meiotic spindle was similar to that observed in control fertilized oocytes after a few minutes of insemination when they were still at the metaphase stage. Conclusions: Here we show that sperm–oocyte interaction can result in the activation of Ca2-independent pathways leading to PKC stimulation and that δ-PKC, during the metaphase–anaphase transition, gradually associates with the meiotic spindle, a major site for localization of elements that control meiosis resumption. Since PKC isotypes are known to translocate to new intracellular sites on activation, δ-PKC may be one of the Ca2-independent PKC isotypes physiologically recruited at fertilization. Taken together, present findings suggest that PKC activity at fertilization can result from the activation of both Ca2-dependent and Ca2-independent isotypes. Therefore, even though the fertilization Ca2 signal is the trigger for oocyte activation, the regulation of this event may also involve the participation of sperm-induced Ca2-independent signalling pathways.