An in vivo model to study blood-testis barrier dynamics

描述(申请人提供)：在精子发生过程中，在成年大鼠睾丸的上皮周期的第VIII阶段，位于生精上皮基底室的发育中的细线期前精母细胞必须穿过血-睾丸屏障(BTB)，进入腺泡室才能进一步发育。如果发育中的生殖细胞没有这种及时的跨越BTB的移动，精子发生将被扰乱，导致不育。虽然这种与精子发生相关的细胞现象几十年前就已为人所知，但调控结缔组织动力学以促进生殖细胞穿越结缔组织的机制(S)却完全未知。这在很大程度上是由于缺乏合适的体内模型来研究BTB的动力学。在这项申请中，P.I.建议开发并广泛描述一种新的模型来满足这一需求。简而言之，在成年大鼠睾丸中，局部给予22个氨基酸的合成肽，其基础是闭合素的第二细胞外环，这是一种紧密连接(TJ)完整的膜蛋白，可诱导生精上皮中BTB的可逆性破坏。此外，这一事件还与几种目标蛋白(如转化生长因子-3、转化生长因子-β3和肿瘤坏死因子α，TNFa)在体外模拟Sertoli细胞TJ屏障重建事件的表达变化有关。也许最重要的是，这种局部封闭多肽治疗还导致生精上皮可逆性生殖细胞丢失(特别是精子细胞和精母细胞，但不是精原细胞)。P.I.试图通过描绘生精上皮细胞、分子和生化变化的详细时间线来广泛表征这一新的活体模型，这些变化与精子发生的状态和BTB的完整性相关。这些研究结果将为该领域的研究人员提供一个可靠的研究模型，以了解精子发生过程中BTB重组的机制和调节。我们的主要目标是开发一种创新的体内模型来研究BTB的动力学，这与目前使用毒物(如镉和甘油)的研究模型有很大不同。首先，能够诱导BTB重组的多肽是非细胞毒性的。第二，也可能是最重要的是，被破坏的BTB可以重新密封，使这个模型独特地适合于研究BTB在精子发生过程中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.