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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:PA1 Participates in the Maintenance of Blood-Testis Barrier Integrity via Cooperation with JUN in the Sertoli Cells of Mice
| PRJNA778398 | ENA
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:BackgroundRNA N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood-testis barrier (BTB) through cell junctions between adjacent Sertoli cells.MethodsALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation-quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation-qPCR, western blot, coimmunoprecipitation, and polysome fractionation-qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure.ResultsALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice.ConclusionsOur study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.
| S-EPMC9682758 | 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:Carcinoma in situ (CIS) is the noninvasive precursor of most human testicular germ cell tumors. In normal seminiferous epithelium, specialized tight junctions between Sertoli cells constitute the major component of the blood-testis barrier. Sertoli cells associated with CIS exhibit impaired maturation status, but their functional significance remains unknown. The aim was to determine whether the blood-testis barrier is morphologically and/or functionally altered. We investigated the expression and distribution pattern of the tight junction proteins zonula occludens (ZO) 1 and 2 in normal seminiferous tubules compared to tubules showing CIS. In normal tubules, ZO-1 and ZO-2 immunostaining was observed at the blood-testis barrier region of adjacent Sertoli cells. Within CIS tubules, ZO-1 and ZO-2 immunoreactivity was reduced at the blood-testis barrier region, but spread to stain the Sertoli cell cytoplasm. Western blot analysis confirmed ZO-1 and ZO-2, and their respective mRNA were shown by RT-PCR. Additionally, we assessed the functional integrity of the blood-testis barrier by lanthanum tracer study. Lanthanum permeated tight junctions in CIS tubules, indicating disruption of the blood-testis barrier. In conclusion, Sertoli cells associated with CIS show an altered distribution of ZO-1 and ZO-2 and lose their blood-testis barrier function.
| S-EPMC1783719 | biostudies-literature
Project description:Yaks, a valuable livestock species endemic to China's Tibetan plateau, have a low reproductive rate. Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood-testis barrier (BTB) in the cryptorchidism. Previous studies have shown that CAV1 significantly contributes to the regulation of cell tight junctions and spermatogenesis. Therefore, we hypothesize that CAV1 may play a regulatory role in tight junctions and BTB in Yaks Sertoli cells, thereby influencing the development of cryptorchidism. Additional analysis using immunofluorescence, qRT-PCR, and Western blotting confirmed that CAV1 expression is up-regulated in yak cryptorchidism. CAV1 over-expression plasmids and small RNA interference sequences were then transfected in vitro into yak Sertoli cells. It was furthermore found that CAV1 has a positive regulatory effect on tight junctions and BTB integrity, and that this regulatory effect is achieved through the FAK/ERK signaling pathway. Taken together, our findings, the first application of CAV1 to yak cryptorchidism, provide new insights into the molecular mechanisms of cell tight junctions and BTB. This paper suggests that CAV1 could be used as a potential therapeutic target for yak cryptorchidism and may provide insight for future investigations into the occurrence of cryptorchidism, the maintenance of a normal physiological environment for spermatogenesis and male reproductive physiology in the yak.
| S-EPMC10812639 | biostudies-literature
Project description:Tight junction integral membrane proteins such as claudins and occludin are tethered to the actin cytoskeleton by adaptor proteins, notably the closely related zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3. All three ZO proteins have recently been inactivated in mice. Although ZO-3 knockout mice lack an obvious phenotype, animals deficient in ZO-1 or ZO-2 show early embryonic lethality. Here, we rescue the embryonic lethality of ZO-2 knockout mice by injecting ZO-2(-/-) embryonic stem (ES) cells into wild-type blastocysts to generate viable ZO-2 chimera. ZO-2(-/-) ES cells contribute extensively to different tissues of the chimera, consistent with an extraembryonic requirement for ZO-2 rather than a critical role in epiblast development. Adult chimera present a set of phenotypes in different organs. In particular, male ZO-2 chimeras show reduced fertility and pathological changes in the testis. Lanthanum tracer experiments show a compromised blood-testis barrier. Expression levels of ZO-1, ZO-3, claudin-11, and occludin are not apparently affected. ZO-1 and occludin still localize to the blood-testis barrier region, but claudin-11 is less well restricted and the localization of connexin-43 is perturbed. The critical role of ZO-2 for male fertility and blood-testis barrier integrity thus provides a first example for a nonredundant role of an individual ZO protein in adult mice.
| S-EPMC2762135 | biostudies-literature
Project description:It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.
| S-EPMC7992013 | biostudies-literature