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In this study, we have explored the role of the KATNB1 gene, a microtubule‐severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co‐localize with α‐tubulin showing a unique stage‐specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ‐barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium‐induced blood–testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin‐ and microtubule‐based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin‐ and microtubule‐based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology. [ABSTRACT FROM AUTHOR]

Background: 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. Objectives: Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues. Materials and methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Daily, people are exposed to chemicals and environmental compounds such as bisphenols (BPs). These substances are present in more than 80% of human fluids. Human exposure to BPs is associated with male reproductive health disorders. Some of the main targets of BPs are intercellular junction proteins of the blood‐testis barrier (BTB) in Sertoli cells because BPs alter the expression or induce aberrant localization of these proteins. In this systematic review, we explore the effects of BP exposure on the expression of BTB junction proteins and the characteristics of in vivo studies to identify potential gaps and priorities for future research. To this end, we conducted a systematic review of articles. Thirteen studies met our inclusion criteria. In most studies, animals treated with bisphenol‐A (BPA) showed decreased occludin expression at all tested doses. However, bisphenol‐AF treatment did not alter occludin expression. Cx43, ZO‐1, β‐catenin, nectin‐3, cortactin, paladin, and claudin‐11 expression also decreased in some tested doses of BP, while N‐cadherin and FAK expression increased. BP treatment did not alter the expression of α and γ catenin, E‐cadherin, JAM‐A, and Arp 3. However, the expression of all these proteins was altered when BPA was administered to neonatal rodents in microgram doses. The results show significant heterogeneity between studies. Thus, it is necessary to perform more research to characterize the changes in BTB protein expression induced by BPs in animals to highlight future research directions that can inform the evaluation of risk of toxicity in humans. [ABSTRACT FROM AUTHOR]

Abstract Background Oligoasthenospermia 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. Methods Mice 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. Results Sperm 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. Conclusion The 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.

Sertoli cells contribute to the formation of the blood-testis barrier (BTB), which is necessary for normal spermatogenesis. Recently, microRNAs (miRNAs) have emerged as posttranscriptional regulatory elements in BTB function during spermatogenesis. Our previous study has shown that miR-181c or miR-181d (miR-181c/d) is highly expressed in testes from boars at 60 days old compared with at 180 days old. Herein, we found that overexpression of miR-181c/d via miR-181c/d mimics in murine Sertoli cells (SCs) or through injecting miR-181c/d-overexpressing lentivirus in murine testes perturbs BTB function by altering BTB-associated protein distribution at the Sertoli cell–cell interface and F-actin organization, but this in vivo perturbation disappears approximately 6 weeks after the final treatment. We also found that miR-181c/d represses Sertoli cell proliferation and promotes its apoptosis. Moreover, miR-181c/d regulates Sertoli cell survival and barrier function by targeting platelet-activating factor acetylhydrolase 1b regulatory subunit 1 (Pafah1b1) gene. Furthermore, miR-181c/d suppresses PAFAH1B1 expression, reduces the complex of PAFAH1B1 with IQ motif-containing GTPase activating protein 1, and inhibits CDC42/PAK1/LIMK1/Cofilin pathway which is required for F-actin stabilization. In total, our results reveal the regulatory axis of miR-181c/d-Pafah1b1 in cell survival and barrier function of Sertoli cells and provide additional insights into miRNA functions in mammalian spermatogenesis. [ABSTRACT FROM AUTHOR]

Aim: Depending on the findings resulting from the knock-downing of ezrin and of fascin 1 in vivo, we aim to show the defects or disruption of the blood-testis barrier (BTB) structure and F-actin bundling after Perfluorooctanesulfonate (PFOS) treatment in primary Sertoli cell culture. Methods: Study Design: Primary Sertoli cell isolation was occurred with control and PFOS-treated (20M) groups. Sertoli cells were prepared for both experiments as 0.5 x 106 cell/ml. Method: Dual-labeled immunofluorescence analysis to assess co-localization of fascin 1 with ezrin both in Sertoli cells was performed, and Co-IP, by using lysates of seminiferous tubules, was performed using actin and ezrin proteins to identify specific protein-protein interaction with fascin 1. Results: Firstly, we showed that ezrin and fascin 1, which were components of the ectoplasmic specialization were co-localized in the Sertoli cells and also they were interacted each other. Secondly, we indicated that they were dislocated in the PFOS-treated Sertoli cells in vitro. Because of PFOS (20M), the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory proteins at the cell-cell interface necessary to maintain localization of actin-regulatory at the BTB.Conclusion: In summary, these findings suggest that ezrin and fascin 1 can work together to preserve BTB integrity by regulating F-actin organization in the PFOS-mediated Sertoli cell disruption.

Background Retinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis. Methods Sperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR. Results Rai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups. Conclusion In summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

Blood-testis barrier (BTB) and apical ectoplasmic specialization (ES) serve as structural supports for germ cell (GC) development. We demonstrated that the Sertoli cell (SC)-specific coxsackievirus and adenovirus receptor (CXADR) knockout (SC-CXADR-/-), but not the GC-specific knockout, impaired spermatogenesis. An increase in GC apoptosis and premature loss of elongated spermatids were observed in SC-CXADR-/- testes. The BTB function was compromised in SC-CXADR-/- testes with dysregulation of oocludin and zonula occludens-1 expression at the basal compartment of the seminiferous epithelium. An integrated omics analyses confirmed that altered gene ontology terms identified in SC-CXADR-/- testes are highly associated with spermatid development and differentiation, spermatogenesis, and sperm motility and are considered as unique testicular function terms. Leptin, Nasp, Tektin3, Larp 7, and acrosin, which are highly associated with male fertility, were found to be down-regulated in SC-CXADR-/- testes. Based on the data from the omics analyses, we employed the CXADR-deficient SC model to further investigate the molecular mechanisms involved. We unraveled that SC-CXADRs are required for β-catenin inactivation and cell division cycle protein 42 (Cdc42) activation, resulting in maintaining the integrity and function of the BTB and apical ES as well as inhibiting gene transcription, such as the Myc gene, in the testes. We demonstrated for the first time that CXADR is an important mediator governing β-catenin and Cdc42 signaling that is essential for spermatogenesis. The molecular mechanisms identified herein may provide new insights to unravel the novel functions and signaling cascades of CXADR in other key CXADR-expressing tissues. [ABSTRACT FROM AUTHOR]

Objective: The bioavailability of the non-hormonal male contraceptive adjudin is low in rats due to the blood-testis barrier (BTB). This study was designed to examine if F5-peptide, an endogenously produced reversible BTB modifier, could enhance the bioavailability of adjudin to affect spermatogenesis and provide a contraceptive effect in rats while reducing systemic toxicity.Study Design: We overexpressed F5-peptide in adult male rats (n=10 rats; with 3 or 4 rats for each of the three different experiments noted in the three regimens) by intratesticular injection of a mammalian expression vector pCI-neo (pCI-neo/F5-peptide) vs. empty vector alone (pCI-neo/Ctrl) to be followed by treatment with adjudin by oral gavage at a dose of 10 or 20 mg/kg. The status of spermatogenesis was assessed by histological analysis and dual-labeled immunofluorescence analysis on Day 16. To assess fertility, we allowed treated males (n=3-4 rats) to mate with mature female rats (n=3-4) individually, and assessed the number of pups on Days 23, 36 and 82 to assess fertility and reversibility.Results: All 4 treated rats overexpressed with F5-peptide and low-dose adjudin were infertile by Day 36, and half of these rats were fertile by Day 82, illustrating reversibility. However, overexpression of F5-peptide alone (or low-dose adjudin alone) had no effects on fertility in n=3 rats. These findings were consistent with the histology data that illustrated the BTB modifier F5-peptide promoted the action of adjudin to induce germ cell exfoliation, mediated by changes in cytoskeletal organization of F-actin and microtubules across the epithelium, thereby reducing the systemic toxicity of adjudin.Conclusion: In this proof-of-concept study, it was shown that overexpression of the F5-peptide prior to administration of adjudin to rats at a low (and ineffective dose by itself) was found to induce reversible male infertility.Implications: Overexpression of F5-peptide, an endogenously produced biomolecule in the testis known to induce BTB remodeling, enhanced the contraceptive effect of adjudin in rats, supporting proof of concept studies of BTB disrupters in men. [ABSTRACT FROM AUTHOR]

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis. [ABSTRACT FROM AUTHOR]

Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests.

Testicular varicocele, a dilation of the veins of the pampiniform plexus thought to increase testicular temperature via venous congestion, is commonly associated with male infertility. Significant study has clarified the negative impact of varicocele on semen parameters and more recent work has shed light on its detrimental effects on the molecular and ultrastructural features of sperm and the testicular microenvironment, as well as more clearly defined the positive impacts of treatment on couples′ fertility. The relationship between varicocele and testicular endocrine function, while known for some time based on histologic evaluation, has become more apparent in the clinical setting with a growing link between varicocele and hypogonadism. Finally, in the pediatric setting, while future study will clarify the impact of varicocele on fertility and testicular function, recent work supports a parallel effect of varicocele in adolescents and adults, suggesting a re-evaluation of current treatment approaches in light of the progressive nature of the condition and potential increased risk of future disease.

A natural disaster leading to accumulation of environmental contaminants may have substantial effects on the male reproductive system. Our aim was to compare and assess semen parameters in a normospermic population residing in the Southern Louisiana, USA area pre- and post-Hurricane Katrina. We retrospectively evaluated semen analyses data (n = 3452) of 1855 patients who attended the Tulane University Andrology/Fertility Clinic between 1999 and 2013. The study inclusion criteria were men whose semen analyses showed ≥ 1.5 ml volume; ≥15 million ml -1 sperm concentration; ≥39 million total sperm count; ≥40% motility; >30% morphology, with an abstinence interval of 2-7 days. After the inclusion criteria applied to the population, 367 normospermic patients were included in the study. Descriptive statistics and group-based analyses were performed to interpret the differences between the pre-Katrina (Group 1, 1999-2005) and the post-Katrina (Group 2, 2006-2013) populations. There were significant differences in motility, morphology, number of white blood cell, immature germ cell count, pH and presence of sperm agglutination, but surprisingly there were no significant differences in sperm count between the two populations. This long-term comparative analysis further documents that a major natural disaster with its accompanied environmental issues can influence certain semen parameters (e.g., motility and morphology) and, by extension, fertility potential of the population of such areas.

In this experimental prospective study, we aimed to analyze the effect of transient scrotal hyperthermia on the male reproductive organs, from the perspective of sperm parameters, semen plasma biochemical markers, and oxidative stress, to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis. Two groups of volunteers (10 per group) received testicular warming in a 43°C water bath 10 times, for 30 min each time: group 1: 10 consecutive days; group 2: once every 3 days. Sperm parameters, epididymis and accessory sex gland function, semen plasma oxidative stress and serum sex hormones were tested before treatment and in the 16-week recovery period after treatment. At last, we found an obvious reversible decrease in sperm concentration (P = 0.005 for Group 1 and P= 0.008 for Group 2 when the minimums were compared with baseline levels, the same below), motility (P = 0.009 and 0.021, respectively), the hypoosmotic swelling test score (P = 0.007 and 0.008, respectively), total acrosin activity (P = 0.018 and 0.009, respectively), and an increase in the seminal plasma malondialdehyde concentration (P = 0.005 and 0.017, respectively). The decrease of sperm concentration was greater for Group 2 than for Group 1 (P = 0.031). We concluded that transient scrotal hyperthermia seriously, but reversibly, negatively affected the spermatogenesis, oxidative stress may be involved in this process. In addition, intermittent heat exposure more seriously suppresses the spermatogenesis compared to consecutive heat exposure. This may be indicative for clinical infertility etiology analysis and the design of contraceptive methods based on heat stress.

Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis.

Cell polarity in the adult mammalian testis refers to the polarized alignment of developing spermatids during spermiogenesis and the polarized organization of organelles (e.g., phagosomes, endocytic vesicles, Sertoli cell nuclei, Golgi apparatus) in Sertoli cells and germ cells to support spermatogenesis. Without these distinctive features of cell polarity in the seminiferous epithelium, it is not possible to support the daily production of millions of sperm in the limited space provided by the seminiferous tubules in either rodent or human males through the adulthood. In short, cell polarity provides a novel mean to align spermatids and the supporting organelles (e.g., phagosomes, Golgi apparatus, endocytic vesicles) in a highly organized fashion spatially in the seminiferous epithelium during the epithelial cycle of spermatogenesis. This is analogous to different assembling units in a manufacturing plant such that as developing spermatids move along the “assembly line” conferred by Sertoli cells, different structural/functional components can be added to (or removed from) the developing spermatids during spermiogenesis, so that functional spermatozoa are produced at the end of the assembly line. Herein, we briefly review findings regarding the regulation of cell polarity in the testis with specific emphasis on developing spermatids, supported by an intriguing network of regulatory proteins along a local functional axis. Emerging evidence has suggested that cell cytoskeletons provide the tracks which in turn confer the unique assembly lines in the seminiferous epithelium. We also provide some thought-provoking concepts based on which functional experiments can be designed in future studies. [ABSTRACT FROM AUTHOR]

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier “leaky.” Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons. [ABSTRACT FROM AUTHOR]

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 a3(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 a3(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. [ABSTRACT FROM AUTHOR]

Wuzi-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. The purpose of this study is to investigate whether TAp73 suppression leads to oligoasthenospermia and the application of WZYZW treatment in condition of TAp73 suppression. C57BL/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. Compared 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. By 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. [Display omitted] • Effects of WZYZW on sperm quality, hormone levels and testis tissue in TAp73 inhibition model mice. • WZYZW significantly improved cellular junctions in testicular tissue in TAp73 suppressed mice. • WZYZW significantly upregulated TAp73, Integrin-α6, N-cadherin, Nectin-2 and downregulated Occludin gene and protein expressions. [ABSTRACT FROM AUTHOR]

Sertoli cells isolated from rodents or humans and cultured in vitro are known to establish a functional tight junction (TJ)-permeability barrier that mimics the blood–testis barrier (BTB) in vivo. This model has been widely used by investigators to study the biology of the TJ and the BTB. Studies have shown that environmental toxicants (e.g., perfluorooctanesulfonate (PFOS), bisphenol A (BPA) and cadmium) that exert their disruptive effects to induce Sertoli cell injury using this in vitro model are reproducible in studies in vivo. Thus, this in vitro system provides a convenient approach to probe the molecular mechanism(s) underlying toxicant-induced testis injury but also to provide new insights in understanding spermatogenesis, such as the biology of cell adhesion, BTB restructuring that supports preleptotene spermatocyte transport, and others. Herein, we provide a brief and critical review based on studies using this in vitro model of Sertoli cell cultures using primary cells isolated from rodent testes vs. humans to monitor environmental toxicant-mediated Sertoli cell injury. In short, recent findings have shown that environmental toxicants exert their effects on Sertoli cells to induce testis injury through their action on Sertoli cell actin- and/or microtubule-based cytoskeleton. These effects are mediated via their disruptive effects on actin- and/or microtubule-binding proteins. Sertoli cells also utilize differential spatiotemporal expression of these actin binding proteins to confer plasticity to the BTB to regulate germ cell transport across the BTB. [ABSTRACT FROM AUTHOR]

Ezrin, radixin, moesin and merlin (ERM) proteins are highly homologous actin-binding proteins that share extensive sequence similarity with each other. These proteins tether integral membrane proteins and their cytoplasmic peripheral proteins (e.g., adaptors, nonreceptor protein kinases and phosphatases) to the microfilaments of actin-based cytoskeleton. Thus, these proteins are crucial to confer integrity of the apical membrane domain and its associated junctional complex, namely the tight junction and the adherens junction. Since ectoplasmic specialization (ES) is an F-actin-rich testis-specific anchoring junction-a highly dynamic ultrastructure in the seminiferous epithelium due to continuous transport of germ cells, in particular spermatids, across the epithelium during the epithelial cycle-it is conceivable that ERM proteins are playing an active role in these events. Although these proteins were first reported almost 25 years and have since been extensively studied in multiple epithelia/endothelia, few reports are found in the literature to examine their role in the actin filament bundles at the ES. Studies have shown that ezrin is also a constituent protein of the actin-based tunneling nanotubes (TNT) also known as intercellular bridges, which are transient cytoplasmic tubular ultrastructures that transport signals, molecules and even organelles between adjacent and distant cells in an epithelium to coordinate cell events that occur across an epithelium. Herein, we critically evaluate recent data on ERM in light of recent findings in the field in particular ezrin regarding its role in actin dynamics at the ES in the testis, illustrating additional studies are warranted to examine its physiological significance in spermatogenesis. [ABSTRACT FROM AUTHOR]

In the mammalian testis such as in rats, a unique actin-rich cell-cell adherens junction (AJ) known as ectoplasmic specialization (ES) is found in the seminiferous epithelium. ES is conspicuously found between Sertoli cells near the basement membrane known as the basal ES, which together with tight junction (TJ), gap junction, and desmosome constitute the blood-testis barrier (BTB). The BTB, in turn, anatomically divides the seminiferous epithelium into the basal and the adluminal (apical) compartment. On the other hand, ES is also found at the Sertoli-spermatid interface known as apical ES which is the only anchoring device for developing step 8–19 spermatids during spermiogenesis. One of the most typical features of the ES is the array of actin microfilament bundles that lie perpendicular to the Sertoli cell plasma membrane and are sandwiched in-between the cisternae of endoplasmic reticulum and the Sertoli cell plasma membrane. While these actin filament bundles confer the adhesive strength of Sertoli cells at the BTB and also spermatids in the adluminal compartment, they must be rapidly re-organized from their bundled to unbundled/branched configuration and vice versa to provide plasticity to the ES so that preleptotene spermatocytes and spermatids can be transported across the immunological barrier and the adluminal compartment, respectively, during the epithelial cycle of spermatogenesis. Fascin is a family of actin microfilament cross-linking and bundling proteins that is known to confer bundling of parallel actin microfilaments in mammalian cells. A recent report has illustrated the significance of a fascin protein called fascin 1 in actin microfilaments at the ES, pertinent to its role in spermatogenesis (Gungor-Ordueri et al. Am J Physiol Endocrinol Metab307, E738-753, 2004 (DOI:10.1152/ajpendo.00113.2014). In this Commentary, we critically evaluate these findings in light of the role of fascin in other mammalian cells, providing some insightful information for future investigations. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin complex 1 (mTORC1) is an emerging regulator of blood-tissue barriers that utilizes ribosomal protein S6 (rpS6) as the downstream signaling molecule. To explore the role of rpS6 in blood-testis barrier (BTB) function, a constitutively active quadruple rpS6 phosphomimetic mutant was constructed in mammalian expression vector and overexpressed in Sertoli cells cultured in vitro that mimicked the BTB in vivo. Using this quadruple phosphomimetic mutant, phosphorylated (p)-rpS6 was shown to disrupt IGF-1/insulin signaling, thereby abolishing Akt phosphorylation, which led to an induction of MMP-9. This increase in MMP-9 secretion perturbed the Sertoli cell tight junction permeability barrier by proteolysis-mediated downregulation of tight junction proteins at the BTB. These findings were confirmed by the use of a specific MMP-9 inhibitor that blocked the disruption of the tight junction permeability barrier by the rpS6 mutant. Additionally, RNA interference (RNAi)- mediated Akt silencing was able to mimic the results of rpS6 mutant overexpression in Sertoli cells, further confirming this p-rpS6-Akt- MMP-9 signaling pathway. In conclusion, these data support a new concept of mTORC1-mediated BTB regulation, that is possibly also applicable to other blood-tissue barriers. [ABSTRACT FROM AUTHOR]

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 testisspecific 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. [ABSTRACT FROM AUTHOR]

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed. [ABSTRACT FROM AUTHOR]

Highlights: [•] Remodeling of actin microfilaments at the ectoplasmic specialization (ES) is crucial to confer spermatid transport. [•] ES remodeling is regulated by the coordinated effects of actin bundling and branched actin polymerization-inducing proteins. [•] Spermiation is an F-actin remodeling cellular event. [Copyright &y& Elsevier]

During spermatogenesis, spermatogonia (2n, diploid) undergo a series of mitotic divisions as well as differentiation to become spermatocytes, which enter meiosis I to be followed by meiosis II to form round spermatids (1n, haploid), and then differentiate into spermatozoa (1n, haploid) via spermiogenesis. These events take place in the epithelium of the seminiferous tubule, involving extensive junction restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface to allow the transport of developing germ cells across the epithelium. Although structural aspects of these cell-cell junctions have been studied, the underlying mechanism(s) that governs these events has yet to be explored. Earlier studies have shown that a non-receptor protein tyrosine kinase known as focal adhesion kinase (FAK) is a likely regulator of these events due to the stage-specific and spatiotemporal expression of its various phosphorylated/activated forms at the testis-specific anchoring junctions in the testis, as well as its association with actin regulatory proteins. Recent studies have shown that FAK, in particular its two activated phosphorylated forms p-FAK-Tyr407 and p-FAK-Tyr397, are crucial regulators in modulating junction restructuring at the Sertoli cell-cell interface at the blood-testis barrier (BTB) known as the basal ectoplasmic specialization (basal ES), as well as at the Sertolispermatid interface called apical ES during spermiogenesis via its effects on the filamentous (F)-actin organization at the ES. We herein summarize and critically evaluate the current knowledge regarding the physiological significance of FAK in regulating BTB and apical ES dynamics by governing the conversion of actin filaments at the ES from a "bundled" to a "de-bundled/ branched" configuration and vice versa. We also provide a molecular model on the role of FAK in regulating these events based on the latest findings in the field. [ABSTRACT FROM AUTHOR]

One of the most important but still poorly understood cellular phenomena occurring during spermatogenesis is the movement of preleptotene/leptotene spermatocytes across the blood-testis barrier (BTB), an ultrastructure comprised of tight junctions (TJs), basal ectoplasmic specializations, gap junctions, and desmosomes. Previous studies have shown cytokines and androgens to mediate BTB restructuring, but it is not yet entirely known if germ cells can regulate barrier function, and if yes, how. To address this question, we utilized a previously characterized Sertoli-germ cell coculture model coupled with transepithelial electrical resistance (TE R), immunoblotting, and immunolocalization experiments. When freshly isolated germ cells from adult rat testes were added to Sertoli cells at a Sertoli:germ cell ratio of 1:5 (Sertoli cells were previously cultured at high density on Matrigel™-coated culture inserts for 3 d to allow assembly of a functional permeability barrier that mimicked the Sertoli cell BTB in vivo), there was a significant increase in TE R compared with timematched controls (i.e., Sertoli cells only), illustrating that germ cells promote Sertoli cell barrier function. This increase in barrier function was not likely the result of TJ gene expression by germ cells. Instead, germ cells upregulated the steadystate levels of several TJ proteins, including occludin, tricellulin, claudin, junctional adhesion molecule-A, and coxsackievirus and adenovirus receptor (CAR) in Sertoli cells. These results were corroborated in part by immunofluorescence staining when an increase in occludin at Sertoli-Sertoli cell borders was observed in vitro. Taken collectively, our results illustrate that germ cells contribute to BTB integrity, which is essential for spermatogenesis and fertility. [ABSTRACT FROM AUTHOR]

For non-hormonal male contraceptives F that exert their effects in the testis locally instead of via the hypothalamicpituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII-VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be synthesized can improve their efficacy in downregulating BCRP and perhaps other drug efflux transporters in the testis to improve their efficacy to traverse the BTB by modifying their interacting domains. [ABSTRACT FROM AUTHOR]

RAI14 (retinoic acid induced protein 14) is an actin-binding protein first identified in the liver. In the testis, RAI14 is expressed by both Sertoli and germ cells in the seminiferous epithelium. Besides binding to actin in the testis, RAI14 is also a binding protein for palladin, an actin cross-linking and bundling protein. A recent report has shown that RAI14 displays stage-specific and spatiotemporal expression at the ES [ectoplasmic specialization, a testis-specific flamentous (F)-actin-rich adherens junction] in the seminiferous epithelium of adult rat testes during the epithelial cycle of spermatogenesis, illustrating its likely involvement in F-actin organization at the ES. Functional studies in which RAI14 was knocked down by RNAi in Sertoli cells in vitro and also in testicular cells in vivo have illustrated its role in conferring the integrity of actin flament bundles at the ES, perturbing the Sertoli cell tight junction (TJ)-pemeability barrier function in vitro, and also spermatid polarity and adhesion in vivo, thereby regulating spermatid transport at spermiation. Herein, we critically evaluate these earlier findings and also provide a likely hypothetic model based on the functional role of RAI14 at the ES, and how RAI14 is working with palladin and other actin regulatory proteins in the testis to regulate the transport of (1) spermatids and (2) preleptotene spermatocytes across the seminiferous epithelium and the blood-testis barrier (BTB), respectively, during spermatogenesis. This model should serve as a framework upon which functional experiments can be designed to better understand the biology of RAI14 and other actin-binding and regulatory proteins in the testis. [ABSTRACT FROM AUTHOR]

The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα(IV) NC1 recombinant protein produced in mammalian cells and puriied to apparent homogeneity by ainity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. these indings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis. [ABSTRACT FROM AUTHOR]

Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18-19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis. [ABSTRACT FROM AUTHOR]

In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yetto-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and α-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator. [ABSTRACT FROM AUTHOR]

In the mammalian testis, extensive restructuring takes place across the seminiferous epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface during the epithelial cycle of spermatogenesis, which is important to facilitate changes in the cell shape and morphology of developing germ cells. However, precise communications also take place at the cell junctions to coordinate the discrete events pertinent to spermatogenesis, namely spermatogonial renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation. It is obvious that these cellular events are intimately related to the underlying actin-based cytoskeleton which is being used by different cell junctions for their attachment. However, little is known on the biology and regulation of this cytoskeleton, in particular its possible involvement in endocytic vesicle-mediated trafficking during spermatogenesis, which in turn affects cell adhesive function and communication at the cell-cell interface. Studies in other epithelia in recent years have shed insightful information on the intimate involvement of actin dynamics and protein trafficking in regulating cell adhesion and communications. The goal of this critical review is to provide an updated assessment of the latest findings in the field on how these complex processes are being regulated during spermatogenesis. We also provide a working model based on the latest findings in the field including our laboratory to provide our thoughts on an apparent complicated subject, which also serves as the framework for investigators in the field. It is obvious that this model will be rapidly updated when more data are available in future years. [ABSTRACT FROM AUTHOR]

BACKGROUND During the seminiferous epithelial cycle, restructuring takes places at the Sertoli–Sertoli and Sertoli–germ cell interface to accommodate spermatogonia/spermatogonial stem cell renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation since developing germ cells, in particular spermatids, move ‘up and down’ the seminiferous epithelium. Furthermore, preleptotene spermatocytes differentiated from type B spermatogonia residing at the basal compartment must traverse the blood–testis barrier (BTB) to enter the adluminal compartment to prepare for meiosis at Stage VIII of the epithelial cycle, a process also accompanied by the release of sperm at spermiation. These cellular events that take place at the opposite ends of the epithelium are co-ordinated by a functional axis designated the apical ectoplasmic specialization (ES)—BTB—basement membrane. However, the regulatory molecules that co-ordinate cellular events in this axis are not known. METHODS Literature was searched at http://www.pubmed.org and http://scholar.google.com to identify published findings regarding intercellular adhesion molecules (ICAMs) and the regulation of this axis. RESULTS Members of the ICAM family, namely ICAM-1 and ICAM-2, and the biologically active soluble ICAM-1 (sICAM-1) are the likely regulatory molecules that co-ordinate these events. sICAM-1 and ICAM-1 have antagonistic effects on the Sertoli cell tight junction-permeability barrier, involved in Sertoli cell BTB restructuring, whereas ICAM-2 is restricted to the apical ES, regulating spermatid adhesion during the epithelial cycle. Studies in other epithelia/endothelia on the role of the ICAM family in regulating cell movement are discussed and this information has been evaluated and integrated into studies of these proteins in the testis to create a hypothetical model, depicting how ICAMs regulate junction restructuring events during spermatogenesis. CONCLUSIONS ICAMs are crucial regulatory molecules of spermatogenesis. The proposed hypothetical model serves as a framework in designing functional experiments for future studies. [ABSTRACT FROM AUTHOR]

During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research. [ABSTRACT FROM AUTHOR]

In the seminiferous epithelium of the mammalian testis, the most distinctive ultrastructure is the extensive bundles of actin filaments that lie near the Sertolispermatid interface and the Sertoli- Sertoli cell interface known as the apical ectoplasmic specialization (apical ES) and the basal ES, respectively. These actin filament bundles not only confer strong adhesion at these sites, they are uniquely found in the testis. Recent studies have shown that ES also confers spermatid and Sertoli cell polarity in the seminiferous epithelium during the epithelial cycle. While these junctions were first described in the 1970s, there are few functional studies in the literature to examine the regulation of these actin filament bundles. It is conceivable that these actin filament bundles at the ES undergo extensive re-organization to accommodate changes in location of developing spermatids during spermiogenesis as spermatids are transported across the seminiferous epithelium. Additionally, these actin filaments are rapidly reorganized during BTB restructuring to accommodate the transit of preleptotene spermatocytes across the barrier at stage VIII of the epithelial cycle. Thus, actin binding and regulatory proteins are likely involved in these events to confer changes in F-actin organization at these sites. Interestingly, there are no reports in the field to study these regulatory proteins until recently. Herein, we summarize some of the latest findings in the field regarding a novel actin cross-linker and actin-bundling protein called palladin. We also discuss in this opinion article the likely role of palladin in regulating actin filament bundles at the ES during spermatogenesis, highlighting the significance of palladin and how this protein is plausibly working in concert with other actin-binding/regulatory proteins and components of polarity proteins to regulate the cyclic events of actin organization and re-organization during the epithelial cycle of spermatogenesis. We also propose a hypothetic model by which palladin regulates ES restructuring during the epithelial cycle of spermatogenesis. [ABSTRACT FROM AUTHOR]

The blood-testis barrier (BTB) divides the seminiferous epithelium into the basal and the adluminal compartment. It restricts paracellular diffusion of molecules between Sertoli cells, confers cell polarity, and creates a unique microenvironment in the adluminal compartment for spermatid development. However, it undergoes restructuring during the epithelial cycle so that preleptotene spermatocytes differentiated from type B spermatogonia residing in the basal compartment can traverse the BTB at stage VIII of the cycle, while the immunological barrier is maintained. Herein, coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) integral membrane protein in the testis and multiple epithelia and endothelia, was found to act as a regulatory protein at the BTB, besides serving as a structural adhesion protein. RNAi-mediated knockdown of CAR in a Sertoli cell epithelium with an established TJ-permeability barrier that mimicked the BTB in vivo resulted in a disruption of the TJ barrier and an increase in endocytosis of the TJ-protein occludin. Furthermore, such an enhancement in occludin endocytosis was accompanied by a downregulation of Thr-phosphorylation in occludin and an increase in the association of endocytosed occludin with early endosome antigen-1. These findings were confirmed by overexpressing CAR in Sertoli cells, which was found to "tighten" the Sertoli cell TJ barrier, promoting BTB function. These findings support the emerging concept that CAR is not only a structural protein, it is involved in conferring the phosphorylation status of other adhesion proteins at the BTB (e.g., occludin) possibly mediated via its structural interactions with nonreceptor protein kinases, thereby modulating endocytic vesicle-mediated protein trafficking. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr407 and p-FAK-Try397 display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr407 and Try397. Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr407 phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a Afunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB. [ABSTRACT FROM AUTHOR]

During the seminiferous epithelial cycle of spermatogenesis, the ectoplasmic specialization (ES, a testis-specific adherens junction, AJ, type) maintains the polarity of elongating/elongated spermatids and confers adhesion to Sertoli cells in the seminiferous epithelium, and known as the apical ES. On the other hand, the ES is also found at the Sertoli-Sertoli cell interface at the blood-testis barrier (BTB) known as basal ES, which together with the tight junction (TJ), maintains Sertoli cell polarity and adhesion, creating a functional barrier that limits paracellular transport of substances across the BTB. However, the apical and basal ES are segregated and restricted to the adluminal compartment and the BTB, respectively. During the transit of preleptotene spermatocytes across the BTB and the release of sperm at spermiation at stage VIII of the seminiferous epithelial cycle, both the apical and basal ES undergo extensive restructuring to facilitate cell movement at these sites. The regulation of these events, in particular their coordination, remains unclear. Studies in other epithelia have shown that the tubulin cytoskeleton is intimately related to cell movement, and MArK [microtubule-associated protein (MAp)/microtubule affinity-regulating kinase] family kinases are crucial regulators of tubulin cytoskeleton stability. Herein MArK4, the predominant member of the MARK protein family in the testis, was shown to be expressed by both Sertoli and germ cells. MARK4 was also detected at the apical and basal ES, displaying highly restrictive spatiotemporal expression at these sites, as well as co-localizing with markers of the apical and basal ES. the expression of MARK4 was found to be stage-specific during the epithelial cycle, structurally associating with α-tubulin and the desmosomal adaptor plakophilin-2, but not with actin-based BTB proteins occludin, β-catenin and Eps8 (epidermal growth factor receptor pathway substrate 8, an actin bundling and barbed end capping protein). More importantly, it was shown that the expression of MARK4 tightly associated with the integrity of the apical ES because a diminished expression of MARK4 associated with apical ES disruption that led to the detachment of elongating/elongated spermatids from the epithelium. these findings thus illustrate that the integrity of apical ES, an actin-based and testis-specific AJ, is dependent not only on the actin filament network, but also on the tubulin-based cytoskeleton. [ABSTRACT FROM AUTHOR]

Filamins are a family of actin-binding proteins composed of filamin A, B and C. Besides of their ability to induce perpendicular branching of F-actin filaments via their actin binding domains near the N-terminus, filamins can regulate multiple cellular functions because of their unique ability to recruit more than 90 protein binding partners to their primary sequences which are having highly diversified cellular functions. However, this family of proteins has not been examined in the testis until recently. Herein, we highlight recent findings in the field regarding the role of these proteins in cell epithelia, and based on recent data in the testis regarding their role on spermatogenesis, this review provides the basis for future functional studies. [ABSTRACT FROM AUTHOR]

Tubulobulbar complexes (TBCs) are actin-based structures that help establish close contact between Sertoli-Sertoli cells or Sertoli-mature germ cells (spermatids) in the seminiferous tubules of the testes. They are actin-rich push-through devices that eliminate excess spermatid cytoplasm and prepare mature spermatids for release into the tubular lumen. Just prior to spermiation, the elongated spermatid interacts with the Sertoli cell via an extensive structure comprising various adhesion molecules called the apical ectoplasmic specialization which is partially replaced by the apical TBC, on the concave surface of the spermatid head. The sperm release process involves extensive restructuring, namely the disassembly and reassembly of junctions at the Sertoli-spermatid interface in the seminiferous epithelium. Based on the presence of different classes of molecules in the TBCs or the defects observed in the absence of TBCs, the main functions attributed to TBCs are elimination of excess spermatid cytoplasm, endocytosis and recycling of junctional molecules, shaping of the spermatid acrosome, and forming transient anchoring devices for mature spermatids before they are released. This review summarizes the recent findings that focus on the role of TBCs in cell cytoskeleton restructuring during sperm release in the testes and the molecular mechanism involved. [ABSTRACT FROM AUTHOR]