猪精子α-L-岩藻糖苷酶活性定位及其在精-卵结合及受精中的作用

Localization of α-L-fucosidase within pig spermatozoa with special reference to its function in fertilization and sperm-oocyte interaction

本试验旨在证明猪精子中α-L-岩藻糖苷酶的存在及其在受精中的作用。猪精子α-L-岩藻糖苷酶活性用分光光度计405nm波长测定,并在受精液中添加α-L-岩藻糖苷酶特异抑制因子(deoxyfuconojirimycin hydrochloride,DFM-H)或特异单糖(L-岩藻糖)来检测对该酶活性的影响。结果显示,猪精子含有明显的α-L-岩藻糖苷酶活性。当精子用10μmol/L钙离子载体A23187处理1h后,有70%精子发生顶体反应和72%的酶活性被释放。体外受精液中添加250μmol/LDFM-H或30mmol/L L-岩藻糖,可分别抑制66%或63%的精子α-L-岩藻糖苷酶活性,并相应抑制37%或36%的透明带表面精子结合数及56%或67%的精子侵入卵数。这些结果证明,猪精子α-L-岩藻糖苷酶主要分布于精子顶体中,特异地作用于透明带寡糖链中的α-L-岩藻糖残基,而参与精子穿入透明带的过程。

To identify sperm α-L-fucosidase specific for L-fucose residue existed in the zona pellucida of pig oocytes,the frozen-thawed ejaculated spermatozoa were treated experimentally and assayed for activities of α-L-fucosidase.There are significant α-L-fucosidase activities in it measured by the absorbance at a wave length of 405 nm.When spermatozoa are incubated with calcium ionophore A23187 for 1 h,about 70% of them were acrosome-reacted and about 72% of the activities of α-L-fucosidase lost from spermatozoa.When 250 μmol/L α-L-fucosidase inhibitor(deoxyfuconojirimycin hydrochloride,DFM-H) or 30 mmol/L specific sugar(L-fucose) supplemented in the sperm suspension,the activities of α-L-fucosidase reduced by 66% and 63%,respectively.When cumulus-free pig oocytes matured in culture were inseminated in the presence of 250 μmol/L DFM-H or 30 mmol/L L-fucose,the number of spermatozoa bound to the zona pellucida reduced by 37% and 36%,and the proportion of oocytes penetrated reduced by 56% and 67%,respectively.These results proved that the α-L-fucosidase do exists in boar sperm,mainly in the acrosome matrix,and suggested that it may involve in the pig sperm-oocyte interaction,especially participate in the process of sperm penetrate to zona pellucida by its interaction with the terminal L-fucose residue existed in zona pellucida.