Philippe Durand

Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium

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.

Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model.

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 Durands 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 12days. Exposure to low concentrations of chromium (10μg/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.

Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium

There is evidence that exposure to environmental factors is at least partly responsible for changes in semen quality observed over the past decades. The detection of reproductive toxicants under Registration, Evaluation and Authorisation of Chemicals (REACH) will impact animal use for regulatory safety testing. We first validated a model of culture of rat seminiferous tubules for toxicological studies on spermatogenesis. Then, using this model of culture, we assessed the deleterious effects of 1, 10, and 100 microg/l hexavalent chromium [Cr(VI)] on meiotic cells. The prophase I of meiosis was studied in vivo and ex vivo. Bromo-2-deoxyuridine (BrdU) was used to describe the kinetics of germ cell differentiation. SCP3 labeling allowed to establish the distribution of the stages of the meiotic prophase I and to perform a qualitative study of the pachytene stage in the absence or presence of Cr(VI). The development of the meiotic step of pubertal rats was similar in vivo and ex vivo. The number of total cells appeared not affected by the presence of Cr(VI) irrespective of its concentration. However, the numbers of late spermatocytes and of round spermatids were decreased by Cr(VI) even at the lower concentration. The percentage of synaptonemal complex abnormalities increased slightly with the time of culture and dramatically with Cr(VI) concentrations. This model of culture appears suitable for toxicological studies. This study shows that Cr(VI) is toxic for meiotic cells even at low concentrations, and its toxicity increases in a dose-dependent manner.

Redundancy of the effect of TGFβ1 and β‐NGF on the second meiotic division of rat spermatocytes

We have previously shown that in cocultures of late pachytene/diplotene spermatocytes (PS/DS) with Sertoli cells, β‐nerve growth factor (β‐NGF) or transforming growth factor (TGFβ1) regulates the second meiotic division by blocking secondary spermatocytes in metaphase II, and thereby lowers round spermatid formation. In this study, we raised the question if β‐NGF and TGFβ1 have additional or redundant effects on this step. Hence, we addressed the effect of β‐NGF in combination with TGFβ1, as compared to those of β‐NGF or TGFβ1 separately, on the completion of meiosis by rat late PS/DS. Identification and counting of meiotic cells were performed by cytological methods. Under our culture conditions, some late PS/DS differentiated into round spermatids. When β‐NGF in combination with TGFβ1 was added to the culture medium, the numbers of PS/DS and of secondary spermatocytes were not modified by the treatment. By contrast, the number of round spermatids was about 2‐fold lower in treated cultures than in basal cultures, and an increase in metaphase II, but not metaphase I, over basal values was observed. Similar results were found when either β‐NGF or TGFβ1 was added to the culture medium. These results indicate a redundancy between β‐NGF and TGFβ1 on the negative regulation of the second meiotic division of rat spermatocytes. This suggests that β‐NGF and TGFβ1 share some common pathway of regulation of this step. In addition, these results reinforce the view that the adjustment of the number of round spermatids supported by Sertoli cells is a key event of the spermatogenic process. Microsc. Res. Tech., 2009.

Cytostatic Factor Proteins Are Present in Male Meiotic Cells and β-Nerve Growth Factor Increases Mos Levels in Rat Late Spermatocytes

Background In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells. Methodology/Principal Findings We show here, that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF, and their respective mRNAs, are present in male rat meiotic cells; this was assessed by using Western blotting, immunocytochemistry and reverse transcriptase PCR. We measured the relative cellular levels of Mos, Emi2, Cyclin E and Cdk2 in the meiotic cells by flow cytometry and found that the four proteins increased throughout the first meiotic prophase, reaching their highest levels in middle to late pachytene spermatocytes, then decreased following the meiotic divisions. In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF increased the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes. Conclusion/Significance Our results suggest that CSF is not restricted to the oocyte. In addition, they reinforce the view that NGF, by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells.

Complete Human and Rat Ex Vivo Spermatogenesis from Fresh or Frozen Testicular Tissue

ABSTRACT Until now, complete ex vivo spermatogenesis has been reported only in the mouse. In this species, the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in humans. We performed long-term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8- or 20-day-old male rats or from adult human subjects who had undergone hormone treatments leading to a nearly complete regression of their spermatogenesis before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related to a dramatic increase in the levels of the mRNAs specifically expressed by round spermatids, Transition protein 1, Transition protein 2, and Protamine 3 in rat cultures. From 2% to 3.8% of cells were found to be haploid cells by fluorescence in situ hybridization analysis of human cultures. In this bioreactor, long-term cultures of seminiferous tubule segments from prepubertal rats or from adult men allowed completion of the spermatogenic process leading to morphologically mature spermatozoa. Further studies will need to address the way of optimizing the yield of every step of spermatogenesis by adjusting the composition of the culture medium, the geometry, and the material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells

Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2-week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the motheaten SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied.

Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture.

To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis. A quantitative and comparative method was developed to estimate how known pathways were disturbed by each substance. This pathway-driven analysis revealed a strong alteration of steroidogenesis and an impairment of meiosis in all cases, albeit the initial molecular events were different for both substances. The ex vivo cytogenetic analysis confirmed that both fungicides alter the course of the first meiotic prophase. In addition, the mixture of both substances triggered effects greater than the sum of their cumulative effects and compromised future sperm motility after a shorter time of exposure compared with the fungicides tested separately. The alliance of an ex vivo culture with omics strategies complemented with a physiological examination is a powerful combination of tools for testing substances, separately or in a mixture, for their testicular toxicity. In particular, proteomics allowed the identification of systematically differentially expressed proteins in the secretomes of exposed cultures, such as FUCO and PEBP1, two proteins linked with the motility and fertilizing ability of spermatozoa, respectively. These proteins may be potential biomarkers of testicular dysfunction and infertility.

Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants

Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture. This organotypic culture system demonstrated the ability to successfully model in vivo testicular toxicity (Sertoli cell toxicity and disruption of meiosis) for all four compounds. These findings support the application of this system to study molecules and evaluate mechanisms of testicular toxicity.