A lack of commensal microbiota influences the male reproductive tract intergenerationally in mice

The microbiome encompasses the array of microorganisms inhabiting various niches in the body and is necessary for numerous physiological processes, including normal metabolism and a functioning immune system. Not only does the absence of a microbiome in mice impact the exposed animals but also inherited phenotypes in successive generations of progeny, suggesting that the absence of a microbiome impacts the germline and gametes. Indeed, recent research has identified a role of the gut microbiome in contributing to male fertility, in both healthy and disease states. While this link is beginning to be established, the impact of the microbiome on the male reproductive tract remains understudied. Here, we utilized a germ-free mouse model to examine the influence of the absence of microbes on the male reproductive tract. In contrast to mice with an established microbiome, germ-free mice display decreased testicular weight and the prevalence of an epididymitis-like inflammation phenotype. These histopathological changes are accompanied by transcriptomic dysregulation in the reproductive tract of germ-free mice, particularly in the cauda epididymis. Moreover, such transcriptomic changes are transmitted to the next generation with high correlation of gene expression in the cauda epididymis between germ-free mice and their conventionalized (microbiome-restored) male offspring, when compared to control mice. Ultimately, our findings identify the reproductive sequalae of males without a functional microbiome and additionally in their conventionalized offspring, suggesting that the paternal microbiota is an underappreciated contributor to male reproductive function. To investigate the influence of microbiota on the male reproductive tract we collected testis from specific pathogen free control mice (SPF) and germ-free mice (GF) (n = 3-4) and caput and cauda epididymis tissue from SPF, GF and male offspring from GF x SPF parents for RNA-seq. We also investigated the role of T and B cells in directing gene expression changes by performing RNA-seq on caput and cauda epididymal tissue from Rag2 knockout male mice.微生物组（microbiome）指栖息于机体各类微生态龛位的微生物群落，其对于包括正常代谢与免疫系统功能在内的诸多生理过程不可或缺。小鼠体内微生物组的缺失不仅会对暴露的实验个体产生影响，还会引发子代的遗传表型异常，这提示微生物组缺失会对生殖系与配子造成影响。诚然，近期研究已明确肠道微生物组在健康与疾病状态下均对雄性生育能力具有调控作用。尽管这一关联已逐步得到证实，但微生物组对雄性生殖道的影响仍未得到充分研究。本研究利用无菌小鼠模型（germ-free mouse model）探究了微生物组缺失对雄性生殖道的影响。与定植有成熟微生物组的小鼠相比，无菌小鼠表现出睾丸重量降低以及附睾炎样炎症表型的发生率升高。此类组织病理学变化伴随无菌小鼠生殖道内的转录组失调，尤以附睾尾（cauda epididymis）最为显著。此外，这类转录组变化可传递至子代：与对照组小鼠相比，无菌小鼠与其经微生物组重建（conventionalized）的雄性后代的附睾尾基因表达呈现高度相关性。最终，本研究明确了无功能性微生物组的雄性个体及其微生物组重建后代的生殖系统继发效应，提示父代微生物组是雄性生殖功能中一个未被充分重视的调控因素。为探究微生物组对雄性生殖道的影响，我们采集了无特定病原体对照小鼠（SPF）与无菌小鼠（GF）的睾丸组织（样本量n=3-4），并收集了SPF小鼠、GF小鼠以及GF×SPF亲本的雄性后代的附睾头（caput epididymis）与附睾尾组织进行RNA-seq测序。此外，我们还通过对Rag2基因敲除（Rag2 knockout）雄性小鼠的附睾头与附睾尾组织开展RNA-seq测序，探究了T、B细胞在调控基因表达变化中的作用。