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Release of phospholipase C and [Ca²⁺]_i oscillation-inducing activity during mammalian fertilization

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, USA

Correspondence should be addressed to R A Fissore at Department of Veterinary and Animal Sciences, University of Massachusetts, 411 Paige Laboratories, 161 Holdsworth Way, Amherst, Massachusetts 01003, USA; Email: rfissore{at}vasci.umass.edu

During fertilization of mammalian eggs a factor from the sperm, the sperm factor (SF), is released into the ooplasm and induces persistent [Ca²⁺]_i oscillations that are required for egg activation and embryo development. A sperm-specific phospholipase C (PLC), PLCz, is thought to be the SF. Here, we investigated whether the SF activity and PLCare simultaneously and completely released into the ooplasm soon after sperm entry. To accomplish this, we enucleated sperm heads within 90 min of intracytoplasmic sperm injection (ICSI) and monitored the persistence of the [Ca²⁺]_i oscillations in eggs in which the sperm had been withdrawn. We also stained the enucleatedsperm heads to ascertain the presence/absence of PLC. Our results show that by 90 min all the SF activity had been released from the sperm, as fertilized enucleated eggs oscillated as fertilized controls, even in cases in which oscillations were prolonged by arresting eggs at metaphase. In addition, we found that the released SF activity became associated with the pronucleus (PN), as induction of PN envelope breakdown evoked comparable [Ca²⁺]_i responses in enucleated and non-manipulated zygotes. Lastly, we found that PLCzlocalized to the equatorial area of bull sperm and to the post-acrosomal region of mouse sperm and that by 90 min after ICSI all the sperm’s PLCimmunoreactivity was lost in both species. Altogether, our findings show that during fertilization the SF activity and PLCimmunoreactivity are simultaneously released from the sperm, suggesting that PLCmay be the only [Ca²⁺]_i oscillation-inducing factor of mammalian sperm.