Steroidal Glycosides from Allium tuberosum Seeds and Their Roles in Promoting Testosterone Production of Rat Leydig Cells.

A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1-27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1-10 and 22-26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13-15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 µM, respectively.

Screening of the Active Component Promoting Leydig Cell Proliferation from Lepidium meyenii Using HPLC-ESI-MS/MS Coupled with Multivariate Statistical Analysis.

Lepidium meyenii is now widely consumed as a functional food and medicinal product, which is known as an enhancer of reproductive health. However, the specific chemical composition and mechanism of action for improving sexual function are unclear. The present study aims at screening and determining the potential compounds, which promote mouse leydig cells (TM3) proliferation. The partial least squares analysis (PLS) was employed to reveal the correlation between common peaks of high performance liquid chromatography (HPLC) fingerprint of L. meyenii and the proliferation activity of TM3. The results suggested that three compounds had good activities on the proliferation of TM3 and promoting testosterone secretion, there were N-benzyl-hexadecanamide, N-benzyl-(9z,12z)-octadecadienamide and N-benzyl-(9z,12z,15z)-octadecatrienamide which might be the potential bioactive markers related to the enhancing sexual ability functions of L. meyenii. The first step in testosterone synthesis is the transport of cholesterol into the mitochondria, and the homeostasis of mitochondrial function is related to cyclophilin D (CypD). In order to expound how bioactive ingredients lead to promoting testosterone secretion, a molecular docking simulation was used for further illustration in the active sites and binding degree of the ligands on CypD. The results indicated there was a positive correlation between the binding energy absolute value and testosterone secretion activity. In addition, in this study it also provided the reference for a simple, quick method to screen the promoting leydig cell proliferation active components in traditional Chinese medicine (TCM).

Melatonin appears to protect against steroidogenic collapse in both mice fed with high-fat diet and H2 O2 -treated TM3 cells.

High-fat diets (HFDs) are detrimental to steroidogenesis and male fertility. This study aimed to investigate the protective effects of melatonin (MT) treatment on testicular dysfunction in mice fed with HFD. C57BL/6J male mice were randomly divided into three groups: CTRL, HFD and HFD + MT. MT treatment mitigated the increase in body weight and adipose tissue in HFD-fed mice. Serum levels of sex hormones were improved upon MT supplementation, and the expression of the testosterone synthesis proteins, StAR and P450scc was rescued as well. MT treatment significantly up-regulated the expression of SIRT1, SOD2, and GPx4 and down-regulated the expression of GRP78 and CHOP, indicating an attenuation of oxidative stress (OS) and endoplasmic reticulum (ER) stress. In TM3 cells, MT treatment protected against H2 O2 -induced steroidogenic collapse by improving mitochondrial function and attenuating OS and ER stress. These results indicate that MT treatment can improve steroidogenesis in mice fed with HFD and may have therapeutic value in the treatment of obesity-associated hypogonadism.

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants.

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ±â€¯1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ±â€¯4.0 vs. 16.7 ±â€¯1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 µM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Effect of Stevia rebaudiana Bertoni extract on sexual dysfunction in Streptozotocin-induced diabetic male rats.

Stevia rebaudiana Bertoni has been used locally as a non-calorie sweetener in medicine and diabetic diet which claimed to have aphrodisiac properties, although no scientific data of this function have been reported. The aim of this study was to investigate the effect of S. rebaudiana extract on sexual dysfunction, testosterone levels and number of Leydig cells in Streptozotocin (STZ)-induced diabetic male rats. A total of 28 diabetic male rats were randomly divided into 4 groups: diabetic group without any extract and 3 extract groups (5, 50 and 100 mg/kg). Seven normal control rats were treated with vehicle mount latency and frequency of (ML, MF), intromission latency and frequency (IL, IF), ejaculation latency and frequency of (EL, EF), the mount latency post ejaculation (MPE), the intromission latency post ejaculation (ILE), the intromission frequency post ejaculation (IFE) were recorded during 30 min on days 0, 14, 28. The serum testosterone levels, blood glucose, sex organs weight, number of leydig cells and histology of testicular tissue were measured. The stevia group (5 mg/kg) had a significant (p<0.05) increase in EF and IF. The number of Leydig cells in the diabetic group were significantly (p<0.05) reduced compared to the normal group and diabetic groups with extract (5 and 50 mg/kg). The serum testosterone levels and other sexual behaviors did not show any significant differences. The low- dose stevia extract with attention to antioxidant, vasodilator and anti-diabetic properties can be aphrodisiac in STZ- induced diabetic male rats.

A novel function of geranylgeraniol in regulating testosterone production.

Isoprenoids play widely differing roles in various physiological processes in animals and plants. Geranylgeraniol (GGOH) is an isoprenoid found in plants, and is an important metabolic derivative in the isoprenoid/cholesterol synthesis pathway. Earlier studies focused on GGOH's ability to improve the side effects of bisphosphonate therapy by regulating the mevalonate pathway. More recently, the mevalonate pathway-independent effects of GGOH have been described, including anti-inflammatory, anti-tumorigenic, and neuroprotective activities. It is noteworthy that GGOH regulates the steroidogenesis pathway in testis-derived I-10 tumor cells. Testosterone is a hormone produced via steroidogenesis in testicles and plays a role in fetal development and the male reproductive system. GGOH enhanced testosterone and progesterone (its precursor) levels in I-10 cells by activating adenylate cyclase via cAMP/PKA signaling, without altering phosphodiesterase activity. These findings highlight the potential benefits of GGOH as a therapeutic agent for low testosterone levels, such as late-onset hypogonadism in men.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Protective effect of DHEA on hydrogen peroxide-induced oxidative damage and apoptosis in primary rat Leydig cells.

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement due to its putative anti-aging properties. However, the effect of DHEA in Leydig cells, a major target cell of DHEA biotransformation in male, are not clear. The present study aimed to investigate the preventative effect of DHEA on oxidative damage and apoptosis after H2O2 treatment in Leydig cells. The results showed that DHEA treatment attenuated the reduction of cell viability induced by H2O2. No differences were observed on the superoxide anion (O2-) content, while DHEA treatment decreased reactive oxygen species (ROS) and hydroxyl radical (•OH) content in H2O2-treated Leydig cells. Pre-treatment with DHEA increased peroxidase (POD) activity and decreased glutathione peroxidase (GSH-Px) activity in H2O2-treated Leydig cell. DHEA treatment attenuated DNA damage as indicated by the decreasing of tail moment, comet length and olive tail moment. Total apoptosis ratio and early apoptosis ratio were significantly decreased in H2O2-treated Leydig cell that were pre-treatment with DHEA. DHEA treatment decreased Bax, capase-9 and capase-3 mRNA levels in H2O2-treated Leydig cells. Our results demonstrated that pre-treatment with DHEA prevented the Leydig cells oxidative damage caused by H2O2 through increasing POD activity, which resulted in inhibition of •OH generation. Meanwhile, pre-treatment with DHEA inhibited H2O2-induced Leydig cells early apoptosis which mainly by reducing the pro-apoptotic protein Bax and caspases-9, caspases-3 mRNA levels. This information is important to understand the molecular mechanism of anti-ageing effect and potential application in treatment of oxidative stress induced related diseases of DHEA.

Prepubertal subchronic exposure to soy milk and glyphosate leads to endocrine disruption.

Lactose intolerance is characterized by low or inexistent levels of lactase, and the main treatment consists of dietary changes, especially replacing dairy milk by soy milk. Soy contains phytoestrogens, substances with known estrogenic activity, besides, glyphosate-based herbicides are extensively used in soy crops, being frequently a residue in soy beans, bringing to a concern regarding the consumption of soy-based products, especially for children in breastfeeding period with lactose intolerance. This study evaluated the pubertal toxicity of a soy milk rich feeding (supplemented or not with glyphosate, doses of 50 and 100 mg/kg) during prepubertal period in male rats. Endocrine disruption was observed through decrease in testosterone levels, decrease in Sertoli cell number and increase in the percentage of degenerated Sertoli and Leydig cells in animals receiving soy milk supplemented with glyphosate (both doses) and in animals treated only with soy milk. Animals treated with soy milk with glyphosate (both doses) showed decrease spermatids number and increase of epididymal tail mass compared to control, and decrease in the diameter of seminiferous tubules compared to soy milk control group. Animals receiving soy milk supplemented with 100 mg/kg glyphosate showed decrease in round spermatids and increase in abnormal sperm morphology, compared to control.

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways.

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H2O2-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H2O2-induced Leydig cells. Apoptosis was significant decreased in H2O2-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H2O2 in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H2O2. These data showed that DHEA could ameliorate H2O2-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) increases serum testosterone concentration and enhances steroidogenic ability of Leydig cells in male rats.

Although Lepidium meyenii (maca), a plant growing in Peru's central Andes, has been traditionally used for enhancing fertility and reproductive performance in domestic animals and human beings, effects of maca on reproductive organs are still unclear. This study examined whether feeding the hydroalcoholic extract powder of maca for 6 weeks affects weight of the reproductive organs, serum concentrations of testosterone and luteinising hormone (LH), number and cytoplasmic area of immunohistochemically stained Leydig cells, and steroidogenesis of cultured Leydig cells in 8-week-old male rats. Feeding the extract powder increased weight of seminal vesicles, serum testosterone level and cytoplasmic area of Leydig cells when compared with controls. Weight of prostate gland, serum LH concentration and number of Leydig cells were not affected by the maca treatment. The testosterone production by Leydig cells significantly increased when cultured with 22R-hydroxycholesterol or pregnenolone and tended to increase when cultured with hCG by feeding the extract powder. The results show that feeding the hydroalcoholic extract powder of maca for 6 weeks increases serum testosterone concentration associated with seminal vesicle stimulation in male rats, and this increase in testosterone level may be related to the enhanced ability of testosterone production by Leydig cells especially in the metabolic process following cholesterol.

Involvement of KLF14 and egr-1 in the TGF-beta1 action on Leydig cell proliferation.

Transforming growth factor ß1 (TGF-ß1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-ß1 exerts stimulatory and inhibitory effect depending on the type I receptor involved in the signaling pathway. The aim of the present work was to study the signaling mechanisms and the intermediates involved in the action of TGF-ß1 on TM3 Leydig cell proliferation in the presence or absence of progesterone. The MTT assay showed that the presence of progesterone in the culture media lead to a proliferative effect that was blocked by Ru 486, an inhibitor of progesterone receptor; and ALK-5 did not participate in this effect. TGF-ß1 (1 ng/ml) increased the expression of p15 (an inhibitor of cell cycle) in TM3 Leydig cells, and this effect was blocked by progesterone (1µM). The expression of PCNA presented a higher increase in the cell cultured with TGF-ß1 plus progesterone than in cells cultured only with TGF-ß1. Progesterone induced the gene expression of endoglin, a cofactor of TGF-ß1 receptor that leads to a stimulatory signaling pathway, despite of the absence of progesterone response element in endoglin gene. In addition, the presence of progesterone induced the gene expression of egr-1 and also KLF14, indicating that this steroid channels the signaling pathway into a non-canonical mechanism. In conclusion, these findings suggest that the proliferative action of TGF-ß1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone.

Advantage of Guaraná (Paullinia cupana Mart.) supplementation on cadmium-induced damages in testis of adult Wistar rats.

Paullinia cupana is an Amazonian bush whose seeds have long been used in folk medicine. However, most of the therapeutic properties attributed to this plant are broad and nonspecific, although an antioxidant activity has been reported.  On the other hand, cadmium is a heavy metal known for increasing free radicals, hence resulting in cellular oxidative damages. This study was designed to evaluate whether Paullinia cupana is able to reduce cadmium-induced morphological impairment in Wistar rat testis. Adult male Wistar rats 110 days old were ip injected with cadmium (1.15 mg/kg BW [body weight]) and subsequently treated with P. cupana during 56 days.  Furthermore, groups receiving either P. cupana extract or cadmium are mentioned. After the treatment period, testis samples were subjected to histological and stereological analyses. Moderate to severe testicular impairments were shown by the animals exposed to cadmium. However, the animals supplemented with P. cupana after cadmium exposure showed a significant decrease in the proportion of damaged seminiferous tubules. Also, P. cupana supplementation was effective in maintaining the number of Leydig cells per testis in the animals exposed to cadmium. In conclusion, P. cupana supplementation was partially efficient in preventing cadmium from damaging the testis of adult Wistar rats.

Cytoprotective effects of fruit pulp of Eugenia jambolana on H 2 O 2 -induced oxidative stress and apoptosis in rat Leydig cells in vitro.

This study was undertaken to investigate the cytoprotective effect of the fruit pulp of Eugenia jambolana (50-250 µg ml(-1) ) against the damage induced by H 2 O 2 (100 µm) exposure to Leydig cells in vitro. Cell survival with extract was found comparable to similar effects by N-acetyl-l-cysteine. H 2 O 2 -induced rise in thiobarbituric acid reactive substance formation and decline in the activity and expression of antioxidant enzymes like superoxide dismutase, catalase and glutathione-s-transferase were effectively checked. Cellular glutathione and total antioxidant capacity demonstrated significant improvement. The increase in expression of inducible nitric oxide (NO) synthase leading to NO production was successfully countered. Co-treatment of the extract helped in the down-regulation of caspase-3 and poly-ADP-ribose polymerase resulting in a significant reduction in Leydig cell apoptosis induced by H 2 O 2 . Upstream marker proteins of extrinsic (caspase-8, Fas, FasL) and intrinsic (caspase-9) pathway of metazoan apoptosis were identically down-regulated. The Bcl-2 family of proteins, though, remained unaffected. The extract also positively modulated the other marker proteins like c-Jun NH 2 -terminal kinase, p38, Akt, nuclear factor-κB, c-Fos, cellular FLICE-inhibitory protein, cyclooxygenase-2 and p53. Taken together, the above-mentioned findings establish the anti-oxidative and anti-apoptotic potency of the extract that ameliorates the H 2 O 2 -induced adverse effects on rat Leydig cells in vitro.

Kolaviron biflavanoids of Garcinia kola seeds protect atrazine-induced cytotoxicity in primary cultures of rat Leydig cells.

We sought to explore the mechanism by which kolaviron (Kol) protects against atrazine (ATZ)-induced toxicity of cultured interstitial Leydig cells (ILCs). In our experiments, treatment with Kol improved Leydig cell viability and significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels. Further investigations revealed a reduction in glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione-S-transferase (GST) and elevation of superoxide dismutase 1 (SOD-1) and superoxide dismutase 2 (SOD-2) as measured by messenger RNA (mRNA) expression. Additionally, the ATZ-induced alterations in the mRNA transcript copy numbers of steroidogenesis genes: steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were shifted toward the control values by Kol. Taken together, these findings indicate that Kol protects ILCs from ATZ-induced toxicity via the reduction in ROS and MDA levels and induce normalization of mRNA levels of all the tested genes.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.

Prunus armeniaca L (apricot) protects rat testes from detrimental effects of low-dose x-rays.

Exposure to low x-ray doses damages the spermatozoa, mainly by late-onset (ie, after 3 months) oxidative stress. Antioxidants ameliorate oxidation and prevent tissue damage. Prunus armeniaca L (apricot), rich in carotenoids and vitamins, is a potent natural antioxidant. We hypothesized that an apricot-rich diet might ameliorate the detrimental effects of low-dose x-rays on testis tissue. A 20% apricot diet was composed isoenergetically to the regular rodent diet. The total phenolic content, reducing power, and antioxidant capacity of both diets were determined. Sprague-Dawley rats received apricot-rich diets before and after x-ray exposure. Regular diets were given to controls. Rats were exposed to 0.2 Gy x-rays at the eighth week and were euthanized at the 20th postexposure week. Testicular oxidative status was determined by tissue thiobarbituric acid-reactive substances, reduced glutathione, superoxide dismutase, and catalase activities. For histologic evaluation, qualitative and quantitative microscopic determinations were performed, and Leydig and Sertoli cell counts and Johnsen scores were measured. The control diet group had significant testicular oxidative stress and mild tissue deterioration. Leydig and Sertoli cell counts, tubule diameters, and Johnsen scores were significantly decreased in the exposure groups. Apricot-rich diet significantly ameliorated the oxidative status and prevented the damage in tubular histology. The protective effects were prominent when the diet was maintained throughout the time course and were partially protected when the diet was initiated after exposure. The natural antioxidant activity of apricot ameliorates the delayed detrimental effects of low-dose irradiation on testis tissue. The high total antioxidant capacity of the apricot deserves further investigation.

The effect of midazolam on mouse Leydig cell steroidogenesis and apoptosis.

The peripheral-type benzodiazepine receptor (PBR), a putative receptor in Leydig cells, modulates steroidogenesis. Since benzodiazepines are commonly used in regional anesthesia, their peripheral effects need to be defined. Therefore, this study set out to investigate in vitro effects of the benzodiazepine midazolam (MDZ) on Leydig cell steroidogenesis, and the possible underlying mechanisms. The effects of MDZ on steroidogenesis in primary mouse Leydig cells and MA-10 Leydig tumor cells were determined by radioimmunoassay. PBR, P450scc, 3beta-HSD and StAR protein expression induced by MDZ was determined by Western blotting. Inhibitors of the signal transduction pathway and a MDZ antagonist were used to investigate the intracellular cascades activated by MDZ. In both cell types, MDZ-stimulated steroidogenesis in dose- and time-dependent manners, and induced the expression of PBR and StAR proteins, but had no effect on P450scc and 3beta-HSD expressions. Moreover, H89 (PKA inhibitor) and GF109203X (PKC inhibitor) attenuated MDZ-stimulated steroid production. Interestingly, the MDZ antagonist (flumazenil) did not decrease MDZ-induced steroid production in both cell types. These results highly indicated that MDZ-induced steroidogenesis in mouse Leydig cells via PKA and PKC pathways, along with the expression of PBR and StAR proteins. In addition, MDZ at high dosages induced rounding-up, membrane blebbing, and then death in MA-10 cells. In conclusion, midazolam could induce Leydig tumor cell steroidogenesis, and high dose of midazolam could induce apoptosis in Leydig tumor cells.

Beta-cypermethrin impairs reproductive function in male mice by inducing oxidative stress.

Cypermethrin (CYP), an insecticide, has deleterious effects on male reproductive function. The objective was to identify whether the effects of beta-CYP on male reproductive organs were associated with oxidative stress. Three doses of beta-CYP (1, 10, and 20mg/kg) were administered to male mice for 35 d, with or without vitamin E (20mg/kg). The moderate (10mg/kg) and high (20mg/kg) doses of beta-CYP not only decreased body weight and the weight of the testes, epididymides, seminal vesicles, and prostate (P<0.05) but also reduced serum testosterone concentration and the expression of steroidogenic acute regulatory protein (P<0.05), in addition to damaging the seminiferous tubules and sperm development. Furthermore, moderate and high doses of beta-CYP administration decreased sperm number, sperm motility, and intact acrosome rate (P<0.05). Based on ultrastructural analyses, high doses of beta-CYP produced swelling and degeneration of mitochondria and the smooth endoplasmic reticulum of Leydig cells and caused the formation of concentric circles. These toxic effects of beta-CYP may be mediated by increasing oxidative stress, as the moderate and high doses of this compound increased malondialdehyde and nitric oxide in testes (P<0.05); reduced the activity of catalase, glutathione peroxidase (GSH-Px), and superoxide dismutase (P<0.05); and activated ERK1/2 (P<0.05). Vitamin E reversed the effects of beta-CYP on testosterone production and testis damage (P<0.05 vs. the high-dose group). Therefore, we inferred that beta-CYP damaged the structure of testes and decreased sperm output by inducing oxidative stress.