Cultured Rat Sertoli Cells Research Articles

The Regulation of Sertoli Cell Micro-RNAs by TGFB3.

Most events of spermatogenesis, including meiosis, spermiogenesis and spermiation, take place in a unique adluminal microenvironment created by the blood-testis barrier, which is in part comprised of specialised junctions between Sertoli cells. In the rat testis, restructuring of the blood-testis barrier occurs at stage VIII of the seminiferous epithelial cycle, to allow the movement of preleptotene spermatocytes from the basal to the adluminal compartment to prepare for meiosis. However, the molecular mechanism(s) by which this blood-testis barrier restructuring occurs are poorly understood. It is well known that members of the TGF beta superfamily, including TGFβ2, TGFβ3 and GDF9, are present at stage VIII and can disrupt blood-testis barrier integrity in vivo and in vitro. In other epithelial cell types (e.g. breast, kidney), TGFβ-induced cell junction restructuring is mediated, in part, via micro-RNAs (miRNAs), which are small non-coding RNAs that post-transcriptionally regulate gene expression. We therefore hypothesised that TGFβ3 can regulate miRNAs in Sertoli cells, which in turn control spermatogenesis by targeting junction-associated proteins important in the function of the blood-testis barrier. The aim of this study was to identify TGFβ3-regulated miRNAs in rat Sertoli cells in vitro, and to correlate these with potential junctional targets. Addition of TGFβ3 to rat Sertoli cells in primary culture caused both dose- and time-dependent decreases in Sertoli cell tight junction function; these decreases could be completely ablated by addition of a prodomain antagonist which specifically binds TGFβ3. Comparative microarray analysis was then used to characterise TGFβ3-specific miRNAs produced by rat Sertoli cells. Of a total of 353 miRNAs which were commonly expressed in both TGFβ3- and TGFβ3-antagonist treated cells, 13 were up-regulated by TGFβ3 >5-fold and 12 miRNAs were down- regulated >5-fold. Several of the up-regulated miRNAs (miR-615-3p, miR-139-3p, miR-349) were also present in high concentration in Sertoli cells. Bioinformatic prediction of miRNA-mediated pathways (DIANA LAB micro-RNA pathway analysis software) using the up-regulated miRNAs as a combined group revealed adherens junctions, focal adhesions, and the actin cytoskeleton, to be amongst the four top pathways represented. Each of these pathways has known roles in the specialised junctions between Sertoli cells which contribute to the blood-testis barrier, suggesting that TGFβ-mediated miRNAs may impact on their function. This is supported by recent evidence from our lab which demonstrated that FSH- and androgen-mediated miRNAs in Sertoli cells target cell adhesion pathways in a coordinated manner. We conclude that TGFβ3 mediates a discrete subset of Sertoli cell miRNAs which potentially impact the regulation of Sertoli cell junctions important in the function of the blood-testis barrier. Research supported by the National Health and Medical Research Council (Australia) Program Grant #494802 and Project Grant #1009144. (poster)

Molecular changes induced by bisphenol-A in rat Sertoli cell culture

Bisphenol-A (BPA) is an industrial chemical and is known to act as an endocrine disrupter. This study was designed to evaluate how BPA regulates Sertoli cell (SC) signal molecules. Purified rat SCs were cultured and treated with BPA (200 µmol/l) at various time points. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), nuclear factor kappa B (NF-κB), cyclooxygenase-1,2 (COX-1, 2), estrogen receptor-α (ER-α), and androgen receptor (AR). The levels of transferrin (TF), prostaglandin E2 (PGE2), and prostaglandin F2α (PGF2α) in culture medium were quantified by ELISA. Interleukin (IL)-1β and IL-6 mRNAs were measured by quantitative real-time PCR (QRT-PCR). Compared with the control, BPA activated the phosphorylation of ERK1/2 (p-ERK1/2) through 30 min to 6 h. TF was down-regulated at 6 and 24 h. Furthermore, IL-1β was up-regulated at 30 min and IL-6 was up-regulated at 1 and 24 h. ERK activity inhibitor (PD98059, 10 µmol/l) inhibited these molecular changes. These results reveal the possibility that BPA may have adverse effects on spermatogenesis via ERK1/2.

Evaluation of the effect of Carpolobia alba (Polygalaceae) aqueous extract on male reproductive function in rats

In order to assess the effect of Carpolobia alba on male reproductive function, its aqueous extract was tested on lactate release by rat Sertoli cell cultures. The extract was administered daily to adult male rats. After 60 days of treatment, the animals were sacrificed and weight of organs (liver, kidneys, testes, epididymides, seminal vesicles and prostate) recorded. Liver glutathione levels and aniline hydroxylase activity, epididymal α-glycosidase activity, as well as serum testosterone level and aminotransferase activities were also assessed. Carpolobia alba extract at the doses 0.01 and 0.1 mg/kg significantly increased serum testosterone levels (P<0.01) in male rats, while other investigated parameters on serum and Sertoli cells remained unchanged. These results proved the androgenic effect of C. alba and thereby, sustain its use in the management of male infertility.

P,p′-Dichlorodiphenoxydichloroethylene induced apoptosis of Sertoli cells through oxidative stress-mediated p38 MAPK and mitochondrial pathway

p,p′-DDE, the major metabolite of dichlorodiphenoxytrichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. However, the mechanism underlying its male reproductive toxicity remains limited. Our previous studies have demonstrated that p,p′-DDE could induce mitochondria-mediated apoptosis of cultured rat Sertoli cells. In the present study, we investigated mitogen-activated protein kinase pathways as well as other mitochondria-related molecules including Bax family members and cytochrome c. Results showed that p,p′-DDE could induce oxidative stress-mediated p38 and JNK phosphorylation. In addition, elevated mRNA levels of cytochrome c and ratios of bax/bcl-w and bak/bcl-w were induced by p,p′-DDE treatment, which could be inhibited by RNA synthesis inhibitor (actinomycin D). p,p′-DDE-induced apoptosis was blocked by NAC (N-acetyl-L-cystein) preincubation and attenuated by pretreatment with p38 inhibitor (SB202190) or actinomycin D, but not with JNK inhibitor (SP600125). All of the findings suggested that oxidative stress-mediated p38 MAPK pathway and the balance between pro- and anti-apoptotic bax-gene family might play critical roles in p,p′-DDE-induced apoptosis.

Regulation of Sertoli-Germ Cell Adhesion and Sperm Release by FSH and Nonclassical Testosterone Signaling

Testosterone and FSH act in synergy to produce the factors required to maximize the production of spermatozoa and male fertility. However, the molecular mechanisms by which these hormones support spermatogenesis are not well established. Recently, we identified a nonclassical mechanism of testosterone signaling in cultured rat Sertoli cells. We found that testosterone binding to the androgen receptor recruits and activates Src tyrosine kinase. Src then causes the activation of the epidermal growth factor receptor, which results in the phosphorylation and activation of the ERK MAPK and the cAMP response element-binding protein transcription factor. In this report, we find that FSH inhibits testosterone-mediated activation of ERK and the MAPK pathway in Sertoli cells via the protein kinase A-mediated inhibition of Raf kinase. In addition, FSH, as well as inhibitors of Src and ERK kinase activity, reduced germ cell attachment to Sertoli cells in culture. Using pathway-specific androgen receptor mutants we found that the nonclassical pathway is required for testosterone-mediated increases in germ cell attachment to Sertoli cells. Studies of seminiferous tubule explants determined that Src kinase, but not ERK kinase, activity is required for the release of sperm from seminiferous tubule explants. These findings suggest the nonclassical testosterone-signaling pathway acts via Src and ERK kinases to facilitate the adhesion of immature germ cells to Sertoli cells and through Src to permit the release of mature spermatozoa. In contrast, FSH acts to limit testosterone-mediated ERK kinase activity and germ cell attachment.

Claudin-11 expression increased in spermatogenic defect in human testes

Expression of claudin-11, which builds up blood-testis barrier (BTB), was increased in decreased spermatogenesis, including in hypospermatogenesis, spermatocytic and maturation arrest, and Sertoli cell only (SCO) testes. Increased claudin-11 immunoreactivity was observed at the inter-Sertoli tight junctions in maturation arrest and in the cytoplasm of Sertoli cells in SCO. The late spermatogenic wave may negatively regulate claudin-11 gene activation and the subcellular localization of claudin-11 in Sertoli cells, thus altering the BTB in the human testis.

FSH-stimulated PTEN activity accounts for the lack of FSH mitogenic effect in prepubertal rat Sertoli cells

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.

Activation and Repression Domains Within the Promoter of the Rat Cathepsin L Gene Orchestrate Sertoli Cell-Specific and Stage-Specific Gene Transcription in Transgenic Mice1

In murine testes, only Sertoli cells express the cathepsin L (Ctsl) gene, and this expression is restricted to stages V-VIII of the cycle. Our previous transgenic analysis of Tg (-2065/+977) demonstrated that this expression is regulated by a approximately 2-kb promoter. To begin to elucidate this regulation, we analyzed the in vivo expression of two new transgenes, Tg (-935/+977) and Tg (-451/+977). Tg (-935/+977) was expressed by Sertoli cells but, in contrast to Tg (-2065/+977), was expressed at all stages of the cycle, by spermatocytes, by the vascular endothelium, and by seven other organs. Tg (-451/+977) was not expressed by Sertoli cells but by spermatogenic cells and by the brain. Lack of expression of Tg (-451/+977) by Sertoli cells was not due to a lack of essential cis-acting elements. Transient transfection analysis of primary cultures of mature rat Sertoli cells demonstrated that in mature Sertoli cells, most of the activity of the Ctsl promoter is accounted for by one of two redundant upstream GC motifs and an Initiator that are within 100 bp of the transcription start site. We conclude that transcriptional repressors upstream from nucleotide -935 of the rat Ctsl gene restrict testicular expression of this gene to Sertoli cells at stages V-VIII. At these stages, transcriptional activators located between nucleotides -935 and -452 promote access of the transcriptional machinery to the two GC boxes and to the Initiator. Thus, upstream repressors and activators as well as cis-acting elements near the transcription start site control stage-specific Ctsl transcription by Sertoli cells.

FSH and bFGF stimulate the production of glutathione in cultured rat Sertoli cells

Migration of developing germ cells from the basal to the adluminal compartment of the seminiferous epithelium requires extensive tissue restructuring, resulting in the production of reactive oxygen species. Sertoli cells are involved in this process. Glutathione (GSH), produced by Sertoli cells, has an essential role in cell protection against oxidative stress. Intracellular GSH content is maintained by de novo synthesis, involving glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunits, and by recycling from oxidized GSH, catalysed by glutathione reductase (GR). To assess whether follicle-stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) modulate GSH production in Sertoli cells by regulating the expression of GCLC, GCLM and/or GR, we performed in vitro studies using rat Sertoli cells in primary culture. FSH and bFGF stimulation increased Sertoli cell GSH levels after 24 h incubation. The simultaneous addition of FSH and bFGF did not produce any further effect. GCLM expression was upregulated by FSH and bFGF 6 h. At 24 h, only the FSH-mediated effect was still observed. FSH and bFGF also upregulated GR expression. In conclusion, our results show that FSH and bFGF increase GSH levels in Sertoli cells through stimulation of the de novo synthesis and recycling by upregulating GCLM and GR expression respectively. Therefore, protection of germ cells against oxidative stress seems to be regulated by hormones and germ cell-released growth factors capable of influencing the production of Sertoli cell GSH.

Cytotoxicity and oxidative stress study in cultured rat Sertoli cells with Methyl tert-butyl ether (MTBE) exposure

Cultured Sertoli cells were tested for their cytotoxicity and oxidative stress induced by Methyl tert-butyl ether (MTBE) which has been extensively used as a gasoline additive. In cytotoxic experiments, Sertoli cells were cultured with medium alone (control), 5, 500, or 50,000 μM MTBE. Lactate dehydrogcnase (LDH) leakage assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI), and flow cytometric analyses were used. In oxidative stress experiments, Sertoli cells were cultured with medium alone (control), 0.5, 50, or 5000 μM MTBE. The production of reactive oxygen species (ROS), maleic dialdehyde (MDA) content and the level of superoxide dismutase (SOD) activity in cell supernatants were measured. Meanwhile, the expression level of 8-oxoguanine DNA glycosidase (OGG1) and extracellular form of superoxide dismutase ( SOD EX ) in Sertoli cells were determined by RT-PCR. We also compared the current findings with the previous findings in rat spermatogenic cells exposed to MTBE. The present data indicate that high dose MTBE may exert a direct toxic effect on Sertoli cells. Oxidative stress induced by MTBE is a possible mechanism of cytotoxicity.

Growth Differentiation Factor 9 Is a Germ Cell Regulator of Sertoli Cell Function

Oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFbeta ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

Sertoli Cells Enhance Formation of Capillary-Like Structures in Vitro

Sertoli cells isolated from the testis (referred to as extratesticular Sertoli cells) have been shown to facilitate allo- and xenogeneic cell transplantations. It appears likely that the ability of these cells to enhance the success of cell engraftment is due, in part, to the retention of their intratesticular functions of trophic support and immunoprotection. Sertoli cells also are involved in the regulation of angiogenesis in the testis, which may also contribute to enhanced cell engraftment success facilitated by extratesticular Sertoli cells. Because the maintenance of the cell's intratesticular angiogenic function has not yet been evaluated for extratesticular Sertoli cells, this study examined the cell's ability to enhance angiogenesis in vitro. Sertoli cell conditioned media were derived from isolated rat Sertoli cell cultures and used in a rat aortic model of induced angiogenesis, in endothelial and smooth muscle cell monocultures, and in endothelial smooth muscle cocultures. An angiogenic rat cytokine array identified angiogenic factors in the control and conditioned media. Aorta sections incubated with Sertoli cell conditioned media showed a marked increase in the formation of capillary-like structures when compared to controls. Likewise, endothelial cells incubated in conditioned media organized into capillary-like structures not observed when incubated in control media. In coculture, smooth muscle cells were associated with endothelial cell-derived capillary-like structures only when incubated in conditioned media. Cytokine arrays indicated the presence and a qualitative increase of specific angiogenic growth factors in Sertoli cell conditioned media not observed in control media. Results indicate that extratesticular Sertoli cells retain their intratesticular angiogenic function in vitro.

P,p′-DDE induces mitochondria-mediated apoptosis of cultured rat Sertoli cells

p,p′-Dichlorodiphenoxydichloroethylene (p,p′-DDE), the major metabolite of dichlorodiphenoxytrichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant. However, the mechanism underlying male reproductive toxicity of p,p′-DDE remains limited. In the present study, Sertoli cells were used to investigate the molecular mechanism involved in p,p′-DDE’s male reproductive toxicity. Results showed that p,p′-DDE exposure at over 30 μM showed induction of apoptotic cell death. p,p′-DDE could induce mitochondria-mediated apoptotic changes including elevation in reactive oxygen species (ROS) generation, decrease in mitochondrial membrane potential (Δ Ψ m), and release of cytochrome c into the cytosol, which could be blocked by antioxidant agent N-acetyl- l-cysteine (NAC). In addition, elevated ratios of Bax/Bcl-w and Bak/Bcl-w and cleavages of procaspase-3 and -9 were induced by p,p′-DDE treatment. All of the results suggested that ROS generation may play a critical role in the initiation of p,p′-DDE-induced apoptosis by mediation of the disruption of Δ Ψ m, the release of cytochrome c into the cytosol and further the activation of caspase cascade.

439. GDF9 and BMP15 are germ-free regulators of Sertoli cell function

Oocyte-secreted Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are critical regulatory factors in female reproduction. Together they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study, we show that the expression and stage-specific localisation of both factors are limited to the germ cells of the rat seminiferous epithelium; with GDF9 being principally localised in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of the GDF9 and BMP15 signalling receptors (ALK5, ALK6, and BMPRII). Individual receptor types were expressed throughout the seminiferous epithelium, but co-expression of type I and type II receptors was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFβ ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction formation and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localisation of the junctional proteins claudin-11, occludin and ZO-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 upregulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together these results demonstrate that GDF9 and BMP15 are germ cell specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

17Beta-Estradiol Induces the Translocation of the Estrogen Receptors ESR1 and ESR2 to the Cell Membrane, MAPK3/1 Phosphorylation and Proliferation of Cultured Immature Rat Sertoli Cells1

The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Transient receptor potential vanilloid 1 (TRPV1) channels in cultured rat Sertoli cells regulate an acid sensing chloride channel

Sertoli cells provide a controlled microenvironment for regulation and maintenance of spermatogenesis for which an acidic milieu is crucial for male fertility. Sertoli cells also contribute to protection of spermatogenetic cells. Here, we showed that TRPV1 is expressed in rat Sertoli cells and regulates an acid sensing Cl − channel (ASCC). The expression of TRPV1 in rat Sertoli cells was demonstrated by RT-PCR, immunostaining and calcium measurement experiments. ASCC activity was inhibited by capsaicin (IC 50 = 214.3 ± 1.6 nM), olvanil (IC 50 = 400 ± 1.7 pM) and resiniferatoxin (IC 50 = 9.3 ± 1.5 nM) but potentiated by capsazepine (EC 50 = 5.3 ± 1.3 μM) and ruthenium red (EC 50 = 2.3 ± 1.5 μM). In the human airway epithelial cell line Calu-3 in which ASCC can be detected but not TRPV1, capsaicin and capsazepine were without any effect. Finally the application of the non-steroidal anti-inflammatory drug ibuprofen prevented the control of ASCC by TRPV1. Our study provides the first evidence for a regulation by TRPV1 of an acid sensing chloride channel in rat Sertoli cells. TRPV1 and ASCC may thus be considered as new potential physiological regulators of spermatogenesis and targets for pharmacological treatments of reproductive disorders as cryptorchidism, Sertoli cell tumors or torsion of the spermatic cord.

Mono-(2-ethylhexyl) Phthalate rapidly decreases claudin-11 in Sertoli cells

Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Claudin-11 is involoved in membrane interactions at Sertoli cells tight-junction and with the extracellular matrix. The function of tight-junction is under the control of paracrine and endocrine as well as physiochemical factors in various tissues. So, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and claudin-11 in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP by activating mitogen-activated protein kinases (MAPK) signaling pathways. Controlled animal experiment in vitro. Sertoli cells were isolated and purified from the testes of 18-d-old Sprague Dawley rats and incubated at 34 C. On day 3 ex vivo, Sertoli cells were treated with MEHP (1 μM, 10 μM, or 100 μM). At 0.5, 1, 3, 6, and 24 h after either vehicle (control) or MEHP, whole cell lysates for protein and total RNA were isolated from each replicate. Claudin-11 mRNAs were evaluated by quantitative real-time RT-PCR analysis and phosphorylated proteins (p38, p44/42, and SAPK/JNK) were assayed by Western blot analysis. We also examined nitric oxide synthases (NOSs), the members of the tumor necrosis factor (TNF) ligands (Fas L and TRAIL) and its receptor (Fas) expressions by RT-PCR and Western blot analysis. MEHP treatment led to a significant time- and dose-dependent decrease in Claudin-11 mRNA. Fas, Fas L, TRAIL, and NOSs protein and mRNA were not changed. The MEHP exposure induced the phosphorylation of p44/42, not p38 and SAPK/JNK. When p44/42 MAPK activity inhibitors are used, decrease of claudin-11 by MEHP is prevented, indicating that these effects depend on activation of enzymes. These data show that MEHP exposure rapidly alters claudin-11 in Sertoli cells, which might contribute to defects in intact junctional complex formation of sertoli cells leading to impairment of the integrity of the blood-testis barrier and alterations in endocrine and spermatogenic function.

Evolution of DNA strand-breaks in cultured spermatocytes: The Comet Assay reveals differences in normal and γ-irradiated germ cells

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and γ-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

Octylphenol induces apoptosis in cultured rat Sertoli cells

In this study, the effects of 4- tert-octylphenol (OP) were examined on the viability of rat cultured Sertoli cells using the MTT assay and OP-induced apoptosis was detected by transmission electron microscope (TEM), flow cytometric analysis and Hoechst staining. In addition, RT-PCR was used to analyze the levels of Bcl-2 and Bax mRNA. Bcl-2, Bax and caspase-3 protein expressions were determined by Western blot analysis. Sertoli cells were treated with OP from 30 to 60 μM for 6–24 h. Decreased viability of Sertoli cells and increased apoptosis occurred in a concentration- and a time-dependent manner. The expression of Bcl-2 was down-regulated, while the expression of Bax up-regulated. OP also down-regulated the expression of 32 kDa procaspase-3, which was cleaved to generate active subunit (17 kDa). These results suggest that OP may induce Sertoli cell apoptosis by regulation of Bcl-2/Bax and caspase-3 activation.

Pharmacological profile of inhibition of the chloride channels activated by extracellular acid in cultured rat Sertoli cells

Sertoli cells from mammalian testis are key cells involved in the development and maintenance of stem cell spermatogonia as well as in the secretion of a Cl(-) and K(+)-rich fluid into the lumen of seminiferous tubules. The pharmacology and contribution of Cl(-) channels to the physiology of Sertoli cells were investigated using whole-cell patch clamp and iodide efflux experiments applied to cultured rat Sertoli cells. We characterized an outwardly rectifying Cl(-) current stimulated by various acid species including the physiologically relevant lactic acid. Using the iodide efflux technique, the pharmacological properties of this Cl(-) current, noted ICl(acid), revealed Ca(2+)-independent inhibition by DIDS (IC(50) = 27 microM), glibenclamide (IC(50) = 31 microM) and DPC (IC(50) = 86 microM). ICl(acid) was neither affected by calix[4]arene nor by 9-AC. The order of potency for inhibition of ICl(acid) is DIDS approximately glibenclamide > DPC >> calix[4]arene, 9-AC. For comparison, the inhibitory profile of the swelling- and ATP-activated Cl(-) currents in Sertoli cells is DPC = DIDS >> glibenclamide = 9-AC for ICl(swell) and DPC = 9-AC = DIDS >> glibenclamide for ICl(ATP). This description provides new insights into the physiology and pharmacology of the endogenous Cl(-) channels expressed and potentially involved in fluid secretion in Sertoli cells.