Science.gov

Sample records for rat leydig cells

  1. IMMATURE RAT LEYDIG CELLS ARE INTRINSICALLY LESS SENSITIVE THAN ADULT LEYDIG CELLS TO ETHANE DIMETHANESULFONATE

    EPA Science Inventory

    Leydig cells from immature rat tests appear to be insensitive to doses of ethane-1,2-dimethanesulfonate (EDS) which eliminate Leydig cells from adult rat testes. e sought to determine whether this differential response to EDS is intrinsic to the Leydig cell or mediated by other i...

  2. Estrogenic regulation of Leydig cell development in the rat

    SciTech Connect

    Myers, R.B.

    1989-01-01

    Initial studies demonstrated that treatment of male rats with estradiol for a period of four days resulted in a reduction in {sup 3}H-thymidine incorporation of isolated interstitial cells. Furthermore, {sup 3}H-thymidine incorporation in interstitial cells of 33 day old rats was inhibited by the addition of estradiol in vitro. Subsequent studies were performed in the ethylene dimethanesulphonate (EDS) treated rat. Leydig cells were rapidly destroyed after EDS administration as determined by hCG binding, steroid synthesis and morphological studies. A significant finding was the production of 5{alpha}-androstane-3{alpha},17{beta}-diol by regeneration Leydig cells of the EDS treated rat. In subsequent studies, rats received daily treatment with estradiol and/or hCG/LH after EDS treatment. Estradiol treatment had no effect on Leydig cell degeneration. Leydig cell regeneration, however, did not occur in the estradiol treated rat.

  3. EFFECTS OF ETHANE DIMETHANESULFONATE (EDS) ON ADULT AND IMMATURE RABBIT LEYDIG CELLS: COMPARISON WITH EDS-TREATED RAT LEYDIG CELLS

    EPA Science Inventory

    Ethane-dimethanesulfonate (EDS) has been shown to selectively kill Leydig cells and depress testosterone production in adult rats. ecent study has shown that immature rat leydig cells are less sensitive to EDS exposure. here is evidence that the rabbit metabolizes EDS to methane ...

  4. Ontogenesis of leptin receptor in rat Leydig cells.

    PubMed

    Caprio, Massimiliano; Fabbrini, Elisa; Ricci, Giulia; Basciani, Sabrina; Gnessi, Lucio; Arizzi, Mario; Carta, Anna R; De Martino, Massimo U; Isidori, Andrea M; Frajese, Giovanni V; Fabbri, Andrea

    2003-04-01

    There are still many controversies about the role of leptin in reproductive function and sexual development. We recently demonstrated that leptin receptors are expressed in rodent Leydig cells and that leptin has inhibitory effects on hCG-stimulated testosterone production by adult rat Leydig cells in culture. In this study, we evaluated the expression of leptin receptor (Ob-R) in rat testes from gestational to adult age in comparison with the pattern of expression of relaxin-like factor (RLF), a specific marker of Leydig cell differentiation status. Immunohistochemical analysis showed that, in prenatal life, Ob-R immunoreactivity was absent at early embryonic ages (E14.5) and appeared at a late embryonic age (E19.5); in postnatal life, immunoreactivity was evident only after sexual maturation (35-, 60-, and 90-days old), whereas it was absent in testes from sexually immature rats (7-, 14-, and 21-days old). Immunoreaction was always confined to Leydig cells and no signal of Ob-R was detected within the tubules. The pattern of expression of Ob-R during testicular development was similar with that of RLF immunoreactivity, which was present in mature fetal as well as adult-type Leydig cells. In contrast with the findings in the testis, in the hypothalamus, the immunohistochemical pattern of Ob-R was very similar between pre- and postpubertal life. Reverse transcription-polymerase chain reaction studies showed that Ob-R expression was present in embryonic, prepubertal, and adult rat testes; semiquantitative analysis showed that mRNA levels were much higher in late versus early embryonic testes, as well as in mature adults versus sexually immature testes, with a gradual increase from younger to older ages. Functional studies showed that, while leptin (150 ng/ml) significantly inhibited hCG-stimulated testosterone production in adult rat Leydig cells (46% reduction; P > 0.01), it did not modify prepubertal rat Leydig cells steroidogenic function in vitro. In conclusion

  5. Effects of hypoxia on testosterone release in rat Leydig cells.

    PubMed

    Hwang, Guey-Shyang; Chen, Szu-Tah; Chen, Te-Jung; Wang, Shyi-Wu

    2009-11-01

    The aim of this study was to explore the effect and action mechanisms of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12% O(2) + 88% N(2), 1.5 l/ml) 8 h/day for 4 days. Normoxic rats were used as control. In an in vivo experiment, hypoxic and normoxic rats were euthanized and the blood samples collected. In the in vitro experiment, the enzymatically dispersed rat Leydig cells were prepared and challenged with forskolin (an adenylyl cyclase activator, 10(-4) M), 8-Br-cAMP (a membrane-permeable analog of cAMP, 10(-4) M), hCG (0.05 IU), the precursors of the biosynthesis testosterone, including 25-OH-C (10(-5) M), pregnenolone (10(-7) M), progesterone (10(-7) M), 17-OH-progesterone (10(-7) M), and androstendione (10(-7)-10(-5) M), nifedipine (L-type Ca(2+) channel blocker, 10(-6)-10(-4) M), nimodipine (L-type Ca(2+) channel blocker, 10(-5) M), tetrandrine (L-type Ca(2+) channel blocker, 10(-5) M), and NAADP (calcium-signaling messenger causing release of calcium from intracellular stores, 10(-6)-10(-4) M). The concentrations of testosterone in plasma and medium were measured by radioimmunoassay. The level of plasma testosterone in hypoxic rats was higher than that in normoxic rats. Enhanced testosterone production was observed in rat Leydig cells treated with hCG, 8-Br-cAMP, or forskolin in both normoxic and hypoxic conditions. Intermittent hypoxia resulted in a further increase of testosterone production in response to the testosterone precursors. The activity of 17β-hydroxysteroid dehydrogenase was stimulated by the treatment of intermittent hypoxia in vitro. The intermittent hypoxia-induced higher production of testosterone was accompanied with the influx of calcium via L-type calcium channel and the increase of intracellular calcium via the mechanism of calcium mobilization. These results suggested that the intermittent hypoxia stimulated the secretion of testosterone at least in

  6. Green tea polyphenols inhibit testosterone production in rat Leydig cells

    PubMed Central

    Figueiroa, Marina S; César Vieira, Juliany S B; Leite, Disleide S; Filho, Ruben C O Andrade; Ferreira, Fabiano; Gouveia, Patrícia S; Udrisar, Daniel P; Wanderley, Maria I

    2009-01-01

    This study investigated the acute effects of green tea extract (GTE) and its polyphenol constituents, (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin (EC), on basal and stimulated testosterone production by rat Leydig cells in vitro. Leydig cells purified in a Percoll gradient were incubated for 3 h with GTE, EGCG or EC and the testosterone precursor androstenedione, in the presence or absence of either protein kinase A (PKA) or protein kinase C (PKC) activators. The reversibility of the effect was studied by pretreating cells for 15 min with GTE or EGCG, allowing them to recover for 1 h and challenging them for 2 h with human chorionic gonadotropin (hCG), luteinizing hormone releasing hormone (LHRH), 22(R)-hydroxycholesterol or androstenedione. GTE and EGCG, but not EC, inhibited both basal and kinase-stimulated testosterone production. Under the pretreatment conditions, the inhibitory effect of the higher concentration of GTE/EGCG on hCG/LHRH-stimulated or 22(R)-hydroxycholesterol-induced testosterone production was maintained, whereas androstenedione-supported testosterone production returned to control levels. At the lower concentration of GTE/EGCG, the inhibitory effect of these polyphenols on 22(R)-hydroxycholesterol-supported testosterone production was reversed. The inhibitory effects of GTE may be explained by the action of its principal component, EGCG, and the presence of a gallate group in its structure seems important for its high efficacy in inhibiting testosterone production. The mechanisms underlying the effects of GTE and EGCG involve the inhibition of the PKA/PKC signalling pathways, as well as the inhibition of P450 side-chain cleavage enzyme and 17β-hydroxysteroid dehydrogenase function. PMID:19330017

  7. Developmental exposures of male rats to soy isoflavones impact Leydig cell differentiation.

    PubMed

    Sherrill, Jessica D; Sparks, Morgan; Dennis, John; Mansour, Mahmoud; Kemppainen, Barbara W; Bartol, Frank F; Morrison, Edward E; Akingbemi, Benson T

    2010-09-01

    Testicular Leydig cells, which are the predominant source of the male sex steroid hormone testosterone, express estrogen receptors (ESRs) and are subject to regulation by estrogen. Following ingestion, the two major isoflavones in soybeans, genistin and daidzin, are hydrolyzed by gut microflora to form genistein and daidzein, which have the capacity to bind ESRs and affect gene expression. Thus, the increasing use of soy-based products as nondairy sources of protein has raised concerns about the potential of these products to cause reproductive toxicity. In the present study, perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells. Isoflavones have the capacity to act directly as mitogens in Leydig cells, because genistein treatment induced Leydig cell division in vitro. Genistein action regulating Leydig cell division involved ESRs, acting in concert with signaling molecules in the transduction pathway mediated by protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). Enhanced proliferative activity in the prepubertal period increased Leydig cell numbers, which alleviated deficits in androgen biosynthesis and/or augmented serum and testicular testosterone concentrations in adulthood. Together, these observations indicate that the perinatal exposures of male rats to isoflavones affected Leydig cell differentiation, and they imply that including soy products in the diets of neonates has potential implications for testis function. PMID:20554919

  8. Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate.

    PubMed

    Molenaar, R; de Rooij, D G; Rommerts, F F; Reuvers, P J; van der Molen, H J

    1985-12-01

    Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats. PMID:3000465

  9. Primary culture of purified Leydig cells isolated from adult rat testes.

    PubMed

    Browning, J Y; Heindel, J J; Grotjan, H E

    1983-02-01

    Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function. PMID:6848362

  10. Leydig cells: From stem cells to aging.

    PubMed

    Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R

    2009-07-10

    Leydig cells are the testosterone-producing cells of the testis. The adult Leydig cell population ultimately develops from undifferentiated mesenchymal-like stem cells present in the interstitial compartment of the neonatal testis. Four distinct stages of adult Leydig cell development have been identified and characterized: stem Leydig cells, progenitor Leydig cells, immature Leydig cells and adult Leydig cells. The stem Leydig cells are undifferentiated cells that are capable of indefinite self-renewal, differentiation, and replenishment of the Leydig cell niche. Progenitor Leydig cells are derived from the stem Leydig cells. These spindle-shaped cells are luteinizing hormone (LH) receptor positive, have high mitotic activity, and produce little testosterone but rather testosterone metabolites. The progenitor Leydig cells give rise to immature Leydig cells which are round, contain large amounts of smooth endoplasmic reticulum, and produce some testosterone but also very high levels of testosterone metabolites. A single division of these cells produces adult Leydig cells, which are terminally differentiated cells that produce high levels of testosterone. As men age, serum testosterone levels decline, and this is associated with alterations in body composition, energy level, muscle strength, physical, sexual and cognitive functions, and mood. In the Brown Norway rat, used extensively as a model for male reproductive aging, age-related reductions in serum testosterone result from significant decline in the ability of aged Leydig cells to produce testosterone in response to LH stimulation. This review describes Leydig cell development and aging. Additionally, the molecular mechanisms by which testosterone synthesis declines with aging are discussed. PMID:19481681

  11. Species-specific mechanism in rat Leydig cell tumorigenesis by procymidone.

    PubMed

    Murakami, M; Hosokawa, S; Yamada, T; Harakawa, M; Ito, M; Koyama, Y; Kimura, J; Yoshitake, A; Yamada, H

    1995-04-01

    To clarify the mechanism of species difference in the induction of testicular interstitial cell tumor (ICT, Leydig cell tumor) between rats and mice, male Sprague-Dawley rats and ICR mice were fed procymidone at dietary concentrations of 700, 2000 or 6000 ppm and 1000, 5000, or 10,000 ppm, respectively, for 3 months. The Leydig cell functions were evaluated by serum testosterone and luteinizing hormone (LH) levels, testosterone levels in the testis, LH levels in the pituitary, the capacity of the testis to respond to gonadotropin stimulation, i.e., the production of testosterone in vitro, and by the testicular binding of labeled human chorionic gonadotropin (hCG). Measurement of testosterone and LH levels in rat serum, the testis, or the pituitary showed that both hormones were enhanced throughout the 3-month treatment period. The hypergonadotropism was associated with the increase of interstitial cell response to hCG in vitro for up to 3 months. As with rats, both serum and pituitary LH were increased in mice at 4 weeks but not at 13 weeks. However, in contrast to rats, no significant increase in testosterone was observed in mice either in vivo or ex vivo during the course of the study. This suggests a difference between the rat and mouse in the response of the Leydig cell to the LH stimulation associated with procymidone administration. These differences in the response of interstitial cells to procymidone may be the basis for the distinct species responses to procymidone-induced Leydig cell tumorigenesis. The sustained response of the Leydig cells to stimulation in the rat results in chronic hyperplasia and subsequent benign tumor formation, while the attenuated response of Leydig cells in the mouse is associated with neither hyperplasia nor neoplasia. PMID:7716766

  12. Modulatory effects of leptin on leydig cell function of normal and hyperleptinemic rats.

    PubMed

    Giovambattista, Andrés; Suescun, María O; Nessralla, Claudio C D L; França, Luiz R; Spinedi, Eduardo; Calandra, Ricardo S

    2003-11-01

    Neonatal L-monosodium glutamate (MSG) administration in rats induces several neuroendocrine and metabolic disruptions. Leptin, the adipocyte product, modulates several neuroendocrine systems including the hypothalamic-pituitary-gonadal (HPG) axis in mammals. The aim of the present study was to determine whether MSG-induced chronic hyperleptinemia could play any relevant role in the hypogonadism developed by male rats when examined in adulthood. We found that 120-day-old MSG male rats displayed significant hyperleptinemia, hypogonadism, and undisturbed basic testis structure and spermatogenesis. In vitro studies in purified Leydig cells from normal (CTR) and MSG-damaged rats revealed that basal and human chorionic gonadotropin (hCG)-stimulated 17-hydroxy-progesterone (17-HO-P(4)), Delta(4)-androstenedione (Delta(4)A) and testosterone (T) secretions were significantly lower in MSG than in CTR cells. Exposure to murine leptin (Mleptin, 10(-8)M) significantly inhibited hCG-elicited T secretion by CTR cells after 180 min incubation. While Mleptin significantly inhibited hCG-stimulated Delta(4)A output and the Delta(4)A:17-OH-P(4) ratio of secretion, conversely, it failed to modify the ratio T:Delta(4)A release by CTR Leydig cells. Interestingly, the effects of Mleptin found on CTR Leydig cells were absent in MSG Leydig cells. Finally, endogenous hyperleptinemia was associated with a significant decrease in Leydig cell expression of Ob-Rb mRNA in MSG rats. In summary, this study demonstrates that: (1) Mleptin inhibited testicular steroidogenesis in CTR rats; (2) MSG-treated rats showed lower in vitro 17-OH-P(4), Delta(4)A and T production under basal and post-hCG stimulation conditions; (3) purified Leydig cells from MSG-treated rats displayed resistance to the inhibitory action of Mleptin on T release, and (4) endogenous leptin exerts a modulatory effect on Leydig cell Ob-Rb mRNA expression. The inhibitory effect of leptin on testicular function is thus abrogated in MSG

  13. The mechanism for lindane-induced inhibition of steroidogenesis in cultured rat Leydig cells.

    PubMed

    Ronco, A M; Valdés, K; Marcus, D; Llanos, M

    2001-02-21

    The in vitro effect of the gamma-isomer of hexachlorocyclohexane, lindane, on rat Leydig cell steroidogenesis was studied. Leydig cells from mature male rats were incubated with human chorionic gonadotropin (hCG, 1 IU) for 3 h at 34 degrees C in the presence of different doses of lindane (2-200 microg/ml; 2-200 ppm). Results demonstrate that lindane produces a dose-dependent inhibition of testosterone production in hCG-stimulated Leydig cells. The decreased testosterone synthesis was accompanied with a half-reduced LH/hCG receptor number without any modification in the K(d) value. In addition, lindane also decreased cAMP production. These effects were not due to a detrimental action of lindane on cell viability. Results of this study demonstrate a direct inhibitory action of lindane on testicular steroidogenesis, at least in part, through a reduction in the classical second messenger production involved in this pathway. PMID:11250058

  14. MAINTENANCE OF TESTOSTERONE PRODUCTION BY PURIFIED ADULT RAT LEYDIG CELLS FOR THREE DAYS IN VITRO

    EPA Science Inventory

    Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which we found to optimize testosterone production during 24 h, we sought to maintain optimal testosterone production for 3 d. eydig cells cultured on Cytodex 3 beads at 19% O2 in Dulbecco's ...

  15. Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide.

    PubMed Central

    Janszen, F H; Cooke, B A; van der Molen, H J

    1977-01-01

    The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as 'protein 21"). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as "protein 33"), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed. Images PLATE 4 PLATE 1 PLATE 2 PLATE 3 PMID:849289

  16. MODULATION OF RAT LEYDIG CELL STEROIDOGENIC FUNCTION BY DI(2-ETHYLHEXYL)PHTHALATE

    EPA Science Inventory

    Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate.

    Akingbemi BT, Youker RT, Sottas CM, Ge R, Katz E, Klinefelter GR, Zirkin BR, Hardy MP.

    Center for Biomedical Research, Population Council, New York, New York 10021, USA. benson@popcbr...

  17. Cadmium-induced damage to primary cultures of rat Leydig cells.

    PubMed

    Yang, Jian-Ming; Arnush, Marc; Chen, Qiong-Yu; Wu, Xiang-Dong; Pang, Bing; Jiang, Xue-Zhi

    2003-01-01

    The mechanism of testicular toxicity of cadmium is poorly understood. Previous studies focusing on cadmium-related changes in testicular histopathology have implicated testicular blood vessel damage as the main cause of cadmium toxicity. To further explore the toxic effects of cadmium on testis, we isolated and cultured rat Leydig cells, exposed to 10, 20, and 40 microM of cadmium chloride (base doses). After 24 h of exposure, cells and supernatants were harvested to examine cytotoxicity and genotoxicity of cadmium. The results show that both cell viability and concentration of testosterone excretion in primary Leydig cells are significantly lower in cadmium-exposed groups compared to the controls. Changes in testosterone excretion with human chorionic gonadotropin (hCG) stimulation is especially profound. The contents of malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px) in exposed groups are significantly higher than those in the control group, but the activity of superoxide dismutase (SOD) is lower. The number of cells with DNA single strand breaks and the levels of cellular DNA damage in all three exposure groups are significantly higher than in controls. These results indicate that cadmium is directly toxic to primary Leydig cells, and that the decreased percentage of normal cells and the increased level of DNA damage in cadmium-exposed Leydig cells may be responsible for decreased testosterone secretion. PMID:14555193

  18. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells

    PubMed Central

    LIU, LIN; WANG, DIAN; LI, LONGLONG; DING, XIAO; MA, HAITIAN

    2016-01-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  19. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells.

    PubMed

    Liu, Lin; Wang, Dian; Li, Longlong; Ding, Xiao; Ma, Haitian

    2016-07-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti‑aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose‑dependent manner, whereas it improved cell viability in a time‑dependent and dose‑dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  20. The prenylflavonoid phytoestrogens 8-prenylnaringenin and isoxanthohumol diferentially suppress steroidogenesis in rat Leydig cells in ontogenesis.

    PubMed

    Izzo, Gaia; Söder, Olle; Svechnikov, Konstantin

    2011-08-01

    8-Prenylnaringenin and isoxanthohumol are prenylflavonoids found in the hop plant, Humulus lupulus (Cannabaceae), which is traditionally used to add bitterness and flavor to beer. Flavonoids have previously been reported to exert endocrine disrupting actions. Therefore, we investigated the effects of 8-prenylnaringenin and isoxanthohumol on steroidogenesis activated by human chorionic gonadotropin (hCG) in primary cultures of rat Leydig cells at different stages of their development. The present study is the first to demonstrate that the prenylflavonoids 8-prenylnaringenin and isoxanthohumol exert complex maturation-dependent effects on Leydig cell steroidogenesis. Those compounds inhibited hCG-stimulated androgen production by Leydig cells at all stages of their development, a process that was associated with the reduced ability of the cells to produce cAMP. However, these same compounds up-regulated hCG-activated StAR expression in progenitor (PLC) and immature (ILC) but not adult types of Leydig cells (ALC). Further, 8-prenylnaringenin and isoxanthohumol were not able to suppress androgen production activated by an exogenous analog of cAMP, (Bu)2 cAMP, in ALC and ILC but synergistically stimulated steroidogenesis in PLC. Our data suggest that 8-prenylnaringenin and isoxanthohumol affect cAMP-dependent cellular processes up-stream transport of cholesterol into mitochondria. PMID:21061451

  1. Zinc-induced survival of Leydig cells in Fischer rats (Rattus norvegicus) treated with cadmium chloride.

    PubMed

    Villanueva, Octavio; Vigueras, Rosa María; Hernández, Rafael; Chavira, Roberto; Cárdenas, Mario; Villa, Antonio; Murphy, Eduardo

    2005-12-01

    Zinc is known to prevent cadmium-induced carcinogenesis and Leydig cell destruction in rat testes; however, the mechanism of action is not known, although it has been suggested that pituitary feedback increases the production of luteinizing hormone (LH) in response to low circulating androgen. We therefore examined the biological role of zinc in reducing cadmium toxicity in the Leydig cells of Fischer rats. Two groups of eleven 6-month-old rats were injected subcutaneously with 20 micromol CdCl2/kg weekly for 5 weeks; one of these groups also received 1 mmol/kg zinc acetate weekly for the same 5 weeks. A third group of rats received 1 mmol/kg zinc acetate weekly, and a fourth group was injected with saline weekly for 5 weeks. After 8 months of study, the animals were euthanized by CO2 inhalation. The results indicated that the number of surviving Leydig cells was significantly lower in the cadmium group (7.34% = 0.095 x 10(9)/cm3) than in the cadmium-zinc group (20.85%) or control animals (91.2%). Moreover, the concentrations of serum testosterone and LH were significantly higher in the cadmium group than in any of the other groups. This difference probably was due to the testosterone produced by a small reservoir of surviving Leydig cells and to other endocrine factors. These findings suggest that Fischer rat testis may be a good model system for testing the effects of cadmium and zinc on the production of LH and testosterone and other androgens before spontaneous cancers develop. PMID:16422150

  2. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1989-12-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.

  3. Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway.

    PubMed

    Jing, Jun; Chen, Li; Fu, Hai-Yan; Fan, Kai; Yao, Qi; Ge, Yi-Feng; Lu, Jin-Chun; Yao, Bing

    2015-01-01

    This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway. PMID:25807302

  4. Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes.

    PubMed

    Li, Xiaoheng; Wang, Zhao; Jiang, Zhenming; Guo, Jingjing; Zhang, Yuxi; Li, Chenhao; Chung, Jinyong; Folmer, Janet; Liu, June; Lian, Qingquan; Ge, Renshan; Zirkin, Barry R; Chen, Haolin

    2016-03-01

    Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry. PMID:26929346

  5. AB250. Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway

    PubMed Central

    Yao, Bin

    2016-01-01

    Background This study investigated the effect of annexin V on the proliferation of primary rat Leydig cells and the potential mechanism. Methods The primary rat Leydig cells were cultured in vitro and treated with 1 nmol/L annexin 5 and with siRNA–Ect2 transfection. The cell proliferation rate was measured by MTT assay. Phase distribution of cell cycle was analyzed by flow cytometry. The expression of Ect2 in protein level were detected by western blotting. RhoA activity was measured by Rho activation assay kit. Results Our results showed that annexin V promoted rat Leydig cell proliferation and cell cycle progression in a dose- and time-dependent manner. Increased level of annexin V also enhanced Ect2 protein expression. However, siRNA knockdown of Ect2 attenuated annexin V-induced proliferation of rat Leydig cells. Taken together, these data suggest that increased level of annexin V induced rat Leydig cell proliferation and cell cycle progression via Ect2. Since RhoA activity was increased following Ect2 activation, we further investigated whether Ect2 was involved in annexin V-induced proliferation via the RhoA/ROCK pathway, and the results showed that annexin V increased RhoA activity too, and this effect was abolished by the knockdown of Ect2. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated annexin V-induced proliferation and cell cycle progression. Conclusions We thus conclude that Ect2 is involved in annexin V-induced rat Leydig cell proliferation through the RhoA/ROCK pathway.

  6. Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells

    PubMed Central

    Liu, Hua-Cheng; Zhu, Danyan; Wang, Chan; Guan, Hongguo; Li, Senlin; Hu, Cong; Chen, Zhichuan; Hu, Yuanyuan; Lin, Han; Lian, Qing-Quan; Ge, Ren-Shan

    2015-01-01

    Background Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Methodology Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. Results and Conclusions In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of

  7. Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

    SciTech Connect

    Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

    1986-05-01

    The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions and subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.

  8. Advanced glycation end products inhibit testosterone secretion by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

    PubMed

    Zhao, Yun-Tao; Qi, Ya-Wei; Hu, Chuan-Yin; Chen, Shao-Hong; Liu, You

    2016-08-01

    Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products (AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs (25, 50, 100 and 200 µg/ml). Testosterone production induced by human chorionic gonadotropin (hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitative PCR and western blot analyssi, respectively. Reactive oxygen species (ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate (DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins [C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control (P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control (P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine (NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and

  9. Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells

    PubMed Central

    Li, Xiaoheng; Chen, Xiaomin; Hu, Guoxin; Li, Linxi; Su, Huina; Wang, Yiyan; Chen, Dongxin; Zhu, Qiqi; Li, Chao; Li, Junwei; Wang, Mingcang; Lian, Qingquan; Ge, Ren-Shan

    2016-01-01

    Dicyclohexyl phthalate (DCHP) is one of the phthalate plasticizers. The objective of the present study was to investigate the effects of DCHP on fetal Leydig cell distribution and function as well as testis development. Female pregnant Sprague Dawley dams orally received vehicle (corn oil, control) or DCHP (10, 100, and 500 mg/kg/day) from gestational day (GD) 12 to GD 21. At GD 21.5, testicular testosterone production, fetal Leydig cell number and distribution, testicular gene and protein expression levels were examined. DCHP administration produced a dose-dependent increase of the incidence of multinucleated gonocytes at ≥100 mg/kg. DCHP dose-dependently increased abnormal fetal Leydig cell aggregation and decreased fetal Leydig cell size, cytoplasmic size, and nuclear size at ≥10 mg/kg. DCHP reduced the expression levels of steroidogenesis-related genes (including Star, Hsd3b1, and Hsd17b3) and testis-descent related gene Insl3 as well as protein levels of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and insulin-like 3 (INSL3) at ≥10 mg/kg. DCHP significantly inhibited testicular testosterone levels at ≥100 mg/kg. The results indicate that in utero exposure to DCHP affects the expression levels of fetal Leydig cell steroidogenic genes and results in the occurrence of multinucleated gonocytes and Leydig cell aggregation. PMID:26907321

  10. Protective Effect of Adrenomedullin on Rat Leydig Cells from Lipopolysaccharide-Induced Inflammation and Apoptosis via the PI3K/Akt Signaling Pathway ADM on Rat Leydig Cells from Inflammation and Apoptosis

    PubMed Central

    Zhou, Pang-Hu; Hu, Wei; Zhang, Xiao-Bin; Wang, Wei; Zhang, Li-Jun

    2016-01-01

    This study was carried out to investigate whether ADM can modulate LPS-induced inflammation and apoptosis in rat Leydig cells. Leydig cells were treated with ADM before LPS-induced cytotoxicity. We determined the concentrations of ROS, MDA, GSH, LDH, and testosterone and the MMP. The mRNA levels of IL-1, IL-6, iNOS, and COX-2 were obtained, and the concentrations of IL-1, IL-6, NO, and PGE2 were determined. Apoptosis was assessed by TUNEL and detection of DNA fragmentation. The levels of mRNA and protein were determined for Bcl-2, Bax, caspase-3, and PARP. The protein contents for total and p-Akt were measured. ADM pretreatment significantly elevated the MMP and testosterone concentration and reduced the levels of ROS, MDA, GSH, and LDH. ADM pretreatment significantly decreased the mRNA levels of IL-1, IL-6, iNOS, and COX-2 and the concentrations of IL-1, IL-6, NO, and PGE2. LPS-induced TUNEL-positive Leydig cells were significantly decreased by ADM pretreatment, a result further confirmed by decreased DNA fragmentation. ADM pretreatment decreased apoptosis by significantly promoting Bcl-2 and inhibiting Bax, caspase-3, and PARP expressions. The LPS activity that reduced p-Akt level was significantly inhibited by ADM pretreatment. ADM protected rat Leydig cells from LPS-induced inflammation and apoptosis, which might be associated with PI3K/Akt mitochondrial signaling pathway. PMID:27212810

  11. Protective Effect of Adrenomedullin on Rat Leydig Cells from Lipopolysaccharide-Induced Inflammation and Apoptosis via the PI3K/Akt Signaling Pathway ADM on Rat Leydig Cells from Inflammation and Apoptosis.

    PubMed

    Zhou, Pang-Hu; Hu, Wei; Zhang, Xiao-Bin; Wang, Wei; Zhang, Li-Jun

    2016-01-01

    This study was carried out to investigate whether ADM can modulate LPS-induced inflammation and apoptosis in rat Leydig cells. Leydig cells were treated with ADM before LPS-induced cytotoxicity. We determined the concentrations of ROS, MDA, GSH, LDH, and testosterone and the MMP. The mRNA levels of IL-1, IL-6, iNOS, and COX-2 were obtained, and the concentrations of IL-1, IL-6, NO, and PGE2 were determined. Apoptosis was assessed by TUNEL and detection of DNA fragmentation. The levels of mRNA and protein were determined for Bcl-2, Bax, caspase-3, and PARP. The protein contents for total and p-Akt were measured. ADM pretreatment significantly elevated the MMP and testosterone concentration and reduced the levels of ROS, MDA, GSH, and LDH. ADM pretreatment significantly decreased the mRNA levels of IL-1, IL-6, iNOS, and COX-2 and the concentrations of IL-1, IL-6, NO, and PGE2. LPS-induced TUNEL-positive Leydig cells were significantly decreased by ADM pretreatment, a result further confirmed by decreased DNA fragmentation. ADM pretreatment decreased apoptosis by significantly promoting Bcl-2 and inhibiting Bax, caspase-3, and PARP expressions. The LPS activity that reduced p-Akt level was significantly inhibited by ADM pretreatment. ADM protected rat Leydig cells from LPS-induced inflammation and apoptosis, which might be associated with PI3K/Akt mitochondrial signaling pathway. PMID:27212810

  12. Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: In vivo and ex vivo studies

    PubMed Central

    2010-01-01

    Background The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats. Methods Twelve adult male Wistar rats were treated with rosiglitazone (5 mg/kg) administered by gavage for 15 days. Twelve control animals were treated with the vehicle. The ability of rosiglitazone to directly affect the production of testosterone by Leydig cells ex vivo was evaluated using isolated Leydig cells from rosiglitazone-treated rats. Testosterone production was induced either by activators of the cAMP/PKA pathway (hCG and dbcAMP) or substrates of steroidogenesis [22(R)-hydroxy-cholesterol (22(R)-OH-C), which is a substrate for the P450scc enzyme, and pregnenolone, which is the product of the P450scc-catalyzed step]. Testosterone in plasma and in incubation medium was measured by radioimmunoassay. The StAR and P450scc expression was detected by immunocytochemistry. Results The levels of total circulating testosterone were not altered by rosiglitazone treatment. A decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats. The ultrastructural and immunocytochemical analysis of Leydig cells from rosiglitazone-treated rats revealed cells with characteristics of increased activity as well as increased StAR and P450scc expression, which are key proteins in androgen biosynthesis. However, a number of rosiglitazone-treated cells exhibited significant mitochondrial damage. Conclusion The results revealed that the Leydig cells from rosiglitazone-treated rats showed significant reduction in testosterone production under basal, hCG/dbcAMP- or 22 (R)-OH-C/pregnenolone-induced conditions, although increased labeling of St

  13. Leydig cells contribute to the inhibition of spermatogonial differentiation after irradiation of the rat.

    PubMed

    Shetty, G; Zhou, W; Weng, C C Y; Shao, S H; Meistrich, M L

    2016-05-01

    Irradiation with 6 Gy produces a complete block of spermatogonial differentiation in LBNF1 rats that would be permanent without treatment. Subsequent suppression of gonadotropins and testosterone (T) restores differentiation to the spermatocyte stage; however, this process requires 6 weeks. We evaluated the role of Leydig cells (LCs) in maintenance of the block in spermatogonial differentiation after exposure to radiation by specifically eliminating functional LCs with ethane dimethane sulfonate (EDS). EDS (but not another alkylating agent), given at 10 weeks after irradiation, induced spermatogonial differentiation in 24% of seminiferous tubules 2 weeks later. However, differentiation became blocked again at 4 weeks as LCs recovered. When EDS was followed by treatment with GnRH antagonist and flutamide, sustained spermatogonial differentiation was induced in >70% of tubules within 2 weeks. When EDS was followed by GnRH antagonist plus exogenous T, which also inhibits LC recovery but restores follicle stimulating hormone (FSH) levels, the spermatogonial differentiation was again rapid but transient. These results confirm that the factors that block spermatogonial differentiation are indirectly regulated by T, and probably FSH, and that adult and possibly immature LCs contribute to the production of such inhibitory factors. We tested whether insulin-like 3 (INSL3), a LC-produced protein whose expression correlated with the block in spermatogonial differentiation, was indeed responsible for the block by injecting synthetic INSL3 into the testes and knocking down its expression in vivo with siRNA. Neither treatment had any effect on spermatogonial differentiation. The Leydig cell products that contribute to the inhibition of spermatogonial differentiation in irradiated rats remain to be elucidated. PMID:26991593

  14. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    SciTech Connect

    Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  15. IN VITRO/IN VIVO EFFECTS OF ETHANE DIMETHANESULPHONATE ON LEYDIG CELLS OF ADULT RATS

    EPA Science Inventory

    Ethane dimethanesulphonate (EDS) is studied extensively to date, certain toxicological criteria have not been met. or instance, the does-responsiveness of Leydig cells to EDS, both in vitro and in vivo, is not well established. n addition, the date regarding the cellular site of ...

  16. Time-Course Changes of Steroidogenic Gene Expression and Steroidogenesis of Rat Leydig Cells after Acute Immobilization Stress

    PubMed Central

    Lin, Han; Yuan, Kai-ming; Zhou, Hong-yu; Bu, Tiao; Su, Huina; Liu, Shiwen; Zhu, Qiqi; Wang, Yiyan; Hu, Yuanyuan; Shan, Yuanyuan; Lian, Qing-quan; Wu, Xiao-yun; Ge, Ren-shan

    2014-01-01

    Leydig cells secrete testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid (corticosterone, CORT; in rats), which decreases circulating testosterone levels in part through a direct action by binding to the glucocorticoid receptors (NR3C1) in Leydig cells. The intratesticular CORT level is dependent on oxidative inactivation of glucocorticoid by 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) in Leydig cells. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute immobilization stress in rats. The plasma CORT levels were significantly increased 0.5, 1, 3 and 6 h after immobilization stress, while plasma testosterone levels were significantly reduced 3 and 6 h, after stress and luteinizing hormone (LH) did not change. Immobilization stress caused the down-regulation of Scarb1, Star and Cyp17a1 expression levels in the rat testis starting at the first hour of stress, ahead of the significant decreases of plasma testosterone levels. Other mRNA levels, including Cyp11a1, Hsd3b1 and Hsd17b3, began to decline after 3 h. Hsd11b1 and Nos2 mRNA levels did not change during the course of stress. Administration of glucocorticoid antagonist RU486 significantly restored plasma testosterone levels. In conclusion, Scarb1, Star and Cyp17a1 expression levels are more sensitive to acute stress, and acute immobilization stress causes the decline of the steroidogenic pathway via elevating the levels of glucocorticoid, which binds to NR3C1 in Leydig cells to inhibit steroidogenic gene expression. PMID:25405735

  17. Leydig cell tumours in childhood.

    PubMed

    Mengel, W; Knorr, D

    1983-01-01

    Two cases of Leydig cell tumours in childhood are presented. In one case, delayed diagnosis and operation led to pubertas praecox vera whereas in the other case normal growth and development occurred after early diagnosis and operation. PMID:6878724

  18. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    SciTech Connect

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  19. Anabolic-androgenic steroids induce apoptosis and NOS2 (nitric-oxide synthase 2) in adult rat Leydig cells following in vivo exposure.

    PubMed

    Janjic, Marija M; Stojkov, Natasa J; Andric, Silvana A; Kostic, Tatjana S

    2012-12-01

    Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone (T) predominantly taken as drugs of abuse. Using in vivo treatment of adult male rats we investigated the effects of testosterone enanthate (TE) a widely abused AAS, on apoptosis of Leydig cells. Increased T and decreased luteinizing hormone levels in serum and decreased intra-testicular T values were found in 2 and 10 weeks treated groups. Two weeks of TE-treatment stimulated the expression of inducible nitric oxide synthase (NOS2) followed by increased NO production, decreased mitochondrial membrane potential and increased prevalence of Leydig cell apoptosis. This was prevented by in vivo administration of androgen receptor blocker. The induced NOS2 level and apoptosis returned to control levels after 10 weeks of TE-treatment but testes contained fewer Leydig cells. Overall, AAS in addition to reduced steroidogenesis induce transient increase of Leydig cells apoptotic rate through mechanism associated with androgen receptor, most likely involving NOS2 induction. PMID:23085480

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

    PubMed

    Ohta, Y; Yoshida, K; Kamiya, S; Kawate, N; Takahashi, M; Inaba, T; Hatoya, S; Morii, H; Takahashi, K; Ito, M; Ogawa, H; Tamada, H

    2016-04-01

    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. PMID:26174043

  1. Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells.

    PubMed

    Li, Linxi; Chen, Xiaomin; Hu, Guoxin; Wang, Sicong; Xu, Renai; Zhu, Qiqi; Li, Xiaoheng; Wang, Mingcang; Lian, Qing-Quan; Ge, Ren-Shan

    2016-01-01

    Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05-50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulated Cyp11a1 and Hsd3b1 expression levels at 50 nM. However, 50 μM DBP downregulated Star, Hsd3b1, and Hsd17b3 expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes. PMID:27148549

  2. Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells

    PubMed Central

    Li, Linxi; Chen, Xiaomin; Hu, Guoxin; Wang, Sicong; Xu, Renai; Zhu, Qiqi; Li, Xiaoheng; Wang, Mingcang; Lian, Qing-Quan; Ge, Ren-Shan

    2016-01-01

    Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05–50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulated Cyp11a1 and Hsd3b1 expression levels at 50 nM. However, 50 μM DBP downregulated Star, Hsd3b1, and Hsd17b3 expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes. PMID:27148549

  3. Steroid metabolism by purified adult rat Leydig cells in primary culture

    SciTech Connect

    Browning, J.Y.; Tcholakian, R.K.; Kessler, M.J.; Grotjan, H.E. Jr.

    1982-11-01

    To characterize Leydig cell steroidogensis, we examined the metabolism of (3H)pregnenolone (3 beta-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20-30% of the (3H)pregnenolone was converted to testosterone (17 beta-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3,20-dione) were isolated. The delta 5 intermediates, 17-hydroxypregnenolone (3 beta, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (3H)pregnenolone via the delta 4 pathway. On day 0 of culture, unidentified metabolites considered of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (3H-pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3,20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C17-20 desmolase activity or that hCG acutely stimulates 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 5 isomerase activities.

  4. Steroidogenesis in amlodipine treated purified Leydig cells

    SciTech Connect

    Latif, Rabia; Lodhi, Ghulam Mustafa; Hameed, Waqas; Aslam, Muhammad

    2012-01-01

    Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

  5. Exposure to phytoestrogens in the perinatal period affects androgen secretion by testicular Leydig cells in the adult rat.

    PubMed

    Akingbemi, Benson T; Braden, Tim D; Kemppainen, Barbara W; Hancock, Karen D; Sherrill, Jessica D; Cook, Sarah J; He, Xiaoying; Supko, Jeffrey G

    2007-09-01

    The use of soy-based products in the diet of infants has raised concerns regarding the reproductive toxicity of genistein and daidzein, the predominant isoflavones in soybeans with estrogenic activity. Time-bred Long-Evans dams were fed diets containing 0, 5, 50, 500, or 1000 ppm of soy isoflavones from gestational d 12 until weaning at d 21 postpartum. Male rats in all groups were fed soy-free diets from postnatal d 21 until 90 d of age. The mean +/- SD concentration of unconjugated (i.e. biologically active) genistein and daidzein in serum from the group of dams maintained on the diet containing the highest amount of isoflavones (1000 ppm) were 17 +/- 27 and 56 +/- 30 nM, respectively, at d 21 postpartum. The concentrations were considerably greater in male offspring (genistein: 73 +/- 46 nM; daidzein: 106 +/- 53 nM). Although steroidogenesis was decreased in individual Leydig cells, male rats from the highest exposure group (1000 ppm diet) exhibited elevated serum levels of the sex steroid hormones androsterone at 21 d (control: 15 +/- 1.5 vs.28 +/- 3.5 ng/ml; P < 0.05) and testosterone at 90 d of age (control: 7.5 +/- 1 vs.17 +/- 2 ng/ml; P < 0.05). Testosterone secretion by immature Leydig cells, isolated from 35-d-old male rats, decreased on exposure to 0.1 nm genistein in vitro (control: 175 +/- 5 vs. 117 +/- 3 ng/10(6) cells per 24 h; P < 0.05), indicative of direct phytoestrogen action. Thus, phytoestrogens have the ability to regulate Leydig cells, and additional studies to assess potential adverse effects of dietary soy-based products on reproductive tract development in neonates are warranted. PMID:17569756

  6. Potassium and chloride conductances in rat Leydig cells: effects of gonadotrophins and cyclic adenosine monophosphate.

    PubMed Central

    Duchatelle, P; Joffre, M

    1990-01-01

    1. The effects of gonadotrophins (luteinizing hormone and human chorionic gonadotrophin) and cyclic AMP on ionic conductances were investigated using the tight-seal whole-cell recording technique in Leydig cells freshly isolated from nature rat testis by enzymatic treatment. 2. In resting cells, the predominant ionic conductance is a voltage-dependent K+ conductance resembling the delayed rectifier K+ conductance of T-lymphocytes. This conductance is characterized by: (1) a time-dependent inactivation for potentials more positive than +20 mV, (2) a reversal potential near -65 mV, (3) a sensitivity to intracellular Cs+, and (4) a sensitivity to extracellular TEA and 4-aminopyridine. 3. A Cl- conductance is also present resembling the Cl- background conductance in squid axons and heart cells. In resting cells, this conductance contributes only a small component of the total outward current obtained with depolarizing pulses. 4. Gonadotrophins (human chorionic gonadotrophin, porcine luteinizing hormone and ovine luteinizing hormone) have little effect on the K+ conductance. They transiently increase a Cl- conductance after a delay of up to 30 s. This response does not occur if the hormones are applied late in the whole-cell recording. Gonadoliberine (GnRH) does not affect the Cl- or K+ conductance. 5. Internal cyclic AMP (100 microM) mimics all these effects while internal application of a GTP-ATP mixture induces a similar response, which is, however, sustained rather than transient. 6. The Cl- conductance was studied quantitatively with a GTP-ATP internal solution. This conductance is activated by depolarizing voltage steps to test potentials of -40 mV or more. Under these conditions, the instantaneous current observed as soon as the depolarizing pulse is applied displays outward rectification and reverses near ECl. During the pulses, a strong inactivation is observed for potentials greater than +40 mV. This conductance is independent of external and internal calcium

  7. Leydig cell aging and hypogonadism.

    PubMed

    Beattie, M C; Adekola, L; Papadopoulos, V; Chen, H; Zirkin, B R

    2015-08-01

    Leydig cell testosterone (T) production is reduced with age, resulting in reduced serum T levels (hypogonadism). A number of cellular changes have been identified in the steroidogenic pathway of aged Leydig cells that are associated with reduced T formation, including reductions in luteinizing hormone (LH)-stimulated cAMP production, the cholesterol transport proteins steroidogenic acute regulatory (STAR) protein and translocator protein (TSPO), and downstream steroidogenic enzymes of the mitochondria and smooth endoplasmic reticulum. Many of the changes in steroid formation that characterize aged Leydig cells can be elicited by the experimental alteration of the redox environment of young cells, suggesting that changes in the intracellular redox balance may cause reduced T production. Hypogonadism is estimated to affect about 5 million American men, including both aged and young. This condition has been linked to mood changes, worsening cognition, fatigue, depression, decreased lean body mass, reduced bone mineral density, increased visceral fat, metabolic syndrome, decreased libido, and sexual dysfunction. Exogenous T administration is now used widely to elevate serum T levels in hypogonadal men and thus to treat symptoms of hypogonadism. However, recent evidence suggests that men who take exogenous T may face increased risk of stroke, heart attack, and prostate tumorigenesis. Moreover, it is well established that administered T can have suppressive effects on LH, resulting in lower Leydig cell T production, reduced intratesticular T concentration, and reduced spermatogenesis. This makes exogenous T administration inappropriate for men who wish to father children. There are promising new approaches to increase serum T by directly stimulating Leydig cell T production rather than by exogenous T therapy, thus potentially avoiding some of its negative consequences. PMID:25700847

  8. Lactogen receptors in rat Leydig cells: analysis of their structure with bifunctional cross-linking reagents

    SciTech Connect

    Bonifacino, J.S.; Dufau, M.L.

    1985-04-01

    (/sup 125/I)Iodohuman GH was found to bind to receptors with specificity for lactogenic hormones in a Triton X-100 extract from Leydig cell membranes displaying an affinity constant of 3.8 X 10(/sup 9/) M-1 and a binding capacity of 167 fmol/mg protein. Cross-linking of solubilized (/sup 125/I)iodohuman GH-receptor complexes with disuccinimidyl suberate followed by analysis by sodium dodecyl sulfate-gel electrophoresis in the presence of beta-mercaptoethanol and autoradiography resulted in the appearance of bands with apparent mol wt of 113,000, 103,000, 59,000, and 53,000. The appearance of these bands was prevented by incubation in the presence of lactogenic hormones. By using a two-dimensional electrophoresis technique (first dimension under nonreducing conditions; second dimension under reducing conditions), it was demonstrated that a fraction of the mol wt 59,000 species can be released from the mol wt 103,000 species upon cleavage of disulfide bonds. These results suggest the existence of lactogen receptor species with approximate mol wt of 91,000, 81,000, 37,000, and 31,000 in Triton X-100 extracts from Leydig cell membranes if the contribution of the free hormone (mol wt, 22,000) is subtracted. A fraction of the mol wt 37,000 subunits appears to be contained within the 81,000 species linked through disulfide bonds.

  9. PURIFICATION OF RAT LEYDIG CELLS: INCREASED YIELDS AFTER UNIT-GRAVITY SEDIMENTATION OF COLLAGENASE-DISPERSED INTERSTITIAL CELLS

    EPA Science Inventory

    Abstract

    Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure, that includes a filtration with nylon mesh (100 micron pore size) to separate interstitial cells from the seminiferous tubules, combining centr...

  10. Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

    SciTech Connect

    Didolkar, A.K.; Sundaram, K.

    1987-07-27

    Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to insositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA) could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA augmented hCG induced T secretion, while at higher concentrations they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5,8,11,14 Eicosatetrayenoic acid, a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. The authors conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclo-oxygenase products. 28 references, 4 figures, 2 tables.

  11. Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1)

    SciTech Connect

    Kellokumpu, S.

    1987-02-01

    The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with (/sup 125/I)hCG indicated that the bound hormone was lost much more rapidly from the tumor cells than from the luteal cells. The tumor cells were also found to internalize and degrade the hormone more effectively than the luteal cells. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-(/sup 125/I)hCG complexes (M/sub r/ 96,000 and 74,000) into the medium, although their amount was negligible in MLTC-1 cells. Possibly due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the M/sub r/ 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalized receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumor cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.

  12. Adrenomedullin attenuates interleukin-1β-induced inflammation and apoptosis in rat Leydig cells via inhibition of NF-κB signaling pathway.

    PubMed

    Hu, Wei; Zhou, Pang-hu; Rao, Ting; Zhang, Xiao-bin; Wang, Wei; Zhang, Li-jun

    2015-12-10

    The aim of this paper is to investigate the protective effects of adrenomedullin (ADM) on interleukin-1β (IL-1β)-induced inflammation and apoptosis in rat Leydig cells and its underlying molecular mechanisms. Leydig cells were isolated from adult Sprague-Dawley rats. The cell culture was established by adding ADM 2h prior to 24h treatment with IL-1β-induced cytotoxicity. We detected cell viability and concentrations of testosterone, reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH). Gene expression levels were measured for inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). Concentrations were detected for nitric oxide (NO) and prostaglandin E2 (PGE2). Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Levels of gene expression and protein were detected for Bcl-2, Bax, caspase-3, and poly adenosine diphosphate-ribose polymerase (PARP). Protein levels were measured for nuclear factor kappa B (NF-κB) p65 and IκBα. ADM reduced IL-1β-induced cytotoxicity. ADM pretreatment significantly increased testosterone concentrations and decreased ROS, MDA, and GSH concentrations. ADM pretreatment inhibited IL-1β-induced inflammation in Leydig cells by decreasing the gene expression levels of iNOS and COX-2, as well as the concentrations of NO and PGE2. ADM pretreatment further decreased the number of TUNEL-positive stained Leydig cells, as confirmed by the increase in gene expression and protein levels of Bcl-2 and the decrease of Bax, caspase-3, and PARP levels. Moreover, ADM pretreatment inhibited NF-κB p65 phosphorylation and IκBα phosphorylation and degradation. ADM has potential anti-inflammatory and anti-apoptotic properties in IL-1β-induced rat Leydig cells, which might be related to NF-κB signaling pathway. PMID:26511504

  13. Response to the Svingen Comments on Li et al. Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells. Int. J. Environ. Res. Public Health, 2016, 13, 246.

    PubMed

    Li, Xiaoheng; Chen, Xiaomin; Hu, Guoxin; Li, Linxi; Su, Huina; Wang, Yiyan; Chen, Dongxin; Zhu, Qiqi; Li, Chao; Li, Junwei; Wang, Mingcang; Lian, Qingquan; Ge, Ren-Shan

    2016-01-01

    Referring to the comments of Svingen [1] on our latest publication about Effects of in utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells [2], we would like to give some comments.[...]. PMID:27231929

  14. The functional development of Leydig cells in a marsupial.

    PubMed

    Butler, Christopher M; Shaw, Geoff; Clark, Joan; Renfree, Marilyn B

    2008-01-01

    Leydig cells are the major source of androgen in the male mammal. We describe here for the first time the development of the Leydig cell in a macropodid marsupial, the tammar wallaby, Macropus eugenii. Leydig cells are first recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells. Lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in the tammar testis. Morphologically mature Leydig cells, marked by distinct mitochondria with tubular cristae and an extensive anastomosing network of smooth endoplasmic reticulum, are developed by day 10 after birth - the time of peak testosterone content in perinatal tammar testes. The volume percentage of each cell type in the testis does not change over time so the growth of each cellular component keeps pace with growth of the whole testis. There was no morphological or quantitative evidence of a change from one population of Leydig cells to another in the tammar testis as has been reported in several other species including the rat, mouse and human. Maturation of the testis is also marked by the development of tight junctions between the cell membranes of adjacent Sertoli cells. These appear around day 30 after birth and coincide with the onset of mitotic arrest in male germ cells. Overall, the development of the Leydig cell in the tammar wallaby follows a similar pattern to that seen in other mammals, although the start of Leydig cell differentiation is, like many other organ systems in marsupials, post natal, not fetal and there appears to be only a single population of Leydig cells. PMID:18069991

  15. The functional development of Leydig cells in a marsupial

    PubMed Central

    Butler, Christopher M; Shaw, Geoff; Clark, Joan; Renfree, Marilyn B

    2008-01-01

    Leydig cells are the major source of androgen in the male mammal. We describe here for the first time the development of the Leydig cell in a macropodid marsupial, the tammar wallaby, Macropus eugenii. Leydig cells are first recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells. Lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in the tammar testis. Morphologically mature Leydig cells, marked by distinct mitochondria with tubular cristae and an extensive anastomosing network of smooth endoplasmic reticulum, are developed by day 10 after birth – the time of peak testosterone content in perinatal tammar testes. The volume percentage of each cell type in the testis does not change over time so the growth of each cellular component keeps pace with growth of the whole testis. There was no morphological or quantitative evidence of a change from one population of Leydig cells to another in the tammar testis as has been reported in several other species including the rat, mouse and human. Maturation of the testis is also marked by the development of tight junctions between the cell membranes of adjacent Sertoli cells. These appear around day 30 after birth and coincide with the onset of mitotic arrest in male germ cells. Overall, the development of the Leydig cell in the tammar wallaby follows a similar pattern to that seen in other mammals, although the start of Leydig cell differentiation is, like many other organ systems in marsupials, post natal, not fetal and there appears to be only a single population of Leydig cells. PMID:18069991

  16. Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate

    PubMed Central

    Wakui, Shin; Motohashi, Masaya; Satoh, Takemi; Shirai, Masaru; Mutou, Tomoko; Takahashi, Hiroyuki; Wempe, Michael F.; Endou, Hitoshi; Inomata, Tomoo; Asari, Masao

    2013-01-01

    We recently reported that prenatal rat exposure to di(n-butyl) phthalate (DBP) induced Leydig cell (LC) hyperplasia after nine weeks (wks) of age, yet the number of LCs was similar to that of the vehicle group until seven weeks. Nuclear pleomorphism of hyperplastic LCs is common and is considered to be continuous progressive degeneration. Thus, computer-assisted image cell nuclear analysis of LCs was performed on 5- and 7-wk-old Sprague-Dawley (SD) rats whose dams had been administered DBP (i.g.) at 100 mg/kg/day or vehicle (corn oil) on gestation day 12 to 21. The results of the 5-wk-old DBP group were similar to those of the vehicle group; LC nuclei of the 7-wk-old DBP group showed normal ploidy and similar amounts of DNA. However, the size, elongation and peripheral chromatin aggregation parameters were significantly higher, and the reticular chromatin distribution and isolated chromatin aggregation parameters were significantly lower compared with the vehicle group. The present study quantitatively demonstrated nuclear morphological alterations in rat LCs at 7 wks old (puberty) due to the prenatal DBP administration before apparent LC hyperplasia developed. PMID:24526819

  17. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  18. Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

    PubMed Central

    Cooke, B A; Janszen, F H; Clotscher, W F; van der Molen, H J

    1975-01-01

    Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis. PMID:174545

  19. Genetics Home Reference: Leydig cell hypoplasia

    MedlinePlus

    ... triggers these cells to produce androgens. Androgens, including testosterone, are the hormones that control male sexual development ... or absent Leydig cells and impaired production of testosterone. A lack of testosterone interferes with the development ...

  20. Regulation of NGFI-B/Nur77 gene expression in the rat ovary and in leydig tumor cells MA-10.

    PubMed

    Inaoka, Yoshihiko; Yazawa, Takashi; Uesaka, Miki; Mizutani, Tetsuya; Yamada, Kazuya; Miyamoto, Kaoru

    2008-05-01

    NR4A1, also called NGFI-B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate-early genes. In the endocrine organs, including the gonads, NGFI-B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI-B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo-pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI-B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8-bromoadenosine 3'-5'-cyclic monophosphate (8 Br-cAMP) was examined in mouse Leydig tumor cells MA-10. Luciferase assays using NGFI-B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI-B/Nur77 gene expression was mediated through three of the four activator protein-1 (AP-1)-like sites, namely the -233 AP-1, -213 AP-1 and -69 AP-1 sites adjacent to the transcription start site of the NGFI-B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP-responsive element binding (CREB) proteins bind to the -233 AP-1 site, whereas the main binding protein to the -213 AP-1 site was CREB, and Jun family protein to the -69 AP-1 site, respectively. The rapid induction of NGFI-B/Nur77 gene expression by LH/hCG in MA-10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP-protein kinase A (PKA) pathway. PMID:18163434

  1. Fetal Leydig Cells: Progenitor Cell Review Maintenance and Differentiation

    PubMed Central

    BARSOUM, IVRAYM B.; YAO, HUMPHREY H.-C.

    2012-01-01

    In most eutherian mammals, sexually dimorphic masculinization is established by androgen-producing fetal Leydig cells in the embryonic testis. Fetal Leydig cells, which lack expression of the testis-determining gene SRY, arise after the appearance of SRY-expressing Sertoli cells. Therefore, the appearance and differentiation of fetal Leydig cells are probably regulated by factors derived from Sertoli cells. Results from mouse genetic models have revealed that maintenance and differentiation of fetal Leydig cell population depends upon a balance between differentiation-promoting and differentiation-suppressing mechanisms. Although paracrine signaling via Sertoli cell–derived Hedgehog ligands is necessary and sufficient for fetal Leydig cell formation, cell-cell interaction via Notch signaling and intracellular transcription factors such as POD1 are implicated as suppressors of fetal Leydig cell differentiation. This review provides a model that summarizes the recent findings in fetal Leydig cell development. PMID:19875489

  2. Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production.

    PubMed

    Ronco, A M; Moraga, P F; Llanos, M N

    2002-01-01

    We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of

  3. Binding and internalization in vivo of (/sup 125/I)hCG in Leydig cells of the rat

    SciTech Connect

    Hermo, L.; Lalli, M.

    1988-01-01

    The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ((/sup 125/I)hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of (/sup 125/I)hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the (/sup 125/I)hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of (/sup 125/I)hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval.

  4. Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

    PubMed Central

    Geigerseder, Christof; Doepner, Richard FG; Thalhammer, Andrea; Krieger, Annette; Mayerhofer, Artur

    2004-01-01

    The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment. PMID:15040802

  5. Expression of functional leptin receptors in rodent Leydig cells.

    PubMed

    Caprio, M; Isidori, A M; Carta, A R; Moretti, C; Dufau, M L; Fabbri, A

    1999-11-01

    Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot

  6. Stimulation of cholesterol side-chain cleavage by a luteinizing-hormone-releasing hormone (luliberin) agonist (ICI 118630) in rat Leydig cells.

    PubMed Central

    Sullivan, M H; Cooke, B A

    1983-01-01

    The action of a luliberin (luteinizing-hormone-releasing hormone) agonist (ICI 118630) and lutropin (luteinizing hormone) on the activity of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat Leydig cells has been investigated. This has been carried out by studying the metabolism of exogenous (22R)-22- and 25-hydroxycholesterol to testosterone. It was found that both hydroxycholesterols increased testosterone production to higher levels than achieved by lutropin alone. Addition of luliberin agonist but not lutropin was found to increase further the metabolism of the hydroxycholesterol to testosterone; this occurred in the presence of saturating and subsaturating levels of the hydroxycholesterols. This effect of luliberin agonist was potentiated in the presence of lutropin. The protein synthesis inhibitor, cycloheximide, inhibited the luliberin agonist-induced stimulation of the hydroxycholesterol metabolism. At low calcium levels (1.1 microM), testosterone production was increased by addition of (22R)-22-hydroxycholesterol but the luliberin agonist effect was negated. The calmodulin inhibitor trifluoperazine inhibited (22R)-22-hydroxycholesterol-stimulated steroidogenesis and negated the luliberin agonist effect. These results indicate that luliberin agonist specifically increases the synthesis of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat testis Leydig cells. PMID:6230077

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

    PubMed

    Anand, H; Misro, M M; Sharma, S B; Prakash, S

    2013-06-01

    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. PMID:22731239

  8. Intratesticular alpha1-adrenergic receptors mediate stress-disturbed transcription of steroidogenic stimulator NUR77 as well as steroidogenic repressors DAX1 and ARR19 in Leydig cells of adult rats.

    PubMed

    Stojkov-Mimic, Natasa J; Bjelic, Maja M; Radovic, Sava M; Mihajlovic, Aleksandar I; Sokanovic, Srdjan J; Baburski, Aleksandar Z; Janjic, Marija M; Kostic, Tatjana S; Andric, Silvana A

    2015-09-01

    The aim of the present study was to define the role of testicular α1-adrenergic receptors (α1-ADRs) in stress-triggered adaptation of testosterone-producing Leydig cells of adult rats. Results showed that in vivo blockade of testicular α1-ADRs prevented partial recovery of circulating androgen levels registered after 10× repeated immobilization stress (10 × IMO). Moreover, α1-ADR-blockade diminished 10 × IMO-triggered recovery of Leydig cell androgen production, and abolished mitochondrial membrane potential recovery. In the same cells, 10 × IMO-induced increase in Star transcript was abolished, Lhcgr transcript decreased, while transcription of other steroidogenic proteins was not changed. α1-ADR-blockade recovered stress-induced decrease of Nur77, one of the main steroidogenic stimulator, while significantly reduced 10 × IMO-increased in the transcription of the main steroidogenic repressors, Arr19 and Dax1. In vitro experiments revealed an adrenaline-induced α1-ADR-mediated decrease in Nur77 transcription in Leydig cells. Adrenaline-induced increase of repressor Dax1 also involves ADRs in Leydig cells. Accordingly, α1-ADRs participate in some of the stress-triggered effects on the steroidogenic machinery of Leydig cells. PMID:26003139

  9. Mono-(2-ethylhexyl) Phthalate Directly Alters the Expression of Leydig Cell Genes and CYP17 Lyase Activity in Cultured Rat Fetal Testis

    PubMed Central

    Chauvigné, François; Plummer, Simon; Lesné, Laurianne; Cravedi, Jean-Pierre; Dejucq-Rainsford, Nathalie; Fostier, Alexis; Jégou, Bernard

    2011-01-01

    Exposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to be done to understand the mechanisms underlying the direct action of phthalate within the fetal testis. We aimed to investigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing on Leydig cell steroidogenesis in particular. We used an in vitro system based on the culture for three days, with or without MEHP, of rat fetal testes obtained at 14.5 days post-coitum. Exposure to MEHP led to a dose-dependent decrease in testosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (5α-DHT) (−68%) and androstenedione (−54%) was also inhibited by 10 µM MEHP, whereas 17 alpha-hydroxyprogesterone (17α-OHP) production was found to increase (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the other precursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), converting 17α-OHP to androstenedione. The subsequent gene expression and protein levels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important for testes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which is involved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B. These findings indicate that under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directly inhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target to MEHP explaining the MEHP-induced suppression of steroidogenesis observed. PMID:22087261

  10. Orally applied doxazosin disturbed testosterone homeostasis and changed the transcriptional profile of steroidogenic machinery, cAMP/cGMP signalling and adrenergic receptors in Leydig cells of adult rats.

    PubMed

    Stojkov, N J; Janjic, M M; Kostic, T S; Andric, S A

    2013-03-01

    Doxazosin (Doxa) is an α1-selective adrenergic receptor (ADR) antagonist widely used, alone or in combination, to treat high blood pressure, benign prostatic hyperplasia symptoms, and recently has been suggested as a potential drug for prostate cancer prevention/treatment. This study was designed to evaluate the effect of in vivo Doxa po-application, in clinically relevant dose, on: (i) steroidogenic machinery homeostasis; (ii) cAMP/cGMP signalling; (iii) transcription profile of ADR in Leydig cells of adult rats. The results showed that po-application of Doxa for once (1×Doxa), or for two (2×Doxa) or 10 (10×Doxa) consecutive days significantly disturbed steroidogenic machinery homeostasis in Leydig cells. Doxa po-application significantly decreased circulating luteinizing hormone and androgens levels. The level of androgens in testicular interstitial fluid and that extracted from testes obtained from 1×Doxa/2×Doxa rats decreased, although it remained unchanged in 10×Doxa rats. Similarly, the ex vivo basal androgen production followed in testes isolated from 1×Doxa/2×Doxa rats decreased, while remained unchanged in 10×Doxa rats. Differently, ex vivo testosterone production and steroidogenic capacity of Leydig cells isolated from 1×Doxa/2×Doxa rats was stimulated, while 10×Doxa had opposite effect. In the same cells, cAMP content/release showed similar stimulatory effect, but back to control level in Leydig cells of 10×Doxa. 1×Doxa/2×Doxa decreased transcripts for cAMP specific phosphodiesterases Pde7b/Pde8b, whereas 10×Doxa increased Pde4d. All types of treatment reduced the expression of genes encoding protein kinase A (PRKA) regulatory subunit (Prkar2b), whereas only 10×Doxa stimulated catalytic subunit (Prkaca). Doxa application more affected cGMP signalling: stimulated transcription of constitutive nitric oxide synthases (Nos1, Nos3) in time-dependent manner, whereas reduced inducible Nos2. 10×Doxa increased guanylyl cyclase 1 transcript and

  11. Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells.

    PubMed

    Gak, Igor A; Radovic, Sava M; Dukic, Aleksandra R; Janjic, Marija M; Stojkov-Mimic, Natasa J; Kostic, Tatjana S; Andric, Silvana A

    2015-10-01

    Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies. PMID:26036344

  12. Atypical Leydig cell hyperplasia in adult rats with low T and high LH induced by prenatal Di(n-butyl) phthalate exposure.

    PubMed

    Wakui, Shin; Takahashi, Hiroyuki; Mutou, Tomoko; Shirai, Masaru; Jutabha, Promsuk; Anzai, Naohiko; Wempe, Michael F; Kansaku, Norio; Hano, Hiroshi; Inomata, Tomo; Endou, Hitoshi

    2013-01-01

    The present study describes atypical Leydig cell (LC) hyperplasia in 20-week-old Sprague-Dawley rats with low testosterone and high luteinizing hormone levels after prenatal administration of 100 mg/kg/day di(n-butyl) phthalate on days 12 to 21 postconception. Light microscopy revealed LC hyperplasia surrounded by severely degenerated seminiferous tubules. Aggregated LCs had large ovoid nuclei with nucleoli and abundant eosinophilic cytoplasm. Immunohistochemical analysis showed expression of proliferating cell nuclear antigen and vimentin in many hyperplastic LCs. Electron microscopy revealed atypical nuclei, abundant free ribosomes, stripped rough endoplasmic reticulum, intermediate-size filaments, elongated cytoplasmic filopodia, atypical tight junctions, and cilia formations, but smooth endoplasmic reticulum was scarcely observed. PMID:22968287

  13. Phthalate-induced testicular dysgenesis syndrome: Leydig cell influence.

    PubMed

    Hu, Guo-Xin; Lian, Qing-Quan; Ge, Ren-Shan; Hardy, Dianne O; Li, Xiao-Kun

    2009-04-01

    Phthalates, the most abundantly produced plasticizers, leach out from polyvinyl chloride plastics and disrupt androgen action. Male rats that are exposed to phthalates in utero develop symptoms characteristic of the human condition referred to as testicular dysgenesis syndrome (TDS). Environmental influences have been suspected to contribute to the increasing incidence of TDS in humans (i.e. cryptorchidism and hypospadias in newborn boys and testicular cancer and reduced sperm quality in adult males). In this review, we discuss the recent findings that prenatal exposure to phthalates affects Leydig cell function in the postnatal testis. This review also focuses on the recent progress in our understanding of how Leydig cell factors contribute to phthalate-mediated TDS. PMID:19278865

  14. Malignant Leydig cell tumour of the testis.

    PubMed

    Powari, Manish; Kakkar, Nandita; Singh, S K; Rai, R S; Jogai, Sanjay

    2002-01-01

    A case of malignant Leydig cell tumour is presented. It is a rare primary malignant tumour of the testis and occurs exclusively in adults. The present case is of interest because it occurred at the young age of 25 years which is rare. Histologically it showed almost all features which suggest malignancy and also had metastases to the lungs and liver. The clinical details and pathology of this tumour are discussed. PMID:11803271

  15. Toxic mechanisms of 3-monochloropropane-1,2-diol on progesterone production in R2C rat leydig cells.

    PubMed

    Sun, Jianxia; Bai, Shun; Bai, Weibin; Zou, Feiyan; Zhang, Lei; Su, Zhijian; Zhang, Qihao; Ou, Shiyi; Huang, Yadong

    2013-10-16

    3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been shown to impede the male reproductive function. However, its mechanism of action remains to be elucidated. In this study, the effects of 3-MCPD on progesterone production were investigated using R2C Leydig cells. 3-MCPD caused concentration-dependent inhibition of cell viability at the IC25, IC50, and IC75 levels of 1.027, 1.802, and 3.160 mM, respectively. Single cell gel/comet assay and atomic force microscopy assay showed that 3-MCPD significantly induced early apoptosis. In addition, 3-MCPD significantly reduced progesterone production by reducing the expression of cytochrome P450 side-chain cleavage enzyme, steroidogenic acute regulatory protein, and 3β-hydroxysteroid dehydrogenase in R2C cells. The change in steroidogenic acute regulatory protein expression was highly consistent with progesterone production. Furthermore, the mitochondrial membrane potential and cAMP significantly decreased. PMID:24040863

  16. Lutropin/choriogonadotropin (LH/CG) stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the ERK1/2 cascade*

    PubMed Central

    Shiraishi, Koji; Ascoli, Mario

    2007-01-01

    Primary cultures of progenitor and immature rat Leydig cells were established from the testes of 21 and 35 day old rats, respectively. The cell population remained homogeneous after 4–6 days in culture as judged by staining for 3β-hydroxysteroid dehydrogenase but the cells were unable to bind 125I-hCG or to respond to hCG with classical LH receptor (LHR)-mediated responses including cAMP and inositol phosphate accumulation, steroid biosynthesis or the phosphorylation of the extracellular regulated kinases 1/2 (ERK1/2). Infection of primary cultures with recombinant adenovirus coding for β-galactosidase showed that ~65% of the cells are infected. Infection with adenovirus coding for the human LHR (hLHR) allowed for expression of the hLHR at a density of ~25,000 receptors/cell and allowed the cells to respond to hCG with increases in cAMP and inositol phosphate accumulation, steroid biosynthesis and the phosphorylation of ERK1/2. Although progenitor and immature cells were able to respond to hCG with an increase in progesterone, only the immature cells responded with an increase in testosterone. In addition to these classical LHR-mediated responses the primary cultures of progenitor or immature rat Leydig cells expressing the recombinant hLHR proliferated robustly when incubated with hCG and this proliferative response was sensitive to an inhibitor of ERK1/2 phosphorylation. These studies establish a novel experimental paradigm that can be used to study the proliferative response of Leydig cells to LH/CG. We conclude that activation of the LHR-provoked Leydig cell proliferation requires activation of the ERK1/2 cascade. PMID:17412805

  17. Apoptosis Process in Mouse Leydig Cells during Postnatal Development

    NASA Astrophysics Data System (ADS)

    Salles Faria, Maria José; Simões, Zilá Paulino; Luz; Orive Lunardi, Laurelucia; Hartfelder, Klaus

    2003-02-01

    The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.

  18. Sertoli-Leydig cell tumor

    MedlinePlus

    ... the testes, release a male sex hormone called testosterone . These cells are also found in a woman's ... the levels of female and male hormones, including testosterone . An ultrasound or another imaging test will likely ...

  19. A METABOLITE OF METHOXYCHLOR,2,2-BIS(P-HYDOXYPHENYL)-1,1,1- TRICHLOROETHANE REDUCES TESTOSTERONE BIOSYNTHESIS IN RAT LEYDIG CELLS THROUGH SUPPRESSION OF STEADY-STATE MESSENGER RIBONUCLEIC ACID LEVELS OF THE CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME

    EPA Science Inventory

    Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the ma...

  20. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells.

    PubMed

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  1. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells

    PubMed Central

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  2. Sirtuin 4 Regulates Lipopolysaccharide Mediated Leydig Cell Dysfunction.

    PubMed

    Ramatchandirin, Balamurugan; Sadasivam, Mohanraj; Kannan, Arun; Prahalathan, Chidambaram

    2016-04-01

    Bacterial lipopolysaccharide (LPS) is the most important contributing factor in pathogenesis of bacterial infection in male accessory glands; and it has shown to inhibit testicular steroidogenesis and induce apoptosis. The present study demonstrates that LPS causes mitochondrial dysfunction via suppression of sirtuin 4 (SIRT4); which in turn affects Leydig cell function by modulating steroidogenesis and apoptosis. LC-540 Leydig cells treated with LPS (10 µg/ml) showed impaired steroidogenesis and increased cellular apoptosis. The mRNA and protein expression of SIRT4 were decreased in LPS treated cells when compared to controls. The obtained data suggest that the c-Jun N-terminal kinase (JNK) activation suppresses SIRT4 expression in LPS treated Leydig cells. Furthermore, the overexpression of SIRT4 prevented LPS induced impaired steroidogenesis and cellular apoptosis by improving mitochondrial function. These findings provide valuable information that SIRT4 regulates LPS mediated Leydig cell dysfunction. PMID:26365714

  3. Effects of the Methanol Extract of Basella alba L (Basellaceae) on Steroid Production in Leydig Cells

    PubMed Central

    Nantia, Edouard Akono; Travert, Carine; Manfo, Faustin-Pascal T.; Carreau, Serge; Monsees, Thomas K.; Moundipa, Paul Fewou

    2011-01-01

    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. PMID:21339992

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

    PubMed

    Nantia, Edouard Akono; Travert, Carine; Manfo, Faustin-Pascal T; Carreau, Serge; Monsees, Thomas K; Moundipa, Paul Fewou

    2011-01-01

    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. PMID:21339992

  5. Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells.

    PubMed

    Chimento, A; Casaburi, I; Bartucci, M; Patrizii, M; Dattilo, R; Avena, P; Andò, S; Pezzi, V; Sirianni, R

    2013-01-01

    We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients. PMID:23907461

  6. Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells

    PubMed Central

    Chimento, A; Casaburi, I; Bartucci, M; Patrizii, M; Dattilo, R; Avena, P; Andò, S; Pezzi, V; Sirianni, R

    2013-01-01

    We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients. PMID:23907461

  7. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men

    PubMed Central

    O’Hara, Laura; McInnes, Kerry; Simitsidellis, Ioannis; Morgan, Stephanie; Atanassova, Nina; Slowikowska-Hilczer, Jolanta; Kula, Krzysztof; Szarras-Czapnik, Maria; Milne, Laura; Mitchell, Rod T.; Smith, Lee B.

    2015-01-01

    Leydig cell number and function decline as men age, and low testosterone is associated with all “Western” cardio-metabolic disorders. However, whether perturbed androgen action within the adult Leydig cell lineage predisposes individuals to this late-onset degeneration remains unknown. To address this, we generated a novel mouse model in which androgen receptor (AR) is ablated from ∼75% of adult Leydig stem cell/cell progenitors, from fetal life onward (Leydig cell AR knockout mice), permitting interrogation of the specific roles of autocrine Leydig cell AR signaling through comparison to adjacent AR-retaining Leydig cells, testes from littermate controls, and to human testes, including from patients with complete androgen insensitivity syndrome (CAIS). This revealed that autocrine AR signaling is dispensable for the attainment of final Leydig cell number but is essential for Leydig cell maturation and regulation of steroidogenic enzymes in adulthood. Furthermore, these studies reveal that autocrine AR signaling in Leydig cells protects against late-onset degeneration of the seminiferous epithelium in mice and inhibits Leydig cell apoptosis in both adult mice and patients with CAIS, possibly via opposing aberrant estrogen signaling. We conclude that autocrine androgen action within Leydig cells is essential for the lifelong support of spermatogenesis and the development and lifelong health of Leydig cells.—O’Hara, L., McInnes, K., Simitsidellis, I., Morgan, S., Atanassova, N., Slowikowska-Hilczer, J., Kula, K., Szarras-Czapnik, M., Milne, L., Mitchell, R. T., Smith, L. B. Autocrine androgen action is essential for Leydig cell maturation and function, and protects against late-onset Leydig cell apoptosis in both mice and men. PMID:25404712

  8. Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis.

    PubMed

    Yoon, M J; Roser, J F

    2010-12-01

    Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may have potential as a treatment regime to prevent testicular degeneration. The present study was designed to evaluate the anti-apoptotic effect of IGF-I on cultured equine Leydig cells. Testes were collected from 5 post-pubertal stallions (2-4 years old) during routine castrations. A highly purified preparation of equine Leydig cells was obtained from a discontinuous Percoll gradient. Purity of equine Leydig cells was assessed using histochemical 3β-HSD staining. Equine Leydig cells and selected doses of recombinant human IGF-1 (rhIGF-I; Parlow A.F., National Hormone and Peptide Program, Harbor-UCLA Medical Center) were added to wells of 24 or 96 well culture plates in triplicate and cultured for 24 or 48 h under 95% air:5% CO(2) at 34°C. After 24 or 48 h incubation, apoptotic rate was assessed using a Cell Death Detection ELISA kit. Significantly lower apoptotic rates were observed in equine Leydig cells cultured with 5, 10, or 50ng/ml of rhIGF-I compared with control cells cultured without rhIGF-I for 24h. Exposure to 1, 5, 10 or 50 ng/ml of rhIGF-I significantly decreased apoptotic rate in equine Leydig cells cultured for 48 h. After 48 h incubation, cells were labeled with Annexin V and propodium iodine to determine the populations of healthy, apoptotic, and necrotic cells by counting stained cells using a Nikon Eclipse inverted fluorescence microscope. As a percentage of

  9. Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal testes.

    PubMed

    Shima, Yuichi; Miyabayashi, Kanako; Haraguchi, Shogo; Arakawa, Tatsuhiko; Otake, Hiroyuki; Baba, Takashi; Matsuzaki, Sawako; Shishido, Yurina; Akiyama, Haruhiko; Tachibana, Taro; Tsutsui, Kazuyoshi; Morohashi, Ken-ichirou

    2013-01-01

    Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis. PMID:23125070

  10. Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

    PubMed

    He, Ze; Sun, Qin; Liang, Yuan-Jiao; Chen, Li; Ge, Yi-Feng; Yun, Shi-Feng; Yao, Bing

    2016-01-01

    This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells. PMID:26289400

  11. Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation

    PubMed Central

    Le Tortorec, A.; Denis, H.; Satie, A-P.; Patard, J-J.; Ruffault, A.; Jégou, B.; Dejucq-Rainsford, N.

    2008-01-01

    BACKGROUND The immuno-privileged status of the testis is essential to the maintenance of its functions, and innate immunity is likely to play a key role in limiting harmful viral infections, as demonstrated in the rat. In men mumps virus infects Leydig cells and has deleterious effects on testosterone production and spermatogenesis. The aim of this study was to test whether mumps virus infection of isolated human Leydig cells was associated with an inhibition of their innate antiviral defences. METHODS Leydig cell production of mRNA and protein for interferons (IFNs) and of three antiviral proteins—2′5′ oligoadenylate synthetase (2′5′OAS), double-stranded RNA-activated protein kinase (PKR) and MxA—was investigated, in the absence or presence of mumps virus or viral stimuli including poly I:C, a mimetic of RNA viruses replication product. RESULTS Stimulated or not, human Leydig cells appeared unable to produce routinely detectable IFNs α, β and γ. Although the level of PKR remained unchanged after stimulation, the expression of 2′5′OAS and MxA was enhanced following either mumps virus or poly I:C exposure (P < 0.05 versus control). CONCLUSIONS Overall, our results demonstrate that mumps virus replication in human Leydig cells is not associated with a specific inhibition of IFNs or 2′5′OAS, MxA and PKR production and that these cells display relatively weak endogenous antiviral abilities, as opposed to their rat counterparts. PMID:18567898

  12. Expression of cubilin in mouse testes and Leydig cells.

    PubMed

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes. PMID:26148765

  13. H4 histamine receptors inhibit steroidogenesis and proliferation in Leydig cells.

    PubMed

    Abiuso, Adriana María Belén; Berensztein, Esperanza; Pagotto, Romina María; Pereyra, Elba Nora; Medina, Vanina; Martinel Lamas, Diego José; Besio Moreno, Marcos; Pignataro, Omar Pedro; Mondillo, Carolina

    2014-12-01

    The histamine H4 receptor (HRH4), discovered only 13 years ago, is considered a promising drug target for allergy, inflammation, autoimmune disorders and cancer, as reflected by a steadily growing number of scientific publications and patent applications. Although the presence of HRH4 has been evidenced in the testis, its specific localization or its role has not been established. Herein, we sought to identify the possible involvement of HRH4 in the regulation of Leydig cell function. We first evaluated its expression in MA-10 Leydig tumor cells and then assessed the effects of two HRH4 agonists on steroidogenesis and proliferation. We found that HRH4 is functionally expressed in MA-10 cells, and that its activation leads to the inhibition of LH/human chorionic gonadotropin-induced cAMP production and StAR protein expression. Furthermore, we observed decreased cell proliferation after a 24-h HRH4 agonist treatment. We then detected for the sites of HRH4 expression in the normal rat testis, and detected HRH4 immunostaining in the Leydig cells of rats aged 7-240 days, while 21-day-old rats also presented HRH4 expression in male gametes. Finally, we evaluated the effect of HRH4 activation on the proliferation of normal progenitor and immature rat Leydig cell culture, and both proved to be susceptible to the anti-proliferative effect of HRH4 agonists. Given the importance of histamine (2-(1H-imidazol-4-yl)ethanamine) in human (patho)physiology, continued efforts are directed at elucidating the emerging properties of HRH4 and its ligands. This study reveals new sites of HRH4 expression, and should be considered in the design of selective HRH4 agonists for therapeutic purposes. PMID:25253872

  14. Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells

    PubMed Central

    Miyabayashi, Kanako; Katoh-Fukui, Yuko; Ogawa, Hidesato; Baba, Takashi; Shima, Yuichi; Sugiyama, Noriyuki; Kitamura, Kunio; Morohashi, Ken-ichirou

    2013-01-01

    Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage. PMID:23840809

  15. Clinicopathologic features of ovarian Sertoli-Leydig cell tumors

    PubMed Central

    Zhang, Hai-Yan; Zhu, Jia-Er; Huang, Wen; Zhu, Jin

    2014-01-01

    Background: Ovarian Stertoli-Ledig cell tumor (SLCT) is a rare type of sex cord-stromal tumor of the ovary. The present study was to evaluate clinicalopahologic features and prognosis of patients with Sertoli-Leydig cell tumor treated by surgery and adjuvant chemotherapy during short term follow-up. Methods: A total of sixteen patients with ovarian Sertoli-Leydig cell tumor treated at the Obstetrics and Gynecology Hospital, Shanghai, China, between Jan 2001 and Dec 2011 were reviewed. The clinical data, treatment and prognosis were obtained from medical records. Results: The median age of the patients with ovarian Sertoli-Leydig cell tumor was about 27.5 years old in non-menopausal women, while the median age of menopausal women was about 63 years old. The most common complaint was with hormonal-related symptoms in the form of secondary amenorrhea and infinity, features of virilization, abdominal mass or irregular vaginal bleeding. All of sixteen patients underwent surgical staging and all were found to have stage I disease at the time of diagnosis. Eleven patients with intermediate and two patients with poorly differentiated tumors received adjuvant chemotherapy. There were differences found in operative time, blood loss and postoperative recovery time between laparotomy and laparoscopy. There were no disease-related deaths and all patients were under complete remission at the last follow-up. Conclusions: Ovarian Sertoli-Leydig cell tumors could happen in any period age of women. However, the tumors typically occur in the single side while still at the early stage, a favorable outcome could be achieved by surgery and adjuvant chemotherapy. Laparoscopy has similar surgical effects as laparotomy, but has a number of advantages. PMID:25400781

  16. Leydig cell damage after testicular irradiation for lymphoblastic leukemia

    SciTech Connect

    Shalet, S.M.; Horner, A.; Ahmed, S.R.; Morris-Jones, P.H.

    1985-01-01

    The effect of testicular irradiation on Leydig cell function has been studied in a group of boys irradiated between 1 and 5 years earlier for a testicular relapse of acute lymphoblastic leukemia. Six of the seven boys irradiated during prepubertal life had an absent testosterone response to HCG stimulation. Two of the four boys irradiated during puberty had an appropriate basal testosterone level, but the testosterone response to HCG stimulation was subnormal in three of the four. Abnormalities in gonadotropin secretion consistent with testicular damage were noted in nine of the 11 boys. Evidence of severe Leydig cell damage was present irrespective of whether the boys were studied within 1 year or between 3 and 5 years after irradiation, suggesting that recovery is unlikely. Androgen replacement therapy has been started in four boys and will be required by the majority of the remainder to undergo normal pubertal development.

  17. Reprogramming of Sertoli cells to fetal-like Leydig cells by Wt1 ablation

    PubMed Central

    Zhang, Lianjun; Chen, Min; Wen, Qing; Li, Yaqiong; Wang, Yaqing; Wang, Yanbo; Qin, Yan; Cui, Xiuhong; Yang, Lin; Huff, Vicki; Gao, Fei

    2015-01-01

    Sertoli and Leydig cells, the two major somatic cell types in the testis, have different morphologies and functions. Both are essential for gonad development and spermatogenesis. However, whether these cells are derived from the same progenitor cells and the mechanism regulating the differentiation between these two cell types during gonad development remains unclear. A previous study showed that overactivation of Ctnnb1 (cadherin-associated protein, beta 1) in Sertoli cells resulted in Sertoli cell tumors. Surprisingly, in the present study, we found that simultaneous deletion of Wilms’ Tumor Gene 1 (Wt1) and overactivation of Ctnnb1 in Sertoli cells led to Leydig cell-like tumor development. Lineage tracing experiments revealed that the Leydig-like tumor cells were derived from Sertoli cells. Further studies confirmed that Wt1 is required for the maintenance of the Sertoli cell lineage and that deletion of Wt1 resulted in the reprogramming of Sertoli cells to Leydig cells. Consistent with this interpretation, overexpression of Wt1 in Leydig cells led to the up-regulation of Sertoli cell-specific gene expression and the down-regulation of steroidogenic gene expression. These results demonstrate that the distinction between Sertoli cells and Leydig cells is regulated by Wt1, implying that these two cell types most likely originate from the same progenitor cells. This study thus provides a novel concept for somatic cell fate determination in testis development that may also represent an etiology of male infertility in human patients. PMID:25775596

  18. RODENT LEYDIG CELL TUMORIGENESIS: A REVIEW OF THE PHYSIOLOGY, PATHOLOGY, MECHANISMS, AND RELEVANCE TO HUMANS

    EPA Science Inventory

    Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years bec...

  19. Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death

    PubMed Central

    Morgan, Jessica A.; Lynch, John; Panetta, John C.; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T.; Janke, Laura; Green, Daniel M.; Chemaitilly, Wassim; Schuetz, John D.

    2015-01-01

    Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer. PMID:26576726

  20. Cell interactions and genetic regulation that contribute to testicular Leydig cell development and differentiation.

    PubMed

    Martin, Luc J

    2016-06-01

    Leydig cells, located within the interstitial compartment of the testis, are major contributors of androgen synthesis and secretion, which play an important role in testis development, normal masculinization, maintenance of spermatogenesis, and general male fertility. Accordingly, dysfunction of Leydig cells may lead to various male reproductive maladies, including primary hypogonadism, cryptorchidism, and hypospadias. A better understanding of how cell interactions and gene regulation contribute to testicular Leydig cell development and differentiation may therefore help limit the incidence of such male reproductive pathologies. Several hormones and signaling molecules have been identified as important regulators of Leydig cell differentiation and function. Recent work on the regulation of testis development, especially of Leydig cells, has focused on the Desert hedgehog and platelet-derived growth factor signaling pathways. This review outlines recent findings regarding cell interactions and gene regulation involved in the development and regulation of fetal and adult Leydig cell populations. Mol. Reprod. Dev. 83: 470-487, 2016. © 2016 Wiley Periodicals, Inc. PMID:27079813

  1. Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

    PubMed

    Carney, Colleen M; Muszynski, Jessica L; Strotman, Lindsay N; Lewis, Samantha R; O'Connell, Rachel L; Beebe, David J; Theberge, Ashleigh B; Jorgensen, Joan S

    2014-10-01

    Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  2. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    PubMed Central

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  3. Establishment and evaluation of a stable steroidogenic goat Leydig cell line.

    PubMed

    Zhou, Jinhua; Dai, Rui; Lei, Lanjie; Lin, Pengfei; Lu, Xiaolong; Wang, Xiangguo; Tang, Keqiong; Wang, Aihua; Jin, Yaping

    2016-04-01

    Leydig cells play a key role in synthesizing androgen and regulating spermatogenesis. The dysfunction of Leydig cells may lead to various male diseases. Although primary Leydig cell cultures have been used, their finite lifespan hinders the assessment of long-term effects. In the present study, primary goat Leydig cells (GLCs) were immortalized via the transfection of a plasmid containing the human telomerase reverse transcriptase (hTERT) gene. The expressions of hTERT and telomerase activity were evaluated in transduced GLCs (hTERT-GLCs). These cells steadily expressed the hTERT gene and exhibited longer telomere lengths at passage 55 that were similar to those of HeLa cells. The hTERT-GLCs at passages 30 and 50 expressed genes that encoded key proteins, enzymes and receptors that are inherent to normal Leydig cells, for example, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and LH-receptor (LH-R). Additionally, the immortalized goat Leydig cells secreted detectable quantities of testosterone in response to hCG stimulation. Furthermore, this cell line appeared to proliferate more quickly than the control cells, although no neoplastic transformation occurred in vitro. We concluded that the GLCs immortalized with hTERT retained their original characteristics and might provide a useful model for the study of Leydig cell function. PMID:26462462

  4. Male rats exposed in utero to di(n-butyl) phthalate: Age-related changes in Leydig cell smooth endoplasmic reticulum and testicular testosterone-biosynthesis enzymes/proteins.

    PubMed

    Motohashi, Masaya; Wempe, Michael F; Mutou, Tomoko; Takahashi, Hiroyuki; Kansaku, Norio; Ikegami, Masahiro; Inomata, Tomo; Asari, Masao; Wakui, Shin

    2016-01-01

    This study investigated the age-related (i.e., weeks 5, 7, 9, 14 and 17) morphological changes of Leydig cell smooth endoplasmic reticulum (LCs-ER) and testicular testosterone biosynthesis/protein expression in rats in utero exposed to di(n-butyl) phthalate (DBP) (intragastrically; 100mg/kg/day) on days 12-21 post-conception. Ultrastructural observations revealed the LCs-ER of the DBP group were non-dilated until peri-puberty, and thereafter decreased and disappeared. RT-PCR and Western blotting analyses revealed that StAR and P450scc levels in the DBP group were significantly lower at 5 and 7 weeks compared with the vehicle group but became similar during weeks 9-17. Although 3β-HSD, P450c17, and 17β-HSD levels of mRNA and protein in the DBP group were similar to the vehicle control group at 5 and 7 weeks of age, they were significantly lower during weeks 9-17. In utero DBP exposure results in age-related LCs-ER changes corresponding to reduction of testicular testosterone biosynthesis enzymes/associated proteins. PMID:26706031

  5. Genetic ablation of androgen receptor signaling in fetal Leydig cell lineage affects Leydig cell functions in adult testis.

    PubMed

    Kaftanovskaya, Elena M; Lopez, Carolina; Ferguson, Lydia; Myhr, Courtney; Agoulnik, Alexander I

    2015-06-01

    It is commonly accepted that androgen-producing fetal Leydig cells (FLC) are substituted by adult Leydig cells (ALC) during perinatal testis development. The mechanisms influencing this process are unclear. We used mice with a retinoid acid receptor 2 promoter-Cre recombinase transgene (Rarb-cre) expressed in embryonic FLC precursors, but not in postnatal testis, and a dual fluorescent Cre recombinase reporter to label FLC and ALC in vivo. All FLC in newborn testis had the recombinant, whereas the majority of LC in adult testis had the nonrecombinant reporter. Primary LC cultures from adult testis had either recombinant (20%) or nonrecombinant (80%) cells, demonstrating that the FLC survive in adult testis and their ontogeny is distinct from ALC. Conditional inactivation of androgen receptor (AR) allele using the Rarb-cre transgene resulted in a 50% increase of AR-negative LC in adult testis. The mutant males became infertile with age, with all LC in older testis showing signs of incomplete differentiation, such as a large number of big lipid droplets, an increase of finger-like protrusions, and a misexpression of steroidogenic or FLC- and ALC-specific genes. We propose that the antiandrogenic exposure during early development may similarly result in an increase of FLC in adult testis, leading to abnormal LC differentiation. PMID:25713029

  6. Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone.

    PubMed Central

    Cooke, B A; Lindh, M L; Janszen, F H

    1976-01-01

    The effect of different doses of luteinizing hormone on activation of protein kinases, cyclic AMP and testosterone production was studied in purified rat testis Leydig-cell preparations in the presence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). In addition, the nature of the protein kinases present in these cells and other tissues was investigated. The following results were obtained. 1. With all the amounts of luteinizing hormone used (0.1-1000 ng/ml), both activation of protein kinase and stimulation of testosterone production were demonstrated. With the lowest amount of luteinizing hormone (0.1 ng/ml), an 8.4+/-0.9% (S.E.M.,n=6) stimulation of protein kinase activation occurred, increasing to 100% with 1000 ng/ml, compared with 3.2+/-1.0%(S.E.M.,n=7) and 100% stimulation of testosterone production with 0.1 and 100 ng/ml respectively. 2. With amounts of luteinizing hormone up to 1 ng/ml (which gave half-maximal stimulation of testosterone production) no detectable increases in net cyclic AMP production were obtained. With higher amounts of luteinizing hormone, cyclic AMP production increased, but maximal production was not reached with 1000 ng/ml. 3. Two isoenzymic forms of protein kinase were present in Leydig cells and seminiferous tubules; type I was eluted with 0.075 M-and type II with 0.22-0.25 m-NaCl from DEAE-cellulose columns. 4. The protein kinase activity was not affected by the presence of erythrocytes in the Leydig-cell preparation, but varied depending on the type of histone used as substrate (histone F2b greater than mixed greater than histone F1). PMID:189752

  7. Molecular Mechanisms Elicited by d-Aspartate in Leydig Cells and Spermatogonia

    PubMed Central

    Di Fiore, Maria Maddalena; Santillo, Alessandra; Falvo, Sara; Longobardi, Salvatore; Chieffi Baccari, Gabriella

    2016-01-01

    A bulk of evidence suggests that d-aspartate (d-Asp) regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by d-Asp possibly via binding to the N-methyl-d-aspartate receptor (NMDAR) in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK) pathways. d-Asp treatment enhances gene and protein expression of enzymes involved in the steroidogenic cascade. d-Asp also directly affects spermatogonial mitotic activity. In spermatogonial GC-1 cells, d-Asp induces phosphorylation of MAPK and AKT serine-threonine kinase proteins, and stimulates expression of proliferating cell nuclear antigen (PCNA) and aurora kinase B (AURKB). Further stimulation of spermatogonial GC-1 cell proliferation might come from estradiol/estrogen receptor β (ESR2) interaction. d-Asp modulates androgen and estrogen levels as well as the expression of their receptors in the rat epididymis by acting on mRNA levels of Srd5a1 and Cyp19a1 enzymes, hence suggesting involvement in spermatozoa maturation. PMID:27428949

  8. Molecular Mechanisms Elicited by d-Aspartate in Leydig Cells and Spermatogonia.

    PubMed

    Di Fiore, Maria Maddalena; Santillo, Alessandra; Falvo, Sara; Longobardi, Salvatore; Chieffi Baccari, Gabriella

    2016-01-01

    A bulk of evidence suggests that d-aspartate (d-Asp) regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by d-Asp possibly via binding to the N-methyl-d-aspartate receptor (NMDAR) in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK) pathways. d-Asp treatment enhances gene and protein expression of enzymes involved in the steroidogenic cascade. d-Asp also directly affects spermatogonial mitotic activity. In spermatogonial GC-1 cells, d-Asp induces phosphorylation of MAPK and AKT serine-threonine kinase proteins, and stimulates expression of proliferating cell nuclear antigen (PCNA) and aurora kinase B (AURKB). Further stimulation of spermatogonial GC-1 cell proliferation might come from estradiol/estrogen receptor β (ESR2) interaction. d-Asp modulates androgen and estrogen levels as well as the expression of their receptors in the rat epididymis by acting on mRNA levels of Srd5a1 and Cyp19a1 enzymes, hence suggesting involvement in spermatozoa maturation. PMID:27428949

  9. Disturbance in Testosterone Production in Leydig Cells by Polycyclic Aromatic Hydevrepocarbons

    PubMed Central

    Oh, Seunghoon

    2014-01-01

    Polycyclic aromatic hydevrepocarbons (PAHs), which are ubiquitous in the air, are present as volatile and particulate pollutants that result from incomplete combustion. Most PAHs have toxic, mutagenic, and/or carcinogenic properties. Among PAHs, benzo[a]pyrene (B[a]P) and dimethylbenz[a]anthracene (DMBA) are suspected endocrine disruptors. The testis is an important target for PAHs, yet effects on steroidogenesis in Leydig cells are yet to be ascertained. Particularly, disruption of testosterone production by these chemicals can result in serious defects in male reproduction. Exposure to B[a]P reduced serum and intratesticular fluid testosterone levels in rats. Of note, the testosterone level reductions were accompanied by decreased steroidogenic acute regulatory protein (StAR) and 3β-hydevrepoxysteroid dehydevrepogenase isomerase (3β-HSD) expression in Leydig cells. B[a]P exposure can decrease epididymal sperm quality, possibly by disturbing the testosterone level. StAR may be a key steroidogenic protein that is targeted by B[a]P or other PAHs. PMID:25949189

  10. Evidence for genetic heterogeneity in male pseudohermaphroditism due to Leydig cell hypoplasia.

    PubMed

    Zenteno, J C; Canto, P; Kofman-Alfaro, S; Mendez, J P

    1999-10-01

    Leydig cell aplasia or hypoplasia is a rare form of male pseudohermaphroditism resulting from inadequate fetal testicular Leydig cell differentiation. Affected individuals presented a wide phenotypic spectrum, ranging from complete female external genitalia to males with micropenis. Recessive mutations in the LH receptor gene have been identified as responsible for the condition. The majority of these mutations are point mutations and have been located in exon 11 of the gene. In this study, we report the molecular characterization of the LH receptor gene in three siblings with Leydig cell hypoplasia. After sequencing the 11 exons of the gene, no deleterious mutations were detected in any patient. However, we identified a previously described polymorphism in exon 11. In patients 1 and 3 DNA sequencing revealed a C to T substitution at nucleotide 1065; both patients were homozygous GAT/GAT at codon 355. In contrast, patient 2 was homozygous GAC/GAC, whereas the father and one unaffected sister were heterozygous GAC/GAT at this polymorphic site. These results exclude that Leydig cell hypoplasia in this family is due to a mutation in the LH receptor gene and provide evidence that defects in other loci may also result in failure of Leydig cell differentiation, demonstrating, for the first time, that Leydig cell hypoplasia is a genetically heterogeneous condition. PMID:10523033

  11. Estrogen promotes Leydig cell engulfment by macrophages in male infertility

    PubMed Central

    Yu, Wanpeng; Zheng, Han; Lin, Wei; Tajima, Astushi; Zhang, Yong; Zhang, Xiaoyan; Zhang, Hongwen; Wu, Jihua; Han, Daishu; Rahman, Nafis A.; Korach, Kenneth S.; Gao, George Fu; Inoue, Ituro; Li, Xiangdong

    2014-01-01

    Male infertility accounts for almost half of infertility cases worldwide. A subset of infertile men exhibit reduced testosterone and enhanced levels of estradiol (E2), though it is unclear how increased E2 promotes deterioration of male fertility. Here, we utilized a transgenic mouse strain that overexpresses human CYP19, which encodes aromatase (AROM+ mice), and mice with knockout of Esr1, encoding estrogen receptor α (ERαKO mice), to analyze interactions between viable Leydig cells (LCs) and testicular macrophages that may lead to male infertility. In AROM+ males, enhanced E2 promoted LC hyperplasia and macrophage activation via ERα signaling. E2 stimulated LCs to produce growth arrest–specific 6 (GAS6), which mediates phagocytosis of apoptotic cells by bridging cells with surface exposed phosphatidylserine (PS) to macrophage receptors, including the tyrosine kinases TYRO3, AXL, and MER. Overproduction of E2 increased apoptosis-independent extrusion of PS on LCs, which in turn promoted engulfment by E2/ERα-activated macrophages that was mediated by AXL-GAS6-PS interaction. We further confirmed E2-dependant engulfment of LCs by real-time 3D imaging. Furthermore, evaluation of molecular markers in the testes of patients with nonobstructive azoospermia (NOA) revealed enhanced expression of CYP19, GAS6, and AXL, which suggests that the AROM+ mouse model reflects human infertility. Together, these results suggest that GAS6 has a potential as a clinical biomarker and therapeutic target for male infertility. PMID:24762434

  12. Involution of human fetal Leydig cells. An immunohistochemical, ultrastructural and quantitative study.

    PubMed Central

    Codesal, J; Regadera, J; Nistal, M; Regadera-Sejas, J; Paniagua, R

    1990-01-01

    The testes of stillborn fetuses (from 13 to 28 weeks of gestational age), fetuses born alive (from 29 weeks of gestational age) who died a few days later, and infants dying 1 to 8 months after birth were processed for light and electron microscopy. Paraffin-embedded material was stained with the avidin-biotin peroxidase complex (ABC) method for immunohistochemical detection of testosterone (T) in order to quantify the age-related changes in the number of T-positive interstitial cells. This number decreased progressively from the 24th week of gestation up to birth and remained unchanged up to the second month of postnatal life. During the third month of age, the number of T-positive cells rose markedly but fell again from the fourth month to the end of the study. The ultrastructural study revealed the following types of interstitial cells at all ages studied: fibroblast-like cells, myofibroblast-like cells, developed fetal Leydig cells, degenerating fetal Leydig cells and infantile Leydig cells with a multilobed nucleus and focal cytoplasmic accumulations of smooth endoplasmic reticulum and lipid droplets. Quantitative ultrastructural studies revealed that the changes in the number of fetal Leydig cells with age were similar to those found in the number of T-positive cells although, for each age studied, absolute values were higher in the ultrastructural study. The number of infantile Leydig cells increased with age. Images Figs. 1-4 Figs. 5-9 Figs. 10-11 PMID:2272896

  13. Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells

    PubMed Central

    Kilcoyne, Karen R.; Smith, Lee B.; Atanassova, Nina; Macpherson, Sheila; McKinnell, Chris; van den Driesche, Sander; Jobling, Matthew S.; Chambers, Thomas J. G.; De Gendt, Karel; Verhoeven, Guido; O’Hara, Laura; Platts, Sophie; Renato de Franca, Luiz; Lara, Nathália L. M.; Anderson, Richard A.; Sharpe, Richard M.

    2014-01-01

    Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk. PMID:24753613

  14. Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells.

    PubMed

    Pomara, Cristoforo; Barone, Rosario; Marino Gammazza, Antonella; Sangiorgi, Claudia; Barone, Fulvio; Pitruzzella, Alessandro; Locorotondo, Nicola; Di Gaudio, Francesca; Salerno, Monica; Maglietta, Francesca; Sarni, Antonio Luciano; Di Felice, Valentina; Cappello, Francesco; Turillazzi, Emanuela

    2016-06-01

    Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real-time PCR. The results obtained show that testosterone levels increase at a 3.9 μM concentration of nandrolone and return to the basal level a 15.6 μM dose of nandrolone. Nandrolone-induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a-hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down-regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects. PMID:26626779

  15. Phthalate ester toxicity in Leydig cells: developmental timing and dosage considerations.

    PubMed

    Ge, Ren-Shan; Chen, Guo-Rong; Tanrikut, Cigdem; Hardy, Matthew P

    2007-01-01

    Humans have significant exposures to phthalates, as these chemical plasticizers are ubiquitously present in flexible plastics. Recent epidemiological evidence indicates that boys born to women exposed to phthalates during pregnancy have an increased incidence of congenital genital malformations and spermatogenic dysfunction, signs of a condition referred to as testicular dysgenesis syndrome (TDS). TDS is thought to develop as a result of environmental factors that cause a testicular disturbance at an early fetal stage with a resultant spectrum of clinical testicular dysfunction, ranging from impaired spermatogenesis and genital malformations to increased risk for development of testicular cancer. Proposed environmental factors in the etiology of TDS include endocrine disrupting compounds such as the phthalates. Leydig cells have been classified as one of the main targets for phthalate ester toxicity in the body based on studies in rodents. In support of this hypothesis, two Leydig cell products - insulin-like growth factor 3 (INSL3) and testosterone (T) - are both suppressed after phthalate exposures. Both fetal and adult generations of Leydig cells are affected by phthalate esters, although their sensitivities may differ. In rodent models, when pregnant dams are exposed to phthalate esters, fetal Leydig cells form enlarged clusters that are retained in the testis even after birth, in contrast to untreated controls. Despite the retention of fetal Leydig cells, however, their numbers and average cell volume of total in exposed males are reduced, as are INSL3 production and steroidogenic competence. These alterations are directly associated with clinical features of TDS, including cryptorchidism and impaired spermatogenesis. PMID:17258888

  16. Changes in mouse Leydig cell steroidogenesis following infrared and helium-neon laser irradiation.

    PubMed

    Celani, M F; Grandi, M; Gilioli, G

    1987-03-01

    The effects on mouse Leydig cell steroidogenesis of infrared (IR) laser rays, in the presence or absence of helium-neon (He-Ne) radiations, were investigated. Testosterone (T) production in response to luteinizing hormone (LH) by mouse Leydig cells exposed to IR (4.2 X 10(-3) J/cm2/min) plus He-Ne (8.0 X 10(-7) J/cm2/min) laser radiations was significantly higher than that by control Leydig cells. The Leydig cell responsiveness to LH (T delta %), as well as the secretion of cyclic AMP (cAMP) and androstenedione (A) in response to the highest dose of LH (0.5 mIU), were also significantly increased by the IR plus He-Ne irradiation. In contrast, the He-Ne irradiation (8.0 X 10(-7) J/cm2/min) in the absence of IR rays failed to affect T production by mouse Leydig cells. Similar results were obtained by adding to the He-Ne rays a low dose of IR radiation (3.4 X 10(-3) J/cm2/min), whereas higher doses of IR radiations (4.2 X 10(-3) and 5.1 X 10(-3) J/cm2/min) elicited a similar significant increase of T production by mouse interstitial cells. PMID:3595730

  17. INHIBITION OF TESTICULAR STEROIDOGENESIS BY THE XENOESTROGEN BISPHENOL A IS ASSOCIATED WITH REDUCED PITUITARY LH SECRETION AND DECREASED STEROIDOGENIC ENZYME GENE EXPRESSION IN RAT LEYDIG CELLS

    EPA Science Inventory

    Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and constituent of resins used in food packaging and denistry, is significant. In this report, exposure of rats to 2.4 ug/kg/day (a dose that approximates BPA levels in the environment) from postnatal da...

  18. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  19. Sertoli–Leydig cell tumor of the ovary: A diagnostic dilemma

    PubMed Central

    Liggins, Casandra A.; Ma, Ly T.; Schlumbrecht, Matthew P.

    2015-01-01

    Background Sertoli–Leydig cell tumors are rare sex-cord stromal tumors of the ovary that can present with a variety of histological elements, which may complicate diagnosis and treatment. Case A 40-year-old female presenting with pelvic pain is found to have a large complex right adnexal mass and elevated alpha-fetoprotein. The mass was diagnosed as a Sertoli–Leydig cell tumor with heterologous elements including carcinoid and hepatoid components. She was treated with surgical resection followed by adjuvant chemotherapy and remains clear of disease. Conclusion Prognostic indicators for Sertoli–Leydig cell tumors include degree and type of heterologous element differentiation. Thorough characterization of such elements is crucial for adequate diagnosis and treatment. PMID:26937481

  20. Comments on Li et al. Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells. Int. J. Environ. Res. Public Health 2016, 13, 246

    PubMed Central

    Svingen, Terje

    2016-01-01

    Profiling the expression levels of genes or proteins in tissues comprising two or more cell types is commonplace in biological sciences. Such analyses present particular challenges, however, for example a potential shift in cellular composition, or ‘cellularity’, between specimens. That is, does an observed change in expression level represent what occurs within individual cells, or does it represent a shift in the ratio of different cell types within the tissue? This commentary attempts to highlight the importance of considering cellularity when interpreting quantitative expression data, using the mammalian testis and a recent study on the effects of phthalate exposure on testis function as an example. PMID:27231928

  1. Comments on Li et al. Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells. Int. J. Environ. Res. Public Health 2016, 13, 246.

    PubMed

    Svingen, Terje

    2016-01-01

    Profiling the expression levels of genes or proteins in tissues comprising two or more cell types is commonplace in biological sciences. Such analyses present particular challenges, however, for example a potential shift in cellular composition, or 'cellularity', between specimens. That is, does an observed change in expression level represent what occurs within individual cells, or does it represent a shift in the ratio of different cell types within the tissue? This commentary attempts to highlight the importance of considering cellularity when interpreting quantitative expression data, using the mammalian testis and a recent study on the effects of phthalate exposure on testis function as an example. PMID:27231928

  2. Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells

    PubMed Central

    Wu, Han; Shi, Lili; Wang, Qing; Cheng, Lijing; Zhao, Xiang; Chen, Qiaoyuan; Jiang, Qian; Feng, Min; Li, Qihan; Han, Daishu

    2016-01-01

    Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis. PMID:26776505

  3. GATA4 Is a Key Regulator of Steroidogenesis and Glycolysis in Mouse Leydig Cells

    PubMed Central

    Schrade, Anja; Kyrönlahti, Antti; Akinrinade, Oyediran; Pihlajoki, Marjut; Häkkinen, Merja; Fischer, Simon; Alastalo, Tero-Pekka; Velagapudi, Vidya; Toppari, Jorma; Wilson, David B.

    2015-01-01

    Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are down-regulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4flox/flox adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type. PMID:25668067

  4. Developmental and Endocrine Regulation of Kisspeptin Expression in Mouse Leydig Cells

    PubMed Central

    Adeshina, Ikeoluwa; Chen, Haolin; Zirkin, Barry R.; Hussain, Mehboob A.; Wondisford, Fredric; Wolfe, Andrew

    2015-01-01

    Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function. PMID:25635620

  5. Androgen-Forming Stem Leydig cells: Identification, Function and Therapeutic Potential

    PubMed Central

    Zhang, Yunhui; Ge, Renshan; Hardy, Matthew P.

    2008-01-01

    Leydig cells are the primary source of testosterone in the male, and differentiation of Leydig cells in the testes is one of the primary events in the development of the male body and fertility. Stem Leydig cells (SLCs) exist in the testis throughout postnatal life, but a lack of cell surface markers previously hindered attempts to obtain purified SLC fractions. Once isolated, the properties of SLCs provide interesting clues for the ontogeny of these cells within the embryo. Moreover, the clinical potential of SLCs might be used to reverse age-related declines in testosterone levels in aging men, and stimulate reproductive function in hypogonadal males. This review focuses on the source, identification and outlook for therapeutic applications of SLCs. Separate pools of SLCs may give rise to fetal and adult generations of Leydig cell, which may account for their observed functional differences. These differences should in turn be taken into account when assessing the consequences of environmental pollutants such as the phthalate ester, diethylhexylphthalate (DEHP). PMID:18525122

  6. Modulation of mouse Leydig cell steroidogenesis through a specific arginine-vasopressin receptor

    SciTech Connect

    Tahri-Joutei, A.; Pointis, G.

    1988-01-01

    Characterization of specific vasopressin binding sites was investigated in purified mouse Leydig cells using tritiated arginine-vasopressin. Binding of radioligand was saturable, time- and temperature-dependent and reversible. (/sup 3/H)-AVP was found to bind to a single class of sites with high affinity and low capacity. Binding displacements with specific selection analogs of AVP indicated the presence of V/sub 1/ subtype receptors on Leydig cells. The ability of AVP to displace (/sup 3/H)-AVP binding was greater than LVP and oxytocin. The unrelated peptides, somatostatin and substance P, were less potent, while neurotensin and LHRH did not displace (/sup 3/H)-AVP binding. The time-course effects of AVP-pretreatment on basal and hCG-stimulated testosterone and cAMP accumulations were studied in primary culture of Leydig cells. Basal testosterone accumulation was significantly increased by a 24 h AVP-pretreatment of Leydig cells. This effect was potentiated by the phosphodiesterase inhibitor (MIX) and was concomitantly accompanied by a slight but significant increase in cAMP accumulation. AVP-pretreatment of the cells for 72 h had no effect on basal testosterone accumulation, but exerted a marked inhibitory effect on the hCG-stimulated testosterone accumulation. This reduction of testosterone accumulation occurred even in the presence of MIX and was not accompanied by any significant change of cAMP levels.

  7. Mutational analysis of the luteinizing hormone receptor gene in two individuals with Leydig cell tumors.

    PubMed

    Canto, Patricia; Söderlund, Daniela; Ramón, Guillermo; Nishimura, Elisa; Méndez, Juan Pablo

    2002-03-01

    Inactivating mutations of the luteinizing hormone receptor (LHR) gene in males induce Leydig cell agenesis or hypoplasia, while activating mutations cause testotoxicosis. Recently, it was demonstrated that a somatic heterozygous activating mutation of the LHR gene (Asp578His), limited to the tumor, was the cause of Leydig cell adenomas in three unrelated patients. We describe the molecular study of two unrelated boys with gonadotropin-independent hypersecretion of testosterone due to Leydig cell adenomas. Genomic DNA was extracted from the tumor, the adjacent normal testis tissue, and blood leukocytes. Both individuals exhibited an heterozygous missense mutation, limited only to the tumor, consisting of a guanine (G) to cytosine (C) substitution at codon 578 (GAT to CAT), turning aspartic acid into histidine. The presence of the same mutation in different ethnic groups demonstrates the existence of a mutational hot spot in the LHR gene. Indeed, this mutation occurs at the conserved aspartic acid residue at amino acid 578, where a substitution by glycine is the most common mutation observed in testotoxicosis and where a substitution by tyrosine has been linked to a more severe clinical phenotype where diffuse Leydig cell hyperplasia is found. Our results confirm the fact that somatic activating mutations of gonadotropin receptors are involved in gonadal tumorigenesis. PMID:11857565

  8. EFFECT OF CADMIUM AND OTHER METAL CATIONS ON IN VITRO LEYDIG CELL TESTOSTERONE PRODUCTION

    EPA Science Inventory

    In vivo assessment of toxicant action on Leydig cell function is subject to homeostatic mechanisms which make it difficult to determine whether any changes seen in serum testosterone (T) concentration are due to extragonadal endocrine alternations or to a direct effect on the Ley...

  9. True Precocious Puberty Following Treatment of a Leydig Cell Tumor: Two Case Reports and Literature Review

    PubMed Central

    Verrotti, Alberto; Penta, Laura; Zenzeri, Letizia; Lucchetti, Laura; Giovenali, Paolo; De Feo, Pierpaolo

    2015-01-01

    Leydig cell testicular tumors are a rare cause of precocious pseudopuberty in boys. Surgery is the main therapy and shows good overall prognosis. The physical signs of precocious puberty are expected to disappear shortly after surgical removal of the mass. We report two children, 7.5 and 7.7 year-old boys, who underwent testis-sparing surgery for a Leydig cell testicular tumor causing precocious pseudopuberty. During follow-up, after an immediate clinical and laboratory regression, both boys presented signs of precocious puberty and ultimately developed central precocious puberty. They were successfully treated with gonadotropin-releasing hormone (GnRH) analogs. Only six other cases have been described regarding the development of central precocious puberty after successful treatment of a Leydig cell tumor causing precocious pseudopuberty. Gonadotropin-dependent precocious puberty should be considered in children treated for a Leydig cell tumor presenting persistent or recurrent physical signs of puberty activation. In such cases, therapy with GnRH analogs appears to be the most effective medical treatment. PMID:26579503

  10. A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG.

    PubMed

    Mazaud Guittot, Séverine; Vérot, Adélie; Odet, Fanny; Chauvin, Marie-Agnès; le Magueresse-Battistoni, Brigitte

    2008-04-01

    Extracellular matrix (ECM) proteins have been shown to alter Leydig cell steroidogenesis in vitro, substantiating the hypothesis that Leydig cell steroidogenic activity and matrix environment are interdependent events. However, the nature of the ECM components synthesized by Leydig cells and their regulation by LH/human chorionic gonadotropin (hCG) remain unknown. Here, we examine the occurrence of the 11 laminin subunits and the 6 alpha chains of collagen IV (COL4A1-6) by RT-PCR in Leydig cells cultured with or without LH/hCG. Leydig cells were a tumor Leydig cell line (mLTC-1) or 8-week-old mice Leydig cells. Based on PCR data, it is suggested that normal Leydig cells may synthesize a maximum of 11 laminin heterotrimers and the 6 alpha chains of collagen IV. They also may synthesize various proteases and inhibitors of the metzincin family. The mLTC-1 cells have a limited repertoire as compared with normal Leydig cells. Interestingly, none of the ten proteases and inhibitors monitored is under LH-hCG regulation whereas every protease and inhibitor of the serine protease family yet identified in Leydig cells is under gonadotropin regulation. In addition, a few laminin and collagen subunit genes are regulated by LH/hCG. These are laminins alpha3 and gamma3 (Lama3 and Lamc3), Col4a3, and Col4a6, which are negatively regulated by LH/hCG in both Leydig cell types, and Col4a4, which was downregulated in primary cultures but not in mLTC-1 cells. Collectively, the present study suggests that Leydig cells modulate in a selective fashion their matrix environment in response to their trophic hormone. This may alter the steroidogenic outcome of Leydig cells. PMID:18367508

  11. Intracellular redistribution of SCP2 in Leydig cells after hormonal stimulation may contribute to increased pregnenolone production.

    PubMed

    van Noort, M; Rommerts, F F; van Amerongen, A; Wirtz, K W

    1988-07-15

    Sterol carrier protein2 (SCP2) also designated non specific lipid transfer protein (nsL-TP), added to tumour Leydig cell mitochondria as a pure compound or in cytosolic preparations, stimulates pregnenolone production two- to three-fold. This stimulation can be abolished by addition of anti rat SCP2 but not by preimmune IgG-antibodies. SCP2- levels in the cytosol are increased in less than two minutes after addition of lutropin (LH). This increased SCP2 level may contribute to stimulation of steroid production in intact cells. After hormonal stimulation the subcellular distribution of SCP2 changes. A two-fold increase of SCP2- levels in the supernatant fraction and four-fold decrease in extracts of the particulate fraction was observed 30 min after stimulation of tumour Leydig cells with LH and subsequent fractionation. This apparent shift of SCP2 can be explained by an altered association with membranes or a true relocation of the protein from the particulate to the supernatant fractions under the influence of the hormone. PMID:3395346

  12. Large moderately-differentiated ovarian Sertoli-Leydig cell tumor in a 13-year-old female: A case report

    PubMed Central

    ZHANG, HUI; HAO, JING; LI, CHUN-YAN; LI, TAO; MU, YU-LAN

    2016-01-01

    Sertoli-Leydig cell tumor of the ovary, also known as androblastoma, is a rare neoplasm from the group of sex cord-stromal tumors of the ovary. The tumor accounts for <0.5% of all primary ovarian neoplasms. The clinical signs and symptoms of Sertoli-Leydig cell tumors can be associated with either hormonal production or the presence of a mass-occupying lesion. In the current study, a 13-year-old female was diagnosed with a stage Ic ovarian Sertoli-Leydig cell tumor following abdominal pain and distension. One month after a right oophorectomy, the follow-up magnetic resonance imaging scan was negative for residual or recurrent tumor. The overall 5-year survival rate for moderately-differentiated (grade 2) and poorly-differentiated (grade 3) Sertoli-Leydig cell tumors is 80%, and long-term follow-up is therefore highly advised in this patient. PMID:26893701

  13. Pdgfr-α mediates testis cord organization and fetal Leydig cell development in the XY gonad

    PubMed Central

    Brennan, Jennifer; Tilmann, Christopher; Capel, Blanche

    2003-01-01

    During testis development, the rapid morphological changes initiated by Sry require the coordinate integration of many signaling pathways. Based on the established role of the platelet-derived growth factor (PDGF) family of ligands and receptors in migration, proliferation, and differentiation of cells in various organ systems, we have investigated the role of PDGF in testis organogenesis. Analysis of expression patterns and characterization of the gonad phenotype in Pdgfr-α−/− embryos identified PDGFR-α as a critical mediator of signaling in the early testis at multiple steps of testis development. Pdgfr-α−/− XY gonads displayed disruptions in the organization of the vasculature and in the partitioning of interstitial and testis cord compartments. Closer examination revealed severe reductions in characteristic XY proliferation, mesonephric cell migration, and fetal Leydig cell differentiation. This work identifies PDGF signaling through the α receptor as an important event downstream of Sry in testis organogenesis and Leydig cell differentiation. PMID:12651897

  14. Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction

    SciTech Connect

    Stammel, W.; Thomas, H. ); Staib, W.; Kuehn-Velten, W.K. )

    1991-01-01

    Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds may be mediators of the development of Leydig cell insufficiency. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC{sub 50} values being comparable to those of estradiol, 2-hydroxyestradiol, and the phytoestrogens, coumestrol and genistein; salsolinol and salsoline were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited substrate binding to cytochrome P45OXVII, with similar efficiency as the estrogens did; salsoline and salsolidine were again much less effective.

  15. T-2 toxin-induced cytotoxicity and damage on TM3 Leydig cells.

    PubMed

    Yuan, Zhihang; Matias, Froilan Bernard; Yi, Jin-E; Wu, Jing

    2016-01-01

    T-2 toxin is a highly toxic mycotoxin produced by various Fusarium species, mainly, Fusarium sporotrichoides, and has been reported to have toxic effects on reproductive system of adult male animals. This study investigated the dose-dependent cytotoxicity of T-2 toxin on reproductive cells using TM3 Leydig cells. Specifically, the cytotoxic effect of T-2 toxin was assessed by measuring cell viability; lactate dehydrogenase (LDH); malondialdehyde (MDA); antioxidant activity by measuring superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and DNA damage; and cell apoptosis. Results showed that T-2 toxin is highly cytotoxic on TM3 Leydig cells. However, Trolox-treated TM3 Leydig cells showed significantly reduced oxidative damage, DNA damage, and apoptosis induced by T-2 toxin. This study proves that T-2 toxin can damage the testes and thus affects the reproductive capacity of animals and humans. Furthermore, oxidative stress plays an important role in the cytotoxic effect of T-2 toxin. PMID:26707243

  16. Disruption of NHE8 expression impairs Leydig cell function in the testes.

    PubMed

    Xu, Hua; Chen, Huacong; Li, Jing; Zhao, Yang; Ghishan, Fayez K

    2015-02-15

    Multiple sodium/hydrogen exchanger (NHE) isoforms are expressed in the testes, and they play various roles in cell volume regulation, intracellular pH regulation, and fluid absorption. NHE8, the most recently characterized NHE family member, is detected in the Leydig cells in humans and mice in great abundance by immunohistochemistry in the current study. Male mice lacking NHE8 expression were infertile. Despite having intact male reproductive organs, male NHE8-/- mice have smaller testes and lacked spermatozoon in the seminiferous tubules and the epididymis. At the age of 39 wk, few spermogonia were seen in the testis in NHE8-/- mice. Although male NHE8-/- mice have normal serum levels of luteinizing hormone and follicle-stimulating hormone, serum testosterone level was significantly reduced. These mice have decreased expression of luteinizing hormone receptor in the testes. In NHE8 small-interfering RNA-transfected mouse Leydig cells (MLTC-1), silencing of NHE8 decreased the expression of luteinizing hormone receptor by ∼70%. Moreover, loss of NHE8 function in Leydig cells resulted in disorganized luteinizing hormone receptor membrane distribution. Therefore, male infertility in NHE8-/- mice is at least partially due to the disruption of luteinizing hormone receptor distribution and consequent low testosterone production, which leads to Sertoli cell dysfunction. Our work identified a novel role of NHE8 in male fertility through its effect on modifying luteinizing hormone receptor function. PMID:25472965

  17. GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

    PubMed

    Bergeron, Francis; Nadeau, Gabriel; Viger, Robert S

    2015-03-01

    GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program. PMID:25504870

  18. Leydig-cell function in children after direct testicular irradiation for acute lymphoblastic leukemia

    SciTech Connect

    Brauner, R.; Czernichow, P.; Cramer, P.; Schaison, G.; Rappaport, R.

    1983-07-07

    To assess the effect of testicular irradiation on testicular endocrine function, we studied 12 boys with acute lymphoblastic leukemia who had been treated with direct testicular irradiation 10 months to 8 1/2 years earlier. Insufficient Leydig-cell function, manifested by a low response of plasma testosterone to chorionic gonadotropin or an increased basal level of plasma luteinizing hormone (or both), was observed in 10 patients, 7 of whom were pubertal. Two of these patients had a compensated testicular endocrine insufficiency with only high plasma concentrations of luteinizing hormone. Testosterone secretion was severely impaired in three pubertal boys studied more than four years after testicular irradiation. A diminished testicular volume indicating tubular atrophy was found in all pubertal patients, including three who had not received cyclophosphamide or cytarabine. These data indicate that testosterone insufficiency is a frequent complication of testicular irradiation, although some patients continue to have Leydig-cell activity for several years after therapy.

  19. Immunofluorescent localization of the StAR protein in mitochondria of mouse Leydig cells in vitro.

    PubMed

    Kotula, M; Kozieł, E; Sadowska, J; Gancarczyk, M; Bilińska, B

    2001-01-01

    The Steroidogenic Acute Regulatory (StAR) protein is assumed to enhance the rate-limiting step of the steroid biosynthesis. Now, it is the most likely candidate, responsible for acutely regulating transfer of cholesterol from the outer to the inner mitochondrial membrane. In this study, the immunoreactive StAR protein was observed in the mitochondria of mouse cultured Leydig cells stimulated by hCG andtesticular macrophage-conditioned medium. Immunocytochemistry was performed using a polyclonal rabbit antibody against the StAR protein. For selective staining of mitochondria in Leydig cells, the Mito Tracker dye was used. Computerized, superimposed images from double-fluorescence staining showed a remarkable degree of similarity in the distribution of the StAR protein and mitochondria, indicating mitochondrial localization of StAR. PMID:11374809

  20. Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis.

    PubMed

    Jones, Steven; Boisvert, Annie; Naghi, Andrada; Hullin-Matsuda, Françoise; Greimel, Peter; Kobayashi, Toshihide; Papadopoulos, Vassilios; Culty, Martine

    2016-04-29

    Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10μM GEN+10μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN+MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10μM combined GEN+MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN+MEHP. We propose a working model for GEN+MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally relevant doses

  1. Endocrine regulation of testosterone production by Leydig cells in the catfish, Clarias batrachus: probable mediators of growth hormone.

    PubMed

    Nee Pathak, N Dubey; Kumar, Pankaj; Lal, Bechan

    2015-03-01

    Growth hormone (GH), in the recent past, has been recognized as a potent steroid stimulating hormone independent of gonadotropin (GtH). However, the mode and mechanism of its steroidogenic action in the testis is not yet elucidated, particularly in fish. The present study was designed to understand the mode and mechanism of steroidogenic action of growth hormone in testis of the catfish, Clarias batrachus through in vivo and in vitro Leydig cell culture studies using the signaling molecule inhibitors. Exogenous administration of GtH, GH and insulin to the male catfish increased testicular and circulating testosterone level. In vitro treatment of Leydig cells with these hormones also increased testosterone production. The steroidogenic action of GH appeared to be indirect and mediated through Leydig cell produced insulin-like growth factor I (IGF-I), as the treatments with actinomycin D, cycloheximide and anti-IGF-I abolished the GH-induced testosterone production by Leydig cells. The GH-induced stimulation in IGF-I production by the isolated Leydig cells further substantiates this notion. GH appears to employ cAMP/PKA and tyrosine kinase signaling pathways to induce IGF-I production, as the adenylyl cyclase inhibitor (SQ 22,536), cAMP-dependent protein kinase (PKA) blocker (H-89) and tyrosine kinase inhibitor (lavendustin A) abolished the GH-induced IGF-I production and in turn testosterone by the Leydig cells. This study suggests that GH exerts independent androgenic effect in the catfish testis indirectly through augmenting the Leydig cell production of IGF-I. PMID:25650168

  2. Overexpression of PRL7D1 in Leydig Cells Causes Male Reproductive Dysfunction in Mice.

    PubMed

    Liu, Yaping; Su, Xingyu; Hao, Jie; Chen, Maoxin; Liu, Weijia; Liao, Xiaogang; Li, Gang

    2016-01-01

    Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice. PMID:26771609

  3. Overexpression of PRL7D1 in Leydig Cells Causes Male Reproductive Dysfunction in Mice

    PubMed Central

    Liu, Yaping; Su, Xingyu; Hao, Jie; Chen, Maoxin; Liu, Weijia; Liao, Xiaogang; Li, Gang

    2016-01-01

    Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice. PMID:26771609

  4. Isolation and Characterization of Fetal Leydig Progenitor Cells of Male Mice.

    PubMed

    Inoue, Miki; Shima, Yuichi; Miyabayashi, Kanako; Tokunaga, Kaori; Sato, Tetsuya; Baba, Takashi; Ohkawa, Yasuyuki; Akiyama, Haruhiko; Suyama, Mikita; Morohashi, Ken-Ichirou

    2016-03-01

    Fetal and adult Leydig cells develop in mammalian prenatal and postnatal testes, respectively. In mice, fetal Leydig cells (FLCs) emerge in the interstitial space of the testis at embryonic day 12.5 and thereafter increase in number, possibly through differentiation from progenitor cells. However, the progenitor cells have not yet been identified. Previously, we established transgenic mice in which FLCs are labeled strongly with enhanced green fluorescent protein (EGFP). Interestingly, fluorescence-activated cell sorting provided us with weakly EGFP-labeled cells as well as strongly EGFP-labeled FLCs. In vitro reconstruction of fetal testes demonstrated that weakly EGFP-labeled cells contain FLC progenitors. Transcriptome from the 2 cell populations revealed, as expected, marked differences in the expression of genes required for growth factor/receptor signaling and steroidogenesis. In addition, genes for energy metabolisms such as glycolytic pathways and the citrate cycle were activated in strongly EGFP-labeled cells, suggesting that metabolism is activated during FLC differentiation. PMID:26697723

  5. A potential role for zinc transporter 7 in testosterone synthesis in mouse Leydig tumor cells.

    PubMed

    Chu, Qingqing; Chi, Zhi-Hong; Zhang, Xiuli; Liang, Dan; Wang, Xuemei; Zhao, Yue; Zhang, Li; Zhang, Ping

    2016-06-01

    Previous studies have demonstrated that zinc (Zn) is an essential trace element which is involved in male reproduction. The zinc transporter (ZnT) family, SLC30a, is involved in the maintenance of Zn homeostasis and in mediating intracellular signaling events; however, relatively little is known regarding the effect of ZnTs on testosterone synthesis. Thus, in the present study, we aimed to determine the effect of Zn transporter 7 (ZnT7) on testosterone synthesis in male CD-1 mice and mouse Leydig cells. The findings of the present study revealed that the concentrations of Zn in the testes and Leydig cells were significantly lower in mice fed a Zn-deficient diet compared with the control mice fed a Zn-adequate diet. In addition, ZnT7 was principally expressed and colocalized with steroidogenic acute regulatory protein (StAR) in the Leydig cells of male CD-1 mice. ZnT7 expression was downregulated in the mice fed a Zn-deficient diet, which led to decreases in the expression of the enzymes involved in testosterone synthesis namely cholesterol side‑chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerase (3β-HSD) as well as decreased serum testosterone levels. These results suggested that Znt7 may be involved in testosterone synthesis in the mouse testes. To examine this hypothesis, we used the mouse Leydig tumor cell line (MLTC-1 cell line) in which the ZnT7 gene had been silenced, in order to gauge the impact of changes in ZnT7 expression on testosterone secretion and the enzymes involved in testosterone synthesis. The results demonstrated that ZnT7 gene silencing downregulated the expression of StAR, P450scc and 3β-HSD as well as progesterone concentrations in the human chorionic gonadotrophin (hCG)-stimulated MLTC-1 cells. Taken together, these findings reveal that ZnT7 may play an important role in the regulation of testosterone synthesis by modulating steroidogenic enzymes, and may represent a therapeutic target in

  6. Astaxanthin protects steroidogenesis from hydrogen peroxide-induced oxidative stress in mouse Leydig cells.

    PubMed

    Wang, Jyun-Yuan; Lee, Yue-Jia; Chou, Mei-Chia; Chang, Renin; Chiu, Chih-Hsien; Liang, Yao-Jen; Wu, Leang-Shin

    2015-03-01

    Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress. PMID:25786065

  7. Astaxanthin Protects Steroidogenesis from Hydrogen Peroxide-Induced Oxidative Stress in Mouse Leydig Cells

    PubMed Central

    Wang, Jyun-Yuan; Lee, Yue-Jia; Chou, Mei-Chia; Chang, Renin; Chiu, Chih-Hsien; Liang, Yao-Jen; Wu, Leang-Shin

    2015-01-01

    Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS) scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST), a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2) to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa) form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress. PMID:25786065

  8. The Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells.

    PubMed

    Di-Luoffo, Mickaël; Brousseau, Catherine; Bergeron, Francis; Tremblay, Jacques J

    2015-12-01

    Testosterone is essential for spermatogenesis and the development of male sexual characteristics. However, steroidogenesis produces a significant amount of reactive oxygen species (ROS), which can disrupt testosterone production. The myocyte enhancer factor 2 (MEF2) is an important regulator of organogenesis and cell differentiation in various tissues. In the testis, MEF2 is present in Sertoli and Leydig cells throughout fetal and adult life. MEF2-deficient MA-10 Leydig cells exhibit a significant decrease in steroidogenesis concomitant with a reduction in glutathione S-transferase (GST) activity and in the expression of the 4 Gsta members (GST) that encode ROS inactivating enzymes. Here, we report a novel role for MEF2 in ROS detoxification by directly regulating Gsta expression in Leydig cells. Endogenous Gsta1-4 mRNA levels were decreased in MEF2-deficient MA-10 Leydig cells. Conversely, overexpression of MEF2 increased endogenous Gsta1 levels. MEF2 recruitment to the proximal Gsta1 promoter and direct binding on the -506-bp MEF2 element were confirmed by chromatin immunoprecipitation and DNA precipitation assays. In MA-10 Leydig cells, MEF2 activates the Gsta1 promoter and cooperates with Ca(2+)/calmodulin-dependent kinases I to further enhance Gsta1 promoter activity. These effects were lost when the -506-bp MEF2 element was mutated or when a MEF2-Engrailed dominant negative protein was used. Similar results were obtained on the Gsta2, Gsta3, and Gsta4 promoters, suggesting a global role for MEF2 factors in the regulation of all 4 Gsta genes. Altogether, our results identify a novel role for MEF2 in the expression of genes involved in ROS detoxification, a process essential for adequate testosterone production in Leydig cells. PMID:26393304

  9. Chemical Shift Images of Organelles in Leydig cells of Mice Testes

    NASA Astrophysics Data System (ADS)

    Ejima, T.; Neichi, Y.; Yanagihara, M.; Kado, M.; Ishino, M.; Yasuda, K.; Tamotsu, S.

    2013-10-01

    Soft X-ray transmission images of Leydig cells of mice testes changing incident wavelength were observed with the use of a contact microscope. After normalization of transmission images, absorbance images were obtained and compared with a visible differential interference image. Some organelles were identified by the image comparison, and absorption spectra of the organelles were obtained from the absorbance images. The absorption spectra show that peak structures are different depending on the observed organelles. The structures and the positions of organelles were clearly identified at C-K absorption.

  10. Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway.

    PubMed

    Lin, Pengfei; Chen, Fenglei; Sun, Jin; Zhou, Jinhua; Wang, Xiangguo; Wang, Nan; Li, Xiao; Zhang, Zhe; Wang, Aihua; Jin, YaPing

    2015-04-01

    Zearalenone (ZEN) is a Fusarium mycotoxin that causes several reproductive disorders and genotoxic effects. This study demonstrated the involvement of endoplasmic reticulum (ER) stress in ZEN-induced mouse Leydig cell death. Our study showed that ZEN reduced cell proliferation in a murine Leydig tumour cell line in a dose-dependent manner. The involvement of apoptosis as a major cause of ZEN-induced cell death was further confirmed by the results of a caspase-3 activity assay, which showed a ZEN dose-dependent increase in cell death. Treatment of MLTC-1 and primary mouse Leydig cells with ZEN upregulated the expression of the ER stress-typical markers GRP78, CHOP and caspase-12 protein. Further, pre-treating the cells with 4-phenylbutyrate or knocking down GRP78 using lentivirus-encoded shRNA significantly diminished ZEN-induced apoptosis and inhibited the expression of CHOP and caspase-12. In summary, these results suggest that the activation of an ER stress pathway plays a key role in ZEN-induced apoptosis in the mouse Leydig cells. PMID:25720297

  11. Effects of polychlorinated biphenyls (PCBs) on in vitro biosynthesis of testosterone and cell viability in mouse Leydig cells

    SciTech Connect

    Johansson, B.

    1989-01-01

    Some PCBs (polychlorinated biphenyls) show a tendency to accumulate in steroid-producing organs such as the adrenals, testes and ovaries. Moreover, some hexachlorobiphenyls are accumulated in the interstitial part of the testis, where the steroid-producing cells are located (Brandt 1977). In an earlier study (Johansson 1987) the authors investigated the in vivo effects of PCBs on mice. They could not find any evidence for effects of Clophen A50 and 2,2',4,4',5,5'-hexachlorobiphenyl on plasma testosterone levels or on the ability of the Leydig cells to respond to luteinizing hormone (LH). Despite these results they wanted to determine whether PCBs have any effect on testosterone synthesis when administered to Leydig cells in vitro, since it has been shown earlier that a substance having no effects on testosterone synthesis when given in vivo can have drastic effects when administered in vitro.

  12. Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

    PubMed

    Bozon, V; Pajot-Augy, E; Vignon, X; Salesse, R

    1998-08-25

    In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars. PMID:9806348

  13. Role of peroxiredoxin 2 in H2O2‑induced oxidative stress of primary Leydig cells.

    PubMed

    Duan, Ting; Fan, Kai; Chen, Shengrong; Yao, Qi; Zeng, Rong; Hong, Zhiwei; Peng, Longping; Shao, Yong; Yao, Bing

    2016-06-01

    Late‑onset hypogonadism is defined as a condition caused by a decline in the levels of testosterone with aging. One of the major factors contributing to the low levels of testosterone is the accumulation of reactive oxygen species (ROS) in Leydig cells during the ageing process. Peroxiredoxin 2 (Prdx2), a member of the peroxiredoxin family, is an antioxidant protein, the predominant function of which is to neutralize ROS. However, its role in Leydig cells remains to be elucidated. In the present study, primary Leydig cells were exposed to low concentrations of hydrogen peroxide (H2O2) to induce oxidative stress. Cell apoptosis was measured using an Annexin V fluorescein isothiocyanate/propidium iodide apoptosis detection kit and flow cytometry. The level of testosterone was determined by radioimmunoassay, and the mRNA and protein expression levels of Prdx2 were detected by reverse transcription‑polymerase chain reaction and western blotting, respectively. The results revealed a significant increase in cell apoptosis and decrease in testosterone production. In addition, the expression of Prdx2 was decreased by H2O2 in a dose‑ and time‑dependent manner, and this decrease may have been caused by the induction of its molecular structure transformation due to H2O2 elimination. The above findings indicated that Prdx2 may prevent H2O2 accumulation in Leydig cells, and may be important in oxidative stress‑induced apoptosis and decreased testosterone production. PMID:27082744

  14. Leydig cell number and sperm production decrease induced by chronic ametryn exposure: a negative impact on animal reproductive health.

    PubMed

    Dantas, T A; Cancian, G; Neodini, D N R; Mano, D R S; Capucho, C; Predes, F S; Pulz, R Barbieri; Pigoso, A A; Dolder, H; Severi-Aguiar, G D C

    2015-06-01

    Ametryn is an herbicide used to control broadleaf and grass weeds and its acute and chronic toxicity is expected to be low. Since toxicological data on ametryn is scarce, the aim of this study was to evaluate rat reproductive toxicity. Thirty-six adult male Wistar rats (90 days) were divided into three groups: Co (control) and T1 and T2 exposed to 15 and 30 mg/kg/day of ametryn, respectively, for 56 days. Testicular analysis demonstrated that ametryn decreased sperm number per testis, daily sperm production, and Leydig cell number in both treated groups, although little perceptible morphological change has been observed in seminiferous tubule structure. Lipid peroxidation was higher in group T2, catalase activity decreased in T1 group, superoxide dismutase activity diminished, and a smaller number of sulphydryl groups of total proteins were verified in both exposed groups, suggesting oxidative stress. These results showed negative ametryn influence on the testes and can compromise animal reproductive performance and survival. PMID:25561257

  15. Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus×Graomys centralis male hybrids.

    PubMed

    Díaz de Barboza, Gabriela; Rodríguez, Valeria; Ponce, Rubén; Theiler, Gerardo; Maldonado, Cristina; Tolosa de Talamoni, Nori

    2014-07-01

    Spermatogenesis is disrupted in Graomys griseoflavus×Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D28k. PMID:24894511

  16. Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

    SciTech Connect

    Iwase, Yumiko . E-mail: Iwase.Yumiko@mg.m-pharma.co.jp; Fukata, Hideki . E-mail: fukata@faculty.chiba-u.jp; Mori, Chisato . E-mail: cmori@faculty.chiba-u.jp

    2006-05-01

    Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17{beta}-estradiol (E{sub 2}, a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 {mu}M DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 {mu}M E{sub 2} and 25 {mu}M GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at {mu}M level between DES and E{sub 2} on GJIC inhibition was observed, but not between GEN and E{sub 2}. DES and E{sub 2} showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 {mu}M was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E{sub 2} (10 pM and 20 {mu}M) and GEN (25 {mu}M) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E{sub 2} and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual nongenotoxic mechanism including PKC pathway on testicular carcinogenesis or development.

  17. ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis.

    PubMed

    Fan, Jinjiang; Li, Xinlu; Issop, Leeyah; Culty, Martine; Papadopoulos, Vassilios

    2016-07-01

    Fatty acid metabolism and steroid biosynthesis are 2 major pathways shared by peroxisomes and mitochondria. Both organelles are in close apposition to the endoplasmic reticulum, with which they communicate via interorganelle membrane contact sites to promote cellular signaling and the exchange of ions and lipids. To date, no convincing evidence of the direct contact between peroxisomes and mitochondria was reported in mammalian cells. Hormone-induced, tightly controlled steroid hormone biosynthesis requires interorganelle interactions. Using immunofluorescent staining and live-cell imaging, we found that dibutyryl-cAMP treatment of MA-10 mouse tumor Leydig cells rapidly induces peroxisomes to approach mitochondria and form peroxisome-mitochondrial contact sites/fusion, revealed by the subcellular distribution of the endogenous acyl-coenzyme A-binding domain (ACBD)2/ECI2 isoform A generated by alternative splicing, and further validated using a proximity ligation assay. This event occurs likely via a peroxisome-like structure, which is mediated by peroxisomal and mitochondrial matrix protein import complexes: peroxisomal import receptor peroxisomal biogenesis factor 5 (PEX5), and the mitochondrial import receptor subunit translocase of outer mitochondrial membrane 20 homolog (yeast) protein. Similar results were obtained using the mLTC-1 mouse tumor Leydig cells. Ectopic expression of the ACBD2/ECI2 isoform A in MA-10 cells led to increased basal and hormone-stimulated steroid formation, indicating that ACBD2/ECI2-mediated peroxisomes-mitochondria interactions favor in the exchange of metabolites and/or macromolecules between these 2 organelles in support of steroid biosynthesis. Considering the widespread occurrence of the ACBD2/ECI2 protein, we propose that this protein might serve as a tool to assist in understanding the contact between peroxisomes and mitochondria. PMID:27167610

  18. Ultrastructural Studies of Germ Cell Development and the Functions of Leydig Cells and Sertoli Cells associated with Spermatogenesis in Kareius bicoloratus (Teleostei, Pleuronectiformes, Pleuronectidae)

    PubMed Central

    Kang, Hee-Woong; Kim, Sung Hwan; Chung, Jae Seung

    2016-01-01

    The ultrastructures of germ cells and the functions of Leydig cells and Sertoli cells during spermatogenesis inmale Kareius bicoloratus (Pleuronectidae) were investigated by electron microscope observation. Each of the well-developed Leydig cells during active maturation division and before spermiation contained an ovoid vesicular nucleus, a number of smooth endoplasmic reticula, well-developed tubular or vesicular mitochondrial cristae, and several lipid droplets in the cytoplasm. It is assumed that Leydig cells are typical steroidogenic cells showing cytological characteristics associated with male steroidogenesis. No cyclic structural changes in the Leydig cells were observed through the year. However, although no clear evidence of steroidogenesis or of any transfer of nutrients from the Sertoli cells to spermatogenic cells was observed, cyclic structural changes in the Sertoli cells were observed over the year. During the period of undischarged germ cell degeneration after spermiation, the Sertoli cells evidenced a lysosomal system associated with phagocytic function in the seminiferous lobules. In this study, the Sertoli cells function in phagocytosis and the resorption of products originating from degenerating spermatids and spermatozoa after spermiation. The spermatozoon lacks an acrosome, as have been shown in all teleost fish spermatozoa. The flagellum or sperm tail of this species evidences the typical 9+2 array of microtubules. PMID:27294207

  19. Subcellular distribution of ( sup 3 H)-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1991-01-01

    The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively.

  20. Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

    SciTech Connect

    Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

    1987-05-01

    It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition of PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.

  1. Ovarian Sertoli-Leydig Cell Tumor with Predominant Heterologous Mucinous Differentiation and Foci of Hepatocytic Differentiation: Case Report and Review of The Literature.

    PubMed

    Liang, Li; Menzin, Andrew; Lovecchio, John Louis; Navarro, Maria D

    2015-01-01

    Sertoli-Leydig cell tumor is a rare ovarian neoplasm and belongs to the group of sex cord stromal tumors. We present a case of a 15-year old girl diagnosed with Sertoli-Leydig cell tumor with heterologous elements consisting predominantly of mucinous epithelium and a sparse Sertoli-Leydig cell component, mimicking mucinous neoplasm. Furthermore, foci of hepatocytic differentiation were also identified. Immunohistochemical stains showed the component of Sertoli cell differentiation was positive for cytokeratin 18 and inhibin. The component of Leydig cell differentiation was strongly positive for inhibin. The component of hepatocytic differentiation was positive for low molecular weight keratin, HepPar1, alpha-fetoprotein and weakly positive for inhibin. Thus, this was a very rare case which created a challenge for pathologists, especially on frozen sections. PMID:26116602

  2. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells

    PubMed Central

    Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.

    2016-01-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  3. In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny.

    PubMed

    Sekaran, Suganya; Jagadeesan, Arunakaran

    2015-07-01

    Phthalates are the largest group of environmental pollutants and are considered toxicant to the endocrine system. The present study was aimed to test the effect of in utero exposure of di(2-ethylhexyl)phthalate (DEHP) on Leydig cell steroidogenesis in F1 male offspring's. Pregnant dams were oral gavaged with different doses (1, 10, and 100 mg/kg/day) of DEHP or olive oil during gestational Day 9-21. Serum testosterone (T) and estradiol (E2) levels were significantly reduced in male offspring at 60 days of age. Our results also demonstrate a coordinate, dose-dependent disruption of genes involved in steroidogenesis. The gene expression of StAR, Cyp11a1, 3β-HSD, 17β-HSD, 5α-reductase and cytochrome P450 19a1 (or) aromatase (Cyp-19) were significantly decreased. The transcription factors like steroidogenic factor-1 (SF-1) and specific protein-1 (Sp-1) showed a significant decrease in 10 and 100 mg DEHP treatment group. DNA methylation analysis using bisulfite specific-methylation PCR shows hypermethylation in the SF-1 and Sp-1 promoter regions. Further to determine whether the DEHP-induced methylation changes were associated with increased DNA methyltransferase (Dnmt) levels, we measured the expression levels of Dnmt3a, Dnmt3b, Dnmt1, and Dnmt3l using real-time PCR and Western blot method. The mRNA and protein expressions of Dnmt3a, Dnmt3b, and Dnmt1 were stimulated in 10 and 100 mg DEHP treatment groups, whereas no significant change was seen in Dnmt3l expression, suggesting that increased Dnmt3a/b, Dnmt1 may cause DNA hypermethylation in testicular Leydig cells. Overall, these data suggest that gestational exposure to DEHP affects adult testicular function via altered methylation patterns. PMID:25649163

  4. Combined steroidogenic characters of fetal adrenal and Leydig cells in childhood adrenocortical carcinoma.

    PubMed

    Fujisawa, Yasuko; Sakaguchi, Kimiyoshi; Ono, Hiroyuki; Yamaguchi, Rie; Kato, Fumiko; Kagami, Masayo; Fukami, Maki; Ogata, Tsutomu

    2016-05-01

    Although childhood adrenocortical carcinomas (c-ACCs) with a TP53 mutation are known to produce androgens, detailed steroidogenic characters have not been clarified. Here, we examined steroid metabolite profiles and expression patterns of steroidogenic genes in a c-ACC removed from the left adrenal position of a 2-year-old Brazilian boy with precocious puberty, using an atrophic left adrenal gland removed at the time of tumorectomy as a control. The c-ACC produced not only abundant dehydroepiandrosterone-sulfate but also a large amount of testosterone via the Δ5 pathway with Δ5-androstenediol rather than Δ4-androstenedione as the primary intermediate metabolite. Furthermore, the c-ACC was associated with elevated expressions of CYP11A1, CYP17A1, POR, HSD17B3, and SULT2A1, a low but similar expression of CYB5A, and reduced expressions of AKR1C3 (HSD17B5) and HSD3B2. Notably, a Leydig cell marker INSL3 was expressed at a low but detectable level in the c-ACC. Furthermore, molecular studies revealed a maternally inherited heterozygous germline TP53 mutation, and several post-zygotic genetic aberrations in the c-ACC including loss of paternally derived chromosome 17 with a wildtype TP53 and loss of maternally inherited chromosome 11 and resultant marked hyperexpression of paternally expressed growth promoting gene IGF2 and drastic hypoexpression of maternally expressed growth suppressing gene CDKN1C. These results imply the presence of combined steroidogenic properties of fetal adrenal and Leydig cells in this patient's c-ACC with a germline TP53 mutation and several postzygotic carcinogenic events. PMID:26940356

  5. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells.

    PubMed

    Sondhi, Varun; Owen, Bryn M; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A; Hughes, Beverly A; Arlt, Wiebke; Mangelsdorf, David J; Auchus, Richard J

    2016-04-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  6. Deficiency of CDKN1A or Both CDKN1A and CDKN1B Affects the Pubertal Development of Mouse Leydig Cells1

    PubMed Central

    Lin, Han; Huang, Yadong; Su, Zhijian; Zhu, Qiqi; Ge, Yufei; Wang, Guimin; Wang, Claire Q.F.; Mukai, Motoko; Holsberger, Denise R.; Cooke, Paul S.; Lian, Qing-Quan; Ge, Ren-Shan

    2015-01-01

    ABSTRACT Cyclin-dependent kinase inhibitors p21Cip1 (CDKN1A) and p27Kip1 (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a−/−) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a−/−, and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a−/− mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a−/− mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a−/− did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis. PMID:25609837

  7. cAMP-Specific Phosphodiesterases 8A and 8B, Essential Regulators of Leydig Cell Steroidogenesis

    PubMed Central

    Shimizu-Albergine, Masami; Tsai, Li-Chun Lisa; Patrucco, Enrico

    2012-01-01

    Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(−/−), PDE8B(−/−) and PDE8A(−/−)/B(−/−) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (−/−)/B(−/−) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (−/−)/B(−/−) cells had higher testosterone production than cells from either PDE8A(−/−) or PDE8B(−/−) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(−/−)/B(−/−) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted. PMID:22232524

  8. Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells

    SciTech Connect

    Bernier, M.; Chatelain, P.; Mather, J.P.; Saez, J.M.

    1986-11-01

    The author have investigated the effects of insulin and somatomedin-C/insulin like growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 ..mu..g/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 ..mu..g/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated (/sup 3/H)-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through the receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.

  9. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics.

    PubMed

    Golkowski, Martin; Shimizu-Albergine, Masami; Suh, Hyong Won; Beavo, Joseph A; Ong, Shao-En

    2016-07-01

    Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion

  10. Precocious Puberty and Leydig Cell Hyperplasia in Male Mice With a Gain of Function Mutation in the LH Receptor Gene

    PubMed Central

    McGee, Stacey R.

    2013-01-01

    The LH receptor (LHR) is critical for steroidogenesis and gametogenesis. Its essential role is underscored by the developmental and reproductive abnormalities that occur due to genetic mutations identified in the human LHR. In males, activating mutations are associated with precocious puberty and Leydig cell hyperplasia. To generate a mouse model for the human disease, we have introduced an aspartic acid to glycine mutation in amino acid residue 582 (D582G) of the mouse LHR gene corresponding to the most common D578G mutation found in boys with familial male-limited precocious puberty (FMPP). In transfected cells, mouse D582G mLHR exhibited constitutive activity with a 23-fold increase in basal cAMP levels compared with the wild-type receptor. A temporal study of male mice from 7 days to 24 weeks indicated that the knock-in mice with the mutated receptor (KiLHRD582G) exhibited precocious puberty with elevated testosterone levels as early as 7 days of age and through adulthood. Leydig cell-specific genes encoding LHR and several steroidogenic enzymes were up-regulated in KiLHRD582G testis. Leydig cell hyperplasia was detected at all ages, whereas Sertoli and germ cell development appeared normal. A novel finding from our studies, not previously reported in the FMPP cases, is that extensive hyperplasia is commonly found around the periphery of the testis. We further demonstrate that the hyperplasia is due to premature proliferation and precocious differentiation of adult Leydig cells in the KiLHRD582G testis. The KiLHRD582G mice provide a mouse model for FMPP, and we suggest that it is a useful model for studying pathologies associated with altered LHR signaling. PMID:23861372

  11. Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

    SciTech Connect

    Iseki, Minoru; Ikuta, Togo; Kobayashi, Tetsuya; Kawajiri, Kaname . E-mail: kawajiri@cancer-c.pref.saitama.jp

    2005-06-17

    Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21{sup Cip1}, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21{sup Cip1} mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21{sup Cip1} mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21{sup Cip1}.

  12. Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

    PubMed

    Kalayou, Shewit; Granum, Cesilie; Berntsen, Hanne Friis; Groseth, Per Kristian; Verhaegen, Steven; Connolly, Lisa; Brandt, Ingvar; de Souza, Gustavo Antonio; Ropstad, Erik

    2016-03-30

    Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions

  13. Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells*

    PubMed Central

    Ahn, Seung Won; Gang, Gil-Tae; Kim, Yong Deuk; Ahn, Ryun-Sup; Harris, Robert A.; Lee, Chul-Ho; Choi, Hueng-Sik

    2013-01-01

    Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states. PMID:23589295

  14. Ovarian Sertoli-Leydig cell tumor with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

    PubMed Central

    Horta, Mariana; Cunha, Teresa Margarida; Marques, Rita Canas; Félix, Ana

    2014-01-01

    Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker. PMID:25926909

  15. Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

    SciTech Connect

    Turner, B.; Vordermark, J.S.; Fechner, P.Y.

    1995-07-03

    We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there is a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.

  16. Prevention of deoxynivalenol- and zearalenone-associated oxidative stress does not restore MA-10 Leydig cell functions.

    PubMed

    Savard, Christian; Nogues, Perrine; Boyer, Alexandre; Chorfi, Younes

    2016-02-01

    The worldwide contamination of grains designated to human and animal feeding with Fusarium mycotoxins is a significant problem. Among Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA) are the most prevalent mycotoxins found in cereals. Co-occurrence of DON and ZEA is also very frequent and indicates that these mycotoxins might be involved in a wide range of synergistic or additive interactions. Both mycotoxins have been linked to various male reproduction problems including downregulation of steroidogenesis. In this study, the impact of DON and ZEA alone or in combination on the viability and steroid production of Leydig cell line MA-10 was determined. The ability of vitamin E, sesamin and their combination to prevent oxidative stress and restore progesterone secretion in DON- and ZEA-exposed cells was also determined. Results showed that MA-10 cells were more sensitive to the effect of DON compared to ZEA. DON and ZEA also significantly reduced MA-10 progesterone secretion after forskolin activation but no significant interactions between DON and ZEA were detected. Preventive treatment with the combination of vitamin E and sesamin significantly reduced ROS production and increased cell survival after exposition to DON and ZEA. However this treatment failed to restore normal progesterone secretion. In conclusion, both DON and ZEA are deleterious to steroidogenesis in Leydig cells. Prevention of oxidative stress caused by DON and ZEA was effective to restore cell viability but failed to restore other functions of Leydig cells suggesting that ROS production is not the main cause of steroidogenic failure in DON and ZEA treated MA-10 cells. PMID:26783879

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

    PubMed

    Gonzalez, C R; Vallcaneras, S S; Calandra, R S; Gonzalez Calvar, S I

    2013-02-01

    Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that modulates cell homeostasis. In Leydig cells, TGF-β1 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-β1 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-β1 (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-β1 plus progesterone than in cells cultured only with TGF-β1. Progesterone induced the gene expression of endoglin, a cofactor of TGF-β1 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-β1 involves endoglin. This co-receptor might be induced by KLF14 which is probably activated by progesterone. PMID:23317878

  18. BLTK1 murine Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and developmental toxicants.

    PubMed

    Forgacs, Agnes L; Ding, Qi; Jaremba, Rosemary G; Huhtaniemi, Ilpo T; Rahman, Nafis A; Zacharewski, Timothy R

    2012-06-01

    Leydig cells are the primary site of androgen biosynthesis in males. Several environmental toxicants target steroidogenesis resulting in both developmental and reproductive effects including testicular dysgenesis syndrome. The aim of this study was to evaluate the effect of several structurally diverse endocrine disrupting compounds (EDCs) on steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells possess a fully functional steroidogenic pathway that produces low basal levels of testosterone (T) and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) elicited concentration- and time-dependent induction of 3',5'-cyclic adenosine monophosphate, progesterone (P), and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3, and Srd5a1 messenger RNA levels. The evaluation of several structurally diverse male reproductive toxicants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl phthalate (MEHP), glyphosate, and RDX in BLTK1 cells suggests different modes of action perturb steroidogenesis. For example, prochloraz and triclosan antifungals reduced rhCG induction of T, consistent with published in vivo data but did not alter basal T levels. In contrast, atrazine and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T levels, whereas TCDD, glyphosate, and RDX had no effect on basal or rhCG induction of T in BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This model can also be used to elucidate the different mechanisms underlying toxicant-mediated disruption of steroidogenesis. PMID:22461451

  19. The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells

    PubMed Central

    Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

    2016-01-01

    Background: Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors’ production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. Methods: The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey’s post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. Results: With increasing drug concentration, cellular viability decreased significantly (p<0.05) and in contrast, PDGF levels increased (p<0.05). Different imatinib concentrations had no significant effect on SCF level. Increasing the duration of treatment from 2 to 6 days had no obvious effect on cellular viability, PDGF and SCF levels. Conclusion: Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug. PMID:27141462

  20. Male pseudohermaphoditism with Leydig cell agenesis and persistent muellerian ducts associated with partial deletion of chromosome 13

    SciTech Connect

    Potocki, L.; Oyer, C.E.; Tantravahi, U.

    1994-09-01

    Two chromosomally male infants with partial monosomy 13q were found to have Leydig cell agenesis (LCA) and persistent muellerian ducts (PMD). Post mortem examination in each case revealed cardiovascular, gastrointestinal, genitourinary, musculoskeletal, and central nervous system abnormalities, characteristic of monosomy 13q. Histologic examination confirmed the presence of muellerian derivatives within the pelvis, and the absence of Leydig cells within the testes. Sertoli cells were present. Karyotypes revealed partial monosomy 13q secondary to an unbalanced translocation, der(13)t(1;13)(q43;q21), in one infant, and to a ring chromosome 13 involving a deletion of 13q31-qter, in the other. The etiology of male pseudohermaphroditism is heterogeneous and included PMD due to absence of antimuellerian hormone (AMH) and LCA. Genitourinary abnormalities such as undescended testicles, hypospadias and micropenis have been described in monosomy 13q; however, testicular pathology in these cases has not been described. The cases presented here are the first reported cases in which male pseudohermaphroditism due to LCA and PMD is associated with monosomy 13q. This suggests the genetic locus involved in Leydig cell development may be located on the long arm of chromosome 13. The gene for AMH has been mapped to 19p13.3-13.2. The presence of muellerian structures and Sertoli cells, in the absence of abnormalities of chromosome 19p. suggests there may be genes on 13q coding for an enzyme in the pathway of AMH synthesis or for the AMH receptor. Based on these two cases, the critical region could possibly involve 13q13-qter.

  1. [INVESTIGATIONS OF SUBMICROSCOPIC ARCHITECTONICS SERTOLI AND LEYDIG CELLS AFTER HYDROCHLORIDE SEROTONIN DESTRUCTIVE IMPACT AND THE POSSIBILITY OF CORRECTION BY STIMULANTS OF METABOLIC PROCESSES].

    PubMed

    Brechka, N; Nevzorov, V; Bondarenko, V; Malova, N; Selyukova, N

    2015-01-01

    The results of study of ultrastructural changes in the Sertoli cells and Leydig's cells organelles after destructive influence of the serotonin hydrochloride and under influence bioglobin-U have been presented. It was shown that serotonin hydrochloride causes mitochondrial dysfunction and activates intracellular catabolic processes on the intracellular level. Bioglobin-U increases the activity and reparative synthetic reactions, reduced the degree of mitochondrial dysfunction and catabolic processes and activate the Leydig cell metabolism, and significantly reduces the number of foci destruction membranes of the endoplasmic reticulum, mitochondrial, and membranes of nucleus on the background of serotonin hydrochloride. PMID:26552310

  2. A virilizing Leydig cell tumor of the ovary associated with stromal hyperplasia under gonadotropin control.

    PubMed

    Marcondes, J A; Nery, M; Mendonça, B B; Hayashida, S A; Halbe, H W; Carvalho, F M; Wajchenberg, B L

    1997-12-01

    A 34-yr-old nulliparous black woman presented with hair loss, facial hirsutism, irregular menses and infertility associated with greatly increased serum total testosterone levels. The adrenal glands and the ovaries were normal on radiological and ultrasonographic investigation. Catheterization of the veins draining from the adrenal glands and the ovaries yielded testosterone levels of 20.3 nmol/L and 20.0 nmol/L in the right and the left adrenal veins, respectively, and 17.9 nmol/L and 27.4 nmol/L in the right and left ovaries venous plexus, respectively. Sequencial dexamethasone and ethynyl estradiol suppression test showed a decrease in cortisol level with no change in total testosterone level on dexamethasone while an increase in testosterone from 10.5 nmol/L to 20.1 nmol/L was observed ten days after ethynil estradiol had been associated to dexamethasone. When a gonadotropin-releasing hormone agonist (gonadorelin 3.5 mg i.m.) was administered for 2 months, serum gonadotropins levels decreased to less than 2 IU/L, total testosterone to 3.8 nmol/L and estradiol to less than 36 pmol/L. The patient was submitted to a pelvic exploratory laparotomy and a left salpingo-oophorectomy was performed. A solid and circumscribed ovarian tumor of 1.0 cm in diameter was found. The pathological diagnosis was a Leydig cell tumor with surrounding stromal hyperplasia. These findings may suggest that this tumor was gonadotropin-dependent being indirectly stimulated by ethynil estradiol, through a sensitization of the pituitary gonadotropes and increase in gonadotropin levels and suppressed by a gonadotropin-releasing hormone agonist. PMID:9492110

  3. Atrazine-Mediated Disruption of Steroidogenesis in BLTK1 Murine Leydig Cells.

    PubMed

    Karmaus, Agnes L; Zacharewski, Timothy R

    2015-12-01

    Atrazine (ATR) is a broad-spectrum triazine herbicide that disrupts steroidogenesis resulting in reproductive and developmental toxicity at high doses. Mouse BLTK1 Leydig cells were used as a steroidogenic model to investigate the effects of ATR on testosterone (T) biosynthesis. Induction of steroidogenesis by 3 ng/ml recombinant human chorionic gonadotropin (rhCG) induced intracellular 3',5' cyclic adenosine monophosphate (cAMP) approximately 20-fold and T approximately 3-fold at 4 h. Co-treatment with 300 μM ATR super-induced cAMP levels 100-fold yet antagonized rhCG-mediated induction of T approximately 20% at 4 h. ATR inhibited cAMP-specific phosphodiesterase (cPDE) with an IC50 of ≥98 μM, suggesting cPDE inhibition contributes to the super-induction of cAMP. However, concentrations of up to 3 mM db-cAMP did not antagonize rhCG induction of T levels, suggesting cAMP super-induction alone does not decrease T biosynthesis. Western analysis of cAMP-activated protein kinase A (PKA) target proteins identified ATR-mediated concentration-dependent alterations in phosphorylation including phospho-CREB. These results suggest the cPDE inhibition by ATR and super-induction of cAMP are independent of effects on T levels, and that altered phosphorylation of key steroidogenic regulatory proteins may underlie ATR-mediated disruption of steroidogenesis. PMID:26377646

  4. Increasing insulin resistance is associated with a decrease in Leydig cell testosterone secretion in men.

    PubMed

    Pitteloud, Nelly; Hardin, Megan; Dwyer, Andrew A; Valassi, Elena; Yialamas, Maria; Elahi, Dariush; Hayes, Frances J

    2005-05-01

    Insulin resistance is associated with low testosterone (T) levels in men, the mechanism of which is unclear. Thus, the aim of this study was to evaluate the hypothalamic-pituitary-gonadal axis in men with a spectrum of insulin sensitivity. Twenty-one men (aged 25-65 yr) had a glucose tolerance test and assessment of insulin sensitivity using a hyperinsulinemic-euglycemic clamp. Insulin sensitivity, expressed as the M value (milligrams per kilograms(-1) per minute(-1)), was calculated from the glucose disposal rate during the final 30 min of the clamp. Eighteen subjects had blood sampling every 10 min for 12 h to assess LH pulsatility. Hypogonadism was then induced with a GnRH antagonist, followed by sequential stimulation testing with GnRH (750 ng/kg, iv) and human chorionic gonadotropin (hCG; 1000 IU, im) to assess pituitary and testicular responsiveness, respectively. Nine subjects had normal glucose tolerance, nine had impaired glucose tolerance, and three had diabetes mellitus. There was a positive relationship between M and T levels (r = 0.46; P < 0.05). No relationship was seen between M and parameters of LH secretion, including mean LH levels, LH pulse amplitude, LH pulse frequency, and LH response to exogenous GnRH administration. In contrast, a strong correlation was observed between M and the T response to hCG (r = 0.73; P < 0.005). Baseline T levels correlated with the increase in T after hCG administration (r = 0.47; P < 0.05). During the clamp, T levels increased from a baseline level of 367 +/- 30 to 419 +/- 38 ng/dl during the last 30 min (P < 0.05). From these data we conclude that insulin resistance is associated with a decrease in Leydig cell T secretion in men. Additional studies are required to determine the mechanism of this effect. PMID:15713702

  5. KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells.

    PubMed

    Tremblay, Maxime A; Mendoza-Villarroel, Raifish E; Robert, Nicholas M; Bergeron, Francis; Tremblay, Jacques J

    2016-04-01

    Insulin-like 3 (INSL3), a Leydig cell-specific hormone, is essential for testis descent during foetal life and bone metabolism in adults. Despite its essential roles in male reproductive and bone health, very little is known regarding its transcriptional regulation in Leydig cells. To date, few transcription factors have been shown to activate INSL3 promoter activity: the nuclear receptors AR, NUR77, COUP-TFII and SF1. To identify additional regulators, we have isolated and performed a detailed analysis of a 1.1 kb human INSL3 promoter fragment. Through 5' progressive deletions and site-directed mutagenesis, we have mapped a 10 bp element responsible for about 80% of INSL3 promoter activity in Leydig cells. This element is identical to the CPE element of the placental-specific glycoprotein-5 (PSG5) promoter that is recognized by the developmental regulator Krüppel-like factor 6 (KLF6). Using PCR and western blotting, we found that KLF6 is expressed in several Leydig and Sertoli cell lines. Furthermore, immunohistochemistry on adult mouse testis revealed the presence of KLF6 in the nuclei of both Leydig and Sertoli cells. KLF6 binds to the 10 bp KLF element at -108 bp and activates the -1.1 kb human, but not the mouse, INSL3 promoter. KLF6-mediated activation of the human INSL3 promoter required an intact KLF element as well as Leydig/Sertoli-enriched factors because KLF6 did not stimulate the human INSL3 promoter activity in CV-1 fibroblast cells. Consistent with this, we found that KLF6 transcriptionally cooperates with NUR77 and SF1. Collectively, our results identify KLF6 as a regulator of human INSL3 transcription. PMID:26874000

  6. Bilateral Laparoscopic Gonadectomy in a Patient With Complete Androgen Insensitivity Syndrome and Bilateral Sertoli-Leydig Cell Tumor: A Case Report and Brief Review of the Literature

    PubMed Central

    Asl Zare, Mohammad; Kalantari, Mahmood Reza; Asadpour, Amir Abbas; Kamalati, Ali

    2014-01-01

    Introduction: Complete androgen insensitivity syndrome (previously called testicular feminization) is specified by a 46 XY karyotype and negative sex chromatin, bilateral undescended testes, female genitalia appearance, and lack of mullerian derivatives. Case Presentation: A 28-year-old woman with complete (severe) androgen resistance underwent prophylactic laparoscopic bilateral gonadectomy because of the eventually increased risk of gonadal malignancy. Although the gonads appeared grossly normal, microscopic examination revealed bilateral well differentiated sertoli–leydig cell tumor (SLCT). Discussion: Our Medline search revealed that this is the first reported case of bilateral sertoli–leydig cell tumor (SLCT) in androgen insensitivity syndrome. PMID:25032133

  7. In vitro production of cyclic AMP and steroids from an ovarian Sertoli-Leydig cell tumor. Notes on clinical management.

    PubMed

    Abrahamsson, G; Dahlgren, E; Hahlin, M; Knutson, F; Norström, A; Janson, P O

    1995-04-01

    A 27 year old nulliparous woman with a history of chronic anovulation and signs of virilization with a markedly elevated serum level of testosterone, underwent a laparotomy with peroperative bilateral ovarian vein catheterization and bilateral bisection of both ovaries. A solid, 1.5 cm, well delimited tumor located centrally in the right ovary, was excised. Testosterone levels in ovarian venous blood from the tumor bearing side, were 88.4 nmol/l and from the contralateral ovary 3.9 nmol/l. Histopathological examination showed a Sertoli-Leydig cell tumor which was radically extirpated. Postoperatively, the serum levels of androgen normalized, the woman had regular cycles, became pregnant and delivered a normal female baby. Pieces of tumor tissue were incubated for 2 h, with and without addition of gonadotropins and adrenocorticotropic hormone (ACTH). Human chorionic gonadotropin (CG), follicle stimulating hormone (FSH) and adrenocorticotropic hormone (ACTH) caused significant increases in cyclic monophosphate (cAMP) production in tumor tissue in vitro, as compared to controls. Furthermore, ACTH also significantly stimulated 17 beta-estradiol production. In tumor cells cultured for 48 h, FSH slightly, but not significantly, increased the production of progesterone. In the cell culture, [3H]-thymidine incorporation into deoxyribonucleic acid (DNA) was stimulated by IGF1 alpha but not by hCG and FSH. It is concluded that Sertoli-Leydig cell tumors may be sensitive to gonadotropins and ACTH and that their small size, solid shape and intra-ovarian localization can cause diagnostic difficulties. PMID:7732806

  8. Sub-acute intravenous administration of silver nanoparticles in male mice alters Leydig cell function and testosterone levels

    PubMed Central

    GARCIA, THOMAS X.; COSTA, GUILHERME M. J.; FRANÇA, LUIZ R.; HOFMANN, MARIE-CLAUDE

    2014-01-01

    The aim of this study was to determine whether short-term, in vivo exposure to silver nanoparticles (AgNPs) could be toxic to male reproduction. Low dose (1 mg/kg/dose) AgNPs were intravenously injected into male CD1 mice over 12 days. Treatment resulted in no changes in body and testis weights, sperm concentration and motility, fertility indices, or follicle-stimulating hormone and luteinizing hormone serum concentrations; however, serum and intratesticular testosterone concentrations were significantly increased 15 days after initial treatment. Histologic evaluation revealed significant changes in epithelium morphology, germ cell apoptosis, and Leydig cell size. Additionally, gene expression analysis revealed Cyp11a1 and Hsd3b1 mRNA significantly upregulated in treated animals. These data suggest that AgNPs do not impair spermatogonial stem cells in vivo since treatment did not result in significant decreases in testis weight and sperm concentrations. However, AgNPs appear to affect Leydig cell function, yielding increasing testicular and serum testosterone levels. PMID:24447867

  9. Glucocorticoid Receptor as a Potential Target to Decrease Aromatase Expression and Inhibit Leydig Tumor Growth.

    PubMed

    Panza, Salvatore; Malivindi, Rocco; Chemi, Francesca; Rago, Vittoria; Giordano, Cinzia; Barone, Ines; Bonofiglio, Daniela; Gelsomino, Luca; Giordano, Francesca; Andò, Sebastiano; Catalano, Stefania

    2016-05-01

    Leydig cell tumors are the most frequent interstitial neoplasms of the testis with increased incidence in recent years. They are hormonally active and are considered one of the steroid-secreting tumors. Although usually benign, the malignant phenotype responds poorly to conventional chemotherapy or radiation, highlighting the need to identify new therapeutic targets for treatment. Here, we identified a novel glucocorticoid-mediated mechanism that controls cell growth in Leydig cell tumors. We found that a synthetic glucocorticoid receptor agonist, dexamethasone, reduces cell proliferation in rat Leydig tumor cells by decreasing the expression and the enzymatic activity of the estrogen-producing enzyme aromatase. This inhibitory effect relies on the ability of activated glucocorticoid receptor to regulate the aromatase gene transcriptional activity through the recruitment of nuclear receptor corepressor protein and silencing mediator of retinoid and thyroid hormone receptors to a newly identified putative glucocorticoid responsive element within the aromatase promoter II. Our in vivo studies reveal a reduction of tumor growth, after dexamethasone treatment, in animal xenografts. Tumors from dexamethasone-treated mice exhibit a decrease in the expression of the proliferation marker Ki-67 and the aromatase enzyme. Our data demonstrate that activated glucocorticoid receptor, decreasing aromatase expression, induces Leydig tumor regression both in vitro and in vivo, suggesting that glucocorticoid receptor might be a potential target for the therapy of Leydig cell tumors. PMID:26968343

  10. ISOLATION AND CULTURE OF LEYDIG CELLS FROM ADULT RATS

    EPA Science Inventory

    Testosterone is essential for quantitatively normal sperm production in the testis, normal sperm maturation in the epididymis, maintenance of the accessory sex organs, and effective sexual behavior. ariety of xenobiotics can result in a significant decrease in spermatogenesis, sp...

  11. Characterization of lipid droplets in steroidogenic MLTC-1 Leydig cells: Protein profiles and the morphological change induced by hormone stimulation.

    PubMed

    Yamaguchi, Tomohiro; Fujikawa, Noriyuki; Nimura, Satomi; Tokuoka, Yutaro; Tsuda, Sonoka; Aiuchi, Toshihiro; Kato, Rina; Obama, Takashi; Itabe, Hiroyuki

    2015-10-01

    Lipid droplets (LDs) are functional subcellular organelles involved in multiple intracellular processes. LDs are found in nearly all types of eukaryotic cells, but their properties are highly variable in different types of tissues. Steroidogenic cells synthesize steroid hormones de novo from the cholesterol deposited in cytosolic LDs. However, the roles of LD proteins in steroidogenesis under pituitary hormone stimulation have not been well elucidated. The protein profile of isolated LDs from the mouse Leydig tumor cell line MLTC-1 was distinct from that of hepatic cells or macrophages. By proteomic analysis of the components using mass spectrometry, two enzymes for steroidogenesis, 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) and 17 β-hydroxysteroid dehydrogenase type 11 (17βHSD11), were identified in two strong bands in the LD fractions. The LD fraction of MLTC-1 cells also included CYP11A1 and CYP17, suggesting that the LDs contain all the enzymes needed for testosterone synthesis. The steroidogenesis in Leydig cells is activated by luteinizing hormone through a PKA-dependent pathway. Stimulation of MLTC-1 cells with luteinizing hormone or 8-bromo-cAMP caused drastic changes in the morphology of the LDs in the MLTC-1 cells. Upon stimulation, large perinuclear LDs are turned into much smaller LDs and dispersed throughout the cytosol. These results raise the possibility that LDs are involved in a regulatory pathway of steroidogenesis, not just by serving as a storage depot for cholesterol esters, but also by providing enzymes and generating sites for enzymatic activity. PMID:26143378

  12. Observation of Organelles in Leydig Cells by Contact Soft X-Ray Microscopy with a Laser Plasma X-Ray Source

    NASA Astrophysics Data System (ADS)

    Kado, M.; Ishino, M.; Tamotsu, S.; Yasuda, K.; Kishimoto, M.; Nishikino, M.; Kinjo, Y.; Shinohara, K.

    2011-09-01

    We observed the same biological specimens for comparison of the images by contact soft x-ray microscopy with a laser plasma x-ray source with those by confocal laser microscopy. Images of wet Leydig cells were directly comparable for organelles and showed that actin filaments and mitochondria were clearly identified in the soft x-ray images.

  13. MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells.

    PubMed

    Daems, Caroline; Di-Luoffo, Mickaël; Paradis, Élise; Tremblay, Jacques J

    2015-07-01

    In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca(2+)/CAMK (Ca(2+)/calmodulin-dependent kinase)-myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at -232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action. PMID:25860031

  14. Steroidogenic differential effects in neonatal porcine Leydig cells exposed to persistent organic pollutants derived from cod liver oil.

    PubMed

    Granum, Cesilie; Anchersen, Sara; Karlsson, Camilla; Berg, Vidar; Olsaker, Ingrid; Verhaegen, Steven; Ropstad, Erik

    2015-11-01

    Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects. PMID:26055946

  15. Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats.

    PubMed

    Surampudi, P; Chang, I; Lue, Y; Doumit, T; Jia, Y; Atienza, V; Liu, P Y; Swerdloff, R S; Wang, C

    2015-05-01

    Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I-VI) and late stages (IX-XIV) but not at middle stages (VII-VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII-VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low. PMID:25891800

  16. Midazolam regulated caspase pathway, endoplasmic reticulum stress, autophagy, and cell cycle to induce apoptosis in MA-10 mouse Leydig tumor cells

    PubMed Central

    So, Edmund Cheung; Chen, Yung-Chia; Wang, Shu-Chun; Wu, Chia-Ching; Huang, Man-Chi; Lai, Meng-Shao; Pan, Bo-Syong; Kang, Fu-Chi; Huang, Bu-Miin

    2016-01-01

    Purpose Midazolam is widely used as a sedative and anesthetic induction agent by modulating the different GABA receptors in the central nervous system. Studies have also shown that midazolam has an anticancer effect on various tumors. In a previous study, we found that midazolam could induce MA-10 mouse Leydig tumor cell apoptosis by activating caspase cascade. However, the detailed mechanism related to the upstream and downstream pathways of the caspase cascade, such as endoplasmic reticulum (ER) stress, autophagy, and p53 pathways plus cell cycle regulation in MA-10 mouse Leydig tumor cells, remains elusive. Methods Flow cytometry assay and Western blot analyses were exploited. Results Midazolam significantly decreased cell viability but increased sub-G1 phase cell numbers in MA-10 cells (P<0.05). Annexin V/propidium iodide double staining further confirmed that midazolam induced apoptosis. In addition, expressions of Fas and Fas ligand could be detected in MA-10 cells with midazolam treatments, and Bax translocation and cytochrome c release were also involved in midazolam-induced MA-10 cell apoptosis. Moreover, the staining and expression of LC3-II proteins could be observed with midazolam treatment, implying midazolam could induce autophagy to control MA-10 cell apoptosis. Furthermore, the expressions of p-EIF2α, ATF4, ATF3, and CHOP could be induced by midazolam, indicating that midazolam could stimulate apoptosis through ER stress in MA-10 cells. Additionally, the expressions of cyclin A, cyclin B, and CDK1 could be inhibited by midazolam, and the phosphorylation of p53, P27, and P21 could be adjusted by midazolam, suggesting that midazolam could manage cell cycle through the regulation of p53 pathway to induce apoptosis in MA-10 cells. Conclusion Midazolam could induce cell apoptosis through the activation of ER stress and the regulation of cell cycle through p53 pathway with the involvement of autophagy in MA-10 mouse Leydig tumor cells. PMID:27175086

  17. DICER1 mutations in Familial Multi-Nodular Goiter with and without Ovarian Sertoli-Leydig Cell Tumors

    PubMed Central

    Frio, Thomas Rio; Bahubeshi, Amin; Kanellopoulou, Chryssa; Hamel, Nancy; Niedziela, Marek; Sabbaghian, Nelly; Pouchet, Carly; Gilbert, Lucy; O’Brien, Paul K.; Serfas, Kim; Broderick, Peter; Houlston, Richard S.; Lesueur, Fabienne; Bonora, Elena; Muljo, Stefan; Schimke, R. Neil; Soglio, Dorothée Bouron-Dal; Arseneau, Jocelyne; Schultz, Kris Ann; Priest, John R.; Nguyen, Van-Hung; Harach, H. Ruben; Livingston, David M.; Foulkes, William D.; Tischkowitz, Marc

    2012-01-01

    Context Non-toxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported and there are five such published families which also contain individuals with Sertoli-Leydig cell tumors of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have recently been identified in families affected pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCT. Objective To determine whether familial MNG with or without SLCT in the absence of PPB was caused by mutations in DICER1. Design, Setting and Patients From September 2009 to September 2010, we studied two MNG families and three MNG/SLCT families. We screened affected probands for mutations in the DICER1 gene. We investigated blood lymphocytes, MNG and SLCT tissue from family members for loss of the wild-type allele (loss of heterozygosity), DICER1 expression and microRNA dysregulation. Main Outcome Measure(s) Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. Results We identified and characterized germline DICER1 mutations in all five families. Molecular analysis of the three SLCTs showed no loss of heterozygosity at DICER1, and IHC analysis in two available samples showed strong expression of DICER1 in Sertoli cells, but weak staining of Leydig cells. MicroRNA profiling of RNA derived from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. Conclusions DICER1 mutations predispose to both familial MNG and MNG with SLCT, independent of PPB and germline DICER1 mutations lead to dysregulation of miRNA. This could be investigated further as a possible novel mechanism of tumorigenesis. PMID:21205968

  18. Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor.

    PubMed

    Roelofs, Maarke J E; van den Berg, Martin; Bovee, Toine F H; Piersma, Aldert H; van Duursen, Majorie B M

    2015-03-01

    Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 μM, 60 μM, and 22 nM for GR, and 39 μM, 20 μM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular Δ4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable. PMID:25576683

  19. Cordycepin induced MA-10 mouse Leydig tumor cell apoptosis by regulating p38 MAPKs and PI3K/AKT signaling pathways

    PubMed Central

    Pan, Bo-Syong; Wang, Yang-Kao; Lai, Meng-Shao; Mu, Yi-Fen; Huang, Bu-Miin

    2015-01-01

    The p38 MAPKs play important roles in the regulation of balance between cell survival and cell death on the development of various cancers. However, the roles of p38 MAPKs regulating apoptotic effects on Leydig tumor cells remain unclear. In the present study, we showed that cordycepin (3′-deoxyadenosine) selectively induced apoptosis in MA-10 mouse Leydig tumor cells through regulating the p38 MAPK and PI3K/AKT signaling pathways. Cordycepin reduced viability in MA-10, TM4, and NT2/D1 cells, but not cause cell death of primary mouse Leydig cells on moderate concentration. Cordycepin increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding, activation of caspase-3, and cleavage of PARP. Inhibition of p38 MAPKs activity by SB203580 significantly prevented cordycepin-induced apoptosis in MA-10 cells. Co-treatment with wortmannin or the autophagy inhibitor 3-methyladenine (3-MA) elevated levels of apoptosis in cordycepin-treated MA-10 cells. Moreover, cordycepin activated p53, p21 and TGFß; and downregulated CDK2. The antitumour activity of cordycepin-treated MA-10 cells was significantly distinct in severe combined immunodeficiency (SCID) mice in vivo. These results suggested that cordycein is a highly selective treatment to induce MA-10 cells apoptosis via p38 MAPKs signaling. PMID:26303320

  20. Apoptotic effect of cordycepin combined with cisplatin and/or paclitaxel on MA-10 mouse Leydig tumor cells

    PubMed Central

    Kang, Fu-Chi; Chen, Pei-Jung; Pan, Bo-Syong; Lai, Meng-Shao; Chen, Yung-Chia; Huang, Bu-Miin

    2015-01-01

    Background Chemotherapy is not limited to a single treatment, and the evidence demonstrates that different drug combinations can have positive results in patients. In this study, we sought to determine whether cordycepin combined with cisplatin and/or paclitaxel would have an additive effective on inducing apoptosis in mouse Leydig tumor cells, and the mechanisms were also briefly examined. Methods The additive effects of cordycepin combined with cisplatin and/or paclitaxel on apoptosis in MA-10 cells were investigated by monitoring changes in morphological characteristics and examining cell viability, flow cytometry assays, and Western blot analyses. Results Combination of cordycepin plus cisplatin and/or paclitaxel for 12 and 24 hours induced apoptotic features in MA-10 cells. The MTT assay showed that the combination treatment reduced the viability of MA-10 cells in a dose-dependent manner, with additive effects. Cell cycle analysis showed that combination treatment significantly increased subG1 phase cell numbers in MA-10 cells, indicating apoptosis. Moreover, cordycepin plus cisplatin and/or paclitaxel significantly induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase, and phosphorylation of c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, p38, and p53 proteins in MA-10 cells. Conclusion Cordycepin plus cisplatin and/or paclitaxel can have an additive effect on apoptosis in MA-10 cells, with activation of caspase, mitogen-activated protein kinase, and p53 signal pathways. PMID:26366090

  1. Light and electron microscopical observations on the Leydig cells of the scrotal and abdominal testes of naturally unilateral cryptorchid West African dwarf goats.

    PubMed Central

    Ezeasor, D N

    1985-01-01

    The structure of interstitial cells of Leydig in the scrotal and abdominal testes of adult West African dwarf goats was studied utilising light and electron microscopy. The Leydig cells in both testes were scattered singly, in cords or clusters in the intertubular connective tissue in close proximity to vascular elements. The intertubular connective tissue in the abdominal testes was however much wider because of the hypoplasia of the seminiferous tubules. While the cells of the scrotal testes exhibited non-granular, pale staining cytoplasm, those of the abdominal testes were darkly staining and the majority contained coarse intracytoplasmic osmiophilic granules Interspersed amongst these cells were adipose cells occasionally distributed overall. With the electron microscope, it was found that agranular endoplasmic reticulum, Golgi apparatus and mitochondria were more prominently developed in the scrotal testes. In marked contrast, there were numerous lipid droplets in the cytoplasm of the Leydig cells in the abdominal testes. Furthermore, the cytoplasm of several of these cells showed evidence of degeneration. It is concluded that, contrary to observations in the experimentally induced condition, abdominal retention of testes in natural unilateral cryptorchidism induces alterations in the light microscopical and ultrastructural features of the Leydig cells of West African dwarf goats, changes which possibly can be ascribed to the chronic decline in testicular blood flow and the elevated temperature of the abdominal environment. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 PMID:2867081

  2. Observation of Actin Filaments in Leydig Cells with a Contact-type Soft X-ray Microscope with Laser Plasma X-ray Source

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito; Shinohara, Kunio

    Actin filaments in Leydig cells from mouse testes have been observed with a contact-type soft x-ray microscope with laser plasma x-ray source. The Leydig cells were fixed with paraformaldehyde, stained with Phalloidin, and observed with a confocal laser microscope prior to the observation with x-ray microscope. Obtained images by both of the confocal laser microscopy and the x-ray microscopy were directly compared and revealed that not only position of actin filaments but also the shapes can be identified each other. The actin filaments in the x-ray images were clearly recognized and their structures were obtained in more detail compared to those in the confocal laser microscope images.

  3. The co-occurrence of an ovarian Sertoli-Leydig cell tumor with a thyroid carcinoma is highly suggestive of a DICER1 syndrome.

    PubMed

    Durieux, Emeline; Descotes, Françoise; Mauduit, Claire; Decaussin, Myriam; Guyetant, Serge; Devouassoux-Shisheboran, Mojgan

    2016-05-01

    The DICER1 gene encodes an endoribonuclease involved in the production of mature microRNAs which regulates gene expression through several mechanisms. Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Pleuropulmonary blastoma is the most frequent lesion seen in this syndrome. Thyroid abnormalities are also a common finding, essentially concerning multinodular goiter. However, differentiated thyroid carcinoma is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 RNase IIIb catalytic domain in several tumor types, including ovarian Sertoli-Leydig cell tumors. We report two cases of differentiated thyroid carcinoma associated with ovarian Sertoli-Leydig cell tumor and with a heterozygous DICER1 gene mutation, occurring in two unrelated young girls without pleuropulmonary blastoma. Both thyroid carcinomas showed an E1813 mutation in exon 25 while the ovarian tumors harboured a somatic mutation in E1705 in exon 24 and a D1709 mutation in exon 25. Our observations confirm that the occurrence of an ovarian Sertoli-Leydig cell tumor with a thyroid carcinoma is highly suggestive of a DICER1 syndrome. We contend that the possibility of a relationship between sporadic thyroid carcinoma in young patients and somatic DICER1 gene mutation needs further investigation. PMID:26983701

  4. Sustained in vivo blockade of α₁-adrenergic receptors prevented some of stress-triggered effects on steroidogenic machinery in Leydig cells.

    PubMed

    Stojkov, Natasa J; Janjic, Marija M; Baburski, Aleksandar Z; Mihajlovic, Aleksandar I; Drljaca, Dragana M; Sokanovic, Srdjan J; Bjelic, Maja M; Kostic, Tatjana S; Andric, Silvana A

    2013-07-15

    This study was designed to systematically analyze and evaluate the effects of in vivo blockade of α₁-adrenergic receptors (α₁-ADRs) on the stress-induced disturbance of steroidogenic machinery in Leydig cells. Parameters followed 1) steroidogenic enzymes/proteins, transcription factors, and cAMP/testosterone production; 2) the main hallmarks of stress (epinephrine, glucocorticoids); and 3) transcription profiles of ADRs and oxidases with high affinity to inactivate glucocorticoids. Results showed that sustained blockade of α₁-ADRs prevented stress-induced 1) decrease of the transcripts/proteins for main steroidogenic CYPs (CYP11A1, CYP17A1); 2) decrease of Scarb1 and Hsd3b1 transcripts; 3) decrease of transcript for Nur77, one of the main activator of the steroidogenic expression; and 4) increase of Dax1 and Arr19, the main steroidogenic repressors in Leydig cells. In the same cells, the expression of steroidogenic stimulatory factor Creb1, StAR, and androgen receptor increased. In this signaling scenario, stress-induced stimulation of Adra1a/Adra1b/Adrbk1 and Hsd11b2 (the unidirectional oxidase with high affinity to inactivate glucocorticoids) was not changed. Blockade additionally stimulated stress-increased transcription of the most abundantly expressed ADRs Adra1d/Adrb1/Adrb2 in Leydig cells. In the same cells, stress-decreased testosterone production, the main marker of Leydig cells functionality, was completely prevented, while reduction of cAMP, the main regulator of androgenesis, was partially prevented. Accordingly, the presented data provide a new molecular/transcriptional base for "fight/adaptation" of steroidogenic cells and new molecular insights into the role of α₁-ADRs in stress-impaired Leydig cell steroidogenesis. The results are important in term of wide use of α₁-ADR selective antagonists, alone/in combination, to treat high blood pressure, nightmares associated with posttraumatic stress disorder, and disrupted sexual health. PMID

  5. Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

    PubMed Central

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J.

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis. PMID:21829656

  6. Long duration exposure to cadmium leads to increased cell survival, decreased DNA repair capacity, and genomic instability in mouse testicular Leydig cells.

    PubMed

    Singh, Kamaleshwar P; Kumari, Ragini; Pevey, Christina; Jackson, Desiree; DuMond, James W

    2009-06-28

    Epidemiological and experimental studies have shown that cadmium is carcinogenic to human and experimental animals, however, the mechanism of cadmium-induced carcinogenesis is not clear. The aberrant expression of cell cycle and DNA repair genes resulting in increased cell proliferation and genomic instability are the characteristic features of cancer cells. The purpose of this study was to determine if exposure to cadmium can perturb cell proliferation/survival and causes genomic instability in TM3 cells, a mouse testicular Leydig cell line. The results of this study revealed that short-duration exposure to lower doses of cadmium significantly increase the growth of TM3 cells, whereas, higher doses are toxic and cause cell death. The long duration exposure to higher doses of cadmium, however, results in increased cell survival and acquisition of apoptotic resistance. Gene expression analysis by real-time PCR revealed increased expression of the anti-apoptotic gene Bcl-2, whereas decreased expression of pro-apoptotic gene Bax. Decreased expression of genes for maintenance of DNA methylation, DNMT1, and DNA repair, OGG1 and MYH, was also observed in cells exposed to cadmium for 24h. The random amplified polymorphic DNA (RAPD) assay revealed genomic instability in cells with chronic exposure to cadmium. The findings of this study indicate that mouse testicular Leydig cells adapt to chronic cadmium exposure by increasing cell survival through increased expression of Bcl-2, and decreased expression of Bax. The increased proliferation of cells with genomic instability may result in malignant transformation, and therefore, could be a viable mechanism for cadmium-induced cancers. PMID:19232459

  7. Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

    PubMed Central

    Rone, Malena B.; Midzak, Andrew S.; Martinez-Arguelles, Daniel B.; Fan, Jinjiang; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    ABSTRACT Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation. PMID:25210128

  8. MEF2 and NR2F2 cooperate to regulate Akr1c14 gene expression in mouse MA-10 Leydig cells.

    PubMed

    Di-Luoffo, M; Brousseau, C; Tremblay, J J

    2016-03-01

    Leydig cells are essential for male reproductive development and health throughout life. Production of androgens [testosterone, dihydrotestosterone (DHT)] as well as intermediate steroids [progesterone, dihydroprogesterone (DHP)] is tightly regulated. In the mouse, the 3α-hydroxysteroid dehydrogenase enzyme (3α-HSD, AKR1C14) catalyses the interconversion of DHP and DHT into less potent steroids. Despite its importance, nothing is currently known regarding the regulation of Akr1c14 expression in Leydig cells. Recently, the transcription factors MEF2 and NR2F2 were identified in the mouse testis including in Leydig cells where they were found to regulate expression of genes involved in steroidogenesis. Analyses of transcriptomic data from MEF2- or NR2F2-deficient MA-10 Leydig cells revealed a significant decrease in Akr1c14 mRNA levels. Using qPCR, we confirmed that Akr1c14 mRNA levels were decreased in MEF2- and in NR2F2-deficient conditions. Conversely, overexpression of MEF2A or/and NR2F2 in MA-10 Leydig cells led to an increase in endogenous Akr1c14 mRNA levels. Recruitment of MEF2 and NR2F2 to the Akr1c14 promoter was confirmed by ChIP while DNA precipitation assays revealed direct binding of MEF2 but not NR2F2 to this region. In functional promoter studies, NR2F2 was found to activate the Akr1c14 promoter while MEF2A on its own had no effect. Combination of both NR2F2 and MEF2A led to a cooperative activation of the Akr1c14 promoter and this required intact MEF2 and NR2F2 elements. Finally, co-immunoprecipitation experiments showed that MEF2 and NR2F2 are present in the same protein complex. In conclusion, our results identify a novel cooperation between MEF2 factors and NR2F2 in the expression of the Akr1c14 gene involved in the regulation of DHP/DHT levels. PMID:26748576

  9. Mono-2-ethylhexyl phthalate stimulates androgen production but suppresses mitochondrial function in mouse leydig cells with different steroidogenic potential.

    PubMed

    Savchuk, Iuliia; Söder, Olle; Svechnikov, Konstantin

    2015-05-01

    Numerous studies have reported on testicular toxicity of phthalates in different experimental paradigms and showed that Leydig cells (LCs) were one of the main targets of phthalate actions. Adverse effects of phthalates on LCs steroidogenesis have been attributed to their metabolites, monophthalates. This study focuses on investigation whether LCs responsiveness to monophthalates action is associated with their potential to produce androgens. We found that of 3 monophthalates investigated [ie, mono-2-ethylhexyl phthalate (MEHP), mono-n-butyl phthalate, and mono-n-benzyl phthalate] only MEHP caused biological effects on the mouse LCs function. This monophthalate stimulated basal steroidogenesis associated with upregulation of StAR protein expression with no effect on hCG-stimulated androgen production by LCs from CBA/Lac and C57BL/6j mouse genotypes were observed. Further, MEHP attenuated ATP production and increased superoxide generation by both phenotypes of mouse LCs that indicated on mitochondrial dysfunction induced by the monophthalate. All together, our data indicate that MEHP-mediated stimulation of steroidogenesis and perturbation in mitochondrial function are not associated with the capacity of the LCs to synthesize androgens. We suggest that this effect of MEHP observed in LCs of rodent origin needs to be taken into consideration in analysis of earlier start of puberty in boys and may highlight a possible influence of phthalates on reproductive health in males. PMID:25677926

  10. A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia.

    PubMed

    Vezzoli, Valeria; Duminuco, Paolo; Vottero, Alessandra; Kleinau, Gunnar; Schülein, Ralf; Minari, Roberta; Bassi, Ivan; Bernasconi, Sergio; Persani, Luca; Bonomi, Marco

    2015-11-01

    The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient. PMID:26246498

  11. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

    PubMed Central

    Li, Nancy C.; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  12. Increased Proliferation but Decreased Steroidogenic Capacity in Leydig Cells from Mice Lacking Cyclin-Dependent Kinase Inhibitor 1B1

    PubMed Central

    Lin, Han; Hu, Guo-Xin; Dong, Lei; Dong, Qiang; Mukai, Motoko; Chen, Bing-Bing; Holsberger, Denise R.; Sottas, Chantal M.; Cooke, Paul S.; Lian, Qing-Quan; Li, Xiao-Kun; Ge, Ren-Shan

    2009-01-01

    Proliferating cells express cyclins, cell cycle regulatory proteins that regulate the activity of cyclin-dependent kinases (CDKs). The actions of CDKs are regulated by specific inhibitors, the CDK inhibitors (CDKIs), which are comprised of the Cip/Kip and INK4 families. Expression of the Cip/Kip CDKI 1B (Cdkn1b, encoding protein CDKN1B, also called p27kip1) in developing Leydig cells (LCs) has been reported, but the function of CDKN1B in LCs is unclear. The goal of the present study was to determine the effects of CDKN1B on LC proliferation and steroidogenesis by examining these parameters in Cdkn1b knockout (Cdkn1b−/−) mice. LC proliferation was measured by bromodeoxyuridine incorporation. Testicular testosterone levels, mRNA levels, and enzyme activities of steroidogenic enzymes were compared in Cdkn1b−/− and Cdkn1b+/+ mice. The labeling index of LCs in Cdkn1b−/− mice was 1.5% ± 0.2%, almost 7-fold higher than 0.2% ± 0.08% (P < 0.001) in the Cdkn1b+/+ control mice. LC number per testis in Cdkn1b−/− mice was 2-fold that seen in the Cdkn1b+/+ control mice. However, testicular testosterone levels, mRNA levels of steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), and 3beta-hydroxtsteroid dehydrogenase 6 (Hsd3b6), and their respective proteins, were significantly lower in Cdkn1b−/− mice. We conclude that deficiency of CDKN1B increased LC proliferation, but decreased steroidogenesis. Thus, CDKN1B is an important regulator of LC development and function. PMID:19211806

  13. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

    PubMed

    Li, Nancy C; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  14. Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene

    PubMed Central

    Lumbroso, Serge; Verhoef-Post, Miriam; Richter-Unruh, Annette; Looijenga, Leendert H. J.; Funaro, Ada; Beishuizen, Auke; van Marle, André; Drop, Stenvert L. S.; Themmen, Axel P. N.

    2011-01-01

    Context: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. Objective: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. Patients: We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. Results: The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. Conclusions: Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant. PMID:21490077

  15. Localized irradiation of testes with carcinoma in situ: Effects on Leydig cell function and eradication of malignant germ cells in 20 patients

    SciTech Connect

    Giwercman, A.; von der Maase, H.; Berthelsen, J.G.; Rorth, M.; Bertelsen, A.; Skakkebaek, N.E. )

    1991-09-01

    Twenty men (median age, 31 yr) previously treated for unilateral testicular cancer received localized irradiation in a dose of 20 Gray in 10 fractions for carcinoma in situ of the remaining testis. Follow-up testicular biopsies performed 3 (n = 19) and 24 (n = 14) months after the treatment showed in all cases a Sertoli cell-only pattern. Hormonal evaluation was performed before as well as 3, 12, 24, and 36 months after radiation treatment. Endocrine parameters were followed for a median of 30 months (3-36 months). Baseline serum testosterone values decreased during the follow-up period from 13.3 {plus minus} 6.0 to 10.8 {plus minus} 6.4 nmol/L (mean {plus minus} SD), although the decrease was not statistically significant (P = 0.06). Serum LH values increased during the first 3 months of follow-up from 10.4 {plus minus} 5.4 to 15.6 {plus minus} 7.3 IU/L (P less than 0.0001) and then remained unchanged. Significant decreases in GnRH- and hCG-stimulated testosterone levels also indicated an impairment of Leydig cell function. FSH levels increased (P less than 0.0001) during the first 3 months of follow-up from 21.8 {plus minus} 11.1 to 33.2 {plus minus} 13.2 IU/L. The authors conclude that localized irradiation of 20 Gray eradicated carcinoma in situ germ cells. Development of a second testicular cancer has until now been prevented. Leydig cell function was partially impaired by the radiation dose given.

  16. Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements.

    PubMed

    Abdou, Houssein S; Robert, Nicholas M; Tremblay, Jacques J

    2016-04-01

    The nuclear receptor NR4A1 is expressed in steroidogenic Leydig cells where it plays pivotal roles by regulating the expression of several genes involved in steroidogenesis and male sex differentiation including Star, HSD3B2, and Insl3 Activation of the cAMP and Ca(2+) signaling pathways in response to LH stimulation leads to a rapid and robust activation of Nr4a1 gene expression that requires the Ca(2+)/CAMKI pathway. However, the downstream transcription factor(s) have yet to be characterized. To identify potential Ca(2+)/CaM effectors responsible for hormone-induced Nr4a1 expression, MA-10 Leydig cells were treated with forskolin to increase endogenous cAMP levels, dantrolene to inhibit endoplasmic reticulum Ca(2+) release, and W7 to inhibit CaM activity. We identified Ca(2+)-responsive elements located in the discrete regions of the Nr4a1 promoter, which contain binding sites for several transcription factors such as AP1, CREB, and MEF2. We found that one of the three AP1/CRE sites located at -255 bp is the most responsive to the Ca(2+) signaling pathway as are the two MEF2 binding sites at -315 and -285 bp. Furthermore, we found that the hormone-induced recruitment of phospho-CREB and of the co-activator p300 to the Nr4a1 promoter requires the Ca(2+) pathway. Lastly, siRNA-mediated knockdown of CREB impaired NR4A1 expression and steroidogenesis. Together, our data indicate that the Ca(2+) signaling pathway increases Nr4a1 expression in MA-10 Leydig cells, at least in part, by enhancing the recruitment of coactivator most likely through the MEF2, AP1, and CREB transcription factors thus demonstrating an important interplay between the Ca(2+) and cAMP pathways in regulating Nr4a1 expression. PMID:26647388

  17. Hydrodynamic properties of the gonadotropin receptor from a murine Leydig tumor cell line are altered by desensitization

    SciTech Connect

    Rebois, R.V.; Bradley, R.M.; Titlow, C.C.

    1987-10-06

    The murine Leydig tumor cell line 1 (MLTC-1) contains gonadotropin receptors (GR) that are coupled to adenylate cyclase through the stimulatory guanine nucleotide binding protein (G/sub s/). The binding of human choriogonadotropin (hGC) causes MLTC-1 cells to accumulate cAMP. With time, the ability of MLTC-1 cells to respond to hCG is attenuated by a process called desensitization. The hydrodynamic properties of GR from control and desensitized MLTC-1 cells were studied. Sucrose density gradient sedimentation in H/sub 2/O and D/sub 2/O and gel filtration chromatography were used to estimate the Stokes radius (a), partial specific volume (v/sub c/), sedimentation coefficient (s/sub 20,w/), and molecular weight (M/sub r/) of the detergent-solubilized hormone-receptor complex (hCG-GR). (/sup 125/I)hCG was bound to MLTC-1 cells under conditions that allow (37/sup 0/C) or prevent (0/sup 0/C) desensitization, and hCG-GR was solubilized in Triton X-100. In the absence of desensitization, control hCG-GR had a M/sub r/ of 213,000, whereas desensitized hCG-GR had a M/sub r/ of 158,000. Deglycosylated hCG (DG-HCG) is an antagonist that binds to GR with high affinity but fails to stimulate adenylate cyclase or cause desensitization. (/sup 125/I)DG-hCG was bound to MLTC-1 cells and DG-hCG-GR solubilized in Triton X-100. The hydrodynamic properties of DG-hCG-GR were the same as that for control hCG-GR. There was no evidence for the association of adenylate cyclase or G/sub s/ with GR in Triton X-100 solubilized preparations. When hCG was cross-linked to GR and solubilized with sodium dodecyl sulfate (SDS), the M/sub r/ was found to be 116,000, which was similar to that determined by SDS-polyacrylamide gel electrophoresis and less than that of the Triton X-100 solubilized control hCG-GR.

  18. Interaction of putative estrogens and the estrogen receptor system in Leydig cells in the BALB/c mouse testis resulting in the initiation of DNA synthesis

    SciTech Connect

    Juriansz, R.L.

    1986-01-01

    Continuous administration of estrogens for 7-9 months, both steroidal and nonsteroidal, to male BALB/c mice, leads to the formation of testicular Leydig cell tumors. Three days following the subcutaneous implantation of a pellet of estrogen in cholesterol, there is a peak in the incorporation of /sup 3/H-thymidine into the DNA of the interstitial cells. These effects are hypothesized to be mediated by the estrogen receptor system in the Leydig cell. Common experimental techniques for the measurement of hormone binding, such as dextran coated charcoal treatment, proved to be impossible to employ in this system, therefore a procedure was developed using hydroxyapatite to obtain binding data. The cytosolic estrogen receptor was found to have a dissociation constant for estradiol-17..beta.. of 6.5 x 10/sup -8/ M, while that of the nuclear estrogen receptor was 1.25 x 10/sup -8/ M. Competition assays were utilized to determine the cytosolic estrogen receptor's affinity for nonsteroidal estrogens, steroidal estrogens, and triphenylethylene.

  19. Transgenerational Effects of Di(2-ethylhexyl) Phthalate in the SD Male Rat

    EPA Science Inventory

    In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis and other androgen-dependent tissues. In ...

  20. A Rare Case of Intra-Endometrial Leiomyoma of Uterus Simulating Degenerated Submucosal Leiomyoma Accompanied by a Large Sertoli-Leydig Cell Tumor.

    PubMed

    Jeong, Kyungah; Lee, Sa Ra; Park, Sanghui

    2016-03-01

    A 50-year-old peri-menopausal woman presented with hard palpable mass on her lower abdomen and anemia from heavy menstrual bleeding. Ultrasonography showed a 13×12 cm sized hypoechoic solid mass in pelvis and a 2.5×2 cm hypoechoic cystic mass in uterine endometrium. Abdomino-pelvic computed tomography revealed a hypodense pelvic mass without enhancement, suggesting a leiomyoma of intraligamentary type or sex cord tumor of right ovary with submucosal myoma of uterus. Laparoscopy revealed a large Sertoli-Leydig cell tumor of right ovary with a very rare entity of intra-endometrial uterine leiomyoma accompanied by adenomyosis. The final diagnosis of ovarian sex-cord tumor (Sertoli-Leydig cell), stage Ia with intra-endometrial leiomyoma with adenomyosis, was made. Considering the large size of the tumor and poorly differentiated nature, 6 cycles of chemotherapy with Taxol and Carboplatin regimen were administered. There is neither evidence of major complications nor recurrence during 20 months' follow-up. PMID:26847310

  1. A Rare Case of Intra-Endometrial Leiomyoma of Uterus Simulating Degenerated Submucosal Leiomyoma Accompanied by a Large Sertoli-Leydig Cell Tumor

    PubMed Central

    Jeong, Kyungah; Park, Sanghui

    2016-01-01

    A 50-year-old peri-menopausal woman presented with hard palpable mass on her lower abdomen and anemia from heavy menstrual bleeding. Ultrasonography showed a 13×12 cm sized hypoechoic solid mass in pelvis and a 2.5×2 cm hypoechoic cystic mass in uterine endometrium. Abdomino-pelvic computed tomography revealed a hypodense pelvic mass without enhancement, suggesting a leiomyoma of intraligamentary type or sex cord tumor of right ovary with submucosal myoma of uterus. Laparoscopy revealed a large Sertoli-Leydig cell tumor of right ovary with a very rare entity of intra-endometrial uterine leiomyoma accompanied by adenomyosis. The final diagnosis of ovarian sex-cord tumor (Sertoli-Leydig cell), stage Ia with intra-endometrial leiomyoma with adenomyosis, was made. Considering the large size of the tumor and poorly differentiated nature, 6 cycles of chemotherapy with Taxol and Carboplatin regimen were administered. There is neither evidence of major complications nor recurrence during 20 months' follow-up. PMID:26847310

  2. DISTRIBUTION OF [14C]ETHANE DIMENTHANESULFONATE IN IMMATURE AND ADULT MALE RATS FOLLOWING AN ACUTE EXPOSURE

    EPA Science Inventory

    In the adult rat, ethane dimethanesulphonate (EDS) reduces testosterone (T) production by killing Leydig cells. Studies have also shown that acute EDS administration produces transient infertility and epididymal effects. Although these later effects were believed to be indirect r...

  3. TPP and TCEP induce oxidative stress and alter steroidogenesis in TM3 Leydig cells.

    PubMed

    Chen, Guanliang; Zhang, Songbin; Jin, Yuanxiang; Wu, Yan; Liu, Ling; Qian, Haifeng; Fu, Zhengwei

    2015-11-01

    Effects of triphenyl phosphate (TPP) and tris-(2-chloroethyl) phosphate (TCEP) exposure on induction of oxidative stress and endocrine disruption were investigated in TM3 cells. After 24h exposure, cell growth declined and morphology changed in TPP and TCEP treated groups with high dosages. Significant increases in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione S-transferase (GST) activities and their respective gene expressions in a dose-dependent and/or time-dependent manner in TPP or TCEP groups. Moreover, the expression of main genes related to testosterone (T) synthesis including cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17α-hydroxysteroid dehydrogenase (P450-17α), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) were dramatically reduced by TPP and TCEP treatments, especially with the high dosage for 24h. TPP and TCEP treatments for 24h caused significant decreases in T levels in the medium. Furthermore, co-treatments of hCG with TPP or TCEP could inhibit hCG-induced changes in the expression of P450scc, P450-17α and 17β-HSD and T levels. Taken together, TPP and TCEP could induce oxidative stress and endocrine disruption in TM3 cells. PMID:26049154

  4. Woman with virilizing congenital adrenal hyperplasia and Leydig cell tumor of the ovary.

    PubMed

    Fernández-García Salazar, Rosario; Muñoz-Darias, Carmen; Haro-Mora, Juan Jesús; Almaraz, M Cruz; Audí, Laura; Martínez-Tudela, Juana; Yahyaoui, Raquel; Esteva, Isabel

    2014-08-01

    We report the case of a 36-year-old woman with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, and corticosteroid replacement therapy since birth. She manifested persistent virilization and high testosterone levels that were attributed to nonadherence to medical treatment. The patient was referred to our gender unit for genitoplastic surgery. We recommended the patient for left oophorectomy after detecting an ovarian mass. Pathologic findings confirmed an ovarian hilus cell tumor. Testosterone levels fell back to normal and masculinization disappeared but ACTH remained elevated. This case represents a very rare type of primary ovarian tumor that must be considered in persistent virilizing symptoms in women with CAH. PMID:24702195

  5. Low reversibility of intracellular cAMP accumulation in mouse Leydig tumor cells (MLTC-1) stimulated by human Luteinizing Hormone (hLH) and Chorionic Gonadotropin (hCG).

    PubMed

    Klett, Danièle; Meslin, Philippine; Relav, Lauriane; Nguyen, Thi Mong Diep; Mariot, Julie; Jégot, Gwenhaël; Cahoreau, Claire; Combarnous, Yves

    2016-10-15

    In order to study the intracellular cAMP response kinetics of Leydig cells to hormones with LH activity, we used MLTC-1 cells transiently expressing a chimeric cAMP-responsive luciferase so that real-time variations of intracellular cAMP concentration could be followed using oxiluciferin luminescence produced from catalyzed luciferin oxidation. The potencies of the different LHs and CGs were evaluated using areas under the curves (AUC) of their kinetics over 60 min stimulation. All mammalian LHs and CGs tested were found to stimulate cAMP accumulation in these cells. The reversibility of this stimulation was studied by removing the hormone from the culture medium after 10 min of incubation. The ratios of kinetics AUC after removing or not the hormone were used to evaluate the stimulation reversibility of each hormone. Natural and recombinant hLHs and hCGs were found to exhibit slowly reversible activation compared to pituitary rat, ovine, porcine, camel and equine LHs, serum-derived eCG (PMSG) and recombinant eLH/CGs. Carbohydrate side chains are not involved in this phenomenon since natural and recombinant homologous hormones exhibit the same reversibility rates. It is still unknown whether only one human subunit, α or β, is responsible for this behaviour or whether it is due to a particular feature of the hLH and hCG quaternary structure. PMID:27373440

  6. A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

    PubMed Central

    Demirbas, Didem; Wyman, Arlene R.; Shimizu-Albergine, Masami; Cakici, Ozgur; Beavo, Joseph A.; Hoffman, Charles S.

    2013-01-01

    A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC50 value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems. PMID:23967182

  7. Leydig cell tumor

    MedlinePlus

    ... health care provider if you have symptoms of testicular cancer. Prevention Performing testicular self-examination (TSE) each month may help detect testicular cancer at an early stage, before it spreads. Finding ...

  8. Perfluorooctane Sulfonate Concentrations in Amniotic Fluid, Biomarkers of Fetal Leydig Cell Function, and Cryptorchidism and Hypospadias in Danish Boys (1980–1996)

    PubMed Central

    Toft, Gunnar; Jönsson, Bo A.G.; Bonde, Jens Peter; Nørgaard-Pedersen, Bent; Hougaard, David M.; Cohen, Arieh; Lindh, Christian H.; Ivell, Richard; Anand-Ivell, Ravinder; Lindhard, Morten S.

    2015-01-01

    Background Exposure to perfluorooctane sulfonate (PFOS) may potentially disturb fetal Leydig cell hormone production and male genital development. Objectives We aimed to study the associations between levels of amniotic fluid PFOS, fetal steroid hormone, and insulin-like factor 3 (INSL3) and the prevalence of cryptorchidism and hypospadias. Methods Using the Danish National Patient Registry, we selected 270 cryptorchidism cases, 75 hypospadias cases, and 300 controls with stored maternal amniotic fluid samples available in a Danish pregnancy-screening biobank (1980–1996). We used mass spectrometry to measure PFOS in amniotic fluid from 645 persons and steroid hormones in samples from 545 persons. INSL3 was measured by immunoassay from 475 persons. Associations between PFOS concentration in amniotic fluid, hormone levels, and genital malformations were assessed by confounder-adjusted linear and logistic regression. Results The highest tertile of PFOS exposure (> 1.4 ng/mL) in amniotic fluid was associated with a 40% (95% CI: –69, –11%) lower INSL3 level and an 18% (95% CI: 7, 29%) higher testosterone level compared with the lowest tertile (< 0.8 ng/mL). Amniotic fluid PFOS concentration was not associated with cryptorchidism or hypospadias. Conclusions Environmental PFOS exposure was associated with steroid hormone and INSL3 concentrations in amniotic fluid, but was not associated with cryptorchidism or hypospadias in our study population. Additional studies are needed to determine whether associations with fetal hormone levels may have long-term implications for reproductive health. Citation Toft G, Jönsson BA, Bonde JP, Nørgaard-Pedersen B, Hougaard DM, Cohen A, Lindh CH, Ivell R, Anand-Ivell R, Lindhard MS. 2016. Perfluorooctane sulfonate concentrations in amniotic fluid, biomarkers of fetal Leydig cell function, and cryptorchidism and hypospadias in Danish boys (1980–1996). Environ Health Perspect 124:151–156; http://dx.doi.org/10.1289/ehp.1409288

  9. 1,3-Dichloro-2-propanol inhibits progesterone production through the expression of steroidogenic enzymes and cAMP concentration in Leydig cells.

    PubMed

    Sun, Jianxia; Bai, Shun; Bai, Weibin; Zou, Feiyan; Zhang, Lei; Li, Guoqiang; Hu, Yunfeng; Li, Mingwei; Yan, Rian; Su, Zhijian; Huang, Yadong

    2014-07-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a well-known food processing contaminant that has been shown to impede male reproductive function. However, its mechanism of action remains elusive. In this study, the effects of 1,3-DCP on progesterone production were investigated using the R2C Leydig cell model. 1,3-DCP significantly reduced cell viability from 7.48% to 97.4% at doses comprised between 0.5 and 6mM. Single cell gel/comet assays and atomic force microscopy assays showed that 1,3-DCP induced early phase cell apoptosis. In addition, 1,3-DCP significantly reduced progesterone production detected by radioimmunoassay (RIA). The results from quantitative polymerase chain reaction and western blotting demonstrated that the mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase were significantly down-regulated in R2C cells. Particularly, the change rhythm of Star expression was highly consistent with progesterone production. Furthermore, the cyclic adenosine monophosphate (cAMP) and the mitochondrial membrane potential mediated by ROS, which are involved in regulating progesterone synthesis were also decreased in response to the 1,3-DCP treatment. Overall, the data presented here suggested that 1,3-DCP interferes with the male steroidogenic capacity mainly by down-regulating the level of cAMP and the key enzymes involved in the androgen synthesis pathway. PMID:24518350

  10. Effect of cAMP on the cholesterol side-chain cleavage enzyme complex (CSCC) in MA-10 Leydig tumor cell mitochondria

    SciTech Connect

    Chaudhary, L.R.; Stocco, D.M.

    1986-05-01

    The rate limiting step in steroid biosynthesis is catalyzed by the cholesterol side-chain cleavage complex (CSCC) which is located on the matrix side of the inner mitochondrial membrane. It has been shown that addition of cAMP or LH/hCG to MA-10 mouse Leydig tumor cells in culture increases the production of progesterone as the major steroid. To examine the effect of cAMP on CSCC activity, cells were grown in culture flasks in the presence or absence of 10/sup -3/M cAMP for 3 h. Cells were harvested and mitochondria were isolated. Reaction conditions were optimized and contained 465000 DPM (26-/sup 14/C)cholesterol, 10mM NaCN, 0.5mM NADPH, 5mM CaCl/sub 2/, 60mM KCl and mitochondria. Reactions were stopped by the addition of ethanol and water and liberated (26-/sup 14/C)isocaproic acid was separated from uncleaved cholesterol by extraction with hexane and chloroform resulting in the retention of isocaproic acid in the aqueous layer. These experiments demonstrated a significant increase in (/sup 14/C)isocaproic acid production by mitochondria isolated from the cells grown in the presence of cAMP when compared to controls indicating that cAMP enhances the production of progesterone by increasing the activity of CSCC. Whether cAMP brings about this increase primarily through phosphorylation/dephosphorylation reactions or through some other mechanism is not clear at this time.

  11. Prepubertal Di-n-Butyl Phthalate Exposure Alters Sertoli and Leydig Cell Function and Lowers Bone Density in Adult Male Mice.

    PubMed

    Bielanowicz, Amanda; Johnson, Rachelle W; Goh, Hoey; Moody, Sarah C; Poulton, Ingrid J; Croce, Nic; Loveland, Kate L; Hedger, Mark P; Sims, Natalie A; Itman, Catherine

    2016-07-01

    Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 μM, but not 5 μM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis. PMID:27058814

  12. Gonadotropin-regulated Testicular RNA Helicase (GRTH/DDX25), a Negative Regulator of Luteinizing/Chorionic Gonadotropin Hormone-induced Steroidogenesis in Leydig Cells

    PubMed Central

    Fukushima, Masato; Villar, Joaquin; Tsai-Morris, Chon-Hwa; Dufau, Maria L.

    2011-01-01

    Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH−/−) mice. However, testosterone production was enhanced in LCs of GRTH−/− mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH−/− mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH−/− mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH−/− mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH−/− mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male. PMID:21719703

  13. Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser58 phosphorylation and 14-3-3γ Lys49 acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser58 or Lys49. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys49 acetylation is important for the cAMP-dependent induction of these interactions. PMID:25086053

  14. 7,12-dimethylbenz[a]anthracene induces sertoli-leydig-cell tumors in the follicle-depleted ovaries of mice treated with 4-vinylcyclohexene diepoxide.

    PubMed

    Craig, Zelieann R; Davis, John R; Marion, Samuel L; Barton, Jennifer K; Hoyer, Patricia B

    2010-02-01

    Ovarian cancer is associated with high mortality due to its late onset of symptoms and lack of reliable screening methods for early detection. Furthermore, the incidence of ovarian cancer is higher in postmenopausal women. Mice rendered follicle-depleted through treatment with 4-vinylcyclohexene diepoxide (VCD) are a model of ovary-intact menopause. The present study was designed to induce ovarian neoplasia in this model by treating mice with 7,12-dimethylbenz[a]anthracene (DMBA). Female B6C3F1 mice (age, 28 d) received intraperitoneal sesame oil (vehicle; VCD- groups) as a control or VCD (160 mg/kg; VCD+ groups) daily for 20 d to cause ovarian failure. Four months after the onset of dosing, mice from each group received a single injection of DMBA (VCD-DMBA+ and VCD+DMBA+ groups, n = 15 per group) or vehicle control (VCD-DMBA-, n = 15; VCD+ DMBA-, n = 14) under the bursa of the right ovary. Ovaries were collected 3 or 5 mo after injection and processed for histologic evaluation. Immunohistochemistry was used to confirm classification of neoplasms. None of the animals in the VCD-DMBA- and VCD-DMBA+ groups (that is, mice still undergoing estrus) had tumors at either time point. At the 3-mo time point, 12.5% of the VCD+DMBA+ mice had ovarian tumors; at 5 mo, 57.1% of the VCD+DMBA+ and 14.3% of VCD+DMBA- ovaries had neoplasms. Neoplasms stained positively for inhibin alpha (granulosa cells) and negatively for keratin 7 (surface epithelium), thus confirming classification of the lesions as Sertoli-Leydig cell tumors. These findings provide evidence for an increased incidence of DMBA-induced ovarian neoplasms in the ovaries of follicle-depleted mice compared with that in age-matched cycling controls. PMID:20158943

  15. Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation to steroid production

    SciTech Connect

    Anakwe, O.O.; Payne, A.H. )

    1987-09-01

    The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase P-450(17 alpha) was investigated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-450(17 alpha) decreased to less than 5% by day 4 and was undetectable between days 7 and 11. In contrast, the amount of immunoreactive P-450scc remained relatively constant throughout the same period. Treatment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-450(17 alpha) with relatively little effect on the amount of P-450scc. The rate of de novo synthesis of each of the P-450 enzymes was studied by determining (35S)methionine incorporation into newly synthesized protein. In the absence of cAMP, de novo synthesis of P-450(17 alpha) ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-450(17 alpha) on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells. The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-450(17 alpha) ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture.

  16. Effects of Prenatal Leydig Cell Function on the Ratio of the Second to Fourth Digit Lengths in School-Aged Children

    PubMed Central

    Mitsui, Takahiko; Araki, Atsuko; Imai, Ayako; Sato, Sakiko; Miyashita, Chihiro; Ito, Sachiko; Sasaki, Seiko; Kitta, Takeya; Moriya, Kimihiko; Cho, Kazutoshi; Morioka, Keita; Kishi, Reiko; Nonomura, Katsuya

    2015-01-01

    Prenatal sex hormones can induce abnormalities in the reproductive system and adversely impact on genital development. We investigated whether sex hormones in cord blood influenced the ratio of the second to fourth digit lengths (2D/4D) in school-aged children. Of the 514 children who participated in a prospective cohort study on birth in Sapporo between 2002 and 2005, the following sex hormone levels were measured in 294 stored cord blood samples (135 boys and 159 girls); testosterone (T), estradiol (E), progesterone, LH, FSH, inhibin B, and insulin-like factor 3 (INSL3). A total of 350 children, who were of school age and could be contacted for this survey, were then requested via mail to send black-and-white photocopies of the palms of both the left and right hands. 2D/4D was calculated in 190 children (88 boys and 102 girls) using photocopies and derived from participants with the characteristics of older mothers, a higher annual household income, higher educational level, and fewer smokers among family members. 2D/4D was significantly lower in males than in females (p<0.01). In the 294 stored cord blood samples, T, T/E, LH, FSH, Inhibin B, and INSL3 levels were significantly higher in samples collected from males than those from females. A multivariate regression model revealed that 2D/4D negatively correlated with INSL3 in males and was significantly higher in males with <0.32 ng/mL of INSL3 (p<0.01). No correlations were observed between other hormones and 2D/4D. In conclusion, 2D/4D in school-aged children, which was significantly lower in males than in females, was affected by prenatal Leydig cell function. PMID:25746668

  17. Steroid-mediated inhibition of cAMP induced de novo synthesis of cytochrome P-450/sub 17 / in Leydig cell cultures

    SciTech Connect

    Hales, D.B.; Sha, L.; Payne, A.H.

    1987-05-01

    The present study was designed to investigate the mechanism by which testosterone (T), produced during cAMP induction of P-450/sub 17 /, modulates the rate of its de novo synthesis. Purified Leydig cells (LC) were maintained in culture for 7 days prior to the initiation of treatment. De novo synthesis was determined by TVS-methionine incorporation, immunoprecipitation with specific antibody, separation by SDS-gel electrophoresis and quantitation by laser densitometry. Treatment of LC with 0.05 mM 8-Br-cAMP (cA) results in a time-dependent increase in the rate of de novo synthesis of P-450/sub 17 / which is increased 2 fold when T production is inhibited by aminoglutethimide (AG). The addition of increasing concentrations of the androgen receptor antagonist, hydroxyflutamide (1-10 M), to cA treated LC enhances the rate of synthesis similar to that seen in cA-treated LC in which T production was inhibited by AG. The addition of increasing concentrations of T (0.05-5 M) or the androgen agonist, mibolerone (1-5 M), to cA + AG treated LC causes a dose-dependent reversal of the AG-enhanced increase in the rate of cA-induced de novo synthesis of P-450/sub 17 /. Addition of estradiol (1 M) or dexamethasone (1 M) was without effect. These data indicate that T produced during cA induction of P-450/sub 17 / negatively regulates the rate of synthesis of this cytochrome P-450 enzyme by an androgen receptor mediated mechanism.

  18. Inutero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis.

    PubMed

    Li, Linxi; Bu, Tiao; Su, Huina; Chen, Zhichuan; Liang, Yuyuan; Zhang, Gaolong; Zhu, Danyan; Shan, Yuanyuan; Xu, Renai; Hu, Yuanyuan; Li, Junwei; Hu, Guoxin; Lian, Qingquan; Ge, Ren-Shan

    2015-01-22

    Diisononyl phthalate (DINP) is a synthetic material that has been widely used as a substitute for other plasticizers prohibited due to reproductive toxicity in consumer products. Some phthalates have been associated with testicular dysgenesis syndrome in male fetus when female pregnant dams were exposed to them. The present study investigated effects of DINP on fetal Leydig cell function and testis development. Female pregnant Sprague Dawley rats received control vehicle (corn oil) or DINP (10, 100, 500, and 1000 mg/kg) by oral gavage from gestational day (GD) 12 to 21. At GD 21.5, testicular testosterone production, fetal Leydig cell numbers and distribution, testicular gene and protein expression levels were examined. DINP showed dose-dependent increase of fetal Leydig cell aggregation with the low observed adverse-effect level (LOAEL) of 10 mg/kg and multinucleated gonocyte with LOAEL of 100 mg/kg. At 10 mg/kg, DINP also significantly increased fetal Leydig cell size, but inhibited insulin-like 3 and 3β-hydroxysteroid dehydrogenase gene expression and protein levels. DINP inhibited testicular testosterone levels at 1000 mg/kg. The results indicate that in utero exposure to DINP affects the expression levels of some fetal Leydig cell steroidogenic genes, gonocyte multinucleation and Leydig cell aggregation. PMID:25445723

  19. Expression Analysis of Gnrh1 and Gnrhr1 in Spermatogenic Cells of Rat

    PubMed Central

    Ciaramella, Vincenza; Pariante, Paolo; Fasano, Silvia; Pierantoni, Riccardo

    2015-01-01

    Hypothalamic Gonadotropin Releasing Hormone (GnRH), via GnRH receptor (GnRHR), is the main actor in the control of reproduction, in that it induces the biosynthesis and the release of pituitary gonadotropins, which in turn promote steroidogenesis and gametogenesis in both sexes. Extrabrain functions of GnRH have been extensively described in the past decades and, in males, local GnRH activity promotes the progression of spermatogenesis and sperm functions at several levels. The canonical localization of Gnrh1 and Gnrhr1 mRNA is Sertoli and Leydig cells, respectively, but ligand and receptor are also expressed in germ cells. Here, we analysed the expression rate of Gnrh1 and Gnrhr1 in rat testis (180 days old) by quantitative real-time PCR (qPCR) and by in situ hybridization we localized Gnrh1 and Gnrhr1 mRNA in different spermatogenic cells of adult animals. Our data confirm the testicular expression of Gnrh1 and of Gnrhr1 in somatic cells and provide evidence that their expression in the germinal compartment is restricted to haploid cells. In addition, not only Sertoli cells connected to spermatids in the last steps of maturation but also Leydig and peritubular myoid cells express Gnrh1. PMID:25861269

  20. Structural organization of the porcine and human genes coding for a leydig cell-specific insulin-like peptide (LEY I-L) and chromosomal localization of the human gene (INSL3)

    SciTech Connect

    Burkhardt E.; Adham, I.M.; Brosig, B.; Gastmann, A.; Engel, W. ); Mattei, M.G. )

    1994-03-01

    Leydig insulin-like protein (LEY I-L) is a member of the insulin-like hormone superfamily. The LEY I-L gene (designated INSL3) is expressed exclusively in prenatal and postnatal Leydig cells. The authors report here the cloning and nucleotide sequence of porcine and human LEY I-L genes including the 5[prime] regions. Both genes consist of two exons and one intron. The organization of the LEY I-L gene is similar to that of insulin and relaxin. The transcription start site in the porcine and human LEY I-L gene is localized 13 and 14 bp upstream of the translation start site, respectively. Alignment of the 5[prime] flanking regions of both genes reveals that the first 107 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 80%. This conserved region contains a consensus TATAA box, a CAAT-like element (GAAT), and a consensus SP1 sequence (GGGCGG) at equivalent positions in both genes and therefore may play a role in regulation of expression of the LEY I-L gene. The porcine and human genome contains a single copy of the LEY I-L gene. By in situ hybridization, the human gene was assigned to bands p13.2-p12 of the short arm of chromosome 19. 25 refs., 6 figs.

  1. Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

    SciTech Connect

    Erkekoglu, Pinar; Rachidi, Walid; Giray, Belma; Favier, Alain; Hincal, Filiz

    2010-10-01

    Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates' testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 {mu}M selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.

  2. Characterization of beta-adrenergic receptors in dispersed rat testicular interstitial cells

    SciTech Connect

    Poyet, P.; Labrie, F.

    1987-01-01

    Recent studies have shown that beta-adrenergic agents stimulate steroidogenesis and cyclic AMP formation in mouse Leydig cells in culture. To obtain information about the possible presence and the characteristics of a beta-adrenergic receptor in rat testicular interstitial cells, the potent beta-adrenergic antagonist (/sup 125/I)cyanopindolol (CYP) was used as ligand. Interstitial cells prepared by collagenase dispersion from rat testis were incubated with the ligand for 2 h at room temperature. (/sup 125/I)cyanopindolol binds to a single class of high affinity sites at an apparent KD value of 15 pM. A number of sites of 6,600 sites/cell is measured when 0.1 microM (-) propranolol is used to determine non-specific binding. The order of potency of a series of agonists competing for (/sup 125/I)cyanopindolol binding is consistent with the interaction of a beta 2-subtype receptor: zinterol greater than (-) isoproterenol greater than (-) epinephrine = salbutamol much greater than (-) norepinephrine. In addition, it was observed that the potency of a large series of specific beta 1 and beta 2 synthetic compounds for displacing (/sup 125/I)cyanopindolol in rat interstitial cells is similar to the potency observed for these compounds in a typical beta 2-adrenergic tissue, the rat lung. For example, the potency of zinterol, a specific beta 2-adrenergic agonist, is 10 times higher in interstitial cells and lung than in rat heart, a typical beta 1-adrenergic tissue. Inversely, practolol, a typical beta 1-antagonist, is about 50 times more potent in rat heart than in interstitial cells and lung.

  3. Primary rat Sertoli and interstitial cells exhibit a differential response to cadmium

    SciTech Connect

    Clough, S.R.; Welsh, M.J.; Payne, A.H.; Brown, C.D.; Brabec, M.J. )

    1990-01-01

    Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of 109Cd, reduction of the vital tetrazolium dye MTT, incorporation of 3H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl2, were less affected. The 72-hr LC50's for Sertoli cells and interstitial cells were 4.1 and 19.6 microM CdCl2, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.

  4. Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

    PubMed Central

    Jarazo Dietrich, Sabrina; Fass, Mónica Irina; Jacobo, Patricia Verónica; Sobarzo, Cristian Marcelo Alejandro; Lustig, Livia; Theas, María Susana

    2015-01-01

    Background Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO. Objective The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion. Method and Results EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect. Conclusions We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular

  5. Sensitivity to cadmium-induced genotoxicity in rat testicular cells is associated with minimal expression of the metallothionein gene.

    PubMed

    Shiraishi, N; Hochadel, J F; Coogan, T P; Koropatnick, J; Waalkes, M P

    1995-02-01

    Cadmium is a carcinogenic metal. Although the mechanism of tumor induction is unknown, DNA/metal interactions may be involved. Metallothionein can protect against cadmium toxicity in our previous work it was shown to reduce cadmium genotoxicity in cultured cells. To extend these results, the genotoxicity of cadmium was studied in R2C cells, a rat testicular Leydig cell line. The R2C cells were very sensitive to cadmium-induced single-strand DNA damage (SSD), as measured by alkaline elution. SSD occurred in R2C cells after treatment with 25 and 50 microM CdCl2 for 2 hr. Prior work showed other cells required much higher levels of cadmium (approximately 500 microM) to induce genotoxicity. The genotoxic levels of cadmium (25-50 microM) were not cytotoxic in R2C cells as assessed by a metabolic activity (MTT) assay. Pretreatment of R2C cells with a low cadmium dose (2 microM, 24 hr) had no effect on cadmium-induced SSD, in contrast to prior work in other cells where such pretreatments reduced SSD through metallothionein gene activation. In fact, cadmium or zinc treatments resulted in little or no increase in metallothionein gene expression in R2C cells as determined by Northern blot analysis for metallothionein mRNA using cDNA or oligonucleotide probes and radioimmunoassay for metallothionein protein production. Basal metallothionein mRNA was essentially nondetectable. Induction of a cadmium-binding protein in R2C cells did occur, as determined by Cd-heme assay, but did not induce tolerance to SSD. In vivo, the Leydig cell is a target for cadmium carcinogenicity and its cadmium-binding protein is thought not to be a true metallothionein. These results indicate that R2C cells are sensitive to cadmium-induced genotoxicity and that this sensitivity is associated with minimal expression of the metallothionein gene. PMID:7871536

  6. EXPOSURE TO DIETHYL HEXYL PHTHALATE (DEHP) DELAYS PUBERTY AND REDUCES ANDROGEN-DEPENDENT TISSUE WEIGHTS IN LONG EVANS HOODED AND SPRAGUE DAWLEY MALE RATS

    EPA Science Inventory

    DEHP is a plasticizer that alters sexual differentiation in the male rat by reducing fetal Leydig cell testosterone synthesis and insl3 mRNA levels. When exposure includes the pubertal stage of life, DEHP and other phthalates delay puberty and reduce androgen-dependent tissue wei...

  7. CHRONIC EXPOSURE TO DI(2-ETHYLHEXYL) PHTHALATE (DEHP) DELAYS PUBERTY AND REDUCES ANDROGEN-DEPENDENT TISSUE WEIGHTS IN THE MALE RAT

    EPA Science Inventory

    DEHP, dibutyl (DBP)-, and benzyl butyl (BBP)- phthalate are plasticizers that cause adverse developmental reproductive effects in laboratory animals. They alter sexual differentiation in the rat by reducing fetal Leydig cell testosterone synthesis and insl3 mRNA levels, which in ...

  8. Fertility-sparing management and obstetric outcomes in a 20-year-old patient with a Sertoli-Leydig cell tumor of the ovary: A case report and review of the literature

    PubMed Central

    Stavrakis, Thomas; Kalogiannidis, Ioannis; Petousis, Stamatios; Tsompanidou, Chrisoula; Delkos, Dimitris; Prapas, Nikolaos; Rousso, David

    2016-01-01

    Sertoli-Leydig cell tumors (SLCTs) are an uncommon subtype of sex-cord stromal tumors of the ovary, which most commonly arise in women of reproductive age, creating an issue with regard to the preservation of fertility. The clinical manifestation of SLCTs varies widely, ranging from an asymptomatic clinical profile to extreme virilization. Correct diagnosis of SLCT is crucial and is primarily based on histopathological results. The current study presents the case of a 20-year-old woman who underwent unilateral salpingo-oophorectomy and adjuvant chemotherapy due to the diagnosis of an SLCT of the left ovary. Almost 2 years after the initial surgery, during the follow-up period, the patient conceived normally. Pregnancy was uneventful and the patient vaginally delivered a healthy infant at 38 weeks of gestation. A total of 1 year after delivery (3 years after the initial diagnosis), follow-up of the patient did not reveal any disease recurrence. In conclusion, SLCTs may be adequately treated by fertility-sparing surgery and chemotherapy in young women who wish to preserve their fertility. Natural conception, an uncomplicated pregnancy and a vaginal delivery are possible. PMID:27446397

  9. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology. PMID:27608010

  10. Benzene-induced histopathological changes and germ cell population dynamics in testes of Sprague Dawley rats.

    PubMed

    Singh, R K; Bansode, F W

    2011-11-01

    Benzene has been considered as an occupational hematotoxin and leukemogen. The present study was conducted to determine the effects of oral administration of benzene on reproductive organs and testicular spermatogenesis in rats. Adult rats were divided into three weight matched groups (Gr. I-III) containing 6 each. Gr. I rats received vehicle only and served as control. Rats in Gr. II and III were fed orally with 0.5 and 1 ml kg(-1) dose of benzene for 14 and 9 days, respectively and autopsy was done on 15th and 10th day. Food and water intake and gross behavioral changes were recorded daily during the entire treatment. Results showed no significant change in reproductive organ weights viz. testis, epididymis and ventral prostate in benzene-treated (0.5 or 1 ml kg(-1)) rats than that in controls. But, caused a significant decrease (p < 0.005) in weights of seminal vesicles in rats treated with both 0.5 and 1 ml kg(-1) doses compared to control. In contrast, at higher dose (1 ml kg(-1)) of benzene, significant (p < 0.001) decline in body weight and 100% mortality was observed on day 10 of autopsy. In treated rats, testicular cytotoxicity was marked by multinucleated giant cells formation, cytoplasmic vacuolization, pyknosis of nuclei, chromatolysis, desquamation and dissolution of germ cells in tubular lumen. The quantitative analysis of spermatogenesis showed a significant (p < 0.001) decrease in number ofA-spermatogonia (in 1 ml kg(-1) dose only), primary spermatocytes (non-pachytene and pachytene) and spermatids (round and elongated) in treated as compared to control rats. The diameters of testicular tubules and Leydig cells nuclei were also significantly (p < 0.001) reduced in treated rats. A steady loss in food and water intake recorded and signs of ill health were observed in treated (0.5 or 1 ml kg(-1)) rats. Results of the study indicated antitesticular lantispermatogenic effects of benzene at 0.5 and 1 ml kg(-1) dose in rats. PMID:22471202