Dynamic changes of intracellular zinc ion level during maturation, fertilization, activation, and development in mouse oocytes.

Although it is well known that calcium oscillations are required for fertilization in all mammalian species studied to date, recent studies also showed the ejection of zinc into the extracellular milieu in a series of coordinated events, called "zinc spark," during mammalian fertilization. These results led us to the hypothesis that a zinc ion-dependent signal is important for oocyte maturation, fertilization (activation), and further embryonic development. In this study, we evaluated the amounts and localization of intracellular zinc ions during maturation, fertilization, activation, and embryonic development in mouse oocytes. Our results show that abundant zinc ions are present in both immature and mature oocytes. After in vitro fertilization, the amounts of zinc ions were dramatically decreased at the pronuclear (PN) stage. Artificial activation by cycloheximide, SrCl2 , and TPEN also reduced the amounts of zinc ions in the PN stage. On the other hand, PN embryos derived from sperm injection still showed high level of zinc ions. However, the amounts of zinc ions rapidly increased at the blastocysts regardless of activation method. We showed here that the amounts of zinc ions dramatically changed during maturation, fertilization, activation, and development in mouse oocytes.

Carboxylated ε-poly-L-lysine, a cryoprotective agent, is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages.

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.

Phospholipase Cζ (PLCζ) versus postacrosomal sheath WW domain-binding protein (PAWP): Which molecule will survive as a sperm factor?

During mammalian fertilization, sperm is fused with the oocyte's membrane, triggering the resumption of meiosis from the metaphase II arrest, the extrusion of the second polar body, and the exocytosis of cortical granules; these events are collectively called 'oocyte activation.' In all species studied to date, the transient rise in the cytosolic level of calcium (in particular, the repeated calcium increases called 'calcium oscillations' in mammals) is required for these events. Researchers have focused on identifying the factor(s) that can induce calcium oscillations during fertilization. Sperm-specific phospholipase C, i.e., PLC zeta (PLCζ), is a strong candidate of the factor(s), and several research groups using different species obtained evidence that PLCζ is a sperm factor that can induce calcium oscillations during fertilization. However, postacrosomal sheath Tryptophan-Tryptophan (WW)-domain-binding protein (PAWP) was recently shown to have a pivotal role in inducing calcium oscillations in some species. In this review, we focus on PLCζ and PAWP as sperm factors, and we discuss this controversy: Which of these two molecules survives as a sperm factor?