The biology of blood-testis barrier dynamics

描述(申请人提供)：这项建议的长期目标是以成年大鼠睾丸为研究模型，了解精子发生过程中血-睾丸屏障(BTB)动态的生物学和调节。具体地说，它试图揭开在生精上皮周期的第VIII阶段发生的前细线期和细线期精母细胞穿过btb时，维持btb完整性的机制(S)。这项研究很重要，因为它回答了该领域一个悬而未决的问题，即睾丸利用生化和/或分子机制(S)调节BTB的及时“重组”，以允许精母细胞转运，同时维持BTB的免疫屏障功能。这些信息还有助于开发创新的方法来设计新的药物(例如，通过破坏生精上皮中的生殖细胞功能来发挥作用的男性避孕药)，这些药物可以针对BTB背后的生精上皮。最近的研究表明，细胞因子(如肿瘤坏死因子、肿瘤坏死因子和转化生长因子-2/-3、转化生长因子-2或转化生长因子-3)对BTB的完整性有破坏作用，而睾酮则促进BTB的功能，说明这些分子的相反作用对于维持BTB在精子发生过程中的及时“打开”和“关闭”，同时促进生殖细胞跨越屏障是至关重要的。既然两人都是肿瘤坏死因子？和转化生长因子-β3是支持细胞和生殖细胞的产物，睾酮是间质细胞的产物(但雄激素受体主要存在于支持细胞)，在睾丸中，这些分子可能在精子发生过程中释放到BTB微环境中，调节BTB的动态。在这项申请中，私家侦探将检验这一假设，即肿瘤坏死因子？BTB通过对基底膜蛋白(如胶原蛋白、蛋白水解酶和蛋白水解酶抑制物)的作用来调节BTB的动态变化，而基底膜蛋白又反过来调节BTB的整体膜蛋白的稳态水平。这将确定BTB的地位，以促进或禁止细线期前期/细线期精母细胞通过屏障(具体目标1)。此外，P.I.将测试这一假设，即细胞因子和睾酮通过对BTB的内吞作用、循环作用、内吞体介导胞内降解和BTB上完整的膜蛋白的跨细胞/重新定位(特定目标2)的不同作用来调节它们对BTB的相反作用。拟议的研究将使用生物化学、分子生物学和细胞生物学的尖端技术进行。P.I.还将开发和表征一种新的体外模型，使用支持-生殖细胞共培养来研究生殖细胞跨越BTB的运动规律，这将成为该领域研究人员的一个有价值的研究工具。简而言之，这项建议解决了精子发生中一个长期存在的谜团，即关于BTB在上皮周期中的生物学和调控。公共卫生相关性：本项目以成年大鼠睾丸为研究模型，试图了解在生精上皮周期中调节血-睾丸屏障(BTB)动态的机制(S)。BTB必须在精子发生过程中短暂地“开放”或“重组”，以促进精母细胞通过屏障。这反过来又提供了一个独特的机会，将一种药物，如破坏细胞黏附的男性避孕药，运送到支架桥后面发挥其作用，如果调节支架桥适时打开的机制(S)是已知的话。简而言之，这项建议试图了解BTB在精子发生过程中的动态生物学。它还提供了对：(I)开发男性避孕的新方法，以及(Ii)不明原因的男性不育的原因的新见解。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the biology and regulation of blood-testis barrier (BTB) dynamics during spermatogenesis using the adult rat testis as a study model. Specifically, it seeks to unravel the mechanism(s) that maintains BTB integrity as preleptotene and leptotene spermatocytes traverse the BTB that occurs at stage VIII of the seminiferous epithelial cycle of spermatogenesis. This study is important since it answers an open question in the field regarding the biochemical and/or molecular mechanism(s) utilized by the testis that regulates the timely 'restructuring' of the BTB to permit spermatocyte transit while maintaining the immunological barrier function at the BTB. This information is also helpful to develop innovative approaches to design new drugs (e.g., male contraceptives that exert their effects by disrupting germ cell function in the seminiferous epithelium) that can be targeted to the seminiferous epithelium behind the BTB. Recent studies have shown that cytokines (e.g., tumor necrosis factor ?, TNF?, and transforming growth factor-?2/-3, TGF-?2 or -?3) have a disruptive effect on BTB integrity, whereas testosterone promotes BTB function, illustrating that the opposing effects of these molecules are crucial to maintain the timely "opening" and "closing" of the BTB during spermatogenesis while facilitating germ cell movement across the barrier. Since both TNF? and TGF-?3 are products of Sertoli and germ cells, and testosterone is a product of Leydig cells (but the androgen receptor resides mostly on the Sertoli cell), in the testis, it is likely that these molecules are released into the BTB microenvironment during spermatogenesis to regulate BTB dynamics. In this application, the P.I. will examine the hypothesis that TNF? regulates BTB dynamics via its effects on basement membrane proteins, such as collagens, proteases and protease inhibitors, which, in turn, regulates the steady-state levels of integral membrane proteins at the BTB. This will determine the status of the BTB to facilitate or prohibit the transit of preleptotene/leptotene spermatocytes across the barrier (Specific Aim 1). Furthermore, the P.I. will test the hypothesis that cytokines and testosterone mediate their opposing effects on the BTB via their differential actions on endocytosis, recycling, endosome-mediated intracellular degradation and transcytosis/relocation of integral membrane proteins at the BTB (Specific Aim 2). The proposed studies will be performed using cutting-edge techniques of biochemistry, molecular biology, and cell biology. The P.I. will also develop and characterize a novel in vitro model using Sertoli-germ cell cocultures to investigate the regulation of germ cell movement across the BTB, which can become a valuable research tool for investigators in the field. In short, this proposal addresses a long-standing mystery in spermatogenesis regarding the biology and regulation of the BTB during the epithelial cycle. PUBLIC HEALTH RELEVANCE: This project seeks to understand the mechanism(s) that regulates blood-testis barrier (BTB) dynamics during the seminiferous epithelial cycle of spermatogenesis using the adult rat testis as a study model. The BTB must `open' or `restructure' transiently during spermatogenesis to facilitate the transit of spermatocytes across the barrier. This, in turn, poses a unique opportunity to transport a drug, such as a male contraceptive that disrupts cell adhesion, behind the BTB to exert its effects, if the mechanism(s) that regulates the timely opening of the BTB is known. In short, this proposal seeks to understand the biology of BTB dynamics during spermatogenesis. It also offers new insight into: (i) developing novel approaches for male contraception, and (ii) the causes of unexplained male infertility.