p-FAK-Tyr³⁹⁷ regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

Abstract

Wan HT, Mruk DD, Li SY, Mok KW, Lee WM, Wong CK, Cheng CY. p-FAK-Tyr 397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization. Am J Physiol Endocrinol Metab 305: E687-E699, 2013. First published July 23, 2013; doi:10.1152/ajpendo.00254.2013.-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. spermatogenesis; focal adhesion kinase; focal adhesion kinase mutant; adherens junction; actin filament bundles; testis IN THE RAT TESTIS, step 1 spermatids derived from secondary spermatocytes via meiosis undergo extensive morphological changes during spermiogenesis (8,10,20). Besides changes in cell shape via 19 steps in which round spermatids (step 1) transform into elongated spermatids (step 19), step 1 spermatids residing in the adluminal compartment but near the basal compartment and adjacent to the basement membrane must traverse back-and-forth the seminiferous epithelium during the epithelial cycle of spermatogenesis (8,19,22). As such, fully developed spermatids (i.e., spermatozoa) can be lined up at the luminal edge of the tubule lumen at late stage VIII of the epithelial cycle for spermiation (10,20,24). During spermiogenesis, a testis-specific adherens junction (AJ) appears at the Sertoli-spermatid (step 8) interface at stage VIII of the cycle known as apical ectoplasmic specialization (apical ES) (6, 27, 32). Once apical ES forms, it replaces d...

rpS6 regulates blood-testis barrier dynamics via its effects on MMP-9 mediated by Akt signaling

“…This in vitro system is widely used by investigators to study BTB dynamics (Janecki et al, 1992;Lie et al, 2012;Nicholls et al, 2009;Qiu et al, 2013). Furthermore, findings obtained by using this in vitro system have been reproduced in studies in vivo (Lui et al, 2003;Qiu et al, 2013;Su et al, 2012;Wan et al, 2013), illustrating its physiological relevance. As such, the effect of p-rpS6 on the Sertoli cell tight junction barrier was first investigated by quantifying changes in the tight junction permeability across the Sertoli cell epithelium following the overexpression of different constructs.…”

Sertoli cells are the target of environmental toxicants in the testis – a mechanistic and therapeutic insight

“…In short, toxicants induce restructuring of the apical ES in early and elongating spermatids by disrupting the actin microfilament organization, pushing immature spermatids to undergo ‘spermiation’; toxicants also induce similar changes at the basal ES/BTB, forcing the BTB to enter a stage VIII-like restructuring, reversing the timely events of the epithelial cycle (Figure 2). These changes in the localization of actin regulatory proteins following toxicant-induced germ cell defoliation from the testis may be regulated, at least in part, by FAK, in particular p-FAK-Tyr 407 and p-FAK-Tyr 397 , based on recent studies [56,100]. For instance, p-FAK-Tyr 397 is known to confer apical ES integrity and its presence is detected at the apical ES until spermiation at late stage VIII [56,101,102].…”

Regulation of blood–testis barrier by actin binding proteins and protein kinases

“…Studies in vitro (Lie, et al 2012) and in vivo (Wan, et al 2013) based on the use of phosphomimetic vs. non-phosphorylatable mutants have shown that the two phosphorylated (activated) forms of FAK, namely p-FAK-Tyr 407 and p-FAK-Tyr 397 , are crucial regulators of ES including the basal ES/BTB and the apical ES in the rat testis (for reviews, see (Wan, et al 2014, Xiao, et al 2014b)). For instance, p-FAK-Tyr 407 is highly expressed at the basal ES/BTB in the testis based on studies using dual-labeled immunofluorescence analysis and confocal microscopy (Lie, et al 2012).…”