Phospholipase C zeta characterization and egg activation ability of donkey sperm

Abstract

Assisted reproduction in horses has rapidly increased in the last few years. This has opened the opportunity to explore its application in other equids, such as donkeys, considered vulnerable or endangered. Up to date, no in vitro blastocyst production has been reported in donkeys. Our work aimed to describe the localization of donkey phospholipase C zeta (PLCζ), considered to be the physiological molecule responsible for generating calcium oscillations that induce egg activation, and to compare the ability of the donkey sperm to induce the pronuclear formation and recruit maternal SMARCA4 when injected into pig eggs through ICSI. Frozen ejaculated sperm cells from two jacks and three stallions were used. IF using PLCζ and SMARCA4 antibodies were performed in sperm and zygotes respectively. Zygotes were fixed 18 h after injection. Four different main patterns of PLCζ expression were observed after analyzing a total of 590 sperm cells: F, flagellum only; A, acrosomal; PA, post-acrosomal, and A+PA: acrosomal and post-acrosomal. Surprisingly, around 60% of donkey sperm expressed F and A PLCζ localization, whereas in horses, PA and A+PA were the most common patterns (∼80%). All sperm cells showed flagellum localization of PLCζ. Interestingly, pig oocytes were significantly less activated after injection with donkey sperm than horse sperm (Table 1),although SMARCA4 pronuclear localization levels in the zygotes were similar among groups. Our results describe for the first time the expression pattern of PLCζ in the donkey sperm. Moreover, we observed that horse sperm has a significantly increased ability to induce pig oocyte activation compared to the donkey. Since PLCζ is a crucial molecule for oocyte activation, we hypothesize that the different patterns of localization described for donkey sperm could be associated with low activation rates observed when injected into the egg. Therefore, strategies to improve the in vitro development of donkey ICSI embryos, such as assisted activation, might be required. More research is needed to confirm these preliminary results.