An in vivo model to study blood-testis barrier dynamics

描述（由申请人提供）：在精子发生过程中，在成年大鼠睾丸上皮周期的第八阶段，位于精原上皮基底室的发育中的preleptotene精母细胞必须穿过血睾丸屏障（BTB），进入输卵管室进行进一步发育。如果没有发育中的生殖细胞在BTB中的这种及时运动，精子发生将被破坏，导致不育。虽然这种与精子发生有关的细胞现象已经知道了几十年，但调节BTB动力学以促进生殖细胞穿越BTB的机制是完全未知的。这在很大程度上是由于缺乏合适的体内模型来研究BTB动力学。在这个应用程序中，P.I.建议开发和广泛表征一个新的模型来满足这一需求。简而言之，在成年大鼠睾丸局部注射一种22个氨基酸的合成肽，该肽基于闭塞蛋白的第二细胞外环，封闭蛋白是BTB的紧密连接(TJ)-整体膜蛋白，可诱导精系上皮BTB的可逆破坏。此外，这一事件与几种靶蛋白（如转化生长因子-3、TGF- 3和肿瘤坏死因子a、TNFa）表达的变化有关，这些蛋白在体外模拟了Sertoli细胞tj屏障重组事件。也许最重要的是，这种局部闭塞肽治疗也诱导了精子上皮中可逆性的生殖细胞损失（特别是精细胞和精母细胞，但不包括精原细胞）。P.I.试图通过描述与精子发生状态和BTB完整性相关的精原上皮细胞，分子和生化变化的详细时间表，广泛表征这种新的体内模型。这些研究结果将为研究人员了解精子发生过程中BTB重组的机制和调控提供一个可靠的研究模型。我们的主要目标是开发一种创新的体内模型来研究BTB动力学，这与目前使用毒物（例如镉和甘油）的研究模型有很大不同。首先，可以诱导BTB重组的肽是非细胞毒性的。其次，可能也是最重要的一点，被破坏的BTB可以被“重新密封”，这使得该模型非常适合研究精子发生过程中BTB重新组装的生物学和调控。

DESCRIPTION (provided by applicant): During spermatogenesis, developing preleptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at stage VIII of the epithelial cycle in adult rat testes, entering the adluminal compartment for further development. Without this timely movement of developing germ cells across the BTB, spermatogenesis will be disrupted, leading to infertility. While this cellular phenomenon pertinent to spermatogenesis is known for decades, the mechanism(s) that regulates BTB dynamics to facilitate germ cells to traverse the BTB is entirely unknown. This by and large is due to the lack of a suitable in vivo model to study BTB dynamics. In this application, the P.I. proposes to develop and extensively characterize a novel model to meet this need. In brief, local administration of a 22-amino acid synthetic peptide based on the second extracellular loop of occludin, a tight junction (TJ)-integral membrane protein at the BTB, to adult rat testes was shown to induce reversible disruption of BTB in the seminiferous epithelium. Also, this event was associated with changes in the expression of several target proteins (e.g., transforming growth factor ¿-3, TGF-¿3, and tumor necrosis factor a, TNFa) that mimicked the Sertoli cell TJ-barrier restructuring events in vitro. Perhaps the most important of all, this local occludin peptide treatment also induced reversible germ cell loss (in particular spermatids and spermatocytes, but not spermatogonia) from the seminiferous epithelium. The P.I. seeks to extensively characterize this novel in vivo model by delineating the detailed timeline of cellular, molecular and biochemical changes in the seminiferous epithelium correlating with the status of spermatogenesis and the integrity of the BTB. Results of these studies will yield a reliable study model for investigators in the field to understand the mechanism and regulation of BTB restructuring during spermatogenesis. Our primary goal is to develop an innovative in vivo model to study BTB dynamics which is significantly different from currently available study models using toxicants (e.g., cadmium and glycerol). First, the peptide that can induce BTB restructuring is non-cytotoxic. Second and perhaps most importantly, the disrupted BTB can be "resealed", making this model uniquely suitable to study the biology and regulation of BTB re-assembly during spermatogenesis.