Par3/Par6 polarity complex coordinates apical ectoplasmic specialization and blood–testis barrier restructuring during spermatogenesis

Wong, Elissa W.P.; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

doi:10.1073/pnas.0801527105

Cited by 117 publications

(227 citation statements)

References 25 publications

(40 reference statements)

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“…TGF-β3 is a known regulator of BTB (2). In order to test the possibility that Cdc42 is a downstream mediator of TGF-β3, we first examined if TGF-β3 affects the activation of Cdc42 in a Sertoli cell culture system known to establish functional and structural barrier that mimics the BTB in vivo (9). As Cdc42 switches between active (i.e., GTP-bound) and inactive (i.e., GDP-bound) states, we performed a pull-down assay using GSTp21-binding domain (i.e., PBD) of PAK-1 to precipitate active Cdc42 (Cdc42-GTP).…”

Section: Resultsmentioning

confidence: 99%

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Wong

Mruk

Lee

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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101

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In the testis, the blood-testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood-tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium behind the BTB. Cytokines (e.g., TGF-β3) are known to regulate BTB dynamics by enhancing the endocytosis of integral membrane proteins and their intracellular degradation. This thus reduces the levels of proteins above the spermatocytes in transit at the BTB, causing its disruption after testosterone-induced new tight junction (TJ) fibrils are formed behind these cells. By using Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo, Cdc42 was shown to be a crucial regulator that mediated the TGF-β3-induced BTB disruption. TGF-β3 was shown to activate Cdc42 to its active GTP-bound form. However, an inactivation of Cdc42 by overexpressing its dominant-negative mutant T17N in Sertoli cell epithelium was shown to block the TGF-β3-induced acceleration in protein endocytosis. Consequently, this prevented the disruption of Sertoli cell TJ permeability barrier and redistribution of TJ proteins (e.g., CAR and ZO-1) from the cell-cell interface to cell cytosol caused by TGF-β3. In summary, Cdc42 is a crucial regulatory component in the TGF-β3-mediated cascade of events that leads to the disruption of the TJ fibrils above the preleptotene spermatocytes to facilitate their transit.cytokines | GTPases | protein endocytosis | seminiferous epithelial cycle | spermatogenesis

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Section: Resultsmentioning

confidence: 99%

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Wong

Mruk

Lee

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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101

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“…Furthermore, polarity proteins such as Par6 and 14-3-3 (also known as Par5) are highly expressed at the apical ES but at the convex side of the elongating spermatids. These polarity proteins are also integrated components of this anchoring device (25,26) and function to maintain proper spermatid orientation (25). It is likely that the concerted effects of polarity proteins Par6 and 14-3-3 at the convex side and N-WASP-Arp2/3 complex at the concave side of the spermatid head provide the necessary machineries to maintain spermatid orientation.…”

Section: Discussionmentioning

confidence: 99%

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

Lie

Chan²,

Mruk³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood-testis barrier (BTB) must remain intact during the transit of spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with spermatid movement and proper spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of spermatid transit plus a loss of proper orientation in the epithelium, and a "tightened" Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.actin dynamics | ectoplasmic specialization | seminiferous epithelial cycle | spermiogenesis

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“…For histological analysis, testes were fixed in Bouin's fixative, embedded in paraffin, sectioned with a microtome, and stained with hematoxylin-eosin. Images were acquired using an Olympus BX61 Fluorescence Microscope equipped with a DP71 digital camera, as described earlier (37,38). Images in TIFF format were analyzed using Adobe PhotoShop in Creative Suite (version 3.0).…”

Section: Animals and Antibodiesmentioning

confidence: 99%

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

Wan

Mruk

et al. 2013

American Journal of Physiology-Endocrinology and Metabolism

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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...

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Par3/Par6 polarity complex coordinates apical ectoplasmic specialization and blood–testis barrier restructuring during spermatogenesis

Cited by 117 publications

References 25 publications

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

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

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Par3/Par6 polarity complex coordinates apical ectoplasmic specialization and blood–testis barrier restructuring during spermatogenesis

Cited by 117 publications

References 25 publications

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

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

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