Disrupted development and altered hormone signaling in male Padi2/Padi4 double knockout mice

Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice for a range of reproductive defects. Results from controlled breeding trials found that DKO breeding pairs appeared to take longer to have their first litter than WT controls and that DKO male weanlings weighed significantly less than their WT counterparts. Additionally, DKO males took significantly longer than WT males to reach puberty and also had lower serum testosterone levels. Furthermore, DKO males had smaller testes than WT males and also had increased rates of germ cell apoptosis. The DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling. The RNAseq study consisted of 3 different genotypes (WT, Padi2 single knockout and Padi2/4 double knockout) each with 2 biological replicates.肽基精氨酸脱亚胺酶（Peptidylarginine deiminase, PAD）家族可将精氨酸残基转化为瓜氨酸，该过程被称为瓜氨酸化（citrullination）或脱亚胺化（deimination）。近期研究发现两种PAD家族成员——PAD2与PAD4——可在体外参与激素信号通路调控，本研究旨在体内验证PAD2/PAD4与激素信号通路之间的关联。对Padi2与Padi4单基因敲除（single knockout, SKO）小鼠的初步分析未发现明显生殖缺陷，我们推测这一现象可能源于遗传补偿效应。为验证该假说，我们构建了Padi2/Padi4双基因敲除（double knockout, DKO）小鼠模型，并对该模型小鼠开展了一系列生殖缺陷相关检测。可控繁育实验结果显示，与野生型（Wild Type, WT）对照组相比，DKO繁育组首次产仔的周期更长；且DKO雄性离乳幼鼠的体重显著低于同窝WT雄性幼鼠。此外，DKO雄性小鼠达到性成熟的时间显著长于WT雄性小鼠，且其血清睾酮水平更低。进一步观察发现，DKO雄性小鼠的睾丸体积显著小于WT雄性小鼠，且生殖细胞凋亡率升高。本研究构建的DKO小鼠模型为PAD功能研究提供了全新工具，本研究结果也首次在体内层面证实了PAD家族与激素信号通路之间的关联。本次RNA测序（RNA sequencing, RNA-seq）实验涵盖3种基因型组别——WT、Padi2单基因敲除组以及Padi2/4双基因敲除组，每组均设置2次生物学重复。