180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes

Abstract

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (> 70%), DNA integrity (<8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P<0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P<0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.