Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

Abstract BackgroundThe blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown. ResultsHere, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1. ConclusionOverall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.

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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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00254.2013 ◽

2013 ◽

Vol 305 (6) ◽

pp. E687-E699 ◽

Cited By ~ 37

Author(s):

Hin-Ting Wan ◽

Dolores D. Mruk ◽

Stephen Y. T. Li ◽

Ka-Wai Mok ◽

Will M. Lee ◽

...

Keyword(s):

Actin Polymerization ◽

Early Stage ◽

Regulatory Protein ◽

Growth Factor Receptor ◽

Actin Organization ◽

Rat Testis ◽

Mammalian Expression ◽

Ectoplasmic Specialization ◽

Stage Viii

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-Tyr397, 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-Tyr397 that regulates spermatid adhesion at the apical ES in vivo.

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Formin 1 Regulates Microtubule and F-Actin Organization to Support Spermatid Transport During Spermatogenesis in the Rat Testis

Endocrinology ◽

10.1210/en.2016-1133 ◽

2016 ◽

Vol 157 (7) ◽

pp. 2894-2908 ◽

Cited By ~ 16

Author(s):

Nan Li ◽

Dolores D. Mruk ◽

Elizabeth I. Tang ◽

Will M. Lee ◽

Chris K. C. Wong ◽

...

Keyword(s):

Sertoli Cells ◽

Protein Trafficking ◽

Cell Shape ◽

Binding Protein ◽

Cell Movement ◽

Seminiferous Epithelium ◽

Intracellular Protein ◽

Down Regulation ◽

Actin Organization ◽

Support Cell

Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.

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PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis

Journal of Histology ◽

10.1155/2014/634290 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9

Author(s):

Junya Suzuki ◽

Sadaki Yokota

Keyword(s):

Sertoli Cells ◽

Rna Silencing ◽

Binding Proteins ◽

Spermatogenic Cells ◽

P Bodies ◽

Cytoplasmic Rna ◽

Dsrna Binding ◽

Chromatoid Bodies ◽

Pachytene Spermatocytes ◽

Ectoplasmic Specialization

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including PRKRA, TRBP, and Dicer. RISC localizes to P-bodies. The nuage of the spermatogenic cells has function similar to the P-bodies. We study whether PRKRA localizes to nuage of spermatogenic cells of rat and mouse. PRKRA localized to four types of nuage structures, including aggregates of 60–90 nm particles, irregularly-shaped perinuclear granules, and intermitochondrial cement of pachytene spermatocytes, and chromatoid bodies of round spermatids. In addition, PRKRA is associated with dense material surrounding tubulobulbar complexes and with the ectoplasmic specialization. The results suggest that PRKRA functions in the nuage as an element of RNA silencing system and plays unknown role in the ectoplasmic specialization and at the tubulobulbar complexes of Sertoli cells attaching the head of late spermatids.

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Hormonal regulation of spermatid binding

Journal of Cell Science ◽

10.1242/jcs.100.3.623 ◽

1991 ◽

Vol 100 (3) ◽

pp. 623-633

Author(s):

D.F. Cameron ◽

K.E. Muffly

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Hormonal Regulation ◽

Unit Area ◽

Junctional Complex ◽

Cell Cytoplasm ◽

Coculture Model ◽

Ectoplasmic Specialization

A Sertoli-spermatid coculture model is described in which a large percentage (greater than 76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD = the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH + testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described ‘Sertoli ectoplasmic specialization’ observed adjacent to post-step 8 spermatids in vivo. FSH + testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH + testosterone and is associated with the peripheral distribution of actin and vinculin.

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Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes

Reproduction ◽

10.1530/rep-10-0464 ◽

2011 ◽

Vol 141 (5) ◽

pp. 571-580 ◽

Cited By ~ 48

Author(s):

Ka-Wai Mok ◽

Dolores D Mruk ◽

Pearl P Y Lie ◽

Wing-Yee Lui ◽

C Yan Cheng

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Sertoli Cells ◽

Seminiferous Epithelium ◽

Self Renewal ◽

Male Contraceptive ◽

Cellular Target ◽

Apical Compartment ◽

Junction Type ◽

Ectoplasmic Specialization

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood–testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.

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The Dynamic of the Apical Ectoplasmic Specialization between Spermatids and Sertoli Cells: The Case of the Small GTPase Rap1

BioMed Research International ◽

10.1155/2014/635979 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 14

Author(s):

Giovanna Berruti ◽

Chiara Paiardi

Keyword(s):

Sertoli Cells ◽

Human Population ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Small Gtpase ◽

Therapeutic Approaches ◽

Tubule Lumen ◽

Male Patients ◽

Ectoplasmic Specialization

Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception.

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rpS6 Regulates Blood-Testis Barrier Dynamics By Affecting F-Actin Organization and Protein Recruitment

Endocrinology ◽

10.1210/en.2012-1665 ◽

2012 ◽

Vol 153 (10) ◽

pp. 5036-5048 ◽

Cited By ~ 45

Author(s):

Ka-Wai Mok ◽

Dolores D. Mruk ◽

Bruno Silvestrini ◽

C. Yan Cheng

Keyword(s):

Sertoli Cell ◽

Actin Filament ◽

Sertoli Cells ◽

Plasma Membranes ◽

Permeability Barrier ◽

Actin Organization ◽

Filament Bundles ◽

Mtorc1 Pathway ◽

Protein Recruitment ◽

Blood Testis Barrier

Abstract During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII–IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB.

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The Candidate Male Contraceptive Agent, Gamendazole, Affects Actin Organization and Vinculin Based Focal Contact Distribution in Sertoli Cells In Vitro.

Biology of Reproduction ◽

10.1093/biolreprod/78.s1.206b ◽

2008 ◽

Vol 78 (Suppl_1) ◽

pp. 206-207

Author(s):

Jeffrey Cotitta ◽

Sedide Ozturk ◽

Terri Kinzy ◽

Sudhakar Jakkaraj ◽

Ramappa Chakrasali ◽

...

Keyword(s):

Sertoli Cells ◽

Focal Contact ◽

Actin Organization ◽

Male Contraceptive ◽

Contact Distribution ◽

Contraceptive Agent

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Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00113.2014 ◽

2014 ◽

Vol 307 (9) ◽

pp. E738-E753 ◽

Cited By ~ 14

Author(s):

N. Ece Gungor-Ordueri ◽

Ciler Celik-Ozenci ◽

C. Yan Cheng

Keyword(s):

Mammalian Cells ◽

Early Stage ◽

Seminiferous Epithelium ◽

Permeability Barrier ◽

Actin Organization ◽

Rat Testis ◽

Ectoplasmic Specialization ◽

Stage Viii

In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation.

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