褪黑素及其受体调控绵羊精原干细胞分化为单倍体精子细胞机制的研究

We have established an in vitro sheep SSCs/testis somatic cells co-culture system to produce haploid sperms, which were microinjected into mature oocytes and hatched blastocysts. However, the efficiency of sheep haploid sperm production solution was much low. Studies have shown that melatonin receptor present in the testicular cell of ovine (a short day breeding species). The present study was designed to explore the molecular mechanism of MLT effect on sheep SSCs/testis somatic cell system differentiation efficiency in vitro in presence or absence of MLT or its receptor inhibitors. The key point is to confirm cell source of MLT/MLT receptor in the testis and in the co-culture system, and to clarify the involvement of signaling pathways. These studies will provide academic basis for the mechanism of ovine spermatogenesis, and the transgenic studies using the germ cells produced by the in vitro SSCs/testis somatic cell differentiation system.

本研究组建立了绵羊SSCs/睾丸体细胞离体分化系统，可分化出功能性单倍体精子(显微注射成熟卵母细胞后，可获得囊胚)。但绵羊SSCs分化为单倍体精子的效率较低。研究表明短日照动物绵羊睾丸中存在褪黑素(MLT)受体。本研究通过添加外源MLT可提高SSCs体外分化为精子细胞的能力，但其作用机制不明确。阐明精子发生机制是调控SSCs体外分化为单倍体精子的关键。本研究将重点探索MLT及其受体调控绵羊精子发生的分子机制。确定MLT及其受体在睾丸和SSCs分化系统中的来源与定位。通过在建立的分化系统中添加MLT及其受体抑制剂，检测睾丸体细胞内睾酮与cAMP含量、相关激素受体表达与抗氧化凋亡相关因子变化；并通过活体埋植MLT，进行睾丸转录组测序，分析体内精子发生相关基因的变化，明晰MLT通过什么信息通路调控精子发生。从而为阐明绵羊精子发生机制和应用SSCs体外分化出单倍体精细胞进行转基因研究提供理论依据。

精原干细胞(SSCs)具有自我更新和分化的能力，能将遗传信息传递给下一代。本研究首次成功在体外诱导分化获得羊功能性单倍体精子细胞。探索了绵羊睾丸体细胞相互作用机制，褪黑素是一种神经内分泌激素，发现褪黑素能够调控Sertoli cells分泌功能，影响Leydig cells睾酮分泌。通过添加褪黑素到羊体外诱导分化体系中发现，其通过核膜受体RORα上调StAR表达，促进睾酮分泌，提高SSCs分化为单倍体的效率2倍多。并建立了褪黑素调控短日照动物类固醇激素调控的分子机制。对SSCs定向诱导分化，可为探讨精子的发生机制，治疗雄性不育提供参考。