Src蛋白激酶家族成员Src和Yes差异性调控大鼠生精过程中细胞连接动态变化的分子机制

Src family kinases are key mediators of protein phosphorylation in multiple cell signaling pathways, which are known to regulate cell differentiation, proliferation, migration and adhesion. Src and Yes are the two most ubiquitously expressed and best studied members, but whether they perform overlapping or discrete cellular functions is not clear. Interestingly, our previous work has shown that Src and Yes are not only the targets of a novel male contraceptive named Adjudin, but also play crucial roles in spermatogenesis. Although the underlying mechanism has not been fully elucidated, Src and Yes were found to be involved in conferring cell adhesion between Sertoli and germ cells, as well as that between adjacent Sertoli cells (i.e., the blood-testis barrier) by modulating the phosphorylation status of integral membrane proteins at the site, which ensures the successful development and differentiation of germ cells. Herein, we aim to provide evidence for the differential regulation of cell junction dynamics by Src and Yes during rat spermatogenesis, unraveling their specific roles in endocytosis and endosome sorting/trafficking of integral membrane proteins in Sertoli cells. In this application, we also propose to study the function of Src in phagocytic activity of Sertoli cells since studies have shown that Src is tightly associated with ultrastructures of the residual body of degenerating spermatids. We believe this study will merit better understanding of the molecular basis of male infertility and spermatogenic failure, some of which are also applicable to studies in cancer and epithelial cells.

Src家族激酶介导的蛋白磷酸化是细胞中多种信号转导途径的关键，在细胞的分化、增殖、迁移和黏附中起重要作用。Src和Yes是其中分布最广、人们研究最多的两个成员，但它们功能上的冗余性或特异性还有待考察。我们前期工作发现，Src和Yes不但是新型男用避孕药Adjudin的靶标，还是精子发生的重要调控者，可通过改变细胞连接中膜整合蛋白的磷酸化水平来调节生精细胞和睾丸支持细胞、以及相邻支持细胞之间（即所形成的血睾屏障结构）的相互作用，从而保证生精细胞的正常发育和分化；不过它们的具体作用机制尚未得到阐明。本研究将围绕大鼠生精过程中细胞连接的动态变化，探讨Src和Yes对支持细胞中膜整合蛋白的内吞、胞内体分选和运输所具有的差异性调控能力，并在此基础上揭示Src参与支持细胞吞噬行为的作用机制。本研究不仅有助于深入理解男性不育及睾丸生精障碍的分子机理，对其它系统（如癌症、上皮细胞）的研究也颇具启发意义。

血睾屏障是哺乳动物血-组织屏障中最坚固的屏障之一，它处于不停的动态变化和重组中。一方面，“旧”的血睾屏障要解体，以使细线前期的精母细胞通过基底小室进入近腔小室继续完成后续发育；另一方面，血睾屏障要保有其完整性，以使精子发生能顺利进行，因此，“新”的血睾屏障需要在细线前期的精母细胞后组建。这一切变化都是通过内吞小泡介导的蛋白运输过程进行，而这些事件具体的分子机制尚不明确。根据前期实验的结果，我们提出假设，Src和Yes很可能在旧血睾屏障解体和新血睾屏障组建中发挥不同的作用。我们利用siRNA介导的基因沉默技术，分别敲低Src和Yes在体外培养的大鼠睾丸支持细胞中的表达，随后利用生物化学等多方面的检测手段，来探明这2个Src家族主要的蛋白激酶，在内吞小泡介导的蛋白运输过程中的差异化作用，包括细胞内吞、膜蛋白的回收和再利用，以及支持细胞的吞噬作用。我们发现，Yes可加速内吞后膜蛋白的胞转作用和膜蛋白的回收再利用，以形成新的血睾屏障；而Src的作用则是使血睾屏障组分蛋白通过核内体发生降解，以使旧血睾屏障解体。利用荧光标记的小球模拟凋亡的生精细胞，我们发现支持细胞的吞噬作用需有Src的参与。最后，我们提出了一个相关的Src和Yes调节血睾屏障动态变化机制的模型，以供未来研究参考之用。