Gonzalez-Castro Raul A, Amoroso-Sanches Fabio, Stokes JoAnne E, Graham James K, Carnevale Elaine M
Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA.
Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA; and Corresponding author. Email:
Reprod Fertil Dev. 2019 Jan;31(12):1778-1792. doi: 10.1071/RD19217.
Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.
当受精的精子将精子携带的卵母细胞激活因子输送到卵母细胞胞质中时，卵母细胞激活就开始了。卵母细胞激活的候选因子包括两种蛋白质，即磷脂酶Cζ1（PLCZ1）和顶体后WW结合蛋白（PAWP；也称为WBP2 N端样蛋白（WBP2NL））。我们在种马精子中定位了PLCZ1和WBP2NL/PAWP，并研究了PLCZ1含量、精子参数以及胞质内精子注射（ICSI）后的卵裂情况。PLCZ1被鉴定为一种71 kDa的蛋白质，存在于顶体和顶体后区域、中段以及尾部的主段。抗WBP2NL抗体在顶体后区域、中段和尾部主段鉴定出两条WBP2NL条带（28 kDa和32 kDa）。精子头部中PLCZ1和WBP2NL的表达呈正相关（P = 0.04）。对PLCZ1的流式细胞术评估显示，种马之间荧光强度和阳性标记精子百分比存在很大差异。PLCZ1在活精子中的表达明显高于非活精子，并且DNA片段化与PLCZ1表达和阳性标记精子百分比呈负相关（P<0.05）。使用被认为PLCZ1含量高与低的马精子群体，在对牛和马的卵母细胞进行ICSI后，卵裂率显著更高，这支持了PLCZ1对卵母细胞激活的重要性。
