mTOR通路对蒙古马血睾屏障建立的调控作用及其机制

The establishment of the blood-testis barrier is one of the earlier events in sexual maturation in mammals. As an important immune barrier in the body, it is necessary for the normal spermtogenesis. At present, studies on the regulation of blood-testis barrier mainly focus on rodent animal models, and largely on the dynamic opening-closing mechanism of the blood testis barrier in adult testes. Our previous RNAseq for the comparison of testes transcriptomes between prepubertal and postpubertal Mongolian stallions showed that between the two groups, there were significant expressional differences in the mTOR pathway-related factors, which are reported as main regulators in the dynamic changes of the blood-testis barrier of adult mice, and these changes well corresponded to the testis state of before and after barrier formation. We speculate that the mTOR pathway play an important role not only in the dynamics of adult blood-testis barrier, as evidenced by other experiments, but also in barrier initial formation. Therefore, this project will further identify the spatiotemporal relationship between the mTOR pathway factors and the blood-testis barrier establishment-related molecules with the method of immuno-histochemistry/fluorescence, and then prove the regulatory function of mTOR on the barrier establishment via histochemistry and cell biology experiments, which followed by the experiments to identify the downtream factors, on which mTOR mainly exerts effects to regulate barrier establishment as well as the mTOR upstream factors such as testosterone that control barrier establishment through mTOR, so that finally figure out a more complete and detailed molecular process of the mTOR signaling pathway mediated blood-testis barrier establishment in Mongolian horses.

血睾屏障的建立是哺乳动物性成熟过程中较早事件之一，作为机体内重要免疫屏障，是精子正常生成必需的。目前血睾屏障调控研究集中在啮齿模式动物，且主要关注成体睾丸血睾屏障动态开合机理，对初情期阶段血睾屏障初建机制研究不多。前期我们RNAseq技术测序对比初情期前后蒙古马睾丸转录组表达发现曾报道的成体鼠血睾屏障动态变化相关mTOR通路多个因子在初情期后有显著增高。由此我们认为mTOR通路不仅如其他实验证明对成体血睾屏障动态变化重要，可能对马性成熟阶段血睾屏障初建也有重要作用。所以本课题将延续前期工作通过免疫组化/荧光法细致鉴定蒙古马睾丸内mTOR通路和血睾屏障初建相关分子之间时空表达关系；通过组织细胞实验解析mTOR对屏障初建的调控作用；体外支持细胞实验向下确定mTOR介导屏障初建主要作用点和向上探寻睾酮等潜在mTOR上游因子，结合体内外数据以期阐明较完整的mTOR通路介导蒙古马血睾屏障初建机制。

鉴于血睾屏障的正确组织方式对雄性动物的生殖是至关重要的，而我们目前对血睾屏障的分子层面的了解几乎全部来自于小鼠模型，为更加清楚的了解马的雄性性成熟过程，本课题原定基于小鼠上所揭示的核心调控机制mTOR信号通路进一步了解马的血睾屏障的建立和调控机制。然而，在研究中，我们发现小鼠和马作为进化上较远的两个物种，竟然在申请人原以为保守的分子表达规律上不尽相同，使课题的开展方向被迫与原有计划产生了不同，我们将以小鼠机制为核心进行研究的思路改为了重头就马论马对马的未成熟和成熟支持细胞进行了原始转录组的探索。结果表明，转录组结果不但确定了我们前期在不同年龄马睾丸组织中核心分子的表达情况，并且为我们进一步围绕马自身的血睾屏障分子机制的研究奠定了基础。实验总体进程虽然受到了三年疫情的严重影响，仍有一部分后续的研究，虽已准备好样品和实验材料却未能按时完成，但是目前已获得的结果为课题进一步按照新的思路挖掘马特有的血睾屏障建立与调控机制提供了重要的基础。课题开展期间发表相关学术论文3篇（1篇SCI，2篇 中文核心），目前正在投稿SCI 论文1篇（预计2023年6月发表），申请人参与撰写《马科学》专著1部，申请相关专利2项（目前实质审查阶段），培养博士研究生1人，硕士毕业生2人，核心研究成员硕士研究生伊敏娜同学2020年保送直博，目前仍在该方向上开展进一步研究。