TM4 Sertoli Cells Research Articles

The transcription factors NR5A1 and JUNB cooperate to activate the Axl promoter in mouse Sertoli cell lines.

The Axl gene is a receptor tyrosine kinase essential for male fertility. With other Tyro3 family members, it regulates cell apoptosis and preserves the organization of seminiferous tubules. However, the regulation of the expression of Axl in testicular Sertoli cells is not entirely understood. The transcription factors NR5A1 and JUNB are involved in several male fertility mechanisms such as sex development and steroidogenesis. We hypothesize that Axl promoter activity is regulated by cooperation between JUNB and NR5A1 in Sertoli cells. Following transfections of TM4 Sertoli cells with DsiRNA interference against Axl, our results show that cell morphology may be regulated by AXL. Using transfections of expression plasmids and reporter plasmids containing the Axl promoter, we report that Axl expression is highly activated by cooperation between NR5A1 and JUNB in TM4 and 15P-1 Sertoli cells. Chromatin immunoprecipitation and luciferase reporter assays with 5' promoter deletions demonstrate that JUNB and NR5A1 are being recruited to DNA regulatory elements in the proximal region of the Axl promoter. The fourth intronic region of Axl also participates in the recruitment of JUNB. Thus, Axl expression is regulated by a cooperation between the transcription factors JUNB and NR5A1 and influences the morphology of TM4 Sertoli cells.

Potential role of mitochondria and endoplasmic reticulum in the response elicited by D-aspartate in TM4 Sertoli cells.

D-Aspartic Acid (D-Asp) affects spermatogenesis by enhancing the biosynthesis of the sex steroid hormones acting either through the hypothalamus-pituitary-testis axis or directly on Leydig cells. Recently, in vitro studies have also demonstrated the direct effects of D-Asp on the proliferation and/or activity of germ cells. However, although D-Asp is present in Sertoli cells (SC), the specific role of the amino acid in these cells remains unknown. This study investigated the effects of D-Asp on the proliferation and activity of TM4 SC, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). We found that D-Asp enhanced the proliferation and activity of TM4 cells as evidenced by the activation of ERK/Akt/PCNA pathway and the increase in the protein levels of the androgen receptor. Furthermore, D-Asp reduced both the oxidative stress and apoptotic process. An increase in mitochondrial functionality and dynamics, as well as a reduction in ER stress, were also found in D-Asp-treated TM4 cells. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions and lipid transfer from ER to the mitochondria, and vice versa. The data demonstrated that D-Asp induced stabilization of MAM in TM4 cells. In conclusion, this study is the first to demonstrate a direct effect of D-Asp on SC activity and to clarify the cellular/molecular mechanism underlying these effects, suggesting that D-Asp could stimulate spermatogenesis by improving the efficiency of SC.

Integrative analysis of transcriptomics and metabolomics reveals the protective effect and mechanism of salidroside on testicular ischemia-reperfusion injury.

Testicular torsion is a critical urologic condition for which testicular detorsion surgery is considered irreplaceable as well as the golden method of reversal. However, the surgical treatment is equivalent to a blood reperfusion process, and no specific drugs are available to treat blood reperfusion injuries. Salidroside (SAL) is one of the main effective substances in rhodiola, which has been shown to have antioxidant and antiapoptosis activities. This study was designed to determine whether SAL exerted a protective effect on testicular ischemia-reperfusion (I/R) injury. In this study, the I/R injury model of the testes and reoxygenation (OGD/R) model were used for verification, and SAL was administered at doses of 100mg/kg and 0.05mmol/L, respectively. After the experiments, the testicular tissue and TM4 Sertoli cells were collected for histopathologic and biochemical analyses. The results revealed that SAL improves the structure of testicular tissue and regulates the oxidation-antioxidation system. To further understand the molecular mechanisms of SAL in treating testicular I/R injuries, transcriptomics and metabonomics analyses were integrated. The results show that the Nfr2/HO-1/GPX4/ferroptosis signaling pathway is enriched significantly, indicating that it may be the main regulatory pathway for SAL in the treatment of testicular I/R injuries. Thereafter, transfection with Nrf2 plasmid-liposome was used to reverse verify that the Nfr2/HO-1/GPX4/ferroptosis signaling pathway was the main pathway for SAL anti-testicular I/R injury treatment. Thus, it is suggested that SAL can protect against testicular I/R injuries by regulating the Nfr2/HO-1/GPX4 signaling pathway to inhibit ferroptosis and that SAL may be a potential drug for the treatment of testicular I/R injuries.

Assessment of the toxicity of different antiretroviral drugs and their combinations on Sertoli and Leydig cells

The human immunodeficiency virus continues to pose a significant global public health challenge, affecting millions of individuals. The current treatment strategy has incorporated the utilization of combinations of antiretroviral drugs. The administration of these drugs is associated with many deleterious consequences on several physiological systems, notably the reproductive system. This study aimed to assess the toxic effects of abacavir sulfate, ritonavir, nevirapine, and zidovudine, as well as their combinations, on TM3 Leydig and TM4 Sertoli cells. The cell viability was gauged using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays. Reactive oxygen species (ROS) production was assessed via the 2’,7’-dichlorofluorescein diacetate (DCFDA) test, and DNA damage was determined using the comet assay. Results indicated cytotoxic effects at low drug concentrations, both individually and combined. The administration of drugs, individually and in combination, resulted in the production of ROS and caused damage to the DNA at the tested concentrations. In conclusion, the results of this study suggest that the administration of antiretroviral drugs can lead to testicular toxicity by promoting the generation of ROS and DNA damage. Furthermore, it should be noted that the toxicity of antiretroviral drug combinations was shown to be higher compared to that of individual drugs.

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

Prenatal dexamethasone exposure impairs rat blood-testis barrier function and sperm quality in adult offspring via GR/KDM1B/FSTL3/TGFβ signaling.

Both epidemiological and animal studies suggest that adverse environment during pregnancy can change the offspring development programming, but it is difficult to achieve prenatal early warning. In this study we investigated the impact of prenatal dexamethasone exposure (PDE) on sperm quality and function of blood-testis barrier (BTB) in adult offspring and the underlying mechanisms. Pregnant rats were injected with dexamethasone (0.1, 0.2 and 0.4 mg·kg-1·d-1, s.c.) from GD9 to GD20. After weaning (PW4), the pups were fed with lab chow. At PW12 and PW28, the male offspring were euthanized to collect blood and testes samples. We showed that PDE significantly decreased sperm quality (including quantity and motility) in male offspring, which was associated with impaired BTB and decreased CX43/E-cadherin expression in the testis. We demonstrated that PDE induced morphological abnormalities of fetal testicle and Sertoli cell development originated from intrauterine. By tracing to fetal testicular Sertoli cells, we found that PDE dose-dependently increased expression of histone lysine demethylases (KDM1B), decreasing histone 3 lysine 9 dimethylation (H3K9me2) levels of follistatin-like-3 (FSTL3) promoter region and increased FSTL3 expression, and inhibited TGFβ signaling and CX43/E-cadherin expression in offspring before and after birth. These results were validated in TM4 Sertoli cells following dexamethasone treatment. Meanwhile, the H3K9me2 levels of FSTL3 promoter in maternal peripheral blood mononuclear cell (PBMC) and placenta were decreased and its expression increased, which was positively correlated with the changes in offspring testis. Based on analysis of human samples, we found that the H3K9me2 levels of FSTL3 promoter in maternal blood PBMC and placenta were positively correlated with fetal blood testosterone levels after prenatal dexamethasone exposure. We conclude that PDE can reduce sperm quality in adult offspring rats, which is related to the damage of testis BTB via epigenetic modification and change of FSTL3 expression in Sertoli cells. The H3K9me2 levels of the FSTL3 promoter and its expression in the maternal blood PBMC can be used as a prenatal warning marker for fetal testicular dysplasia.

Novel findings from arsenic‑lead combined exposure in mouse testicular TM4 Sertoli cells based on transcriptomics

Arsenic (As) and lead (Pb) exist widespread in daily life, and they are common harmful substances in the environment. As and Pb pollute the environment more often in combination than in isolation. The TM4 Sertoli cell line is one of the most common normal mouse testicular Sertoli cell lines. In vitro, we found that the type of combined action of As and Pb on TM4 Sertoli cells was additive action by using the isobologram analysis. To further investigate the combined toxicity of As and Pb, we performed mRNA and miRNA sequencing on TM4 Sertoli cells exposed to As alone (4 μM NaAsO2) and AsPb combined (4 μM NaAsO2 and 150 μM PbAc), respectively. Compared with the control group, 1391 differentially expressed genes (DEGs) and 6 differentially expressed miRNAs (DEMs) were identified in the As group. Compared with the control group, 2384 DEGs and 44 DEMs were identified in the AsPb group. Compared with the As group, 387 DEGs and 4 DEMs were identified in the AsPb group. Through data analysis, we discovered for the first time that As caused the dysfunction of cholesterol synthesis and energy metabolism, and disrupted cyclic adenosine monophosphate signaling pathway and wingless/integrated (Wnt) signaling pathway in TM4 Sertoli cells. In addition to affecting cholesterol synthesis and energy metabolism, AsPb combined exposure also up-regulated the antioxidant reaction level of TM4 Sertoli cells. Meanwhile, the Wnt signaling pathway of TM4 Sertoli cells was relatively normal when exposed to AsPb. In conclusion, at the transcription level, the combined action of AsPb is not merely additive effect, but involves synergistic and antagonistic effects. The new discovery of the joint toxic mechanism of As and Pb breaks the stereotype of the combined action and provides a good theoretical basis and research clue for future study of the combined-exposure of harmful materials.

Testosterone restores TM3 and TM4 cell viability, reduces reactive oxygen species generation, and protects against atrazine-induced stereological changes in rat testes.

In this study, we performed the stereological examination of rat testes and evaluated the protective effect of testosterone against atrazine (ATZ) toxicity in TM3 Leydig and TM4 Sertoli cells. Testosterone intake in rats increased the volumetric density of the seminiferous tubules; tubular diameter; germinal epithelial height; number of spermatogonia, primary and secondary spermatocytes, round spermatids, Sertoli cells, and Leydig cells; and Johnsen scores compared with the values after ATZ treatment (p < 0.05). Furthermore, testosterone increased the viability of TM3 cells and reduced reactive oxygen species (ROS) generation in TM4 cells compared to theATZ-treated group. In conclusion, exogenous testosterone intake maintains testicular morphometry and spermatogenesis in rats, and minimizes cell death and ROS generation in testicular cell lines exposed to ATZ. However, TM4 cells are more responsive to testosterone-mediated regulation of ROS generation induced by ATZ than TM3 cells.

Testosterone modulates atrazine-induced cytotoxicity in testicular cell lines

Testosterone greatly influences healthy growth and development of male sex organs. Testosterone has been demonstrated to possess antioxidant and anti-cytotoxic effects in cells. The present study examine the protective effect of testosterone on ATZ-induced cytotoxicity using TM3 Leydig and TM4 Sertoli cell lines as testicular in vitro models. TM3 Leydig and TM4 Sertoli cell lines were treated with atrazine (ATZ) 232 μM with varying concentrations of testosterone (0.1. 1, 10, 20 nM) for 6 h, 12 h and 48 h. Administration of testosterone increased the viability of the cell lines for 12 h and 48 h exposure compared to ATZ values (p&lt; 0.005). Furthermore, testosterone reduced TM3 cell and TM4 cell GSH levels. Exogenous testosterone intake minimizes cell death and GSH levels in testicular cell lines exposed to ATZ. However, the lowest dose of testosterone attenuates the ATZ-induced increase of GSH levels in both TM3 and TM4 cells (p&lt; 0.005).

Dual Activation of Peroxymonosulfate Using MnFe2O4/g-C3N4 and Visible Light for the Efficient Degradation of Steroid Hormones: Performance, Mechanisms, and Environmental Impacts.

Single activation of peroxymonosulfate (PMS) in a homogeneous system is sometimes insufficient for producing reactive oxygen species (ROS) for water treatment applications. In this work, manganese spinel ferrite and graphitic carbon nitride (MnFe2O4/g-C3N4; MnF) were successfully used as an activator for PMS under visible light irradiation to remove the four-most-detected-hormone-contaminated water under different environmental conditions. The incorporation of g-C3N4 in the nanocomposites led to material enhancements, including increased crystallinity, reduced particle agglomeration, amplified magnetism, improved recyclability, and increased active surface area, thereby facilitating the PMS activation and electron transfer processes. The dominant active radical species included singlet oxygen (1O2) and superoxide anions (O2•-), which were more susceptible to the estrogen molecular structure than testosterone due to the higher electron-rich moieties. The self-scavenging effect occurred at high PMS concentrations, whereas elevated constituent ion concentrations can be both inhibitors and promoters due to the generation of secondary radicals. The MnF/PMS/vis system degradation byproducts and possible pathways of 17β-estradiol and 17α-methyltestosterone were identified. The impact of hormone-treated water on Oryza sativa L. seed germination, shoot length, and root length was found to be lower than that of untreated water. However, the viability of both ELT3 and Sertoli TM4 cells was affected only at higher water compositions. Our results confirmed that MnF and visible light could be potential PMS activators due to their superior degradation performance and ability to produce safer treated water.

Development of a model for studying the developmental consequences of oxidative sperm DNA damage by targeting redox-cycling naphthoquinones to the Sertoli cell population.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations.

From animal to cell model: Pyroptosis targeted-fibrosis is a novel mechanism of lead-induced testicular toxicity.

Lead (Pb) exists widely in soil and seriously threatens agricultural soil and food crops. Pb can cause serious damage to organs. In this study, the animal model of Pb-induced rat testicular injury and the cell model of Pb-induced TM4 Sertoli cell injury were established to verify whether the testicular toxicity of Pb was related to pyroptosis-mediated fibrosis. The results of experiment in vivo showed that Pb could cause oxidative stress and up-regulated the expression levels of inflammation, pyroptosis, and fibrosis-related proteins in the testis of rats. The results of experiments in vitro showed that Pb induced the cell damage, enhanced the reactive oxygen species level in the TM4 Sertoli cells. After using nuclear factor-kappa B inhibitor and Caspase-1 inhibitor, the elevation of TM4 Sertoli cell inflammation, pyroptosis, and fibrosis-related proteins induced by Pb exposure was significantly decreased. Taken together, Pb can cause pyroptosis-targeted fibrosis and ultimately issues in testicular damage.

Col3a1 delivered via extracellular vesicles of Sertoli cells is essential for mice Sertoli cell proliferation

It is generally believed that Sertoli cells can proliferate only before sexual maturity. In this study, we found that extracellular vesicles of Sertoli cells derived from prepubertal mice (SEVs) have the ability to promote the proliferation of Sertoli cell population. In addition, via proteomic analysis, we compared the functional components of extracellular vesicles derived from Sertoli cells of mice at 12–14 days and 8 weeks. The functional profiling of SEVs suggested important developmental roles, and this was confirmed by analysis comparing the transcriptomic changes in Sertoli cells treated with DMSO and GW4869. The following analysis pointed to Col3a1 as a key factor in SEVs, which was further validated using primary Sertoli cells and TM4 cell line. The present study suggests a possible role for Col3a1 in promoting the proliferation of cultured Sertoli cells and provides a new perspective on the function of extracellular vesicles in Sertoli cell development.

Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models.

Sertoli cells are essential for germ cell development and function. Their disruption by endocrine disrupting chemicals (EDCs) or drugs could jeopardize spermatogenesis, contributing to male infertility. Perinatal exposure to EDCs and acetaminophen (APAP) disrupts male reproductive functions in animals and humans. Infants can be exposed simultaneously to the dietary soy phytoestrogen genistein (GEN) and APAP used for fever or pain relief. Our goal was to determine the effects of 10-100 µM APAP and GEN, alone or mixed, on immature Sertoli cells using mouse TM4 Sertoli cell line and postnatal-day 8 rat Sertoli cells, by measuring cell viability, proliferation, prostaglandins, genes and protein expression, and functional pathways. A value of 50 µM APAP decreased the viability, while 100 µM APAP and GEN decreased the proliferation. Sertoli cell and eicosanoid pathway genes were affected by GEN and mixtures, with downregulation of Sox9, Cox1, Cox2, and genes relevant for Sertoli cell function, while genes involved in inflammation were increased. RNA-seq analysis identified p53 and TNF signaling pathways as common targets of GEN and GEN mixture in both cell types. These results suggest that APAP and GEN dysregulate immature Sertoli cell function and may aid in elucidating novel EDC and drug targets contributing to the etiology of male infertility.

Toxicity of methomyl insecticides to testicular cells and protective effect of folic acid.

Methomyl is a carbamate insecticide with confirmed testicular toxicity. This study intended to observe the effect of methomyl on testicular cells and the protective effect of folic acid through in vitro experiments. The GC-1 spermatogonia, TM4 Sertoli cells, and TM3 Leydig cells were treated with methomyl (0, 250, 500, and 1000μM) with or without folic acid (0, 10, 100, and 1000nM) for 24h. It was found that methomyl increased cytotoxicity to testicular cells in a dose-dependent manner. In spermatogonia, methomyl significantly inhibited the expression of proliferation genes Ki67 and PCNA at 1000μM, and increased the expression of apoptosis genes Caspase3 and Bax at each dose. In Sertoli cells, methomyl dose-dependently inhibited the expression of blood-testis barrier function genes TJP1, Cx43, and N-cadherin, but did not affect Occludin and E-cadherin. In Leydig cells, methomyl inhibited the expression of steroid synthase P450scc, StAR, Hsd3b1 and down-regulated the level of testosterone, but did not affect Cyp17a1 and Hsd17b1. Further, folic acid could basically reduce the damage caused by methomyl. This study provided new insights into the toxicity of methomyl and the protective effect of folic acid.

PM2.5 mediates mouse testis Sertoli TM4 cell damage by reducing cellular NAD+

Objective This study aims to explore the mechanism of PM2.5 damage to the reproductive system of male mice. Methods Mouse testis Sertoli TM4 cells were divided into four groups: a control group (no additional ingredients except for medium), PM2.5 group (medium containing 100 μg/mL PM2.5), PM2.5 + NAM group (medium containing 100 μg/mL PM2.5 and 5 mM NAM), and NAM group (medium containing 5 mM nicotinamide) and cultured in vitro for 24 or 48 h. The apoptosis rate of TM4 cells was measured using flow cytometry, the intracellular levels of NAD+ and NADH were detected using an NAD+/NADH assay kit, and the protein expression levels of SIRT1 and PARP1 were determined by western blotting. Results Mouse testis Sertoli TM4 cells exposed to PM2.5 demonstrated an increase in the apoptosis rate and PARP1 protein expression, albeit a decrease in NAD+, NADH, and SIRT1 protein levels (p = 0.05). These changes were reversed in the group treated with a combination of PM2.5 and nicotinamide (p = 0.05). Conclusion PM2.5 can cause Sertoli TM4 cell damage in mouse testes by decreasing intracellular NAD+ levels.

Oxidative Stress, Cytotoxic and Inflammatory Effects of Azoles Combinatorial Mixtures in Sertoli TM4 Cells.

Triazole and imidazole fungicides are an emerging class of contaminants with an increasing and ubiquitous presence in the environment. In mammals, their reproductive toxicity has been reported. Concerning male reproduction, a combinatorial activity of tebuconazole (TEB; triazole fungicide) and econazole (ECO; imidazole compound) in inducing mitochondrial impairment, energy depletion, cell cycle arrest, and the sequential activation of autophagy and apoptosis in Sertoli TM4 cells (SCs) has recently been demonstrated. Given the strict relationship between mitochondrial activity and reactive oxygen species (ROS), and the causative role of oxidative stress (OS) in male reproductive dysfunction, the individual and combined potential of TEB and ECO in inducing redox status alterations and OS was investigated. Furthermore, considering the impact of cyclooxygenase (COX)-2 and tumor necrosis factor-alpha (TNF-α) in modulating male fertility, protein expression levels were assessed. In the present study, we demonstrate that azoles-induced cytotoxicity is associated with a significant increase in ROS production, a drastic reduction in superoxide dismutase (SOD) and GSH-S-transferase activity levels, and a marked increase in the levels of oxidized (GSSG) glutathione. Exposure to azoles also induced COX-2 expression and increased TNF-α production. Furthermore, pre-treatment with N-acetylcysteine (NAC) mitigates ROS accumulation, attenuates COX-2 expression and TNF-α production, and rescues SCs from azole-induced apoptosis, suggesting a ROS-dependent molecular mechanism underlying the azole-induced cytotoxicity.

The transcription factors Creb1 and CEBPB regulate Sox9 promoter activity in TM4 Sertoli cells

In Sertoli cells, the Sox9 gene is essential for testicular development and normal spermatogenesis. SOX9 is critical for postnatal Sertoli cells differentiation and proliferation in the testis. However, the molecular mechanisms that specifically regulate its expression are not entirely understood. Sox9 expression is regulated by CREB1 and CEBPB in other biological contexts such as during chondrogenesis and in rat thyroid follicular cells. We hypothesized that Sox9 promoter activity is regulated by CREB1 and CEBPB in Sertoli cells. Our results show that Sox9 expression is dependent on the activation of these transcription factors by the cAMP/PKA signaling pathway in TM4 Sertoli cells. Chromatin immunoprecipitation and promoter/reporter luciferase assays with 5′ promoter deletions and site-directed mutagenesis demonstrated that CREB1 is being recruited to a DNA regulatory element at -141 bp of the Sox9 promoter region. Such regulation is dependent on the cAMP/PKA signaling pathway, resulting in phosphorylation of CREB1. Activation of Sox9 expression by CEBPB may involve its recruitment to the proximal promoter region by protein–protein interaction with CREB1. Thus, we have shown that the Sox9 promoter is being regulated by the transcription factors CREB1 and CEBPB in TM4 Sertoli cells and involve their recruitment to the proximal promoter region.

Regulation of Cdh2 by the AP-1 family transcription factor Junb in TM4 Sertoli cells

Cadherins are transmembrane proteins that mediate cell-to-cell adhesion and various cellular processes. In Sertoli cells of the testis, Cdh2 contributes to the development of the testis and the formation of the blood-testis barrier, being essential for germ cells' protection. Analyses of chromatin accessibility and epigenetic marks in adult mouse testis have shown that the region from −800 to +900 bp respective to Cdh2 transcription start site (TSS) is likely the active regulatory region of this gene. In addition, the JASPAR 2022 matrix has predicted an AP-1 binding element at about −600 bp. Transcription factors of the activator protein 1 (AP-1) family have been implicated in the regulation of the expression of genes encoding cell-to-cell interaction proteins such as Gja1, Nectin2 and Cdh3. To test the potential regulation of Cdh2 by members of the AP-1 family, siRNAs were transfected into TM4 Sertoli cells. The knockdown of Junb led to a decrease in Cdh2 expression. ChIP-qPCR and luciferase reporter assays with site-directed mutagenesis confirmed the recruitment of Junb to several AP-1 regulatory elements in the proximal region of the Cdh2 promoter in TM4 cells. Further investigation with luciferase reporter assays showed that other AP-1 members can also activate the Cdh2 promoter albeit to a lesser extent than Junb. Taken together, these data suggest that in TM4 Sertoli cells, Junb is responsible for the regulation of Cdh2 expression which requires its recruitment to the proximal region of the Cdh2 promoter.

Effect of duloxetine as antidepressant on TM4 Sertoli cells: Evaluation of Bax and Cx43 gene expression

هدف: از چالش­های عمده بشریت افزایش افسردگی و اختلال عملکرد جنسی ناشی از داروهای ضدافسردگی است. با توجه به نقش سلول­های سرتولی در اسپرماتوژنز، پژوهش حاضر، اثر داروی دولوکستین را بر زنده­مانی، آپوپتوزیس و بیان ژن­های Bax و­ (Connexin 43) Cx43 در سلول­های سرتولی بررسی کرده است. مواد و روش‌‌ها: سلول­های TM4 در محیط DMEM/F12 حاوی %5/2 FBS، 5% سرم اسب و %1 پنی سیلین-استرپتومایسین کشت شدند. دولوکستین با دوزهای 30،60، 15، 5/7، 75/3 میکرو­گرم/ میلی­لیتر در زمان­های 24 تا 72 ساعت روی سلول­ها اثر داده شد. آزمون MTT جهت ارزیابی زنده­مانی سلول­ها، فلوسایتومتری جهت ارزیابی آپوپتوزیس و RT-qPCR جهت بررسی بیان ژن Bax (عامل پیش­برندۀ آپوپتوزیس) و Cx43 (ضروری برای اسپرماتوژنز) انجام شد. نتایج: دولوکستین در یک الگوی وابسته به دوز و زمان، بقای سلولی را کاهش داد. بر اساس داده­های MTT دوز IC50 15 میکروگرم/ میلی­لیتر در 48 ساعت محاسبه گردید (p≤0.05). آپوپتوزیس سلول­های TM4 نسبت به گروه کنترل در دوز میانه مهاری 15 میکروگرم/ میلی­لیتر دولوکستین، افزایش یافت (p≤0.01). RT-qPCR حاکی از افزایش بیان ژن­های Cx43 (p≤0.05) و Bax (p≤0.01) تحت تاثیر دولوکستین بود. نتیجه­گیری: با توجه به داده­های فلوسایتومتری و افزایش بیان ژن Bax، دولوکستین با القای آپوپتوزیس در سلول­های سرتولی می­تواند عاملی منفی و مخرب در پیشبرد اسپرماتوژنز در نظر گرفته شود. از سوی دیگر، دولوکستین با افزایش سطح بیان ژن Cx43، نقل و انتقالات مولکولی توسط اتصالات شکافدار را بین سلول­های سرتولی افزایش داده و می‫تواند باعث انتقال سیگنال­های پیش برنده آپوپتوزیس به سلول­های دودومان اسپرماتوژنیک و در نتیجه کاهش کیفیت اسپرماتوژنز ­شود.