PLCζ can stably regulate Ca2+ fluctuations in early embryo

Abstract

Phospholipase C zeta (PLCζ) is an important inducer of Ca²⁺ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca²⁺ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca²⁺ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca²⁺ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca²⁺ in early embryos at different stages, Ca²⁺ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca²⁺ fluctuations differed. PLCζ resulted in Ca²⁺ peaks that appeared at the cleavage and morula stages of early embryos, and Ca²⁺ returned to normal levels at the morula stage. However, the Ca²⁺ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca²⁺ fluctuations at different stages during early embryonic development.

Keywords: Ca(2+) wave; Early embryo; Kazakh sheep; PLCζ; Parthenogenetic activation.