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International audience; Atrazine (ATZ), a widely used agricultural pesticide and benzo[a]pyrene (BaP), a ubiquitous environmental human carcinogen can induce alterations of spermatogenesis. In the present study, we showed first that our seminiferous tubule culture model, in bicameral chambers, allowed the settlement of the blood-testis barrier (BTB) in 8-day-old male rat cultures and the differentiation of spermatogonia into round spermatids.The effect of a mixture of 1 μg/L of ATZ and 1 μg/L of BaP was then investigated either during or after the establishment of the BTB by using 8- or 20-22-day-old rats. Cultures were performed over a 3-week period. Our results show that claudin-11 and connexin 43 two proteins of the BTB, were impaired by the mixture which also reduced the number of round spermatids (the direct precursors of spermatozoa), by targeting the middle to late pachytene spermatocytes. These effects were observed in 8- and 20-22-day -old rat seminiferous tubule cultures. However, the decrease of the number of round spermatids was faster and more marked in the 8-day- than in the 20-22-day -old rat seminiferous tubule cultures. Our study emphasizes the possible influence of the age of an individual on the effect of (a) toxicant(s) on spermatogenesis.

International audience; It has been shown that non-cytotoxic doses of Carbendazim (CBZ), a broad-spectrum benzimidazole fungicide, possess endocrine-disrupting (androgen-like) actions, ex vivo, on the pubertal rat seminiferous epithelium. Iprodione (IPR), a dicarboximide fungicide, is also known to be an endocrine-disrupter (anti-androgen). The effect of a mixture of these two pesticides was investigated in the validated rat seminiferous tubule culture model. Cultures were performed in the absence or presence of CBZ 50 nM or IPR 50 nM either alone or in mixture (Mix), over a 3-week period. Mix exerted a dramatic effect on two proteins (Connexin 43 and Claudin-11) of the blood-testis barrier and possessed similar effects to IPR on some germ cell populations. The presence of IPR together with CBZ (Mix) cancelled the effect of CBZ on the increase of the androgen-dependent TP1 and TP2 mRNAs and on the decrease of ER alpha, ER beta mRNAs. Nevertheless, CBZ alone or IPR alone or Mix induced toxicity on spermatogenesis resulting in a decrease of round spermatids (the precursors of spermatozoa). These results strongly suggest that, even at these low concentrations, the effects of IPR and of CBZ are not solely dependent on their respective anti-androgenic and androgen-like effects and should involve several mechanisms of action.

Reproductive organs are complex and well-structured tissues essential to perpetuate the species. In mammals, the male and female reproductive organs vary on their organization, morphology and function. Connectivity between cells in such tissues plays pivotal roles in organogenesis and tissue functions through the regulation of cellular proliferation, migration, differentiation and apoptosis. Connexins and pannexins can be seen as major regulators of these physiological processes. In the present review, we assembled several lines of evidence demonstrating that these two families of proteins are essential for male and female reproduction.

Connexins (Cx), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore alterations of Cx expression and degradation lead to the development of physiopathological disorders. Due to the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear. By using a combination of time-lapse fluorescence microscopy and high resolution transmission electron microscopy we evidenced new specific cellular events concerning gap junction degradation and recycling. Indeed, by time lapse video microscopy we demonstrated, for the first time to our knowledge, that an entire annular gap junction can be fully recycled back to the plasma membrane. Moreover, we dissected the degradative processes of gap junction by electron microscopy approaches. Interestingly, in addition to canonical autophagy and heterophagy pathways, previously described, we discovered that both pathways could sometimes intermingle. Strikingly, our results also highlighted a new lysosome-based autophagy pathway that could play a pivotal role in common autophagy degradation. The present investigation reveals that AGJ degradation is a more complex process that it was previously thought. First, a complete recycling of the gap junction plaque after its internalization could occur. Second, the degradation of this peculiar double membrane structure is possible through autophagy, heterophagy, hetero-autophagy or by lysosomal-based autophagy. Altogether, this work underlines novel aspects of gap junction degradation that could be extended to other cell biology processes. This article is protected by copyright. All rights reserved.

Cell death is a fundamental process for organogenesis, immunity and cell renewal. During the last decades a broad range of molecular tools were identified as important players for several different cell death pathways (apoptosis, pyroptosis, necrosis, autosis…). Aside from these direct regulators of cell death programs, several lines of evidence proposed connexins and pannexins as potent effectors of cell death. In the present review we discussed the potential roles played by connexins, pannexins and innexins in the different cell death programs at different scales from gap junction intercellular communication to protein-protein interactions. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Background NRD convertase, also termed Nardilysin, is a Zn++ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined. Methods To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa. Results Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme. Conclusions The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

Many recent epidemiological, clinical and experimental findings support the hypothesis that environmental toxicants are responsible for the increasing male reproductive disorders (congenital malformations, declining sperm counts and testicular cancer) over the past 20 years. It has also been reported that exposure to these toxicants, during critical periods of development (fetal and neonatal), represents a more considerable risk for animals and humans than exposure during adulthood. However, the molecular targets for these chemicals have not been clearly identified. Recent studies showed that a family of transmembranous proteins, named connexins, regulates numerous physiological processes involved in testicular development and function, such as Sertoli and germ cell proliferation, differentiation, germ cell migration and apoptosis. In the testis, knockout strategy revealed that connexin 43, the predominant connexin in this organ, is essential for spermatogenesis. In addition, there is evidence that many environmental toxicants could alter testicular connexin 43 by dysregulation of numerous mechanisms controlling its function. In the present work, we propose first to give an overview of connexin expression and intercellular gap junction coupling in the developing fetal and neonatal testes. Second, we underline the impact of maternally chemical exposure on connexin 43 expression in the perinatal developing testis. Lastly, we attempt to link this precocious effect to male offspring fertility.

Résumé Les jonctions communicantes et leurs protéines constitutives, les connexines (Cxs), sont des constituants nécessaires à la cohésion tissulaire et reconnus comme suppresseurs de tumeurs. Le but de la présente revue est de faire le point sur l’organisation et le rôle des Cxs au sein du testicule et d’analyser leur expression en physiopathologie testiculaire. Organisées en structures hexamèriques formant un canal reliant directement les cytoplasmes des cellules adjacentes, les Cxs sont impliquées dans de nombreux processus physiologiques tels que la prolifération et la différenciation cellulaires. Le maintien d’une balance entre prolifération, différenciation et apoptose est un équilibre primordial évitant une prolifération cellulaire anarchique, risque de cancer. La spermatogenèse est un modèle sophistiqué de prolifération et de différenciation des cellules germinales dans lequel les Cxs jouent un rôle essentiel. Il est acquis qu’une altération de l’expression membranaire des Cxs est l’un des signes avant-coureurs de la cinétique tumorale germinale, et il a été suggéré que les toxiques environnementaux qui, dans leur grande majorité, affectent l’expression de ces protéines, puissent être impliqués dans le développement de cette pathologie. La recherche de molécules capables de freiner les effets délétères de toxiques carcinogènes sur les Cxs semble être à l’heure actuelle une voie intéressante ouvrant de nouvelles perspectives en santé humaine.

Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell–cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli–Sertoli cell functional synchronization and the Sertoli–germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation.

The role of gap junctions in proliferation, differentiation and apoptosis has been recently highlighted. Nevertheless, the molecular mechanisms that control these physiological events by acting on gap junction channels are still unknown. We have recently demonstrated that heteromeric gap junction plaques composed by Cx43 and Cx33 are unstable at the cell boundary and are rapidly internalized by endocytosis. In the present study, we analyze the phosphorylation status of Cx43 in homomeric (Cx43/Cx43) and heteromeric (Cx33/Cx43) complexes and their association with the tyrosine kinase c-Src. Our data show that c-Src interaction and P2 phosphorylation of Cx43, which are essential for homomeric Cx43 complex endocytosis, were altered in the heteromeric Cx33/Cx43 complex: lack of association between Cx33 and activated c-Src and disappearance of the P2 phosphorylated Cx43 isoform. The present findings demonstrate that the interaction of Cx33 with Cx43 within a same heteromeric complex may conduce to channel instability through alteration of the phosphorylation status of Cx43 independently of the control of the c-Src kinase. The data described here emphasize a new mechanism of Cx43 internalization Src kinase-independent.

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERα and only express the cytoplasmic β isoform. ERβ localization was not modified by either steroid. The threshold level was surprisingly low, around 10 −16 M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell-cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P(1) phosphorylated and P(0) unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.

The gap junction protein connexin 43 (Cx43) exhibits dynamic trafficking that is altered in most tumor cells and in response to carcinogen exposure. A number of connexin (Cx)-binding proteins are known to be involved in endocytic internalization of gap junctions. Here, we analyzed the discrete molecular interactions that occur between Src, ZO-1 and Cx43 during Cx43 internalization in response to the non-genomic carcinogen γ-hexachlorocyclohexane (HCH). Internalization of the Cx43 gap junction plaque was significantly accelerated in Cx43-GFP transfected 42GPA9 Sertoli cells that were exposed to the carcinogen. HCH induced the rapid recruitment of Src to the plasma membrane, activation of Src within 3 minutes and the efficient inhibition of gap junctional coupling, but had no effect in the presence of the Src inhibitor PP2. Immunoprecipitation experiments demonstrated that HCH increased Cx43-Src interaction and concomitantly decreased Cx43–ZO-1 association. ZO-1 was detected on both sides of the gap junction plaques in untreated cells, but appeared to be mainly localized on one side during HCH-induced internalization. The dissociation of ZO-1 from Cx43 appears to occur specifically on the side of the plaque to which Src was recruited. These findings provide mechanistic evidence by which internalization of the Cx43 gap junction plaque might be initiated, suggesting that Src-mediated dissociation of ZO-1 from one side of the plaque initiates endocytic internalization of gap junctions and that this process is amplified in response to exposure to HCH.

Stability of cell-to-cell interactions and integrity of junctional membrane proteins are essential for biological processes including cancer prevention. The present study shows that DDT, a non-genomic carcinogen used at a non-cytotoxic dose (1 μM), rapidly disrupted the cell–cell contacts and concomitantly induced the formation of cytoplasmic vacuoles close to the plasma membrane in the SerW3 Sertoli cell line. High-resolution deconvolution microscopy reveals that this vacuolization process was clathrin-dependent since a hyperosmotic media (0.2 M sucrose) blocked rhodamine–dextran endocytosis. In response to DDT, junctional proteins such as Cx43, N-Cadherin and ZO-1 were internalized and present in vacuoles. In Cx43-GFP transfected cells, time lapse videomicroscopy demonstrates that DDT rapidly enhanced fragmentation of the gap junction plaques and abolished the gap junction coupling without major modification of Cx43 phosphorylation status. Repeated exposure to DDT resulted in chronic gap junction coupling injury. The present results demonstrate that one of the early effect of DDT is to interfere with the plasma membrane and to perturb its function, specifically its ability to establish cell–cell junctions that are essential for tissue homeostasis and control of cell proliferation and differentiation. Such an alteration may play a specific role during carcinogenesis.

Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated. © 2007 Wiley-Liss, Inc.

(IF : 3,54); International audience; Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during developmental and differentiation processes. In bone, the involvement of the gap junctional protein, connexin-43 (Cx43), and of GJIC in osteoblastic differentiation and mineralization of the extracellular matrix has been previously demonstrated. Former studies have shown that endothelin-1 (ET-1) was also implicated in the control of osteoblastic proliferation and differentiation. However, depending on the cellular models, ET-1 has been shown to decrease or increase osteoblastic differentiation markers. As no data were available on the ET-1 effect on GJIC and Cx43 expression in osteoblastic cells, we analyzed here the possible crosstalk between Cx43 and ET-1 in a human cell line (hFOB 1.19) which displays different Cx43 expression levels and phenotypes when cultured at 33.5 or 39 degrees C. The presence of ET-1 (10(-8) M) for 2-12 days of culture did not significantly alter the proliferation rate of hFOB cells whatever their phenotype. In contrast, ET-1 induced a differential inhibitory effect on the biochemical differentiation markers (alkaline phosphatase activity and osteocalcin expression) with a significant reduction in the differentiated phenotype at 39 degrees C, whereas no effects were measured at 33.5 degrees C. The inhibitory effect was linked to a decrease of GJIC and of Cx43 both at transcriptional and protein levels. Altogether, our results suggest that Cx43 expression level could influence the action of ET-1 on human osteoblastic cell differentiation. Our data also indicate that the gap junctional protein could play a pivotal role in the response of osteoblasts to mitogenic factors implicated in bone pathologies.

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

Connexins, the constitutive proteins of gap junctions, are considered to be tumour suppressive agents and are often impaired in the tumourigenic processes. In the present study, the expression of connexin 43 (Cx43), which is involved in the control of spermatogenesis through Sertoli/germ cell coupling, has been investigated in human testicular seminoma cells (tumours and the JKT-1 cell line). Cx43 was immunolocalized in the Golgi apparatus without membrane expression and was detected by immunoblotting in JKT-1 as exclusive 70 kD bands. No mutation could be found by sequencing the transcript obtained by RT-PCR. Transfection with a Cx43-V5 vector reproduced the same gel shift, identifying these 70 kD bands as Cx43. The Cx43-70 kD bands were also expressed in normal testicular tissue, associated with the classical 43 kD isoforms. Stable transfection of JKT-1 with a Cx43-GFP vector allowed restoration of Cx43 membrane expression, functional cell coupling, and inhibition of the cell proliferation rate. Storage of Cx43 in the Golgi apparatus may correspond during spermatogenesis to an intermittent physiological process that becomes permanent in malignant seminoma cells as a result of the tumourigenic process. By preventing Cx43 membrane expression, this disrupted traffic may itself participate in tumour promotion.

International audience; During pregnancy, the villous trophoblast develops from the fusion of cytotrophoblastic cells (CT) into a syncytiotrophoblast (ST), supporting the main physiological functions of the human placenta. Connexin43 (Cx43) is demonstrated in situ and in vitro in the villous trophoblast between CT and between CT and ST. Moreover, the presence of a gap junctional intercellular communication (GJIC) during in vitro trophoblast differentiation was previously demonstrated. Because the exchange of molecules through gap junctions is considered to play a major role in the control of cell and tissue differentiation, we studied the effects of a gap junctional uncoupler, heptanol, on morphological and functional trophoblast differentiation and on GJIC measured by the fluorescence recovery after photobleaching method. We found that when the GJIC was interrupted, CT still aggregated but fused poorly. This morphological effect was associated with a significant decrease of trophoblastic-specific gene expression (␤ human chorionic gonadotropin and human chorionic soma-tomammotropin). This blocking action was reversible as demonstrated by recovery of GJIC and trophoblast differentiation process after heptanol removal. Moreover, the inhibition of the trophoblast differentiation did not affect Cx43 transcript expression and Cx43 protein expression. These data suggest that the molecular exchanges through gap junctions preceding cellular fusion are essential for trophoblast differentiation generating the multifunctional syncytiotrophoblast. human chorionic gonadotropin, placenta, signal transduction, syncytiotrophoblast, trophoblast

Gap junctions are intercellular channels formed of connexins (Cx) at appositional plasma membranes between adjacent cells that have been involved in the control of cell proliferation and differentiation. Altered Cx expression is implicated consistently in several human diseases and in tumorigenesis. Although Cx43 plays a critical role in Sertoli cell control of spermatogenesis, there is no evidence of its altered expression in human testicular pathologies. We show here that Cx43 mRNA expression was significantly reduced in testes of infertile patients with secretory azoospermia (p < 0.05) compared with testes displaying normal spermatogenesis (excretory azoospermic patients). In Sertoli cell-only syndrome, in situ hybridization and immunohistochemistry analyses indicated that Cx43 mRNA and protein were undetectable in Sertoli cells but were still present in the interstitial compartment. In a rat model of Sertoli cell-only syndrome, the lack of Cx43 in Sertoli cells was associated with an impairment of gap junction intercellular communication between adjacent Sertoli cells. These results reveal that Cx43 mRNA and protein expression are markedly impaired in Sertoli cells of infertile patients. This defect could be a new functional marker of undifferentiated Sertoli cells and could be related to the increased risk of testicular cancer recently described in the population of infertile men.

Lindane (gamma-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell-cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 microM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Gap junctions, through their constitutive proteins, connexins (Cx), are involved in several processes including regulation of cellular proliferation, tissue differentiation, homeostasis and neoplasic transformation. Internalization of the gap junction plaque to form annular gap junction is a dynamic process, which present similarities with endocytosis, and participates in the control of gap junction coupling. Cx43 exhibits dynamic trafficking that needs sequential implication of a large number of protein partners. We have recently shown that ZO-1 localized in both sides of the gap junction plaque was restricted to one side during internalization. The dissociation between ZO-1 and Cx43 particularly occurred on the face where c-Src specifically associated with Cx43 and was abnormally accelerated in response to a carcinogen. In this addendum we summarize and further discuss these results.

The plasminogen activator (PA) system is thought to play a major role in the proteolytic events associated with spermatogenesis. The mechanisms controlling the expression of PA and of its major physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1), in the seminiferous epithelium are still unknown. In the present study we analyzed the expression of PA and PAI-1 in a murine Sertoli cell line (42GPA9) in response to stimulation by lipopolysaccharides (LPS) used to activate the phagocytic activity of these cells. Immortalized Sertoli cells cultured under basal conditions secreted predominantly tissue-type PA (tPA) as demonstrated by zymographic analysis and the presence of tPA transcripts. In zymographic experiments a larger molecular weight proteolytic band corresponding to the formation of PA–PAI-1 complex was also observed. The stimulation of immortalized Sertoli cells by LPS resulted in both alteration of the apparent tPA molecular weight to a higher form and transient increase in PAI-1 biosynthesis. The phorbol ester TPA stimulates similarly PAI-1 synthesis in the Sertoli cell line, while 8-bromo-cAMP has no effect. These results suggest for the first time the existence of a direct linkage between molecular events triggered by phagocytosis and regulation of tPA and PAI-1 in Sertoli cells.

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.

In the present study we report the isolation and characterization of a clonal Sertoli cell line (42GPA9) from sexually mature polyoma virus large T (PyLT) transgenic mice. The cells multiplied indefinitely and expressed large T antigen. The 42GPA9 cell line expressed biochemical features associated with normal Sertoli cells. Transferrin, sulfated glycoprotein-2, and the ligand of c-kit were detected by reverse transcription-polymerase chain reactions and Western blot analyses. Zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained FSH receptors as suggested by their specific responsiveness to the gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the detection of FSH receptor mRNAs. Another original aspect of the 42GPA9 cell line is its ability to form tight junctions at confluency as demonstrated by electron microscopic study and immunolocalization of the tight junction-associated protein zonula occludens 1. The 42GPA9 cell line, which has retained several important hallmarks of normal Sertoli cells, may prove useful for further studies on Sertoli cell behavior and on Sertoli-germ cell interactions in the mature testis.

Gap junction protein connexins (Cxs) play essential roles in cell homeostasis, growth, differentiation and death. Therefore, Cx dysfunction has been associated with many diseases and with tumor development. Cxs control cell apoptosis through different molecular mechanisms. First, gap junction channels classically facilitate the influx and flux of apoptotic signals between adjacent cells and hemichannels between the intracellular and extracellular environments. Second, recent studies demonstrate that Cx proteins, independently from their functional role through channels or hemichannels and in conjunction with their intracytoplasmic localization, may act as signaling effectors able to activate the canonical mitochondrial apoptotic pathway. In the present review, we dissected both functions of Cx in apoptosis, providing new avenues for apoptosis-mediated cancer therapy.

International audience; Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 mu g/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood testis barrier dynamic is postulated. (c) 2013 Elsevier Inc. All rights reserved.

Gap junction channels link cytoplasms of adjacent cells. Connexins, their constitutive proteins, are essential in cell homeostasis and are implicated in numerous physiological processes. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival, and apoptosis, in which a connexin isotype, connexin 43, plays a crucial role as evidenced by genomic approaches based on gene deletion. The balance between cell proliferation/differentiation/apoptosis is a prerequisite for maintaining levels of spermatozoa essential for fertility and for limiting anarchic cell proliferation, a major risk of testis tumor. The present review highlights the emerging role of connexins in testis pathogenesis, focusing specifically on two intimately interconnected human testicular diseases (azoospermia with impaired spermatogenesis and testicular germ cell tumors), whose incidence increased during the last decades. This work proposes connexin 43 as a potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.

Connexins, through gap junctional intercellular communication, are known to regulate many physiological functions involved in developmental processes such as cell proliferation, differentiation, migration and apoptosis. Strikingly, alterations of connexin expression and trafficking are often, if not always, associated with human developmental diseases and carcinogenesis. In this respect, disrupted trafficking dynamics and aberrant intracytoplasmic localization of connexins are considered as typical features of functionality failure leading to the pathological state. Recent findings demonstrate that interactions of connexins with numerous protein partners, which take place throughout connexin trafficking, are essential for gap junction formation, membranous stabilization and degradation. In the present study, we give an overview of the physiological molecular machinery and of the specific interactions between connexins and their partners, which are involved in connexin trafficking, and we highlight their changes in pathological situations.

A dramatical decline in human male reproductive function has been reported for the past 20 years. Many recent epidemiological, clinical and experimental findings suggest that the reproductive dysfunction could result from prenatal and neonatal chemical compound exposure. Even if numerous studies argue for a relationship between male infertility and environmental and/or occupational exposure, the molecular mechanisms by which these anti-reproductive compounds act are still unclear. Recent findings showed that a family of transmembranous proteins, connexins, regulates numerous physiological functions involved in the development such as cell proliferation, differentiation, migration and apoptosis. In the testis and the ovary, connexins are known to be essential for the establishment and the maintenance of spermatogenesis in males and oogenesis and folliculogenesis in females. Moreover, mutation of connexin genes leads to several developmental human diseases (myelin-related diseases, hearing loss, congenital cataract, skin disorders or more complex syndromes such as the oculodendrodigital dysplasia....) and altered connexin expression, trafficking and degradation are often associated with the tumoral process. We propose, in the present work, to give an overview of connexin expression and intercellular gap junction coupling during development: in preimplantation, implantation and postimplantation embryos. Moreover, we underline the impact of maternal chemical exposure on connexin expression during fetal gonad development and we link this effect to future offspring fertility.

International audience; Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.

The presence of arginine vasopressin (AVP)-like peptide(s) and AVP receptors has been previously demonstrated in the testis of several species. In this study we examined the immunocytochemical localization of AVP and of its associated neurophysin (NPII) within the mouse testis, and the testicular immunoreactive (IR)-AVP content in normal pubertal and adult mice and in unilaterally cryptorchid animals. Immunostaining was conducted on fresh frozen adult testicular sections and on cultured testicular cells using the avidin-biotin immunoperoxidase technique. Immunoreactivities for AVP and NPII were localized at the periphery of the seminiferous tubules. Staining for AVP and NPII was reduced in testes showing impaired spermatogenesis. AVP and NPII immunoreactivities were present in cultured Sertoli cells, but staining was undetectable in cultured Leydig cells. Negative controls were obtained by applying antiserum that had been preabsorbed with excess peptides in place of primary antiserum. Mouse testes were found to contain IR-AVP, which co-eluted with synthetic AVP on HPLC and diluted in parallel in a specific RIA for AVP. Levels of IR-AVP expressed as pg/mg wet-weight testis were significantly higher (p < 0.05) in pubertal (36.6 ± 1.5) than in adult animals (27.4 ± 1.5). Experimental unilateral cryptorchidism in adult animals resulted 2 wk later in a marked reduction (p < 0.01) in IR-AVP levels in the abdominal testis (18.1 1.0 pg/mg testis) as compared to the contralateral scrotal testis (29.8 ± 1.1 pg/mg testis). The present study provides, for the first time, information on the probable site of production of IR-AVP in the mouse testis and indicates that the testicular IR-AVP content is influenced by the maturational and functional status of the testis. These results are consistent with a paracrine role for AVP-like peptide(s) in the regulation of testicular function.

The effects of intratesticular injection of naloxone, a universal opioid antagonist, on testicular immunoreactive (IR)-arginine vasopressin (AVP) content and on in vitro testosterone production by Leydig cells were investigated in the mouse. Bilateral intratesticular injection of increasing doses of naloxone (0.1-10 pg/testis) resulted 24 h later in a dose-dependent increase in testosterone production by Leydig cells incubated for 3 h in the presence or absence of hCG (100 ng/ml). Unilateral intratesticular injection of naloxone (10 pg) similarly enhanced basal and hCG-stimulated testosterone production by Leydig cells, but production was not modified in Leydig cells from the contralateral vehicle-injected testis, nor was it changed when the same dose was injected subcutaneously. Unilateral intratesticular injection of 10 pg naloxone led to a dose-dependent increase in the hCG-responsiveness without altering the slope of the hCG dose-response curve. In vitro exposure of Leydig cells to increasing doses of naloxone (10 - 9 to 10 - 7 M) did not alter either basal or hCG-stimulated testosterone production. Testicular IR-AVP content declined in a dose-dependent manner in naloxone-injected testis, but remained unchanged in the contralateral vehicleinjected testis and in testis from animals that received similar doses of naloxone subcutaneously. Since AVP has been shown to locally exert a negative control on testosterone production within the testis, it might be hypothesized that the increased Leydig cell activity after local naloxone administration results from reduced intratesticular IR-AVP levels. The possibility that endogenous opioid peptides may locally control testosterone biosynthesis via testicular AVP system regulation is discussed.

International audience; In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

International audience; Follicle-stimulating hormone (FSH) is required for initiation and maintenance of spermatogenesis, a dynamic process of cell proliferation and maturation. By using FSH-gold particles and pulse-chase experiments, we analyzed the kinetics of FSH endocytosis in Sertoli and germ cells during development. Ultrastructural time-dependent analysis demonstrates that FSH was first located on plasma membrane, before being accumulated within the endosomal compartment, in the early endosomes, identified by morphological criteria and Rab-5 colocalization. Thereafter, FSH-gold was routed to the degradation pathway. The FSH endocytosis kinetic was similar in Sertoli cells, spermatogonia and spermatocytes. However, quantitative analysis of gold particles revealed differences in the dynamic of FSH accumulation in the endosomes between immature and mature rats. This age-dependent kinetic of FSH endocytosis, mostly detectable by ultrastructural analysis associated with quantitative data, argues for a potential new regulatory mechanism of the FSH signalling pathway that could occur during maturation of testicular cells.

Several studies suggest that exposure to environmental pollutants is partly responsible for testicular pathologies that have considerably increased over the last decades (cryptorchidism, hypospadias, cancer, decrease in the number of ejaculated spermatozoa). However, the cellular and molecular mechanisms involved in this reprotoxicity remain mostly unknown. One of the challenges of the european regulation REACH is to improve the knowledge on the chemical, toxic and ecotoxic properties of substances used in everyday life. As for the testicular toxicity, the few in vivo models used are not always the most appropriate for mechanistic studies. Our laboratory has developed and validated on a physiological point of view, coculture systems of germ cells in bicameral chambers, which reproduce a blood-testis barrier, allowing the determination of the mechanisms responsible for the toxicity of organic or mineral compounds on spermatogenesis, while reducing greatly the number of animals required.

Cx43 gap junctions are essential for proliferation, differentiation, and apoptosis of germ cells during spermatogenesis. However, only few and indirect observations have been reported on the distribution of Cx43, the predominant Cx within the seminiferous tubules. In the present study, we developed an innovative method that allows visualization of the three- dimensional localization of Cx43 associated with gap junctions and their functionality in isolated spermatogenic stage-specific seminiferous tubules. Cx43 gap junctions were present between myoid cells, between Sertoli cells, and between Sertoli and germ cells. Cx43 levels and coupling were stage-dependent with higher values at stages VI-VIII of spermatogenesis and markedly reduced at stages IX-X. Short-term exposure of seminiferous tubule fragments at stages VI-VIII and of the 42GPA9 Sertoli cell line transfected with a Cx43-GFP vector, to FSH, cAMP, DHT, and 17beta-E(2) significantly altered Cx43 distribution as well as gap junction coupling. These observations highlight a nongenomic effect of these testicular effectors on Cx43 gap junction.

International audience; The gap junction proteins, connexins (Cx), are present in the testis and among them Cx43 play an essential role in spermatogenesis. By using an in vitro proliferation model of germ cells and Sertoli cells, we tempted here to clarify the role of Cx43 in the control of Sertoli and germ cell proliferation and apoptosis. Cx43 was detected in purified preparations of Sertoli cells and spermatogonia and immunolocalized in both cell types identified by vimentin and c-kit, respectively. Inhibition of gap junction coupling by the gap junction inhibitor alpha-GA significantly enhanced BrdU incorporation in Sertoli cells and reduced the number of activated caspase-3 positive germ cells. Similarly, inhibitory Cx43 and pan-Cx mimetic inhibitory peptides increased proliferation of Sertoli cells and stimulated survival of germ cells. Cx32 mimetic inhibitory peptide also stimulated Sertoli cell proliferation without altering germ cell proliferation and apoptosis. The present results reveal that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation. (C) 2008 Elsevier Ltd. All rights reserved.

Male fertility is a highly controlled process that requires proliferation, meiosis, and differentiation of male germ cells in the testis, maturation of spermatozoa in the epididymis, and functional male accessory glands. In addition to endocrine and paracrine controls, gap junctions and their constitutive proteins, connexins, are essential for male fertility. The predominant testicular connexin, Cx43, is distributed in the interstitial compartment, between Leydig cells and in the seminiferous tubule compartment. Experiments in which the Cx43 gene is deleted show that in the seminiferous epithelium, Cx43 participates in Sertoli cell functional synchronization, Sertoli to germ cell dialogue, and germ cell proliferation and differentiation. Connexins are also present in other structures of the male genital tract where they participate in coordinating cell-specific activities and in modulating initiation and maintenance of smooth muscle tone. In addition, connexins appear to play important roles in pathology of the testis and prostate.

International audience; Spermatogenesis involves the realization of a particular genetic program which requires a specific environment ("niche"). Multiplication, differentiation and apoptosis of male germ cells are finely regulated by pituitary hormones (mainly LH and FSH), and by a complex network of factors originating from both the somatic cells and the germ cells of the testis. It is becoming clear that hormones and intra-testicular regulatory factors can compensate, at least in part, for the absence of some hormones or factors including FSH and LH or androgen receptors. Since, most of the growth factors, cytokines and neurotrophins produced within the testis are widely expressed in the organism, the attempts to understand their role in spermatogenesis by "classical" knock-out strategies have been often disappointing. Therefore an important aspect of our previous work was to settle and characterize carefully two systems of cocultures of testicular germ cells with somatic cells in bicameral chambers. For instance, we showed for the first time that the whole meiotic step could be performed in vitro in a mammalian species (the rat). Moreover, all our data indicate that our co-culture systems enable to highlight mechanisms pertinent to the physiological processes. Sperm parameters have been deteriorated considerably during the past 4-5 decades. There is now evidence that chemical exposure is at least partly responsible for these testicular diseases. If a large number of environmental pollutants are able to affect male fertility and to exert carcinogenic effects, their cellular and molecular mechanisms are still unidentified. The cultures in bicameral chambers that we settled can be used to study the effects of a toxicant when added in the basal compartment of the culture chamber, which appears relevant to the in vivo situation. Taken together our results indicate that our in vitro culture systems, which allow screening for the effect of biological activity of different physiological factors, can be also helpful to study that of any chemicals on both survival and multiplication/differentiation of somatic and/or spermatogenic cells on a relatively long time period.

Gap junctions, intercellular channels structured by the connexin protein family, have been implicated in the control of cell homeostasis, proliferation, differentiation and death. A loss of the gap junction intercellular communication and/or connexin dysfunction are typical features of cancer per se and have been associated with the effect of many carcinogens. Indeed, many early human neoplasia of various organs and human tumor cell lines exhibit deficient connexin-mediated communication expression mainly related, in a large number of observations, with an aberrant cytoplasmic localization of this membranous protein. Restoration of normal phenotype in transformed cells by restoration of exogenous connexin gave rise to the concept that connexins may act as tumor suppressors. However, the mechanisms by which connexins mediate such a tumor suppressor effect are multiple. They may result from: formation of functional channels; hemichannels or are directly associated with connexin expression. In addition, the literature shows that they may be dependent upon the cell type and the connexin type. In the present review, we analyze all these aspects of connexin/gap junction involvement in the carcinogenesis process, in human cancers and discuss the possibility of using connexins as potential anti-oncogenic targets for cancer chemoprevention and/or chemotherapy.

Connexins, that have their main function as part of gap junction channels, are proteins expressed in a large number of tissues such as endocrine, nervous, vascular, and muscular tissues. Gap junctions are implicated in tissue homeostasis and control of cell proliferation and differentiation. Interestingly, mutations of connexin genes have been reported in several human diseases (peripheral neuropathies, cardiovascular and dermatological diseases, hereditary cataract, and deafness) and altered expression of connexins have been associated with tumoral progression. Today, several lines of study argue for a critical role of gap junctions in corporal smooth muscle relaxation and erectile response. The present review highlights the emerging role of connexin43, one of these membranous proteins, in the physiology and physiopathology of human erectile function and its possible medical application.

The gap junction proteins, connexins (Cxs), are present in the testis, and among them, Cx43 play an essential role in spermatogenesis. In the present study, we investigated the testicular expression and regulation of another Cx, Cx33, previously described as a negative regulator of gap junction communication. Cx33 mRNA was present in testis and undetectable in heart, liver, ovary, and uterus. In the mature testis, Cx33 was specifically immunolocalized in the basal compartment of the seminiferous tubules, whereas Cx43 was present in both seminiferous tubule and interstitial compartments. During stages IX and X of spermatogenesis, characterized by Sertoli cell phagocytosis of residual bodies, Cx43 was poorly expressed within seminiferous tubules, while Cx33 signal was strong. To evaluate the role of phagocytosis in the control of Cx33 and Cx43 expression, the effect of LPS was analyzed in the Sertoli cell line 42GPA9. We show herein that phagocytosis activation by LPS concomitantly stimulated Cx33 and inhibited Cx43 mRNA levels. These effects appear to have been mediated through IL-1α, because the exposure of Sertoli cells to the IL-1 receptor antagonist partly reversed these effects. IL-1α enhanced and reduced, respectively, the levels of Cx33 and Cx43 mRNA in a time- and dose-dependent manner. These data reveal that Cx33 and Cx43 genes are controlled differently within the testis and suggest that these two Cxs may exert opposite and complementary effects on spermatogenesis.

Spermatogenesis is a highly controlled process that allows proliferation and differentiation of male germ cells. This is under classical endocrine and paracrine controls. There is also evidence that gap junctions between Leydig cells, between Sertoli cells and between Sertoli and germ cells participate in the local regulation of spermatogenesis. Recent studies reveal that connexin 43 (Cx43), the predominant gap junction protein in the testis, is essential for the initiation and maintenance of spermatogenesis. In this review, we focus on the identification, distribution and control of connexins in the mammalian testis. The implication of connexin-based gap junctions in testicular physiology and in pathological disorders of spermatogenesis (spermatogenic arrest and testis cancer) is also discussed.

In the testis, Sertoli cells establish intercellular junctions that are essential for spermatogenesis. The SerW3 Sertoli cell line displays some features of native Sertoli cells. Western blot and immunofluorescence analyses showed that SerW3 Sertoli cells expressed typical components of tight (occludin and zonula occludens-1), anchoring (N-cadherin) and gap (connexin 43) junctions. Testicular toxicants (DDT, pentachlorophenol, dieldrin, dinitrobenzene, cadmium chloride, cisplatin, gossypol, bisphenol A and tert-octylphenol) affected intercellular junctions by either reducing the amount or inducing aberrant intracellular localization of these membranous proteins. Phosphodiesterase inhibitors (isobutyl methylxantine, rolipram, zaprinast, zardaverine) did not alter junctional-complex component levels but caused a rapid and reversible redistribution of these proteins to the cytoplasmic compartment. The present study showed that occludin, ZO-1, N-cadherin and specifically Cx43 could be early targets for testicular toxicants. The SerW3 cell line therefore appears as a useful in vitro model to evaluate molecules with potential anti-reproductive effects.