Apical Ectoplasmic Specialization Research Articles

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

BDE-209 disrupted the blood-testis barrier integrity by inhibiting estrogen receptorα signaling pathway in Sprague-Dawley rats.

Deca brominated diphenyl ether (BDE-209) is a widely used flame retardant with endocrine-disrupting activity which reportedly caused sperm quality decline and damaged blood-testis barrier (BTB). However, whether BDE-209 exposure led to BTB integrity dysfunction through affecting microtubule cytoskeletal organization and junctions was not well-elucidated. This study aimed to investigate the role of estrogen receptor α (ERα) in BDE-209-mediated perturbation of BTB integrity. Male rats and primary culture Sertoli cells were co-treated with BDE-209 and propylpyrazoletriol (PPT). The data demonstrated that BDE-209 impaired BTB integrity by reducing crucial tight junction-related proteins with ZO-1 and Occludin. Furthermore, the data suggested that BDE-209 diminished the apical ectoplasmic specialization markers with Eps8 and Formin1. In addition, BDE-209 damaged BTB ultrastructure including tight junctions and ectoplasmic specialization structures with broken tight junctions and the absence of actin microfilaments. Further experiments revealed that ERα was triggered in BDE-209-treated Sertoli cells. Unexpectedly, we found that PPT rescued BDE-209-mediated disruption of BTB integrity including tight junction and apical ectoplasmic specialization by activating ERα in Sertoli cells. Taken together, these findings indicated that intratesticular BDE-209 exposure perturbed BTB integrity and destroyed BTB structure by blocking ERα pathway. Our findings provide a new therapeutic target for male reproductive dysfunction.

Identification of active spermatogenesis using a multiphoton microscope.

The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for identifying seminiferous tubules with spermatozoa. Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues. Testicular tissues of 10-week-old normal rats were fixed with 4% paraformaldehyde. Fluorescent labeling of seminiferous tubule nuclei and F-actin was performed, and the specimens were observed without sectioning using a multiphoton microscope. Cryptorchid rats were used as a model lacking elongated spermatids. Multiphoton images were compared with images of normal seminiferous tubules. In addition, seminiferous tubules of 10-week-old normal rats were labeled by testicular interstitial injection of fluorescent probes and observed by a multiphoton microscope without fixation. Terminal deoxynucleotidyl transferase dUTP nick end labeling-stained images of normal and probe-injected testes were compared. In fixed seminiferous tubules, elongated spermatids were identified. In addition, F-actin of apical ectoplasmic specialization was observed around elongated spermatids. Furthermore, spermatogenic stages were identified by an array of nuclei or F-actin. In cryptorchid testes, elongated spermatids and F-actin of the apical ectoplasmic specialization were not observed. In testes injected with fluorescent probes, F-actin of the apical ectoplasmic specialization was observed, and spermatogenic stages were identified without fixation. There was no significant difference in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells per seminiferous tubule between normal and probe-injected testes. Seminiferous epithelium could be observed without sectioning of tissues by fluorescent probes and a multiphoton microscope. Active spermatogenesis was observed by labeling F-actin with and without fixation. Moreover, the toxicity of fluorescent probes was limited. Our method has a potential for live imaging of testicular tissue.

Wuzi-Yanzong-Wan prevents oligoasthenospermia due to TAp73 suppression by affecting cellular junction remodeling in testicular tissue in mice

Ethnopharmacological relevanceWuzi-Yanzong-Wan (WZYZW) is a classic Chinese herbal preparation, which has a significant clinical efficacy in tonifying the kidney and benefiting the sperm, and is widely used in the treatment of oligoasthenospermia with a long history. TAp73 inhibition results in the decrease of sperm quality, but the therapeutic mechanism of WZYZW on oligoasthenospermia caused by TAp73 gene inhibition remains elusive. Aims of studyThe purpose of this study is to investigate whether TAp73 suppression leads to oligoasthenospermia and the application of WZYZW treatment in condition of TAp73 suppression. MethodologyC57BL/6 male mice were injected with Pifithrin-α (2.5 mg/kg) intraperitoneally for 30 days to induce TAp73 suppression model, with WZYZW at 1.0, 2.0 and 4.0 g/kg were administrated in parallel. The blood, testis and epididymis were collected, with organ coefficient calculated. Makler sperm counter was used to analyze the density, motility, survival and malformation rate of sperm. Apoptosis of sperm was analyzed by flow cytometry. Serum hormone levels were determined using ELISA. HE staining and transmission electron microscopy (TEM) were used to observe histopathological changes of testis in blood-testis barrier (BTB), ectoplasmic specialization (ES) and other cell junctions. Expressions of cell adhesion factors including TAp73, Integrin-α6, N-cadherin, Nectin-2 and Occludin were determined by RT-PCR and western blotting. ResultsCompared to control mice, TAp73 inhibition dramatically decreased the epididymal coefficient, sperm quality, and serum testosterone (T) level, while increasing apoptosis in sperm in mice. HE staining and TEM showed that the tight junction (TJ) and apical ES structure were seriously abnormal in the testis in mice with TAp73 inhibition. Additionally, the expression of Occludin protein was elevated, while that of TAp73, Integrin-α6, N-cadherin, and Nectin-2 reduced in model mice. WZYZW treatment ameliorated testicular spermatogenic dysfunctions in TAp73 suppressed mice, restoring the decreased sperm quality, serum T level and testicular histopathological changes of TJ and ES, as well as decreasing sperm malformation rate and apoptosis. Moreover, WZYZW reversed the expressions of Occludin, TAp73, Integrin-α6, N-cadherin and Nectin-2 in TAp73 suppressed mice. ConclusionsBy impairing spermatogenesis and maturation, TAp73 inhibition led to oligoasthenospermia in mice. WZYZW could rescue the oligoasthenospermia associated with TAp73 inhibition via affecting the dynamic remodeling of cellular junctions in testicular tissues in mice.

FYN regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization via its effect on Arp3 in the mouse testis.

FYN is a non-receptor tyrosine kinase of the SRC family that facilitates virus entry across epithelial tight junctions. However, the role of FYN in mammalian testes in maintaining the blood-testis barrier (BTB) integrity and the adhesion of germ cells to Sertoli cells are not well defined. Here, we show that FYN is a component of the BTB and the apical ectoplasmic specialization (ES) at Sertoli-Sertoli and Sertoli-spermatid interfaces, respectively, and is expressed extensively in mouse testes during postnatal development. FYN was shown to be structurally linked to the actin and microtubule-based cytoskeletons. An in vivo model was used to explore the modulatory effect of FYN on BTB and apical ES dynamics within the testes when adult mice were treated intraperitoneally with CdCl2 (3 mg/kg body weight). The CdCl2-induced epithelial restructuring was associated with a transient increase in the interaction between FYN and the actin branching/nucleation protein Arp3, as well as an induction of Arp3 phosphorylation, which possibly lead to actin cytoskeleton remodeling, resulting in BTB damage and germ cell loss in the seminiferous epithelium. Based on the results, we propose a model in which FYN and Arp3 form a protein complex that is responsible for junction reorganization events at the apical ES and the BTB. It is also possible for viruses to break through the BTB and enter the immunoprivileged testicular microenvironment via this mechanism.

PA1 participates in the maintenance of blood\u2013testis barrier integrity via cooperation with JUN in the Sertoli cells of mice

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.

Cell-Cell Interaction-Mediated Signaling in the Testis Induces Reproductive Dysfunction-Lesson from the Toxicant/Pharmaceutical Models.

Emerging evidence has shown that cell-cell interactions between testicular cells, in particular at the Sertoli cell-cell and Sertoli-germ cell interface, are crucial to support spermatogenesis. The unique ultrastructures that support cell-cell interactions in the testis are the basal ES (ectoplasmic specialization) and the apical ES. The basal ES is found between adjacent Sertoli cells near the basement membrane that also constitute the blood-testis barrier (BTB). The apical ES is restrictively expressed at the Sertoli-spermatid contact site in the apical (adluminal) compartment of the seminiferous epithelium. These ultrastructures are present in both rodent and human testes, but the majority of studies found in the literature were done in rodent testes. As such, our discussion herein, unless otherwise specified, is focused on studies in testes of adult rats. Studies have shown that the testicular cell-cell interactions crucial to support spermatogenesis are mediated through distinctive signaling proteins and pathways, most notably involving FAK, Akt1/2 and Cdc42 GTPase. Thus, manipulation of some of these signaling proteins, such as FAK, through the use of phosphomimetic mutants for overexpression in Sertoli cell epithelium in vitro or in the testis in vivo, making FAK either constitutively active or inactive, we can modify the outcome of spermatogenesis. For instance, using the toxicant-induced Sertoli cell or testis injury in rats as study models, we can either block or rescue toxicant-induced infertility through overexpression of p-FAK-Y397 or p-FAK-Y407 (and their mutants), including the use of specific activator(s) of the involved signaling proteins against pAkt1/2. These findings thus illustrate that a potential therapeutic approach can be developed to manage toxicant-induced male reproductive dysfunction. In this review, we critically evaluate these recent findings, highlighting the direction for future investigations by bringing the laboratory-based research through a translation path to clinical investigations.

Autophagy: a multifaceted player in the fate of sperm.

BACKGROUNDAutophagy is an intracellular catabolic process of degrading and recycling proteins and organelles to modulate various physiological and pathological events, including cell differentiation and development. Emerging data indicate that autophagy is closely associated with male reproduction, especially the biosynthetic and catabolic processes of sperm. Throughout the fate of sperm, a series of highly specialized cellular events occur, involving pre-testicular, testicular and post-testicular events. Nonetheless, the most fundamental question of whether autophagy plays a protective or harmful role in male reproduction, especially in sperm, remains unclear.OBJECTIVE AND RATIONALEWe summarize the functional roles of autophagy in the pre-testicular (hypothalamic–pituitary–testis (HPG) axis), testicular (spermatocytogenesis, spermatidogenesis, spermiogenesis, spermiation) and post-testicular (sperm maturation and fertilization) processes according to the timeline of sperm fate. Additionally, critical mechanisms of the action and clinical impacts of autophagy on sperm are identified, laying the foundation for the treatment of male infertility.SEARCH METHODSIn this narrative review, the PubMed database was used to search peer-reviewed publications for summarizing the functional roles of autophagy in the fate of sperm using the following terms: ‘autophagy’, ‘sperm’, ‘hypothalamic–pituitary–testis axis’, ‘spermatogenesis’, ‘spermatocytogenesis’, ‘spermatidogenesis’, ‘spermiogenesis’, ‘spermiation’, ‘sperm maturation’, ‘fertilization’, ‘capacitation’ and ‘acrosome’ in combination with autophagy-related proteins. We also performed a bibliographic search for the clinical impact of the autophagy process using the keywords of autophagy inhibitors such as ‘bafilomycin A1’, ‘chloroquine’, ‘hydroxychloroquine’, ‘3-Methyl Adenine (3-MA)’, ‘lucanthone’, ‘wortmannin’ and autophagy activators such as ‘rapamycin’, ‘perifosine’, ‘metformin’ in combination with ‘disease’, ‘treatment’, ‘therapy’, ‘male infertility’ and equivalent terms. In addition, reference lists of primary and review articles were reviewed for additional relevant publications. All relevant publications until August 2021 were critically evaluated and discussed on the basis of relevance, quality and timelines.OUTCOMES(i) In pre-testicular processes, autophagy-related genes are involved in the regulation of the HPG axis; and (ii) in testicular processes, mTORC1, the main gate to autophagy, is crucial for spermatogonia stem cell (SCCs) proliferation, differentiation, meiotic progression, inactivation of sex chromosomes and spermiogenesis. During spermatidogenesis, autophagy maintains haploid round spermatid chromatoid body homeostasis for differentiation. During spermiogenesis, autophagy participates in acrosome biogenesis, flagella assembly, head shaping and the removal of cytoplasm from elongating spermatid. After spermatogenesis, through PDLIM1, autophagy orchestrates apical ectoplasmic specialization and basal ectoplasmic specialization to handle cytoskeleton assembly, governing spermatid movement and release during spermiation. In post-testicular processes, there is no direct evidence that autophagy participates in the process of capacitation. However, autophagy modulates the acrosome reaction, paternal mitochondria elimination and clearance of membranous organelles during fertilization.WIDER IMPLICATIONSDeciphering the roles of autophagy in the entire fate of sperm will provide valuable insights into therapies for diseases, especially male infertility.

E3 ubiquitin ligase ASB17 is required for spermiation in mice.

BackgroundA major goal of spermiation is to degrade the apical ectoplasmic specialization (ES) junction between Sertoli cells and elongating spermatids in preparation for the eventual disengagement of spermatids into the lumen. E3 ubiquitin ligases mediate the process of ubiquitination and the subsequent proteasomal degradation, but their specific role during spermiation remains largely unexplored.MethodsAnkyrin repeat and SOCS box protein 17 (Asb17)-knockout mice were generated via a CRISPR/Cas9 approach. Epididymal sperm parameters were assessed by a computer-assisted sperm analysis (CASA) system, and morphological analysis of testicular tissues were performed based on histological and immunostaining staining, and transmission electron microscopy (TEM). The interactions between ASB17 and Espin (ESPN) were predicted by HawkDock server and validated through protein pull-down and immunoprecipitation assays.ResultsWe report that ASB17, an E3 ligase, is required for the completion of spermiation and that mice lacking Asb17 are oligozoospermic owing to spermiation failure. ASB17-deficient mice are fertile; however, spermatids exhibit a disorganized ES junction, resulting in retention within the seminiferous epithelium. Mechanistically, ASB17 deficiency leads to excess accumulation of ESPN, an actin-binding essential structural component of the ES. We determined that ASB17 regulates the removal of the ES through ubiquitin mediated protein degradation of ESPN.ConclusionsIn summary, our study describes a role for ASB17 in the regulation of cell-cell junctions between germ cells and somatic cells in the testis. These findings establish a novel mechanism for the regulatory role of E3 ligases during spermatogenesis.

Establishment of an oligoasthenospermia mouse model based on TAp73 gene suppression.

BackgroundOligoasthenospermia is one of the main causes of male infertility. Researchers usually use chemical drugs to directly damage germ cells to prepare oligoasthenospermia models, which disregards the adhesion and migration between spermatogenic cells and Sertoli cells. TAp73 is a critical regulator of the adhesin of germ cell; thus, we sought to explore a novel oligoasthenospermia model based on TAp73 gene suppression.MethodsMice in the Pifithrin‐α group were injected intraperitoneally with 2.5 mg/kg Pifithrin‐α (TAp73 inhibitor) daily for 30 consecutive days. Reproductive hormone levels and epididymal sperm quality, as well as the network morphology of Sertoli cells were tested.ResultsSperm density, motility, and the relative protein and mRNA expression of TAp73 and Nectin 2 were obviously decreased in the Pifithrin‐α group compared with the normal control group. No significant distinction was observed in the relative mRNA and protein expression of ZO‐1. Furthermore, the tight junctions (TJs) and apical ectoplasmic specialization (ES) were destroyed in the Pifithrin‐α group.ConclusionThe above results indicate that we successfully established a new oligoasthenospermia mouse model. This study provides a foundation for further exploration of the roles of TAp73 genes during spermatogenesis and provides new research objects for further oligospermia research and future drug discovery.

AKAP9 supports spermatogenesis through its effects on microtubule and actin cytoskeletons in the rat testis.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

Wnt5a Regulates Junctional Function of Sertoli cells Through PCP-mediated Effects on mTORC1 and mTORC2.

The blood-testis barrier (BTB) and apical ectoplasmic specialization (ES), which are synchronized through the crosstalk of Sertoli cells and Sertoli germ cells, are required for spermatogenesis and sperm release. Here, we show that Wnt5a, a noncanonical Wnt signaling pathway ligand, is predominately expressed in both the BTB and apical ES and has a specific expression pattern during the seminiferous epithelium cycle. We employed siRNA to knockdown Wnt5a expression in testis and Sertoli cells, and then identified elongated spermatids that lost their polarity and were embedded in the seminiferous epithelium. Moreover, phagosomes were found near the tubule lumen. These defects were due to BTB and apical ES disruption. We also verified that the expression level and/or location of BTB-associated proteins, actin binding proteins (ABPs), and F-actin was changed after Wnt5a knockdown in vivo and in vitro. Additionally, we demonstrated that Wnt5a regulated actin dynamics through Ror2-mediated mTORC1 and mTORC2. This study clarified the molecular mechanism of Wnt5a in Sertoli cell junctions through the planar cell polarity (PCP) signaling pathway. Our findings could provide an experimental basis for the clinical diagnosis and treatment of male infertility caused by Sertoli cell junction impairment.

KIF15 Supports Spermatogenesis Via Its Effects on Sertoli Cell Microtubule, Actin, Vimentin, and Septin Cytoskeletons

Throughout spermatogenesis, cellular cargoes including haploid spermatids are required to be transported across the seminiferous epithelium, either toward the microtubule (MT) plus (+) end near the basement membrane at stage V, or to the MT minus (-) end near the tubule lumen at stages VI to VIII of the epithelial cycle. Furthermore, preleptotene spermatocytes, differentiated from type B spermatogonia, are transported across the Sertoli cell blood-testis barrier (BTB) to enter the adluminal compartment. Few studies, however, have been conducted to explore the function of MT-dependent motor proteins to support spermatid transport during spermiogenesis. Herein, we examined the role of MT-dependent and microtubule plus (+) end-directed motor protein kinesin 15 (KIF15) in the testis. KIF15 displayed a stage-specific expression across the seminiferous epithelium, associated with MTs, and appeared as aggregates on the MT tracks that aligned perpendicular to the basement membrane and laid across the entire epithelium. KIF15 also tightly associated with apical ectoplasmic specialization, displaying strict stage-specific distribution, apparently to support spermatid transport across the epithelium. We used a loss-of-function approach by RNAi to examine the role of KIF15 in Sertoli cell epithelium in vitro to examine its role in cytoskeletal-dependent Sertoli cell function. It was noted that KIF15 knockdown by RNAi that reduced KIF15 expression by ~70% in Sertoli cells with an established functional tight junction barrier impeded the barrier function. This effect was mediated through remarkable changes in the cytoskeletal organization of MTs, but also actin-, vimentin-, and septin-based cytoskeletons, illustrating that KIF15 exerts its regulatory effects well beyond microtubules.

Bisphenol S perturbs Sertoli cell junctions in male rats via alterations in cytoskeletal organization mediated by an imbalance between mTORC1 and mTORC2

Bisphenol S (BPS) is now used as an alternative of bisphenol A (BPA), but has been implicated in male reproductive dysfunction—including diminished sperm number and quality and altered hormonal concentrations. However, the mechanisms of action subserving these effects remains unclear. In the present study, BPS at doses of 50 mg/kg bw and 100 mg/kg bw caused defects in the integrity of the blood-testis barrier (BTB) and apical ectoplasmic specialization (ES), and we also delineated an underlying molecular mechanism of action. BPS induced F-actin and α-tubulin disorganization in seminiferous tubules, which in turn led to the truncation of actin filaments and microtubules. Additionally, BPS was found to perturb the expression of the actin-binding proteins Arp3 and Eps8, which are critical for the organization of the actin filaments. mTORC1 and mTORC2 manifest opposing roles in Sertoli cell junctional function, and we demonstrated that mTORC1/rpS6/Akt/MMP9 signaling was increased and that mTORC2/rictor activity was also attenuated. In summary, we showed that BPS-induced disruption of the BTB and apical ES perturbed normal spermatogenic function that was mediated by mTORC1 and mTORC2. The imbalance in mTORC1 and mTORC2, in turn, altered the expression of actin-binding proteins, resulting in the impairment of F-actin and MT organization, and inhibited the expression of junctional proteins at the BTB and apical ES.

The Non-hormonal Male Contraceptive Adjudin Exerts its Effects via MAPs and Signaling Proteins mTORC1/rpS6 and FAK-Y407.

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell-cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.

Microcystin-leucine-arginine induces apical ectoplasmic specialization disassembly

Microcystin-leucine-arginine (MC-LR) has been identified to be a hazardous material to cause hepatotoxicity. In this study, mice were exposed to MC-LR dissolved in drinking water at doses of 1, 10, 20 and 30 μg/L for 90 and 180 days, respectively. We validated MC-LR accelerated spermatid exfoliation and caused large vacuoles in testes, reducing sperm count and increasing percentage of morphologically abnormal sperm. Furthermore, we found MC-LR induced the apical ectoplasmic specialization (ES) disassembly by disrupting F-actin organization. Further studies identified that downregulation of Palladin, the actin crosslinking protein, might be associated with disassembly of the apical ES in mice testis following MC-LR exposure. We also confirmed that MC-LR disrupted the interaction between Palladin and other actin-related proteins and thus impeded the F-actin organization. Additionally, we found that autophagy initiated by AMPK/ULK1 signaling pathway mediated the degradation of Palladin in Sertoli cells challenged with MC-LR. Following exposure to MC-LR, reduced PP2A activity and upregulated expression of LKB1 and CAMKK2 could activate AMPK. In conclusion, these results revealed MC-LR induced the degradation of Palladin via AMPK/ULK1-mediated autophagy, which might result in the apical ES disorder and spermatid exfoliation from spermatogenic epithelium. Our work may provide a new perspective to understand MC-LR-induced male infertility.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator: lesson from the testis

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood–testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell–cell and also Sertoli–spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Filamentous actin disorganization and absence of apical ectoplasmic specialization disassembly during spermiation upon interference with retinoid signaling†

Spermiation is a multiple-step process involving profound cellular changes in both spermatids and Sertoli cells. We have observed spermiation defects, including abnormalities in spermatid orientation, translocation and release, in mice deficient in the retinoic acid receptor alpha (RARA) and upon treatment with a pan-RAR antagonist. To elucidate the role of retinoid signaling in regulating spermiation, we first characterized the time course of appearance of spermiogenic defects in response to treatment with the pan-RAR antagonist. The results revealed that defects in spermiation are indeed among the earliest abnormalities in spermatogenesis observed upon inhibition of retinoid signaling. Using fluorescent dye-conjugated phalloidin to label the ectoplasmic specialization (ES), we showed for the first time that these defects involved improper formation of filamentous actin (F-actin) bundles in step 8–9 spermatids and a failure of the actin-surrounded spermatids to move apically to the lumen and to disassemble the ES. The aberrant F-actin organization is associated with diminished nectin-3 expression in both RARA-deficient and pan-RAR antagonist-treated testes. An abnormal localization of both tyrosinated and detyrosinated tubulins was also observed during spermatid translocation in the seminiferous epithelium in drug-treated testes. These results highlight a crucial role of RAR receptor-mediated retinoid signaling in regulating microtubules and actin dynamics in the cytoskeleton rearrangements, required for proper spermiation. This is critical to understand in light of ongoing efforts to inhibit retinoid signaling as a novel approach for male contraception and may reveal spermiation components that could also be considered as new targets for male contraception.

MTORC1/rpS6 and spermatogenic function in the testis—insights from the adjudin model

mTORC1/rpS6 signaling complex promoted Sertoli blood-testis barrier (BTB) remodeling by perturbing Sertoli cell-cell adhesion site known as the basal ectoplasmic specialization (ES). mTORC1/rpS6 complex also promoted disruption of spermatid adhesion at the Sertoli-spermatid interface called the apical ES. Herein, we performed analyses using the adjudin (a non-hormonal male contraceptive drug under development) model, wherein adjudin was known to perturb apical and basal ES function when used at high dose. Through direct administration of adjudin to the testis, adjudin at doses that failed to perturb BTB integrity per se, overexpression of an rpS6 phosphomimetic (i.e., constitutively active) mutant (i.e., p-rpS6-MT) that modified BTB function considerably potentiated adjudin efficacy. This led to disorderly spatial expression of proteins necessary to maintain the proper cytoskeletal organization of F-actin and microtubules (MTs) across the seminiferous epithelium, leading to germ cell exfoliation and aspermatogenesis. These findings yielded important insights regarding the role of mTORC1/rpS6 signaling complex in regulating BTB homeostasis.