An in vivo model to study blood-testis barrier dynamics

描述（由申请方提供）：在精子发生期间，位于生精上皮基底室中的发育中的细线期前精母细胞必须在成年大鼠睾丸上皮周期的第VIII阶段穿过血睾屏障（BTB），进入近腔室进行进一步发育。如果没有这种发育中的生殖细胞穿过BTB的及时运动，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过BTB的机制完全未知。这主要是由于缺乏合适的体内模型来研究BTB动力学。在本申请中，P.I.建议开发和广泛表征一种新的模型，以满足这一需求。简而言之，对成年大鼠睾丸局部施用基于闭合蛋白（BTB处的紧密连接（TJ）-整合膜蛋白）的第二细胞外环的22-氨基酸合成肽显示出诱导生精上皮中BTB的可逆破坏。此外，该事件与几种靶蛋白表达的变化有关（例如，转化生长因子-β 3，TGF-β 3和肿瘤坏死因子α，TNF α），模拟体外支持细胞TJ-屏障重组事件。也许最重要的是，这种局部闭合蛋白肽处理还诱导生精上皮的可逆性生殖细胞损失（特别是精子细胞和精母细胞，但不是精原细胞）。私家侦探试图通过描绘与精子发生状态和BTB完整性相关的生精上皮细胞、分子和生化变化的详细时间轴来广泛表征这种新型体内模型。这些研究结果将为该领域的研究人员了解精子发生过程中BTB重组的机制和调控提供可靠的研究模型。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，该模型与目前可用的使用毒物的研究模型（例如，镉和甘油）。首先，可以诱导BTB重组的肽是非细胞毒性的。第二，也许是最重要的，破坏的BTB可以“重新密封”，使该模型特别适合于研究生物学和调节BTB重组精子发生过程中。

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.