Tsga8 is required for spermatid morphogenesis and male fertility in mice

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility. RNA-seq was performed using Tsga8 control (Tsga8+/Y) and knockout (Tsga8-/Y) mice (2 or 3 replicate animals). Collection of tissues enriched for spermatids was performed using laser capture microdissection (LCM).在精子发生（spermatogenesis）过程中，复杂的基因表达由表观遗传调控因子（epigenetic modifiers）协同调控，这些因子对于存在于未分化精原细胞（undifferentiated spermatogonia）中的精原干细胞（spermatogonial stem cells, SSCs）的分化不可或缺。我们先前的研究表明，KMT2B可在未分化精原细胞的二价与单价启动子区域介导组蛋白H3赖氨酸4三甲基化（H3K4me3）修饰的沉积。由于此类基因的表达发生于精子发生后期或胚胎发育阶段，我们推测其中多数基因可能由KMT2B预先编程，以实现后续的表达激活。本研究证实，作为KMT2B靶向基因之一的Tsga8，在精子细胞形态发生过程中发挥关键作用。小鼠体内Tsga8基因缺失会导致雄性不育，伴随圆形精子细胞染色体分布异常、延长型精子细胞头部畸形以及精子释放障碍。Tsga8敲除会引发数千个基因的表达失调，其中包括在精子细胞中重新激活的X染色体基因；同时还会导致核浓缩不足，伴随组蛋白-鱼精蛋白替换的关键因子过渡蛋白1（TNP1）与鱼精蛋白1（PRM1）的表达水平下调。对精子细胞实施卵胞浆内单精子注射（ICSI）可挽救该不育表型，表明精子细胞的基因组具备受精能力。综上，Tsga8是KMT2B的靶向基因，对于精子形成和生育能力至关重要。本研究通过2至3只生物学重复的Tsga8野生型（Tsga8+/Y）与基因敲除型（Tsga8-/Y）小鼠样本开展RNA测序（RNA-seq）；精子细胞富集组织的收集则采用激光捕获显微切割（LCM）技术完成。