Search

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.