Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3-, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.Fil: Tateno, Hiroyuki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hino,Toshiaki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Darszon, Alberto. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Yanagimachi, Ryuzo. University of Hawaii Medical School. Institute for Biogenesis Research; Estados UnidosFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

BACKGROUND: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives