The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, New York;
Am J Physiol Endocrinol Metab. 2013 Sep 15;305(6):E687-99. doi: 10.1152/ajpendo.00254.2013. Epub 2013 Jul 23.
During spermatogenesis, the molecular mechanism that confers spermatid adhesion to the Sertoli cell at the apical ectoplasmic specialization (apical ES), a testis-specific F-actin-rich adherens junction, in the rat testis remains elusive. Herein, the activated form of focal adhesion kinase (FAK), p-FAK-Tyr(397), a component of the apical ES that was expressed predominantly and stage specifically in stage VII-early stage VIII tubules, was found to be a crucial apical ES regulator. Using an FAK-Y397E phosphomimetic mutant cloned in a mammalian expression vector for its transfection vs. FAK and vector alone in adult rat testes in vivo, its overexpression was found to cause defects in spermiation. These defects in spermiation were manifested by entrapment of spermatids in the seminiferous epithelium in late stage VIII-X tubules and were mediated by a disruption on the spatiotemporal expression and/or mislocalization of actin regulatory protein actin-related protein 3, which induces branched actin polymerization, epidermal growth factor receptor pathway substrate 8 (an actin barbed end capping and bundling protein), and palladin (an actin cross-linking and bundling protein). This thus perturbed changes of F-actin organization at the apical ES to facilitate spermiation, which also led to a concomitant alteration in the distribution and upregulation of adhesion proteins nectin-2 and nectin-3 at the apical ES. As such, nectin-2 and -3 remained at the apical ES to anchor step 19 spermatids on to the epithelium, delaying spermiation. These findings illustrate a mechanistic pathway mediated by p-FAK-Tyr(397) that regulates spermatid adhesion at the apical ES in vivo.
在精子发生过程中，赋予精子顶端质膜下特化（apical ES）处精子黏附到支持细胞的分子机制仍然难以捉摸，这是一种睾丸特异性富含 F-肌动蛋白的黏附连接。在此，发现顶端 ES 的激活形式的粘着斑激酶（FAK），p-FAK-Tyr（397），作为顶端 ES 的一个组成部分，在 VII 期至早期 VIII 期小管中主要且特异表达，是一个关键的顶端 ES 调节剂。使用 FAK-Y397E 磷酸模拟突变体克隆在哺乳动物表达载体中，用于其在成年大鼠睾丸中的转染与 FAK 和载体单独转染进行比较，发现其过表达会导致精子发生缺陷。这些精子发生缺陷表现为晚期 VIII-X 期小管中的精子被束缚在生精上皮中，这是由肌动蛋白调节蛋白肌动蛋白相关蛋白 3 的时空表达和/或定位错误介导的，肌动蛋白相关蛋白 3 诱导分支肌动蛋白聚合、表皮生长因子受体途径底物 8（肌动蛋白帽端封闭和束状蛋白）和 paladin（肌动蛋白交联和束状蛋白）。这从而扰乱了顶端 ES 处 F-肌动蛋白组织的变化，以促进精子发生，这也导致了顶端 ES 处黏附蛋白 nectin-2 和 nectin-3 的分布和上调发生伴随性改变。因此，nectin-2 和 -3 仍然位于顶端 ES 处，将第 19 步精子锚定在生精上皮上，从而延迟精子发生。这些发现说明了一个由 p-FAK-Tyr（397）介导的机制途径，该途径调节体内顶端 ES 处的精子黏附。
