Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling [scRNAseq_Female]

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral phenotypes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational phenotypes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline. To interrogate the role of the glucocorticoid receptor (GR) in female germ cells, we performed single cell RNA-seq on germ cells following genetic deletion of GR. To generate the GR deletion allele, mouse strain Nr3c1tm1.1Jda harboring a floxed allele of GR exon 3 was crossed to beta-actin cre mice (Tmem163Tg(ACTB-cre)2Mrt) to generate a heterozygous deletion of GR. Mice with heterozygous deletion of GR were crossed to OCT4-GFP mice (Tg(Pou5f1-EGFP)2Mnn) to facilitate germ cell labeling. As the GR deletion allele is homozygous lethal at birth, mice were maintained as heterozygotes, and crossed to each other to generate embryos with homozygous deletion of GR for the experiment. To enrich for germ cells, fetal ovaries from GR deletion and WT controls were collected at E15.5, pooled according to genotype (n=2 GR deletion embryos, n=4 WT embryos), and were digested and FACS sorted for the OCT4-GFP transgene. OCT4-GFP positive germ cells were mixed back in with sorted OCT4-GFP negative somatic cells at a ratio of 60:40, and samples were submitted for single cell RNA-seq using the 10X Illumina platform.长期以来，学界已明确生理应激可通过激素失调损害哺乳动物的生殖能力；而日益增多的证据显示，妊娠前或妊娠期间经历的应激还可对子代健康产生负面影响。妊娠期生理应激的啮齿动物模型可诱导持续长达三代的神经与行为表型异常，这提示应激信号能够在生殖系中诱导持久的表观遗传改变。使用糖皮质激素类激素进行处理，足以重现生理应激模型中观察到的跨代表型。此类激素可结合并激活糖皮质激素受体（glucocorticoid receptor, GR）——一种配体诱导型转录因子，因此GR介导的信号通路可能参与了应激诱导表型的跨代遗传。 本研究揭示了小鼠生殖系中GR表达的动态时空调控模式，观察到GR在胎儿卵母细胞、围产期及成体精原细胞中均有表达。功能实验结果显示，胎儿卵母细胞本身对GR信号变化存在内在缓冲机制：无论是GR基因敲除还是用地塞米松（dexamethasone）进行GR激动处理，均未改变胎儿卵母细胞的转录组特征或减数分裂进程。与之相反，雄性生殖系对糖皮质激素介导的信号通路敏感，具体表现为可调控精原细胞内的RNA剪接，但这并不会影响生育能力。 综上，本研究表明GR在生殖系中存在性别二态性功能，为理解应激如何通过生殖系调控遗传信息传递的机制迈出了重要一步。 为探究糖皮质激素受体（GR）在雌性生殖细胞中的作用，我们对GR基因敲除后的生殖细胞开展了单细胞RNA测序（single cell RNA-seq）。为构建GR敲除等位基因，我们将携带GR第3外显子floxed等位基因的小鼠品系Nr3c1tm1.1Jda与β-actin-Cre小鼠（Tmem163Tg(ACTB-cre)2Mrt）杂交，获得GR杂合缺失小鼠。将GR杂合缺失小鼠与OCT4-GFP小鼠（Tg(Pou5f1-EGFP)2Mnn）杂交，以实现生殖细胞的荧光标记。由于GR敲除等位基因在出生时即纯合致死，我们将小鼠维持为杂合子品系，并通过杂合子互交获得用于实验的GR纯合缺失胚胎。 为富集生殖细胞，我们在胚胎发育第15.5天（E15.5）收集GR敲除组与野生型（wild type, WT）对照组的胎卵巢，按基因型分组（n=2只GR敲除胚胎，n=4只WT胚胎），经消化后通过荧光激活细胞分选（FACS）分选OCT4-GFP阳性的生殖细胞。随后将OCT4-GFP阳性细胞与分选得到的OCT4-GFP阴性体细胞以60:40的比例混合，使用10X Illumina平台对样本进行单细胞RNA测序。