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

ABSTRACT: The Par3/Par6/aPKC and the CRB3/Pals1/PATJ polarity complexes are involved in regulating apical ectoplasmic specialization (ES) and blood-testis barrier (BTB) restructuring in the testis. Par6 was a component of the apical ES and the BTB. However, its level was considerably diminished at both sites at stage VIII of the cycle. Par6 also formed a stable complex with Pals1 and JAM-C (a component of the apical ES) in normal testes. When rats were treated with adjudin to induce apical ES restructuring without compromising the BTB, Par6 staining virtually disappeared at the apical ES in misaligned spermatids before their depletion. Additionally, the Par6/Pals1 complex became tightly associated with Src kinase, rendering a loss of association of the Par6/Pals1 complex with JAM-C, thereby destabilizing apical ES to facilitate spermatid loss. Primary Sertoli cell cultures with established functional BTB, but without apical ES, were next used to assess the Par6-based complex on BTB dynamics. When either Par6 or Par3 was knocked down by RNAi in Sertoli cell epithelium, a significant loss of the corresponding protein by approximately 60% in cells vs. controls was detected, alongside with a decline in aPKC after Par6, but not Par3, knockdown. This Par3 or Par6 knockdown also led to a transient loss of selected BTB proteins at the cell-cell interface, thereby compromising the BTB integrity. These findings illustrate that the Par6/Par3-based polarity complex likely coordinates the events of apical ES and BTB restructuring that take place concurrently at the opposing ends of adjacent Sertoli cells in the seminiferous epithelium during spermatogenesis.

SUBMITTER: Wong EW

PROVIDER: S-EPMC2474483 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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

Wong Elissa W P EW Mruk Dolores D DD Lee Will M WM Cheng C Yan CY

Proceedings of the National Academy of Sciences of the United States of America 20080710 28

The Par3/Par6/aPKC and the CRB3/Pals1/PATJ polarity complexes are involved in regulating apical ectoplasmic specialization (ES) and blood-testis barrier (BTB) restructuring in the testis. Par6 was a component of the apical ES and the BTB. However, its level was considerably diminished at both sites at stage VIII of the cycle. Par6 also formed a stable complex with Pals1 and JAM-C (a component of the apical ES) in normal testes. When rats were treated with adjudin to induce apical ES restructurin ...[more]

PMID: 18621709

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Project description:Ectoplasmic specialization (ES) is an actin-rich adherens junction in the seminiferous epithelium of adult mammalian testes. ES is restricted to the Sertoli-spermatid (apical ES) interface, as well as the Sertoli cell-cell (basal ES) interface at the blood-testis barrier (BTB). ES is typified by the presence of an array of bundles of actin microfilaments near the Sertoli cell plasma membrane. These actin microfilament bundles require rapid debundling to convert them from a bundled to branched/unbundled configuration and vice versa to confer plasticity to support the transport of 1) spermatids in the adluminal compartment and 2) preleptotene spermatocytes at the BTB while maintaining cell adhesion. Plastin 3 is one of the plastin family members abundantly found in yeast, plant and animal cells that confers actin microfilaments their bundled configuration. Herein, plastin 3 was shown to be a component of the apical and basal ES in the rat testis, displaying spatiotemporal expression during the epithelial cycle. A knockdown (KD) of plastin 3 in Sertoli cells by RNA interference using an in vitro model to study BTB function showed that a transient loss of plastin 3 perturbed the Sertoli cell tight junction-permeability barrier, mediated by changes in the localization of basal ES proteins N-cadherin and β-catenin. More importantly, these changes were the result of an alteration of the actin microfilaments, converting from their bundled to branched configuration when examined microscopically, and validated by biochemical assays that quantified actin-bundling and polymerization activity. Moreover, these changes were confirmed by studies in vivo by plastin 3 KD in the testis in which mis-localization of N-cadherin and β-catenin was also detected at the BTB, concomitant with defects in the transport of spermatids and phagosomes and a disruption of cell adhesion most notably in elongated spermatids due to a loss of actin-bundling capability at the apical ES, which in turn affected localization of adhesion protein complexes at the site. In summary, plastin 3 is a regulator of actin microfilament bundles at the ES in which it dictates the configuration of the filamentous actin network by assuming either a bundled or unbundled/branched configuration via changes in its spatiotemporal expression during the epithelial cycle.

Project description:Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin ?3 or ?3 chains) derived from the ?6?1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on ?1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and ?1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis.

Project description:Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

Dataset Information

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

Publications

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

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