Department of Anatomical, Histological, Forensic and Orthopedic Sciences - Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.
Department of Surgery "P. Valdoni", Sapienza university of Rome, Rome, Italy.
J Cell Physiol. 2018 Apr;233(4):3093-3104. doi: 10.1002/jcp.26146. Epub 2017 Sep 20.
We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida-deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization.
我们之前的研究表明，谷胱甘肽过氧化物酶 4（GPx4）的核形式（nGPx4）在精子头部具有独特的分布，定位于核基质和顶体，并且缺乏 nGPx4 的精子在体外更容易去浓缩。在这项研究中，我们假设精子保留乙酰化组蛋白和 nGPx4 参与受精时父本染色质去浓缩和雄性原核形成。事实上，通过免疫荧光和 Western blot 分析，在 nGPx4-KO 精子中观察到显著更高水平的乙酰化组蛋白 H4 和乙酰化组蛋白 H3。在缺乏透明带的卵母细胞中，WT 精子在 Trichostatin（TSA）存在的情况下进行体外受精，也表明父本组蛋白乙酰化与受精时精子核去浓缩的时间呈负相关。相比之下，TSA 对 nGPx4-KO 精子没有影响，这表明它们的组蛋白乙酰化已达到最大值。此外，与 WT 精子相比，印记基因 Igf2/H19 在 KO 精子中被低甲基化。nGPx4 的缺乏对雄性生育力产生负面影响，导致总幼仔和妊娠分娩数量显著减少，体外受精试验中的原核（PN）胚胎数量显著减少，体内卵子受精时间延迟约 2 小时。由于 nGPx4-KO 和 WT 精子的透明带结合和融合到卵母细胞膜相似，nGPx4 精子的亚生育力反映了精子通过卵丘/透明带的进展减少，这表明顶体存在缺陷，与顶体 nGPx4 的定位一致。我们得出结论，父本乙酰化组蛋白和顶体 nGPx4 直接参与受精。
