At fertilization, the sperm induces a series of species-specific intracellular Ca2+ rises ([Ca2+]i) that are important for oocyte activation and embryonic development in all mammalian species studied to date. These have not been investigated in the horse, where in vitro assisted reproduction techniques generally provide suboptimal results. In this study, we characterize the activity of equine sperm extracts (sperm factor; eSF) in mouse oocytes, and demonstrate their ability to induce [Ca 2+]i transients and parthenogenetic activation in equine oocytes. However, sperm injected (ICSI) horse oocytes do not consistently mount [Ca2+]i responses, and this failure is not the result of inadequate sperm factor release into the oocyte. This research may explain suboptimal results reported for ICSI in the equine and suggests potential differences in fertilization induced-signaling mechanisms amongst different mammalian species.

The mechanisms by which the sperm generates long lasting [Ca2+ ]i oscillations in mammalian oocytes is discussed in the first chapter. In Chapter 2, a study was undertaken to determine if injection of porcine sperm factor(s) (pSF) can be used to activate bovine oocytes during nuclear transfer. It was found that injection of 5 mg/ml pSF triggered [Ca 2+]i oscillations that resembled those associated with fertilization. This concentration of pSF supported in vitro and in vivo development up to 60–90 d of gestation. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because it was unable to support oscillations pass 3 to 5 h post-injection. Likewise, a single injection of pSF failed to trigger down-regulation of the inositol 1,4,5-trisphosphate receptor-1 sub-type (IP3R-1). These results demonstrate that pSF can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited due to the premature cessation of the induced oscillations. In chapter 3 we evaluated the temporal release of the Ca2+-active factor during mouse fertilization and its possible association with the perinuclear theca (PT) of mammalian sperm. Between 15–60 min post sperm entry a significant portion of the Ca2+ releasing activity became dissociated from the sperm head. The loss of the Ca2+ releasing activity coincided with exposure and solubilization of the sperm's PT, although disassembly of the PT did not appear to be required for the release of the Ca2+ factor. Lastly, the conditions in the egg that promote release of the sperm's Ca2+ factor do not appear to be cell cycle specific. Finally, in chapter 4 we evaluated the impact of the IP3R-1 on the generation of Ca2+ release in bovine oocytes. Bovine oocytes with fewer numbers of IP3R-1 were produced by injection of adenophostin A, a potent antagonist of the IP3R-1 that triggers degradation of the receptors. Western blot analysis revealed that >80% of the receptors were degraded. The ability of IP3R-1 deficient oocytes to trigger Ca2+ release was examined by injecting IP3 and pSF, two agonists of the IP3R-1. IP3-induced Ca2+ release was partially blocked in IP3R-1 deficient oocytes. Moreover, injection of pSF into IP3R-1 deficient oocytes failed to establish persistent Ca2+ responses. These results suggest that IP3R-1 number has significant impact on the generation of Ca2+ release in bovine oocytes.

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