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The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

During fertilization of mammalian eggs a factor from the sperm, the sperm factor (SF), is released into the ooplasm and induces persistent [Ca2+]ioscillations 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 PLCζare 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sperm injection (ICSI) and monitored the persistence of the [Ca2+]ioscillations in eggs in which the sperm had been withdrawn. We also stained the enucleatedsperm heads to ascertain the presence/absence of PLCζ. 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+]iresponses 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 PLCζimmunoreactivity was lost in both species. Altogether, our findings show that during fertilization the SF activity and PLCζimmunoreactivity are simultaneously released from the sperm, suggesting that PLCζmay be the only [Ca2+]ioscillation-inducing factor of mammalian sperm.

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]ioscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active bothin vivoandin vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]ioscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]irelease. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]ioscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]ioscillations.

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

Mammalian oocytes undergo significant growth during oogenesis and experience extensive cytoplasmic and nuclear modifications immediately before ovulation in a process commonly referred to as oocyte maturation. These changes are intended to maximize the developmental success after fertilization. Entry of a spermatozoon into the oocyte, which occurs a few hours after ovulation, initiates long-lasting oscillations in the free intracellular calcium ([Ca(2+)](i)) that are responsible for all events of oocyte activation and the initiation of the developmental programme that often culminates in the birth of young. Nevertheless, the cellular and molecular changes that occur during maturation to optimize development are transient, and exhibit rapid deterioration. Moreover, fertilization of oocytes after an extended residence in the oviduct (or in culture) initiates a different developmental programme, one that is characterized by fragmentation, programmed cell death, and abnormal development. Inasmuch as [Ca(2+)](i) oscillations can trigger both developmental programmes in mammalian oocytes, this review addresses one of the mechanism(s) possibly used by spermatozoa to initiate these persistent [Ca(2+)](i) responses, and the cellular and molecular changes that may underlie the postovulatory cellular fragmentation of ageing mammalian oocytes.