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Regulation of the blood-testis barrier by a local axis in the testis: role of laminin ?2 in the basement membrane.

ABSTRACT: Laminin ?2 is one of the constituent components of the basement membrane (BM) in adult rat testes. Earlier studies that used a mouse genetic model have shown that a deletion of laminin ?2 impedes male fertility by disrupting ectoplasmic specialization (ES; a testis-specific, actin-rich anchoring junction) function along the length of Sertoli cell in the testis. This includes ES at the Sertoli cell-elongating/elongated spermatid interface, which is known as apical ES and possibly the Sertoli-Sertoli cell interface, known as basal ES, at the blood-testis barrier (BTB). Studies have also illustrated that there is a local regulatory axis that functionally links cellular events of spermiation that occur near the luminal edge of tubule lumen at the apical ES and the basal ES/BTB remodeling near the BM at opposite ends of the seminiferous epithelium during the epithelial cycle, known as the apical ES-BTB-BM axis. However, the precise role of BM in this axis remains unknown. Here, we show that laminin ?2 in the BM serves as the crucial regulator in this axis as laminin ?2, likely its 80-kDa fragment from the C terminus, was found to be transported across the seminiferous epithelium at stages VIII-IX of the epithelial cycle, from the BM to the luminal edge of the tubule, possibly being used to modulate apical ES restructuring at these stages. Of more importance, a knockdown of laminin ?2 in Sertoli cells was shown to induce the Sertoli cell tight junction permeability barrier disruption via changes in localization of adhesion proteins at the tight junction and basal ES at the Sertoli cell BTB. These changes were found to be mediated by a disruption of F-actin organization that was induced by changes in the spatiotemporal expression of actin binding/regulatory proteins. Furthermore, laminin ?2 knockdown also perturbed microtubule (MT) organization by considerable down-regulation of MT polymerization via changes in the spatiotemporal expression of EB1 (end-binding protein 1), a +TIP (MT plus-end tracking protein). In short, laminin ?2 in the BM seems to play a crucial role in the BTB-BM axis by modulating BTB dynamics during spermatogenesis.-Gao, Y., Mruk, D., Chen, H., Lui, W.-Y., Lee, W. M., Cheng, C. Y. Regulation of the blood-testis barrier by a local axis in the testis: role of laminin ?2 in the basement membrane.

SUBMITTER: Gao Y

PROVIDER: S-EPMC5240664 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Regulation of the blood-testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane.

Gao Ying Y Mruk Dolores D Chen Haiqi H Lui Wing-Yee WY Lee Will M WM Cheng C Yan CY

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20161104 2

Laminin α2 is one of the constituent components of the basement membrane (BM) in adult rat testes. Earlier studies that used a mouse genetic model have shown that a deletion of laminin α2 impedes male fertility by disrupting ectoplasmic specialization (ES; a testis-specific, actin-rich anchoring junction) function along the length of Sertoli cell in the testis. This includes ES at the Sertoli cell-elongating/elongated spermatid interface, which is known as apical ES and possibly the Sertoli-Sert ...[more]

PMID: 27815338

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Project description:Spermatogenesis takes place in the epithelium of the seminiferous tubules of the testes, producing millions of spermatozoa per day in an adult male in rodents and humans. Thus, multiple cellular events that are regulated by an array of signaling molecules and pathways are tightly coordinated to support spermatogenesis. Here, we report findings of a local regulatory axis between the basement membrane (BM), the blood-testis barrier (BTB), and the apical ectoplasmic specialization (apical ES; a testis-specific, actin-rich adherens junction at the Sertoli cell-spermatid interface) to coordinate cellular events across the seminiferous epithelium during the epithelial cycle. In short, a biologically active fragment, noncollagenous 1 (NC1) domain that is derived from collagen chains in the BM, was found to modulate cell junction dynamics at the BTB and apical ES. NC1 domain from the collagen ?3(IV) chain was cloned into a mammalian expression vector, pCI-neo, with and without a collagen signal peptide. We also prepared a specific Ab against the purified recombinant NC1 domain peptide. These reagents were used to examine whether overexpression of NC1 domain with high transfection efficacy would perturb spermatogenesis, in particular, spermatid adhesion (i.e., inducing apical ES degeneration) and BTB function (i.e., basal ES and tight junction disruption, making the barrier leaky), in the testis in vivo We report our findings that NC1 domain derived from collagen ?3(IV) chain-a major structural component of the BM-was capable of inducing BTB remodeling, making the BTB leaky in studies in vivo Furthermore, NC1 domain peptide was transported across the epithelium via a microtubule-dependent mechanism and is capable of inducing apical ES degeneration, which leads to germ cell exfoliation from the seminiferous epithelium. Of more importance, we show that NC1 domain peptide exerted its regulatory effect by disorganizing actin microfilaments and microtubules in Sertoli cells so that they failed to support cell adhesion and transport of germ cells and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium. This local regulatory axis between the BM, BTB, and the apical ES thus coordinates cellular events that take place across the seminiferous epithelium during the epithelial cycle of spermatogenesis.-Chen, H., Mruk, D. D., Lee, W. M., Cheng, C. Y. Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane-derived noncollagenous 1 domain peptide.

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:BACKGROUND: Functional inactivation of the tumor suppressor Smad4 in colorectal and pancreatic carcinogenesis occurs coincident with the transition to invasive growth. Breaking the basement membrane (BM) barrier, a prerequisite for invasive growth, can be due to tumor induced proteolytic tissue remodeling or to reduced synthesis of BM molecules by incipient tumor cells. Laminin-332 (laminin-5), a heterotrimeric BM component composed of alpha 3-, beta 3- and gamma 2-chains, has recently been identified as a target structure of Smad4 and represents the first example for expression control of an essential BM component by a tumor and invasion suppressor. Biochemically Smad4 is a transmitter of signals of the TGFbeta superfamily of cytokines. We have reported previously, that Smad4 functions as a positive transcriptional regulator of constitutive and of TGFbeta-induced transcription of all three genes encoding Laminin-332, LAMA3, LAMB3 and LAMC2. METHODS: Promoter-reporter constructs harboring 4 kb upstream regions, each of the three genes encoding Laminin-322 as well as deletion and mutations constructs were established. Promoter activities and TGFbeta induction were assayed through transient transfections in Smad4-negative human cancer cells and their stable Smad4-positive derivatives. Functionally relevant binding sites were subsequently confirmed through chromatin immunoprecipitation. RESULTS: Herein, we report that Smad4 mediates transcriptional regulation through three different mechanisms, namely through Smad4 binding to a functional SBE site exclusively in the LAMA3 promoter, Smad4 binding to AP1 (and Sp1) sites presumably via interaction with AP1 family components and lastly a Smad4 impact on transcription of AP1 factors. Whereas Smad4 is essential for positive regulation of all three genes, the molecular mechanisms are significantly divergent between the LAMA3 promoter as compared to the LAMB3 and LAMC2 promoters. CONCLUSION: We hypothesize that this divergence in modular regulation of the three promoters may lay the ground for uncoupled regulation of Laminin-332 in Smad4-deficient tumor cells in response to stromally expressed cytokines acting on budding tumor cells.

Dataset Information

Regulation of the blood-testis barrier by a local axis in the testis: role of laminin ?2 in the basement membrane.

Publications

Regulation of the blood-testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane.

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