蛋白棕榈酰硫酯1(PPT-1)调节Sertoli细胞紧密连接的机制研究

Spermatogenesis occurs in immune-privileged seminiferous tubules that are protected by the blood-testis barrier (BTB), and BTB disruption results in sperm damage and male infertility. The tight junction (TJ) between the Sertoli cells is a important structure of BTB that anatomically divided seminiferous epithelium into the basal and the adluminal compartments. One of the crucial cellular events during spermatogenesis is to allow the transport of preleptotene spermatocytes across the immunological barrier at stage VIII of the epithelial cycle, and during which the “old” BTB that lie above the preleptotene spermatocytes are disassembled while the “new” BTB underneath the preleptotene spermatocytes connected. Yet the molecular mechanisms underlying these events remain unknown. . Our previous study on obesity and male subfertility showed that high fat diet induced obesity could affect the Sertoli cell junctions, especially the TJ, and lead to poor sperm quality. Moreover, we found a protein, Palmitoyl protein thioesterase 1 (PPT-1), expressed in Sertoli cell, might involve in the maintenance of BTB. However, as reported before, PPT-1 participated in the endocytosis and the regeneration of synaptic vesicles as well as in the recycling of the vesicle components. In addition, the blood-brain barrier (BBB), mainly consisted of cell junctions similar to BTB, was disrupted in Ppt1-KO mice. Therefore, we supposed that PPT-1 in the Sertoli cell might participate in the formation and the recycling of endosomes during endocytosis of the TJ proteins, thus, reuse of the TJ proteins in endosomes fused with the membrane to form new BTB.. Thus, in this study, we will assess the effects of PPT-1 on Sertoli cell using the Ppt-1 conditional knockout animal model. Further, proteins interacted with PPT-1 in Sertoli cell will be investigated by immunoprecipitation. Finally, the variation of mRNA and protein files in the Sertoli cells with PPT-1 deficiency will be identified by microarray and comparative proteomic analysis, and the potential functions of these variant proteins in Sertoli cell as well as their relationship with TJ will be investigated. In conclusion, this applying project is focus on the function of PPT-1 in Sertoli cell and will further improve the mechanism of the BTB dynamics.

血睾屏障为睾丸免疫豁免和精子发生提供稳定的微环境，而Sertoli细胞紧密连接结构是血睾屏障的关键组成。我们前期研究发现：高脂饮食诱发肥胖可以影响雄性生育；且睾丸Sertoli细胞紧密连接分子及结构发生改变。其中，我们首次发现棕榈酰蛋白硫酯酶1（PPT-1）在Sertoli表达且参与维持细胞紧密连接。综合已报道的PPT-1参与神经突触前膜内吞、囊泡运输以及参与维持血脑屏障紧密连接功能，我们推测：PPT-1参与Sertoli细胞对紧密连接蛋白的内吞和内体再循环过程，从而维持细胞紧密连接结构的稳定。本课题将通过：构建Sertoli细胞特异的Ppt-1条件敲除小鼠模型研究PPT-1在Sertoli细胞中的功能；通过PPT-1相互作用蛋白的筛选、表达谱芯片分析、比较蛋白质组学分析，探索PPT-1参与调控细胞紧密连接的分子机制，从而完善Sertoli细胞紧密连接的调控机制。

支持细胞（Sertoli cell）是构成生精上皮的一种重要细胞，通过向发育中的生殖细胞提供营养和能量支持，对维持精子发生至关重要。支持细胞功能紊乱可导致男性精子发生紊乱和生育能力下降。在本项研究中，我们着重研究了棕榈酰蛋白硫酯酶1（PPT1）在支持细胞中的表达和功能。我们发现经胆固醇处理的支持细胞中PPT1的表达显著上升，并且在小鼠支持细胞中特异性敲除ppt1基因，导致敲除小鼠精子质量明显下降和精子畸形率增高，生育能力降低。具体地说，Ppt1缺乏导致支持细胞结构异常并伴随着异常的溶酶体积累和支持细胞胆固醇水平的升高。此外，Ppt1缺乏导致发育中的生殖细胞与生精上皮中的支持细胞粘附性降低，这可能是由于精子数量和运动能力下降以及精子头部畸形发生率增高而导致男性生育能力下降的原因。总之，PPT1通过调节支持细胞的溶酶体功能和胆固醇代谢影响精子质量和男性生育能力。