Primary Cultures Of Sertoli Cells Research Articles

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of Gi. AKT activation could be detected 5min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was Gi- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to Gi and activation of both MEK/ERK1/2 and PI3K/AKT pathways.

Focal adhesion kinase-Tyr 407 and -Tyr 397 exhibit antagonistic effects on blood–testis barrier dynamics in the rat

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr(407) and p-FAK-Tyr(397) display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr(407) and Tyr(397). Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr(407) phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.

Insufficient androgen and FSH signaling may be responsible for the azoospermia of the infantile primate testes despite exposure to an adult-like hormonal milieu

In humans, as well as in other higher primates, the infantile testis is exposed to an adult-like hormonal milieu, but spermatogenesis is not initiated at this stage of primate development. In the present study, we examined the molecular basis of this intriguing infertile state of the primate testis. The integrity of androgen receptor (AR) and FSH receptor (FSHR) signaling pathways in primary cultures of Sertoli cells (Scs) harvested from azoospermic infant and spermatogenic pubertal monkey testes were investigated under identical in vitro hormonal conditions. In order to synchronously harvest Scs from early pubertal testis, the activation of testicular puberty was timed experimentally by prematurely initiating gonadotrophin secretion in juvenile animals with an intermittent infusion of gonadotrophin-releasing hormone. While qRT-PCR demonstrated that AR and FSHR mRNA expression in Scs from infant and pubertal testes were comparable, androgen-binding and FSH-mediated cAMP production by infant Scs was extremely low. Compromised AR and FSHR signaling in infant Scs was further supported by the finding that testosterone (T) and FSH failed to augment the expression of the T responsive gene, claudin 11, and the FSH responsive genes, inhibin-βB, stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF) in Scs harvested at this stage of development. These results indicate that compromised AR and FSHR signaling pathways in Scs underlie the inability of the infant primate testis to respond to an endogenous hormonal milieu that later in development, at the time puberty, stimulates the initiation of spermatogenesis. This finding may have relevance to some forms of idiopathic infertility in men.

Tubulobulbar Complexes Formed In Sertoli Cells In Primary Cultures Associate With Intercellular Junction Elements

In the seminiferous epithelium, unique double membrane structures termed tubulobulbar complexes (TBCs) develop in association with basal junction complexes. These actin‐related structures are proposed to internalize intercellular junctions during the movement of spermatocytes from basal to adluminal compartments of the epithelium. To study the role of basal TBCs in junction turnover, we use a primary Sertoli cell culture system in which TBCs develop in the vicinity of junction complexes between Sertoli cells. We immunoprobed cultured Sertoli cells for the intercellular junction elements Claudin‐11, Connexin‐43 and Nectin‐2 and discovered rod‐like structures that protrude into Sertoli cells from the cell periphery. These protrusions resemble TBCs that form in the intact seminiferous epithelium. These protrusions also co‐localize with F‐actin, which is known to associate with the proximal tubular part of TBCs and can be used as a marker for TBCs. Significantly, cultured Sertoli cells that express GFP‐tagged Claudin‐11 and Connexin‐43 extend similar protrusions into neighboring non‐transfected Sertoli cells. Our results are consistent both with the conclusion that junction elements from neighboring Sertoli cells are found together within TBCs, and with the hypothesis that tubulobulbar complexes may play a role in the turnover of intercellular junctions. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source : NSERC

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

Expression of ATP-Binding Cassette Membrane Transporters in Rodent and Human Sertoli Cells: Relevance to the Permeability of Antiretroviral Therapy at the Blood-Testis Barrier

The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [³H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [³H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [³H]atazanavir, BCECF, and [³H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB.

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells culturedin vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100nM E2 (17β-oestradiol) or 100nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2pmol/cell) at similar rates during the 50h. After 25h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.

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)

Defective CFTR-Dependent CREB Activation Results in Impaired Spermatogenesis and Azoospermia

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO3 − transport and the expression of the HCO3 − sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO3 − could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

Tubulobulbar complexes in Sertoli cell primary cultures

In the seminiferous epithelium, unique double membrane structures termed tubulobulbar complexes (TBCs) develop in association with basal junction complexes between Sertoli cells. These actin‐related structures are thought to internalize intercellular junctions during the movement of spermatocytes from basal to adluminal compartments of the epithelium. We are establishing a primary Sertoli cell culture system to study the role of basal TBCs in junction turnover. We confirm that Sertoli cells isolated from 20 day‐old rat pups morphologically differentiate when grown on Matrigel in Transwells. The cells are high cuboidal to columnar in shape with basal nuclei, and with basal junction complexes similar to the appearance of junction complexes between Sertoli cells in vivo. Significantly, TBCs and TBC‐like structures develop in association with intercellular junctions. Double‐membrane coated‐pits, together with double‐membrane tubular structures capped by coated‐pits are observed in regions related to ectoplasmic specializations. When immunostained for integral junction proteins, “rod‐like” structures are observed to extend into the cell from the periphery. These observations indicate that TBCs are able to develop in differentiated Sertoli cells in culture, making further functional analyses of TBCs possible in vitro. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source: NSERC

Hormonal Regulation of Sertoli Cell Micro-RNAs at Spermiation

Spermatogenesis is absolutely dependent on FSH and androgens; suppression of these hormones inhibits germ cell development and thus sperm production. The final release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to hormone suppression. To define the molecular mechanisms that mediate FSH and androgen effects in the Sertoli cell, we investigated the expression and regulation of micro-RNAs (miRNAs), small noncoding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs up-regulated more than 2-fold after hormone suppression in vivo and in vitro in primary Sertoli cell cultures. The regulation of four of these miRNAs (miR-23b, -30c, -30d, and -690) was confirmed by quantitative RT-PCR. Bioinformatic analysis of potential targets of hormonally regulated miRNAs identified genes important for focal adhesion and regulation of the actin cytoskeleton, processes known to be intimately associated with adhesion of spermatids to Sertoli cells. Two of the identified genes, Pten, an intracellular phosphatase, and Eps15, a mediator of endocytosis, were down-regulated by the withdrawal of hormones in vivo and possess miR-23b target sites in their 3'-untranslated regions. Overexpression of miR-23b in vitro resulted in decreased translation of PTEN and EPS15 protein as assessed by Western blot and luciferase analysis. We conclude that FSH and androgens act on Sertoli cells in stage VIII to control the expression of miRNAs that operate in a coordinated manner to regulate cell adhesion pathways and male fertility and that miRNA transcription is a new paradigm in the hormone dependence of spermiation.

An in vitro system to study Sertoli cell blood-testis barrier dynamics.

The use of an in vitro system based on primary cultures of Sertoli cells isolated from rat testes has greatly facilitated the study of the blood-testis barrier in recent years. Herein, we summarize the detailed procedures on the isolation of undifferentiated Sertoli cells from 20-day-old rat testes, the culture of these cells as a monolayer on Matrigel-coated bicameral units, the characterization of these cultured cells, and the use of the Sertoli cell epithelium for monitoring the integrity of the Sertoli cell blood-testis barrier. This information is based on the routine use of this system in our laboratory to study the Sertoli cell blood-testis barrier in the past two decades, which should be helpful for investigators in the field.

Connexin 43 is critical to maintain the homeostasis of the blood–testis barrier via its effects on tight junction reassembly

In mammalian testes, the blood-testis barrier (BTB) or Sertoli cell barrier created by specialized junctions between Sertoli cells near the basement membrane confers an immunological barrier by sequestering the events of meiotic division and postmeiotic germ cell development from the systemic circulation. The BTB is constituted by coexisting tight junctions (TJs), basal ectoplasmic specializations, desmosomes, and gap junctions. Despite being one of the tightest blood-tissue barriers, the BTB has to restructure cyclically during spermatogenesis. A recent study showed that gap junction protein connexin 43 (Cx43) and desmosome protein plakophilin-2 are working synergistically to modulate the BTB integrity by regulating the distribution of TJ-associated proteins at the Sertoli-Sertoli cell interface. However, the precise role of Cx43 in regulating the cyclical restructuring of junctions remains obscure. In this report, the calcium switch and the bisphenol A (BPA) models were used to induce junction restructuring in primary cultures of Sertoli cells isolated from rat testes that formed a TJ-permeability barrier that mimicked the BTB in vivo. The removal of calcium by EGTA perturbed the Sertoli cell tight junction barrier, but calcium repletion allowed the "resealing" of the disrupted barrier. However, a knockdown of Cx43 in Sertoli cells by RNAi significantly reduced the kinetics of TJ-barrier resealing. These observations were confirmed using the bisphenol A model in which the knockdown of Cx43 by RNAi also perturbed the TJ-barrier reassembly following BPA removal. In summary, Cx43 is crucial for TJ reassembly at the BTB during its cyclic restructuring throughout the seminiferous epithelial cycle of spermatogenesis.

Bisphenol A effect on glutathione synthesis and recycling in testicular Sertoli cells

Controversial effects of bisphenol A (BPA) have been reported on testicular function. These differences might reflect dissimilar exposure conditions. Dose responses to toxicants may be non-linear, e.g. U-shaped, with effects at low and at high levels of exposure and lower or inexistent effects at intermediate levels. Sertoli cells produce high levels of glutathione (GSH) as a cell defense mechanism. In this study, we addressed the question whether the exposure to different doses of BPA could influence Sertoli cell GSH synthesis and recycling. Primary Sertoli cell cultures were exposed to various doses of BPA (0.5 nM-100 μM). Cell viability was measured as an outcome of toxic effect. GSH cell content was determined to evaluate cell response to toxicant exposure. Glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunit expression were assessed to estimate GSH synthesis, and GSH reductase (GR) expression to estimate GSH recycling. BPA 100 μM, but not lower doses, decreased cell viability. BPA 10 and 50 μM, but not lower doses, induced an increment in Sertoli cell GSH levels, due to a rapid upregulation of GCLC and GR and a slower upregulation of GCLM. High doses of BPA are deleterious for Sertoli cells. Intermediate doses do not affect Sertoli cell viability and increase cell content of GSH owing to increased GSH synthesis and recycling enzyme expression. Lower doses of BPA are not capable of eliciting a cell defense response. These observations may explain a non-linear dose response of Sertoli cells to BPA.

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.

Effect of microwave radiation on primary cultured Sertoli cells

To explore whether microwave radiation may cause injury of primary cultured Sertoli cells. The model of primary cultured Sertoli cells in vitro was established, which was radiated by microwave with average power density 0, 30 and 100 mW/cm(2) for five minutes. The changes of cell cycle, apoptosis and death, and intracellular Ca2+ concentration in the Sertoli cells were measured at sixth hours through Annexin V-PI double labeling and Fluo-3-AM labeling, flow cytometry combined with laser scanning confocal microscopy after microwave exposure. The numbers of Sertoli cells were obviously reduced in G0-G1 and G2-M phase (62.57% +/- 3.22% and 8.25% +/- 1.75%) and increased in S phase (29.17% +/- 4.87%) compared with the control groups (79.18% +/- 0.24%, 11.17% +/- 0.50% and 9.64% +/- 0.62%) (P < 0.05 or P < 0.01), but the changes of rate of apoptosis and death and intracellular Ca2+ concentration showed no difference at 6 h after exposure to 30 mW/cm(2) microwave. There was a significant increase in the Sertoli cell counts of G0-G1 phase (87.69% +/- 1.32%), and decrease in the Sertoli cell counts of G2-M and S phase (7.41% +/- 0.60% and 4.87% +/- 0.91%) (P < 0.01). There was also a significant increase in intracellular Ca2+ concentration and rate of apoptosis and death (P < 0.05 or P < 0.01) at 6 h after exposure to 100 mW/cm(2) microwave. 100 mW/cm(2) microwave radiation may cause growth inhibition and increase of apoptosis and death in the primary cultured Sertoli cells. The increase of intracellular Ca2+ concentration is one of the injury mechanisms.

Connexin 43 and plakophilin-2 as a protein complex that regulates blood–testis barrier dynamics

The blood-testis barrier (BTB) formed by adjacent Sertoli cells is composed of coexisting tight junction (TJ), basal ectoplasmic specialization (ES), and desmosome-like junction. Desmosome-like junctions display structural features of desmosome and gap junctions, but its function at the BTB remains unknown. Herein, we demonstrate that connexin 43 (Cx43), a gap junction integral membrane protein, structurally interacts with desmosomal protein plakophilin-2 (PKP2), basal ES proteins N-cadherin and beta-catenin, and signaling molecule c-Src, but not with the TJ proteins occludin and ZO-1 in the seminiferous epithelium of adult rats. The localization of Cx43 in the seminiferous epithelium during (i) the normal epithelial cycle of spermatogenesis and (ii) anchoring junction restructuring at the Sertoli-spermatid interface induced by adjudin which mimics junction restructuring events during spermatogenesis have suggested that Cx43 is involved in cell adhesion. The knockdown of Cx43 by RNAi technique using specific siRNA duplexes was performed in primary Sertoli cell cultures with an established TJ permeability barrier that mimicked the BTB in vivo. This knockdown of Cx43 affected neither the TJ barrier function nor the steady-state levels of junction proteins of TJ, basal ES, and desmosome-like junction. However, after the knockdown of both Cx43 and PKP2, the Sertoli cell TJ barrier function was perturbed transiently. This perturbation was concomitant with a mislocalization of occludin and ZO-1 from the cell-cell interface. In summary, Cx43 and PKP2 form a protein complex within the desmosome-like junction to regulate cell adhesion at the BTB, partly through its effects on the occludin/ZO-1 complex, so as to facilitate the transit of primary preleptotene spermatocytes.

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.

Enzymatic properties of adult human Sertoli cells in vitro.

The adult human Sertoli cells produced lactate, estradiol-17beta, transferrin and inhibin; germ cells modulate synthesis of these compounds. In order to study the functional features of human Sertoli cells in vitro, the aim of this study was to measure the lactic dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and creatine kinase activities (CK) in primary cultures of Sertoli cells prepared from young men (mean age: 29 years, n = 11). Five LDH isozymes have been found in Sertoli cells, the main fractions being the LDH3 and LDH4; each of them represented 30% of the total LDH activity. Furthermore, CK and ALP activities were measured in Sertoli cells. It is of note that the Sertoli cell ALP activity was 50% lower than that of germ cells. Whatever the Sertoli cell parameter measured herein, there is a great variability between patients and FSH (dbc AMP, as well as retinol, insulin, testosterone) is poorly effective in improving these enzymatic activities in vitro. We have confirmed that GGT was exclusively present in Sertoli cells and thus may be considered as a specific marker. LDH is involved in Sertoli cell glucose transformation and thus provided energetic substrates for germ cells. In contrast, the roles of CK and ALP remains to be clarified. In conclusion, we have demonstrated the existence of several enzymes namely LDH, GGT, ALP and CK in Sertoli cells prepared from adult human testis.