Testis Cells Research Articles

Dibutyl phthalate-induced oxidative stress and apoptosis in swine testis cells and therapy of naringenin via PTEN/PI3K/AKT signaling pathway.

Dibutyl phthalate (DBP) is a phthalic acid ester (PAE) that has posed a health hazard to the organisms. Naringenin (NRG) is a flavanone compound that has shown protection against several environmental chemicals through suppression of oxidative stress and activation of phosphatidylinositol 3-kinase/threonine kinase (PI3K/AKT) signaling pathway. Herein, swine testis (ST) cells were treated with 1.8μM of DBP or/and 25.39 nM of NRG for 24 h, we described the discovery path of NRG inhibition on apoptosis in DBP-exposed ST cells through targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN). We first found that the anti-apoptosis effect of NRG is dependent on mitochondrial pathway through flow cytometry and related gene/protein expression, and then we detected PI3K/AKT pathway-related gene/protein expression, and established a computational docking assay between NRG and PTEN. We found that NRG specifically binds to three basic residues (His93, Lys125, Lys128) of P loop in PTEN, as well as phosphatase domains (Asp92, His93, Cys124, Lys125, Ala126, Lys128, and Arg130) in active dephosphorylation pockets, thereby reducing PTEN level and activating PI3K/AKT signaling pathway, and further inhibiting oxidative stress and mitochondrial pathway apoptosis. Taken together, our results push forward that NRG deserves further attention as a potential antagonistic therapy against DBP through targeting PTEN to inhibit oxidative stress and activate PI3K/AKT signaling pathway.

Assessment of Contagious Ecthyma Virus in Camels of Wasit Province, Iraq.

Camel contagious ecthyma (CCE) is an infectious disease caused by the Paravox virus (PPV) of the family <i>Poxviridae</i>. Due to the importance of the camel breeding industry in tropical and subtropical regions, the present study aimed to isolate the causative agent of camel contagious ecthyma (CCE) using cell culture and molecular confirmation of virus isolate. A total of 210 camels aged 6 months to 4 years were selected from different districts in Wasit province (Iraq) from August 2017 to April 2019. These animals, which included 117 females and 93 males, displayed signs of papules, blisters, pustules, and scabs on the skin. To isolate the CCE virus, primary and secondary cell cultivation was performed using the lamb testis (LT) cells. The findings pointed out that there were cytopathic effects during the second passage of the virus, characterized by rounding and cells aggregation after 72 h. Furthermore, there were dramatic changes, including sloughing off and detachment from the surface of the monolayer, in monolayer cells after 48-72 h. The titration values of the isolated Orf virus in LT cells were obtained at 10^-5 TCI50 /0.05 and 10^-6 TCID 50 / 0.05 ml in the third and fourth passages, respectively. As expected, the <i>B2L gene</i> of affected camels was amplified from a skin biopsy DNA sample to produce nearly 594 base pairs. In conclusion, the results of the current study focused on epidemiological and virological characteristics of CCE in Wasit province; moreover, the virus was confirmed by a specific gene called the <i>B2L gene</i>.

Long-term exposure to low concentrations of MC-LR induces blood-testis barrier damage through the RhoA/ROCK pathway

Microcystin-leucine arginine (MC-LR), an emerging water pollutant, produced by cyanobacteria, has an acute testicular toxicity. However, little is known about the chronic toxic effects of MC-LR exposure on the testis at environmental concentrations and the underlying molecular mechanisms. In this study, C57BL/6 J mice were exposed to different low concentrations of MC-LR for 6, 9 and 12 months. The results showed that MC-LR could cause testis structure loss, cell abscission and blood-testis barrier (BTB) damage. Long-term exposure of MC-LR also activated RhoA/ROCK pathway, which was accompanied by the rearrangement of α-Tubulin. Furthermore, MC-LR reduced the levels of the adherens junction proteins (N-cadherin and β-catenin) and the tight junction proteins (ZO-1 and Occludin) in a dose- and time-dependent way, causing BTB damage. MC-LR also reduced the expressions of Occludin, ZO-1, β-catenin, and N-cadherin in TM4 cells, accompanied by a disruption of cytoskeletal proteins. More importantly, the RhoA inhibitor Rhosin ameliorated these MC-LR-induced changes. Together, these new findings suggest that long-term exposure to MC-LR induces BTB damage through RhoA/ROCK activation: involvement of tight junction and adherens junction changes and cytoskeleton disruption. This study highlights a new mechanism for MC-LR-induced BTB disruption and provides new insights into the cause and treatment of BTB disruption.

Prohibitin-1 plays a regulatory role in Leydig cell steroidogenesis

SummaryMitochondria are essential for steroidogenesis. In steroidogenic cells, the initiation of steroidogenesis from cholesterol occurs on the matrix side of the inner mitochondrial membrane by the enzyme P450scc. This requires cholesterol import from the cytoplasm through the outer mitochondrial membrane, facilitated by the StAR protein. The subsequent steps leading to P450scc remain elusive. Here we report that the male transgenic mice that expressed a mutant form of a mitochondrial protein prohibitin-1 (PHB1Tyr114Phe) from the Fabp-4 gene promoter displayed smaller testes, higher testosterone, and lower gonadotropin levels compared with PHB1-expressing and wild-type mice. A subsequent analysis of the testis and Leydig cells from the mice revealed that PHB1 played a previously unknown regulatory role in Leydig cell steroidogenesis. This includes a role in coordinating cell signaling, cholesterol homeostasis, and mitochondrial biology pertaining to steroidogenesis. The implications of our finding are broad as the initial stages of steroidogenesis are indistinguishable across steroidogenic cells.

The Potential Impact of COVID-19 on Male Reproductive Health

Since the beginning of 2020, the whole world is trying to face the coronavirus disease 2019 (COVID-19) pandemic. In the last years, SARS-CoV-2 mutations showed an increased contagion and infection profile, affecting a higher proportion of young men during the last pandemic outbreaks. SARS-CoV-2 penetrates host cells by binding to Angiotensin-Converting Enzyme (ACE2) receptors, highly expressed by testis cells, and a significant hyper-gonadotropic hypogonadism rate in COVID-19 patients has been observed, leading to the hypothesis of a possible impact of this disease on male fertility. Indeed, recent founding demonstrated that 25% of patients healed by COVID-19 showed azoo/oligozoo-spermia. Moreover, a statistically significant decrease of sperm concentration, total number and sperm motility was detected in men with moderate infections, when compared with mild symptomatic men or controls. Thus, pathological levels of inflammatory cytokines in semen of men healed from COVID-19 were reported in a major percentage of studied men, with a significant correlation between oligo-crypto-azoospermia and disease severity. Even if the whole mechanisms of male reproductive damages are still unclear, autoimmune self-maintained inflammation may be one of the main determinants of male reproductive system impairment.

Amyloids facilitate DNA transfection in vivo

Amyloid fibril deposits are a main source of pathology in neurodegenerative diseases. Normal proteins such as tau, alpha-synuclein, TDP-43 and others could form specific conformational fibrils called amyloid, which deposited in the brains of neurodegenerative diseases. Although the pathological roles of amyloids in cell death have been discussed a lot, their other functions have not been investigated well. Here, we studied the effect of amyloids on DNA transfection in vivo. We injected quantum dot labeled or non-labeled amyloid-preformed fibrils (PFFs) and a green fluorescent protein (EGFP) expression vector into organs including brain, testis, liver and calf muscle. GFP expression patterns were examined by immunohistochemistry and western blotting. At 24 h after injection, EGFP was predominantly expressed in the neurons in the cortex and the striatum, Leydig cells in testis, hepatocytes in the liver and muscle cells. EGFP expression was inhibited by an endocytosis inhibitor, sertraline in the brain and testis. The amyloid-PFFs potentiated Ca2+ transients shown by calcium imaging and EGFP expression in the brain was blocked by Ca blocker, cilnidipine. Our results show that amyloid-PFFs facilitate DNA transfection and can be used for a new gene delivery system in vivo.

Ontogeny of mouse Sertoli, Leydig and peritubular myoid cells from embryonic day 10 to adulthood

We present a comprehensive description of the differentiating somatic cell types (Sertoli, Leydig, and peritubular myoid cells) of the mouse testis from embryonic day 10.5 (E10.5) to adulthood, postnatal day 60 (P60). Immunohistochemistry was used to analyze expression of: Sox9 (a Sertoli cell marker), 3βHSD-1 (a fetal Leydig cell marker), 3βHSD-6 (an adult Leydig cell marker), α-actin (a peritubular myoid cell marker), and androgen receptor (a marker of all three somatic cell types). The temporal-spatial expression of these markers was used to interrogate findings of earlier experimental studies on the origin of Sertoli, Leydig and peritubular myoid cells, as well as extend previous descriptive studies across a broader developmental period (E10.5-P60). Such comparisons demonstrate inconsistencies that require further examination and raise questions regarding conservation of developmental mechanisms across higher vertebrate species.

Mast Cells in the Mammalian Testis and Epididymis-Animal Models and Detection Methods.

Mast cells (MCs) are an evolutionary well-conserved type of cells, mediating and modulating allergic responses in innate immunity and tissue remodeling after chronic inflammation. Among other tissues, they inhabit both the testis and epididymis. In the testis, MCs usually appear in the interstitial compartment in humans, but not in other standard experimental models, like rats and mice. MCs seem to be responsible for testicular tissue fibrosis in different causes of infertility. Although experimental animal models follow the effect on MC activation or penetration to the interstitial tissue like in humans to some extent, there is an inconsistency in the available literature regarding experimental design, animal strain, and detection methods used. This comprehensive review offers an insight into the literature on MCs in mammalian testes and epididymides. We aimed to find the most suitable model for research on MC and offer recommendations for future experimental designs. When using in vivo animal models, tunica albuginea incorporation and standard histological assessment need to be included. Domesticated boar strains kept in modified controlled conditions exhibit the highest similarity to the MC distribution in the human testis. 3D testicular models are promising but need further fine-tuning to become a valid model for MC investigation.

Review of SARS-CoV-2 infects, replicates, elevates angiotensin II and activates immune cells in human testes

Review of SARS-CoV-2 infects, replicates, elevates angiotensin II and activates immune cells in human testes

Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch.

JAK1 depletion or downregulation was previously reported to account for coronavirus inhibition. Here, we found that AG1024, an IR (insulin receptor) and IGF-1R (insulin-like growth factor 1 receptor) inhibitor, diminishes JAK1 protein levels and exerts anti-coronaviral activities with EC50 values of 5.2 ± 0.3 μM against transmissible gastroenteritis coronavirus (TGEV) and 4.3 ± 0.3 μM against human flu coronavirus OC43. However, although the IR and IGF-1R signaling pathways are activated by insulin or IGF-1 in swine testis cells, they are not triggered upon TGEV infection. AG1024, therefore, inhibits coronaviral replication and downregulates JAK1 protein levels independently of IR and IGF-1R. Moreover, JAK1 proteolysis caused by AG1024 was found through activation of upstream Ndfip1/2 and its effector NEDD4-like E3 ligase Itch. In addition, ouabain, which was reported to mediate JAK1 proteolysis causing anti-coronaviral activity by activation of Ndfip1/2 and NEDD4 E3 ligase, additively inhibited anti-coronaviral activity and JAK1 diminishment in combination with AG1024. This study provides novel insights into the pharmacological effects of AG1024 and Itch E3 ligase mediated JAK1 proteolysis and identified Ndfip1/2 as a cognate effector for JAK1 proteolysis via the diversified E3 ligases NEDD4 and NEDD4-like Itch. These findings are expected to provide valued information for the future development of anti-viral agents.

Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report

To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA). A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded invitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted invitro control. Academic medical center. A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome. Three-dimensional bioprinting and invitro culturing of patient-derived testis cells. Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of invitro culture. Testicular cultures were expandable invitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold. This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of invitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.

Brief exposure of neonatal testis cells to EGF or GDNF alters the regenerated tissue.

We have previously shown that implantation of testis cell aggregates under the back skin of immunodeficient mice results in de novo regeneration of testis tissue. We used this unique model to investigate the effects of epidermal growth factor (EGF) and glial cell-derived neurotrophic factor (GDNF) on testis cord development. Neonatal piglet testis cells were briefly (<1 h) exposed to either low (L: 0.02 μg/mL) or high (H: 2 μg/mL) doses of EGF, GDNF, or vehicle (control), before implantation in recipient mice. Randomly selected implants were removed from each mouse at 1, 2, 4, and 8 weeks post-implantation. GDNF-L implants showed increased testis cord development over time, and EGF-L implants had increased cross-sectional area. The ratio of regular cords decreased over time in EGF-H and GDNF-H implants and was replaced by a higher ratio of irregular cords in GDNF-H. EGF-L and GDNF-H implants were quickest to display rete testis-like structures. Overall, the lower dose of each growth factor was more effective than its higher dose in improving the implantation outcomes. This is the first comprehensive assessment of these key growth factors on de novo formation (regeneration) of testis tissue.Lay summaryIn recent decades, testicular cancer rates have quadrupled in young men while sperm counts have dropped by half. Both conditions may be related to exposure of fetuses or infants to noxious substances causing disruption of normal testis development. To study the effects of any putative factor on testis development, we established an animal model of testis tissue regeneration. We collected newborn piglet testes after routine castration, used enzymes to completely dissociate testis cells, exposed the cells to two key growth factors (EGF or GDNF), and implanted the cells under the back skin of recipient mice, acting as live incubators. We then examined implant samples after 1, 2, 4, or 8 weeks and assessed testis regeneration. Overall, the high dose of each growth factor had adverse effects on the formation of normal testis. Therefore, this novel implantation model may also be used to study the effects of potentially harmful substances on testis development.

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glialcell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10ng/mL bFGF and 10ng/mL bFGF + 100ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on thegrowth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

Reproductive Function of Male Rats and Motor Activity of Their Offspring in Fire Emissions Modeling.

We studied the reproductive function of albino male rats after long-term exposure to wildfire smoke and the species-specific behavior of their sexually mature offspring. After the end of exposure, the mean number of spermatogonia and the relative number of Leydig cells in rat testes decreased and the index of spermatogenesis decreased. The offspring of exposed males mated with intact females demonstrated impaired cognitive abilities and disturbances of species-specific behavior. The severity of these changes depended on time interval between the end of the inhalation exposure and mating.

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.

The complete genome sequence of Indian sheeppox vaccine virus and comparative analysis with other capripoxviruses

Sheeppox virus (SPPV) is responsible for a significant economic loss to sheep husbandry in enzootic regions of Africa, the Middle East, and Asia including the Indian subcontinent. In this study, we present the complete genome sequence of SPPV vaccine strain SPPV-Srin38/00 from India determined by next-generation sequencing (NGS) using Illumina technology. The attenuated Srinagar vaccine strain of SPPV (SPPV-Srin38/00) was developed by serial passaging the virus initially in lamb testes (LT) cells followed by Vero cell line. The SPPV-Srin38/00 virus has a genome size of 150, 103bp, which encodes for 147 functional putative genes and consists of a central coding region flanked by two identical 2353bp inverted terminal repeats (ITRs). Comparative phylogenetic analysis based on complete genome sequences of Capripoxviruses formed three distinct groups each for SPPV, GTPV, and LSDV with clustering of SPPV-Srin38/00 strain with SPPV-A strain. Nine ORFs of SPPV-Srin38/00 namely SPPV-Srin_002/SPPV-Srin_155, SPPV-Srin_004/SPPV-Srin_153, SPPV-Srin_009, SPPV-Srin_013, SPPV-Srin_026, SPPV-Srin_132, and SPPV-Srin_136 were found to be fragmented as compared to LSDV, whereas only one ORF (such as SPPV-Srin_136) was found to be fragmented as compared to GTPV. SPPV genomes, including the SPPV-Srin38/00 strain, shared 99.78-99.98% intraspecies nucleotide identity, indicating that SPPV strains have extremely low genetic diversity. The strain shared 96.80-97.08% and 97.11-97.61% nt identity with GTPV and LSDV strains, respectively. Its ORFs 016, 021, 022, 130 and 138 are the least identical ORFs among three species of the genus Capripoxvirus with 72.5-93% aa identity to GTPV and LSDV strains and may be potentially used for differentiation of CaPV species. This study may contribute to a better understanding of the epidemiology and evolution of capripoxviruses as well as the development of specific detection methods, better expression vectors, and vaccines with improved safety and efficacy.

MicroRNA ssa-mir-196a-4 deceases lgr8 expression in testis development of Chinese tongue sole (Cynoglossus semilaevis).

MicroRNAs (miRNAs) contribute to gonadal development in animals. However, there is little information about miRNA regulation function involved in gonadal development in fish. Our group previously identified sex-related miRNAs of Chinese tongue sole (Cynoglossus semilaevis) during sex determination and differentiation by small RNA sequencing. In the present study, we characterized ssa-mir-196a-4 and its expression in testis and verified its interaction with lgr8. miRNA ssa-mir-196a-4 precursor was predicted to have a typical hairpin structure and highly conserved among various fish species. Fluorescence in situ hybridization (FISH) of ssa-mir-196a-4 in the testis of Chinese tongue sole showed that it is mainly expressed in the cytoplasm of Sertoli cells. We determined that ssa-mir-196a-4 interacted with lgr8 by bioinformatics analysis using miRanda software. According to the dual-luciferase gene reporter assay, lgr8 is a direct target of ssa-mir-196a-4. Overexpression of ssa-mir-196a-4 in the cells of the testis cell line of Chinese tongue sole decreased the expression levels of lgr8 messenger RNA (mRNA) and protein by targeting its coding sequence (CDS) region. These results suggest that ssa-mir-196a-4 acts as a post-transcriptional regulator of lgr8 and plays an important role in developing testes of Chinese tongue sole.

Comprehensive Analysis of the Association Between Human Non-obstructive Azoospermia and Plasticisers via Single-Cell and Traditional RNA Sequencing Methods

A father’s lifetime experience is a major risk factor for a range of diseases in an individual. The influences of exposure can also be transmitted to offspring. Previous studies demonstrated that plasticisers can damage the male offspring reproductive system, but the link between mammalian research and human non-obstructive azoospermia remains underexplored. Here, we analysed reproduction-related genes from four publicly available single-cell RNA-Seq datasets and RNA-Seq datasets on GEO databases to investigate the correlation between human non-obstructive azoospermia and exposure to plasticisers during pregnancy. The R software was used in statistical analyses. A total of 9 co-upregulated genes and 1 co-downregulated gene were found. The Gene Ontology enrichment analyses were performed using the “clusterProfiler” package. Single-cell analyses were conducted to visualise the gene expression pattern in testis cell subgroups. Immunohistochemical images were used to evaluate the level of protein in testicular organs from The Human Protein Atlas. GSVA results provided further functional annotations. Three signature genes, i.e. COL1A1, CYP17A1 and KIF11, might serve as promising diagnostic biomarkers in non-obstructive azoospermia caused by plasticisers. Our results provided a potential new protocol to evaluate the feasibility of life or clinical intervention in patients with non-obstructive azoospermia. We believe that these observations will improve our understanding of the potential mechanisms of plasticiser contributions to human non-obstructive azoospermia and help identify potential targets for immunotherapy in the future.

Transgenerational Transcriptomic and DNA Methylome Profiling of Mouse Fetal Testicular Germline and Somatic Cells after Exposure of Pregnant Mothers to Tributyltin, a Potent Obesogen.

Obesogens such as tributyltin (TBT) are xenobiotic compounds that promote obesity, in part by distorting the normal balance of lipid metabolism. The obesogenic effects of TBT can be observed in directly exposed (F1 and F2 generations) and also subsequent generations (F3 and beyond) that were never exposed. To address the effects of TBT exposure on germ cells, we exposed pregnant transgenic OG2 mouse dams (F0), which specifically express EGFP in germline cells, to an environmentally relevant dose of TBT or DMSO throughout gestation through drinking water. When fed with a high-fat diet, F3 male offspring of TBT-exposed F0 dams (TBT-F3) accumulated much more body fat than did DMSO-F3 males. TBT-F3 males also lost more body fluid and lean compositions than did DMSO-F3 males. Expression of genes involved in transcriptional regulation or mesenchymal differentiation was up-regulated in somatic cells of TBT-F1 (but not TBT-F3) E18.5 fetal testes, and promoter-associated CpG islands were hyper-methylated in TBT-F1 somatic cells. Global mRNA expression of protein-coding genes in F1 or F3 fetal testicular cells was unaffected by F0 exposure to TBT; however, expression of a subset of endogenous retroviruses was significantly affected in F1 and F3. We infer that TBT may directly target testicular somatic cells in F1 testes to irreversibly affect epigenetic suppression of endogenous retroviruses in both germline and somatic cells.

Involvement of TauT/SLC6A6 in Taurine Transport at the Blood-Testis Barrier.

Taurine transport was investigated at the blood–testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.