Generation of calcium oscillations in mammalian eggs: Impact on activation and development and implications for cloning

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{09} 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.