Cultured Leydig Cells Research Articles

TGF-β1 regulation of estrogen production in mature rat Leydig cells.

BackgroundBesides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.Methodology/Principal FindingsPrimary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-β1, and E2 treatment in turn restored the inhibition of TGF-β1 on GJIC.ConclusionsOur results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.

Prepubertal Buffalo (Bubalus bubalis) Leydig Cells: Isolation, Culture and Characterization

Water buffalo (Bubalus bubalis) is an economically important livestock species in India. Male buffaloes display delayed sexual maturity as compared to the bulls (Bos taurus). Serum testosterone level, the key regulator of sexual maturity of males, is reported to be low in male buffaloes in comparison to bulls. Testosterone secretion and progression of spermatogenesis is mediated essentially by Leydig cells in the males. Establishment of primary culture for buffalo Leydig cells can provide an excellent tool to investigate the factors which regulate testicular steroidogenesis. Therefore, the objectives of the present study were to isolate, culture and characterize buffalo Leydig cells. Immunohistological analysis revealed that cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) specifically mark the Leydig cells in prepubertal buffalo testis. Using enzymatic digestion and Percoll density gradient centrifugation, a cell population that consisted of approximately 95% pure Leydig cells was obtained as indicated by CYP11A1 staining. Purified Leydig cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS) for 72 h. The cultured Leydig cells proliferated, expressed Leydig-cell specific transcripts (STAR, HSD3B1, HSD3B6, and CYP17A1) and proteins (CYP11A1, HSD3B and LHCGR), and secreted testosterone. It was concluded from the present study that buffalo Leydig cells can be maintained in culture for 72 h. The primary culture of buffalo Leydig cells can be used for studying acute responses, biochemical properties and other factors regulating testicular steroidogenesis, independent of other testicular cell types.

Effects of T-2 toxin on testosterone biosynthesis in mouse Leydig cells

To investigate the effects of T-2 toxin on testosterone biosynthesis in mouse Leydig cells. Leydig cells isolated from clean and healthy Kunming male mice, whose concentration was adjusted to 5×10(5)/mL and the purity identified by the modified 3β-hydroxysteroid dehydrogenase staining method, were used to establish a primary Leydig cell culture model. Blank control group (treated with 0ng/mL human chorionic gonadotropin (hCG) and 0mol/L T-2 toxin), inductive control group (treated with 10ng/mL hCG and 0mol/L T-2 toxin), low-dose T-2-toxin-exposure group (treated with 10ng/mL hCG and 10(-9)mol/L T-2 toxin), middle-dose T-2 toxin-exposure group (treated with 10ng/mL hCG and 10(-8)mol/L T-2 toxin) and high-dose T-2-toxin-exposure group (treated with 10ng/mL hCG and 10(-7)mol/L T-2 toxin) were designed. The testosterone level was measured after 24h incubation. After 24h culture in liquid medium containing serum, the fresh isolated Leydig cells grew well and the purity exceeded 90%. By inducing 10ng/mL hCG, the testosterone level of Leydig cells increased significantly and the difference compared with the blank control was of statistical sense. Compared with the inductive control group, the testosterone level of Leydig cells decreased, and the difference was of statistical sense in all T-2-toxin-exposure groups. Furthermore, the decrease was due to the increase in the dosage of T-2 toxin. T-2 toxin can directly decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis.

Quercetin decreases steroidogenic enzyme activity, NF-κB expression, and oxidative stress in cultured Leydig cells exposed to atrazine

Quercetin has been reported to protect testicular cells from oxidative damage induced by environmental chemicals. In this study, we isolated interstitial Leydig cells (ILCs) from immature rats, set-up ILCs culture, co-treated cells with atrazine (ATZ) and quercetin (QT), evaluated toxicity, and measured the expression levels of antioxidant enzymes and nuclear factor-kappaB (NF-κB) and levels of steroidogenic enzymes. ATZ decreased ILCs viability at concentrations higher than 10 μg/mL and increased reactive oxygen species, malondialdehyde (MDA), and glutathione levels. ATZ also increased glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and decreased superoxide dismutase-1 (sod1) and superoxide dismutase-2 (sod2) messenger RNA (mRNA) levels which were prevented by QT. The changes in the MDA levels and lactate dehydrogenase leakage induced by ATZ (50 μg/mL) were also prevented on co-treatment with QT (50 μM). Furthermore, ATZ-induced 3β- and 17β-hydroxysteroid dehydrogenase activities and NF-κB-expressions at the mRNA and protein levels were also recovered to control value on co-treatment with QT. These data showed that QT protected against ATZ-induced ILCs toxicity by restoring the expression of NF-κB and steroidogenic activity and by preventing the oxidative stress.

Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency.

Recent data suggest a potential role of testis in vitamin D activation, where Leydig cells could represent key players in this process since they express the highest amount of CYP2R1, a key enzyme involved in vitamin D 25 hydroxylation. To evaluate bone status in unilateral orchiectomy and to assess in vivo and in vitro LH-dependency of Vitamin D 25 hydroxylation. 125 normotestosteronemic patients with testicular cancer (TC), featured by unilateral orchiectomy and 41 age-matched healthy male controls were studied in the Center for Human Reproduction Pathology at the University of Padova. To evaluate LH-dependency of Vitamin D 25 hydroxylation in vitro, Leydig cell cultures were stimulated with hCG and assessed for CYP2R1 expression, whereas in vivo 10 hypogonadotropic hypogonadal (HH) patients were evaluated before and after treatment with gonadotropins for bone metabolism markers. Hormonal pattern and bone metabolism markers were measured in all subjects, whereas 105 patients and 41 controls underwent bone densitometry by DEXA. In TC patients 25-hydroxyvitamin D levels were significantly lower compared to controls. Furthermore, 23.8% of patients with TC displayed low bone density (Z-score <-2 SD). None of the 41 control subjects showed any significant alteration of BMD. In vitro and in vivo studies revealed that CYP2R1 expression in Leydig cells appeared to be hCG dependent. Our data show an association between TC and alteration of the bone status, despite unvaried androgen and estrogen levels, suggesting the evaluation of bone status and possible vitamin D deficiency in TC survivors.

Transient Rise of Serum Testosterone Level after Single Sildenafil Treatment of Adult Male Rats

Phosphodiesterase type 5 (PDE5) inhibitors have been established in therapy for a variety of physiological disorders including erectile dysfunction. Despite its popularity and wide usage in erectile dysfunction treatment, the short-term effect of PDE5 inhibition on Leydig cell functionality and testosterone dynamics is missing. This study was designed to assess the acute in vivo effects of sildenafil citrate (Viagra) treatment on testosterone production. Male adult rats were given sildenafil (1.25 mg/kg BW) per os, and testosterone production were analyzed 30, 60, 120, and 180 minutes after treatment. Additionally, in vitro effect of sildenafil extract on Leydig cell steroidogenesis was estimated. The formation of testicular interstitial fluid (TIF), and testosterone, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) content was followed. Occurrence and phosphorylation of mature steroidogenic acute regulatory protein (StAR) and interaction with protein kinase G 1 (PRKG1) were assessed by immunoprecipitation and Western blot. Serum testosterone was increased 60 and 120 minutes after sildenafil treatment. In 60 minutes, TIF volume was doubled and stayed increased till the end of the experimental period. cGMP and testosterone content in TIF were increased 30 minutes after treatment, and cAMP decreased in 60 minutes. Further, sildenafil-induced stimulation of testosterone production was abolished by ex vivo addition of PRKG1 inhibitor but not by protein kinase A inhibitor. Sildenafil treatment increased the level of phosphorylated and total StAR protein. Moreover, co-immunoprecipitation of StAR and PRKG1 was increased following sildenafil treatment suggesting the active role of this kinase in initiation of testosterone synthesis. Additionally, sildenafil extract applied in vitro on primary Leydig cell culture increased cGMP accumulation and testosterone production in time- and dose-dependent manner without effect on cAMP level. Acute sildenafil treatment enlarged TIF volume but also stimulated testosterone production which may be significant considering the positive testosterone effect in regulation of sexual activity.

Comparative evaluation of nonylphenol isomers on steroidogenesis of rat Leydig Cells

Nonylphenol (NP) has been proven to be one of the most investigated xenohormones interacting with the estrogen receptor. Technical nonylphenol (t-NP) contains at least 20 para-substituted isomers. It has been shown that NP isomers vary in their estrogenic potency. So the use of mixtures or impure substances can lead to misinterpretations and unsatisfying conclusions. In the present study, experiments were performed to examine effects of NP isomers on steroidogenesis of rat Leydig cells. Primary cultured Leydig cells were exposed to NP isomers (p33-NP, p262-NP, p353-NP, p363-NP) at the optimized inhibitory concentration 5μmol/L for 6h. NP isomers showed various degrees of inhibition of testosterone biosynthesis, with p363-NP leading to the most significant decrease and others sharing the similar efficacy. The expression of 3b-HSD, Cyp11a1, Star and the apoptosis of Leydig cells were further measured to investigate the underlying mechanisms. We demonstrated that NP isomers can affect the steroidogenesis of rat Leydig cells, at least in part, through their influence on gene expression and cell apoptosis, but varied in their individual degree. However, the final results were not completely coincident with their estrogenic potency tested in vitro, which implies that effects of NP isomers on steroidogenesis appear to be mediated through some other underlying mechanisms besides their various estrogenic potency.

Comment on “Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression” by Romano et al. 2012

Romano et al. (2012) recently published the results of a study in which pregnant rats were treated with a glyphosate-based herbicide formulation from gestational day 18 to postnatal day 5. Various parameters related to sexual behavior and reproductive development were assessed in the resulting male offspring. Based on their results, the study authors claim that glyphosate exposure in the perinatal period causes changes in males that are associated with hypersecretion of androgens. There are a number of issues with this study, however, that preclude such conclusions. First, as a premise for this study, the authors incorrectly assert that glyphosate is a ‘‘potential endocrine chemical disruptor.’’ As evidence, they cite three in vitro studies of altered aromatase activity conducted in a single laboratory (Benachour et al. 2007; Gasnier et al. 2009; Richard et al. 2005), and their own earlier work suggesting reduced serum testosterone concentrations in response to treatment (Romano et al. 2010). These studies, however, were conducted using glyphosate-based herbicide formulations rather than the active ingredient alone. Exposures were at high concentrations; for instance, in the study by Benachour et al. (2007), the concentration of glyphosate in the tissue culture medium was reported to be 1.27 mM (approximately 200,000 lg/L), which is not relevant to concentrations in target tissue concentrations or environmental media. Further, although sporadic positive responses were reported, aromatase activity was generally unaffected in the few experiments that tested glyphosate by itself. It should be additionally noted that the EPA OPPTS guideline for conducting the aromatase assay (U.S. Environmental Protection Agency 2009a) clearly warns that all glassware and apparatus used need to be free of detergent residue; this is because microsomes are extremely sensitive to the surfactants found in detergents. Surfactants are a major component of the various herbicide formulations tested in these studies and are the likely cause for the observed aromatase inhibition. Therefore, the claim that glyphosate is an endocrine disruptor is not substantiated. Second, it is not clear to what agent(s) the animals were exposed. The study authors indicate that Roundup Transorb (480 g/L glyphosate, 648 g/L isopropylamine salt, and 594 g/L inert ingredients) was the product administered. However, they report no additional details regarding the composition of the herbicide formulation used such as the identity and amount of surfactant(s) in the formulation. This is important because in other studies, Levine et al. (2007) have shown that steroidogenesis in Leydig cell cultures is inhibited by the presence of surfactants, which perturb membrane function of mitochondria. An appropriate control group that was exposed to the surfactant(s) only was not included in the study nor was there a group exposed to glyphosate only. As such, the study results are confounded. Third, the authors did not describe any methods to control for potential litter effects, and it is not clear whether this was done or not. Because the dams were the unit of exposure in these experiments, the exposed litter (the dam) is the appropriate unit of analysis. This is particularly important when assessing these experiments because dosing of the dams occurred from gestational day 18 through postnatal day 5; however, glyphosate does not cross well

Luteinizing hormone receptor splicing variants in bovine Leydig cells

The luteinizing hormone receptor (LHR) plays a key role in testosterone production through its interaction with the gonadotropins, LH and chorionic gonadotropin. We examined the LHR splicing pattern in bovine Leydig cells; LH-induced expression of eight cloned splicing variants was detected by real-time PCR. Luteinizing hormone applied to cultured Leydig cells resulted in expression of full-length LHR and the A and B isoforms, as well as secretion of testosterone, which first increased, then declined, and then increased further, with increased LH levels. The secretion of testosterone progressively increased with increasing LH, but the expression levels of LHR (FL, A, and B) did not increase correspondingly. We conclude that the LHR splicing pattern is complex in bovine Leydig cells, and that expression of full-length LHR and isoforms A and B changes when induced with LH.

Effects of Norepinephrine and Acetylcholine on the Development of Cultured Leydig Cells in Mice

Few data have suggested how norepinephrine (NE) and acetylcholine (Ach) regulate the development of Leydig cells in mice at prepuberty, except for data indicating endocrine effects. The present study aims to elucidate the roles of NE and Ach on the differentiation and proliferation of Leydig cells. Firstly, the expression of adrenergic receptors and muscarinic acetylcholine receptors in Leydig cells was investigated. It was found that adrenergic receptors (β1AR, β2AR, and α1D) and muscarinic acetylcholine receptors (M1 and M3) mRNA are expressed in adult Leydig cells. Then, the effects of NE and Ach on the differentiation and proliferation of Leydig cells were analyzed. The results showed that NE and Ach at 10 μM significantly increased the number of 3β-hydroxysteroid-dehydrogenase- (3β-HSD-) positive Leydig cells and improved the expression of proliferating cell nuclear antigen (PCNA) in Leydig cells on postnatal day (PD) 15 (P < 0.05). NE and Ach at 10 μM had no impact on the expression of PCNA mRNA (P > 0.05), but reduced the expression of 3β-HSD mRNA in adult Leydig cells and a murine Leydig tumor cell line (MLTC-1) (P < 0.05). Therefore, a conclusion may be reached that NE and Ach participated in stimulating the development of Leydig cells in mice from prepuberty to adult stage.

A synergistic effect of insulin-like growth factor (IGF-I) on equine luteinizing hormone (eLH)-induced testosterone production from cultured Leydig cells of horses

Localization of IGF-I and IGF-IR were observed in Leydig cells of horses using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. Previous studies in other species have indicated that IGF-I increases basal and/or LH/hCG-induced testosterone production. The objectives of this study were to (1) test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and (2) determine if this effect is reproductive stage-dependent. Testes were collected from five pubertal (1.1 ± 0.1 year; 1–1.5 year) and eight post-pubertal (2.88 ± 0.35 years; 2–4 years) stallions during routine castrations at the UC Davis Veterinary Hospital. Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated without (control) or with increasing concentrations of purified pituitary-derived eLH and/or recombinant human IGF-I (rhIGF-I) and incubated under 95% air: 5% CO 2 at 32 °C for 24 h. After 24 h, culture media was collected and frozen at −20 °C until analyzed for testosterone by a validated radioimmunoassay (RIA). In pubertal stallions, treatment with both increasing concentrations of rhIGF-I and 5 ng/ml of eLH failed to demonstrate a significant difference in testosterone production compared with 5 ng/ml of eLH only. However, in post-pubertal stallions, a significant increase in the concentration of testosterone in culture media was observed from Leydig cells treated with various concentrations of rhIGF-I and 1 or 5 ng/ml of eLH compared with 1 or 5 ng/ml of eLH only. In conclusion, IGF-I has a synergistic effect on eLH-induced testosterone production in cultured equine Leydig cells from post-pubertal but not pubertal stallions.

Effects of sleep deprivation on serum testosterone concentrations in the rat

Sleep deprivation (SD) leads to decreases in circulating levels of testosterone with unknown mechanisms. We tested the hypothesis that decreased testosterone levels associated with SD may be caused by serotonin-mediated inhibition of its production. Male rats were subjected to SD for 24 or 48 h using the dish-over-water-method with a Rechtschaffen apparatus. Serum testosterone, corticosterone and serotonin (5-HT) concentrations were assessed thereafter, as were testicular StAR and 5-HT 2 receptor levels. SD, regardless of duration led to significant decreases in serum testosterone levels and testicular steroid acute regulatory protein (StAR) protein expression, while 5-HT levels were significantly elevated (all P < 0.05). Corticosterone concentrations were significantly increased in 48 h SD rats ( P < 0.05). In primary Leydig cell cultures, 5-HT decreased chorionic gonadotropin-induced testosterone secretion and StAR expression, which appeared to be dependent on 5-HT 2 receptor activation but independent of cyclic AMP signaling. These findings suggest that decreased serum testosterone levels in SD rats may be the result of 5-HT-related inhibition of testosterone production and decreased testicular expression of StAR protein.

Kolaviron Protects Primary Cultures of Rat Leydig Cells from Atrazine Induced Damages

BACKGROUND: Atrazine (ATZ) induced testicular impairment has been reported in vivo in rats. The current study examined the cytotoxic effect of ATZ and the protective potentials of kolaviron (KV) in vitro employing interstitial Leydig cells (ILC) in culture. ILC isolated from juvenile rats were co-cultured with 232μM ATZ in the presence or absence of KV (60μM). Expression of steroidogenic acute regulatory proteins (StAR), cytochrome P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase-1(SOD-1) and superoxide dismutase-2(SOD-2) were monitored by real-time PCR following 6 h of treatment.

Impact of methoxyacetic acid on mouse Leydig cell gene expression

BackgroundMethoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function.MethodsCultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling.ResultsA total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling.ConclusionsThese findings on the progressive changes in gene expression induced by MAA in a cultured Leydig cell model may help elucidate signaling pathways that lead to the testicular pathophysiological responses induced by MAA exposure and may identify useful biomarkers of MAA toxicity.

Immunolocalization of humanin in human sperm and testis

We have discovered, by immunocytochemistry and immunoelectronmicroscopy, that humanin (HN) is expressed in human ejaculated sperm and testis. In sperm, the HN immunolabeling pattern depends on sperm morphology; in particular, HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology. We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm. Because it has been established that HN has antiapoptotic properties, it is reasonably hypothesized that HN may play an important role in preventing apoptosis in human sperm and testis. Thus, the examination of the HN localization in normal and abnormal sperm could be proposed as an auxiliary test to better define sperm quality.

Hepatocyte Growth Factor (HGF) Modulates Leydig Cell Extracellular Matrix Components

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays multiple roles during mammalian development. We previously demonstrated that in the postnatal testes, the HGF receptor, c-met, is expressed by Leydig cells and HGF increases the steroidogenetic activity of the cells. In the present article, we report that HGF modifies the composition of the extracellular matrix of cultured Leydig cells. We show that HGF increases the metabolic activity of isolated Leydig cells; in particular, the factor increases urokinase plasminogen activator and matrix metalloproteinase 2 secretion. We have also shown that the levels of active transforming growth factor beta are increased by HGF. On the contrary, using the Western blotting technique, a strong reduction in the amount of fibronectin present in the culture medium of cells cultured in the presence of HGF has been detected. The presented data demonstrate that HGF modulates several functional activities of Leydig cells, further supporting the hypothesis that this factor has a relevant role in the regulation of mammalian spermatogenesis.

CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists

Cytochrome P450 1B1 (CYP1B1) is highly expressed in testis, but there is conflicting information regarding the inducibility of testicular CYP1B1 by aryl hydrocarbon receptor (AhR) agonists.To assess AhR-mediated regulation, testicular CYP1B1 expression was measured following treatment of adult rats with 3-methylcholanthrene and various dosages of benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The effect of TCDD on CYP1B1 expression in R2C rat Leydig and MA-10 mouse Leydig cells in culture was also determined.Immunoblot analysis showed that treatment with benzo[a]pyrene at dosages up to 200 mg/kg/day and 3-methylcholanthrene at 25 mg/kg/day did not induce testicular CYP1B1 expression. Treatment with TCDD at dosages of 1, 5 or 100 μg/kg had no effect, but testicular CYP1B1 protein levels were increased by approximately 50% at dosages of 10 and 50 μg/kg.CYP1B1 mRNA levels in MA-10 and CYP1B1 protein levels in R2C cells were not induced by exposure to TCDD (10–1000 nM).Overall, the results indicate that rodent testicular CYP1B1 is not inducible by AhR agonists.

177. ANTIBODIES AGAINST BMPR-IB UNCOVERS A PARACRINE FUNCTION FOR THE RECEPTOR IN MALE MOUSE LEYDIG CELL TESTOSTERONE PRODUCTION IN VITRO

The male reproductive system is regulated by pituitary gonadotrophins and local testicular factors including the transforming growth factor-β superfamily members which up-regulate and modulate testosterone production respectively. Type I and type II bone morphogenetic protein (BMP) receptors have been identified in both steroidogenic and non-steroidogenic cells in the testis and have been reported to impact steroid production via their effect on steroidogenic enzymes. In this study we investigated if BMPR-IB had an autocrine or paracine role in testosterone production using BMPR-IB antibodies in testis tissue and Leydig cell culture in vitro. Immature (21d) and mature (60d) mice were sacrificed by CO2 asphyxiation and the testis removed, decapsulated and cultured in basal and equine chorionic gonadotrophin (eCG)-conditioned media for three hours at 32 °C in 5 % CO2, in the presence and absence of anti-BMPR-IB. Additionally, Leydig cells were Percoll purified from adult mouse testicular interstitial cells and cultured for three hours with and without anti-BMPR-IB under human chorionic gonadotrophin (hCG) stimulated or unstimulated conditions. After three hours of incubation the culture media was aspirated into labeled vials and assayed for testosterone using a radioimmunoassay. In adult testicular slice culture, treatment with anti-BMPR-IB resulted in a significant decrease in basal and eCG-stimulated testosterone production by 37 % and 41 % respectively, while having no significant effect on basal or eCG-stimulated testosterone production by the immature testis. In purified Leydig cell culture from adult male mice BMPR-IB immunization had no effect on testosterone production in basal or hCG-stimulated conditions. In conclusion, anti-BMPR-IB significantly reduced testosterone production in adult testicular slice culture but not in cell culture, demonstrating that BMP paracrine signalling from the seminiferous tubules is likely to be important in modulating testosterone production by Leydig cells. Additionally, the paracrine signalling appears to be developmentally regulated only occurring in the adult testis.

CSD mRNA expression in rat testis and the effect of taurine on testosterone secretion

In the present study, the cysteine sulfinate decarboxylase (CSD) mRNA expression was detected in rat testis by RT-PCR. The results showed that CSD mRNA was expressed in rat testis, and the putative encoded-amino acid sequence was exactly the same as that in rat liver which was already known. At the same time, the effects of taurine on testosterone secretion were investigated both in vivo and in vitro. In vivo, taurine were administered to male rats by tap water. The results showed that taurine obviously stimulated the secretion of FSH, LH and testosterone in serum, but showed no significant effect on the secretion of estradiol. Taurine administered in water could significantly increase the concentration of taurine in the blood and testis of rats. In vitro, cultured Leydig cells were treated with taurine independently or incubated with human chorionic gonadotropin (HCG) and progesterone. The results showed that taurine had biphasic effects on basal testosterone secretion in cultured Leydig cells. Low concentrations of taurine (0.1-100 microg/ml) could stimulate testosterone secretion, whereas high concentration of taurine (400 microg/ml) could inhibit testosterone secretion. Testosterone secretion stimulated by HCG was significantly increased by 10 and 100 microg/ml of taurine administration, and obviously decreased by treating with 400 microg/ml of taurine. Testosterone secretion induced by progesterone was significantly stimulated by treating with 1.0 and 10 microg/ml of taurine, however, it was significantly inhibited when treated with 400 microg/ml of taurine. Meanwhile, the effect of silencing CSD mRNA by siRNA on testosterone secretion was analyzed. The results showed that testosterone secretion was obviously decreased after the inhibition of CSD mRNA expression in cultured Leydig cells. These results indicated that taurine can be synthesized in rat testis by CSD pathway, and it plays important roles in testosterone secretion both in vivo and in vitro which need to be further investigated.

Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses.

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation.