Mouse Leydig Cells Research Articles

Syce1 and Syce3 regulate testosterone and dihydrotestosterone synthesis via steroidogenic pathways in mouse Sertoli and Leydig cells

Testosterone (T) and dihydrotestosterone (DHT) are the main hormones regulating reproduction and development of male animals. Although their synthesis and secretion are regulated by the endocrine system [hypothalamic-pituitary-gonadal (adrenal) axis], it is also possible to synthesize T and DHT from the induction of two proteins: Syce1 and Syce3. As central elements of the synaptonemal complex (SC), Syce1 and Syce3 play a key role in the association of homologous chromosomes during meiosis. However, Syce1 and Syce3 also promote the synthesis of T and DHT, although potential mechanisms have yet to be revealed. In this study, Leydig and Sertoli cells, which are responsible for the production and regulation of steroid hormones in testis, were transfected with recombinant Syce1/Syce3 and silence sequence. Our results revealed the highest expression of Syce1 and Syce3 in spermatogenic cells of the testis. Moreover, overexpression or knockdown of Syce1 and Syce3 in Sertoli and Leydig cells resulted in activation or suppression of steroidogenic genes Star and Hsd3b, which are involved in a steroidogenic pathway that upregulates T synthesis. Upregulated expression of Syce1 resulted in a significant increase in Srd5a1, which can promote DHT secretion. Interestingly, Syce1 and Syce3 overexpression synergistically promoted each other’s abundance. Our results define a previously unknown mechanism of Syce1 and Syce3 dependent activation of steroidogenic signaling in Sertoli and Leydig cells.

1-Nitropyrene disrupts testosterone biogenesis via AKAP1 degradation promoted mitochondrial fission in mouse Leydig cell

Previous study found 1-NP disrupted steroidogenesis in mouse testis, but the underlying mechanism remained elusive. The current work aims to explore the roles of ROS-promoted AKAP1 degradation and excessive mitochondrial fission in 1-NP-induced steroidogenesis disruption in MLTC-1 cells. Transmission electron microscope analysis found 1-NP promoted excessive mitochondrial fission. Further data showed 1-NP disrupted mitochondrial function. pDRP1 (Ser637), a negative regulator of mitochondrial fission, was reduced in 1-NP-treated MLTC-1 cells. Mechanistically, 1-NP caused degradation of AKAP1, an upstream regulator of pDRP1 (Ser637). MG132, a proteasome inhibitor, attenuated 1-NP-induced AKAP1 degradation and downstream pDRP1 (Ser637) reduction, thereby ameliorating 1-NP-downregulated steroidogenesis. Further analysis found that cellular ROS was elevated and NOX4, HO-1 and SOD2 were upregulated in 1-NP-exposed MLTC-1 cells. NAC, a well-known commercial antioxidant, alleviated 1-NP-induced excessive ROS and oxidative stress. 1-NP-induced AKAP1 degradation and subsequent downregulation of pDRP1 (Ser637) were prevented by NAC pretreatment. Moreover, NAC attenuated 1-NP-resulted T synthesis disturbance in MLTC-1 cells. The present study indicates that ROS mediated AKAP1 degradation and subsequent pDRP1 (Ser637) dependent mitochondrial fission is indispensable in 1-NP caused T synthesis disruption. This study provides a new insight into 1-NP-induced endocrine disruption, and offers theoretical basis in public health prevention.

Activation of MT1/MT2 to Protect Testes and Leydig Cells against Cisplatin-Induced Oxidative Stress through the SIRT1/Nrf2 Signaling Pathway.

There is growing concern that chemotherapy drugs can damage Leydig cells and inhibit the production of testosterone. Increasing evidence shows that melatonin benefits the reproductive process. This study mainly explores the protective effect and possible molecular mechanism of melatonin regarding cisplatin-induced oxidative stress in testicular tissue and Leydig cells. We found that there were only Leydig and Sertoli cells in the testes of gastrointestinal tumor patients with azoospermia caused by platinum chemotherapeutic drugs. Melatonin (Mel) receptor 1/melatonin receptor 2 (MT1/MT2) was mainly expressed in human and mouse Leydig cells of the testes. We also observed that the melatonin level in the peripheral blood decreased and oxidative stress occurred in mice treated with cisplatin or gastrointestinal tumor patients treated with platinum-based chemotherapeutic drugs. iTRAQ proteomics showed that SIRT1/Nrf2 signaling and MT1 proteins were downregulated in cisplatin-treated mouse testes. The STRING database predicted that MT1 might be able to regulate the SIRT1/Nrf2 signaling pathway. Melatonin reduced oxidative stress and upregulated SIRT1/Nrf2 signaling in cisplatin-treated mouse testes and Leydig cells. Most importantly, after inhibiting MT1/MT2, melatonin could not upregulate SIRT1/Nrf2 signaling in cisplatin-treated Leydig cells. The MT1/MT2 inhibitor aggravated the cisplatin-induced downregulation of SIRT1/Nrf2 signaling and increased the apoptosis of Leydig cells. We believe that melatonin stimulates SIRT1/Nrf2 signaling by activating MT1/MT2 to prevent the cisplatin-induced apoptosis of Leydig cells.

T2DM-elicited oxidative stress represses MTA3 expression in mouse Leydig cells.

Metastasis-associated protein 3 (MTA3) functions as a versatile coregulator in cancers and in physiological contexts. A predominant expression of MTA3 in interstitial Leydig cells (LCs) and its role as a local modulator of testicular steroidogenesis have recently emerged. Incubation with insulin decreased MTA3 expression in a concentration- and exposure time-dependent manner in LCs. This raises the possibility of additional endocrine actions of insulin in the direct control of MTA3 expression, which remains so far unexplored. Herein, we reported that type 2 diabetes mellitus (T2DM)-mediated inhibition of MTA3 was associated with an increase in testicular oxidative stress. In contrast, a gavage of the strong antioxidant melatonin effectively ameliorated oxidative stress and restored the expression of MTA3, but failed to change serum insulin levels in the diabetic mice with testosterone deficiency (TD). Using multiple biochemical approaches, we demonstrated that oxidative stress suppressed MTA3 expression via repression of nuclear receptor subfamily 4 group A member 1 (NR4A1)-mediated transactivation of MTA3 in mouse LCs. By contrast, ectopic expression of NR4A1 ameliorated oxidative stress-impaired MTA3 expression in LCs. By employing an effective in vivo gene transfer method with microinjection of lentiviral plasmids, we showed that replenishment of MTA3 expression in vivo partially restored testicular steroidogenesis and improved male fertility in diabetic mice with TD. Thus, we have unveiled a central regulatory hub, involving oxidative stress-impaired NR4A1-driven transactivation of MTA3 in stimulated LCs, as a potential mechanism regulating crosstalk between hyperinsulinemia and male infertility associated with TD.

Mouse-human species differences in early testicular development and its implications

The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes.

VDR mediated HSD3B1 to regulate lipid metabolism and promoted testosterone synthesis in mouse Leydig cells.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis wherein the VDR transcriptionally regulates expression of the genes involved in this complex process. 3β-Hydroxysteroid dehydrogenase-1 (HSD3B1) is an absolutely necessary enzyme for androgen synthesis. The purpose of the present study was to explore the molecular mechanism of VDR mediated HSD3B1 regulation of lipid metabolism and testosterone synthesis. The levels of VDR, HSD3B1 and lipid metabolism associated protein were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). Targeted relationship between VDR and Hsd3b1 was evaluated by dual-luciferase reporter assay. Based on the data analysis of mouse testicular proteome, we found that the expression of HSD3B1 was significantly reduced after VDR deletion. Here, we identified that Hsd3b1 was widely expressed in different tissues of mice by RT-qPCR, and was highly expressed in testis, and mainly located in testicular Leydig cells. Dual-luciferase assay confirmed that VDR could bind candidate vitamin D responsive elements (VDREs) in upstream region of Hsd3b1, and enhance gene expression. Furthermore, over-expression VDR and HSD3B1 significantly increased testosterone synthesis in mice Leydig cells. Meanwhile, Lpl expression was significantly down-regulated and Angptl4 expression was significantly up-regulated in the present of HSD3B1 overexpression. Both LPL and ANGPTL4 play important roles in regulating lipid metabolism. The present study unveiled VDR mediated HSD3B1 to regulate lipid metabolism and promoted testosterone synthesis in mouse Leydig cells. These findings will greatly help us to understand the roles of VDR and HSD3B1 in testosterone synthesis and lipid metabolism.

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.

Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modified methods for LC isolation and compared their performance with that of the regular method. Modified method 1 integrated the crude LCs into the 50% Percoll solution before centrifugation. Modified method 2 sequentially used 50 and 60% Percoll solutions to isolate LCs. The purity of LCs was approximately 88.4, 91.3, and 79.7% derived from the regular, modified 1, and modified 2 methods, respectively. The yields of LCs in the same respective order were approximately 1.7 × 105, 3.9 × 105, and 11.9 × 105 cells per 108 interstitial cells input. Modified method 1 attained higher purity and yields than those of the regular method. Although the purity of LCs was relatively low for modified method 2, it could be used before further purification by, for example, fluorescence-activated or magnetic-activated cell sorting, owing to its simplicity and high yields. Therefore, our study provided alternative methods to facilitate LC isolation in mice.

The Effects of Organophosphate Esters Used as Flame Retardants and Plasticizers on Granulosa, Leydig, and Spermatogonial Cells Analyzed Using High-Content Imaging.

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making.

Leukemia inhibitory factor-receptor signalling negatively regulates gonadotrophin-stimulated testosterone production in mouse Leydig Cells

Testicular Leydig cells (LCs) are the principal source of circulating testosterone in males. LC steroidogenesis maintains sexual function, fertility and general health, and is influenced by various paracrine factors. The leukemia inhibitory factor receptor (LIFR) is expressed in the testis and activated by different ligands, including leukemia inhibitory factor (LIF), produced by peritubular myoid cells. LIF can modulate LC testosterone production in vitro under certain circumstances, but the role of consolidated signalling through LIFR in adult LC function in vivo has not been established. We used a conditional Lifr allele in combination with adenoviral vectors expressing Cre-recombinase to generate an acute model of LC Lifr-KO in the adult mouse testis, and showed that LC Lifr is not required for short term LC survival or basal steroidogenesis. However, LIFR-signalling negatively regulates steroidogenic enzyme expression and maximal gonadotrophin-stimulated testosterone biosynthesis, expanding our understanding of the intricate regulation of LC steroidogenic function.

Autophagy modulation in resveratrol protective effects on steroidogenesis in high-fat diet-fed mice and H2O2-challenged TM3 cells.

Autophagy dysregulation and oxidative stress play critical pathophysiological roles in developing obesity-related metabolic health disorders. This study aims to investigate how autophagy modulationis related to resveratrol (RSV) antioxidant activities and preventive effects on steroidogenesis decline associated with a high-fat diet (HFD) and oxidative damage. Eight-week-old C57BL/6J male mice were fed with HFD with or without supplement RSV (400mg/kg/day) by gavage for 16weeks. The control group was fed with a standard diet with no RSV or the same amount of RSV. Mouse Leydig cell line TM3 cell was used for in vitro studies. Oxidative stress was induced in TM3 cells with H2O2, followed by RSV treatment plus autophagy activator rapamycin or autophagy inhibitor 3-methyladenine, respectively. RSV supplement could upregulate proteins level of StAR and mitochondrial proteins COX4 and mtTFA, indicating the amelioration of steroidogenesis decline and mitochondrial dysfunction caused by HFD. Antioxidants such as GPx4 and SOD2 were improved by RSV as well. The observation of autophagosomes and the changes in expressions of LC3II/I, Beclin1, and Atg7 indicated that RSV could reverse the autophagy defect associated with HFD. 3-methyladenine inhibition of autophagy partially abolished RSV protection on mitochondrial function and steroidogenesis in H2O2-challenged TM3 cells. However, the combination use of rapamycin and RSV did not improve protection on Leydig cells against oxidative damage. The stimulation of autophagy by RSV is closely linked to its antioxidant actions and positive impact on steroidogenesis in HFD mice. The findings suggest RSV is protective against obesity-related Leydig cell impairment.

Melatonin inhibits autophagy in TM3 cells via AKT/FOXO1 pathway.

Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear. In this study, we used the TM3 cell line as the research object, and used H2O2 to induce autophagy. After adding 10 ng/ml melatonin, we used qRT-PCR and western-blot to detect autophagy-related gene and protein expression, and flow cytometry to detect cellular ROS level. The results showed that melatonin can significantly inhibit the occurrence of autophagy, accompanied by a significant decrease in the expression of Becn1, LC3, and FOXO1 (P < 0.05), a significant increase in the expression of p62 and pAKT (P < 0.05), and a significant decrease in ROS level (P < 0.05). After added the inhibitor of AKT perifosine, the effect of melatonin on inhibiting autophagy was reversed. On this basis, we used small RNA interference technology to knock down the expression of FOXO1, and found that there was no significant change of the expression of genes and proteins related to autophagy and ROS level. In summary, melatonin can inhibit H2O2-induced autophagy in TM3 cells through the AKT/FOXO1 pathway.

Bisphenol S exposure induces cytotoxicity in mouse Leydig cells

Bisphenol S (BPS), an increasingly used alternative to bisphenol A, has been linked to testosterone deficiency and male reproductive dysfunction in laboratory animals. This study aimed to examine the cytotoxicity of BPS exposure to Leydig cells and to investigate its possible mechanisms. After treatment with BPS (100, 200 and 400 μM) for 48 h in vitro, TM3 mouse Leydig cells exhibited a dose-dependent decrease in the viability. Furthermore, BPS challenge triggered oxidative stress manifested by compromised activities of superoxide dismutase and catalase with exaggerated formation of reactive oxygen species. Especially, BPS exposure resulted in augmented mitochondrial permeability transition pore opening, dissipated mitochondrial membrane potential and reduced ATP generation, along with an altered energy metabolism. Moreover, BPS stimulation enhanced BAX expression and caspase-3 activity and inhibited BCL-2 expression. In addition, BPS-treated TM3 cells showed an accumulation of autophagic vacuoles, together with increased Beclin1 and P62 expression and elevated LC3B-II/LC3B-I ratio. These results demonstrated that in vitro exposure to BPS exerted cytotoxicity to TM3 Leydig cells through inducing oxidative stress, mitochondrial impairment, autophagic disturbance and apoptosis.

Therapeutic Effects of Edaravone on Azoospermia: Free Radical Scavenging and Autophagy Modulation in Testicular Tissue of Mice.

Background:Chemotherapeutic agents such as cyclophosphamide and busulfan have been shown to have a negative impact on the spermatogenesis process. Based on this fact, the objective of this study was to investigate the effects of edaravone on spermatogenesis in busulfan-induced mice.Methods:Forty adult male mice were equally divided into the four groups: 1) control, 2) edaravone, 3) busulfan, and 4) busulfan + edaravone. Then, the sperm parameters, histopathological examinations, and serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also assessed. Caspase-3, Beclin-1, and ATG-7 mRNA levels were also determined using real-time PCR.Results:Our results revealed that treatment of mice with edaravone in busulfan-induced azoospermia significantly improves sperm parameters, including total count, morphology, and viability (p<0.05). Furthermore, edaravone administration led to a significant increase in serum testosterone (p<0.0001) and FSH (p<0.001) levels, as well as testis weight (p<0.05) and volume (p<0.01). Edaravone also prevented a decrease in the number of testicular cells including spermatogonia (p<0.0001), primary spermatocytes (p<0.001), round spermatids (p<0.0001), Sertoli (p<0.01), and Leydig cells (p<0.0001) in busulfan-treated mice. Additionally, in busulfan-induced azoospermia, edaravone significantly reduced the percentage of sperm with immature chromatin (p<0.0001). Following treatment with edaravone, a decrease in reactive oxygen species (ROS) and an increase in glutathione (GSH) production were noted compared to busulfan-treated mice. Furthermore, caspase-3 (p<0.05), Beclin-1, and ATG-7 (p<0.001) genes expression decreased significantly in treatment groups compared to busulfan-induced azoospermia.Conclusion:According to our findings, edaravone can improve spermatogenesis in busulfan-induced azoospermia through free radical scavenging and autophagy modulation in testicular tissue.

Aspirin Alleviates Particulate Matter Induced Asymptomatic Orchitis of Mice via Suppression of cGAS-STING Signaling.

As an important source of air pollutant, airborne particulate matter (PM) has become a major threat to public health. Orchitis is characterized by acute or chronic testicular inflammation and is a primary cause of male infertility. Although accumulating evidence indicates that PM exposure is associated with increased male infertility rates, the mechanism by which PM is involved is not well understood. Here, we found that short-term PM exposure activated NF-κB signaling in mouse Leydig cells and testes and leading to asymptomatic orchitis. Analyzing the mitochondrial abundance and cGAMP levels in PM exposed mouse Leydig cells, we found that PM exposure induced mitochondrial injury and mtDNA release, leading to inflammation via the cGAS-STING axis. We also found that aspirin-induced acetylation of cGAS inhibited the inflammation in mice after PM exposure, especially in the testes. Moreover, aspirin pretreatment rescued offspring growth in PM-exposed mice. In summary, our study not only provides evidence that PM-induced asymptomatic orchitis in mice may be amenable to aspirin pre-treatment by acetylating cGAS, but also provides a potential explanation for male infertility caused by air pollutants.

Long noncoding RNA MIR22HG promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-125a-5p that targets N-Myc downstream-regulated gene 2 in late-onset hypogonadism

Leydig cells (LCs) apoptosis is responsible for the deficiency of serum testosterone in Late-onset hypogonadism (LOH), while its specific mechanism is still unknown. This study focuses on the role of long noncoding RNA (lncRNA) MIR22HG in LC apoptosis and aims to elaborate its regulatory mechanism. MIR22HG was up-regulated in the testicular tissues of mice with LOH and H2O2-treated TM3 cells (mouse Leydig cell line). Interference of MIR22HG ameliorated cell apoptosis and upregulated miR-125a-5p expression in H2O2-treated TM3 cells. Then, the interaction between MIR22HG and miR-125a-5p was confirmed with RIP and RNA pull-down assay. Further study showed that miR-125a-5p downregulated N-Myc downstream-regulated gene 2 (NDRG2) expression by targeting its 3′-UTR of mRNA. What's more, MIR22HG overexpression aggravated cell apoptosis and reduced testosterone production in TM3 cells via miR-125a-5p/NDRG2 pathway. MIR22HG knockdown elevated testosterone levels in LOH mice. In conclusion, MIR22HG up-regulated NDRG2 expression through targeting miR-125a-5p, thus promoting LC apoptosis in LOH.Leydig cell apoptosis is responsible for the deficiency in serum testosterone in late-onset hypogonadism. This study reveals that lncRNA MIR22HG upregulates NDRG2 expression by targeting miR-125a-5p, thus promoting Leydig cell apoptosis in late-onset hypogonadism.

TRPV1 activation induces cell death of TM3 mouse Leydig cells

Eun-Jin Kim, Long Cao Dang, Marie Merci Nyiramana, Adrian S. Siregar, Min-Seok Woo, Chang-Woon Kim and Dawon Kang. Journal of Animal Reproduction and Biotechnology 2021;36:145-53. https://doi.org/10.12750/JARB.36.3.145

Cytotoxic effects of bisphenol A alternatives in TM3 mice Leydig cells in vitro

Cytotoxic effects of bisphenol A alternatives in TM3 mice Leydig cells in vitro

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

Differential Effects of Viral Nucleic Acid Sensor Signaling Pathways on Testicular Sertoli and Leydig Cells.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon β promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.