Biology of the Blood-Testis Barrier

描述(申请人提供)：在哺乳动物的睾丸中，精子发生在生精小管的上皮中，生精小管是每天产生数百万精子的功能单位。血-睾丸屏障(BTB)位于生精上皮基底膜(BM)附近，具有把关和围栏的功能，分别限制有毒物质等有害物质的跨细胞和跨细胞运输，以维持精子的发生。BTB还在物理上将生精上皮分为基生室和腺腔室；因此，减数分裂I和II以及减数分裂后精子细胞的发育都发生在BTB后面的专门微环境中(即管腔室)。BTB是哺乳动物中最紧密的血-组织屏障之一，因为BTB的细胞连接被垂直于相对的Sertoli细胞质膜的肌动蛋白细丝束加强，而Sertoli细胞膜只存在于睾丸中。然而，BTB是一个动态的结构，在上皮周期的第VIII阶段进行广泛的重组，以便前纤毛虫精母细胞可以穿过BTB进入腺体间室进一步发育。现在公认的是，有功能的BTB对精子发生至关重要。在过去的资助期间完成的研究表明，BTB的功能受生物活性多肽的调节：(1)在顶端外质特化(顶端ES，支持-精子细胞界面上的睾丸特异性粘连连接)；(2)在基底膜(BM)，由称为顶端ES-BTB-BM轴的功能轴介导。简而言之，层粘连蛋白和胶原链的生物活性片段在根尖释放 ES和BM分别作为自分泌因子调节BTB在这一轴上的功能。在这里，我们将继续这一重要的研究，以了解调控BTB功能的潜在分子机制(S)。首先，我们将揭示这些片段通过作用于支持细胞的F-肌动蛋白网络(如肌动蛋白结合和调节蛋白)来调节支持细胞黏附和支持细胞重组的机制(S)。我们还将使用最先进的技术和方法研究内体介导的蛋白质运输和小调节RNA在这些事件中的参与。其次，我们将确定生物分子，如细胞因子，类固醇和基质金属蛋白酶，在调节这些生物活性多肽在上皮细胞周期中特定阶段的产生方面的生物学意义。 精子发生。这些发现将对理解BTB在精子发生中的生物学作用产生重大影响。更重要的是，这些发现将为开发新型可逆的男性非激素避孕药提供新的线索。例如，来自顶端ES和BM的多肽可以被开发成避孕药，扰乱BTB功能并损害精子发生。此外，这些发现可能导致开发新的治疗方法来干预毒物引起的男性生殖功能障碍，这是男性生育力下降的主要原因之一。例如，环境毒物镉调节其在BTB中的作用，导致男性生殖功能障碍；这些发现为设计和合成小分子提供了关键信息，这些小分子可以逆转或阻止镉诱导的BTB中断，从而导致男性不育。

DESCRIPTION (provided by applicant): In the mammalian testis, spermatogenesis takes place in the epithelium of the seminiferous tubule, the functional unit that produces millions of spermatozoa daily. The blood-testis barrier (BTB), located near the basement membrane (BM) of the seminiferous epithelium, confers gate-keeper and the fence function, which restricts the paracellular and transcellular transport of harmful substances such as toxicants across the barrier, respectively, to maintain spermatogenesis. The BTB also physically divides the seminiferous epithelium into a basal and an adluminal compartment; thus, meiosis I and II, and post-meiotic spermatid development take place in a specialized microenvironment (i.e., the adluminal compartment) behind the BTB. The BTB is one of the tightest blood-tissue barriers in mammals since cell junctions at the BTB are reinforced by bundles of actin filaments that lie perpendicular to apposing Sertoli cell plasma membranes, which are only found in the testis. However, the BTB is a dynamic structure, undergoing extensive restructuring at stage VIII of the epithelial cycle so that preleptotene spermatocytes can cross the BTB and enter the adluminal compartment for further development. It is now well established that a functional BTB is crucial for spermatogenesis. Studies completed in the past grant period have shown that BTB function is regulated by biologically active peptides released (i) at the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction at the Sertoli-spermatid interface), and (ii) at th basement membrane (BM) mediated by a functional axis designated the apical ES-BTB-BM axis. In short, biologically active fragments of laminin and collagen chains released at the apical ES and the BM, respectively, act as autocrine factors to regulate BTB function in this axis. Herein, we will continue this important research to understand the underlying molecular mechanism(s) that regulates BTB function. First, we will unravel the mechanism(s) by which these fragments regulate Sertoli cell adhesion and BTB restructuring via their effects on the F-actin network at the BTB, such as actin binding and regulatory proteins. We will also examine the involvement of endosome-mediated protein trafficking and small regulatory RNAs in these events using state-of-the-art techniques and approaches. Second, we will identify the biological significance of biomolecules, such as cytokines, steroids and matrix metalloproteases, in regulating the stage-specific production of these bioactive peptides during the epithelial cycle of spermatogenesis. These findings will have a significant impact in understanding the biology of BTB in spermatogenesis. More important, these findings will shed new light on the development of novel reversible non-hormonal contraceptives for men. For instance, peptides derived from the apical ES and BM can be developed into contraceptives to perturb BTB function and impair spermatogenesis. Additionally, these findings can lead to the development of new therapeutic approaches to interfere with toxicant-induced male reproductive dysfunction, which is one of the leading causes of the decline in male fertility. For instance, the environmental toxicant cadmium mediates its effects at the BTB to induce male reproductive dysfunction; these findings provide crucial information in the design and synthesis of small molecules that either reverse or block cadmium-induced BTB disruption that leads to male infertility.