Long-Term Maintenance of Viable Adult Rat Sertoli Cells Able to Establish Testis Barrier Components and Function in Response to Androgens.
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ABSTRACT:
SUBMITTER: Kabbesh H
PROVIDER: S-EPMC8467871 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Androgens are well known to be necessary for spermatogenesis. The purpose of this study was to determine Sertoli cell responsiveness to androgens according to age from birth to puberty. RESULTS:Testicular tissue samples were studied in a population of 84 control boys classified into seven groups according to age: group 1 (1-30 days), group 2 (1-3 months), group 3 (3-6 months), group 4 (0.5-3 years), group 5 (3-6 years), group 6 (6-12 years), and group 7 (12-16 years). We compared these data with those of 2 situations of pathology linked to androgens: 1/premature secretion of testosterone: 4 cases of Leydig cell tumor (LCT) in childhood; and 2 /defect of androgen receptors (AR): 4 cases of complete form of insensitivity to androgen syndrome (CAIS). In control boys, AR immunoreactivity (ir) in Sertoli cells appeared between 4.6 and 10.8 years of age, Anti-Mullerian Hormone (AMH) ir in Sertoli cells disappeared between 9.2 and 10.2 years of age. Connexin 43 (Cx43) ir in Sertoli cells and histological features of the onset of spermatogenesis appeared between 10.8 and 13,8 years of age. Cx43 ir was significantly higher in 12-16 year-olds than in younger boys. In case of CAIS, no spermatogenesis was observed, both AR and Cx43 ir were undetectable and AMH ir was elevated in Sertoli cells even at pubertal age. In the vicinity of LCTs, spermatogenesis occurred and both AR and Cx43 ir were strongly positive and AMH ir in Sertoli cells was low for age. CONCLUSIONS:Androgen action on Sertoli cells is required for onset of spermatogenesis and premature androgen secretion by LCT can induce spermatogenesis in the vicinity of the tumor. AR ir appeared earlier than onset of spermatogenesis, with large interindividual variability. The timing and mechanisms of Sertoli cell responsiveness to androgens are important issues for understanding the induction of spermatogenesis at puberty.
| S-EPMC8670046 | biostudies-literature
Project description:In adult rat testes, the blood-testis barrier (BTB) in the seminiferous epithelium must "open" (or "disassemble") to accommodate the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. However, the molecule(s) and/or mechanism(s) that regulate this event are unknown. In this report, C-type natriuretic peptide (CNP) was shown to be a regulator of BTB dynamics. Although Sertoli and germ cells contributed to the pool of CNP in the seminiferous epithelium, its receptor, natriuretic peptide receptor B, resided almost exclusively in Sertoli cells. CNP also expressed stage-specifically and localized predominantly at the BTB in the seminiferous epithelium at stage VIII of the epithelial cycle. A synthetic CNP-22 peptide, when added to Sertoli cell cultures, was shown to perturb Sertoli cell tight junction in vitro, causing disappearance of BTB-associated proteins (JAM-A, occludin, N-cadherin, and beta-catenin) from the cell-cell interface. This inhibitory effect of CNP on the tight junction was confirmed by transient overexpression of CNP in these cells, which was mediated, at least in part, by accelerating the internalization of BTB integral membrane proteins. To validate these in vitro findings, CNP-22 was administered to testes at a dose of 0.35 or 3.5 mug per testis, which was shown to perturb the BTB integrity In vivo when the barrier function was assessed by monitoring the diffusion of a small molecular probe across the BTB. In summary, CNP secreted by Sertoli and germ cells into the BTB microenvironment regulates BTB dynamics during spermatogenesis.
| S-EPMC1820671 | biostudies-literature
Project description:BackgroundThe blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown.ResultsHere, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1.ConclusionOverall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.
| S-EPMC8981650 | biostudies-literature
Project description:The conformation and function of a subset of serine and threonine-phosphorylated proteins are regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Pin1 is intensely expressed in Sertoli cells, but its function in this post mitotic cell remains unclear. Our aim was to investigate the role of Pin1 in the Sertoli cells. Lack of Pin1 caused disruption of the blood-testis barrier. We next investigated if the activin pathways in the Sertoli cells were affected by lack of Pin1 through immunostaining for Smad3 protein in testis tissue. Indeed, lack of Pin1 caused reduced Smad3 expression in the testis tissue, as well as a reduction in the level of N-Cadherin, a known target of Smad3. Pin1-/- testes express Sertoli cell marker mRNAs in a pattern similar to that seen in Smad3+/- mice, except for an increase in Wt1 expression. The resulting dysregulation of N-Cadherin, connexin 43, and Wt1 targets caused by lack of Pin1 might affect the mesenchymal-epithelial balance in the Sertoli cells and perturb the blood-testis barrier. The effect of Pin1 dosage in Sertoli cells might be useful in the study of toxicant-mediated infertility, gonadal cancer, and for designing male contraceptives.
| S-EPMC5539286 | biostudies-literature
Project description:Spermatogenesis is a complex process that establishes male fertility and involves proper communication between the germline (spermatozoa) and the somatic tissue (Sertoli cells). Many factors that are important for spermatozoa production are also required for Sertoli cell function. Recently, we showed that the transcriptional cofactor ubiquitously expressed transcript (UXT) encodes a protein that is essential in germ cells for spermatogenesis and fertility. However, the role of UXT within Sertoli cells and how it affects Sertoli cell function was still unclear. Here we describe a novel role for UXT in the Sertoli cell's ability to support spermatogenesis. We find that the conditional deletion of Uxt in Sertoli cells results in smaller testis size and weight, which coincided with a loss of germ cells in a subset of seminiferous tubules. In addition, the deletion of Uxt has no impact on Sertoli cell abundance or maturity, as they express markers of mature Sertoli cells. Gene expression analysis reveals that the deletion of Uxt in Sertoli cells reduces the transcription of genes involved in the tight junctions of the blood-testis barrier (BTB). Furthermore, tracer experiments and electron microscopy reveal that the BTB is permeable in UXT KO animals. These findings broaden our understanding of UXT's role in Sertoli cells and its contribution to the structural integrity of the BTB.
| S-EPMC7569350 | biostudies-literature
Project description:In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.
| S-EPMC4398761 | biostudies-literature
Project description:Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.
| S-EPMC4891789 | biostudies-literature
Project description:The blood testis barrier (BTB) is often studied with isolated immature Sertoli cells (SCs), transepithelial resistance (TER) measurements and FITC dextran diffusion assays. Recently, it was found that even in the absence of SCs, only few immune cells enter the seminiferous tubules. Thus, in this study, we evaluated the testicular immunological barrier (TIB) in vitro by transmigration of macrophages through SCs with and without peritubular cells (PCs) and with or without matrigel (MG). Primary PCs were isolated from adult rat testis and kept in mono- or co-cultures with the conditionally reprogrammed primary adult Sertoli cell line (PASC1) from rat that has been recently generated by our group. Rat monocytes isolated from fresh blood were differentiated into M0 macrophages, and after polarization to M1 or M2 macrophages characterized by gene expression of CXCL11 and TNF-α for M1, or CCL17 and CCL22 for M2. Transmigration of LeukoTracker-labeled M0, M1, and M2 macrophages through mono- and co-cultures of PCs/SCs with and without MG demonstrated that SCs are the main constituent of the TIB in vitro with only a negligible contribution of PCs or MG. Moreover, M2 macrophages showed less migration activity compared to M0 or M1. Treatment of SCs with testosterone (T) showed positive effects on the barrier in contrast to negative effects by interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α). The new transmigration model is suitable to evaluate transmigration of macrophages through a barrier consisting of testicular cells and can be applied to study the integrity of testicular barriers with respect to immunological aspects.
| S-EPMC9870002 | biostudies-literature
Project description:Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.
| S-EPMC4480425 | biostudies-literature
Project description:Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10-20 μM (∼5-10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr(407) was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr(407) was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr(407) and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB.
| S-EPMC3868803 | biostudies-literature