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Sample records for rat leydig cells

  1. [Leydig cell function in experimental cryptorchism and varicocele in rats].

    PubMed

    Hernández-Yánez, L; Marín-López, G; Vílchez-Martínez, J; Bishop, W

    1999-06-01

    Leydig cells were isolated from testes of normal, cryptorchid and induced- varicocele rats. These cells were counted and coincubated with and without human Chorionic Gonadotropin (hCG) during 3 hours; thereafter, steroids were measured in the incubation media. Cryptorchid animals showed the lowest number of Leydig cells, the highest Progesterone response to hCG, a slight increment of testosterone and a decrease of estradiol. On the contrary, both left and right testes from varicocele induced rats showed a higher cell number (per g of tissue), lower progesterone response, slightly higher response testosterone and lower testosterone response. These results demonstrate that these conditions of testicular hyperthermia do not affect the number and function of Leydig cells to the same degree. This may be due to differences in the testicular temperature reached with each procedure.

  2. 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

  3. Hepatocyte growth factor-modulated rat Leydig cell functions.

    PubMed

    Del Bravo, Jessica; Catizone, Angela; Ricci, Giulia; Galdieri, Michela

    2007-01-01

    Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

  4. Stem Leydig cell differentiation: gene expression during development of the adult rat population of Leydig cells.

    PubMed

    Stanley, Erin L; Johnston, Daniel S; Fan, Jinjiang; Papadopoulos, Vassilios; Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R; Jelinsky, Scott A

    2011-12-01

    Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.

  5. Glucocorticoid suppresses steroidogenesis in rat progenitor Leydig cells.

    PubMed

    Xiao, Ye-Chen; Huang, Ya-Dong; Hardy, Dianne O; Li, Xiao-Kun; Ge, Ren-Shan

    2010-01-01

    Glucocorticoid (GC) inhibits testosterone production in adult Leydig cells by the glucocorticoid receptor (GR). However, whether GC affects the development of Leydig cells is unclear. The goal of the present study is to investigate the effects of GC on steroidogenesis of rat progenitor Leydig cells (PLCs) in vitro. Dexamethasone (DEX) inhibited androsterone (AO) production in PLCs. The GR antagonist RU38486 reversed the DEX-induced inhibition of AO, whereas the mineralocorticoid receptor antagonist RU28318 did not. RU38486 also reversed DEX-induced reductions in steady-state mRNA levels of steroidogenic acute regulatory protein (Star) and 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1). Steroidogenic acute regulatory protein (StAR) protein expression and 3β-hydroxysteroid dehydrogenase (3βHSD) enzyme activity were affected similarly. These results show that GCs inhibit steroidogenesis of PLCs by suppression of StAR and 3βHSD via a GR-mediated mechanism.

  6. Steroidogenic fate of the Leydig cells that repopulate the testes of young and aged Brown Norway rats after elimination of the preexisting Leydig cells.

    PubMed

    Chen, Haolin; Guo, Jingjing; Ge, Renshan; Lian, Qingquan; Papadopoulos, Vassilios; Zirkin, Barry R

    2015-12-01

    The capacity of Brown Norway rat Leydig cells to produce testosterone (T) decreases with aging. In a previous study, we reported that a new generation of Leydig cells can be restored in both young and old rat testes after a single injection of ethane dimethanesulfonate (EDS), and that the abilities of the new Leydig cells in young and old rats to produce T were equivalent. Our objective herein was to compare the steroidogenic fate of the new Leydig cells over time. Young (3 month-old) and old (18 month-old) rats were injected with EDS to eliminate the existing Leydig cells. Ten weeks after EDS, Leydig cells had been restored and T production by the new Leydig cells isolated from young and old rat testes was equivalent. Thirty weeks after EDS treatment of young rats, the ability of the new Leydig cells to produce T had not diminished from 10 weeks post-EDS. In contrast, at 30 weeks post-EDS, T production by new cells in old rat testes was reduced significantly from the 10-week level. Serum T levels at 10 and 30 weeks were consistent with Leydig cell T production. Serum LH levels did not differ in any group. Thus, although the Leydig cells restored to both young and old rats after EDS initially produced T at high, equivalent levels, the cells in the old testes did not maintain this ability. These results suggest that: 1) the cells from which new populations of Leydig cells are derived may differ depending upon the age of the rat; and/or 2) factors extrinsic to the new Leydig cells in young and old testes differ, and it is these differences that are responsible for reductions in T by the newly formed Leydig cells in the testes of old rats.

  7. Regulation of development of rat stem and progenitor Leydig cells by activin.

    PubMed

    Li, L; Wang, Y; Li, X; Liu, S; Wang, G; Lin, H; Zhu, Q; Guo, J; Chen, H; Ge, H-S; Ge, R-S

    2017-01-01

    Stem Leydig cells have been demonstrated to differentiate into adult Leydig cells via intermediate stages of progenitor and immature Leydig cells. However, the exact regulatory mechanisms are unclear. We hypothesized that the development of stem or progenitor Leydig cells depends upon locally produced growth factors. Microarray analysis revealed that the expression levels of activin type I receptor (Acvr1) and activin A receptor type II-like 1 (Acvrl1) were stem > progenitor = immature = adult Leydig cells. This indicates that their ligand activin might play an important role in stem and progenitor Leydig cell proliferation and differentiation. When seminiferous tubules were incubated with 1 or 10 ng/mL activin A for 3 days, it concentration-dependently increased EdU incorporation into stem Leydig cells by up to 20-fold. When progenitor Leydig cells were incubated with 1 or 10 ng/mL activin A for 2 days, it concentration-dependently increased (3) H-thymidine incorporation into progenitor Leydig cells by up to 200%. Real-time PCR analysis showed that activin A primarily increased Pcna expression but reduced Star, Hsd3b1, and Cyp17a1 expression levels. Activin A also significantly inhibited the basal and luteinizing hormone-stimulated androgen production. In conclusion, activin A primarily stimulates the proliferation of stem and progenitor Leydig cells, but inhibits the differentiation of stem and progenitor Leydig cells into the Leydig cell lineage in rat testis.

  8. Chronic stress induces ageing-associated degeneration in rat Leydig cells

    PubMed Central

    Wang, Fei-Fei; Wang, Qian; Chen, Yong; Lin, Qiang; Gao, Hui-Bao; Zhang, Ping

    2012-01-01

    Several studies have suggested that stress and ageing exert inhibitory effects on rat Leydig cells. In a pattern similar to the normal process of Leydig cell ageing, stress-mediated increases in glucocorticoid levels inhibit steroidogenic enzyme expression that then results in decreased testosterone secretion. We hypothesized that chronic stress accelerates the degenerative changes associated with ageing in Leydig cells. To test this hypothesis, we established a model of chronic stress to evaluate stress-induced morphological and functional alterations in Brown Norway rat Leydig cells; additionally, intracellular lipofuscin levels, reactive oxygen species (ROS) levels and DNA damage were assessed. The results showed that chronic stress accelerated ageing-related changes: ultrastructural alterations associated with ageing, cellular lipofuscin accumulation, increased ROS levels and more extensive DNA damage were observed. Additionally, testosterone levels were decreased. This study sheds new light on the idea that chronic stress contributes to the degenerative changes associated with ageing in rat Leydig cells in vivo. PMID:22609820

  9. Effects of the Janus Kinase Inhibitor, Tofacitinib, on Testicular Leydig Cell Hyperplasia and Adenoma in Rats, and on Prolactin Signaling in Cultured Primary Rat Leydig Cells.

    PubMed

    Chapin, Robert E; Ball, Douglas J; Radi, Zaher A; Kumpf, Steven W; Koza-Taylor, Petra H; Potter, David M; Mark Vogel, W

    2017-01-01

    Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis. Tofacitinib preferentially inhibits receptor signaling through JAK3 and JAK1, relative to JAK2. In the 2-year rat carcinogenicity study, there were tofacitinib, dose-related increases in the incidences of testicular Leydig cell hyperplasia and benign adenomas in male rats, and decreased incidences of mammary tumors and duct dilatation/galactocele in female rats. Such findings in rats are typical of agents, such as dopamine agonists, which decrease prolactin (PRL) activity. Since prolactin signals through the JAK2 pathway, we hypothesized that these findings were off-target effects due to inhibition of PRL signaling via JAK2. The studies reported here were designed to investigate the interruption of PRL signaling pathways in Leydig cells. In isolated primary rat Leydig cells, PRL increased phosphorylated Signal Transducer and Activator of Transcription-5 protein, and mRNA levels for luteinizing hormone receptor. Tofacitinib, at concentrations observed in the rat carcinogenicity study, dose-dependently inhibited these effects. These observations illustrate a novel mechanism, the inhibition of prolactin signaling by which modulation of JAK activity can modulate PRL signaling pathways to induce Leydig cell tumors in rats. Since human Leydig cells lack this PRL dependence for normal function, these rodent tumors do not indicate a health risk to human patients.

  10. Effect of beta adrenergic antagonist on the production of testosterone by rat's Leydig cells.

    PubMed

    Khan, Umar Ali; Aslam, Muhammad; Saeed, S Abdul

    2004-01-01

    The Leydig cell is the source of the male sex steroids, or androgens, which are essential for the maintenance of the male phenotype, the male gonads. and spermatogenesis. It has been reported that patients taking beta-blockers experience sexual dysfunction. The purpose of this study was to explore the direct mechanism by which beta adrenergic antagonist exert its' effect on testosterone production by rat Leydig cells. Enzymatic dispersion of rat Leydig cell was done. About 85000 cells per tube were taken. After removal of endogenous testosterone by preincubation, The rat Leydig cells were incubated with varying concentrations of Atenolol: [Selective Beta-Adrenergic Antagonist] (10(-6), 10(-7) and 10(-9) M) with or with out LH 250 IU for three hours to measure the testosterone release by RIA. Atenolol, in varying concentrations caused a significant (P<0.05) reduction in testosterone release by the rat Leydig cells as compared to the basal release of testosterone in a dose-dependent fashion. Atenolol decreased the testosterone release by LH stimulated Leydig cells more significantly (P<0.001) as compared to the effects of Atenolol produced on non-stimulated Leydig cells. The current data indirectly suggest that Atenolol inhibits testosterone releasee via mechanism involving decrease production of cAMP but not affecting the enzyme activities of steroidogenesis.

  11. Stem Leydig Cell Differentiation: Gene Expression During Development of the Adult Rat Population of Leydig Cells1

    PubMed Central

    Stanley, Erin L.; Johnston, Daniel S.; Fan, Jinjiang; Papadopoulos, Vassilios; Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R.; Jelinsky, Scott A.

    2011-01-01

    ABSTRACT Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct. PMID:21832170

  12. 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.

  13. Effects of Estradiol and Methoxychlor on Leydig Cell Regeneration in the Adult Rat Testis

    PubMed Central

    Chen, Bingbing; Chen, Dongxin; Jiang, Zheli; Li, Jingyang; Liu, Shiwen; Dong, Yaoyao; Yao, Wenwen; Akingbemi, Benson; Ge, Renshan; Li, Xiaokun

    2014-01-01

    The objective of the present study is to determine whether methoxychlor (MXC) exposure in adulthood affects rat Leydig cell regeneration and to compare its effects with estradiol (E2). Adult 90-day-old male Sprague-Dawley rats received ethane dimethane sulfonate (EDS) to eliminate the adult Leydig cell population. Subsequently, rats were randomly assigned to four groups and gavaged with corn oil (control), 0.25 mg/kg E2 and 10 or 100 mg/kg MXC daily from days 5 to 30 post-EDS treatment. The results showed that MXC and E2 reduced serum testosterone levels on day 58 post-EDS treatment. qPCR showed Hsd17b3 mRNA levels were downregulated 7–15 fold by E2 and MXC, indicating that development of the new population of Leydig cells was arrested at the earlier stage. This observation was supported by the results of histochemical staining, which demonstrated that Leydig cells in MXC-treated testis on day 58 post-EDS treatment were mostly progenitor Leydig cells. However, Pdgfb mRNA levels were downregulated, while Lif transcript levels were increased by MXC. In contrast, E2 did not affect gene expression for these growth factors. In conclusion, our findings indicated that both MXC and E2 delayed rat Leydig cell regeneration in the EDS-treated model, presumably acting by different mechanisms. PMID:24806340

  14. Tungstate treatment improves Leydig cell function in streptozotocin-diabetic rats.

    PubMed

    Ballester, Joan; Domínguez, Jorge; Muñoz, M Carmen; Sensat, Meritxell; Rigau, Teresa; Guinovart, Joan J; Rodríguez-Gil, Joan E

    2005-01-01

    Oral administration of sodium tungstate to adult male streptozotocin-diabetic rats for 3 months normalized serum levels of glucose, insulin, luteinizing hormone, and follicle-stimulating hormone. These effects were accompanied by an increase in reproductive performance, which was related to a strong improvement in Leydig cell function markers, such as the recovery of the number of Leydig cells and serum testosterone levels. Moreover, this in vivo recovery was related to a concomitant increase in the cell expression of insulin receptors. Tungstate treatment did not modify Leydig cell function in healthy rats. Furthermore, the addition of tungstate or insulin to the mTLC-1 cell line from Leydig cell origin increased the phosphorylation states of MAP-kinase and glycogen synthase kinase-3. Our results indicate that tungstate treatment in diabetic rats leads to a recovery of reproductive performance by increasing the number of Leydig cells. This increase contributes to the recovery of their functionality, thereby improving the overall function of these cells. We propose that this improvement is caused by the combined effect of the tungstate-induced normalization of insulin glucose and luteinizing hormone serum levels and a direct action of the effector on Leydig cells through modulation of at least MAP-kinase and glycogen synthase kinase-3 activities.

  15. Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells.

    PubMed

    Salva, A; Klinefelter, G R; Hardy, M P

    2001-01-01

    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 centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.

  16. A role of KIT receptor signaling for proliferation and differentiation of rat stem Leydig cells in vitro.

    PubMed

    Liu, Shiwen; Chen, Xiaomin; Wang, Yiyan; Li, Linxi; Wang, Guimin; Li, Xiaoheng; Chen, Haolin; Guo, Jingjing; Lin, Han; Lian, Qing-Quan; Ge, Ren-Shan

    2017-03-15

    In the testis, KIT ligand (KITL, also called stem cell factor) is expressed by Sertoli cells and its receptor (c-kit, KIT) is expressed by spermatogonia and Leydig cells. Although KITL-KIT signaling is critical for the spermatogenesis, its roles in Leydig cell development during puberty are not clear. In the present study, we investigated effects of KITL on stem Leydig cell proliferation and differentiation. Using an in vitro culture system of seminiferous tubules from Leydig cell-depleted testis, we found that KITL increased the proliferation activity of putative stem Leydig cells at higher concentration (10 and 100 ng/ml). Low concentration (1 ng/ml) of KITL significantly induced the differentiation of stem Leydig cells via increasing the expression level of steroidogenic acute regulatory protein (Star). In contrast, higher concentration (100 ng/ml) of KITL inhibited the differentiation of stem Leydig cells via inhibiting the steroidogenic enzyme (Cyp11a1, Cyp17a1, and Hsd17b3) expression levels. We cultured rat progenitor Leydig cells with KITL for 48 h and did not find any influence of KITL on the proliferation and androgen production of these cells. In conclusion, KITL is a growth factor that regulates the development of the stem Leydig cell.

  17. 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...

  18. Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo

    PubMed Central

    Guo, Jing-Jing; Ma, Xue; Wang, Claire QF; Ge, Yu-Fei; Lian, Qing-Quan; Hardy, Dianne O; Zhang, Yu-Fei; Dong, Qiang; Xu, Yun-Fei; Ge, Ren-Shan

    2013-01-01

    Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone (LH) or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague–Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin-releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7α-methyl-nortestosterone (MENT), which is an androgen resistant to metabolism by 5α-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. 3H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igf1, Lifr, Pdgfra, Bcl2, Ccnd3 and Pcna were upregulated by MENT, and those of Pdgfra, Ccnd3 and Pcna were upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells. PMID:23792342

  19. Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo.

    PubMed

    Guo, Jing-Jing; Ma, Xue; Wang, Claire Q F; Ge, Yu-Fei; Lian, Qing-Quan; Hardy, Dianne O; Zhang, Yu-Fei; Dong, Qiang; Xu, Yun-Fei; Ge, Ren-Shan

    2013-09-01

    Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone (LH) or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague-Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin-releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7α-methyl-nortestosterone (MENT), which is an androgen resistant to metabolism by 5α-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. (3)H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igf1, Lifr, Pdgfra, Bcl2, Ccnd3 and Pcna were upregulated by MENT, and those of Pdgfra, Ccnd3 and Pcna were upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells.

  20. [Post-natal development of interstitial Leydig cells in rat testicle].

    PubMed

    Quiros, A; López, A; Larran, J; Castiñeiras, J; Vilches, J

    1992-01-01

    The existence of different stages of postnatal activities in Leydig's cells, as demonstrated in hormone studies, appears to be fully admitted. Such occurrence should have repercussions on the population of those elements primarily entrusted with their production, either in the number of elements or their activities. The present article studies Leydig's cells rate in Wistar rats over the postnatal stage, from birth to adulthood. During this postnatal stage, Leydig's cells population does not actually disappear. There is a decrease immediately after birth and up to the age of 21 days when gonadal drop occurs in this species, and from there on a considerable increase takes place for in a two-month period they multiply by ten. On the other hand, it is highly surprising the great speed of testicle growth up to the age of 60 days, reaching maximum size at 90 days. The increase is due to the growth of the tubular compartment.

  1. [TGF-beta1 reduces connexin43-mediated gap junctional intercellular communication in rat Leydig cells].

    PubMed

    Liu, Man-Li; Zhang, Zhi-Hong; Wang, Zong-Ren; Ma, Jing

    2012-02-01

    To observe the effects of TGF-beta on the expression of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) in rat Leydig cells, and investigate the association of its effects on Leydig cells with its ability of changing GJIC. Primarily cultured purified Leydig cells were divided into a blank control group, a positive control group (treated with the GJIC inhibitor Carbenoxolone), and four TGF-beta1 groups (treated with TGF-beta1 at the concentration of 1, 2, 5 and 10 ng/ml, respectively, for 20 hours). The localization and expression of Cx43 were detected by immunofluorescence and Western blot, and the changes in GJIC analyzed by FRAP assay. Cx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells. TGF-beta1 significantly elevated the expression of Cx43 in the cytoplasm, but caused no evident change in the membrane. Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-beta1 as compared with that of the blank control group (P < 0.05). After 20 hours of treatment with TGF-beta1 at 5 ng/ml, the fluorescence intensity of Leydig cells was markedly reduced (P < 0.01), with a mean fluorescence recovery rate of merely (43.58 +/- 1.87)%. TGF-beta1 could significantly down-regulate GJIC between adjacent Leydig cells, and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.

  2. Histochemical changes in the Leydig cells of rats drinking the aqueous hollyhock extract.

    PubMed

    Papiez, M

    2001-01-01

    The effect of aqueous hollyhock flower (Althaea rosea Cav. var. nigra) extract on the rat Leydig cell metabolism and morphology was studied using histochemical, morphometric and radioimmunological methods. The rats were drinking the extract for 30 days (group A1) and for 180 days (group A2). Leydig cells of group A1 manifested marked increase in the 3beta-HSD, G6PD and NADPD activities and in the Khanolkar reaction intensity. These findings were accompanied by the increase in the volume of Leydig cells and their nuclei. In group A2 Leydig cells, statistically insignificant changes in the G6PD and NADPD activities were observed, however, the significant increase in the 3beta-HSD activity and the Khanolkar reaction intensity indicated compensatory changes. The statistically significant elevation of the androgen level accompanied by a decrease in estrogen content in homogenates of group A2 testes pointed to weak antiestrogenic effect of the extract. The obtained results indicate an influence of the hollyhock extract on steroid metabolism.

  3. 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.

  4. Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.

    PubMed

    Chen, Chih-Chieh; Chen, Chien-Wei; Lin, Po-Han; Chou, Jou-Chun; Weng, Ting-Chun; Jian, Cai-Yun; Hu, Sindy; Lai, Wei-Ho; Lieu, Fu-Kong; Wang, Shyi-Wu; Wang, Paulus S

    2017-08-01

    The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10(-9) -10(-4)  M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Effect of cyclic AMP on the calcium-dependent potassium conductances of rat Leydig cells.

    PubMed

    Desaphy, J F; Joffre, M

    1998-06-01

    Two potassium conductances have been isolated in rat Leydig cells by their sensitivity to cytosolic calcium and to K+ channel blockers. We used the whole-cell configuration of the patch-clamp technique to investigate their sensitivity to cyclic AMP, the main messenger of luteinizing hormone, which stimulates Leydig cell steroidogenesis. The voltage-dependent potassium conductance is not modified by exposing the cell to 1 mM chlorophenylthio-cyclic AMP (CPT-cAMP), a membrane-permeant analogue of cAMP. By contrast, the large, calcium-activated potassium conductance is upregulated by CPT-cAMP. Furthermore, the latter is potentiated by the chloride channel blocker 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid, sodium salt (SITS).

  6. The albumin fraction of rat testicular fluid stimulates steroid production by isolated Leydig cells.

    PubMed

    Melsert, R; Hoogerbrugge, J W; Rommerts, F F

    1988-10-01

    Rat testicular fluid (rTF), but not rat serum (rS) or plasma (rP) can further increase within 4 h maximally luteinizing hormone (LH)-stimulated or 22 R-hydroxycholesterol-supported pregnenolone production by immature rat Leydig cells in vitro. This effect of rTF is dose dependent (0.05-1.2% protein, w/v) with an increase up to 4-fold. The objective of the present study was to isolate and characterize the bioactive factor(s) in rTF. After sequential ammonium sulfate fractionation, gel filtration chromatography on Superose 12 and affinity chromatography on concanavalin A-Sepharose it was shown that the albumin fraction was a major biologically active fraction in rTF. The relative specific activity in these fractions was never greater than 1.3-1.4, which is in agreement with the purification factor required to obtain pure albumin from rTF. Commercially obtained albumin fractions from human, bovine and rat sera, up to 99% purity, also increased Leydig cell steroid production more than 3-fold when added in concentrations between 0.1 and 1% w/v in combination with LH or 22R-hydroxycholesterol. Other proteins such as hemoglobin and ovalbumin were not effective in stimulation of steroid production. Bovine serum albumin (bSA, fraction V) at concentrations of 0.25 and 1.0% (w/v), had no or minor effects on LH-stimulated steroid production by rat granulosa cells or adrenocorticotrophic hormone (ACTH)-stimulated steroid production by rat adrenal cells. These findings indicate that albumin itself or minor compounds copurified with albumin represent the main biologically active component in rTF for short-term stimulation of Leydig cell steroid production. Since bioactivity could not be demonstrated in serum which contains similar amounts of albumin as rTF, inhibitory compounds may be present in rat serum.

  7. 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.

  8. 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

    2015-03-24

    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.

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

    PubMed Central

    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

  10. The subcellular localization of phospholipase D activities in rat Leydig cells.

    PubMed

    Strand, A M; Lauritzen, L; Vinggaard, A M; Hansen, H S

    1999-06-25

    Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg2+ -activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP2-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPgammaS. The peak of oleate Mg2+ -PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP2-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester-stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [14C]-palmitate and then stimulated for 15 min with 100 nM 4-beta-phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [14C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [14C]-phosphatidylbutanol could be inhibited dose-dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.

  11. Nickel sulfate induced apoptosis via activating ROS-dependent mitochondria and endoplasmic reticulum stress pathways in rat Leydig cells.

    PubMed

    Zou, Lingyue; Su, Li; Sun, Yifan; Han, Aijie; Chang, Xuhong; Zhu, An; Liu, Fangfang; Li, Jin; Sun, Yingbiao

    2017-07-01

    Nickel can induce apoptosis of testicular Leydig cells in mice, whereas the mechanisms remain unclear. In this study, we investigated the role of nickel-induced reactive oxygen species (ROS) generation in mitochondria and endoplasmic reticulum stress (ERS) mediated apoptosis pathways in rat Leydig cells. Fluorescent DCF and Annexin-V FITC/PI staining were performed to measure the production of ROS and apoptosis in Leydig cells. RT-qPCR and Western blot were conducted to analyze the key genes and proteins involved in mitochondria and ERS apoptotic pathways. The results showed that nickel sulfate induced ROS generation, consequently resulted in nucleolus deformation and apoptosis in testicular Leydig cells, which were then attenuated by ROS inhibitors of N-acetylcysteine (NAC) and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). Nickel sulfate-triggered Leydig cells apoptosis via mitochondria and ERS pathways was characterized by the upregulated mRNA and proteins expression of Bak, cytochrome c, caspase 9, caspase 3, GRP78, GADD153, and caspase 12, which were inhibited by NAC and TEMPO respectively. The findings indicated that nickel-induced ROS generation was involved in apoptosis via mitochondria and ERS pathways in rat Leydig cells. © 2017 Wiley Periodicals, Inc.

  12. 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

    2016-03-08

    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.

  13. 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.

  14. Goliath, a ring-H2 mitochondrial protein, regulated by luteinizing hormone/human chorionic gonadotropin in rat leydig cells.

    PubMed

    Guais, A; Solhonne, B; Melaine, N; Guellaen, G; Bulle, F

    2004-01-01

    We have cloned the rat homologue of the ring-H2 protein Goliath involved in Drosophila development. The rat Goliath mRNA (1.85 kb) was translated as a major ubiquitous protein species of 28-kDa and three larger isoforms (50, 46, and 36 kDa) expressed mainly in liver, lung, stomach, heart, and thymus and barely detectable in other tissues (kidney, skeletal muscle, brain, testis, intestine, and spleen). By immunohistochemistry on rat testis sections, we localized the protein in interstitial tissue and seminiferous tubules. In tubules, Goliath was expressed mainly in postmeiotic germ cells and to a much lesser extent in Sertoli cells. In the interstitium, Goliath was exclusively present in Leydig cells. Using a series of immunolabeling, cellular fractionation, and electron microscopy experiments, we established that Goliath is present in mitochondria of the R2C Leydig cell line. Using short-term hypophysectomized animals, we showed that Goliath is regulated by LH/hCG in Leydig cells but not in germ cells. This regulation in Leydig cells concerned only the 50-kDa isoform. This report is the first description of a differential regulation of the Goliath protein between germ cells and Leydig cells.

  15. Aging and luteinizing hormone effects on reactive oxygen species production and DNA damage in rat Leydig cells.

    PubMed

    Beattie, Matthew C; Chen, Haolin; Fan, Jinjiang; Papadopoulos, Vassilios; Miller, Paul; Zirkin, Barry R

    2013-04-01

    We observed previously that after long-term suppression of luteinizing hormone (LH) and thus of Leydig cell steroidogenesis, restimulation of the Leydig cells by LH resulted in significantly higher testosterone production than by age-matched cells from control rats. These studies suggest that stimulation over time may elicit harmful effects on the steroidogenic machinery, perhaps through alteration of the intracellular oxidant-to-antioxidant balance. Herein we compared the effects of LH stimulation on stress response genes, formation of intracellular reactive oxygen species (ROS), and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and in aged cells. Microarray analysis indicated that LH stimulation resulted in significant increases in expression of genes associated with stress response and antiapoptotic pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in transiently increased ROS levels compared to controls. Aged Leydig cells also showed increased ROS soon after LH stimulation. However, in contrast to the young cells, ROS production peaked later and the time to recovery was increased. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage but significantly more so in the aged cells. DNA damage levels in response to LH and the levels of intracellular ROS were highly correlated. Taken together, these results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells and that while DNA damage occurs in cells of both ages, there is greater damage in the aged cells.

  16. 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.

  17. Effect of ETBE on reproductive steroids in male rats and rat Leydig cell cultures.

    PubMed

    de Peyster, Ann; Stanard, Bradley; Westover, Christian

    2009-10-08

    These experiments were conducted to follow up on a report of testis seminiferous tubular degeneration in Fischer 344 rats treated with high doses of ethyl t-butyl ether (ETBE). Also, high doses of a related compound, methyl t-butyl ether (MTBE), had been shown to reduce circulating testosterone (T) in rats. Isolated rat Leydig cells were used to compare hCG-stimulated T production following exposure to ETBE, MTBE, and their common main metabolite, TBA. In addition, male Fischer 344 rats were gavaged daily with 600 mg/kg, 1200 mg/kg or 1800 mg/kg ETBE in corn oil (n=12) for 14 days, the 1200 mg/kg dose chosen for comparison with a prior 14-day MTBE gavage experiment. In cell culture experiments, TBA was more potent than either ETBE or MTBE, both of which caused similar inhibition of T production at equimolar concentrations. In the in vivo study, no significant plasma T reduction was seen 1h after the final 1200 mg/kg ETBE dose, whereas 1200 mg/kg MTBE had significantly lowered T when administered similarly to Sprague-Dawley rats. Some rats treated with 1800 mg/kg ETBE had noticeably lower T levels, and the group average T level was 66% of corn oil vehicle control (p>0.05) with high variability also evident in ETBE-treated rats. 17beta-Estradiol had been increased by 1200 mg/kg MTBE, and was elevated in the 1200 and 1800 mg/kg ETBE dose groups (p<0.05), both groups also experiencing significantly reduced body weight gain. None of these effects were seen with 600 mg/kg/day ETBE. No definitive evidence of androgen insufficiency was seen in accessory organ weights, and no testicular pathology was observed after 14 days in a small subset of 1800 mg/kg ETBE-treated animals. Like MTBE, ETBE appears to be capable of altering reproductive steroid levels in peripheral blood sampled 1h after treatment, but only with extremely high doses that inhibit body weight gain and may produce mortality.

  18. Genetics Home Reference: Leydig cell hypoplasia

    MedlinePlus

    ... Home Health Conditions Leydig cell hypoplasia Leydig cell hypoplasia Enable Javascript to view the expand/collapse boxes. ... PDF Open All Close All Description Leydig cell hypoplasia is a condition that affects male sexual development. ...

  19. A high-fat diet fed during different periods of life impairs steroidogenesis of rat Leydig cells.

    PubMed

    Pinto-Fochi, Maria Etelvina; Pytlowanciv, Eloísa Zanin; Reame, Vanessa; Rafacho, Alex; Ribeiro, Daniele Lisboa; Taboga, Sebastião Roberto; Góes, Rejane Maira

    2016-12-01

    This study evaluated the impact of a high-fat diet (HFD) during different stages of rat life, associated or not with maternal obesity, on the content of sex steroid hormones and morphophysiology of Leydig cells. The following periods of development were examined: gestation (O1), gestation and lactation (O2), from weaning to adulthood (O3), from lactation to adulthood (O4), gestation to adulthood (O5), and after sexual maturation (O6). The HFD contained 20% unsaturated fat, whereas the control diet had 4% fat. Maternal obesity was induced by feeding HFD 15 weeks before mating. All HFD groups presented increased body weight, hyperinsulinemia and reduced insulin sensitivity. Except for O1, all HFD groups exhibited a higher adiposity index, hyperleptinemia, reduced testosterone and estradiol testicular levels, and decreased testicular 17β-HSD enzyme . Morphometrical analyses indicated atrophy of Leydig cells in the O2 group. Myelin vesicles were observed in the mitochondrial matrix of Leydig cells in O3, O4, O5 and O6, and autophagosomes containing mitochondria were found in O5 and O6. In conclusion, HFD feeding, before or after sexual maturation, reduces the functional capacity of rat Leydig cells. Maternal obesity associated with HFD during pregnancy/lactation prejudices Leydig cell steroidogenesis and induces its atrophy in adulthood, even if it is replaced by a conventional diet at later stages of life. Regardless of the life period of exposure to HFD, deregulation of leptin is the main factor related to steroidogenic impairment of Leydig cells, and, in groups exposed for longer periods (O3, O4, O5 and O6), this is worsened by structural damage and mitochondrial degeneration of these cells. © 2016 Society for Reproduction and Fertility.

  20. 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

  1. Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells.

    PubMed

    Chung, J Y; Chen, H; Midzak, A; Burnett, A L; Papadopoulos, V; Zirkin, B R

    2013-06-01

    Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4'-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

  2. 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.

  3. 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

  4. Inhibition of 3beta- and 17beta-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid.

    PubMed

    Zhao, Binghai; Chu, Yanhui; Hardy, Dianne O; Li, Xiao-kun; Ge, Ren-Shan

    2010-01-01

    Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) activities in rat testis microsomes and Leydig cells. The IC(50)s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3beta-HSD with an IC(50) of 53.2+/-25.9 microM and 17beta-HSD3 with an IC(50) 17.7+/-6.8 microM. PFOA inhibited intact Leydig cell 3beta-HSD with an IC(50) of 146.1+/-0.9 microM and 17beta-HSD3 with an IC(50) of 194.8+/-1.0 microM. The inhibitions of 3beta-HSD and 17beta-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3beta-HSD and 17beta-HSD3 in rat Leydig cells.

  5. 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

  6. Effect of dibutyl phthalate on expression of connexin 43 and testosterone production of leydig cells in adult rats.

    PubMed

    Zhang, Jing; Jin, Shuguang; Zhao, Jinchang; Li, Huan

    2016-10-01

    To investigate the adverse effect of dibutyl phthalate (DBP) on Leydig cells and its mechanism related to gap junction, Leydig cells isolated from adult rats were treated with 0.1% dimethylsulfoxide (DMSO), 50mg/L DBP, 50mg/L DBP+10μM prostaglandin E2 (PGE2) and 40μM flutamide respectively. Radioimmunoassay, semi-quantitative RT-PCR, immunofluorescence and Western blot were applied to determine the expression of testosterone and Connexin 43 (Cx43) in Leydig cells. The expression of testosterone and Cx43 were both decreased in DBP group (P<0.05). While Cx43 was up-regulated after administered to PGE2, there was no significant change in testosterone. However, testosterone was down-regulated with a significant decrease of Cx43 in flutamide group. The results indicated that the inhibitory effect of DBP on testosterone production was not through the down-regulation of Cx43. On the contrary, the change of testosterone can influence the expression of Cx43 in Leydig cells.

  7. 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

  8. Effects of catechin, epicatechin and epigallocatechin gallate on testosterone production in rat leydig cells.

    PubMed

    Yu, Po-Ling; Pu, Hsiao-Fung; Chen, Sung-Yun; Wang, Shyi-Wu; Wang, Paulus S

    2010-05-15

    Catechins have been reported to have many pharmacological properties such as the effects of anti-oxidative, anti-inflammatory, anti-carcinogenic, anti-ultraviolet, and reduction of blood pressure as well as glucose and cholesterol levels. However, the effect of catechins on the reproductive mechanism is still unknown. In the present study, the effects of catechins on testosterone secretion in rat testicular Leydig cells (LCs) were explored. Both in vivo and in vitro investigations were performed. Purified LCs were incubated with or without catechin (CCN), epicatechin (EC), epigallocatechin gallate (EGCG, 10(-10)-10(-8) M) under challenge with human chorionic gonadotropin (hCG, 0.01 IU/ml), forskolin, SQ22536 (an adenylyl cyclase inhibitor), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), A23187 (a calcium ionophore), and nifedipine (10(-5) M), respectively. To study the effects of catechins on steroidogenesis, steroidogenic precursors-stimulated testosterone release was examined. The functions of the steroidogenic enzymes including protein expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were investigated and expressed by Western blotting. Catechins increased plasma testosterone in vivo in male rats. In vitro, low-dose concentration of catechins increased gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release by anterior pituitary gland and hCG-stimulated testosterone release by LCs of male rats. These results suggested that catechins stimulated testosterone production by acting on rat LCs via the mechanism of increasing the action of cAMP, but not P450scc, StAR protein or the activity of intracellular calcium. EC, one of the catechins increased the testosterone secretion by rat LCs via the enzyme activities of 17beta-hydroxysteroid dehydrogenase (17beta-HSD). (c) 2010 Wiley-Liss, Inc.

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

    PubMed

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

    2013-06-28

    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 information for developing new potential therapies for PADAM (Partial Androgen Deficiency in the Aging Male). Copyright © 2013 Elsevier Inc. All rights reserved.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Inter-relationship between testicular dysgenesis and Leydig cell function in the masculinization programming window in the rat.

    PubMed

    van den Driesche, Sander; Kolovos, Petros; Platts, Sophie; Drake, Amanda J; Sharpe, Richard M

    2012-01-01

    The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the 'masculinization programming window' (MPW; e15.5-e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5-e18.5 (MPW), e13.5-e20.5 or e19.5-e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R² = -0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R² = -0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The

  16. Experimental control of the differentiation of Leydig cells in the rat fetal testis.

    PubMed Central

    Jost, A; Perlman, S; Valentino, O; Castanier, M; Scholler, R; Magre, S

    1988-01-01

    In the developing fetal testis, in vitro as well as in vivo, two kinds of endocrine cells differentiate successively: Sertoli cells, which produce the Müllerian inhibitor (or anti-Müllerian hormone) and aggregate with germ cells into seminiferous cords; and Leydig cells, which release androgens. Serum added to the synthetic culture medium prevents the morphogenesis of the seminiferous cords but not the cytodifferentiation of the endocrine cells. L-Azetidine 2-carboxylic acid (LACA), a proline competitor, introduced into the medium also prevents differentiation of seminiferous cords. In the present experiments, the effects of LACA on the endocrine cells were studied. It did not suppress production of the Müllerian inhibitor, but it opposed differentiation of Leydig cells. Histochemically detectable 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was virtually absent and the release of testosterone, delta 4-androstenedione, 17-hydroxyprogesterone, or progesterone into the medium became undetectable. Moreover, dibutyryl cAMP added to the medium during the final day in vitro had very little effect on the parameters of steroidogenesis. An excess of proline added to the LACA-containing medium permitted normal morphogenesis of seminiferous cords, normal steroidogenesis, and normal response to cAMP. LACA did not prevent the appearance of 3 beta-HSD activity in the adrenals, nor did it reduce the expression of laminin and fibronectin (data not shown) in the mesonephric structures as much as in the testes. The differentiation of the testis and especially of the Leydig cells appears to have special requirements for proline. Images PMID:3186712

  17. Gene expression during development of fetal and adult Leydig cells.

    PubMed

    Dong, Lei; Jelinsky, Scott A; Finger, Joshua N; Johnston, Daniel S; Kopf, Gregory S; Sottas, Chantal M; Hardy, Matthew P; Ge, Ren-Shan

    2007-12-01

    In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

  18. 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.

  19. Long-term feeding of hydroalcoholic extract powder of Lepidium meyenii (maca) enhances the steroidogenic ability of Leydig cells to alleviate its decline with ageing in male rats.

    PubMed

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

    2017-03-10

    This study examined whether feeding hydroalcoholic extract of Lepidium meyenii (maca) to 8-week-old (sexually maturing) or 18-week-old (mature) male rats for more than a half year affects serum testosterone concentration and testosterone production by Leydig cells cultured with hCG, 22R-hydroxycholesterol or pregnenolone. Testosterone concentration was determined in the serum samples obtained before and 6, 12, 18 and 24 weeks after the feeding, and it was significantly increased only at the 6 weeks in the group fed with the maca extract to maturing rats when it was compared with controls. Testosterone production by Leydig cells significantly increased when cultured with hCG by feeding the maca extract to maturing rats for 27 weeks (35 weeks of age) and when cultured with 22R-hydroxycholesterol by feeding it to mature rats for 30 weeks (48 weeks of age). Overall testosterone production by cultured Leydig cells decreased to about a half from 35 to 48 weeks of age. These results suggest that feeding the maca extract for a long time to male rats may enhance the steroidogenic ability of Leydig cells to alleviate its decline with ageing, whereas it may cause only a transient increase in blood testosterone concentration in sexually maturing male rats.

  20. [The ultrastructure of Leydig cells under the influence of drinking mineral water and electromagnetic radiation under the stress conditions in the rats].

    PubMed

    Geniatulina, M S; Korolev, Yu N; Nikulina, L A

    The objective of the present study was elucidate the peculiar features of low-intensity electromagnetic radiation (LI EMR) and mineral water (MW) on the ultrastructure of rat Leydig cells under conditions of immobilization stress. The experiments were carried out on outbred male rats with the use of electron microscopy. It has been demonstrated that the prophylactic consumption of drinking sulfate-containing mineral water and the application low-intensity electromagnetic radiation (with the flow power density of 1 mcW/cm2 and frequency around 1,000 Hz) or the combination of these two modalities under conditions of immobilization stress reduced the degree of ultrastructural derangement in the rat Leydig cells and stimulated the development of regenerative processes. In the cases of the single-factor impact, drinking mineral water exerted more pronounced action than low-intensity electromagnetic radiation on mitochondrial regeneration. In case of the simultaneous application of the two factors their protective action on the Leydig cells was much more conspicuous than that of either of them applied alone. It is concluded that drinking sulfate-containing mineral water in combination with the application of low-intensity electromagnetic radiation enhances resistance of the rat Leydig cells to stress.

  1. An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells : A Complementary Screen for Steroidogenesis in the Testis

    EPA Science Inventory

    An Evaluation of LH-Stimulated Testosterone Production by Highly Purified Rat Leydig Cells: A Complementary Screen for Steroidogenesis in the Testis. 1Botteri, N., 2Suarez, J., 2Laws, S., 2Klinefelter, G.1Oak Ridge Institute for Science and Education, Oak Ridge, TN, 2 U.S. Env...

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

    PubMed

    Ding, Xiao; Yu, Lei; Ge, Chongyang; Ma, Haitian

    2017-03-07

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

  3. Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring.

    PubMed

    Ivell, Richard; Heng, Kee; Nicholson, Helen; Anand-Ivell, Ravinder

    2013-03-01

    Maternal exposure to estrogenic xenobiotics or phthalates has been implicated in the distortion of early male reproductive development, referred to in humans as the testicular dysgenesis syndrome. It is not known, however, whether such early gestational and/or lactational exposure can influence the later adult-type Leydig cell phenotype. In this study, Sprague-Dawley rats were exposed to dibutyl phthalate (DBP; from gestational day (GD) 14.5 to postnatal day (PND) 6) or diethylstilbestrol (DES; from GD14.5 to GD16.5) during a short gestational/lactational window, and male offspring subsequently analysed for various postnatal testicular parameters. All offspring remained in good health throughout the study. Maternal xenobiotic treatment appeared to modify specific Leydig cell gene expression in male offspring, particularly during the dynamic phase of mid-puberty, with serum INSL3 concentrations showing that these compounds led to a faster attainment of peak values, and a modest acceleration of the pubertal trajectory. Part of this effect appeared to be due to a treatment-specific impact on Leydig cell proliferation during puberty for both xenobiotics. Taken together, these results support the notion that maternal exposure to certain xenobiotics can also influence the development of the adult-type Leydig cell population, possibly through an effect on the Leydig stem cell population.

  4. 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...

  5. 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...

  6. 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.

  7. Modulation of K+ conductances by Ca2+ and human chorionic gonadotrophin in Leydig cells from mature rat testis.

    PubMed Central

    Desaphy, J F; Rogier, C; Joffre, M

    1996-01-01

    1. Although the control of steroidogenic activity of the Leydig cell by the peptides luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) is clearly mediated by cAMP, the extent to which Ca2+ controls the Leydig cell function is less well defined. In the present study, the whole-cell configuration of the patch-clamp technique was used to investigate the modulation of potassium conductances by calcium and hCG, in the Leydig cells from mature rat testis. 2. In symmetrical glutamate solutions, depolarizations elicited outwardly rectifying currents, which were mainly carried by potassium and were blocked by tetraethylammonium and 4-aminopyridine. For values of [Ca2+]i below 10(-8) M, transient currents of low amplitudes, insensitive to charybdotoxin (CTX) and iberiotoxin (IBTX), were activated above -40 mV. For [Ca2+]i values of 10(-7) M and above, noisy currents with slow activation kinetics were activated above 0 mV. These currents were sustained and were sensitive to CTX and IBTX. 3. Both current types were modulated by intracellular calcium. Ionomycin and a [Ca2+]i elevation in the range from 10(-9) to 10(-7) M, both inhibited the CTX-insensitive currents, whereas a rise in the calcium concentration above 10(-7) M increased the amplitude and shifted the threshold of activation of the CTX-sensitive currents to less positive levels. 4. hCG (1-50 i.u. ml-1), in conditions where the chloride currents were strongly inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), induced a partial inhibition of the CTX-insensitive currents but was unable to increase the CTX-sensitive currents. 5. No voltage-sensitive calcium current was recorded in control or hCG-stimulated cells. 6. The results indicate that hCG inhibits one kind of Ca(2+)-modulated channel, perhaps as a result of a moderate [Ca2+]i rise, but is unable to increase the intracellular Ca2+ concentration to the range in which large conductance Ca(2+)-dependent channels are

  8. Modulation of K+ conductances by Ca2+ and human chorionic gonadotrophin in Leydig cells from mature rat testis.

    PubMed

    Desaphy, J F; Rogier, C; Joffre, M

    1996-08-15

    1. Although the control of steroidogenic activity of the Leydig cell by the peptides luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) is clearly mediated by cAMP, the extent to which Ca2+ controls the Leydig cell function is less well defined. In the present study, the whole-cell configuration of the patch-clamp technique was used to investigate the modulation of potassium conductances by calcium and hCG, in the Leydig cells from mature rat testis. 2. In symmetrical glutamate solutions, depolarizations elicited outwardly rectifying currents, which were mainly carried by potassium and were blocked by tetraethylammonium and 4-aminopyridine. For values of [Ca2+]i below 10(-8) M, transient currents of low amplitudes, insensitive to charybdotoxin (CTX) and iberiotoxin (IBTX), were activated above -40 mV. For [Ca2+]i values of 10(-7) M and above, noisy currents with slow activation kinetics were activated above 0 mV. These currents were sustained and were sensitive to CTX and IBTX. 3. Both current types were modulated by intracellular calcium. Ionomycin and a [Ca2+]i elevation in the range from 10(-9) to 10(-7) M, both inhibited the CTX-insensitive currents, whereas a rise in the calcium concentration above 10(-7) M increased the amplitude and shifted the threshold of activation of the CTX-sensitive currents to less positive levels. 4. hCG (1-50 i.u. ml-1), in conditions where the chloride currents were strongly inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), induced a partial inhibition of the CTX-insensitive currents but was unable to increase the CTX-sensitive currents. 5. No voltage-sensitive calcium current was recorded in control or hCG-stimulated cells. 6. The results indicate that hCG inhibits one kind of Ca(2+)-modulated channel, perhaps as a result of a moderate [Ca2+]i rise, but is unable to increase the intracellular Ca2+ concentration to the range in which large conductance Ca(2+)-dependent channels are

  9. 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.

  10. Effect of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on testosterone release from rat Leydig cells.

    PubMed

    Hsia, Shih-Min; Tseng, Yi-Wen; Wang, Shyi-Wu; Kuo, Yueh-Hsiung; Huang, Din-Wen; Wang, Paulus S; Chiang, Wenchang

    2009-05-01

    Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3',5'-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17beta-HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion.

  11. Atrazine activates multiple signaling pathways enhancing the rapid hCG-induced androgenesis in rat Leydig cells.

    PubMed

    Pogrmic-Majkic, Kristina; Fa, Svetlana; Samardzija, Dragana; Hrubik, Jelena; Kaisarevic, Sonja; Andric, Nebojsa

    2016-08-10

    Atrazine (ATR) is an endocrine disruptor that affects steroidogenic process, resulting in disruption of reproductive function of the male and female gonads. In this study, we used the primary culture of peripubertal Leydig cells to investigate the effect of ATR on the rapid androgen production stimulated by human chorionic gonadotropin (hCG). We demonstrated that ATR activated multiple signaling pathways enhancing the rapid hCG-stimulated androgen biosynthesis in Leydig cells. Low hCG concentration (0.25ng/mL) caused cAMP-independent, but ERK1/2-dependent increase in androgen production after 60min of incubation. Co-treatment with ATR for 60min enhanced the cAMP production in hCG-stimulated cells. Accumulation of androgens was prevented by addition of U0126, N-acetyl-l-cysteine and AG1478. Co-treatment with hCG and ATR for 60min did not alter steroidogenic acute regulatory protein (Star) mRNA level in Leydig cells. After 120min, hCG further increased androgenesis in Leydig cells that was sensitive to inhibition of the cAMP/PKA, ERK1/2 and ROS signaling pathways. Co-treatment with ATR for 120min further enhanced the hCG-induced androgen production, which was prevented by inhibition of the calcium, PKC and EGFR signaling cascades. After 120min, ATR enhanced the expression of Star mRNA in hCG-stimulated Leydig cells through activation of the PKA and PKC pathway. Collectively, these data suggest that exposure to ATR caused perturbations in multiple signaling pathways, thus enhancing the rapid hCG-dependent androgen biosynthesis in peripubertal Leydig cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

    PubMed

    Pinon-Lataillade, G; Viguier-Martinez, M C; Touzalin, A M; Maas, J; Jégou, B

    1991-01-01

    Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.

  13. 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.

  14. Chronic hypothyroidism only marginally affects adult-type Leydig cell regeneration after EDS administration.

    PubMed

    Rijntjes, Eddy; van Kesteren-Buiting, Anita; Keijer, Jaap; Teerds, Katja J

    2010-02-01

    Chronic prenatally induced dietary hypothyroidism delays adult-type Leydig cell development, but does not block this process. Using a chemical model to induce hypothyroidism, it was suggested that development of a new population of Leydig cells was completely inhibited following the addition of the cytotoxic compound ethane-1,2-dimethyl sulphonate (EDS). In this study, we used a dietary approach to induce hypothyroidism and reinvestigated the regeneration of the Leydig cell population following EDS administration. Eighty-four day old euthyroid and chronically hypothyroid rats received an injection of EDS and were killed directly before or at regular intervals up to 77 days after EDS. In some control and hypothyroid animals, the first progenitor-type Leydig cells were observed at day 12 after EDS. At day 16, Leydig cell progenitors were present in all rats. The percentage of proliferating Leydig cells peaked in the euthyroid animals at day 21 after EDS. In the hypothyroid testis such a peak was not observed, although the percentage of proliferating regenerating Leydig cells was significantly higher from days 35 to 56 compared with the controls. This suggested that the wave of Leydig cell proliferation was delayed in the hypothyroid animals as compared with the euthyroid controls. On the day of EDS injection, the Leydig/Sertoli cell ratio was 37% lower in the hypothyroid rats compared with the controls. The Leydig/Sertoli cell ratio remained lower in the EDS-treated hypothyroid animals compared with the controls at all time points investigated. At day 77 after EDS, the Leydig cell population had returned to its pre-treatment size in both groups. Plasma testosterone production was reduced to below detectable levels immediately after EDS injection, and started to increase again on day 16, reaching pre-treatment values on day 21 in both groups. Taken together, severely reduced thyroid hormone levels did not block the regeneration of the adult-type Leydig cell population

  15. Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

    PubMed

    Baburski, Aleksandar Z; Sokanovic, Srdjan J; Andric, Silvana A; Kostic, Tatjana S

    2017-05-01

    The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

  16. 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.

  17. Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis.

    PubMed

    Onoda, M; Djakiew, D; Papadopoulos, V

    1991-05-01

    The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW greater than 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

  18. 11{beta}-Hydroxysteroid dehydrogenase 2 in rat leydig cells: its role in blunting glucocorticoid action at physiological levels of substrate.

    PubMed

    Ge, Ren-Shan; Dong, Qiang; Niu, En-Mei; Sottas, Chantal M; Hardy, Dianne O; Catterall, James F; Latif, Syed A; Morris, David J; Hardy, Matthew P

    2005-06-01

    Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of

  19. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    EPA Science Inventory

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  20. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    EPA Science Inventory

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  1. Leydig Cell Hyperplasia Revealed by Gynecomastia

    PubMed Central

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably. PMID:18660859

  2. Leydig cell hyperplasia revealed by gynecomastia.

    PubMed

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably.

  3. Leydig cell hyperplasia and Leydig cell tumour in postmenopausal women: report of two cases.

    PubMed

    Hofland, Maarten; Cosyns, Stef; De Sutter, Philippe; Bourgain, Claire; Velkeniers, Brigitte

    2013-03-01

    Leydig cell hyperplasia and Leydig cell tumours of the ovary are rare. We present two cases in which patients had increased blood levels of testosterone and frank hirsutism. Imaging showed minimal abnormalities. After adrenal disease had been ruled out, they underwent a bilateral oophorectomy. One case showed a Leydig cell hyperplasia, the other a Leydig cell tumour. An androgen producing tumour should be excluded in every woman with evidence of hirsutism or frank virilization and markedly elevated testosterone levels. Adrenal disease with androgen hypersecretion can be suspected by detailed clinical, laboratory and radiologic imaging. Although DHEAS has a good sensitivity in the detection of adrenal origin of hyperandrogenism (and hence a good negative predictive value) it is not specific (specificity ranging from 85 to 98%). Imaging of the ovaries can be helpful but does not rule out ovarian disease if normal. Indeed, diffuse stromal Leydig cell hyperplasia and Leydig cell tumours (usually small) may escape imaging and in some cases diagnosis can only be made on pathology. As these clinical entities represent a diagnostic and therapeutic challenge, oophorectomy should be considered in postmenopausal women with hirsutism and elevated testosterone levels, after the exclusion of adrenal causes. The procedure is relatively safe and effective. Follow-up remains indicated.

  4. Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis.

    PubMed

    Poon, Song Ling; Leu, Sew-Fen; Hsu, Hseng-Kuang; Liu, Ming-Yie; Huang, Bu-Miin

    2005-02-11

    Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 20 alpha-hydroxylase and 17 beta-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.

  5. Insights into the Development of the Adult Leydig Cell Lineage from Stem Leydig Cells.

    PubMed

    Ye, Leping; Li, Xiaoheng; Li, Linxi; Chen, Haolin; Ge, Ren-Shan

    2017-01-01

    Adult Leydig cells (ALCs) are the steroidogenic cells in the testes that produce testosterone. ALCs develop postnatally from a pool of stem cells, referred to as stem Leydig cells (SLCs). SLCs are spindle-shaped cells that lack steroidogenic cell markers, including luteinizing hormone (LH) receptor and 3β-hydroxysteroid dehydrogenase. The commitment of SLCs into the progenitor Leydig cells (PLCs), the first stage in the lineage, requires growth factors, including Dessert Hedgehog (DHH) and platelet-derived growth factor-AA. PLCs are still spindle-shaped, but become steroidogenic and produce mainly androsterone. The next transition in the lineage is from PLC to the immature Leydig cell (ILC). This transition requires LH, DHH, and androgen. ILCs are ovoid cells that are competent for producing a different form of androgen, androstanediol. The final stage in the developmental lineage is ALC. The transition to ALC involves the reduced expression of 5α-reductase 1, a step that is necessary to make the cells to produce testosterone as the final product. The transitions along the Leydig cell lineage are associated with the progressive down-regulation of the proliferative activity, and the up-regulation of steroidogenic capacity, with each step requiring unique regulatory signaling.

  6. Insights into the Development of the Adult Leydig Cell Lineage from Stem Leydig Cells

    PubMed Central

    Ye, Leping; Li, Xiaoheng; Li, Linxi; Chen, Haolin; Ge, Ren-Shan

    2017-01-01

    Adult Leydig cells (ALCs) are the steroidogenic cells in the testes that produce testosterone. ALCs develop postnatally from a pool of stem cells, referred to as stem Leydig cells (SLCs). SLCs are spindle-shaped cells that lack steroidogenic cell markers, including luteinizing hormone (LH) receptor and 3β-hydroxysteroid dehydrogenase. The commitment of SLCs into the progenitor Leydig cells (PLCs), the first stage in the lineage, requires growth factors, including Dessert Hedgehog (DHH) and platelet-derived growth factor-AA. PLCs are still spindle-shaped, but become steroidogenic and produce mainly androsterone. The next transition in the lineage is from PLC to the immature Leydig cell (ILC). This transition requires LH, DHH, and androgen. ILCs are ovoid cells that are competent for producing a different form of androgen, androstanediol. The final stage in the developmental lineage is ALC. The transition to ALC involves the reduced expression of 5α-reductase 1, a step that is necessary to make the cells to produce testosterone as the final product. The transitions along the Leydig cell lineage are associated with the progressive down-regulation of the proliferative activity, and the up-regulation of steroidogenic capacity, with each step requiring unique regulatory signaling. PMID:28701961

  7. 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

  8. Mono-(2-ethylhexyl) phthalate directly alters the expression of Leydig cell genes and CYP17 lyase activity in cultured rat fetal testis.

    PubMed

    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.

  9. High-fat diet aggravates 2,2',4,4'-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats.

    PubMed

    Zhang, Zhan; Yu, Yongquan; Xu, Hengsen; Wang, Chao; Ji, Minghui; Gu, Jun; Yang, Lu; Zhu, Jiansheng; Dong, Huibin; Wang, Shou-Lin

    2017-03-12

    Growing evidence has revealed that a high-fat diet (HFD) could lead to disorders of glycolipid metabolism and insulin-resistant states, and HFDs have been associated with the inhibition of testicular steroidogenesis. Our previous study demonstrated that 2,2',4,4'-tetrabromodiphenyl ether (BDE47) could increase the risk of diabetes in humans and reduce testosterone production in rats. However, whether the HFD affects BDE47-inhibited testosterone production by elevating insulin levels and inducing related pathways remains unknown. In male rats treated with BDE47 by gavage for 12 weeks, the HFD significantly increased the BDE47 content of the liver and testis and increased the weight of the adipose tissue; increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin; further aggravated the disruption of the seminiferous epithelium; and lowered the level of testosterone, resulting in fewer sperm in the epididymis. Of note, the HFD enhanced BDE47-induced DAX-1 expression and decreased the expression levels of StAR and 3β-HSD in the testicular interstitial compartments in rats. In isolated primary Leydig cells from rats, BDE47 or insulin increased DAX-1 expression, decreased the expression of StAR and 3β-HSD, and reduced testosterone production, which was nearly reversed by knocking down DAX-1. These results indicated that the HFD aggravates BDE47-inhibited testosterone production through hyperinsulinemia, and the accumulation of testicular BDE47 that induces the up-regulation of DAX-1 and the subsequent down-regulation of steroidogenic proteins, i.e., StAR and 3β-HSD, in Leydig cells.

  10. Endocrine Disruptors and Leydig Cell Function

    PubMed Central

    Svechnikov, K.; Izzo, G.; Landreh, L.; Weisser, J.; Söder, O.

    2010-01-01

    During the past decades, a large body of information concerning the effects of endocrine disrupting compounds (EDCs) on animals and humans has been accumulated. EDCs are of synthetic or natural origin and certain groups are known to disrupt the action of androgens and to impair the development of the male reproductive tract and external genitalia. The present overview describes the effects of the different classes of EDCs, such as pesticides, phthalates, dioxins, and phytoestrogens, including newly synthesized resveratrol analogs on steroidogenesis in Leydig cells. The potential impact of these compounds on androgen production by Leydig cells during fetal development and in the adult age is discussed. In addition, the possible role of EDCs in connection with the increasing frequency of abnormalities in reproductive development in animals and humans is discussed. PMID:20862379

  11. Endocrine disruptors and Leydig cell function.

    PubMed

    Svechnikov, K; Izzo, G; Landreh, L; Weisser, J; Söder, O

    2010-01-01

    During the past decades, a large body of information concerning the effects of endocrine disrupting compounds (EDCs) on animals and humans has been accumulated. EDCs are of synthetic or natural origin and certain groups are known to disrupt the action of androgens and to impair the development of the male reproductive tract and external genitalia. The present overview describes the effects of the different classes of EDCs, such as pesticides, phthalates, dioxins, and phytoestrogens, including newly synthesized resveratrol analogs on steroidogenesis in Leydig cells. The potential impact of these compounds on androgen production by Leydig cells during fetal development and in the adult age is discussed. In addition, the possible role of EDCs in connection with the increasing frequency of abnormalities in reproductive development in animals and humans is discussed.

  12. Steroidogenic Factor 1 Differentially Regulates Fetal and Adult Leydig Cell Development in Male Mice1

    PubMed Central

    Karpova, Tatiana; Ravichandiran, Kumarasamy; Insisienmay, Lovella; Rice, Daren; Agbor, Valentine; Heckert, Leslie L.

    2015-01-01

    The nuclear receptor steroidogenic factor 1 (SF-1, AD4BP, NR5A1) is a key regulator of the endocrine axes and is essential for adrenal and gonad development. Partial rescue of Nr5a1−/− mice with an SF-1-expressing transgene caused a hypomorphic phenotype that revealed its roles in Leydig cell development. In contrast to controls, all male rescue mice (Nr5a1−/−;tg+/0) showed varying signs of androgen deficiency, including spermatogenic arrest, cryptorchidism, and poor virilization. Expression of various Leydig cell markers measured by immunohistochemistry, Western blot analysis, and RT-PCR indicated fetal and adult Leydig cell development were differentially impaired. Whereas fetal Leydig cell development was delayed in Nr5a1−/−;tg+/0 embryos, it recovered to control levels by birth. In contrast, Sult1e1, Vcam1, and Hsd3b6 transcript levels in adult rescue testes indicated complete blockage in adult Leydig cell development. In addition, between Postnatal Days 8 and 12, peritubular cells expressing PTCH1, SF-1, and CYP11A1 were observed in control testes but not in rescue testes, indicating SF-1 is needed for either survival or differentiation of adult Leydig cell progenitors. Cultured prepubertal rat peritubular cells also expressed SF-1 and PTCH1, but Cyp11a1 was expressed only after treatment with cAMP and retinoic acid. Together, data show SF-1 is needed for proper development of fetal and adult Leydig cells but with distinct primary functions; in fetal Leydig cells, it regulates differentiation, whereas in adult Leydig cells it regulates progenitor cell formation and/or survival. PMID:26269506

  13. Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells.

    PubMed

    Akingbemi, Benson T; Sottas, Chantal M; Koulova, Anna I; Klinefelter, Gary R; Hardy, Matthew P

    2004-02-01

    Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 microg/kg.d (a dose that approximates BPA levels in the environment) from postnatal d 21-35 suppressed serum LH (0.21 +/- 0.05 ng/ml; vs. control, 0.52 +/- 0.04; P < 0.01) and testosterone (T) levels (1.62 +/- 0.16 ng/ml; vs. control, 2.52 +/- 0.21; P < 0.05), in association with decreased LHbeta and increased estrogen receptor beta pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nm BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17alpha-hydroxylase/17-20 lyase. BPA decreased serum 17beta-estradiol levels from 0.31 +/- 0.02 ng/ml (control) to 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.23 +/- 0.03 ng/ml in rats exposed to 2.4 microg, 10 microg, or 100 mg/kg.d BPA, respectively, from 21-35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 +/- 34 (control) to 261 +/- 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility.

  14. Replacement of surgical castration by GnRH-inhibition or Leydig cell ablation in the male rat Hershberger antiandrogen assay.

    PubMed

    Ashby, J; Lefevre, P A; Deghenghi, R; Wallis, N

    2001-10-01

    An obstacle to the widespread adoption of the Hershberger antiandrogen assay is the surgical castration procedure required to produce androgen deficiency in the test animals. Here we describe two chemical treatments that produce similar effects to surgical castration. The first is use of ethane dimethane sulphonate (EDS), a specific toxin to the testosterone-producing Leydig cells of the mature testes. The second class of compound is the decapeptide inhibitors of the gonadotrophin-releasing hormone (GnRH), compounds such as Antarelix and Antide. Administration of either EDS or the GnRH inhibitors results in loss of weight of the testes, epididymides, and sex-associated tissues. Co-administration of testosterone to these animals leads to reversal of the induced effects. The basic test protocol for both of these assay modifications is described. Flutamide was used as a representative potent antiandrogen, and DDE as an example of a weakly active antiandrogen. The 5alpha-reductase inhibitor finasteride was used to inhibit the transformation of testosterone to dihydrotestosterone. It is shown that the EDS assay is sensitive to the antiandrogen flutamide, but that it fails to detect the weaker antiandrogen DDE. In contrast, the Antarelix assay performs as well as does the classical castration assay, leading to the detection as antiandrogens of flutamide, DDE, and finasteride. It is concluded that the GnRH inhibition Hershberger assay is more convenient to conduct than the original surgical castration assay, and it involves less stress to the test animals. Copyright 2001 Academic Press.

  15. Prenatal induced chronic dietary hypothyroidism delays but does not block adult-type Leydig cell development.

    PubMed

    Rijntjes, Eddy; Swarts, Hans J M; Anand-Ivell, Ravinder; Teerds, Katja J

    2009-02-01

    Transient hypothyroidism induced by propyl-2-thiouracyl blocks postpartum Leydig cell development. In the present study, the effects of chronic hypothyroidism on the formation of this adult-type Leydig cell population were investigated, using a more physiological approach. Before mating, dams were put on a diet consisting of an iodide-poor feed supplemented with a low dose of perchlorate and, with their offspring, were kept on this diet until death. In the pups at day 12 postpartum, plasma thyroid-stimulating hormone levels were increased by 20-fold, whereas thyroxine and free tri-iodothyronine levels were severely depressed, confirming a hypothyroid condition. Adult-type progenitor Leydig cell formation and proliferation were reduced by 40-60% on days 16 and 28 postpartum. This was followed by increased Leydig cell proliferation at later ages, suggesting a possible slower developmental onset of the adult-type Leydig cell population under hypothyroid conditions. Testosterone levels were increased 2- to 10-fold in the hypothyroid animals between days 21 and 42 postpartum compared with the age-matched controls. Combined with the decreased presence of 5alpha-reductase, this implicates a lower production capacity of 5alpha-reduced androgens. In 84-day-old rats, after correction for body weight-to-testis weight ratio, plasma insulin-like factor-3 levels were 35% lower in the hypothyroid animals, suggestive of a reduced Leydig cell population. This is confirmed by a 37% reduction in the Sertoli cell-to-Leydig cell ratio in hypothyroid rats. In conclusion, we show that dietary-induced hypothyroidism delays but, unlike propyl-2-thiouracyl, does not block the development of the adult-type Leydig cell population.

  16. 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.

  17. Cimetidine-induced Leydig cell apoptosis and reduced EG-VEGF (PK-1) immunoexpression in rats: Evidence for the testicular vasculature atrophy.

    PubMed

    Beltrame, Flávia L; Cerri, Paulo S; Sasso-Cerri, Estela

    2015-11-01

    The antiulcer drug cimetidine has shown to cause changes in the testicular microvasculature of adult rats. Since Leydig cells (LCs) produce the pro-angiogenic factor, EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK-1), this study examined the effect that cimetidine might have on LCs in testes with damaged vasculature. Rats received intraperitoneal injections of 100mg/kg of cimetidine (cimetidine group) or saline vehicle (control group) for 50 days. Serum testosterone levels were measured by chemiluminescence immunoassay and testicular sections were subjected to TUNEL and immunohistochemical reactions for caspase-3, 17β-HSD6, CD163 (ED2 macrophage), PK-1 and androgen receptor (AR). LCs in the cimetidine group showed TUNEL and caspase-3 positive labeling and apoptotic ultrastructural features. Moreover, the presence of 17β-HSD6-positive inclusions inside macrophages and the reduced number of LCs, AR immunoreactivity and serum testosterone levels correlated with a decrease in either the number of PK-1-immunostained LCs or PK-1 immunoreactivity. Although it is not clear which cell type is the primary target of cimetidine in the testicular interstitial compartment, these findings support a direct link between cimetidine-induced testicular vascular atrophy and LCs damage.

  18. [The role of gonadotropins, cyclic AMP, 22-R-OH-cholesterol and cofactors in regulating endocrine functions of the Leydig cells in rats. III. Mechanisms responsible for "desensitization" of the Leydig cells of rats caused by high doses of hCG].

    PubMed

    Grochowski, D; Szamatowicz, M

    1989-05-01

    Two groups of rats (a control group and the group examined) were administered intraperitoneally supraphysiological doses of hCG in order to induce a "down regulation" effect on the level of receptors LH and to achieve the desensibilization of Leydig cells. The authors tried to find out at which stage of sequence of changes from receptor stimulation to hormone production there appears a state of cellular resistance to further stimulation. Sections of the nucleus were incubated with various substances influencing steridogenesis (LH, hCG, dbcAMP, 22-R-OH-cholesterol, NAD + NADP + G-6-P + G-6-PDH). An index of the influence of the above substances on the synthesis of androgens were amounts of pregnenolon as the first and testosterone as the final stage of hormonal changes marked radioimmunologically in nucleus homogenates and incubating media. It was shown that the resistance of Leydig cells to further stimulation in the group of animals that were given high doses of hCG is the result of enzymatic blocks in testosterone synthesis. The first block is "late" block of 17 alpha-hydroxylase and 17-20 desmolase, disturbing transforming of 21-carbon steriods into 19-carbon androgens. When the dose of hCG increases, there appears the second block, the so called "early" block, disturbing mitochondrial synthesis of pregnenolon. It was found that exogenic cofactors are in a position, at least partially, to restore the activity of blocked enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. [Stromal leydig cell tumor coexisting with ascites].

    PubMed

    Czupkałło, G; Jakowicki, J A; Baranowski, W

    2001-10-01

    The stromal Leydig cell tumour (SLCT), a very rare, benign neoplasm was described in 75-year old woman. The patient presented the typical signs of virilisation (hirsutism, masculine alopecia, moderate clitoris enlargement, deep voice) as well as hypertension and insulin independent type of diabetes mellitus (IIDM). Additionally, she had marked ascites (3400 ml as established during surgery). The serum concentration of testosterone before the surgery was elevated up to 7.6 ng/ml. FSH and LH were at very low range (2.5 mIU/ml, 3.4 mIU/ml, respectively) whereas 17 beta-oestradiol was elevated (56 pg/ml). Total abdominal hysterectomy with salpingo-oophorectomy (TAH/BSO) and omentectomy were performed. The histopathological findings revealed stromal Leydig cell tumour with Reinke crystalloids. The postoperative follow-up was complicated by venous thrombosis. Five weeks after the surgery only slight regression of the signs of virilisation was observed. Hormonal findings were adequate to the patient age range (FSH--16.7 mIU/ml, LH--21.1 mIU/ml, testosterone--0.19 ng/ml, 17 beta-oestradiol concentration below 10 pg/ml).

  20. Male Sprague-Dawley rats exposed to in utero di(n-butyl) phthalate: dose dependent and age-related morphological changes in Leydig cell smooth endoplasmic reticulum.

    PubMed

    Shirai, Masaru; Wakui, Shin; Wempe, Michael F; Mutou, Tomoko; Oyama, Noriko; Motohashi, Masaya; Takahashi, Hiroyuki; Kansaku, Norio; Asari, Masao; Hano, Hiroshi; Endou, Hitoshi

    2013-01-01

    When 100 mg/kg/day of di(n-butyl) phthalate (DBP) was intragastrically administered to pregnant Sprague-Dawley rats throughout gestation days 12 to 21, the male pups had similar body weights with no apparent physical differences (e.g., litter size, sex ratio) compared to that of the vehicle group. However, prominent age-related morphological alterations in the smooth endoplasmic reticulum (sER) of testicular Leydig cells (LCs) were observed once these animals reached puberty. At weeks 5 to 7, the abundant sER with non-dilated cisternae was distributed in LCs. Subsequently, although the number of LCs significantly increased, the amount of sER was significantly decreased at 9 to 14 weeks of age and had disappeared at 17 weeks. In contrast, the number of LCs and the amount of sER in LCs of the lower dose groups (10, 30, and 50 mg/kg/day) were similar to those of the vehicle group. Further, serum testosterone levels in the 100 mg/kg dose group were significantly lower during 5 to 17 weeks of age. While their luteinizing hormone (LH) level was significantly lower at 5 to 7 weeks of age, it became significantly higher during 9 to 17 weeks. The amount of sER in LCs decreased with age with the increase in LCs proliferation and serum LH levels in rat exposed in utero to DBP in a dose-dependent manner.

  1. Switch of cadherin expression as a diagnostic tool for Leydig cell tumours.

    PubMed

    F, Bremmer; S, Schweyer; M, Martin-Ortega; B, Hemmerlein; A, Strauss; Hj, Radzun; Cl, Behnes

    2013-10-01

    Leydig cell tumours comprise about 3% of all testicular tumours. The pathogenesis of Leydig cell tumours is still poorly understood. We investigated testis with Leydig cell hyperplasia and Leydig cell tumours for their expression pattern of P- and N-cadherin. We could show a switch of expression of P- and N-cadherin in Leydig cell hyperplasia and Leydig cell tumours in comparison with normal Leydig cells. Cadherins could be established as a new immunohistochemical marker for this testicular tumour entity; their possible role in tumour genesis will be discussed. © 2013 The Authors APMIS © 2013 APMIS.

  2. Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis.

    PubMed Central

    Dix, C J; Habberfield, A D; Sullivan, M H; Cooke, B A

    1984-01-01

    The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol

  3. 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...

  4. 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...

  5. Immunohistochemical analysis of androgen effects on androgen receptor expression in developing Leydig and Sertoli cells.

    PubMed

    Shan, L X; Bardin, C W; Hardy, M P

    1997-03-01

    Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P < 0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced

  6. 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

  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

    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

    2015-03-01

    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.

  8. Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development.

    PubMed

    Ge, R S; Hardy, M P

    1997-09-01

    Postnatal development of Leydig cells can be divided into three distinct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7alpha-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5alpha-reductase, or E2 (50 nM). Proliferative capacity was measured by assaying [3H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A2 and G1, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), intermediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). Similarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), intermediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A2 mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.14), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G1 mRNA levels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 +/- 0.07), and lowest in PLC (0.12 +/- 0.02). The

  9. Prolonged in vivo administration of testosterone-enanthate, the widely used and abused anabolic androgenic steroid, disturbs prolactin and cAMP signaling in Leydig cells of adult rats.

    PubMed

    Bjelic, Maja M; Stojkov, Natasa J; Radovic, Sava M; Baburski, Aleksandar Z; Janjic, Marija M; Kostic, Tatjana S; Andric, Silvana A

    2015-05-01

    This study was designed to systematically analyze and define the effects of 1-day, 2-weeks, 10-weeks intramuscular administration of testosterone-enanthate, widely used and abused anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic genes expression in testosterone-producing Leydig cells of adult rats. The results showed that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane potential of Leydig cells were significantly reduced. This was followed by decreased expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2, Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19 increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased prolactin together with stimulated transcription of Jak2/Jak3 could account for increased Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1, Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in Leydig cells, could be the possible mechanism that contribute to the establishment of a new adaptive response to maintain homeostasis and prevent loss of steroidogenic function. Presented data provide new molecular insights into the relationship between disturbed testosterone homeostasis and mammalian reproduction and are important in terms of wide use and abuse of AASs and human reproductive health. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development.

    PubMed

    Rijntjes, Eddy; Gomes, Marcos L M; Zupanič, Nina; Swarts, Hans J M; Keijer, Jaap; Teerds, Katja J

    2017-01-01

    Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

  11. Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development

    PubMed Central

    Rijntjes, Eddy; Gomes, Marcos L. M.; Zupanič, Nina; Swarts, Hans J. M.; Keijer, Jaap; Teerds, Katja J.

    2017-01-01

    Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations. PMID:28588502

  12. Cyclic GMP signaling in rat urinary bladder, prostate, and epididymis: tissue-specific changes with aging and in response to Leydig cell depletion.

    PubMed

    Müller, Dieter; Mukhopadhyay, Amal K; Davidoff, Michail S; Middendorff, Ralf

    2011-08-01

    Aging of the male reproductive system leads to changes in endocrine signaling and is frequently associated with the emergence of prostate hyperplasia and bladder dysfunctions. Recent reports highlight prostate and bladder as promising targets for therapeutic interventions with inhibitors of the cyclic GMP (cGMP)-degrading phosphodiesterase 5 (PDE5). However, the cGMP signaling system in these organs is as yet poorly characterized, and the possibility of age-related alterations has not been addressed. This study investigates key proteins of cGMP pathways in bladder, prostate, and epididymis of young (3 months) and old (23-24 months) Wistar rats. Local differences in the abundance of PDE5, soluble guanylyl cyclase (sGC) and particulate guanylyl cyclases (GC-A, GC-B), endothelial nitric oxide synthase, and cGMP-dependent protein kinase I (PRKG1 (cGKI)) revealed pronounced tissue-specific peculiarities. Although cGMP-generating enzymes were not affected by age in all organs, we recognized age-related decreases of PDE5 expression in bladder and a selective diminishment of membrane-associated PRKG1 in epididymis. In disagreement with published data, all cGMP pathway proteins including PDE5 are poorly expressed in prostate. However, prostatic PRKG1 expression increases with aging. Androgen withdrawal during temporary Leydig cell elimination induced a massive (>12-fold) upregulation of PRKG1 in prostate but not in other (penis and epididymis) androgen-dependent organs. These findings identify PRKG1 as a key androgen-sensitive signaling protein in prostate of possible importance for growth regulation. The elucidated effects may have significance for age-associated pathologies in the male lower-urinary tract.

  13. 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

  14. 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.

  15. Autoantibodies against Leydig cells in patients after spermatic cord torsion.

    PubMed Central

    Zanchetta, R; Mastrogiacomo, I; Graziotti, P; Foresta, C; Betterle, C

    1984-01-01

    This study is aimed at searching for the presence of circulating antibodies against frozen sections of human testis, ovary and trophoblast in patients that had spermatic cord torsion. Sixty-eight sera samples were studied. Nine patients (13.2%) were positive for organ specific anti-testis autoantibodies. Six patients were positive for antibodies against Leydig cells: five were positive only with the indirect immunofluorescence technique of complement fixing (ITT/CF), the sixth patient was positive only with the indirect immunofluorescence technique (ITT). The other three patients were positive for antibodies against germ line cells: two patients were positive with both techniques, the third was positive only with indirect immunofluorescence technique. Eight of these patients were negative for antibodies against adrenal cortex while only one case was positive with indirect immunofluorescence technique both on adrenal cortex and Leydig cells. Human lyophilized testis absorbed the reactive antibodies against Leydig cells and germ line cells, while adrenal cortex and lyophilized testosterone were ineffective. This study shows the identification of a specific antibody against Leydig cells and germ line cells in patients after spermatic cord torsion. PMID:6362937

  16. Circadian rhythm of the Leydig cells endocrine function is attenuated during aging.

    PubMed

    Baburski, Aleksandar Z; Sokanovic, Srdjan J; Bjelic, Maja M; Radovic, Sava M; Andric, Silvana A; Kostic, Tatjana S

    2016-01-01

    Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups. Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor (Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-month group. The majority of examined clock genes displayed circadian behavior in expression but only a few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore, aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell's endocrine function decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance during aging.

  17. Immunolocalization of aromatase in stallion Leydig cells and seminiferous tubules.

    PubMed

    Sipahutar, Herbert; Sourdaine, Pascal; Moslemi, Safa; Plainfossé, Bruno; Séralini, Gilles-Eric

    2003-03-01

    High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome p450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (2-5 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidin-peroxidase complex. Immunoreactivity was detected with diaminobenzidine. Immunofluorescence detection, using fluoroisothiocyanate-conjugated goat anti-rabbit antibodies, was also applied. Specific aromatase immunoreactivity was observed intensely in Leydig cells but also for the first time, to a lesser extent, in the cytoplasm surrounding germ cells at the junction with Sertoli cells. Interestingly, the immunoreactivity in Sertoli cells appears to vary with the spermatogenic stages in the basal compartment (with spermatogonia) as well as in the adluminal one (with spermatids). Relative staining intensity in Leydig and Sertoli cells and testicular microsomal aromatase activity increased with age. The present study in stallions indicates that in addition to Leydig cells, Sertoli cells also appear to participate in estrogen synthesis, and this could play a paracrine role in the regulation of spermatogenesis.

  18. Roscovitine protects murine Leydig cells from lipopolysaccharide-induced inflammation.

    PubMed

    Xie, Tiancheng; Hu, Guanghui; Dong, Binbin; Yan, Yangye; Liu, Min; Yao, Xudong; Zheng, Junhua; Xu, Yunfei

    2017-05-01

    Roscovitine is a cyclin-dependent kinase inhibitor, which has been previously investigated for its anticancer effects. It has also been confirmed that roscovitine can downregulate the expression of myeloid cell leukemia-1 protein to inhibit inflammation. In the present study, roscovitine was used to treat inflammation in lipopolysaccharide (LPS)-induced model mice. At the cellular level, Leydig cells isolated from mouse testis were assessed for inflammatory factors. It was revealed that roscovitine successfully reduced inflammation-associated injury induced by LPS pretreatment. At the molecular level, roscovitine was found to exert this effect through promotion of adenosine monophosphate-activated protein kinase phosphorylation. To the best of our knowledge, the present study was the first to suggest that roscovitine has a protective role in Leydig cells through its anti-inflammatory action.

  19. 4-Nitrophenol induces Leydig cells hyperplasia, which may contribute to the differential modulation of the androgen receptor and estrogen receptor-α and -β expression in male rat testes.

    PubMed

    Zhang, Yonghui; Piao, Yuanguo; Li, Yansen; Song, Meiyan; Tang, Pingli; Li, Chunmei

    2013-11-25

    4-Nitrophenol (PNP) is generally regarded as an environmental endocrine disruptor capable of estrogenic and anti-androgenic activities. To investigate PNP-induced reproductive effects, immature male rats were injected subcutaneously with PNP (0.1, 1, 10mg/kg body weight or vehicle) daily for 4 weeks. We assessed reproductive tract alterations, sex hormone balance in the serum and estrogen receptor (ER)-α, -β and androgen receptor (AR) expression in testes. Although no significant difference was observed in body weight or testes weights of PNP-treated rats compared with the controls, the serum concentrations of testosterone in the 10mg/kg PNP-treated group were significantly elevated. This effect was accompanied by Leydig cells hyperplasia in the testes. Conversely, there was a significant decrease in estradiol concentration and aromatase expression in the testes of the 10mg/kg PNP-treated group. Furthermore, we observed a significant increase in ERα expression in the testes of the 10mg/kg PNP-treated group compared with the control group. Conversely, ERβ expression displayed a significant reduction. Moreover, AR expression was significantly increased in the 10mg/kg PNP-treated group compared with the control group. The existence of AR, ER-α and -β in the testes suggests that estradiol and testosterone directly affect germ cells and that differential modulation of AR, ER-α and -β in the testis may be involved in the direct effects of PNP or either the indirect effects of PNP-induced disruption of the estradiol-to-testosterone balance or the Leydig cells hyperplasia. Thus, the measurement of many endpoints is necessary for good risk assessment.

  20. The Ultrastructural Changes of the Sertoli and Leydig Cells Following Streptozotocin Induced Diabetes

    PubMed Central

    Kianifard, Davoud; Sadrkhanlou, Rajab Ali; Hasanzadeh, Shapour

    2012-01-01

    Objective(s) This investigation was conducted to evaluate the effects of diabetes on the structure and function of testicular tissue. Materials and Methods Diabetes was induced in male adult rats by a single intraperitoneal injection of streptozotocin. Body and testicular weight, hormonal analyses, histological and ultrastructural analyses were measured. Results The body and testicular weights were dropped significantly (P< 0.05) in diabetic rats in comparison with control rats. On the other hand, in diabetic rats, the blood glucose level increased significantly (P< 0.05). The blood plasma levels of testosterone, 17-β estradiol, progesterone, FSH and LH were reduced in diabetic rats. Histomorphological studies were revealed reduction in diameter of seminiferous tubules and germinal epithelium height, edema in interstitial tissue, germ cell depletion, decrease in cellular population and activity with disruption of spermatogenesis in diabetic rats. Ultrastructural study showed the mitochondrial change and reduction of smooth endoplasmic reticulum in Sertoli and presence of lipid droplets in Leydig cells of diabetic rat’s testes. Conclusion The results of the present study confirmed that, the ultrastructural changes of Sertoli and Leydig cells, brought about by streptozotocin induced diabetes, because of the alterations in pituitary gonadotropins, and these changes influence the normal spermatogenesis in rats. PMID:23493249

  1. Quantitative evaluation of Leydig cells in testicular biopsies of men with varicocele.

    PubMed

    Francavilla, S; Bruno, B; Martini, M; Moscardelli, S; Properzi, G; Francavilla, F; Santiemma, V; Fabbrini, A

    1986-01-01

    A quantitative analysis of Leydig cells was performed in 23 testicular biopsies of men with left varicocele and sperm count ranging from zero to 95,000 sperm/mm3. The oligozoospermic patients had more Leydig cells and higher FSH and LH serum levels than the patient group with more than 10,000 sperm/mm3. The Leydig cell density appeared tightly correlated (p less than 0.01) with the serum level of LH. In oligozoospermic subjects, an altered Leydig cell function could trigger an increased LH secretion; this seems likely to be responsible for the stimulation of interstitial cells resulting in an exaggerated recruitment of mature Leydig cells from their precursors. The comparative analysis of left and right testes failed to show differences in Leydig cell density and spermatogenesis in normozoospermic and oligozoospermic patients. This suggests that the two testes are equally involved by a possible, although unknown, detrimental effect of left side varicocele.

  2. 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.

  3. Leydig cell hyperplasia in the setting of Klinefelter syndrome.

    PubMed

    Sterbis, Joseph; E-Nunu, Toritsetimiyin

    2015-07-24

    A man in his 20's with Klinefelter syndrome presented to the urology clinic with a recent history of left-sided orchalgia. Ultrasound evaluation demonstrated multiple small hypoechoic lesions bilaterally, with the largest lesion measured at 5 mm × 6 mm × 8 mm. Testis cancer tumour markers, chest radiographs and abdominal CT imaging were negative. A partial orchiectomy was performed on the largest lesion, demonstrating the presence of Leydig cell hyperplasia.

  4. 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

  5. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis

    PubMed Central

    Yao, Humphrey Hung-Chang; Whoriskey, Wendy; Capel, Blanche

    2002-01-01

    Establishment of the steroid-producing Leydig cell lineage is an event downstream of Sry that is critical for masculinization of mammalian embryos. Neither the origin of fetal Leydig cell precursors nor the signaling pathway that specifies the Leydig cell lineage is known. Based on the sex-specific expression patterns of Desert Hedgehog (Dhh) and its receptor Patched 1 (Ptch1) in XY gonads, we investigated the potential role of DHH/PTCH1 signaling in the origin and specification of fetal Leydig cells. Analysis of Dhh−/− XY gonads revealed that differentiation of fetal Leydig cells was severely defective. Defects in Leydig cell differentiation in Dhh−/− XY gonads did not result from failure of cell migration from the mesonephros, thought to be a possible source of Leydig cell precursors. Nor did DHH/PTCH1 signaling appear to be involved in the proliferation or survival of fetal Leydig precursors in the interstitium of the XY gonad. Instead, our results suggest that DHH/PTCH1 signaling triggers Leydig cell differentiation by up-regulating Steroidogenic Factor 1 and P450 Side Chain Cleavage enzyme expression in Ptch1-expressing precursor cells located outside testis cords. PMID:12050120

  6. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis.

    PubMed

    Yao, Humphrey Hung-Chang; Whoriskey, Wendy; Capel, Blanche

    2002-06-01

    Establishment of the steroid-producing Leydig cell lineage is an event downstream of Sry that is critical for masculinization of mammalian embryos. Neither the origin of fetal Leydig cell precursors nor the signaling pathway that specifies the Leydig cell lineage is known. Based on the sex-specific expression patterns of Desert Hedgehog (Dhh) and its receptor Patched 1 (Ptch1) in XY gonads, we investigated the potential role of DHH/PTCH1 signaling in the origin and specification of fetal Leydig cells. Analysis of Dhh(-/-) XY gonads revealed that differentiation of fetal Leydig cells was severely defective. Defects in Leydig cell differentiation in Dhh(-/-) XY gonads did not result from failure of cell migration from the mesonephros, thought to be a possible source of Leydig cell precursors. Nor did DHH/PTCH1 signaling appear to be involved in the proliferation or survival of fetal Leydig precursors in the interstitium of the XY gonad. Instead, our results suggest that DHH/PTCH1 signaling triggers Leydig cell differentiation by up-regulating Steroidogenic Factor 1 and P450 Side Chain Cleavage enzyme expression in Ptch1-expressing precursor cells located outside testis cords.

  7. The Role of Foxo3 in Leydig Cells

    PubMed Central

    Choi, Young Suk; Song, Joo Eun; Kong, Byung Soo; Hong, Jae Won; Novelli, Silvia

    2015-01-01

    Purpose Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. Materials and Methods Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. Results Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. Conclusion FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function. PMID:26446641

  8. 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...

  9. 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...

  10. Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction

    PubMed Central

    Jiang, Mei Hua; Cai, Bing; Tuo, Ying; Wang, Jiancheng; Zang, Zhi Jun; Tu, Xiang'an; Gao, Yong; Su, Zhijian; Li, Weiqiang; Li, Guilan; Zhang, Min; Jiao, Jianwei; Wan, Zi; Deng, Chunhua; Lahn, Bruce T; Xiang, Andy Peng

    2014-01-01

    The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency. PMID:25418539

  11. 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

  12. Neuregulin 1 Regulates Proliferation of Leydig Cells to Support Spermatogenesis and Sexual Behavior in Adult Mice.

    PubMed

    Umehara, Takashi; Kawashima, Ikko; Kawai, Tomoko; Hoshino, Yumi; Morohashi, Ken-Ichirou; Shima, Yuichi; Zeng, Wenxian; Richards, JoAnne S; Shimada, Masayuki

    2016-12-01

    Adult Leydig cells are derived from proliferating stem/progenitor Leydig cells in the infant testis and subsequent differentiation to steroidogenic cells in adult mice. Leydig cell proliferation in the infant testis occurs primarily in response to increased levels of LH that induce Leydig cell expression of neuregulin 1 (NRG1). Depletion of NRG1 in Nrg1 mutant mice (Nrg1(flox;flox);Cyp19a1Cre mice) dramatically reduces Leydig cell proliferation in the infant testes, leading to a reduction of testis weight, epididymial weight, and serum T in the adult mutant mice. The mutant mice are subfertile due to impaired sexual behavior and abnormal elongation of the spermatogenic cells. These defects were reversed by T treatment of the mutant mice in vivo. Furthermore, NRG1 alone induces the proliferation of Leydig cells in cultures of infant (d 10) testes obtained from mutant mice. Collectively these results show that LH induction of NRG1 directly drives the proliferation of Leydig cells in the infant testis, leading to an obligatory number of adult Leydig cells required for the production of sufficient androgen to support and maintain spermatogenesis and sexual behavior of adult male mice.

  13. Ultrastructure of human Leydig cells at early gonadal embryogenesis.

    PubMed

    Makabe, S; Naguro, T; Heyn, R; Motta, P M

    1995-01-01

    The ultrastructure of human Leydig cells at different stages of the testicular prenatal development is described by means of transmission and scanning electron microscopy. Between 5 and 7 weeks of gestation (w.g.) the interstitial tissue of the gonad is filled with small undifferentiated mesenchymal cells, migrating primordial germ cells and blood vessels. When the embryo is 7 to 8 weeks-old Leydig cells (LC) appear in basically two morphological patterns, light and dark cells. Their most significative feature is the development of the smooth endoplasmic reticulum (SER) as a dense tubulo-vesicular network and the presence of numerous pleomorphic mitochondria with mainly lamellar cristae. At 14 and 16 w.g. the testicular interstitium reaches the maximum development; the cytoplasm of the LC shows a widespread network of anastomosing tubules of the SER and mitochondria with tubular cristae. Fetal LC show a partial cell coat, lack the crystals of Reinke, have few lipid droplets and show no signs of massive cell degeneration, at least until 16 w.g. These ultrastructural modifications in fetal LC are in accordance with the changes in both steroidogenic activity and hCG levels reported by the literature to occur at this stage of development. Junctional complexes were often observed among LC from 7 to 8 w.g. onwards.

  14. Identification of Stem Leydig Cells Derived from Pig Testicular Interstitium

    PubMed Central

    Yu, Shuai; Zhang, Pengfei; Dong, Wuzi; Zeng, Wenxian

    2017-01-01

    Stem Leydig cells (SLCs), located in the testicular interstitial compartment in the mammalian testes, are capable of differentiating to testosterone-synthesizing Leydig cells (LCs), thus providing a new strategy for treating testosterone deficiency. However, no previous reports have identified and cultured SLCs derived from the pig. The aim of the current study was to isolate, identify, and culture SLCs from pigs. Haematoxylin and eosin staining and immunochemical analysis showed that SLCs were present and that PDGFRα was mainly expressed in the pig testicular interstitium, indicating that PDGFRα was a marker for SLCs in the neonatal pig. In addition, reverse transcription-PCR results showed that SLC markers were expressed in primary isolated LCs, indicating that they were putative SLCs. The putative SLCs were subsequently cultured with a testicular fluid of piglets (pTF) medium. Clones formed after 7 days and the cells expressed PDGFRα. However, no clones grew in the absence of pTF, but the cells expressed CYP17A1, indicating that pTF could sustain the features of porcine SLCs. To summarize, we isolated porcine SLCs and identified their basic characteristics. Taken together, these results may help lay the foundation for research in the clinical application of porcine SLCs. PMID:28243257

  15. 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

  16. 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.

  17. 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.

  18. 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. Copyright © 2016. Published by Elsevier Inc.

  19. Differentiation of Leydig cells in the Mongolian gerbil.

    PubMed

    Pinto, Maria Etelvina; Egydio, Fernanda De Mattos; Taboga, Sebastião Roberto; Mendis-Handagama, S M L Chamindrani; Góes, Rejane Maira

    2010-02-01

    Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiationin this rodent, from birth to adulthood. Gerbil testes at 1 day, 1-7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxysteroid steroid dehydrogenase 1 (11beta-HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3beta-HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11beta-HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11beta-HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity.

  20. In utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats.

    PubMed

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

    2016-04-01

    Female pregnant Sprague-Dawley rats were intragastrically (ig) administered di(n-butyl) phthalate (DBP) at four doses (0, 10, 50 and 100 mg/kg) during gestation days (GD) 12-21 (n = 5 per group). The age-related morphological changes of Leydig cell mitochondrion (LC-Mt) and testosterone biosynthesis enzymes/associated genes/proteins expression levels were investigated. As compared to the control (no DBP), the 10 mg, and 50 mg DBP dose groups, the 100 mg DBP dose group at weeks 5 and 7 showed a significant amount of small LC-Mt. Thereafter, from weeks 9 to 17, the LC-Mt size and quantity in the 100 mg DBP dose group increased and became statistically similar to the other dose groups; hence, dose and time-dependent LC-Mt changes were observed. Throughout the study, the 100 mg DBP dose group had significantly lower testosterone levels. In addition, the 100 mg DBP dose group displayed lower StAR (StAR, steroidogenic acute regulatory protein) and P450scc (CYP11a1, cholesterol side-chain cleavage enzyme) levels at weeks 5 and 7, but they became statistically similar to all other dose groups at weeks 9 to 17; in contrast, the SR-B1 (Sarb1, scavenger receptor class B member 1) levels were similar for all DBP dose groups. The rats in utero 100 mg DBP /kg/day (GD 12-21) exposure results from this study indicate a dose-dependent, age-related morphological change in LC-Mt which are linked to reductions in testosterone biosynthesis genes / proteins expression, specifically StAR and P450scc.

  1. Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism

    PubMed Central

    Peak, Taylor C; Haney, Nora M; Wang, William; DeLay, Kenneth J; Hellstrom, Wayne J

    2016-01-01

    The production of testosterone occurs within the Leydig cells of the testes. When production fails at this level from either congenital, acquired, or systemic disorders, the result is primary hypogonadism. While numerous testosterone formulations have been developed, none are yet fully capable of replicating the physiological patterns of testosterone secretion. Multiple stem cell therapies to restore androgenic function of the testes are under investigation. Leydig cells derived from bone marrow, adipose tissue, umbilical cord, and the testes have shown promise for future therapy for primary hypogonadism. In particular, the discovery and utilization of a group of progenitor stem cells within the testes, known as stem Leydig cells (SLCs), has led not only to a better understanding of testicular development, but of treatment as well. When combining this with an understanding of the mechanisms that lead to Leydig cell dysfunction, researchers and physicians will be able to develop stem cell therapies that target the specific step in the steroidogenic process that is deficient. The current preclinical studies highlight the complex nature of regenerating this steroidogenic process and the problems remain unresolved. In summary, there appears to be two current directions for stem cell therapy in male primary hypogonadism. The first method involves differentiating adult Leydig cells from stem cells of various origins from bone marrow, adipose, or embryonic sources. The second method involves isolating, identifying, and transplanting stem Leydig cells into testicular tissue. Theoretically, in-vivo re-activation of SLCs in men with primary hypogonadism due to age would be another alternative method to treat hypogonadism while eliminating the need for transplantation. PMID:27822338

  2. Effects of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells.

    PubMed

    Lo, H-C; Chen, Y-W; Chien, C-H; Tseng, C-Y; Kuo, Y-M; Huang, B-M

    2005-01-01

    Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as food and crude medicine to improve several kinds of symptoms in Chinese society for a long time. Recent studies have illustrated that the fractions of fruiting bodies of TM exhibit a significant hypoglycemic activity in diabetic mouse models, which usually suffer from sexual dysfunction. In a previous study, we showed that TM reduced plasma testosterone production in normal rats without any positive effect in diabetic rats. It evolved a question of TM directly regulating Leydig cell steroidogenesis. In this study, MA-10 mouse Leydig tumor cells were treated with vehicle, different dosages of TM with or without human chorionic gonadotropin (hCG 50 ng/ml) to clarify the effects. Results showed that TM at different dosages (0.01-10 mg/ml) did not have any effect on MA-10 cell steroidogenesis (p > 0.05). In the presence of hCG, there was an inhibitory trend that TA suppressed MA-10 cell progesterone production at 3 hr treatment with a statistically significant difference by the 10 mg/ml TM (p < 0.05). In time course effect, TM alone did not have any effect on MA-10 cell steroidogenesis from at 1, 2, 3, 6 and 12 hr (p > 0.05). However, TM did reduce hCG-treated MA-10 cell progesterone production at 1, 2 and 3 hr (p < 0.05), respectively. To determine whether TM would have adverse effects on MA-10 cell steroidogenesis in the presence of hCG, MTT assay and recovery studies were conducted. MTT assay indicated that TM had no effect on surviving cells. In addition, with the removal of TM, and then the addition of hCG (2 and 4 hr), progesterone levels were restored within 4 hr. Taken together, present studies suggested that TM suppressed hCG-treated steroidogenesis in MA-10 cells without any toxicity effect.

  3. Seasonal and experimental reactivation of Leydig cells of the bat Tadarida brasiliensis.

    PubMed

    Aoki, A

    1997-04-01

    The Leydig cells of the bat Tadarida brasiliensis, exhibit two well-defined periods of secretory activity that are intimately associated to the bat reproductive cycle. During the breeding season (August-September, late Winter and early Spring in the southern hemisphere), the interstitial tissue contains hypertrophic Leydig cells characterized ultrastructurally by the presence of pleomorphic mitochondria, depletion of lipid droplets, proliferation of membranes of agranular endoplasmic reticulum (AER) and enlargement of the Golgi complexes. By contrast, from Spring to Fall concurrent with regression of seminiferous tubules, the Leydig cells acquire a quiescent appearance with reduction in size and volume of AER membranes, atrophy of the Golgi complex and a massive storage of lipid droplets. The changes occurring in Leydig cells during the breeding season can be duplicated experimentally in non-breeding bats with exogenous stimulation with hCG. The gonadotropic treatment induces rapid changes in both interstitial cells and seminiferous tubules. The latter present evident signs of reactivation including proliferation of the spermatogenic cell line, permeation of the tubular lumen and depletion of lipid droplets. The Leydig cells display similar features to those found in the bat during the mating season at the peak of secretory activity. The bat T. brasiliensis is an excellent model to correlate the morphological organization of the Leydig cells with either seasonal fluctuations of its secretory activity or after experimental stimulation with gonadotropins.

  4. ESR1 inhibits hCG-induced steroidogenesis and proliferation of progenitor Leydig cells in mice.

    PubMed

    Oh, Yeong Seok; Koh, Il Kyoo; Choi, Bomi; Gye, Myung Chan

    2017-03-07

    Oestrogen is an important regulator in reproduction. To understand the role of oestrogen receptor 1 (ESR1) in Leydig cells, we investigated the expression of ESR1 in mouse Leydig cells during postnatal development and the effects of oestrogen on steroidogenesis and proliferation of progenitor Leydig cells (PLCs). In Leydig cells, the ESR1 expression was low at birth, increased until postnatal day 14 at which PLCs were predominant, and then decreased until adulthood. In foetal Leydig cells, ESR1 immunoreactivity increased from birth to postnatal day 14. These suggest that ESR1 is a potential biomarker of Leydig cell development. In PLCs, 17β-estradiol and the ESR1-selective agonist propylpyrazoletriol suppressed human chorionic gonadotropin (hCG)-induced progesterone production and steroidogenic gene expression. The ESR2-selective agonist diarylpropionitrile did not affect steroidogenesis. In PLCs from Esr1 knockout mice, hCG-stimulated steroidogenesis was not suppressed by 17β-estradiol, suggesting that oestrogen inhibits PLC steroidogenesis via ESR1. 17β-estradiol, propylpyrazoletriol, and diarylpropionitrile decreased bromodeoxyuridine uptake in PLCs in the neonatal mice. In cultured PLCs, 17β-estradiol, propylpyrazoletriol, and diarylpropionitrile reduced hCG-stimulated Ki67 and Pcna mRNA expression and the number of KI67-positive PLCs, suggesting that oestrogen inhibits PLC proliferation via both ESR1 and ESR2. In PLCs, ESR1 mediates the oestrogen-induced negative regulation of steroidogenesis and proliferation.

  5. ESR1 inhibits hCG-induced steroidogenesis and proliferation of progenitor Leydig cells in mice

    PubMed Central

    Oh, Yeong Seok; Koh, Il Kyoo; Choi, Bomi; Gye, Myung Chan

    2017-01-01

    Oestrogen is an important regulator in reproduction. To understand the role of oestrogen receptor 1 (ESR1) in Leydig cells, we investigated the expression of ESR1 in mouse Leydig cells during postnatal development and the effects of oestrogen on steroidogenesis and proliferation of progenitor Leydig cells (PLCs). In Leydig cells, the ESR1 expression was low at birth, increased until postnatal day 14 at which PLCs were predominant, and then decreased until adulthood. In foetal Leydig cells, ESR1 immunoreactivity increased from birth to postnatal day 14. These suggest that ESR1 is a potential biomarker of Leydig cell development. In PLCs, 17β-estradiol and the ESR1-selective agonist propylpyrazoletriol suppressed human chorionic gonadotropin (hCG)-induced progesterone production and steroidogenic gene expression. The ESR2-selective agonist diarylpropionitrile did not affect steroidogenesis. In PLCs from Esr1 knockout mice, hCG-stimulated steroidogenesis was not suppressed by 17β-estradiol, suggesting that oestrogen inhibits PLC steroidogenesis via ESR1. 17β-estradiol, propylpyrazoletriol, and diarylpropionitrile decreased bromodeoxyuridine uptake in PLCs in the neonatal mice. In cultured PLCs, 17β-estradiol, propylpyrazoletriol, and diarylpropionitrile reduced hCG-stimulated Ki67 and Pcna mRNA expression and the number of KI67-positive PLCs, suggesting that oestrogen inhibits PLC proliferation via both ESR1 and ESR2. In PLCs, ESR1 mediates the oestrogen-induced negative regulation of steroidogenesis and proliferation. PMID:28266530

  6. Disruption of LH-induced testosterone biosynthesis in testicular Leydig cells by triclosan: probable mechanism of action.

    PubMed

    Kumar, Vikas; Balomajumder, Chandrajeet; Roy, Partha

    2008-09-04

    Triclosan (TCS) is an antimicrobial chemical widely used in different commercial preparations. The present study demonstrated the mechanism of action of TCS-induced anti-androgenicity in rat Leydig cells. Treatment of purified cells with increasing concentrations of TCS (0.001, 0.01, 0.1, 1 and 10 microM) resulted in a significantly decreased activity of adenylyl cyclase enzyme which was followed by a decreased synthesis of cAMP. This decreased cAMP level resulted in the disruption of entire steroidogenic cascade causing a depressed synthesis of testosterone. However, TCS-induced decrease in the production of testosterone returned to normalcy when cells were treated with forskolin (an adenylyl cyclase activator). Transcription followed by translational of four prominent steroidogenic enzyme/proteins, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and steroidogenic acute regulatory (StAR) protein, also decreased in a dose-dependent manner in TCS-treated Leydig cells as determined by RT-PCR, enzyme assay and Western blot. These results suggested that the disruption of the activity of adenylyl cyclase enzyme by TCS in turn leads to the disruption of intermediate steroidogenic cascade causing a depressed testosterone production. The study further confirmed the anti-androgenic activity of TCS in Leydig cells with highest effective concentration at 1 microM.

  7. High calcium activates the EGF receptor potentially through the calcium-sensing receptor in Leydig cancer cells.

    PubMed

    Tfelt-Hansen, Jacob; Yano, Shozo; John Macleod, R; Smajilovic, Sanela; Chattopadhyay, Naibedya; Brown, Edward M

    2005-06-01

    Epidermal growth factor (EGF) plays an important role in the physiology and pathophysiology of the Leydig cell. In H-500 rat Leydig cancer cells, a model for humoral hypercalcemia of malignancy (HHM), we previously showed that the calcium-sensing receptor (CaR) stimulates PTHrP release and proliferation, both involving multiple mitogen-activated protein kinases. An emerging concept of signaling by G-protein coupled receptors (GPCR) is that it occurs via transactivation of receptor tyrosine kinases. Therefore, we investigated whether stimulation with calcium activates the EGFR in H-500 Leydig cancer cells. We show that treatment of H-500 cells with Ca(2+) results in EGFR phosphorylation. The CaR-induced activation of ERK1/2, induction of PTHrP release and stimulation of cellular proliferation in H-500 cells are likewise mediated, in large part, through the EGFR. In conclusion, the calcium activates the EGFR, possibly through the CaR, to regulate downstream signaling events and important biological functions in a model of HHM.

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

    PubMed

    So, Edmund Cheung; Chang, Ya-Ting; Hsing, Chung-His; Poon, Paul Wai-Fung; Leu, Sew-Fen; Huang, Bu-Miin

    2010-02-01

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

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

    PubMed Central

    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

    2015-01-01

    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. J. Cell. Physiol. 231: 1385–1391, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:26626779

  10. Morphometric studies of Leydig cells in chinchillas (Chinchilla lanigera) during high and low fertility seasons.

    PubMed

    Surmacki, Piotr; Sulik, Małgorzata; Seremak, Beata

    2011-01-01

    The aim of this study was to examine morphometric data of Leydig cells of 10 male chinchillas. Testes, cut into 5-microm thick sections, were stained using the p.a.S. and Masson's methods. Some 3800 Leydig cells have been evaluated. Their dimensions, as well as the diameters of their nuclei and the distances of the nuclei from the boundaries of the cells, have been measured. The areas of the surface and volumes of the nuclei of Leydig cells have been calculated, as well as the areas of the surface of the Leydig cells themselves. The following data have been obtained. The Nuclei of Leydig Cells. The largest diameters: longer cells - 12 microm; shorter cells - 10 microm. Mean diameters: longer cells - 5.67 +/- 3.44 microm, shorter cells - 4.45 +/- 3.44 microm. The largest surface area - 120 microm2, the mean surface area - 28.27 +/- 11.21 microm2. The largest volume - 1200 microm3, the mean volume of nucleus - 171.8 +/- 65.82 microm3. Mean distances of Leydig cell nuclei from the opposite boundaries of the cells amounted to 1.29 +/- 1.41 microm, 4.24 +/- 2.39 microm, 4.09 +/- 2.23 microm, 6.12 +/- 2.33 microm. Leydig cells. The largest diameters: longer cells - 24 microm, shorter cells - 22 microm. Mean dimensions: longer cells - 13.86 +/- 2.76 microm, shorter cells - 10.89 +/- 2.44 microm. The largest area of surface - 528 microm2, the mean area of surface - 155.44 +/- 59.78 microm2. Morphometric analysis confirmed cytologic observations that the shape of the nuclei of Leydig cells is somewhat ellipsoidal. The nuclei are located off-centre and are not situated in the greatest agglomeration of cytoplasm. The shape of Leydig cells is irregular. The obtained results may provide insight on the infertility of chinchilla males, as well as in research on the annual cyclic fertility of these animals. They may be put to use in practice for the purpose of improving breeding of this species.

  11. Novel Targets for the Transcription Factors MEF2 in MA-10 Leydig Cells.

    PubMed

    Di-Luoffo, Mickaël; Daems, Caroline; Bergeron, Francis; Tremblay, Jacques J

    2015-07-01

    Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) are transcription factors recently identified in somatic cells of the male gonad. In other tissues, MEF2 factors are essential regulators of organogenesis and cell differentiation. So far in the testis, MEF2 factors were found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MEF2-depleted MA-10 Leydig cells, and the results were analyzed using Partek and Ingenuity Pathway Analysis software. Several genes were differentially expressed in MEF2-depleted Leydig cells, and 16 were validated by quantitative RT-PCR. A large number of these genes are known to be involved in fertility, gonad morphology, and steroidogenesis. These include Ahr, Bmal1, Cyp1b1, Hsd3b1, Hsd17b7, Map2k1, Nr0b2, Pde8a, Por, Smad4, Star, and Tsc22d3, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2-binding sites within the first 2 kb upstream of the transcription start site of the Por, Bmal1, and Nr0b2 promoters, suggesting direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, small interfering RNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1, and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction.

  12. Deletion of the Igf1 Gene: Suppressive Effects on Adult Leydig Cell Development

    PubMed Central

    HU, GUO-XIN; LIN, HAN; CHEN, GUO-RONG; CHEN, BING-BING; LIAN, QING-QUAN; HARDY, DIANNE O.; ZIRKIN, BARRY R.; GE, REN-SHAN

    2014-01-01

    Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3β-hydroxysteroid dehydrogenase–positive cells) were seen in testes of postnatal day (PND) 14–90 Igf1−/− mice compared with age-matched Igf1+/+ controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1−/− and Igf1+/+ mice. In contrast, the labeling index of PLCs was reduced in the Igf1−/− mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1−/− mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1+/+ mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1−/− mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1−/− mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1−/− testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult

  13. Deletion of the Igf1 gene: suppressive effects on adult Leydig cell development.

    PubMed

    Hu, Guo-Xin; Lin, Han; Chen, Guo-Rong; Chen, Bing-Bing; Lian, Qing-Quan; Hardy, Dianne O; Zirkin, Barry R; Ge, Ren-Shan

    2010-01-01

    Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3β-hydroxysteroid dehydrogenase-positive cells) were seen in testes of postnatal day (PND) 14-90 Igf1(-/-) mice compared with age-matched Igf1(+/+) controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1(-/-) and Igf1(+/+) mice. In contrast, the labeling index of PLCs was reduced in the Igf1(-/-) mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1(-/-) mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1(+/+) mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1(-/-) mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1(-/-) mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1(-/-) testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult testes of Igf1

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

    PubMed

    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

    2014-05-06

    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.

  15. Germ cells influence cord formation and Leydig cell gene expression during mouse testis development.

    PubMed

    Rios-Rojas, Clarissa; Spiller, Cassy; Bowles, Josephine; Koopman, Peter

    2016-04-01

    It is widely accepted that, during the development of testes in the mammalian embryo, male germ cells are influenced by signals from the surrounding somatic cells, but not vice versa, so that germ cells are dispensable for the formation of testes. We now demonstrate that development of the mouse fetal testis is compromised in the absence of germ cells. Using two- and three-dimensional imaging techniques, we reveal that W(e)/W(e) mutant testes devoid of germ cells have misshapen and poorly organized cords. We also found that mutant gonads have fewer Sertoli cells than normal and that the Leydig cells express key markers at higher than normal levels. These observations point to the existence of germ cell-derived signals that directly or indirectly affect the Sertoli and Leydig cell populations, and provide a new paradigm for the organogenesis of the mammalian testes. © 2015 Wiley Periodicals, Inc.

  16. Low-dose testosterone treatment decreases oxidative damage in TM3 Leydig cells

    PubMed Central

    Hwang, Thomas IS; Liao, Tien-Ling; Lin, Ji-Fan; Lin, Yi-Chia; Lee, Shu-Yu; Lai, Yen-Chun; Kao, Shu-Huei

    2011-01-01

    Testosterone replacement therapy has benefits for aging men and those with hypogonadism. However, the effects of exogenous testosterone on Leydig cells are still unclear and need to be clarified. In this report, we demonstrate that testosterone supplementation can reduce oxidative damage in Leydig cells. The TM3 Leydig cell line was used as an in vitro cell model in this study. Cytoprotective effects were identified with 100-nmol l−1 testosterone treatment, but cytotoxic effects were found with ≥500-nmol l−1 testosterone supplementation. Significantly reduced reactive oxygen species (ROS) generation, lipid peroxide contents and hypoxia induction factor (HIF)-1α stabilization and activation were found with 100-nmol l−1 testosterone treatment. There was a 1.72-fold increase in ROS generation in the 500-nmol l−1 compared to the 100-nmol l−1 testosterone treatment. A 1.58-fold increase in steroidogenic acute regulatory protein (StAR) expression was found in 50-nmol l−1 testosterone-treated cells (P<0.01). Chemically induced hypoxia was attenuated by testosterone supplementation. Leydig cells treated with low-dose testosterone supplementation showed cytoprotection by decreasing ROS and lipid peroxides, increasing StAR expression and relieving hypoxia stress as demonstrated by HIF-1α stabilization. Increased oxidative damage was found with ≥500-nmol l−1 testosterone manipulation. The mechanism governing the differential dose effects of testosterone on Leydig cells needs further investigation in order to shed light on testosterone replacement therapy. PMID:21297653

  17. Circadian clock and steroidogenic-related gene expression profiles in mouse Leydig cells following dexamethasone stimulation.

    PubMed

    Chen, Huatao; Gao, Lei; Xiong, Yongjie; Yang, Dan; Li, Cuimei; Wang, Aihua; Jin, Yaping

    2017-01-29

    Previous studies have shown that circadian clock genes are expressed in mammalian testes; however, it remains unclear if the expression patterns of these genes are cyclic. Furthermore, it is unknown whether Leydig cells, the primary androgen secreting cells in the testis, play a role in the rhythmicity of circadian clock and steroidogenic-related gene transcription. Here, we examine the circadian clock of mouse Leydig cells, and the link to steroidogenic-related gene transcription. We confirm, via sampling over a full circadian time (CT) period, a lack of circadian rhythmicity in mouse testes in comparison with the robust gene expression cycling of circadian clock genes in mouse livers. Immunofluorescence imaging of mouse testes collected at CT0 and CT12 show that the BMAL1 protein is exclusively expressed in mouse Leydig cells, and clearly linked to the circadian oscillation. Furthermore, dexamethasone treatment synchronized the expression of several of these canonical circadian clock and steroidogenic-related genes. Bioinformatic analyses revealed the presence of several circadian clock-related sequence motifs in the promoters of these steroidogenic-related genes. Our results suggest mouse Leydig cells may contain a functional circadian oscillator and the circadian clockwork in mouse Leydig cells regulates steroidogenic-related gene transcription by binding to the E-box, RORE, and D-box motifs in their promoters. However, additional research is required to determine the specific molecular mechanisms involved. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Influence of flavonols and quercetin derivative compounds on MA-10 Leydig cells steroidogenic genes expressions.

    PubMed

    Cormier, Michelle; Ghouili, Firas; Roumaud, Pauline; Martin, Luc J; Touaibia, Mohamed

    2017-10-01

    Androgen are mainly synthesized and secreted from testicular Leydig cells and play critical roles in testis development, normal masculinization, spermatogenesis, and male fertility. The rate-limiting step in testosterone biosynthesis involves the import of cholesterol inside mitochondria by the steroidogenic acute regulatory (Star) protein. Cholesterol is then converted to pregnenolone by the steroidogenic enzyme Cyp11a1, followed by a chemical transformation to testosterone using other steroidogenic enzymes. Interestingly, levels of Star protein within adult Leydig cells decrease during aging, resulting in defective mitochondrial cholesterol transfer and reduced testosterone production. Such decline may be delayed by increasing Star and/or Cyp11a1 gene expressions using supplementation with flavonoids, a group of the polyphenolic compounds widely distributed in fruits and vegetables. In this study, we examined whether the distribution of hydroxyl groups and/or acetylation or methylation of flavonols could influence their potency to stimulate steroidogenesis within Leydig cells. Low levels of quercetin, myricetin and pentaacetylquercetin (10μM) stimulated cAMP-dependent Star, Cyp11a1 and Fdx1 promoters' activations and may increase steroidogenesis within Leydig cells. Indeed, pentaacetylquercetin successfully increased cAMP-dependent accumulation of progesterone from MA-10 Leydig cells, possibly through activation of Star and Cyp11a1 transcriptions. Thus, dietary supplementation of pentaacetylquercetin could be potentially effective to maintain testosterone production within aging males. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells*

    PubMed Central

    Ahn, Seung Won; Gang, Gil-Tae; Tadi, Surendar; Nedumaran, Balachandar; Kim, Yong Deuk; Park, Ji Hoon; Kweon, Gi Ryang; Koo, Seung-Hoi; Lee, Keesook; Ahn, Ryun-Sup; Yim, Yong-Hyeon; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2012-01-01

    Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis. PMID:23074219

  20. 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

  1. 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...

  2. 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...

  3. Changes in mouse Leydig cells ultrastructure and testosterone secretion after diethylcarbamazine administration.

    PubMed

    Saraiva, Karina Lidianne Alcântara; Silva, Valdemiro Amaro Da; Torres, Dilênia De Oliveira Cipriano; Donato, Mariana Aragão Matos; Peres, Newton Gil; Souza, José Roberto Botelho De; Peixoto, Christina Alves

    2008-07-01

    Diethylcarbamazine (DEC) has been proven to be highly effective against lymphatic filariasis, although its effect on vertebrate cells remains uncertain. Mice Leydig cells after treatment with 200mg/kg of DEC for 12 days showed numerous lipid droplets, degenerated mitochondria, residual bodies and several giant whorl-like smooth endoplasmic reticulum, some of them encircling large lipids droplets. Treatment with lower dosages showed similar alterations on Leydig cells and the morphological effects decreased directly proportional to the drug concentration. Serum testosterone levels were significantly lower only in 200 mg/kg DEC-treated group when compared to the controls. However, no significant changes were observed in the pregnancy rates and offspring number of DEC-treated male-mated female mice in any doses studied. The results obtained in the present study are consistent with the hypothesis that DEC has some effects on mice Leydig cells, although they were not sufficient enough to interfere with the rodent fertility.

  4. A rare ovarian tumor, leydig stromal cell tumor, presenting with virilization: a case report

    PubMed Central

    Aminimoghaddam, Soheila; Hashemi, Forough

    2012-01-01

    Leydig stromal cell tumor is a rare ovarian tumor that belongs to the group of sex-cord stromal tumors. They produce testosterone leading to hyperandrogenism. We present a 41yr old woman with symptoms of virilization and a mass of right adenex via ultra Sonography, and a rise of total and free serum testosterone. An ovarian source of androgen was suspected and a surgery performed. A diagnosis of leydig-stromal cell tumor was confirmed. Our report is a reminder that although idiopathic hirsutism and other benign androgen excess disorder like Polycystic Ovarian Syndrome (PCOs) are common, ovarian mass should be considered in differential diagnosis. PMID:23482693

  5. Role of 11β-OH-C(19) and C(21) steroids in the coupling of 11β-HSD1 and 17β-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells.

    PubMed

    Latif, Syed A; Shen, Mae; Ge, Ren-Shan; Sottas, Chantal M; Hardy, Matthew P; Morris, David J

    2011-06-01

    Here we describe further experiments to support our hypothesis that bidirectional 11β-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17β-HSD3, incubation of Leydig cells with corticosterone (B) or several C(19)- and C(21)-11β-OH-steroids, in the presence of [(3)H]-11-dehydro-corticosterone (A), stimulated 11β-HSD1-reductase activity. However, in presence of 30 μM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11β-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p<0.05) to 224-267 picomole/25,000 cells/30 min when 10 μM 11β-OH-steroids (in addition to 30 μM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 μM), and B or A (500 nM). Incubations of 0.3-6.0 μM of corticosterone (plus or minus 30 μM AD) were then performed to test the effectiveness of 17β-HSD3 as a possible NADP(+) regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11β-HSD1 is enzymatically coupled to 17β-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.

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

    PubMed

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

    2016-01-18

    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.

  7. 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

  8. Effects of T-2 toxin on the regulation of steroidogenesis in mouse Leydig cells.

    PubMed

    Yang, Jian Ying; Zhang, Yong Fa; Li, Yuan Xiao; Guan, Gui Ping; Kong, Xiang Feng; Liang, Ai Min; Ma, Kai Wang; Da Li, Guang; Bai, Xue Fei

    2016-10-01

    T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of testosterone secretion in primary Leydig cells derived from mouse testis. The previous study demonstrated T-2 toxin decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis directly. In this study, we further examined the direct biological effects of T-2 toxin on the process of steroidogenesis, primarily in Leydig cells of mice. Leydig cells of mature mouse were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine the decrease in T-2 toxin-induced testosterone secretion, we measured the transcription level of three key steroidogenic enzymes including 3β-HSD-1, cytochrome P450 side-chain cleavage (P450scc) enzyme, and steroidogenic acute regulatory (StAR) protein in T-2 toxin/human chorionic gonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7), 10(-8), and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with a decrease in the level of transcription of 3β-HSD-1, P450scc, and StAR (p < 0.05).

  9. RiboTag Analysis of Actively Translated mRNAs in Sertoli and Leydig Cells In Vivo

    PubMed Central

    Sanz, Elisenda; Evanoff, Ryan; Quintana, Albert; Evans, Elizabeth; Miller, Jeremy A.; Ko, Chemyong; Amieux, Paul S.; Griswold, Michael D.; McKnight, G. Stanley

    2013-01-01

    Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the “translatome” (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell. PMID:23776628

  10. Nandrolone and stanozolol induce Leydig cell tumor proliferation through an estrogen-dependent mechanism involving IGF-I system.

    PubMed

    Chimento, Adele; Sirianni, Rosa; Zolea, Fabiana; De Luca, Arianna; Lanzino, Marilena; Catalano, Stefania; Andò, Sebastiano; Pezzi, Vincenzo

    2012-05-01

    Several substances such as anabolic androgenic steroids (AAS), peptide hormones like insulin-like growth factor-I (IGF-I), aromatase inhibitors and estrogen antagonists are offered via the Internet, and are assumed without considering the potential deleterious effects that can be caused by their administration. In this study we aimed to determine if nandrolone and stanozolol, two commonly used AAS, could have an effect on Leydig cell tumor proliferation and if their effects could be potentiated by the concomitant use of IGF-I. Using a rat Leydig tumor cell line, R2C cells, as experimental model we found that nandrolone and stanozolol caused a dose-dependent induction of aromatase expression and estradiol (E2) production. When used in combination with IGF-I they were more effective than single molecules in inducing aromatase expression. AAS exhibited estrogenic activity and induced rapid estrogen receptor (ER)-dependent pathways involving IGF1R, AKT, and ERK1/2 phosphorylation. Inhibitors for these kinases decreased AAS-dependent aromatase expression. Up-regulated aromatase levels and related E2 production increased cell proliferation as a consequence of increased cyclin E expression. The observation that ER antagonist ICI182,780 was also able to significantly reduce ASS- and AAS + IGF-induced cell proliferation, confirmed a role for estrogens in AAS-dependent proliferative effects. Taken together these data clearly indicate that the use of high doses of AAS, as it occurs in doping practice, enhances Leydig cell proliferation, increasing the risk of tumor development. This risk is higher when AAS are used in association with IGF-I. To our knowledge this is the first report directly associating AAS and testicular cancer.

  11. 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.

  12. Chloroethylmethanesulfonate-induced effects on the epididymis seem unrelated to altered Leydig cell function.

    PubMed

    Klinefelter, G R; Laskey, J W; Kelce, W R; Ferrell, J; Roberts, N L; Suarez, J D; Slott, V

    1994-07-01

    Decades ago it was reported that when male rats were exposed to chloroethylmethanesulfonate (CEMS) for 5 days prior to weekly matings with untreated females, the second mating resulted in reduced litter size. Since fertility was not assessed at earlier time points, it was not possible to determine whether CEMS exerted any effects on sperm in the epididymis. In this study, we used a 4-day exposure and assessed multiple reproductive endpoints on Day 5 to characterize effects of CEMS exposure (6.25-25 mg/kg) on Leydig cells and the epididymis. Exposure to CEMS caused a dose-related decline in serum testosterone (T) levels. This occurred at a dose lower than that required to decrease T production in vitro by testicular parenchyma. The in vitro decline was not attributed to a decrease in maximal hCG-stimulated T production, but to a decrease in unstimulated T production. CEMS was 5-fold less sensitive than ethane dimethanesulfonate (EDS) in reducing maximal hCG-stimulated T production. To control for alterations in the epididymis resulting from decreased serum T alone, T was implanted in CEMS-treated animals to maintain serum T at a concentration similar to that found in normal rats. This exogenous T failed to prevent the CEMS-induced decrease in the weight of the caput/corpus epididymidis but did prevent the CEMS-induced decrease in seminal vesicle weight. Implantation of T failed to prevent the CEMS-induced reduction in sperm reserves in the cauda epididymidis, and it failed to prevent the CEMS-induced alterations in the histology of both the corpus and proximal cauda epididymidis. The height of the epithelium in both of these regions was increased, and clear cells disappeared from the proximal cauda epididymidis. These results demonstrate that CEMS might alter the ability of the Leydig cell to respond to LH stimulation in vivo, and that alterations in the structure and function of the epididymis occur even when the serum concentration of T is maintained.

  13. 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

  14. Knockout of the transcription factor Nrf2: Effects on testosterone production by aging mouse Leydig cells.

    PubMed

    Chen, Haolin; Jin, Shiying; Guo, Jingjing; Kombairaju, Ponvijay; Biswal, Shyam; Zirkin, Barry R

    2015-07-05

    Aging in rodents and men is associated with reduced serum levels of testosterone and Leydig cell testosterone productions. To further investigate the mechanism by which Leydig cell testosterone production declines, the effect of knocking out Nrf2, a master regulator of phase 2 antioxidant genes, was examined. In wild-type mice, testosterone production and serum testosterone levels remained unchanged through middle age (8 months), but then were reduced significantly by old age (21-24 months). In contrast, serum testosterone levels and Leydig cell testosterone production were reduced significantly in the Nrf2-/- mice as early as middle age, and were reduced further in the aged mice. Reduced steroidogenesis in the knockout mice was associated with reduced antioxidant capacity, and increased expression of protein nitrotyrosine residues, a marker of ROS. These results support the hypothesis that, over time, increases in oxidative stress contribute to or cause the reduced testosterone production that characterizes Leydig cell aging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Isolation and culture of highly enriched populations of Leydig cells from guinea-pig (Cavia porcellus) testes.

    PubMed

    Kukucka, M A; Misra, H P

    1994-01-01

    Leydig cells were isolated from adult male guinea-pig testes using a multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally stimulating dose of ovine LH (100 ng ml-1) and human CG (200 mIU ml-1). Leydig cells incubated in a shaking (120 cycles min-1) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.

  16. Gene expression of luteinizing hormone receptor and steroidogenic enzymes during Leydig cell development.

    PubMed

    Abney, T O; Zhai, J

    1998-02-01

    Testicular Leydig cells (LC) are rapidly and selectively destroyed by an injection of ethane dimethane sulfonate (EDS). LC regeneration occurs in the testis of the EDS-treated rats from the differentiation of the precursor Leydig cells (PLC). This study was designed to investigate the patterns of change in the mRNAs for the luteinizing hormone receptor (LHR) and the steroidogenic enzymes, cholesterol side chain cleavage (P-450scc) and 17 alpha-hydroxylase (P-450(17 alpha)) during LC regeneration from PLCs. Mature (60 days of age) Sprague-Dawley male rats received a single intraperitoneal injection of EDS and were killed at different times between days 2 and 60 post-treatment. PLC- and LC-enriched fractions were isolated from the testes of the EDS-treated rats and age-matched control rats using a collagenase digestion-Percoll gradient method. Total RNA was extracted from these cell populations and subjected to Northern blot analysis. The LC fraction isolated from testes of control rats expressed four major transcripts of the LHR, sized 1.8, 2.5, 4.2 and 7.0 kb. The undifferentiated PLC fraction from controls expressed only a truncated form, the 1.8 kb transcript. This truncated LHR transcript was also the only LHR mRNA species detected in PLCs at day 2 post-EDS treatment. In contrast, all four transcripts of the LHR were detected in the PLC fraction at day 10 post-EDS treatment. The levels of the full length 7.0 kb transcript increased thereafter and reached a peak between days 24 and 36 post-EDS treatment in the PLC fraction. Concomitant with the increase in the 7.0 kb transcript, the truncated 1.8 kb transcript decreased in amount and reached a nadir between days 16 and 36 post-treatment. The changes observed in this cell fraction reflect the process of differentiation of PLCs into LCs. At day 45 post-EDS treatment, the level of the 7.0 kb transcript decreased while the 1.8 kb form increased in the PLC fraction, reflecting the completion of LC regeneration from

  17. CEUS Time Intensity Curves in the Differentiation Between Leydig Cell Carcinoma and Seminoma: A Multicenter Study.

    PubMed

    Drudi, F M; Valentino, M; Bertolotto, M; Malpassini, F; Maghella, F; Cantisani, V; Liberatore, M; De Felice, C; D'Ambrosio, F

    2016-04-01

    Ultrasound (US) is the main imaging technique in the assessment of testicular masses, as it has proved to be highly accurate in the visualization of these pathologies. Identification of a Leydig cell tumor is essential since the lesion is benign in 90% of cases. The aim of this multicenter study is to assess the effectiveness of contrast-enhanced ultrasound (CEUS) in differentiating Leydig cell tumors from seminoma using qualitative and quantitative features. From February 2011 to December 2013, 31 patients (mean age: 34 years; range: 25 - 52) were recruited for this prospective study. Three of them were monorchid. Therefore, a total of 59 testicles were assessed. All patients underwent grayscale US, color Doppler ultrasound (CDUS), CEUS and orchiectomy. The paired one-tailed Student's t-test was carried out to differentiate between Leydig cell tumors and seminomas. 31 lesions suspicious for malignancy were hypoechoic on grayscale US while they did not show a typical pattern on CDUS. CEUS qualitative analysis, based on contrast enhancement pattern, during the arterial and venous phases, did not allow discrimination of Leydig cell tumors from seminoma. Quantitative analysis of time-intensity curves (TICs) demonstrated that only three parameters presented statistical significance, i. e. wash-in rate (WiR) p = 0.014, peak enhancement (PE) p = 0.001 and time to peak (TTP) p = 0.003. The vascular bed of a Leydig cell tumor is wider and the blood flow velocity is higher than that of a seminoma due to more regular neovascularization. In contrast, a seminoma presents large areas of necrosis due to irregular neovascularization. This explains the different PE and WiR values. Further studies involving larger patient populations are mandatory to confirm these encouraging preliminary results. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Concomitant Sertoli and Leydig Cell Tumor of the Testis: A Case Report

    PubMed Central

    Tazi, Mohammed Fadl; Ahallal, Youness; Khallouk, Abdelhak; Elfatemi, Hinde; Bendahou, Mohcine; Tazi, Elmehdi; El Fassi, Mohammed Jamal; Farih, Moulay Hassan

    2011-01-01

    A rare intratubular gonadal stromal tumor was present in the testis of a 45-year-old man who was admitted to our hospital with the chief complaint of gradual enlargement of the left testis. Tumoral markers were negative and no extension was observed. The tumor comprised an intratubular mixture of two types of tumor cells with intercellular junctions: the predominant tumor cells were consistent with a Sertoli cell origin and cells comprising the minor population consistent with a Leydig cell origin. The patient is disease free after 6-month follow-up. The case is considered to be a testicular mixed tubular Sertoli-Leydig cell tumor. It highlights a rare type of primary tumor of the testis that features a good prognosis. PMID:22114547

  19. HPLC determination of an oxytocin-like peptide produced by isolated guinea pig Leydig cells: stimulation by ascorbate.

    PubMed

    Kukucka, M A; Misra, H P

    1992-01-01

    Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/mL LH for 24 h in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum, and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high-performance liquid chromatography with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells stimulated with 0, 25, and 50 microM ascorbate produced and secreted 40.1 +/- 1.23, 77.4 +/- 13.8, 74.2 +/- 26.3 pg of an oxytocin-like peptide, respectively, per 1 x 10(6) cells. These results indicate that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo and that low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro.

  20. MCL1 is a key regulator of steroidogenesis in mouse Leydig cells.

    PubMed

    Guang-Yu, Li; Hai-Yan, Lan; Ji-Hong, Liang; Yun-Cong, Mo; Xue-Lian, Deng; Chun-Yu, Lin; Wen-Yong, Su

    2016-03-01

    Myeloid cell leukemia-1 (MCL1), an anti-apoptotic member of the BCL2 family, is expressed abundantly in the testis. Previous characterization revealed that MCL1 is expressed exclusively in the Leydig cells in the mouse testis, yet what it does in these cells remains unknown. We therefore analyzed testosterone biosynthesis in isolated primary Leydig cells and the MA-10 cell line, in which MCL1 was knocked down using an siRNA strategy. The mRNA abundance of the steroidogenic genes Star, Cyp11a1, Cyp17a1, Hsd3b1, Srd5a, and the luteinizing hormone/choriogonadotropin receptor Lhcgr were significantly reduced following MCL1 knockdown. Of the two enzymes required for testosterone biosynthesis, STAR and P450 SCC (encoded by Cyp11a1) enzyme abundance was also reduced following Mcl1 siRNA treatment, possibly leading to the reduced production of sex steroid precursors, and testosterone in these knockdown cells. Despite its classification as an anti-apoptosis protein, Mcl1 siRNA treatment did not affect cell survival. Collectively, our findings indicate that MCL1 plays a pivotal role in Leydig-cell steroidogenesis, and might provide novel insights into metabolic regulation in this cell. Mol. Reprod. Dev. 83: 226-235, 2016. © 2016 Wiley Periodicals, Inc.

  1. TGF-beta1 system in Leydig cells. Part II: TGF-beta1 and progesterone, through Smad1/5, are involved in the hyperplasia/hypertrophy of Leydig cells.

    PubMed

    Gonzalez, Candela R; Gonzalez, Betina; Rulli, Susana B; Dos Santos, Mara L; Mattos Jardim Costa, Guilherme; França, Luiz R; Calandra, Ricardo S; Gonzalez-Calvar, Silvia I

    2010-08-01

    Several reports indicate that transforming growth factor beta1 (TGF-beta1) participates in the regulation of cell cycle progression. In this work, we analyzed the in vitro effect of TGF-beta1 on Leydig cell proliferation markers and the in vivo effect of this cytokine in Leydig cell hyperplasia/hypertrophy. The in vitro effect of TGF-beta1 (1 ng/ml) plus progesterone (10(-6) M) on purified Leydig cells from 3 week-old mice increased the immunocytochemically detected PCNA and stimulated the phosphorylation of Smad 1/5. Progesterone (10(-6) M) in the presence or absence of TGF-beta1 diminished the ratio Bax/Bcl-2. Morphometric testicular studies of mice treated with progesterone (s.c.) plus TGF-beta1 (intratesticular), showed an increase in interstitial volume and a decrease in tubular volume. Furthermore, the cytoplasmic volume of Leydig cells showed an increment in this experimental group with a diminution in nuclear volume. Thus, it turned out that the administration of progesterone and TGF-beta1 augmented the volume of Leydig cells. These results indicate a clear effect of TGF-beta1 in the hypertrophy/hyperplasia of Leydig cells.

  2. 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

  3. 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

  4. Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

    PubMed

    Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

    2017-10-02

    Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

  5. T-2 toxin inhibits gene expression and activity of key steroidogenesis enzymes in mouse Leydig cells.

    PubMed

    Yang, Jian Ying; Zhang, Yong Fa; Meng, Xiang Ping; Li, Yuan Xiao; Ma, Kai Wang; Bai, Xue Fei

    2015-08-01

    T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of the testosterone secretion in the primary Leydig cells derived from the mouse testis. The previous study demonstrated the effects of T-2 toxin through direct decrease of the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis. In this study, we further examined the direct biological effects of T-2 toxin on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine T-2 toxin-induced testosterone secretion decrease, we measured the transcription levels of 3 key steroidogenic enzymes and 5 enzyme activities including 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD in T-2 toxin/human chorionicgonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7) M, 10(-8) M and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with the decreases in the levels of transcription of 3β-HSD-1, P450scc, and StAR (P<0.05) and also in enzyme activities of 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD (P<0.05).

  6. Toxic effects of sodium fluoride on cell proliferation and apoptosis of Leydig cells from young mice.

    PubMed

    Song, Guo hua; Wang, Rui Li; Chen, Zhao Yang; Zhang, Bin; Wang, Hai Long; Liu, Mao Lin; Gao, Ji Ping; Yan, Xiao Yan

    2014-09-01

    The biological effects of fluoride on human health are often extensive, either beneficial or detrimental. Among the various effects of fluoride exposure in different organs, the reproductive tract is particularly susceptible to disruption by fluoride at a sufficient concentration. It has attracted much attention to the effect of sodium fluoride on male fertility, gestational female, and offspring. Herein, we applied a widespread natural compound sodium fluoride (NaF) and investigated the effects of acute NaF exposure on Leydig cells, including their proliferation, apoptosis, and signal pathway changes. Our results demonstrated that high dosage of NaF could inhibit cell proliferation by stress-induced apoptosis, which was confirmed by cellular and molecular evidences. We found that fluoride exposure affected the expression levels of stress response factors, signal transduction components, and apoptosis-related proteins, including caspase-3/caspase-9, B-cell lymphoma 2 (Bcl-2), and Bax. This study suggests that the complex effects of fluoride on Leydig cells are closely related to its dosage.

  7. 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.

  8. 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.

  9. Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations.

    PubMed

    Fowler, Paul A; Abramovich, David R; Haites, Neva E; Cash, Phillip; Groome, Nigel P; Al-Qahtani, Ahmed; Murray, Tessa J; Lea, Richard G

    2007-11-01

    Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis. Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated. Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals. Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.

  10. Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

    PubMed Central

    Chai, Wei-Ran; Chen, Yong; Wang, Qian; Gao, Hui-Bao

    2008-01-01

    Background Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. Results Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In

  11. Perfluorododecanoic acid-induced steroidogenic inhibition is associated with steroidogenic acute regulatory protein and reactive oxygen species in cAMP-stimulated Leydig cells.

    PubMed

    Shi, Zhimin; Feng, Yixing; Wang, Jianshe; Zhang, Hongxia; Ding, Lina; Dai, Jiayin

    2010-04-01

    Perfluorododecanoic acid (PFDoA) can be detected in environmental matrices and human serum and has been shown to inhibit testicular steroidogenesis in rats. However, the mechanisms that are responsible for the toxic effects of PFDoA remain unknown. The aims of this study were to investigate the mechanism of steroidogenesis inhibition by PFDoA and to identify the molecular target of PFDoA in Leydig cells. The effects of PFDoA on steroid synthesis in Leydig cells were assessed by radioimmunoassay. The expression of key genes and proteins in steroid biosynthesis was determined by real-time PCR and Western blot analysis. Reactive oxygen species (ROS) and hydrogen peroxide (H(2)O(2)) levels were determined using bioluminescence assays. PFDoA inhibited adenosine 3',5'-cyclophosphate (cAMP)-stimulated steroidogenesis in mouse Leydig tumor cells (mLTC-1) and primary rat Leydig cells in a dose-dependent manner. However, PFDoA (1-100 microM) did not exhibit effects on cell viability and cellular ATP levels in mLTC-1 cells. PFDoA inhibited steroidogenic acute regulatory protein (StAR) promoter activity and StAR expression at the messenger RNA (mRNA) and protein levels but did not affect mRNA levels of peripheral-type benzodiazepine receptor, cholesterol side-chain cleavage enzyme, or 3beta-hydroxysteroid dehydrogenase in cAMP-stimulated mLTC-1 cells. PFDoA treatment also resulted in increased levels of mitochondrial ROS and H(2)O(2). After excessive ROS and H(2)O(2) were eliminated in PFDoA-treated mLTC-1 cells by MnTMPyP (a superoxide dismutase analog), progesterone production was partially restored and StAR mRNA and protein levels were partially recovered. These data show that PFDoA inhibits steroidogenesis in cAMP-stimulated Leydig cells by reducing the expression of StAR through a model of action involving oxidative stress.

  12. 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.

  13. Histological and stereological studies on Leydig cells in the testes of Gallus domesticus from pre-hatching to sexual maturity.

    PubMed

    González-Morán, María Genoveva; Soria-Castro, Elizabeth

    2010-07-01

    The present study was aimed at evaluating the sequence of the histological and quantitative changes that occur in diverse components of the interstitial tissue, as well as the variations in the number and volume of Leydig cells of the testes of Gallus domesticus from 8-day-old embryo to 28-week-old chickens. Results indicate an increase in total volume of all components of the interstitial tissue: Leydig cells, blood vessels, and interstitium of chicken testes with advancing development. The proportion of each component differs markedly according to the age of the animal. Data indicate that Leydig cells are present in the chicken testes from pre-hatching to sexual maturity, and show an increase in the total volume, number per testis, and individual cell volume along the age of the animal. Values of P<0.05 were considered statistically significant. The results indicate that the total volume of Leydig cells is the dominant component of the interstitial tissue in the testes of 6-week-old chickens, but in testes of chicken embryos correspond to the lowest percentage of interstitial tissue. Results also reveal that hyperplasia is more important than cell hypertrophy in the increase of the total volume of Leydig cells during advancing development. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Sertoli Cell Number Defines and Predicts Germ and Leydig Cell Population Sizes in the Adult Mouse Testis.

    PubMed

    Rebourcet, Diane; Darbey, Annalucia; Monteiro, Ana; Soffientini, Ugo; Tsai, Yi Ting; Handel, Ian; Pitetti, Jean-Luc; Nef, Serge; Smith, Lee B; O'Shaughnessy, Peter J

    2017-09-01

    Sertoli cells regulate differentiation and development of the testis and are essential for maintaining adult testis function. To model the effects of dysregulating Sertoli cell number during development or aging, we have used acute diphtheria toxin-mediated cell ablation to reduce Sertoli cell population size. Results show that the size of the Sertoli cell population that forms during development determines the number of germ cells and Leydig cells that will be present in the adult testis. Similarly, the number of germ cells and Leydig cells that can be maintained in the adult depends directly on the size of the adult Sertoli cell population. Finally, we have used linear modeling to generate predictive models of testis cell composition during development and in the adult based on the size of the Sertoli cell population. This study shows that at all ages the size of the Sertoli cell population is predictive of resulting testicular cell composition. A reduction in Sertoli cell number/proliferation at any age will therefore lead to a proportional decrease in germ cell and Leydig cell numbers, with likely consequential effects on fertility and health.

  15. Steroidogenesis in primary cultures of neonatal porcine Leydig cells from Duroc and Norwegian Landrace breeds.

    PubMed

    Lervik, S; von Krogh, K; Karlsson, C; Olsaker, I; Andresen, Ø; Dahl, E; Verhaegen, S; Ropstad, E

    2011-10-01

    Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action.

  16. Leydig Cell Loss and Spermatogenic Arrest in Platelet-Derived Growth Factor (Pdgf)-a–Deficient Mice

    PubMed Central

    Gnessi, Lucio; Basciani, Sabrina; Mariani, Stefania; Arizzi, Mario; Spera, Giovanni; Wang, Chiayeng; Bondjers, Cecilia; Karlsson, Linda; Betsholtz, Christer

    2000-01-01

    Platelet-derived growth factor (PDGF)- A–deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Rα expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A−/− mice, combined with the normal developmental expression of PDGF-A and PDGF-Rα, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man. PMID:10831606

  17. The Impact of 4-Nonylphenol on the Viability and Hormone Production of Mouse Leydig Cells.

    PubMed

    Jambor, T; Lukáčová, J; Tvrdá, E; Kňažická, Z; Forgács, Z; Lukáč, N

    2016-01-01

    Exogenous substances altering the function of the endocrine system and exhibiting adverse health effects on the organism are defined as endocrine disruptors. Nonylphenol is one of the most abundant alkylphenol ethoxylate derivatives, being detected in food products. Diverse studies have classified nonylphenol as hazardous to the health, especially to male reproduction. This in vitro study aimed to examine the effects of 4-nonylphenol on androstenedione and testosterone production as well as on the viability of Leydig cells of NMRI mice. The cells were cultured for 44 h with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/ml of 4-nonylphenol and compared to the control. Quantification of testosterone and androstenedione directly from aliquots of the medium was performed by enzyme-linked immunosorbent assay. Cell viability was measured by the metabolic activity assay for mitochondrial functional activity. Androstenedione production significantly (P < 0.001) increased with 1.0; 2.5 and 5.0 μg/ml 4-nonylphenol. Although cAMP-stimulated testosterone production was not significantly affected by 4-nonylphenol, a tendency to attenuate the level of testosterone in the Leydig cells treated with 2.5 and 5.0 μg/ml 4-nonylphenol was observed. The viability of mouse Leydig cells was slightly increased at the lowest doses of 4-nonylphenol (0.04 and 0.2 μg/ml). We also observed an increase at higher concentrations of the substance (1.0; 2.5 and 5.0 μg/ml), but this increase was not significant. Further investigations are required to establish the biological significance and possible reproductive implications.

  18. Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis.

    PubMed

    Allen, John A; Shankara, Tristan; Janus, Paul; Buck, Steve; Diemer, Thorsten; Hales, Karen Held; Hales, Dale B

    2006-08-01

    The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3',5'-cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (Deltapsim), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Deltapsim. Dissipating Deltapsim with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on Deltapsim. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Deltapsim. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3beta-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Deltapsim, mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid

  19. 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

    2015-03-16

    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.

  20. Initial characterization of a subclone of the MA-10 mouse Leydig tumor cell line.

    PubMed

    Kilgore, M W; Stocco, D M

    1989-03-01

    Cloned cell lines have proven to be useful models in understanding the regulation of endocrine cells and steroid synthesis. In this study we report the isolation and characterization of a subclone of the MA-10 Leydig tumor cell line. Whereas there was no difference in basal steroid production between the clone (MA-10 LP) and the parent stock (MA-10), MA-10 LP produces very low levels of progesterone after stimulation by hCG or (Bu)2cAMP. In both cell populations, hCG stimulation resulted in the accumulation of comparable amounts of cAMP in the presence of a phosphodiesterase inhibitor, and similar levels of cAMP were measured at 30 min without inhibitor. Measurement of cholesterol side-chain cleavage activity using two separate methods demonstrated that the low steroid production in MA-10 LP could not be accounted for by a decrease in the activity of this enzyme complex. Additionally, no difference in 3 beta-hydroxysteroid dehydrogenase activity could be demonstrated between the two cell populations. Since the lesion that attenuates the ability of MA-10 to synthesize progesterone is somewhere after the production of cAMP and before cholesterol side-chain cleavage activity, this system may provide a useful model for understanding the regulatory mechanisms controlling steroid biosynthesis in Leydig cells.

  1. A novel androgen receptor mutation resulting in complete androgen insensitivity syndrome and bilateral Leydig cell hyperplasia.

    PubMed

    Singh, Rajender; Shastry, Prabhakar K; Rasalkar, Avinash A; Singh, Lalji; Thangaraj, K

    2006-01-01

    Androgens drive male secondary sexual differentiation and maturation. Mutations in the androgen receptor (AR) gene cause a broad spectrum of abnormal phenotypes in humans, ranging from mild through partial to complete androgen insensitivity. We have analyzed the AR gene by using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing and have studied gonads histologically in a familial case of complete androgen insensitivity syndrome. Sequence analysis of the AR gene showed a novel C2578T missense mutation, resulting in the replacement of a highly conserved leucine residue with phenylalanine (L859F) in ligand-binding domain of the receptor. The residue L859, located in helix 10 of the androgen receptor, plays a significant role in overall architecture of ligand-binding pocket. The mutation was absent from the father, normal brother of the patients, and 100 normal males recruited in this study as controls. The inheritance of the mutation in the family clearly shows that C2578T is the underlying mutation for the eventual phenotype in the patients. Histology of patient's gonads showed Leydig cell hyperplasia, with a few or no spermatogonium. It is thought that AR gene mutations result in hormonal imbalance, resulting in the high levels of luteinizing hormone (LH) and ultimately Leydig cell hyperplasia or tumor formation. In the present study, we have reported a rare familial case of Leydig cell hyperplasia despite consistently normal LH levels. The finding will help in giving counseling to this family and prevent the transmission of the mutated X chromosome to the coming generations.

  2. Fine structure of the epidermal Leydig cells in the axolotl Ambystoma mexicanum in relation to their function.

    PubMed Central

    Jarial, M S

    1989-01-01

    The fine structure of the Leydig cells in the epidermis of the strictly aquatic adult axolotl Ambystoma mexicanum resembles that of similar cells in larval salamanders. The major finding of this study is that the mucous secretion of the Leydig cells is released into the intercellular spaces from which it is discharged through pores onto the surface of the epidermis where it forms a mucous layer to protect the skin. Images Figs. 1-2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Figs. 11-13 PMID:2630544

  3. Effects of hypocholesterolemia and chronic hormonal stimulation on sterol and steroid metabolism in a Leydig cell tumor.

    PubMed

    Ascoli, M

    1981-11-01

    The studies presented herein were done to investigate the effects of drug-induced hypocholesterolemia and chronic hormonal stimulation on cholesterol metabolism and steroid biosynthesis in a functional Leydig cell tumor. It was found that 4-aminopyrazolo(3,4-d)-pyrimidine (4-APP)-induced hypocholesterolemia had no effect on a) the amount of cholesterol present in the tumor, b) cholesterol biosynthesis, and c) steroid production. Chronic stimulation with choriogonadotropin also had no effect on the amount of cholesterol present in the tumor, but it increased steroid production and cholesterol biosynthesis. These results suggest that the Leydig tumor cells primarily use intracellular cholesterol for steroid biosynthesis. Other data show that 4-APP treatment reduces gonadotropin binding in the Leydig tumor cells.

  4. Recurrent ovarian Sertoli–Leydig cell tumor in a child with Peutz–Jeghers syndrome

    PubMed Central

    Bellfield, Edward J.; Alemzadeh, Ramin

    2016-01-01

    We present a female child with Peutz–Jeghers syndrome (PJS) with a recurrent ovarian Sertoli–Leydig cell tumor (SLCT). SLCTs are relatively rare sex cord neoplasms that can occur in PJS. The patient was an African-American female who first presented at the age of 3 years with precocious puberty, and then at the age of 17 years with abdominal pain and irregular menses. In each case, she had resection of the mass, which included oophorectomy. To our knowledge, this is the first reported case in a child with PJS to have a recurrent ovarian SLCT. PMID:28101370

  5. Two distinct origins for Leydig cell progenitors in the fetal testis

    PubMed Central

    DeFalco, Tony; Takahashi, Satoru; Capel, Blanche

    2011-01-01

    During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad-mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell-cell interactions among cells in the interstitium are involved in testis morphogenesis. PMID:21255566

  6. 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.

  7. Postnatal development of Leydig cells in the opossum (Monodelphis domestica): an immunocytochemical and endocrinological study.

    PubMed

    Xie, Q; Mackay, S; Ullmann, S L; Gilmore, D P; Payne, A P; Gray, C

    1998-03-01

    This study involved characterization of Leydig cells of the opossum Monodelphis domestica, functionally by immunocytochemical identification of the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and by measurement of testosterone levels using RIA. Immunostaining for 3 beta-HSD was first detected in a few Leydig cells on Day 16, was increased by Day 24, reached a peak at 4 mo, and was present even in senescent (3 yr) animals. Plasma testosterone was first measurable (0.35 nM) at prepuberty (3.5 mo). Prior to that, plasma testosterone concentrations were uniformly below the level of detection (< 0.3 nM) in both sexes from Day 5 to 2.5 mo. By 4 mo (puberty), plasma testosterone levels in males had risen significantly to 1.53 +/- 0.35 nM, continuing to increase to 1.79 +/- 0.4 nM at 6 mo and peaking at 2.71 +/- 0.29 nM in the adult (1-2 yr). Ovarian testosterone concentrations were consistently lower than those in the testis, as were those of adrenals of both sexes. Thus the testis would appear to be the major source of androgen production throughout life in this species. Our immunocytochemical study suggests that in Monodelphis, puberty is reached at 4 mo, and this was further supported by a rise in circulating testosterone levels at this time.

  8. Sertoli-Leydig cell tumor of the ovary: a clinicopathologic study of 10 cases.

    PubMed

    Ayhan, A; Tuncer, Z S; Hakverdi, A U; Yüce, K; Ayhan, A

    1996-01-01

    Ten patients with Sertoli-Leydig cell tumors of the ovary treated consecutively were analyzed retrospectively for clinicopathological characteristics, treatment modalities and survival. All patients were subjected to a surgical procedure including unilateral salpingoophorectomy (USO) in 5, total abdominal hysterectomy, bilateral salpingoophorectomy (TAH+BSO) in 2, TAH+BSO and omentectomy and pelvic and paraaortic lymphadenectomy and appendicectomy in 3 patients. Following surgery, 5 patients were subjected to adjuvant VAC (vincristine+actinomycine-D+cyclophosphamide) chemotherapy. Sertoli-Leydig cell tumors constitued 0.6% of all ovarian neoplasms in our institution (10/1621). The mean age at diagnosis was 28.2 years. Of the patients, 8 had stage IA, one had IB and one had stage III disease at surgery. Bilaterality was observed in two patients (20.0%). Four patients had poorly-differentiated tumors, of whom 1 had a retiform pattern and 1 had a heterologue element, with marked mitosis whereas the others had well--or moderately--differentiated tumors. Only one patient presented with recurrence during pregnancy and died 16 months after initial surgery. Since most of the patients present with stage IA disease, a favorable outcome can be achieved in most of them and conservative surgery seems to be the treatment of choice in younger patients. However, late recurrences even in early stages, especially in patients with poorly-differentiated tumors, can be detected, resulting in a fatal outcome despite any form of aggressive therapy.

  9. Prolactin (PRL) induction of cyclooxygenase 2 (COX2) expression and prostaglandin (PG) production in hamster Leydig cells.

    PubMed

    Matzkin, María Eugenia; Ambao, Verónica; Carino, Mónica Herminia; Rossi, Soledad Paola; González, Lorena; Turyn, Daniel; Campo, Stella; Calandra, Ricardo Saúl; Frungieri, Mónica Beatriz

    2012-01-02

    Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression-recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1β and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1β and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Antagonistic effects of testosterone and the endocrine disruptor mono-(2-ethylhexyl) phthalate on INSL3 transcription in Leydig cells.

    PubMed

    Laguë, Eric; Tremblay, Jacques J

    2008-09-01

    Insulin-like 3 (INSL3) is a small peptide produced by testicular Leydig cells throughout embryonic and postnatal life and by theca and luteal cells of the adult ovary. During fetal life, INSL3 regulates testicular descent in males, whereas in adults, it acts as an antiapoptotic factor for germ cells in males and as a follicle selection and survival factor in females. Despite its considerable roles in the reproductive system, the mechanisms that regulate Insl3 expression remain poorly understood. There is accumulating evidence suggesting that androgens might regulate Insl3 expression in Leydig cells, but transcriptional data are still lacking. We now report that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. We also show that testosterone activates the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. We have mapped the testosterone-responsive element to the proximal Insl3 promoter region. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Finally we show that mono-(2-ethylhexyl) phthalate, a widely distributed endocrine disruptor with antiandrogenic activity previously shown to inhibit Insl3 expression in vivo, represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Insl3 transcription in Leydig cells and the mode of action of phthalates.

  11. Organophosphate Flame Retardants Act as Endocrine-Disrupting Chemicals in MA-10 Mouse Tumor Leydig Cells.

    PubMed

    Schang, Gauthier; Robaire, Bernard; Hales, Barbara F

    2016-04-01

    The organophosphate flame retardants (OPFRs) have emerged as alternatives to banned brominated flame retardants but little is known about their possible activity as endocrine disruptors. Our goal was to compare the effects of 7 commonly used OPFRsin vitroon MA-10 mouse Leydig tumor cells to those of a major brominated flame retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). The effects of OPFRs and BDE-47 on mitochondrial activity, cell counts, oxidative stress, steroid secretion and gene expression were investigated. BDE-47 and all 7 OPFRs tested significantly reduced MA-10 cell mitochondrial activity (concentrations ≥50 μM) and cell number (concentrations ≥10 μM). All of the OPFRs significantly increased (10 μM, 1.7-4.4-fold) superoxide production whereas BDE-47 had no significant effect. Basal progesterone production was significantly increased (10 μM, 1.5 to 3-fold) by 2-ethylhexyl diphenyl phosphate, isodecyl diphenyl phosphate, isopropylated triphenyl phosphate, tert-butylphenyl diphenyl phosphate, and tricresyl phosphate, while BDE-47, triphenyl phosphate and tri-o-cresyl phosphate had no effect. Interestingly, isopropylated triphenyl phosphate enhanced dbcAMP-stimulated steroid production (∼2-fold), while tri-o-cresyl phosphate decreased (∼2/3) LH-stimulated steroid production. Several OPFRs affected the expression of genes involved in the biosynthesis of progesterone. In conclusion, all the OPFRs tested affected mitochondrial activity, cell survival, and superoxide production. Basal or stimulated steroid secretion was affected by all of the OPFRs except triphenyl phosphate; BDE-47 had no effect. Hence, the OPFRs currently used as alternatives affect Leydig cells to a greater extent than the brominated flame retardants that they have replaced.

  12. Steroidogenic genes expressions are repressed by high levels of leptin and the JAK/STAT signaling pathway in MA-10 Leydig cells.

    PubMed

    Landry, David A; Sormany, François; Haché, Josée; Roumaud, Pauline; Martin, Luc J

    2017-03-25

    The adipose tissue is an important endocrine organ secreting numerous peptide hormones, including leptin. Increased circulating levels of leptin, as a result of hormonal resistance in obese individuals, may contribute to lower androgen production in obese males. However, the molecular mechanisms involved need to be better defined. Androgens are mainly produced by Leydig cells within the testis. In male rodents, activation of the leptin receptor modulates a cascade of intracellular signal transduction pathways which may lead to regulation of transcription factors having influences on steroidogenesis in Leydig cells. Thus, as a result of high leptin levels interacting with its receptor and modulating the activity of the JAK/STAT signaling pathway, the activity of transcription factors important for steroidogenic genes expressions may be inhibited in Leydig cells. Here we show that Lepr is increasingly expressed within Leydig cells according to postnatal development. Although high levels of leptin (corresponding to obesity condition) alone had no effect on Leydig cells' steroidogenic genes expression, it downregulated cAMP-dependent activations of the cholesterol transporter Star and of the rate-limiting steroidogenic enzyme Cyp11a1. Our results suggest that STAT transcriptional activity is downregulated by high levels of leptin, leading to reduced cAMP-dependent steroidogenic genes (Star and Cyp11a1) expressions in MA-10 Leydig cells. However, other transcription factors such as members of the SMAD and NFAT families may be involved and need further investigation to better define how leptin regulates their activities and their relevance for Leydig cells function.

  13. Retiform Sertoli-Leydig Cell Tumor in a 38-Year-Old Woman: A Case Report, Retrospective Review, and Review of Current Literature

    PubMed Central

    Showalter, Josh A.; Roy, Suvra; Deavers, Michael T.; Zhao, Bihong

    2017-01-01

    Ovarian sex cord-stromal tumors arise from the stromal cells that surround and support the oocytes. Sertoli-Leydig cell tumors belong to this category of ovarian neoplasms. We present the case of a 38-year-old woman who was found to have a right ovarian mass. The mass was resected and diagnosed as Stage I Sertoli-Leydig cell tumor, retiform variant, following histopathologic and immunohistochemical examination. This case is unusual given the rarity of the retiform variant of Sertoli-Leydig cell tumor and the atypically older age of 38 years at presentation. PMID:28316852

  14. 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.

  15. Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

    PubMed

    Liu, L; Smith, J W

    2000-06-01

    A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

  16. Effect of experimental infection with Trypanosoma congolense and scrotal insulation on Leydig cell steroidogenesis in the ram.

    PubMed

    Mutayoba, B M; O'Shaughnessy, P J; Jeffcoate, I A; Eckersall, P D; Cestnik, V; Holmes, P H

    1997-08-01

    Testicular steroid content and Leydig cell steroidogenesis in vitro were investigated in rams on Days 28 and 58 after Trypanosoma congolense infection and were compared with those of rams in which testicular temperature had been raised artificially by insulation of the scrotum for 58 d. Testicular testosterone content increased significantly on Day 28 after infection but was lower than that of controls on Day 58 while it increased in scrotal-insulated rams compared with that of controls by Day 58. Testicular progesterone was undetectable in the control and trypanosome-infected groups throughout the experiment, but it increased in the insulated rams by day 58. Basal (unstimulated) Leydig cell testosterone production in the infected rams was similar to that of control rams on Day 28 but was significantly lower on Day 58. Stimulation of Leydig cell testosterone production with hCG or 22R-hydroxycholesterol (22ROHC) significantly reduced in infected rams at both 28 and 58 d after infection as well as in scrotal-insulated rams on Day 58. It is concluded that the increase in testicular testosterone content in the infected and scrotal-insulated rams on Days 28 and 58, respectively, was induced by elevation of testicular temperature by trypanosome infection, perhaps through an effect on testicular blood flow. Reduced testosterone production by Leydig cells from infected and scrotal-insulated rams in response to hCG and 22ROHC suggests that trypanosome-induced pyrexia might be involved in reducing Leydig cell steroidogenesis and subsequent plasma testosterone levels, possibly by affecting enzymes involved in steroid biosynthesis.

  17. Inhibition of steroidogenesis in Leydig cells by Müllerian-inhibiting substance.

    PubMed

    Fynn-Thompson, Eric; Cheng, Henry; Teixeira, Jose

    2003-12-15

    Müllerian-inhibiting substance (MIS), a member of the transforming growth factor-beta family of cytokines that signal through a heteromeric complex of single-transmembrane serine/threonine kinase receptors, is required for Müllerian duct regression and normal reproductive tract development in the male embryo. However, the continued expression of MIS at high levels in males until puberty and its induction in females after birth suggested other roles for MIS. Additionally, Leydig cell development and steroidogenic capacity and ovarian follicle recruitment were abnormal in MIS-knockout or MIS-overexpressing mice. We have shown that MIS inhibits the cAMP-induced expression of cytochrome P450 C17alpha-hydroxylase/C17-20 lyase (Cyp17) mRNA both in vitro and in vivo. Our current efforts are to understand the molecular mechanisms regulating both MIS type II receptor (MISRII) expression and its signaling in rodent Leydig cell lines. MISRII expression in R2C cells requires both steroidogenic factor-1 and an unknown protein to bind to its proximal promoter in the context of 1.6 kb 5'-flanking DNA. When bound by MIS, signaling by the receptor in MA-10 cells blocks the protein kinase A-mediated induction of Cyp17 expression by a cAMP regulatory element-binding protein independent mechanism. We continue to investigate the molecular mechanisms of MISRII expression and possible interactions between MIS-regulated SMAD activation and cAMP signaling. These studies will provide a better understanding of the role played by MIS during postnatal life.

  18. 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.

  19. Does gonadotropin receptor complex have an amplifying role in cAMP/testosterone production in Leydig cells?

    PubMed

    Browne, E S; Bhalla, V K

    1991-01-01

    The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.

  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. 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

  2. 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.

  3. Effects of the Yangjing Capsule Extract on Steroidogenesis and Apoptosis in Mouse Leydig Cells

    PubMed Central

    Sun, Dalin; Cui, Yugui; Jin, Baofang; Zhang, Xindong; Yang, Xiaoyu; Gao, Chao

    2012-01-01

    Objectives. This study aimed to explore the effect and mechanism of Yangjing capsule on testosterone secretion in mouse Leydig tumor cells (MLTC-1). Methods. MLTC-1 cells were treated with the Yangjing capsule extract for 24 h. The testosterone level in medium was measured by radioimmunoassay. The expression of steroidogenic enzymes (StAR, CYP11A1, and HSD3B) in the cells was examined using real-time RT-PCR and immunoblotting. Additionally, MLTC-1 cells were treated for 48 h in a serum-free medium. The cell viability was measured by MTT assay. The cell cycle and apoptosis were analyzed using flow cytometry. The expression of activated caspase-3 was analyzed using RT-PCR and a colorimetric protease assay. Results. The Yangjing capsule extract increased testosterone production and the expression of StAR, CYP11A1, and HSD3B mRNAs and proteins compared with the control. H89 significantly inhibited these effects. The medicine improved the viability of MLTC-1 cells, decreased the number of cells in G0/G1 phase, and increased the number of cells in S-phase, as well as prevented cell apoptosis by inhibiting caspase-3. Conclusion. The Yangjing capsule can stimulate MLTC-1 cells to secrete testosterone and may be an alternative treatment for diseases characterized by insufficient testosterone production. PMID:23259004

  4. 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

  5. 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.

  6. New insights into melatonin/CRH signaling in hamster Leydig cells.

    PubMed

    Rossi, Soledad P; Matzkin, María E; Terradas, Claudio; Ponzio, Roberto; Puigdomenech, Elisa; Levalle, Oscar; Calandra, Ricardo S; Frungieri, Mónica B

    2012-08-01

    We have previously described that melatonin inhibits androgen production in hamster testes via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. This study attempted to determine the initial events of the melatonin/CRH signaling pathway. In Leydig cells from reproductively active Syrian hamsters, Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a colorimetric assay demonstrated that melatonin and CRH activate tyrosine phosphatases and subsequently reduce the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk), down-regulate the expression of c-jun, c-fos and steroidogenic acute regulatory (StAR), and inhibit the production of testosterone. These effects were prevented by a highly selective CRH antagonist, thus indicating that melatonin does not exert a direct role. Specific mitogen-activated protein kinase kinase (MEK) and jnk blockers inhibited expression of c-jun, c-fos, StAR and the production of testosterone, confirming that these are events triggered downstream of erk and jnk. In Leydig cells from photoperiodically regressed adult hamsters, CRH inhibited the production of androstane-3α,17β-diol (3α-diol), the main androgen produced, through the same signaling pathway. Testicular melatonin concentration was 3-4-fold higher in reproductively inactive hamsters than that detected in active animals. Since melatonin, CRH, and their receptors are present not only in hamster testes but also in testicular biopsies of infertile men, we can conjecture about the relevance of this previously uncharacterized pathway in human fertility disorders. In summary, our study identifies crucial intracellular events triggered by melatonin/CRH in the testis that lead to a down-regulation of the steroidogenic process. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Ovarian Leydig cell tumor in a peri-menopausal woman with severe hyperandrogenism and virilization.

    PubMed

    Nardo, L G; Ray, D W; Laing, I; Williams, C; McVey, R J; Seif, M W

    2005-10-01

    The authors report a case of Leydig cell tumor in a 46-year-old woman who first presented with severe clinical hyperandrogenism and associated complex medical history. Investigations revealed markedly raised serum concentrations of testosterone (28.3 nmol/l) and free androgen index (54.4), whereas sex hormone binding globulin, random cortisol, androstenedione, 17-hydroxyprogesterone and dehydroepiandrosterone sulphate concentrations were all within the normal range. Transabdominal ultrasound and computed tomography scan of the pelvis and abdomen showed a slightly bulky right ovary, but no other abnormalities. An ovarian source of androgens was suspected and surgery was arranged. Following a three-year history of defaulting appointments due to agoraphobia, she underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy and intraoperative selective ovarian venous sampling. Histopathological examination revealed a 2 cm Leydig cell tumor within the right ovary. Successful intraoperative ovarian venous sampling demonstrated significantly elevated testosterone levels (>260 nmol/l) from the right ovarian vein. Hyperandrogenaemia normalized post-operatively. The patient showed significant regression of clinical signs and symptoms, including the anxiety disorder. Clinical presentation, biochemistry and imaging modalities should allow to detect androgen-secreting ovarian tumors, while selective venous sampling should be reserved for patients whom uncertainty remains. The present case confirms that androgen-secreting ovarian tumors represent a diagnostic and therapeutic challenge. They have to be considered in the differential diagnosis of severe hyperandrogenism even in peri-menopausal women. Although selective venous sampling is of diagnostic value, however, its impact on future management should be considered on individual basis.

  8. Expression of somatostatin receptor type-2 (sst2A) in immature porcine Leydig cells and a possible role in the local control of testosterone secretion

    PubMed Central

    Fombonne, Joanna; Csaba, Zsolt; von Boxberg, Ysander; Valayer, Amandine; Rey, Catherine; Benahmed, Mohamed; Dournaud, Pascal; Krantic, Slavica

    2003-01-01

    We recently reported that immature porcine Leydig cells express both somatostatin (SRIF) and SRIF receptor type-2 (sst-2) transcripts. The present study was therefore undertaken to assess whether SRIF might exert autocrine actions on these cells through sst2A receptor, one of the two sst2 isoforms known to exert important neuroendocrine and endocrine functions. Using a polyclonal antibody directed towards the C-terminal tail of the sst2A receptor subtype, receptor immunoreactivity was detected in a subpopulation of Leydig cells and spermatogonia. To address the physiological correlates of this expression we then studied the possible involvement of sst2 receptor in the regulation of testosterone secretion. Functional assays showed that the sst2 agonist octreotide inhibited both basal and hCG-stimulated testosterone secretion by testosterone pretreated Leydig cells. To assess whether sst2 receptor expression might be regulated by testosterone, we performed a semi-quantitative RT-PCR analysis of sst2 mRNA expression in Leydig cells cultured in the presence or in the absence of the androgen. A significant increase in sst2 receptor transcripts was observed in testosterone-treated cells. Taken together, these data suggest that SRIF can inhibit testosterone secretion through the sst2A receptor. The mechanism of the local inhibitory actions of SRIF is probably autocrine since immature porcine Leydig cells express SRIF itself and it might involve testosterone-induced increase of sst2 receptor expression in immature Leydig cells. PMID:12646058

  9. 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.

  10. Basic fibroblast growth factor promotes stem Leydig cell development and inhibits LH-stimulated androgen production by regulating microRNA expression.

    PubMed

    Liu, Hui; Yang, Yan; Zhang, Lei; Liang, Rui; Ge, Ren-Shan; Zhang, Yufei; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2014-10-01

    Leydig cells are the primary source of testosterone in the testes, and their steroidogenic function is strictly controlled by the hypothalamus-pituitary-gonad axis. Emerging evidence has indicated that fibroblast growth factors play a role in regulating stem Leydig cell development and steroidogenesis, but little is known about the regulatory mechanism. Using a seminiferous tubule culture system, we demonstrated that basic fibroblast growth factor (bFGF) can promote stem Leydig cell proliferation and commitment toward differentiation in testosterone-producing Leydig cells. However, these promoting effects decreased with an increase in the bFGF dose. Previous studies have reported that bFGF inhibits luteinizing hormone (LH)-stimulated androgen production by downregulating the mRNA expression of steroidogenic genes in immature Leydig cells. However, the expression levels of 677 microRNAs did not change significantly during the LH-mediated process of testosterone synthesis. Five microRNAs (miR-29a, -29c, -142-3p, -451 and -335) were identified, and their expression in immature Leydig cells was regulated simultaneously by bFGF and LH. These results suggested that the inhibition of LH-stimulated androgen production may be modulated by a change in bFGF-mediated microRNA expression, which further impacts the signaling pathway of testosterone biosynthesis and steroidogenic gene expression.

  11. The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells.

    PubMed

    Chen, Yen-Wen; Lo, Hui-Chen; Yang, Jyuer-Ger; Chien, Chi-Hsien; Lee, Shi-Hsiung; Tseng, Chi-Yu; Huang, Bu-Miin

    2006-07-04

    Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.

  12. An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

    PubMed Central

    Wang, Yang; Newton, Derek C.; Miller, Tricia L.; Teichert, Anouk-Martine; Phillips, M. James; Davidoff, Michail S.; Marsden, Philip A.

    2002-01-01

    Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of β-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. β-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P450scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, β-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of β-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, β-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of

  13. Transcriptional regulation of translocator protein (Tspo) via a SINE B2-mediated natural antisense transcript in MA-10 Leydig cells.

    PubMed

    Fan, Jinjiang; Papadopoulos, Vassilios

    2012-05-01

    Translocator protein (18 kDa; TSPO) is a mitochondrial cholesterol- and drug-binding protein involved in cholesterol import into mitochondria, the rate-limiting step in steroidogenesis. TSPO is expressed at high levels in Leydig cells of the testis, and its expression levels dictate the ability of the cells to form androgen. In search of mechanisms that regulate Tspo expression, a number of transcription factors acting on its promoter region have been identified. We report herein the presence of a mechanism of regulation of Tspo expression via complementation with a natural antisense transcript (NAT). At the Tspo locus, a short interspersed repetitive element (SINE) of the SINE B2 family has the potential for high transcriptional activity. The extension of the SINE B2 element-mediated transcript overlapped with exon 3 of the Tspo gene and formed a NAT specific for Tspo (Tspo-NAT) in MA-10 mouse tumor Leydig cells. The identified Tspo-NAT was also found in testis and kidney tissues. Overexpression of the Tspo-NAT regulated Tspo gene expression and its function in steroid formation in MA-10 cells. Time-course studies have indicated that Tspo-NAT expression is regulated by cAMP and could regulate TSPO levels to maintain optimal steroid production by MA-10 Leydig cells. Taken together, these results suggest a new micro-transcriptional mechanism that regulates Tspo expression and thus steroidogenesis via an intron-based SINE B2-driven NAT specific for the Tspo gene.

  14. Androgen Action via Testicular Arteriole Smooth Muscle Cells Is Important for Leydig Cell Function, Vasomotion and Testicular Fluid Dynamics

    PubMed Central

    Welsh, Michelle; Sharpe, Richard M.; Moffat, Lindsey; Atanassova, Nina; Saunders, Philippa T. K.; Kilter, Sigrid; Bergh, Anders; Smith, Lee B.

    2010-01-01

    Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis. PMID:21049031

  15. Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

    PubMed

    Shi, Lei; Song, Ruigao; Yao, Xiaolei; Ren, Youshe

    2017-04-15

    The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 μmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 μmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 μmol/L group. Appropriate Se level (2.0 μmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3β-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3β-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 μmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the

  16. Testosterone induction of prostaglandin-endoperoxide synthase 2 expression and prostaglandin F(2alpha) production in hamster Leydig cells.

    PubMed

    Matzkin, María E; Gonzalez-Calvar, Silvia I; Mayerhofer, Artur; Calandra, Ricardo S; Frungieri, Mónica B

    2009-07-01

    We have previously observed expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the key enzyme in the biosynthesis of prostaglandins (PGs), in reproductively active Syrian hamster Leydig cells, and reported an inhibitory role of PGF(2alpha) on hamster testicular steroidogenesis. In this study, we further investigated PTGS2 expression in hamster Leydig cells during sexual development and photoperiodic gonadal regression. Since PTGS2 is mostly expressed in pubertal and reproductively active adult hamsters with high circulating levels of LH and androgens, we studied the role of these hormones in the regulation/maintenance of testicular PTGS2/PGF(2alpha). In active hamster Leydig cells, LH/hCG and testosterone induced PTGS2 and PGF(2alpha) production, and their actions were abolished by the antiandrogen bicalutamide (Bi). These results indicate that LH does not exert a direct effect on PG synthesis. Testosterone also stimulated phosphorylation of the mitogen-activated protein kinase isoforms 3/1 (MAPK3/1) within minutes and hours, but the testosterone metabolite dihydrotestosterone had no effect on PTGS2 and MAPK3/1. Because Bi and U0126, an inhibitor of the MAP kinase kinases 1 and 2 (MAP2K1/2), abolished testosterone actions on MAPK3/1 and PTGS2, our studies suggest that testosterone directly induces PTGS2/PGF(2alpha) in hamster Leydig cells via androgen receptors and a non-classical mechanism that involves MAPK3/1 activation. Since PGF(2alpha) inhibits testosterone production, it might imply the existence of a regulatory loop that is setting a brake on steroidogenesis. Thus, the androgen environment might be crucial for the regulation of testicular PG production at least during sexual development and photoperiodic variations in hamsters.

  17. SF-1 deficiency causes lipid accumulation in Leydig cells via suppression of STAR and CYP11A1.

    PubMed

    Hatano, Megumi; Migita, Toshiro; Ohishi, Tomokazu; Shima, Yuichi; Ogawa, Yoshihiro; Morohashi, Ken-Ichirou; Hasegawa, Yukihiro; Shibasaki, Futoshi

    2016-11-01

    Genetic mutations of steroidogenic factor 1 (also known as Ad4BP or Nr5a1) have increasingly been reported in patients with 46,XY disorders of sex development (46,XY disorders of sex development). However, because the phenotype of 46,XY disorders of sex development with a steroidogenic factor 1 mutation is wide-ranging, its precise diagnosis remains a clinical problem. We previously reported the frequent occurrence of lipid accumulation in Leydig cells among patients with 46,XY disorders of sex development with a steroidogenic factor 1 mutation, an observation also reported by other authors. To address the mechanism of lipid accumulation in this disease, we examined the effects of steroidogenic factor 1 deficiency on downstream targets of steroidogenic factor 1 in in vitro and in vivo. We found that lipid accumulation in Leydig cells was enhanced after puberty in heterozygous steroidogenic factor 1 knockout mice compared with wild-type mice, and was accompanied by a significant decrease in steroidogenic acute regulatory protein and CYP11A1 expression. In mouse Leydig cell lines, steroidogenic factor 1 knockdown induced a remarkable accumulation of neutral lipids and cholesterol with reduced androgen levels. Steroidogenic factor 1 knockdown reduced the expression of steroidogenic acute regulatory protein and CYP11A1, both of which are transcriptional targets of steroidogenic factor 1 and key molecules for steroidogenesis from cholesterol in the mitochondria. Knockdown of either steroidogenic acute regulatory protein or CYP11A1 also induced lipid accumulation, and knockdown of both had an additive effect. Our data suggested that lipid accumulation in the Leydig cells of the 46,XY disorders of sex development phenotype with a steroidogenic factor 1 mutation is due, at least in part, to the suppression of steroidogenic acute regulatory protein and CYP11A1, and a resulting increase in unmetabolized cholesterol.

  18. 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

  19. Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells.

    PubMed

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

    2013-05-31

    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.

  20. 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 (10 μM) 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

  1. Sertoli-Leydig cell tumor in a 12-year-old girl: a review article and case report.

    PubMed

    Cabrera-Cantú, Fernanda; Urrutia-Osorio, Marta; Valdez-Arellano, Fernando; Rivadeneyra-Espinoza, Liliana; Papaqui, Alejandro; Soto-Vega, Elena

    2014-10-01

    The Sertoli and Leydig cell tumor is an unusual neoplasm that belongs to the sex cord-stromal tumors. Generally these tumors are associated with good prognosis. These tumors usually present virilizing symptoms such as oligomenorrhea or amenorrhea, hirsutism, voice raucity, laryngeal protuberance and clitoromegaly. A 12 year old girl referred acute abdominal pain with no other clinical manifestations. An abdominal ultrasound showed a semisolid mass suggestive of ovarian tumor. The diagnosis was confirmed by a computed tomography. A unilateral salpingo oophorectomy was performed and the pathologist reported a Sertoli-Leydig tumor with intermediate differentiation. The outcome was excellent. These tumors represent a rare condition in children. However, they can occur at any age, therefore it is important to acknowledge the clinical manifestations, diagnostic approach and therapeutic options. In this case the patient presented unusual symptoms which makes it more interesting.

  2. Steroidogenesis in MA-10 mouse Leydig cells is altered via fatty acid import into the mitochondria.

    PubMed

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

    2014-10-01

    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.

  3. Activation of the Hedgehog Pathway in the Mouse Fetal Ovary Leads to Ectopic Appearance of Fetal Leydig Cells and Female Pseudohermaphroditism

    PubMed Central

    Barsoum, Ivraym B.; Bingham, Nathan C.; Parker, Keith L.; Jorgensen, Joan S.; Yao, Humphrey H-C

    2009-01-01

    Proper cell fate determination in mammalian gonads is critical for the establishment of sexual identity. The Hedgehog (Hh) pathway has been implicated in cell fate decision for various organs, including gonads. Desert Hedgehog (Dhh), one of the three mammalian Hh genes, has been implicated with other genes in the establishment of mouse fetal Leydig cells. To investigate whether Hh alone is sufficient to induce fetal Leydig cell differentiation, we ectopically activated the Hh pathway in Steroidogenic factor 1 (SF1)-positive somatic cell precursors of fetal ovaries. Hh activation transformed SF1-positive somatic ovarian cells into functional fetal Leydig cells. These ectopic fetal Leydig cells produced androgens and insulin-like growth factor 3 (INLS3) that cause virilization of female embryos and ovarian descent. However, the female reproductive system remained intact, indicating a typical example of female pseudohermaphroditism. The appearance of fetal Leydig cells was a direct consequence of Hh activation as evident by the absence of other testicular components in the affected ovary. This study provides not only insights into mechanisms of cell lineage specification in gonads, but also a model to understand defects in sexual differentiation. PMID:19268447

  4. [Gynaecomastia and male infertility as symptoms of a nonpalpable Leydig cell tumour].

    PubMed

    van de Riet, J E; Oelke, M; van der Veen, F; Visser, H

    2006-08-19

    A 35-year-old man and his partner were referred for intracytoplasmic sperm injection treatment (ICSI) because of secondary infertility due to severe oligoasthenoteratospermia. Three years earlier he had presented elsewhere with left unilateral gynaecomastia. A hypertrophic mammary gland had been excised one year later. Histopathological investigation showed benign hypertrophy. One year later he developed gynaecomastia on the other side. Physical examination and incomplete hormonal screening showed no abnormalities. The couple were referred to our tertiary clinic for ICSI treatment. The patient still had unilateral gynaecomastia. Hormonal screening showed not only severe oligoasthenoteratospermia, but also an elevated serum oestrogen level. Scrotal ultrasound revealed a 17 mm mass in his right testicle. Subsequently unilateral orchidectomy was performed. Histology showed a benign Leydig cell tumour for which no further therapy was required. Four months after surgery the gynaecomastia diminished, oestrogen levels became normal and improvement in semen parameters followed. Patients with severe male infertility or gynaecomastia are at a higher risk of developing a testicular neoplasm. Besides history taking, physical examination of breasts and testicles, hormonal screening and scrotal sonography should be performed as some testicular neoplasms are not apparent on palpation.

  5. Effect of Vitamin D on basal and Luteinizing Hormone (LH) induced testosterone production and mitochondrial dehydrogenase activity in cultured Leydig cells from immature and mature rams.

    PubMed

    Huang, Yang; Jin, Hui; Chen, Jianwei; Jiang, Xiaolong; Li, Pengfei; Ren, Youshe; Liu, Wenzhong; Yao, Jianbo; Folger, Joseph K; Smith, George W; Lv, Lihua

    2015-07-01

    The objectives of this study were to investigate the potential effects of 1α,25-(OH)2VD3 (biologically active form of Vitamin D) on basal and LH-induced testosterone production and mitochondrial dehydrogenase activity in Leydig cells from immature and mature rams cultured in vitro. Leydig cells were isolated from testes of immature and mature rams, treated without (control) or with increasing concentrations of LH (1, 10, 100ng/ml) and/or 1α,25-(OH)2VD3 (1, 10, 100nM). After 24h, concentrations of testosterone in culture media were measured. After 96h, mitochondrial dehydrogenase activity in Leydig cells were measured. In immature and mature ram Leydig cells, treatment with 10 and 100ng/ml LH increased testosterone production and mitochondrial dehydrogenase activity. Treatment with 1α,25-(OH)2VD3 in the absence of LH did not increase testosterone production, but 10 and 100nM 1α,25-(OH)2VD3 increased LH induced testosterone production for both immature and mature ram Leydig cells. Treatment with all doses of 1α,25-(OH)2VD3 in the absence of LH and 10 and 100ng/ml LH in the absence of 1α,25-(OH)2VD3 increased mitochondrial dehydrogenase activity for cultured Leydig cells from immature and mature rams and 1 and 10nM 1α,25-(OH)2VD3 treatment enhanced the LH induced increase in mitochondrial dehydrogenase activity. Result demonstrate Vitamin D3 induced regulation of function of Leydig cells from immature and mature rams cultured in the presence or absence of LH and support a potential role for Vitamin D3 in regulation of gonadal function in rams.

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

    PubMed

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

    2016-01-01

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

  7. Morphological and functional maturation of Leydig cells: from rodent models to primates.

    PubMed

    Teerds, Katja J; Huhtaniemi, Ilpo T

    2015-01-01

    Leydig cells (LC) are the sites of testicular androgen production. Development of LC occurs in the testes of most mammalian species as two distinct growth phases, i.e. as fetal and pubertal/adult populations. In primates there are indications of a third neonatal growth phase. LC androgen production begins in embryonic life and is crucial for the intrauterine masculinization of the male fetal genital tract and brain, and continues until birth after which it rapidly declines. A short post-natal phase of LC activity in primates (including human) termed 'mini-puberty' precedes the period of juvenile quiescence. The adult population of LC evolves, depending on species, in mid- to late-prepuberty upon reawakening of the hypothalamic-pituitary-testicular axis, and these cells are responsible for testicular androgen production in adult life, which continues with a slight gradual decline until senescence. This review is an updated comparative analysis of the functional and morphological maturation of LC in model species with special reference to rodents and primates. Pubmed, Scopus, Web of Science and Google Scholar databases were searched between December 2012 and October 2014. Studies published in languages other than English or German were excluded, as were data in abstract form only. Studies available on primates were primarily examined and compared with available data from specific animal models with emphasis on rodents. Expression of different marker genes in rodents provides evidence that at least two distinct progenitor lineages give rise to the fetal LC (FLC) population, one arising from the coelomic epithelium and the other from specialized vascular-associated cells along the gonad-mesonephros border. There is general agreement that the formation and functioning of the FLC population in rodents is gonadotrophin-responsive but not gonadotrophin-dependent. In contrast, although there is in primates some controversy on the role of gonadotrophins in the formation of

  8. 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

  9. 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.

  10. Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

    PubMed

    Wen, Qing; Zheng, Qiao-Song; Li, Xi-Xia; Hu, Zhao-Yuan; Gao, Fei; Cheng, C Yan; Liu, Yi-Xun

    2014-12-15

    Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development. Copyright © 2014 the American Physiological Society.

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

    PubMed

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

    2016-01-01

    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. 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. 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. 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.

  12. 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

  13. Immunocytochemical demonstration of androgen receptors in Leydig cells of the bank vole (Clethrionomys glareolus, Schreber): an in vitro study.

    PubMed

    Bilińska, B; Słomczyńska, M; Kmicikiewicz, I

    1996-04-01

    Androgen receptors of the bank vole Leydig cells in vitro were immunostained using a polyclonal antibody against androgen receptors followed by streptavidine-peroxidase complex or rhodamine-labelled goat anti-rabbit IgG visualization. The immunocytochemical studies revealed localization of androgen receptors in the whole cytoplasm or in the perinuclear area of the cells. Addition of dehydroepiandrosterone into the culture medium resulted in nuclear localization of the androgen receptors. Long (18L : 6D) and short (6L : 18D) photoperiods as well as the age of animals were taken into account. The concentration of androgen receptors was changed dependent on age and status of reproduction.

  14. 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.

  15. Sertoli Cell Wt1 Regulates Peritubular Myoid Cell and Fetal Leydig Cell Differentiation during Fetal Testis Development

    PubMed Central

    Wen, Qing; Wang, Yuqian; Tang, Jixin; Cheng, C. Yan; Liu, Yi-Xun

    2016-01-01

    Sertoli cells play a significant role in regulating fetal testis compartmentalization to generate testis cords and interstitium during development. The Sertoli cell Wilms’ tumor 1 (Wt1) gene, which encodes ~24 zinc finger-containing transcription factors, is known to play a crucial role in fetal testis cord assembly and maintenance. However, whether Wt1 regulates fetal testis compartmentalization by modulating the development of peritubular myoid cells (PMCs) and/or fetal Leydig cells (FLCs) remains unknown. Using a Wt1-/flox; Amh-Cre mouse model by deleting Wt1 in Sertoli cells (Wt1SC-cKO) at embryonic day 14.5 (E14.5), Wt1 was found to regulate PMC and FLC development. Wt1 deletion in fetal testis Sertoli cells caused aberrant differentiation and proliferation of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes α-Sma, Myh11 and Des, and interstitial progenitor cell marker gene Vcam1 were down-regulated, whereas FLC marker genes StAR, Cyp11a1, Cyp17a1 and Hsd3b1 were up-regulated, in neonatal Wt1SC-cKO testes. The ratio of PMC:FLC were also reduced in Wt1SC-cKO testes, concomitant with a down-regulation of Notch signaling molecules Jag 1, Notch 2, Notch 3, and Hes1 in neonatal Wt1SC-cKO testes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1 regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization. PMID:28036337

  16. Sertoli Cell Wt1 Regulates Peritubular Myoid Cell and Fetal Leydig Cell Differentiation during Fetal Testis Development.

    PubMed

    Wen, Qing; Wang, Yuqian; Tang, Jixin; Cheng, C Yan; Liu, Yi-Xun

    2016-01-01

    Sertoli cells play a significant role in regulating fetal testis compartmentalization to generate testis cords and interstitium during development. The Sertoli cell Wilms' tumor 1 (Wt1) gene, which encodes ~24 zinc finger-containing transcription factors, is known to play a crucial role in fetal testis cord assembly and maintenance. However, whether Wt1 regulates fetal testis compartmentalization by modulating the development of peritubular myoid cells (PMCs) and/or fetal Leydig cells (FLCs) remains unknown. Using a Wt1-/flox; Amh-Cre mouse model by deleting Wt1 in Sertoli cells (Wt1SC-cKO) at embryonic day 14.5 (E14.5), Wt1 was found to regulate PMC and FLC development. Wt1 deletion in fetal testis Sertoli cells caused aberrant differentiation and proliferation of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes α-Sma, Myh11 and Des, and interstitial progenitor cell marker gene Vcam1 were down-regulated, whereas FLC marker genes StAR, Cyp11a1, Cyp17a1 and Hsd3b1 were up-regulated, in neonatal Wt1SC-cKO testes. The ratio of PMC:FLC were also reduced in Wt1SC-cKO testes, concomitant with a down-regulation of Notch signaling molecules Jag 1, Notch 2, Notch 3, and Hes1 in neonatal Wt1SC-cKO testes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1 regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization.

  17. Asynchronic steroid activity of Leydig and Sertoli cells related to spermatogenic and testosterone cycle in Phymaturus antofagastensis.

    PubMed

    Boretto, J M; Ibargüengoytía, N R; Jahn, G A; Acosta, J C; Vincenti, A E; Fornés, M W

    2010-05-01

    The severe environments where Phymaturus lizards inhabit in the Andes highlands and in Patagonia, Argentina, impose restrictions on their reproduction, offering a framework for the development of life history strategies to overcome hard weather conditions. Among them, prolonged female cycles, asynchrony between sexes in receptivity, and sperm storage in males, were described. Asynchrony in the reproductive timing between males and females is a consequence of different energy requirements for gametogenesis, and often imply the existence of cellular mechanisms to enhance fertilization, such as the asynchronic steroid synthesis between testicular compartments, allowing gametogenesis independently of mating. In the present study ultrastructural and hormone assays were combined for the first time in liolaemids. Specifically, morphological features of steroid activity in Leydig and Sertoli cells, and serum testosterone concentrations have been studied in the lizard Phymaturus antofagastensis. Leydig and Sertoli cells presented morphological features characteristic of steroid synthesis during the spermatogenesis, and evident asynchronic steroid production between testicular compartments. Active Sertoli cells and inactive Leydig cells were observed in spring and autumn, while in mid-summer their steroid activity was synchronic in coincidence with maximal abundance of spermatozoa in epididymis. Serum testosterone concentration was at its maximum in mid-summer (126-230 ng ml(-1)), and minimum in late spring (4-24 ng ml(-1)) and early autumn (2-17 ng ml(-1)). In view of these results, P. antofagastensis males show an original approach to adjust their reproductive activity to physiological and environmental constraints at high latitudes and altitudes in the Andean highlands of Argentina.

  18. Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

    PubMed Central

    Fumel, Betty; Froment, Pascal; Holzenberger, Martin; Livera, Gabriel; Monget, Philippe; Fouchécourt, Sophie

    2015-01-01

    Background In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial. Methods The TRαAMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter. Findings We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRαAMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRαAMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRαAMI-ARO males showed normal fertility. This phenotype is similar to TRαAMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRαAMI-ARO. Conclusions/Significance We concluded that the presence of a mutant TRαAMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis. PMID:25793522

  19. The Intraovarian Actions of Estrogen Receptor-α (ERα) are Necessary to Repress the Formation of Morphological and Functional Leydig-like Cells in the Female Gonad

    PubMed Central

    COUSE, JOHN F.; YATES, MARIANA M.; RODRIGUEZ, KARINA F.; JOHNSON, JO ANNE; POIRIER, DONALD; KORACH, KENNETH S.

    2006-01-01

    The predisposition of the testis and ovary to primarily synthesize testosterone and estradiol, respectively, is due to gonadal-specific cell types that differentially express the various hydroxysteroid (17β) dehydrogenase (HSD17B) isoforms. In testes, Leydig cells rely on LH stimulation to maintain expression of the type 3 (HSD17B3) isoform, which specifically converts androstenedione to testosterone. In ovaries, thecal-interstitial cells also rely on LH to induce androgen synthesis but lack HSD17B3 and therefore secrete androgens of low biological activity. Therefore, thecal cells may possess a mechanism to repress the Leydig cell phenotype and HSD17B3 expression. Estradiol is known to inhibit experimentally Leydig cell function and proliferation. In the current study, we provide evidence that estradiol prevents the development of functional Leydig-like cells in the murine ovary; and that this action is mediated by estrogen receptor-α (ERα). ERα-null (αERKO) female mice exhibit testis-like levels of Hsd17b3 expression in the ovaries and male-like levels of plasma testosterone. Herein, we demonstrate that a) Hsd17b3 expression in αERKO ovaries is a primary effect of the loss of intraovarian ERα actions, b) αERKO ovarian cells produce substantial levels of testosterone in vitro and this is blocked by a HSD17B3 specific inhibitor, c) Hsd17b3 expression in αERKO ovaries is LH regulated and localized to the secondary/thecal interstitial cells, and d) αERKO secondary/thecal interstitial cells possess Leydig-like ultrastructural features. These data indicate that intraovarian ERα actions are required to repress Hsd17b3 expression in the ovary and may be important to maintaining a female phenotype in secondary/thecal interstitial cells. PMID:16627580

  20. Identifying Molecular Targets for Chemoprevention in a Rat Model

    DTIC Science & Technology

    2007-06-01

    at the 65-week time point. This example shows a relatively large patch of Leydig cells ( Ley ) adjacent to two seminiferous tubules (Tub). PhIP Rat...9, 2006 [16] Weisburger JH, Rivenson A, Reinhardt J, Braley J, Pittman B, and Zang E (2002). On the occurrence of Leydig cell tumors in the F344 rat

  1. Increased steroid hormone secretion in mouse Leydig tumor cells after induction of cholesterol translocation by sphingomyelin degradation.

    PubMed

    Pörn, M I; Tenhunen, J; Slotte, J P

    1991-06-07

    The effects of sphingomyelin degradation on [3H]cholesterol transfer from the cell surface to mitochondria were examined in mouse Leydig tumor cells. These cells were used since they utilize cholesterol for steroid hormone synthesis in the mitochondria, and also possess acyl-CoA: cholesterol acyl transferase (ACAT) activity in the endoplasmic reticulum. Exposure of glutaraldehyde-fixed mouse Leydig tumor cells to sphingomyelinase (50 mU/ml, 60 min) resulted in the degradation of about 50% of cell sphingomyelin, suggesting that only half of the sphingomyelin mass in these cells was located in the exoleaflet of the plasma membrane. The partial sphingomyelin degradation resulted in the translocation of cellular unesterified [3H]cholesterol from plasma membranes (cholesterol oxidase-susceptible) to intracellular compartments (oxidase-resistant). The fraction of [3H]cholesterol that was translocated, i.e., between 20 and 50%, varied with different [3H]cholesterol-labeling methods. Cholesterol translocation induced by sphingomyelin degradation subsequently led to the stimulation of ACAT activity, suggesting that a fraction of cell surface cholesterol was transported to the endoplasmic reticulum. The sphingomyelinase-induced [3H]cholesterol flow from the cell surface to the cell interior was also in part directed to the mitochondria, as evidenced by the increased secretion of [3H]steroid hormones. In addition, the cyclic AMP-induced activation of steroidogenesis was further enhanced by the sphingomyelinase-induced cholesterol translocation. Based on the current results, it seems evident that a significant portion of the translocated [3H]cholesterol made its way from plasma membranes into the mitochondria for steroidogenesis.

  2. Leydig cell hypoplasia: absent luteinizing hormone receptor cell surface expression caused by a novel homozygous mutation in the extracellular domain.

    PubMed

    Richter-Unruh, A; Verhoef-Post, M; Malak, S; Homoki, J; Hauffa, B P; Themmen, A P N

    2004-10-01

    Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied a family with a 46,XY girl due to a new homozygous mutation (V144F) in the extracellular ligand-binding domain. HEK 293 cells transfected with the mutant LH receptor exhibited a marked impairment of human chorionic gonadotropin binding. Using Western blotting of the expressed V144F mutant LH receptor protein showed the absence of the glycosylated cell surface form. Treatment of the mutant LH receptor with N-glycosidase F or endoglycosidase-H demonstrated that the mutant receptor is retained in the endoplasmic reticulum. Expression and study of enhanced green fluorescent protein-tagged receptors confirmed that the mutant LHR-V144F receptors do not migrate to the cell surface, and the fluorescence remains intracellular and colocalizes with an endoplasmic reticulum marker, ER-tracker Blue-white DPX. Comparison of the theoretical molecular models of the extracellular domain of the wild-type and the mutant receptor suggests that the mutation LHR-V144F, located in the outer circumference in a alpha-helix of the leucine-rich repeat 4, may induce a conformational strain on the molecule. F144 of the mutant LH receptor has overlapping interactions with F119, which V144 in the wild-type receptor has not.

  3. The nuclear receptor NR2F2 activates star expression and steroidogenesis in mouse MA-10 and MLTC-1 Leydig cells.

    PubMed

    Mendoza-Villarroel, Raifish E; Robert, Nicholas M; Martin, Luc J; Brousseau, Catherine; Tremblay, Jacques J

    2014-07-01

    Testosterone production is dependent on cholesterol transport within the mitochondrial matrix, an essential step mediated by a protein complex containing the steroidogenic acute regulatory (STAR) protein. In steroidogenic Leydig cells, Star expression is hormonally regulated and involves several transcription factors. NR2F2 (COUP-TFII) is an orphan nuclear receptor that plays critical roles in cell differentiation and lineage determination. Conditional NR2F2 knockout prior to puberty leads to male infertility due to insufficient testosterone production, suggesting that NR2F2 could positively regulate steroidogenesis and Star expression. In this study we found that NR2F2 is expressed in the nucleus of some peritubular myoid cells and in interstitial cells, mainly in steroidogenically active adult Leydig cells. In MA-10 and MLTC-1 Leydig cells, small interfering RNA (siRNA)-mediated NR2F2 knockdown reduces basal steroid production without affecting hormone responsiveness. Consistent with this, we found that STAR mRNA and protein levels were reduced in NR2F2-depleted MA-10 and MLTC-1 cells. Transient transfections of Leydig cells revealed that a -986 bp mouse Star promoter construct was activated 3-fold by NR2F2. Using 5' progressive deletion constructs, we mapped the NR2F2-responsive element between -131 and -95 bp. This proximal promoter region contains a previously uncharacterized direct repeat 1 (DR1)-like element to which NR2F2 is recruited and directly binds. Mutations in the DR1-like element that prevent NR2F2 binding severely blunted NR2F2-mediated Star promoter activation. These data identify an essential role for the nuclear receptor NR2F2 as a direct activator of Star gene expression in Leydig cells, and thus in the control of steroid hormone biosynthesis.

  4. Mamld1 Deficiency Significantly Reduces mRNA Expression Levels of Multiple Genes Expressed in Mouse Fetal Leydig Cells but Permits Normal Genital and Reproductive Development

    PubMed Central

    Miyado, Mami; Nakamura, Michiko; Miyado, Kenji; Morohashi, Ken-ichirou; Sano, Shinichiro; Nagata, Eiko; Fukami, Maki

    2012-01-01

    Although mastermind-like domain containing 1 (MAMLD1) (CXORF6) on human chromosome Xq28 has been shown to be a causative gene for 46,XY disorders of sex development with hypospadias, the biological function of MAMLD1/Mamld1 remains to be elucidated. In this study, we first showed gradual and steady increase of testicular Mamld1 mRNA expression levels in wild-type male mice from 12.5 to 18.5 d postcoitum. We then generated Mamld1 knockout (KO) male mice and revealed mildly but significantly reduced testicular mRNA levels (65–80%) of genes exclusively expressed in Leydig cells (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3) as well as grossly normal testicular mRNA levels of genes expressed in other cell types or in Leydig and other cell types. However, no demonstrable abnormality was identified for cytochrome P450 17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B) protein expression levels, appearance of external and internal genitalia, anogenital distance, testis weight, Leydig cell number, intratesticular testosterone and other steroid metabolite concentrations, histological findings, in situ hybridization findings for sonic hedgehog (the key molecule for genital tubercle development), and immunohistochemical findings for anti-Müllerian hormone (Sertoli cell marker), HSD3B (Leydig cell marker), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (germ cell marker) in the KO male mice. Fertility was also normal. These findings imply that Mamld1 deficiency significantly reduces mRNA expression levels of multiple genes expressed in mouse fetal Leydig cells but permits normal genital and reproductive development. The contrastive phenotypic findings between Mamld1 KO male mice and MAMLD1 mutation positive patients would primarily be ascribed to species difference in the fetal sex development. PMID:23087174

  5. Role of oxygen in the regulation of Leydig tumor derived MA-10 cell steroid production: the effect of cobalt chloride.

    PubMed

    Kumar, Anand; Rani, Lata; Dhole, Bodhana

    2014-04-01

    We have earlier shown that cobalt chloride (CoCl2)-induced hypoxia and second messenger 8-bromoadenosine 3', 5'-cyclic adenosine monophosphate (8-Br-cAMP) stimulates vascular endothelial growth factor (VEGF) production in Leydig tumor cell derived MA-10 cells. Both stimuli follow common signal transduction pathways including protein kinase A (PK-A), extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol-3 kinase/akt (PI3-K/Akt) pathways in the stimulation of VEGF by MA-10 cells. In the present study we investigated the role of CoCl2 and 8-Br-cAMP on steroid production in MA-10 cells. The MA-10 cells were cultured in Waymouth MB 752/1 medium, supplemented with 15% heat inactivated horse serum. Progesterone was estimated by radioimmunoassay (RIA).We report that 8-Br-cAMP stimulated progesterone production by the MA-10 cells whereas CoCl2 inhibited the same. Also, 8-Br-cAMP stimulated steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) mRNAs expression. However, CoCl2 had no effect on StAR mRNA. Cobalt chloride directly inhibited the expression of P450scc mRNA. The decrease in progesterone production could be attributed to three different mechanisms, (1) an increase in production of reactive oxygen species (ROS), (2) an increase in HIF-1α activity, and (3) ultimately a decrease in the level of cytochrome P450 side chain cleavage (CYT P450scc). Hypoxia has an action and mechanism of action similar to that of gonadotropins on VEGF production, whereas they have a contrasting effect on steroidogenesis. This study suggests that hypoxia could be as important as gonadotropins in regulating Leydig cell steroidogenesis.

  6. 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.

  7. 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

  8. 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

    2015-03-02

    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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Mitochondrial structure in steroid-producing cells: three-dimensional reconstruction of human Leydig cell mitochondria by electron microscopic tomography.

    PubMed

    Prince, Frederick P; Buttle, Karolyn F

    2004-05-01

    Mitochondria of human Leydig cells were reconstructed in three dimension utilizing the technique of electron microscopic tomography to obtain a better understanding of the topology of the internal membrane system and the relationship of these cristae to the inner boundary membrane (IBM). Cristae structure, in many respects, is consistent with previous tomographic studies from typical mitochondria, i.e., mitochondria from nonsteroid-producing cells. Cristae are diverse in form, with well-defined lamellar cristae interconnected to pleomorphic and tubular regions. Occasional fenestrations are present in the lamellar regions. Also consistent with other mitochondria studied by tomography, the openings of the cristae to the IBM (referred to as crista junctions) are roughly circular or elliptical and approximately 20-25 nm in diameter. Morphological contact sites between the outer mitochondrial membrane and IBM are also present. Cristae membranes in these steroid-producing mitochondria are often found in close proximity to the IBM. Unique to steroid-producing mitochondria is a form of the cristae in which multiple lamellae are in very close apposition, previously defined as the lamellar association. Tomographic reconstructions of the lamellar association reveal that these well-organized membranes also open to the IBM via crista junctions. These regions of closely apposed lamellar cristae are also interconnected and display small tubular extensions from the lamellae. The current study is the first electron microscopic tomography study of mitochondria from steroid-producing cells. The results show the cristae interconnect to form an extensive internal membrane system, which is perhaps better termed the cristae compartment. This internal membrane system is notable due to the high surface area with few small openings to the IBM. Such a morphology is more analogous to the thylakoid membrane system of chloroplasts than the long-standing view of mitochondrial cristae. The

  10. In vivo blockade of α1-adrenergic receptors mitigates stress-disturbed cAMP and cGMP signaling in Leydig cells.

    PubMed

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

    2014-01-01

    The molecular mechanism of stress-associated reproductive dysfunction is complex and largely unknown. This study was designed to systematically analyze molecular effects of systemic in vivo blockade of α1-adrenergic receptors (α1-ADRs) on stress-induced disturbance of cAMP/cGMP signaling in testosterone-producing Leydig cells using the following parameters (i) level of circulating stress hormones, LH and testosterone; (ii) level of main molecular markers of Leydig cell functionality (testosterone, Insl3, cAMP); (iii) expression of cAMP signaling (cAMP 'producers'/'effectors'/'removers') and (iv) expression of NO-cGMP signaling (NO-cGMP 'producers'/'effectors'/'removers'). The results showed that oral administration of α1-ADR blocker before stress increased cGMP and diminished stress-reduced cAMP production in Leydig cells. In the same cells, stress-induced effects on cAMP/cGMP signaling pathways elements were changed. Sustained in vivo α1-ADR blockade completely abolished stress-increased transcription of most abundantly expressed phosphodiesterase that remove cAMP (Pde4b) and potentiated stress-increased expression of PRKA, the main stimulator of Leydig cell steroidogenesis. In the same Leydig cells, stress-decreased NOS3 expression was abolished, while stress-increased GUCY1 (cGMP 'producer') and PRKG1 (cGMP 'effector') were potentiated. It is possible that all molecules mentioned could contribute, at least in part, in recovery of Leydig cell testosterone production. Presented data provide new role of α1-ADRs in stress-triggered disturbance of cAMP/cGMP signaling, and new molecular insights into the relationship between stress and mammalian reproduction. Regardless of whether the effects of α1-blocker + stress are direct or indirect, the results are important in terms of human reproductive health and the wide use of α1-ADR antagonists, alone or in combination, to treat post-traumatic stress disorders, hypertension, benign prostatic hyperplasia symptoms and

  11. Rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways.

    PubMed

    Sun, Jianhua; Wang, Heng; Liu, Bei; Shi, Wenhao; Shi, Juanzi; Zhang, Zhou; Xing, Junping

    2017-04-01

    Oxidative stress is a primary factor in the pathology of male infertility. The strong antioxidative capacity of rutin has been proven by numerous studies, but a protective role in the context of male reproduction remains to be elucidated. To explore the biological role of rutin in protecting male reproductive function and the potential underlying mechanism, H2O2-induced Leydig cells were used as a cell model of oxidation damage. Our findings showed that rutin at concentrations of 10, 20, and 40μmol/L remarkably increased cell survival rate of H2O2-induced Leydig cells to 70.1%, 86.8%, and 80.3% respectively. Next, rutin with concentrations of 10, 20, and 40μmol/L decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels but increased the levels of glutathione (GSH) and testosterone in H2O2-induced Leydig cells. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were remarkably increased by rutin treatment with concentrations of 20 and 40μmol/L, but glutathione peroxidase (GSH-Px) activity was notably decreased. Moreover, rutin with concentrations of 10, 20, and 40μmol/L increased Bcl-2 protein levels but decreased protein levels of Bax and caspase-3. Furthermore, 20μmol/L rutin significantly abrogated the decrease in levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT) induced by H2O2. Pretreatment with LY294002, a PI3K inhibitor, antagonized protective action of 20μmol/L rutin against H2O2-induced cell activities, intracellular oxidant, testosterone, antioxidant enzyme activities, and the apoptosis related protein expression. Taken together, these results suggest that rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways, providing a promising strategy to decrease oxidative stress associated with male infertility.

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

    PubMed Central

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

    2016-01-01

    SUMMARY 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

  13. PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice.

    PubMed

    Harada, Yukiko; Tanaka, Naoki; Ichikawa, Motoki; Kamijo, Yuji; Sugiyama, Eiko; Gonzalez, Frank J; Aoyama, Toshifumi

    2016-12-01

    It was reported that 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide and a possible endocrine disruptor, can disturb spermatogenesis, but the precise mechanism is not understood. Since 2,4-D is a weak peroxisome proliferator in hepatocytes and peroxisome proliferator-activated receptor α (PPARα) is also expressed in Leydig cells, this study aimed to investigate the link between PPARα and 2,4-D-mediated testicular dysfunction. 2,4-D (130 mg/kg/day) was administered to wild-type and Ppara-null mice for 2 weeks, and the alterations in testis and testosterone/cholesterol metabolism in Leydig cells were examined. Treatment with 2,4-D markedly decreased testicular testosterone in wild-type mice, leading to degeneration of spermatocytes and Sertoli cells. The 2,4-D decreased cholesterol levels in Leydig cells of wild-type mice through down-regulating the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 and reductase, involved in de novo cholesterogenesis. However, the mRNAs encoding the important proteins involved in testosterone synthesis were unchanged by 2,4-D except for CYP17A1, indicating that exhausted cholesterol levels in the cells is a main reason for reduced testicular testosterone. Additionally, pregnancy rate and the number of pups between 2,4-D-treated wild-type male mice and untreated female mice were significantly lower compared with those between untreated couples. These phenomena were not observed in 2,4-D-treated Ppara-null males. Collectively, these results suggest a critical role for PPARα in 2,4-D-induced testicular toxicity due to disruption of cholesterol/testosterone homeostasis in Leydig cells. This study yields novel insights into the possible mechanism of testicular dysfunction and male infertility caused by 2,4-D.

  14. cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Cornejo Maciel, Fabiana; Castilla, Rocío; Duarte, Alejandra; Maloberti, Paula; Paz, Cristina; Podestá, Ernesto J

    2006-11-01

    We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.

  15. Morphofunctional changes in Leydig cells throughout the continuous spermatogenesis of the freshwater teleost fish, Serrasalmus spilopleura (Characiformes, Characidae): an ultrastructural and enzyme study.

    PubMed

    Nóbrega, R H; Quagio-Grassiotto, I

    2007-08-01

    The freshwater fish Serrasalmus spilopleura (piranha) has a continuous type of reproduction; gametes are constantly produced and released during the reproductive cycle. The testes do not undergo seasonal morphological changes but exhibit two constant regions throughout the year: the medullar region (involved with spermatogenesis) and the cortical region (involved with spermiation and sperm storage). We have evaluated the ultrastructure of the Leydig cells and the activity of 3beta-HSD (an essential enzyme related to steroid hormone biosynthesis) and acid phosphatase (AcPase; lysosomal marker enzyme) in these two regions. The activity of 3beta-HSD is stronger in the medullar region, and the Leydig cells in this region have a variety of cytological features that reflect differences in hormone synthesis and/or that could be linked to steroidogenic cells under various degrees of hormonal activity. In the cortical region, 3beta-HSD activity is weak and the Leydig cells exhibit signs of degeneration, as confirmed by their ultrastructure and intense AcPase activity. These degenerative signs are indicative of cytoplasmic remodelling to degrade steroidogenic enzymes, such as 3beta-HSD, that could lead to senescence or even to autophagic cell degeneration. S. spilopleura thus constitutes an interesting model for increasing our understanding of steroidogenesis control in freshwater teleost fish.

  16. Transcriptional Regulation of Translocator Protein (Tspo) via a SINE B2-Mediated Natural Antisense Transcript in MA-10 Leydig Cells1

    PubMed Central

    Fan, Jinjiang; Papadopoulos, Vassilios

    2012-01-01

    ABSTRACT Translocator protein (18 kDa; TSPO) is a mitochondrial cholesterol- and drug-binding protein involved in cholesterol import into mitochondria, the rate-limiting step in steroidogenesis. TSPO is expressed at high levels in Leydig cells of the testis, and its expression levels dictate the ability of the cells to form androgen. In search of mechanisms that regulate Tspo expression, a number of transcription factors acting on its promoter region have been identified. We report herein the presence of a mechanism of regulation of Tspo expression via complementation with a natural antisense transcript (NAT). At the Tspo locus, a short interspersed repetitive element (SINE) of the SINE B2 family has the potential for high transcriptional activity. The extension of the SINE B2 element-mediated transcript overlapped with exon 3 of the Tspo gene and formed a NAT specific for Tspo (Tspo-NAT) in MA-10 mouse tumor Leydig cells. The identified Tspo-NAT was also found in testis and kidney tissues. Overexpression of the Tspo-NAT regulated Tspo gene expression and its function in steroid formation in MA-10 cells. Time-course studies have indicated that Tspo-NAT expression is regulated by cAMP and could regulate TSPO levels to maintain optimal steroid production by MA-10 Leydig cells. Taken together, these results suggest a new micro-transcriptional mechanism that regulates Tspo expression and thus steroidogenesis via an intron-based SINE B2-driven NAT specific for the Tspo gene. PMID:22378763

  17. Compensated reduction in Leydig cell function is associated with lower semen quality variables: a study of 8182 European young men.

    PubMed

    Jørgensen, N; Joensen, U N; Toppari, J; Punab, M; Erenpreiss, J; Zilaitiene, B; Paasch, U; Salzbrunn, A; Fernandez, M F; Virtanen, H E; Matulevicius, V; Olea, N; Jensen, T K; Petersen, J H; Skakkebæk, N E; Andersson, A-M

    2016-05-01

    Is the Leydig cell function of young European men associated with semen quality? Compensated reduction in Leydig cell function, defined as increased LH concentration combined with adequate testosterone production is associated with lower semen quality. Semen quality of young European men shows a heterogeneous pattern. Many have sperm counts below and in the lower WHO reference where there nevertheless is a significant risk of subfecundity. Little is known about differences in Leydig cell function between men with semen quality below and within the WHO reference range. A coordinated, cross-sectional population-based study of 8182 men undertaken in 1996-2010. Young men (median age 19.1 years) were investigated in centres in Denmark, Estonia, Finland, Germany Latvia, Lithuania, and Spain. The men originated from the general populations, all were young, almost all were unaware of their fecundity and each provided a semen and blood sample. Associations between semen parameters and serum levels of testosterone and luteinising hormone (LH), calculated free testosterone, and ratios between serum testosterone and LH were determined. Serum testosterone levels were not associated with sperm concentrations, total sperm counts, or percentage of motile or morphologically normal spermatozoa. There was an inverse association between the semen parameters and serum LH levels, and accordingly a positive association to testosterone/LH ratio and calculated-free-testosterone/LH ratio. The size of the study mitigates the intra-individual variability concern. The distinction between different sub-categories of sperm motility and sperm morphology is subjective despite training. However, inter-observer variation would tend towards non-differential misclassification and would decrease the likelihood of detecting associations between reproductive hormone levels and semen variables, suggesting that the presented associations might in reality be even stronger than shown. Although we adjusted

  18. 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.

  19. Reduced testosterone production in TM3 Leydig cells treated with Aspalathus linearis (Rooibos) or Camellia sinensis (tea).

    PubMed

    Opuwari, C S; Monsees, T K

    2015-02-01

    Flavonoids are major compounds of Aspalathus linearis and Camellia sinensis. They are classified as endocrine disruptors and some have been shown to inhibit testosterone production. TM3 Leydig cell cultures were treated with 250-5000 μg mL(-1) A. linearis (unfermented or fermented rooibos) or Camellia sinensis (green or black tea) for 24 h in the absence or presence of 6 mIU/200 μl human chorionic gonadotropin (hCG). Under nonstimulated conditions, all teas tend to decrease testosterone production (3.9-31.8%). However, under hCG-stimulation, a significant reduction in testosterone production was observed at all concentrations by both rooibos and tea (16.3-37.9%). MTT assay and phase contrast microscopy, revealed that at 250-1000 μg ml(-1) , both plants maintained the viability, proliferation and morphology of the cells, while 5000 μg ml(-1) was cytotoxic to the cells (P < 0.05). In conclusion, the results here demonstrate the anti-androgenic property of A. linearis and C. sinensis.

  20. Hormone-dependent expression of a steroidogenic acute regulatory protein natural antisense transcript in MA-10 mouse tumor Leydig cells.

    PubMed

    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.

  1. Induction of lutropin receptors by lutropin and cyclic AMP in cultured mouse tumour (MA10) Leydig cells.

    PubMed Central

    West, A P; Rose, M P; Cooke, B A

    1990-01-01

    The role of cyclic AMP in the regulation of lutropin (luteinizing hormone, LH) receptors has been investigated in cultured mouse tumour (MA10) Leydig cells. The LH receptors were quantified by measuring the binding of 125I-labelled human chorionic gonadotropin (hCG). LH (0.03 nM) in the presence of 1 mM-dibutyryl-cyclic AMP [(Bu)2cAMP] caused a 3-8-fold increase in subsequent 125I-hCG binding. (Bu)2cAMP (1 mM), cholera toxin (11.9 nM) and forskolin (1 microM) each caused a 2-4-fold increase in binding. In the presence of translation (cycloheximide) and transcription (actinomycin D) inhibitors, there was a loss of detectable binding sites. (Bu)2cAMP increased the rate of recovery of binding sites after trypsin treatment of MA10 cells, with a concomitant 2-fold increase in the level of binding sites. Under conditions where receptor levels were increased by 3-8-fold there was also a significant increase in pregnenolone production. It is concluded that LH and cyclic AMP have positive regulatory effects on LH receptors in MA10 cells by inducing the synthesis of new receptors. These induced receptors are functionally coupled to steroidogenesis. PMID:2169244

  2. 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

  3. Ovarian Sertoli-Leydig cell tumor: a report of seven cases and a review of the literature.

    PubMed

    Xiao, Huiting; Li, Bin; Zuo, Jing; Feng, Xiaoli; Li, Xiaoguang; Zhang, Rong; Wu, Lingying

    2013-03-01

    The aim of this study was to investigate the clinicopathologic features, treatment and outcome of seven patients with an ovarian Sertoli-Leydig cell tumor (SLCT). Five patients presented with feminization, two with accompanying virilization. One presented with amenorrhea alone. Three of the five patients showing feminization symptoms had endocrine-related diseases. Histologically, five tumors were well differentiated, the other two were poorly differentiated. The latter two patients were misdiagnosed as having an ovarian epithelial carcinoma or granulosa cell tumor from frozen sections. Immunohistochemistry showed that the tumors were calretinin-positive in two patients and one was inhibin-positive. Four patients underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy(TAH/BSO) and two were treated by unilateral salpingo-oophorectomy. Among them, two patients received adjuvant chemotherapy. Six patients were free of disease in a follow-up of 2-34 years and one achieved a pregnancy. The remaining patient recurred 4 years later. Feminization as well as virilization might provide important clues for a preoperative diagnosis. Histological misdiagnosis is probable in poorly differentiated tumors. Conservative surgery including retention of fertility can be considered. However, the tendency for recurrence in poorly differentiated tumors should be considered.

  4. Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance.

    PubMed

    Trbovich, Alexander M; Martinelle, Nina; O'Neill, Francis H; Pearson, Elliot J; Donahoe, Patricia K; Sluss, Patrick M; Teixeira, Jose

    2004-10-01

    In addition to causing Müllerian duct regression in fetal males, Müllerian inhibiting substance (MIS) inhibits the expression of the bifunctional cytochrome P450, C17 hydroxylase/C(17-20) lyase (Cyp17), the enzyme that catalyzes the committed step in sex steroid synthesis. To investigate the paracrine effects of MIS on steroidogenic activity, we have performed assays with microsomes from mouse MA-10 Leydig cells. With microsomes from untreated MA-10 cells, progesterone was largely metabolized by 5alpha-reductase and subsequently converted by 3-keto steroid reductases to allopregnanolone and epiallopregnanolone. Addition of cAMP to the cells shifted microsomal steroid production to the Cyp17 product androstenedione and its 5alpha,3beta-reduced form, epiandrosterone. Microsomes from MIS-treated cells were less active with the progesterone substrate than those of untreated cells but co-treatment of the cells with both MIS and cAMP mitigated the cAMP-induced shift of the microsomes to androstenedione production. Quantitative analyses of steroid production by Cyp17 showed that cAMP decreased the amount of 17-hydroxyprogesterone produced relative to the androstenedione, suggesting that cAMP signaling lowers the efficiency of the Cyp17 hydroxylase activity or else increases the efficiency of its lyase activity. Addition of MIS to the cAMP-treated cells partially reversed this effect, as well. These results indicate that cAMP induces MA-10 cells to switch from producing 5alpha-reduced progesterone metabolites to producing androstenedione and its metabolites by increasing Cyp17 expression and its relative lyase activity, both of which are inhibited by MIS.

  5. New enzymes involved in the mechanism of action of epidermal growth factor in a clonal strain of Leydig tumor cells.

    PubMed

    Castilla, Rocío; Gadaleta, Mariana; Castillo, Ana Fernanda; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2008-07-01

    The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.

  6. Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression.

    PubMed

    Wang, Xueting; Pan, Longlu; Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

    2017-03-07

    Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis.

  7. Effects of TBEP on the induction of oxidative stress and endocrine disruption in Tm3 Leydig cells.

    PubMed

    Jin, Yuanxiang; Chen, Guanliang; Fu, Zhengwei

    2016-10-01

    The flame retardant tris (2-butoxyethyl) phosphate (TBEP) is a frequently detected contaminant in the environment. In the cultured TM3 cells (originated from ATCC), effects of TBEP on the induction of oxidative stress and endocrine disruption were evaluated. It was observed that exposure to 100 μg/mL TBEP for 24 h significantly reduced the viability of TM3 cells. The mRNA levels of genes related to oxidative stress including Sod, Gpx1, Cat, and Gsta1 were changed in a dose-dependent and/or time-dependent manner after exposure to 30 and 100 μg/mL TBEP for 6, 12, or 24 h. Moreover, notable decrease in glutathione (GSH) contents and increases in oxidized glutathione (GSSG) contents as well as the antioxidant enzyme activities like superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were found in the group treated with 100 μg/mL TBEP for 24 h, indicating that TBEP induced oxidative stress in TM3 Leydig cells. In addition, the expression of genes related to testosterone (T) synthesis including cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17α-hydroxysteroid dehydrogenase (P450-17α), and 17β-hydroxysteroid dehydrogenase (17β-HSD) and T levels in medium were remarkably declined by the treatment of 100 μg/mL TBEP for 24 h. And TBEP could inhibit the expression of P450-17α and 17β-HSD and T levels up-regulated by hCG in TM3 cells. Taken together, these findings indicated that TBEP can induce oxidative stress and alter steroidogenesis in TM3 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1276-1286, 2016. © 2015 Wiley Periodicals, Inc.

  8. Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro.

    PubMed

    Riccetti, Laura; De Pascali, Francesco; Gilioli, Lisa; Potì, Francesco; Giva, Lavinia Beatrice; Marino, Marco; Tagliavini, Simonetta; Trenti, Tommaso; Fanelli, Flaminia; Mezzullo, Marco; Pagotto, Uberto; Simoni, Manuela; Casarini, Livio

    2017-01-05

    Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.

  9. A co-coculture system reveals the involvement of intercellular pathways as mediators of the lutropin receptor (LHR)-stimulated ERK1/2 phosphorylation in Leydig cells

    PubMed Central

    Shiraishi, Koji; Ascoli, Mario

    2007-01-01

    Co-cultures of lutropin receptor (LHR) positive and negative Leydig cells were used to test the hypothesis that the LHR provokes phosphorylation of the extracellular regulated kinases (ERK1/2) using intracellular and intercellular pathways. Addition of hCG to MA-10 cells (LHR positive) stimulates phosphorylation of the EGF receptor (EGFR) and ERK1/2 whereas addition of hCG to I-10 cells (LHR negative) does not. Addition of hCG to co-cultures of MA-10 and I-10 cells rapidly stimulates the phosphorylation of the EGFR and ERK1/2 in I-10 cells, however. Transfection of interfering constructs show that the LHR-mediated activation of Fyn in MA-10 cells is necessary for the phosphorylation of the EGFR and ERK1/2 in I-10 cells. This pathway can also be demonstrated in MA-10 cells but the phosphorylation of ERK1/2 in MA-10 cells also involves a second pathway mediated by protein kinase A (PKA). We propose that the LHR-mediated stimulation of the ERK1/2 cascade in Leydig cells depends on two independent pathways. One is intracellular and is mediated by PKA. The second is mediated by Fyn and it involves the release of soluble factors that act to phosphorylate the EGFR in an autocrine/paracrine fashion. PMID:17727840

  10. A novel clinicopathological analysis of early stage ovarian Sertoli-Leydig cell tumors at a single institution

    PubMed Central

    Nam, Seon Mi; Kim, Jee Whan; Eoh, Kyung Jin; Kim, Hye Min; Lee, Jung Yun; Nam, Eun Ji; Kim, Sunghoon; Kim, Sang Wun

    2017-01-01

    Objective To evaluate the clinical and pathologic characteristics of patients who were diagnosed with ovarian Sertoli-Leydig cell tumors (SLCTs) in a single institution. Methods The medical records of 11 patients who were pathologically diagnosed with SLCTs beginning in 1995 in a single institute was reviewed. Results The median patient age was 31 years (range, 16 to 70 years). Patient International Federation of Gynecology and Obstetrics stages were IA, IC, and IIB in 3 (27.3%), 6 (54.5%), and 2 (18.2%) patients, respectively. Six patients (54.5%) had grade 3 tumors, 3 patients (27.3%) had grade 2 tumors, and 1 patient (9.1%) had a grade 1 tumor. Four patients without children underwent fertility-sparing surgery, and 7 patients had full staging surgery, including a hysterectomy and bilateral salpingo-oophorectomy, with a laparoscopic approach used in 3. Eight patients underwent pelvic lymph node dissection, and 8 patients were administered adjuvant chemotherapy consisting of bleomycin, etoposide, and cisplatin in 6 cases, a modified bleomycin, etoposide, and cisplatin regimen in 1 case, and a combined paclitaxel and cisplatin regimen in 1 case. Two patients died of disease and were re-diagnosed with Sertoli form endometrioid carcinoma. The other patients remain alive without recurrence at the time of reporting. Conclusion Our findings suggest that regardless of tumor stage or grade, ovarian SLCT patients have a good prognosis. Close observation and unilateral salpingo-oophorectomy would be beneficial for women who still wish to have children, while hysterectomy and bilateral salpingo-oophorectomy with adjuvant chemotherapy would be the optimal treatment in other cases. Furthermore, meticulous pathologic diagnosis is needed to develop a precise treatment strategy. PMID:28217670

  11. 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.

  12. Activating mutation of the stimulatory G protein (gsp) as a putative cause of ovarian and testicular human stromal Leydig cell tumors.

    PubMed

    Fragoso, M C; Latronico, A C; Carvalho, F M; Zerbini, M C; Marcondes, J A; Araujo, L M; Lando, V S; Frazzatto, E T; Mendonca, B B; Villares, S M

    1998-06-01

    Activating mutations of the G protein genes have been associated with the development of several endocrine neoplasms. Such activating mutations, gip2, affecting the alpha-subunit of the G alpha i2 protein were previously described by a single group in 30% of ovarian sex cord stromal tumors. Other activating mutations of the alpha-subunit of the Gs (gsp) have been identified in GH-secreting and nonfunctioning pituitary tumors, autonomous thyroid adenomas, and all affected McCune-Albright tissues, but not in sex cord stromal tumors. In the present study, we investigated the presence of gip2 and gsp mutations in 14 human sex cord stromal tumors. Six Leydig cell tumors (4 ovaries and 2 testes), 2 thecomas, 2 granulosa cell tumors, 3 androblastomas, and 1 gonadoblastoma (sex cord and germ cell) were included in this study. Genomic DNA was obtained from either fresh-frozen tumor tissues or paraffin-embedded sections and in some cases from blood samples. Using PCR, denaturing gradient gel electrophoresis, and direct sequencing, we detected 4 tumors (66.6%) with the gsp mutation (R201C) in our series of ovarian and testicular Leydig cell tumors. In contrast, no gip2 mutations were found in any of the sex cord stromal tumors studied. In conclusion, our findings suggest that the putative oncogene gsp may play a significant role in the molecular mechanism of these tumors.

  13. Testis and epididymis of the Indian wall lizard (Hemidactylus flaviviridis): effects of flutamide on FSH and testosterone influenced spermatogenesis, Leydig cell, and epididymis.

    PubMed

    Rai, U; Haider, S

    1991-08-01

    To determine the separate spermatogenic actions of FSH and testosterone, adult male lizards Hemidactylus flaviviridis with recrudescent testes were administered the non-steroidal antiandrogen flutamide either alone or in combination with FSH or testosterone, and the histology and histochemistry of the testes and ductus epididymides were studied. Flutamide-treated animals displayed a marked hypertrophy of Leydig cells. A few spermatids were also seen in testis of more than half the animals treated with flutamide. Flutamide also produced a significant increase of primary spermatocytes; no spermatids were observed in controls. A significant inhibition of spermatogenesis was noted in lizards treated either with testosterone alone or in combination with flutamide. Ovine FSH treatment caused a significant stimulation of spermatogenesis, as indicated by the increase of primary and secondary spermatocytes and the transformation of secondary spermatocytes into spermatids or, in a few cases, into spermatozoa. A considerable depletion of sudanophilic lipid and moderate delta 5-3 beta-hydroxysteroid dehydrogenase activity was noted in the Leydig cells of FSH-treated animals indicating enhanced steroidogenesis. Similar results were obtained when lizards were treated with flutamide + FSH. The effects of simultaneous treatment of flutamide with FSH or testosterone on ductus epididymidis revealed that flutamide markedly inhibited the epithelial cell height and lumen diameter with a loss of luminal content when compared to FSH or testosterone-treated lizards.

  14. Studies of the pituitary-Leydig cell axis in young men with hypogonadotropic hypogonadism and hyposmia: comparison with normal men, prepuberal boys, and hypopituitary patients

    PubMed Central

    Bardin, C. Wayne; Ross, Griff T.; Rifkind, Arleen B.; Cargille, Charles M.; Lipsett, Mortimer B.

    1969-01-01

    Pituitary and gonadal function was studied in seven chromatin-negative men, ages 15-27 yr, with retarded sexual and somatic development, skeletal anomalies, and hyposmia. These hyposmic patients were compared with normal men, prepuberal boys and hypogonadal patients with hypopituitarism. The urinary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels of hyposmic subjects were the same as those of normal boys and hypopituitary patients but significantly lower than those of normal men. Clomiphene citrate did not cause an increase in plasma FSH and LH levels in either hypogonadal group as it does in normal men. In contrast to hypopituitary patients, thyroid and adrenocortical function and release of growth hormone in the hyposmic subjects were normal. The plasma testosterone levels were equally low in prepuberal, hypopituitary, and hyposmic patients but were increased to a greater extent by human chorionic gonadotropin (HCG) treatment in prepuberal and hypopituitary subjects than in the hyposmic patients. Prolonged treatment with HCG has failed to return plasma testosterone levels to normal in two hyposmic patients. These observations suggest that there are defects of both pituitary and Leydig cell function in men with the syndrome of hypogonadism, skeletal anomalies, and hyposmia. They have impaired secretion of FSH and LH and a Leydig cell insensitivity to gonadotropin. Images PMID:4390462

  15. Roles of leptin, adiponectin and resistin in the transcriptional regulation of steroidogenic genes contributing to decreased Leydig cells function in obesity.

    PubMed

    Roumaud, Pauline; Martin, Luc J

    2015-10-01

    The increase in obesity rate is a major public health issue associated with increased pathological conditions such as type 2 diabetes or cardiovascular diseases. Obesity also contributes to decreased testosterone levels in men. Indeed, the adipose tissue is an endocrine organ which produces hormones such as leptin, adiponectin and resistin. Obesity results in pathological accumulations of leptin and resistin, whereas adiponectin plasma levels are markedly reduced, all having a negative impact on testosterone synthesis. This review focuses on current knowledge related to transcriptional regulation of Leydig cells' steroidogenesis by leptin, adiponectin and resistin. We show that there are crosstalks between the regulatory mechanisms of these hormones and androgen production which may result in a dramatic negative influence on testosterone plasma levels. Indeed leptin, adiponectin and resistin can impact expression of different steroidogenic genes such as Star, Cyp11a1 or Sf1. Further investigations will be required to better define the implications of adipose derived hormones on regulation of steroidogenic genes expression within Leydig cells under physiological as well as pathological conditions.

  16. 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.

  17. Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

    PubMed

    Chemes, H; Cigorraga, S; Bergadá, C; Schteingart, H; Rey, R; Pellizzari, E

    1992-05-01

    Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis

    PubMed Central

    Park, Sun-Ji; Kim, Tae-Shin; Park, Choon-Keun; Lee, Sang-Hee; Kim, Jin-Man; Lee, Kyu-Sun; Lee, In-kyu; Park, Jeen-Woo; Lawson, Mark A; Lee, Dong-Seok

    2014-01-01

    Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells. PMID:23256993

  19. Leydig cell tumor

    MedlinePlus

    ... fetoprotein (AFP), human chorionic gonadotropin (beta HCG), and lactate dehydrogenase (LDH) CT scan of the chest, abdomen and pelvis to check if the cancer has spread Ultrasound of the scrotum An examination ...

  20. Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells.

    PubMed

    Perri, Mariarita; Pingitore, Attilio; Cione, Erika; Vilardi, Emma; Perrone, Valentina; Genchi, Giuseppe

    2010-09-01

    Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems. Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively. RA, at nutraceutic/endogenous doses (10-200 nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500 nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects. Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Age and markers of Leydig cell function, but not of Sertoli cell function predict the success of sperm retrieval in adolescents and adults with Klinefelter's syndrome.

    PubMed

    Rohayem, J; Fricke, R; Czeloth, K; Mallidis, C; Wistuba, J; Krallmann, C; Zitzmann, M; Kliesch, S

    2015-09-01

    Microsurgical testicular sperm extraction (mTESE), combined with intracytoplasmic sperm injection (ICSI) represents a chance for azoospermic men with Klinefelter's syndrome (KS) to father children. The objective of this study was to identify predictive factors for the success of mTESE from adolescents and adults with KS. The clinical data of 50 late pubertal adolescents (13-19 years) and 85 adult patients (20-61 years) with non-mosaic KS, who underwent mTESE, were analysed with respect to factors, potentially predictive of active spermatogenesis; specifically a history of cryptorchidism, age, testicular volumes, serum levels of LH, FSH, testosterone (T) and estradiol at the time of surgery. Inhibin B, AMH and INSL3 were additionally analysed in the adolescents. A younger age and a near-compensated Leydig cell function were associated with higher success of sperm retrieval via mTESE: In adolescents ≥15-19 years, spermatozoa were retrieved in 45%, compared to 31% in adults; in adolescents aged 13-14 years, spermatozoa were collected in only 10%. Adolescents with an LH ≤17.5 U/L, along with a T level ≥7.5 nmol/L had the best success rate (54%), which fell to 44% with higher LH, whereas those with low T (<7.5 nmol/L), irrespective of LH had no sperm retrieval. In adults with T levels above and LH below these thresholds, the success rate was 51%, falling to 19%, if LH was higher. When T was lower than threshold, the rate was 17%. No association between testicular volumes, serum levels of FSH, Inhibin B, AMH, estradiol and mTESE success was found. A history of cryptorchidism was associated with lower retrieval rates. A window of opportunity for an approximate 50% chance to retrieve spermatozoa via mTESE exists for young, late pubertal KS patients between age 15 and young adulthood, when Leydig cell function is at its best. In these cases, referral to a centre of expertise should be considered.

  2. 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 ...

  3. 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 ...

  4. Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode.

    PubMed

    Rikihisa, Y; Lin, Y C; Fukaya, T

    1985-06-01

    In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.

  5. Malformations of the epididymis, incomplete regression of the mesonephric tubules and hyperplasia of Leydig cells in canine persistence of Müllerian duct syndrome.

    PubMed

    Whyte, Ana; Monteagudo, Luis V; Díaz-Otero, Angel; Lebrero, M Eugenia; Tejedor, M Teresa; Falceto, M Victoria; Whyte, Jaime; Gallego, Margarita

    2009-10-01

    Persistence of the Müllerian duct syndrome (PMDS) is a rare form of pseudohermaphroditism characterized by the presence of uterus and oviducts in otherwise normally differentiated SRY-positive 78 XY canine males. Undescended testicles are also common. We report a case of a male PMDS dog with a uterus and bilateral cryptorchidism. The dog had an incomplete regression of the mesonephric tubules. As a consequence of this an abnormally enlarged head of the epididymis was observed. In addition, an extreme reduction in size of both the body and the tail was found. Microscopic examination of both testicles revealed bilateral hyperplasia of Leydig cells. The progesterone blood level was measured by ELISA and was found to be abnormally high (3.18 ng/ml) compared to that of normal male dogs (lower than 1 ng/ml). Three months after surgical removal of the internal genitalia, the serum progesterone, testosterone and oestradiol levels were normal for a castrated male dog.

  6. Ovarian Sertoli-Leydig Cell Tumor with Elevated Inhibin B as a Cause of Secondary Amenorrhea in an Adolescent with Germ Line DICER1 Mutation.

    PubMed

    Luke, Amy M; Moroney, John W; Snitchler, Andrea; Whiteway, Susan L

    2017-05-11

    Ovarian tumors, although uncommon in children, can retain endocrine function that disrupts normal feedback mechanisms leading to amenorrhea. Inheritance of germline DICER1 mutations can lead to increased risk for development of ovarian Sertoli-Leydig cell tumors (SLCTs). We report, to our knowledge, the first case of secondary amenorrhea due to elevated inhibin B levels in a female adolescent with an ovarian SLCT. Ovarian tumors should be included in the differential diagnosis for pediatric patients who present with menstrual irregularities. Early evaluation of the hypothalamic-pituitary-ovarian axis and inhibin levels is appropriate. Our case also emphasizes the need for testing for DICER1 mutations in pediatric patients with ovarian SLCTs. Published by Elsevier Inc.

  7. 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

  8. IN UTERO EXPOSURE TO THE FUNGICIDE PROCYMIDONE AND DIBUTYL PHTHALATE PRODUCE DOSE ADDITIVE DISRUPTIONS OF MALE RAT SEXUAL DIFFERENTIATION

    EPA Science Inventory

    Procymidone (PRO) and dibutyl phthalate (DBP) alter male rat sexual differentiation by disrupting the androgen-signaling pathway via distinctly different cellular mechanisms of toxicity. DBP inhibits fetal Leydig cell androgen production whereas PRO binds AR and blocks androgen a...

  9. Influence of luteinizing hormone and adenosine 3':5'-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours.

    PubMed

    Moyle, W R; Jungas, R L; Greep, R O

    1973-06-01

    1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50-100muCi per animal). Single-cell suspensions were prepared from the tumours 5-9 days after the injection of [(3)H]cholesterol and were incubated at 37 degrees C in foetal calf serum supplemented with 50mm-Tris-HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5mug/ml) or 3':5'-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell.

  10. Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

    PubMed Central

    Kurusu, Shiro; Sapirstein, Adam; Sawada, Harumi; Kawaminami, Mitsumori; Bonventre, Joseph V.

    2015-01-01

    In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a−/− ) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a−/− mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a−/− mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors. PMID:21762109

  11. Interleukin-6 and IL-6 receptor cell expression in testis of rats with autoimmune orchitis.

    PubMed

    Rival, Claudia; Theas, María S; Guazzone, Vanesa A; Lustig, Livia

    2006-06-01

    Experimental autoimmune orchitis (EAO) is an organ-specific model of autoimmunity characterized by an interstitial lymphomononuclear cell infiltrate as well as sloughing and apoptosis of germ cells. EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. Rats injected with saline solution and adjuvants were used as control group. The aim of this work was to study the expression of interleukin-6 (IL-6) and its receptor (IL-6R) in the testis of rats with EAO and analyze whether IL-6 could be involved in germ cell apoptosis. By immunohistochemistry, we detected IL-6 expression in testicular macrophages and Leydig cells of control and EAO rats. Sertoli cells showed IL-6 immunoreactivity in most of the seminiferous tubules of control rats, while a few IL-6+ Sertoli cells were found in the testis of rats with EAO. IL-6R immunoreactivity was observed in macrophages, Leydig and germ cells. A significant increase was noted in the number of IL-6R+ germ cells in rats with EAO compared to control rats. The content of IL-6 (ELISA) in the conditioned media obtained from testicular macrophages of rats with orchitis was significantly higher than in the control group. By immunofluorescence performed on isolated testicular macrophages, IL-6 was shown to be expressed by monocytes recently arrived from circulation (ED1+ cells), while resident macrophages (ED2+ cells) were negative. In vitro experiments (trypan blue and MTS assays) showed that IL-6 (50 ng/ml) reduced germ cell viability. We demonstrated also using the TUNEL technique that IL-6 added to cultures of seminiferous tubule segments induced apoptosis of germ cells. Our results suggest that IL-6 and IL-6R may be involved in the pathogenesis of autoimmune orchitis by promoting testicular inflammation and germ cell apoptosis.

  12. The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

    PubMed Central

    Manna, Pulak R.; Soh, Jae-Won; Stocco, Douglas M.

    2011-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a −83/−67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the

  13. Influence of luteinizing hormone and adenosine 3′:5′-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours

    PubMed Central

    Moyle, William R.; Jungas, Robert L.; Greep, Roy O.

    1973-01-01

    1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-3H]cholesterol (50–100μCi per animal). Single-cell suspensions were prepared from the tumours 5–9 days after the injection of [3H]cholesterol and were incubated at 37°C in foetal calf serum supplemented with 50mm-Tris–HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5μg/ml) or 3′:5′-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell. PMID:16742799

  14. Sertoli-Leydig cell tumors: hormonal profile after dynamic test with GnRH analogue: triptorelin represents a useful tool to evaluate tumoral hyperandrogenism.

    PubMed

    Turra, J; Granzotto, M; Gallea, M; Faggian, D; Conte, L; Litta, P; Vettor, R; Mioni, R

    2015-01-01

    We report the case of a 15-year-old woman with signs of hyperandrogenism affected by a Sertoli-Leydig cell tumor (SLCT). In our patient, blood analysis showed a high testosterone (T) level (T: 8.53 nmol/L; nv < 1.87 nmol/L) while the GnRH-analogue test demonstrated an exaggerated secretion of 17-hydroxyprogesterone (OHP), T, and androstenedione (A) by the ovary after stimulation. We compared the GnRH-analogue test of our patient with that obtained in a group of normal and healthy women (no. 8 subjects, 16-26 years old), men (no. 4 subjects, 18-28 years old), and in a group of PCOS patients with age and body weight compared. We found in our patient a value of OHP, 17-beta estradiol (E2) and T, from 2 to 18 times higher than healthy women. When we compared our patient with healthy men, we differently observed a comparable response of T. The response of our patient was also comparable with that observed in the PCOS group for E2. During the post-surgical follow up, the GnRH-analogue test of our patient showed a response of OHP, T, and E2 comparable with that of the PCOS group. The GnRH-analogue test is a useful tool to characterize steroidogenesis in SLCT.

  15. Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation.

    PubMed

    Alamdar, Ambreen; Xi, Guochen; Huang, Qingyu; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

    2017-07-01

    Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Hormone-induced 14-3-3γ Adaptor Protein Regulates Steroidogenic Acute Regulatory Protein Activity and Steroid Biosynthesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Rone, Malena B.; Blonder, Josip; Ye, Xiaoying; Veenstra, Timothy D.; Hales, D. Buck; Culty, Martine; Papadopoulos, Vassilios

    2012-01-01

    Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation. PMID:22427666

  17. The use of genetic manipulation of MA-10 Leydig tumor cells to demonstrate the role of mitochondrial proteins in the acute regulation of steroidogenesis.

    PubMed

    Stocco, D M; Ascoli, M

    1993-03-01

    The true rate-limiting step in steroidogenesis is the delivery of cholesterol to the inner mitochondrial membrane where it is converted to pregnenolone by the cholesterol side-chain cleavage complex. This process is known to require de novo protein synthesis. We have previously described the synthesis of a family of 37, 32, and 30 kilodalton mitochondrial proteins in response to hormone stimulation in MA-10 mouse Leydig tumor cells and have proposed that these proteins are involved in the acute regulation of steroidogenesis. In this study we have used two subclones of MA-10 cells to further demonstrate the correlation between the quantity of these proteins and the production of steroids in response to hormone treatment. One of these, designated MA-10(K3), has been transfected with a mutant gene of the type 1 regulatory subunit of the cAMP-dependent protein kinase under the control of a metallothionein promoter, whereas the other, designated MA-10(P+29), is a constitutive overproducer of a cAMP-phosphodiesterase (PDE). MA-10 parent cells designated (P), produce large amounts of progesterone in response to LH, human CG, and (Bu)2cAMP. The MA-10(K3) cells, on the other hand, whereas significantly higher than controls, produce much less steroid than the parent cells in response to hormone stimulation. Activation of the mutant gene with Zn+2 results in yet a further decrease in the amount of steroid produced. The MA-10(P+29) cells display greatly reduced progesterone production when stimulated with LH, because of the presence of high amounts of PDE, but return to maximally stimulated levels when a PDE inhibitor is present. Quantitation of the synthesis of the mitochondrial proteins described above in MA-10(K3) cells in the presence and absence of Zn+2 and in MA-10(P+29) cells in the presence and absence of PDE inhibitor clearly demonstrate that the amount of the 30 kilodalton mitochondrial proteins present in these cells closely parallels that of progesterone production

  18. Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.

    PubMed

    Choi, Ji-Young; Lee, Sangmi; Hwang, Soojin; Jo, Sangmee Ahn; Kim, Miji; Kim, Young Ju; Pang, Myung-Geol; Jo, Inho

    2013-01-01

    5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.

  19. Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

    PubMed Central

    Minagawa, Itaru; Fukuda, Masafumi; Ishige, Hisako; Kohriki, Hiroshi; Shibata, Masatoshi; Park, Enoch Y.; Kawarasaki, Tatsuo; Kohsaka, Tetsuya

    2011-01-01

    RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity. PMID:21899516

  20. Prostaglandin (PG) FP and EP1 receptors mediate PGF2alpha and PGE2 regulation of interleukin-1beta expression in Leydig cell progenitors.

    PubMed

    Walch, Laurence; Clavarino, Emanuela; Morris, Patricia L

    2003-04-01

    Prostaglandins (PG) mediate IL-1beta regulation of several interleukin mRNAs in progenitor Leydig cells. PGE(2) and PGF(2alpha) potently reverse indomethacin (INDO; a cyclooxygenase inhibitor) inhibition of IL-1beta autoinduction. IL-1beta increases PGE(2) and PGF(2alpha) production. To determine the PG receptors involved in this regulation, this study established by RT-PCR and Western analyses which specific receptors for PGE(2) (EP receptors) and PGF(2alpha) (FP receptors) are expressed in progenitors. Pharmacological characterization of receptors involved in PGE(2) and PGF(2alpha) regulation of IL-1beta mRNA levels was ascertained using real-time PCR analyses. FP, EP(1), EP(2), and EP(4) receptor mRNAs and proteins, and an EP(3) receptor subtype were detected. IL-1beta treatment (24-h) significantly decreased EP(1) receptor levels; INDO abrogated this down-regulation. FP, EP(2), and EP(4) receptor levels increased after IL-1beta and IL-1beta + INDO. A selective FP agonist, cloprostenol (0.1 micro M), and PGF(2alpha) (10 micro M) had similar effects on IL-1beta mRNA levels in progenitors treated with IL-1beta + INDO. None of the EP(2)/EP(4) agonists [butaprost, misoprostol, or 11-deoxy PGE(1) (10 micro M)] affected IL-1beta mRNA levels. In contrast, EP(1)/EP(3) agonists (17-phenyl trinor PGE(2) and sulprostone) increased IL-1beta mRNAs in a dose-dependent manner. EP(1) receptor subtype-selective antagonist, SC-51322, blocked IL-1beta-induced and [IL-1beta + INDO + 17-phenyl trinor PGE(2)]-induced increases in IL-1beta mRNAs. Taken together, our data demonstrate that FP and EP(1) receptors mediate PGF(2alpha) and PGE(2) induction of progenitor IL-1beta expression.

  1. 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.

  2. Treatment of leydig cell tumours of the testis: Can testis-sparing surgery replace radical orchidectomy? Results of a systematic review.

    PubMed

    Bozzini, G; Ratti, D; Carmignani, L

    2017-04-01

    The gold standard for Leydig cell tumours (LCTs) is still considered radical orchidectomy, but testis sparing surgery (TSS) in conjunction with intraoperative frozen section (FSE) has been recently attempted with promising results. Studies were identified by searching electronic databases. A bibliographic search covering the period from January 1980 to December 2012 was conducted using PubMed/MEDLINE and EMBASE database. Studies were excluded if they were single case reports, meeting abstracts and conference proceedings. The present analysis is based on a total of 13 studies that fulfilled the predefined inclusion criteria. A total of 247 participants were included in the 13 studies examined in this systematic review. 145 were treated with radical orchiectomy and 102 with TSS. In the radical surgery group, the follow-up varied from 6 to 249 months). In the TSS group, the follow-up varied from 6 to 192 months. Frozen section was performed in a total of 96 patients. Sensitivity was 87.5%. None of the patients treated with TSS presented a metastatic recurrence, while in patients treated with radical orchiectomy three patients presented with metastatic recurrence In selected cases radical surgery appears excessive and the potential for a shift to TSS as the standard management is gathering momentum. The results confirm the favourable course of LCT treated with TSS. The results obtained are encouraging and the concept is attractive to become the standard therapy in all patients and not only in people affected by (sub)fertility or with solitary testis. Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  3. Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

    PubMed

    de Michele, F; Poels, J; Weerens, L; Petit, C; Evrard, Z; Ambroise, J; Gruson, D; Wyns, C

    2017-01-01

    Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm(3) tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either

  4. Effect of cholesterol transport inhibitors on steroidogenesis and plasma membrane cholesterol transport in cultured MA-10 Leydig tumor cells.

    PubMed

    Nagy, L; Freeman, D A

    1990-05-01

    These studies were directed toward understanding the cellular actions of inhibitor drugs that affect steroidogenesis and cholesterol transport. We investigated the microfilament inhibitor cytochalasin-D, the microtubule inhibitor colchicine, the calmodulin antagonist trifluoperazine, and the inhibitor of acidic vesicle function nigericin. We found that all of these compounds caused dose-dependent inhibition of progesterone synthesis in the MA-10 cells. Each compound also inhibited (Bu)2cAMP-stimulated pregnenolone synthesis, indicating that each inhibited a fundamental process required for steroidogenesis. Each compound was next evaluated for inhibitory actions on cholesterol transport to and from the plasma membrane. On the basis of inhibitor sensitivity, two different categories of cholesterol transport were defined. Transport of newly synthesized or low density lipoprotein-derived cholesterol from the cell interior to the plasma membrane was inhibitor insensitive. Plasma membrane cholesterol internalization, however, was sensitive to all of the inhibitors and did not result because of any drug effect on the acyl-coenzyme-A-cholesterol acyl transferase. Cycling of cholesteryl ester-derived cholesterol through the plasma membrane appeared to occur before its use for steroidogenesis. Thus, inhibition of plasma membrane internalization would prevent utilization of both plasma membrane cholesterol and cholesteryl ester-derived cholesterol, the two major substrate sources for steroid hormone synthesis. Consistent with this interpretation was the finding that inhibition of plasma membrane cholesterol internalization by each inhibitor paralleled the inhibitor's effect on steroidogenesis.

  5. 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.

  6. 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.

  7. Sertoli-Leydig cell tumor

    MedlinePlus

    ... produce and release a male sex hormone called testosterone. Causes The exact cause of this tumor is ... the levels of female and male hormones, including testosterone . An ultrasound or another imaging test will likely ...

  8. Franz von Leydig (1821-1908), pioneer of comparative histology.

    PubMed

    Schneider, Marlon R

    2012-05-01

    Franz von Leydig, a German histologist and zoologist, is known to every student of human or animal anatomy because of the testicular testosterone-producing cells carrying his name. However, he made many contributions to our knowledge of the fine structure of animal tissues, including more than 200 scientific articles and several books. His most important work, the book Lehrbuch der Histologie des Menschen und der Thiere, established him as a pioneer if not the founder of comparative histology. Leydig taught at three different universities (Würzburg, Tübingen and Bonn) and received many honours from scientific organizations worldwide, including the Royal Society. He died in Rothenburg ob der Tauber, the town of his birth, aged 86 years.

  9. Exogenous arachidonate restores the dimethoate-induced inhibition of steroidogenesis in rat interstitial cells.

    PubMed

    Astiz, Mariana; Hurtado de Catalfo, Graciela; de Alaniz, María J T; Marra, Carlos Alberto

    2012-06-01

    The present work studies the potential restorative effect of polyunsaturated fatty acids (PUFA, 5 μM/24 h) on the dimethoate (DMT)-induced inhibition of testosterone biosynthesis in Leydig cells isolated from rat testes. Various fatty acids (FA) from the n-6 (18:2, 20:3, 20:4, 22:4 and 22:5) and n-3 (18.3, 20:5, 22:5, 22:6) series were assayed in Leydig cells, alone (as delipidated BSA complexes) and in combination with DMT (1 ppm). The n-6 FA stimulated lipid peroxidation (LPO) and inhibited the activities of steroidogenic enzymes (3β- and 17β-hydroxysteroid dehydrogenases). The n-3 FA exerted an anti-oxidant effect, decreasing the production of thiobarbituric-acid reactive substances (TBARS) and inhibiting phospholipase A(2) activity. The biosynthesis of testosterone in DMT-treated cultures was completely normalized by ARA (20:4n-6) and partially restored by the addition of 20:3n-6, increasing ARA content inside the mitochondria. The other FA assayed failed to restore androgenesis. COX-2 protein and prostaglandin F2α and E2 production were stimulated by 20:3n-6, ARA, 18:3n-3 and 20:5 n-3. COX-2 protein decreased upon addition of 22:5n-3 and 22:6n-3. StAR protein was increased by ARA and partially increased by 20:3n-6, likely due to its metabolic conversion into ARA. Both FA increased the mitochondrial cholesterol pool available for testosterone biosynthesis. The rate of androgenesis is likely the result of various regulatory factors acting concomitantly on the physiology of Leydig cells.

  10. Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3 beta-hydroxysteroid dehydrogenase/delta (5-4)-isomerase and 17 alpha-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line.

    PubMed

    Lefèvre, A; Rogier, E; Astraudo, C; Duquenne, C; Finaz, C

    1994-12-01

    Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17 alpha-hydroxylase/C17-20 lyase (P-450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3 beta HSD and P-450 17 alpha were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17 alpha and 3 beta HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17 alpha expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0 kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Identifying Molecular Targets for Chemoprevention in a Rat Model

    DTIC Science & Technology

    2005-12-01

    engineered mice. Am J Pathol 2002;161:727-35. [13] Weisburger JH, Rivenson A, Reinhardt J, Braley J, Pittman B, Zang E. On the occurrence of...relatively large patch of Leydig cells ( Ley ) adjacent to two seminiferous tubules (Tub). Borowsky, et al. PhIP Rat Prostate 15 Figure 1

  12. Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats.

    PubMed

    Biegel, L B; Hurtt, M E; Frame, S R; O'Connor, J C; Cook, J C

    2001-03-01

    Wyeth-14,643 (WY) and ammonium perfluorooctanoate (C8) belong to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation in rodents. From previous work, WY, but not C8, has been shown to produce hepatocellular carcinoma in rats, while C8 has been shown to produce Leydig cell adenomas. In addition, based on a review of bioassay data a relationship appears to exist between peroxisome-proliferating compounds and Leydig cell adenoma and pancreatic acinar cell hyperplasia/adenocarcinoma formation. To further investigate the relationship between peroxisome-proliferating compounds and hepatic, Leydig cell, and pancreatic acinar cell tumorigenesis, a 2-year feeding study in male CD rats was initiated to test the hypothesis that peroxisome proliferating compounds induce a tumor triad (liver, Leydig cell, pancreatic acinar cell), and to examine the potential mechanism for the Leydig cell tumors. The study was conducted using 50 ppm WY and 300 ppm C8. The concentration of WY in the diet was decreased to 25 ppm on test day 301 due to increased mortality. In addition to the ad libitum control, a second control was pair-fed to the C8 group. Interim sacrifices were performed at 1- or 3-month intervals. Peroxisome proliferation measured by beta-oxidation activity and cell proliferation were measured in the liver and testis at all time points and in the pancreas beginning at the 9-month time point (cell proliferation only). Serum hormone concentrations (estradiol, testosterone, LH, FSH, and prolactin) were also measured at each time point. Increased relative liver weights and hepatic beta-oxidation activity were observed in both the WY- and C8-treated rats at all time points. In contrast, hepatic cell proliferation was significantly increased only in the WY-treated group. Neither WY nor C8 significantly altered the rate of Leydig cell beta-oxidation or Leydig cell proliferation when compared to the control groups. Moreover, the basal rate of

  13. The compounds from the hollyhock extract (Althaea rosea Cav. var. nigra) affect the aromatization in rat testicular cells in vivo and in vitro.

    PubMed

    Papiez, Monika; Gancarczyk, Monika; Bilińska, Barbara

    2002-01-01

    Among medicinal plants, extract from the hollyhock flowers is a source of antocyanides and flavonoids. The latter compounds belong, among others, to phytoestrogens (plant-derived dietary estrogens). The important role of estrogens in the testis is now well documented, and phytoestrogens, which may act as estrogen agonists or estrogen antagonists can also alter the reproductive function of the male. The aim of this study was to show whether the exposure of male rats to the aqueous hollyhock extract could affect the process of aromatization in their testes and in cultured Leydig cells. This was investigated by immunocytochemistry and radioimmunological assays. Immunoreactivities for aromatase and estrogen receptor beta were weaker both in testicular sections and cultured Leydig cells after hollyhock extract administration when compared to the controls, while the intensity of immunoreaction for estrogen receptor alpha remained unchanged. A lower level of estradiol secreted by cultured Leydig cells from the experimental group positively correlated with a direct inhibition of aromatase activity. Additionally, a quantitative analysis of flavonoid fraction from the hollyhock extract revealed the presence of quercetin and kaempferol. It seems that a weak antiestrogenic activity of flavonoid compounds present in the hollyhock extract is mediated through aromatase and estrogen receptor beta rather than by estrogen receptor alpha.

  14. Proliferating cell nuclear antigen as a molecular biomarker for spermatogenesis in PTU-induced hypothyroidism of rats.

    PubMed

    Tousson, Ehab; Ali, Ehab M M; Ibrahim, Wafaa; Mansour, Mohammed A

    2011-07-01

    The thyroid hormone has few serious effects on the testes except during the neonatal stage. There is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span and its effect on spermatogenesis. Proliferating cell nuclear antigen (PCNA) is a nuclear matrix protein, which is essential for multiple cell cycle pathways. Here we used PCNA immunohistochemistry as a marker to differentiate between the testes of control and hypothyroid rats. About 20 rats were equally divided into 2 groups; the first group was the control group, while the second group was the experimental group in which rats were fed 0.05% 6-n-propyl thiouracil (PTU) in drinking water for 6 weeks. Immunohistochemistry, using an antibody against PCNA, showed at least 3 differences in the pattern of PCNA immunoreactivity (PCNA-ir). First, PCNA-ir was not detected in Sertoli and Leydig cells in the testes of control rats and detected in some of the hypothyroid rats. Second, in the control group more than 96% of spermatogonia were PCNA-positive cells; however, hypothyroidism caused the reduction to approximately 25% PCNA staining in spermatogonia. The third difference was in the abnormal distribution of spermatogonia seen in the hypothyroid rat testis, not in the control one. These results suggest that prepubertal hypothyroidism affects the proliferation of spermatogenic cells leading to impaired spermatogenesis and that PCNA index is a useful marker for assessing germ cell kinetics and spermatogenesis in prepubertal hypothyroidism.

  15. In vitro effect of 4-nonylphenol on human chorionic gonadotropin (hCG) stimulated hormone secretion, cell viability and reactive oxygen species generation in mice Leydig cells.

    PubMed

    Jambor, Tomáš; Tvrdá, Eva; Tušimová, Eva; Kováčik, Anton; Bistáková, Jana; Forgács, Zsolt; Lukáč, Norbert

    2017-03-01

    Nonylphenol is considered an endocrine disruptor and has been reported to affect male reproductive functions. In our in vitro study, we evaluated the effects of 4-nonylphenol (4-NP) on cholesterol levels, hormone formation and viability in cultured Leydig cells from adult ICR male mice. We also determined the potential impact of 4-NP on generation of reactive oxygen species (ROS) after 44 h of cultivation. The cells were cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/mL of 4-NP in the present of 1 IU/mL human chorionic gonadotropin (hCG) and compared to the control. The quantity of cholesterol was determined from culture medium using photometry. Determination of hormone production was performed by enzyme-linked immunosorbent assay. Metabolic activity assay was used for quantification of cell viability. The chemiluminescence technique, which uses a luminometer to measure reactive oxygen species, was employed. Applied doses of 4-NP (0.04-5.0 μg/mL) slight increase cholesterol levels and decrease production of dehydroepiandrosterone after 44 h of cultivation, but not significantly. Incubation of 4-NP treated cells with hCG significantly (P < 0.001) inhibited androstenedione, but not testosterone, formation at the highest concentration (5.0 μg/mL). The viability was significantly (P < 0.05); (P < 0.001) increased at 1.0; 2.5 and 5.0 μg/mL of 4-NP after 44 h treatment. Furthermore, 44 h treatment of 4-NP (0.04-5.0 μg/mL) caused significant (P < 0.001) intracellular accumulation of ROS in exposed cells. Taken together, the results of our in vitro study reported herein is consistent with the conclusion that 4-nonylphenol is able to influence hormonal profile, cell viability and generate ROS.

  16. Cyanidin-3-O-Glucoside Protects against 1,3-Dichloro-2-Propanol-Induced Reduction of Progesterone by Up-regulation of Steroidogenic Enzymes and cAMP Level in Leydig Cells

    PubMed Central

    Sun, Jianxia; Xu, Wei; Zhu, Cuijuan; Hu, Yunfeng; Jiang, Xinwei; Ou, Shiyi; Su, Zhijian; Huang, Yadong; Jiao, Rui; Bai, Weibin

    2016-01-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a food processing contaminant and has been shown to perturb male reproductive function. Cyanidin-3-O-glucoside (C3G), an anthocyanin antioxidant, is reported to have protective effects on many organs. However, it remains unclear whether C3G protects against chemical-induced reproductive toxicity. The present study was therefore to investigate the intervention of C3G on 1,3-DCP-induced reproductive toxicity in R2C Leydig cells. Results demonstrated that C3G inhibited the 1,3-DCP-induced cytotoxicity and cell shape damage with the effective doses being ranging from 10 to 40 μmol/L. In addition, 1,3-DCP (2 mmol/L) exposure significantly increased the ROS level and mitochondrial membrane potential damage ratio, leading to a decrease in progesterone production, while C3G intervention reduced the ROS level, and increased the progesterone production after 24 h treatment. Most importantly, C3G intervention could up-regulate the cyclic adenosine monophosphate (cAMP) level and protein expression of steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase. It was concluded that C3G is effective in reducing 1,3-DCP-induced reproductive toxicity via activating steroidogenic enzymes and cAMP level. PMID:27867356

  17. Primary amenorrhea in a young Polish woman with complete androgen insensitivity syndrome and Sertoli-Leydig cell tumor: identification of a new androgen receptor gene mutation and evidence of aromatase hyperactivity and apoptosis dysregulation within the tumor.

    PubMed

    Jarzabek, Katarzyna; Philibert, Pascal; Koda, Mariusz; Sulkowski, Stanislaw; Kotula-Balak, Malgorzata; Bilinska, Barbara; Kottler, Marie-Laure; Wolczynski, Slawomir; Sultan, Charles

    2007-09-01

    Primary amenorrhea in 46,XY females can be due to complete androgen insensitivity syndrome (CAIS), pure gonadal dysgenesis, 17-hydroxysteroid dehydrogenase deficiency, or mixed gonadal dysgenesis. The present paper describes a new de novo non-sense mutation in exon 1 (K141Z) of the androgen receptor gene (AR) and the expression in CAIS testis of aromatase, estrogen receptors, as well as proliferation- and apoptosis-associated proteins. CAIS is a rare disease characterized by absent virilization in 46,XY individuals and the development of a female phenotype despite normal or even elevated androgen levels. CAIS is usually caused by a mutation in AR, which leads to organ resistance to androgens. Testicular tumors such as Sertoli-Leydig cell tumor often develop in patients with CAIS. The immunohistochemical findings in the testes of our CAIS patient suggest that the high expression of aromatase and other molecular changes in the testis may be responsible for pubertal breast development and the increased risk of testicular tumor.

  18. Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact.

    PubMed

    Kleymenova, Elena; Swanson, Cynthia; Boekelheide, Kim; Gaido, Kevin W

    2005-09-01

    Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.

  19. 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.

  20. Carcinogenicity study of 3-monochloropropane-1,2-diol in Sprague-Dawley rats.

    PubMed

    Cho, Wan-Seob; Han, Beom Seok; Nam, Ki Taek; Park, Kidae; Choi, Mina; Kim, Seung Hee; Jeong, Jayoung; Jang, Dong Deuk

    2008-09-01

    3-Monochloropropane-1,2-diol (alpha-chlorohydrin, 3-MCPD) is a well-known contaminant, which has been detected in a wide range of foods and ingredients, and is also a suspected cause of cancer. In this study, the carcinogenicity of 3-MCPD in SD rats was investigated. Groups of 50 male and 50 female rats were exposed for two years to drinking water containing 0, 25, 100 or 400ppm 3-MCPD. The body weights and water consumptions of the male and female rats given 400ppm 3-MCPD were significantly lower than those of the controls. The incidences of renal tubule adenomas or carcinomas and Leydig cell tumors occurred with dose-related positive trends in male rats. The incidences of renal tubule carcinomas and Leydig cell tumors were significantly increased in male rats given 400ppm 3-MCPD. The incidence of renal tubule adenomas showed a positive trend in female rats, which was significant in 400ppm 3-MCPD group. In conclusion, there was clear evidence of the carcinogenic activity of 3-MCPD in male SD rats, based on the increased incidences of renal tubule carcinomas and Leydig cell tumors. There was some evidence of the carcinogenic activity of 3-MCPD in female SD rats, based on the increased incidence of renal tubule adenomas.

  1. 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

  2. Effects of hyper- and hypothyroidism on the development and proliferation of testicular cells in prepubertal rats.

    PubMed

    Fadlalla, Mohamed Babo; Wei, Quanwei; Fedail, Jaafar Sulieman; Mehfooz, Asif; Mao, Dagan; Shi, Fangxiong

    2017-08-07

    Thyroid hormones are important in the development and regulation of testes. This study was conducted to determine the effects of hyper- and hypothyroidism on testicular development in prepubertal rats aged 20-70 days. Weaning male rats (20 days old) until day 70 age were randomly divided into four groups: control, hyperthyroid (hyper-T), hypothyroid (hypo-T) and hypothyroid treated with thyroxine (T4) (hypo-T+T4). The results indicated that thyroid hormones caused a significant effect in body and testis weights, and food and water consumption. In addition there were changes in serum concentrations of tri-iodothyronine, T4, thyroid stimulating hormone (TSH) and testosterone. Histomorphology showed a significant decrease in seminiferous tubule diameter in hyper-T compared to the other groups. Leydig cell numbers showed a significant elevation in hyper-T but not in hypo-T groups. Immunostaining indicated that TSH receptor (TSHR), thyroid hormone receptors α/β (TRαβ) and proliferating cell nuclear antigen (PCNA) have the roles in testicular development. Our findings suggest that hyper- and hypo-thyroidism regulate testicular cell proliferation and spermatogenesis in prepubertal rats, indicating that expression of TSHR, TRαβ and PCNA may be regulated by thyroid hormones that are involved in testicular development; and that the administration of T4 to the hypo-T+T4 group leads to an improvement in the testicular condition. © 2017 Japanese Society of Animal Science.

  3. 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...

  4. 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 ...

  5. 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...

  6. 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 ...

  7. [Effects of glycosides of Tripterygium wilfordii, methyltestosterone and zhuanggushenjin capsule on nitric oxide synthase in rat testes].

    PubMed

    Ren, Ya-Ping; Sun, Li; Jiang, Wei; Hu, Chun-Ping

    2005-05-01

    To investigate the effects of glycosides of tripterygium wilfordii (GTW), methyltestosterone and Zhuanggushenjin capsule on nitric oxide synthase (NOS) in rat testes. Forty-five rats were equally divided into 5 groups, and respectively given GTW [10 mg/(kg x d)], methyltestosterone [2 mg/(kg x d)], Zhuanggushenjin capsule [0.3 g/(kg x d)], distilled water plus Tween 80 (control I), and distilled water alone (control II) for 4 weeks. At the end of the 5th week, the immunochemical ABC method was used to observe the effects of the three drugs on the NOS positive Leydig cells of the rats. Compared with control II, the GTW group had a significant decrease in the numbers of nNOS and eNOS positive Leydig cells, the methyltestosterone group showed an increase in the number of nNOS but a decrease in that of eNOS positive Leydig cells, and the Zhuanggushenjin group had an increase in the numbers of both nNOS and eNOS positive Leydig cells. GTW can reduce NO production by inhibiting eNOS and nNOS, and hence influence the spermatogenic process. Zhuanggushenjin capsule plays an important role in improving male sexual function by enhancing nNOS and eNOS expression and NO synthesis.

  8. Erdosteine protects rat testis tissue from hypoxic injury by reducing apoptotic cell death.

    PubMed

    Guven, A; Ickin, M; Uzun, O; Bakar, C; Balbay, E Gulec; Balbay, O

    2014-02-01

    The purpose of this study was to examine the effects of hypobaric hypoxia on testis morphology and the effects of erdosteine on testis tissue. Caspase-3 and hypoxia-inducible factor 1α expressions were detected by immunohistochemistry. Adult male Wistar rats were placed in a hypobaric hypoxic chamber. Rats in the erdosteine group were exposed to the same conditions and treated orally with erdosteine (20 mg kg(-1) daily) at the same time from the first day of hypoxic exposure for 2 weeks. The normoxia group was evaluated as the control. The hypoxia group showed decreased height of spermatogenic epithelium in some seminiferous tubules, vacuolisation in spermatogenic epithelial cells, deterioration and gaps in the basal membrane and an increase in blood vessels in the interstitial area. The erdosteine group showed amelioration of both epithelial cell vacuolisation and basal membrane deterioration. Numbers of hypoxia-inducible factor 1α-immunostained Sertoli and Leydig cells were significantly higher in the hypoxia group than in the erdosteine group. The number of seminiferous tubules with caspase-3-immunostained germ cells was highest in the hypoxia group and decreased in the erdosteine and normoxia groups respectively. Based on these observations, erdosteine protects testis tissue from hypoxic injury by reducing apoptotic cell death.

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

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-09-06

    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.

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

    PubMed Central

    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

  11. Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats.

    PubMed

    Suescun, María O; Rival, Claudia; Theas, María S; Calandra, Ricardo S; Lustig, Livia

    2003-06-01

    We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.

  12. 2-Phenylimidazo[1,2-a]pyridine-containing ligands of the 18-kDa translocator protein (TSPO) behave as agonists and antagonists of steroidogenesis in a mouse leydig tumor cell line.

    PubMed

    Midzak, Andrew; Denora, Nunzio; Laquintana, Valentino; Cutrignelli, Annalisa; Lopedota, Angela; Franco, Massimo; Altomare, Cosimo D; Papadopoulos, Vassilios

    2015-08-30

    Ligands of 18-kDa translocator protein (TSPO) are known for their ability to potently and dose-dependently stimulate steroid biosynthesis in steroidogenic cells. In this study, we investigated a number of 2-phenyl-imidazo[1,2-a]pyridine acetamide derivatives, analogs of alpidem, for their ability to bind TSPO and to affect steroidogenesis in a mouse Leydig tumor cell line. We observed that not only some compounds behaved as agonists, stimulating steroidogenesis (e.g., 3 and 4) with EC50 values (15.9 and 6.99μM) close to that determined for FGIN-1-27 used as positive control (7.24μM), but two compounds, namely 5 and 6, which on the other hand are the most lipophilic ones in the investigated series, behaved as antagonists, by significantly inhibiting steroid production at concentrations at least twenty times lower than the cytotoxic ones. To our surprise, the newly synthesized compound 3, which is a strict analog of alpidem bearing at the para position of the 2-phenyl group a methoxy group instead of chlorine, achieved a ten-fold stimulation of the steroid production (for comparison FGIN-1-27 achieved 1.6-fold stimulation). Within the limits of the examined property space, some unprecedented SARs were unveiled, which can help in understanding the key molecular factors underlying the transition from agonism to antagonism in the steroidogenesis process. Besides the substitution pattern and the physicochemical features (mainly hydrogen bonding potential) of the substituents at the positions C(6) and C(8) of the imidazo[1,2-a]pyridine nucleus, and at the para position of the 2-phenyl group, the structure-activity relationship analysis suggested lipophilicity, whose increase seems to be generally related to steroidogenesis inhibition, and steric hindrance, which appeared as a stimulation-limiting factor, as two main properties to control in the design or optimization of novel imidazo[1,2-a]pyridine-based TSPO ligands endowed with potential in modulating the

  13. Is Percoll innocuous to cells?

    PubMed Central

    Wakefield, J S; Gale, J S; Berridge, M V; Jordan, T W; Ford, H C

    1982-01-01

    Peritoneal macrophages from mice, isolated rat liver Kupffer cells and rat testis Leydig cells ingested large numbers of Percoll particles, a gradient medium widely used for separation of cells and subcellular organelles by density-gradient centrifugation. A decrease in the percentage of macrophages adhering to plastic also occurred after exposure of the cells to Percoll, even at 4 degrees C, a temperature at which Percoll was not ingested. The effect of Percoll on macrophage adherence may involve a loose association between the density medium and the cell surface. Other cell-surface-related phenomena may also be affected by prior exposure of cells to Percoll. Images PLATE 1 PLATE 2 PMID:6284138

  14. Ovarian Sertoli-Leydig Cell Tumor with Elevated Inhibin B As a Cause of Secondary Amenorrhea in Adolescents with Germline DICER1 Mutation

    DTIC Science & Technology

    2017-04-06

    infancy and early childhood.8 The D/CERJ-related disorders have expanded to include other ovarian sex cord-stromal tumors Guveni le granulosa cell...in children and adolescents. Cancer. 1987; 59: 12 14 3. Schultz KA, Harris AK, Schneider OT, et al. Ovarian Sex Cord-Stromal Tumor. Journal of

  15. Influence of germ cells upon Sertoli cells during continuous low-dose rate gamma-irradiation of adult rats.

    PubMed

    Pinon-Lataillade, G; Vélez de la Calle, J F; Viguier-Martinez, M C; Garnier, D H; Folliot, R; Maas, J; Jégou, B

    1988-07-01

    The effects of continuous gamma-irradiation of adult rats at two low-dose rates (7 cGy and 12 cGy/day; up to a total dose of 9.1 Gy and 10.69 Gy 60Co gamma-ray, respectively) were investigated. Over a period of 3-131 days of irradiation, groups of experimental and control animals were killed. Body weight, testis, epididymis, prostate and seminal vesicle weights, the number of germ cells and Sertoli cells, tubular ultrastructure, epididymal and testicular levels of biologically active androgen-binding protein (ABP), and the plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were monitored. Irradiation had no effect on body weight, whereas testicular and epididymal weight began to decrease following 35 and 50 days of irradiation at 7 and 12 cGy, respectively. At 7 cGy the target cells of the gamma-rays were essentially A spermatogonia, whereas at 12 cGy A spermatogonia and preleptotene spermatocytes were primarily affected. This resulted in a progressive and sequential dose-related reduction in the number of pachytene spermatocytes, round spermatids and late spermatids (LS). Under both irradiation procedures the Sertoli cell number remained unchanged whereas partial (7 cGy) or no change (12 cGy) was seen at the Leydig cell level. Whatever the irradiation protocol, from the time LS numbers decreased, vacuolisation of the Sertoli cell cytoplasm progressively occurred, followed by thickening and folding of the peritubular tissue. Moreover, in parallel to the drop in the number of these germ cell types, ABP production fell whereas FSH levels rose. A highly significant positive correlation was found between LS numbers and these Sertoli cell parameters. This study supports our previous concept of a control of certain important aspects of Sertoli cell function by late spermatids in the adult rat.

  16. Comparison of the effects of two fixatives for immunolocalization of testosterone in the testes of the cynomolgus monkey, mouse and rat.

    PubMed

    Liang, J H; Sankai, T; Yoshida, T; Yoshikawa, Y

    2000-10-01

    We compared the effect of two fixatives, Bouin's fixative and neutralized buffered 4% formaldehyde (10% formalin), for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats. In the samples fixed with Bouin's fixative, immunoreactive testosterone was detected as intense deposits in the cytoplasm of Leydig cells of monkeys and mice. Immunoreactive testosterone was detected not only in Leydig cells of rats but also moderately shown within tubules. Immunoreactive testosterone could not be detected in the testes of monkeys, mice or rats fixed with neutralized buffered formalin because of the poor morphology caused by the fixative. It is concluded that Bouin's fixative is a suitable fixative for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats.

  17. Testicular Development in Male Rats Is Sensitive to a Soy-Based Diet in the Neonatal Period1

    PubMed Central

    Napier, India D.; Simon, Liz; Perry, Devin; Cooke, Paul S.; Stocco, Douglas M.; Sepehr, Estatira; Doerge, Daniel R.; Kemppainen, Barbara W.; Morrison, Edward E.; Akingbemi, Benson T.

    2014-01-01

    ABSTRACT Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets. PMID:24451983

  18. Suppression of rat and human androgen biosynthetic enzymes by apigenin: Possible use for the treatment of prostate cancer.

    PubMed

    Wang, Xiudi; Wang, Guimin; Li, Xiaoheng; Liu, Jianpeng; Hong, Tingting; Zhu, Qiqi; Huang, Ping; Ge, Ren-Shan

    2016-06-01

    Apigenin is a natural flavone. It has recently been used as a chemopreventive agent. It may also have some beneficial effects to treat prostate cancer by inhibiting androgen production. The objective of the present study was to investigate the effects of apigenin on the steroidogenesis of rat immature Leydig cells and some human testosterone biosynthetic enzyme activities. Rat immature Leydig cells were incubated for 3h with 100μM apigenin without (basal) or with 1ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and 20μM of the following steroid substrates: 22R-hydroxychloesterol (22R), pregnenolone (P5), progesterone (P4), and androstenedione (D4). The medium levels of 5α-androstane-3α, 17β-diol (DIOL), the primary androgen produced by rat immature Leydig cells, were measured. Apigenin significantly inhibited basal, 8BR, 22R, PREG, P4, and D4 stimulated DIOL production in rat immature Leydig cells. Further study showed that apigenin inhibited rat 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17, 20-lyase, and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 11.41±0.7, 8.98±0.10, and 9.37±0.07μM, respectively. Apigenin inhibited human 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 with IC50 values of 2.17±0.04 and 1.31±0.09μM, respectively. Apigenin is a potent inhibitor of rat and human steroidogenic enzymes, being possible use for the treatment of prostate cancer.

  19. Aphrodisiac and spermatogenic potential of alkaloidal fraction of Hygrophila spinosa T. Ander in rats.

    PubMed

    Vyas, Niraj Y; Raval, Manan A

    2016-12-24

    Seeds of Hygrophila spinosa T. Ander (Acanthaceae) are traditionally used as aphrodisiac and spermatogenic in Indian System of medicine. Preliminary phytochemical screening of plant revealed the presence of alkaloids in seeds. As, alkaloidal fractions of several plants showed aphrodisiac and spermatogenic potential, set of experiments were designed to assess alkaloid enriched fraction of seeds of the plant for spermatogenic and aphrodisiac activity using in vitro and in vivo methods. Alkaloid enriched fraction was prepared and assessed for spermatogenic activity using isolated rat Leydig cells in vitro. The fraction was further evaluated in vivo for spermatogenic and aphrodisiac potential using rat as an experimental animal. Increase in weight of reproductive organs, biochemical evaluation of selected parameters, histological studies of testes and sexual behavioral studies were selected as evaluation parameters for in vivo studies. Isolated rat Leydig cells treated with the fraction showed increased amount of testosterone present in culture media (14.7µg/ml) as compared to that of control (0.8µg/ml). Results of in vivo studies showed increase in serum testosterone level in treated animals (50mg/kg) by (115%), increase in weight of testes (8.0%) as compared to control. Marked improvement in testis histo-architecture of rats evident preliminarily by observing overcrowding of spermatozoa in enlarged lumen of seminiferous tubules in animals treated with testosterone and test fraction. Sertoli cells in treated animals were enlarged with highly granulated cytoplasm. Leydig cells also showed enlarged nucleus and darkly stained cytoplasm as compared to control. Mounting behavior of test animals improved, while latency period was decreased, as observed in behavioral studies. The set studies confirmed the ability of the fraction to stimulate Leydig cells and increased serum testosterone level. Increased testosterone level might be responsible for higher number of

  20. Diabetic rat testes: morphological and functional alterations.

    PubMed

    Ricci, G; Catizone, A; Esposito, R; Pisanti, F A; Vietri, M T; Galdieri, M

    2009-12-01

    Reproductive dysfunction is a consequence of diabetes, but the underlying mechanisms are poorly understood. This study investigated the histological and molecular alterations in the testes of rats injected with streptozotocin at prepuperal (SPI rats) and adult age (SAI rats) to understand whether diabetes affects testicular tissue with different severity depending on the age in which this pathological condition starts. The testes of diabetic animals showed frequent abnormal histology, and seminiferous epithelium cytoarchitecture appeared altered as well as the occludin distribution pattern. The early occurrence of diabetes increased the percentage of animals with high number of damaged tubules. The interstitial compartment of the testes was clearly hypertrophic in several portions of the organs both in SPI and SAI rats. Interestingly, fully developed Leydig cells were present in all the treated animals although abnormally distributed. Besides the above-described damages, we found a similar decrease in plasma testosterone levels both in SPI and SAI rats. Oxidative stress (OS) is involved in the pathogenesis of various diabetic complications, and in our experimental models we found that manganese superoxide dismutase was reduced in diabetic animals. We conclude that in STZ-induced diabetes, the altered spermatogenesis, more severe in SPI animals, is possibly due to the effect of OS on Leydig cell function which could cause the testosterone decrease responsible for the alterations found in the seminiferous epithelium of diabetic animals.

  1. Influence of streptozotocin-induced diabetes and insulin treatment on the pituitary-testicular axis during sexual maturation in rats.

    PubMed

    Sudha, S; Valli, G; Julie, P M; Arunakaran, J; Govindarajulu, P; Balasubramanian, K

    2000-01-01

    Effects of streptozotocin (STZ)-diabetes and insulin treatment on the functioning of pituitary-testicular axis during sexual maturation was studied. Prepubertal (30 days old) and pubertal (50 days old) male Wistar rats were made diabetic by a single injection of STZ. A group of diabetic rats was given insulin (3U/100 g b.wt./day in 2 equally divided doses), 3 days after STZ treatment. Prepubertal and pubertal rats of all groups were killed on postnatal days 51 and 71, respectively. STZ-diabetes caused marked reduction in serum LH, FSH, prolactin, testosterone and testicular interstitial fluid testosterone as well as the activities of Leydig cellular steroidogenic enzymes (3beta-and 17beta-hydroxysteroid dehydrogenases). Insulin treatment to diabetic rats maintained these changes at control range except FSH and prolactin in prepubertal rats. The results indicate that (i) diabetes-induced steroidogenic lesions in Leydig cells represent a direct consequence of dysfunctioning of pituitary-testicular axis, (ii) the adverse effects of diabetes on pituitary-testicular functions are influenced by age of its induction and (iii) optimum insulin level is essential for the acquisition of Leydig cellular steroidogenic efficacy during sexual development.

  2. DNA methylation analysis in rat kidney epithelial cells exposed to 3-MCPD and glycidol.

    PubMed

    Senyildiz, Mine; Alpertunga, Buket; Ozden, Sibel

    2016-11-24

    3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been regarded as a rat carcinogen, which is known to induce Leydig-cell and mammary gland tumors in males, as well as kidney tumors in both genders. 3-MCPD is highly suspected to be a non-genotoxic carcinogen. 2,3-Epoxy-1-propanol (glycidol) can be formed via dehalogenation from 3-MCPD. We aimed to investigate the cytotoxic effects of 3-MCPD and glycidol, then to demonstrate the possible epigenetic mechanisms with global and gene-specific DNA methylation in rat kidney epithelial cells (NRK-52E). IC50 value of 3-MCPD was determined as 48 mM and 41.39 mM, whereas IC50 value of glycidol was 1.67 mM and 1.13 mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100 μM and 1000 μM for 3-MCPD and 100 μM and 500 μM for glycidol were observed after 48 h exposure by using 5-methylcytosine (5-mC) ELISA kit. Methylation changes were detected in promoter regions of c-myc and Rassf1a in 3-MCPD and glycidol treated NRK-52E cells by using methylation-specific PCR (MSP), whereas changes on gene expression of c-myc and Rassf1a were observed by using real-time PCR. However, e-cadherin, p16, VHL and p15 genes were unmethylated in their CpG promoter regions in response to treatment with 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol.

  3. Transplanted Adipose-Derived Stem Cells Ameliorate Testicular Dysfunction In A D-Galactose-Induced Aging Rat Model.

    PubMed

    Yang, Chun; Du, Yi-Kuan; Wang, Jun; Luan, Ping; Yang, Qin-Lao; Huang, Wen-Hua; Yuan, Lin

    2015-10-01

    Glycation product accumulation during aging of slowly renewing tissues may be an important mechanism underlying aging of the testis. Adipose-derived stem cells (ADSCs) have shown promise in a novel tissue regenerative technique and may have utility in treating sexual dysfunction. ADSCs have also been found to be effective in antiaging therapy, although the mechanism underlying their effects remains unknown. This study was designed to investigate the anti-aging effect of ADSCs in a D-galactose (D-gal)-induced aging animal model and to clarify the underlying mechanism. Randomly selected 6-week-old male Sprague-Dawley rats were subcutaneously injected with D-gal daily for 8 weeks. Two weeks after completion of treatment, D-gal-induced aging rats were randomized to receive caudal vein injections of 3 × 10(6) 5-bromo 2'deoxy-uridine-labeled ADSCs or an equal volume of phosphate-buffered saline. Serum testosterone level, steroidogenic enzymes (3-β-hydroxysteroid dehydrogenase), and superoxide dismutase (SOD) activity decreased significantly in aging rats compared with the control group; serum lipid peroxidation, spermatogenic cell apoptosis, and methane dicarboxylic aldehyde (MDA) expression increased significantly. ADSCs increased the SOD level and reduced the MDA level in the aging animal model and restored levels of serum testosterone, steroidogenic enzymes, and spermatogenic cell apoptosis. These results demonstrate that ADSCs can contribute to testicular regeneration during aging. ADSCs also provide functional benefits through glycation suppression and antioxidant effects in a rat model of aging. Although some ADSCs differentiated into Leydig cells, the paracrine pathway seems to play a main role in this process, resulting in the reduction of apoptosis. © 2015 Wiley Periodicals, Inc.

  4. Zinc content in epididymal spermatozoa of metoclopramide-treated rats.

    PubMed

    Kaminska, B; Rozewicka, L; Dominiak, B; Mielnicka, M; Mikulska, D

    1987-01-01

    Zinc content was determined separately in spermatozoa taken from epididymal caput and cauda in rats. It was revealed that spermatozoa transported from the epididymal caput to the cauda reduce about 54% of zinc. This reduction is significantly inhibited in spermatozoa of rats receiving metoclopramide. That is also accompanied by a fall of testosterone level in blood serum and of delta 5, 3 beta-HSD activity in Leydig cells. It was found out that the reduction of zinc in spermatozoa at the time of their passage through the epididymis is the process that depends on androgens.

  5. Effect of Molybdenum Nanoparticles on Blood Cells, Liver Enzymes, and Sexual Hormones in Male Rats.

    PubMed

    Asadi, Fardin; Mohseni, Mehran; Dadashi Noshahr, Karim; Soleymani, Fariba Haj; Jalilvand, Ahmad; Heidari, Azam

    2017-01-01

    Despite an increasing surge in application of nanoparticles in industries, there is a serious lack of information concerning their impact on human health and the environment. The present study investigated effects of molybdenum nanoparticles (Mo NPs) injected intraperitoneally into Sprague-Dawley rats at different doses of Mo NPs (5, 10, and 15 mg/kg BW per day) during a period of 28 days. Hematological and biochemical parameters as well as sexual hormones and histopathological examinations of the liver and testis were assessed and compared with control group. The results showed that the serum levels of testosterone decreased significantly in both groups of 10 and 15 mg (Mo NPs)/kg BW in comparison with the control group (p < 0.05). However, there were insignificant differences observed in luteinizing hormone (LH) levels and hematological parameters when compared with the control group (p > 0.05). The results of liver enzymes showed that serum levels of aspartate aminotransferase (AST) decreased significantly in both dosage groups of 5 and 10 mg/kg BW (Mo NPs) when compared with the control group (p < 0.05), and significant decrease obtained in lactate dehydrogenase (LDH) levels at dose of 5 mg/kg BW in comparison with the control group (p < 0.05). The histopathological examination of testis showed a decrease in number of Leydig cells. Also, the number of chronic inflammatory cells increased in portal triad and parenchyma in liver tissue of rats exposed to Mo NPs.

  6. 4-Nitro-3-phenylphenol has both androgenic and anti-androgenic-like effects in rats

    PubMed Central

    TRISOMBOON, Jiratthiya; LI, ChunMei; SUZUKI, Akira; WATANABE, Gen; TAYA, Kazuyoshi

    2015-01-01

    To investigate the effect of endocrine disruption of 4-nitro-3-phenylphenol (PNMPP) on immature male Wistar-Imamichi rats, the rat pituitary was exposed to PNMPP (10–5–10–9 M) for 24 h with or without gonadotropin-releasing hormone (GnRH) in experiment I. In addition, the Leydig cells (10–5–10–9 M) were exposed to PNMPP for 24 h with or without human chronic gonadotropin (hCG) in experiment II. Our results showed that the PNMPP at 10–5–10–7 M suppressed follicle-stimulating hormone (FSH) and luteinizing hormone (LH) productions from GnRH-stimulated pituitary cells. At the same time, PNMPP 10–5–10–7 M induced an increase in testosterone production from the Leydig cells treated with or without hCG. Based on our results, it can be concluded that that PNMPP might have both androgen agonist action by decreasing FSH and LH production in the pituitary and anti-androgenic action by increasing testosterone production in the Leydig cell. PMID:25736398

  7. 4-Nitro-3-phenylphenol has both androgenic and anti-androgenic-like effects in rats.

    PubMed

    Trisomboon, Jiratthiya; Li, ChunMei; Suzuki, Akira; Watanabe, Gen; Taya, Kazuyoshi

    2015-01-01

    To investigate the effect of endocrine disruption of 4-nitro-3-phenylphenol (PNMPP) on immature male Wistar-Imamichi rats, the rat pituitary was exposed to PNMPP (10(-5)-10(-9) M) for 24 h with or without gonadotropin-releasing hormone (GnRH) in experiment I. In addition, the Leydig cells (10(-5)-10(-9) M) were exposed to PNMPP for 24 h with or without human chronic gonadotropin (hCG) in experiment II. Our results showed that the PNMPP at 10(-5)-10(-7) M suppressed follicle-stimulating hormone (FSH) and luteinizing hormone (LH) productions from GnRH-stimulated pituitary cells. At the same time, PNMPP 10(-5)-10(-7) M induced an increase in testosterone production from the Leydig cells treated with or without hCG. Based on our results, it can be concluded that that PNMPP might have both androgen agonist action by decreasing FSH and LH production in the pituitary and anti-androgenic action by increasing testosterone production in the Leydig cell.

  8. [Pathologic changes of spontaneous tumors in Sprague-Dawley and Wistar rats].

    PubMed

    He, Y N; Zhang, S C; Zhang, H M

    2017-04-08

    Objective: To investigate the spontaneous neoplastic lesions and their incidences in Sprague-Dawley (SD) and Wistar rats, and to accumulate background data for carcinogenicity studies. Methods: One hundred and eighty SD rats and 240 Wistar rats (4-week old) , half in each sex, were used in this study. The rats were housed routinely under specific pathogen-free environment and euthanized after 104 weeks. Histopathological examination was undertaken for all animals including deaths and scheduled euthanasia. The types and incidences of spontaneous tumors were gathered statistically. Results: Total 411 rats (176 SD rats and 235 Wistar rats) were examined in this study. The total tumor incidence of the 411 rats was 57.7%(237/411). The total tumor incidence in SD rats was 55.7%(98/176), benign tumor incidence was 48.9%(86/176) and malignant tumor incidence was 15.9%(28/176). The total tumor incidence in Wistar rats was 59.1%(139/235), benign tumor incidence was 51.5%(121/235) and malignant tumor incidence was 14.5%(34/235). The main benign tumors were pituitary adenoma (23.3% in SD rats, 12.3% in Wistar rats), breast fibroadenoma (21.3% in SD rats, 12.9% in Wistar rats) and breast adenoma (16.9% in SD rats, 9.5% in Wistar rats) in females; testis Leydig cell tumor (0 in SD rats, 14.3% in Wistar rats) in males. The main malignant tumors were breast carcinoma (10.1% in SD rats, 3.4% in Wistar rats) and uterine leiomyosarcoma (0 in SD rats, 2.6% in Wistar rats) in females; squamous cell carcinoma of skin (2.3% in SD rats, 0.9% in Wistar rats); subcutaneous fibrosarcoma (1.1% in SD rats, 2.1% in Wistar rats); brain malignant glioma (1.1% in SD rats, 1.7% in Wistar rats). Conclusions: In the study, a high incidence of spontaneous tumors is reported in both SD and Wistar rats housed for 2 years. The incidence of benign tumors is higher than that of malignant rumors. The benign tumors mainly are pituitary adenoma, breast fibroadenoma and breast adenoma in females, and testis

  9. A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels.

    PubMed

    Clair, Emilie; Mesnage, Robin; Travert, Carine; Séralini, Gilles-Éric

    2012-03-01

    The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48h of Roundup exposure Leydig cells are damaged. Within 24-48h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

    PubMed Central

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-01-01

    ABSTRACT Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR. PMID:28791335

  11. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

    PubMed

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-06-01

    Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn(56) of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

  12. Metachronous Bilateral Testicular Leydig-Like Tumors Leading to the Diagnosis of Congenital Adrenal Hyperplasia (Adrenogenital Syndrome)

    PubMed Central

    Vukina, Josip; Chism, David D.; Sharpless, Julie L.; Raynor, Mathew C.; Milowsky, Matthew I.; Funkhouser, William K.

    2015-01-01

    A 33-year-old male with a history of left testis Leydig cell tumor (LCT), 3-month status after left radical orchiectomy, presented with a rapidly enlarging (0.6 cm to 3.7 cm) right testicular mass. He underwent a right radical orchiectomy, sections interpreted as showing a similar Leydig cell-like oncocytic proliferation, with a differential diagnosis including metachronous bilateral LCT and metachronous bilateral testicular tumors associated with congenital adrenal hyperplasia (a.k.a. “testicular adrenal rest tumors” (TARTs) and “testicular tumors of the adrenogenital syndrome” (TTAGS)). Additional workup demonstrated a markedly elevated serum adrenocorticotropic hormone (ACTH) and elevated adrenal precursor steroid levels. He was diagnosed with congenital adrenal hyperplasia, 3β-hydroxysteroid dehydrogenase deficiency (3BHSD) type, and started on treatment. Metachronous bilateral testicular masses in adults should prompt consideration of adult presentation of CAH. Since all untreated CAH patients are expected to have elevated serum ACTH, formal exclusion of CAH prior to surgical resection of a testicular Leydig-like proliferation could be accomplished by screening for elevated serum ACTH. PMID:26351608

  13. Derivation of embryonic stem cells from Brown Norway rats blastocysts.

    PubMed

    Zhao, Xiaoyang; Lv, Zhuo; Liu, Lei; Wang, Liu; Tong, Man; Zhou, Qi

    2010-07-01

    Knockout Brown Norway (BN) rat could be a useful disease model for human disorders, however, a failure to derive embryonic stem (ES) cells disturbs the further development of the model. In this study, we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley (SD) rats. The BN rat ES cells expressed the key transcription factors, and were able to form embryonic bodies (EBs) when being differentiated in vitro. After injecting the BN rat ES cells into the SD rat blastocysts, high-contribution chimeric rats were generated and could survive to their adulthood. Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.

  14. [Morphofunctional state of reproductive system of ageing male rats in case of using nanocerium].

    PubMed

    Nosenko, N D; Zholobak, N M; Poliakova, L I; Sinitsyn, P V; Lymarieva, A A; Shcherbakov, O V; Spivak, M Ia; Reznikov, O H

    2014-01-01

    The influence of nanocrystalline cerium dioxide (NCD, 1 and 100 mg/kg per os daily for 10 days) on morphofuctional state of reproductive system was investigated in ageing male rats. It has been established that activation of hormone-producing testicular Leydig's cells, as well as of secretory and proliferative processes in prostate, underlies the stimulating effect of NCD at a dose 1 mg/kg on hormonal function of testis and spermatogenesis of ageing male rats. NCD used at a dose 100 mg/kg had no significant effect on the assessed indices of morphofuctional state of reproductive system.

  15. Starvation is more efficient than the washing technique for purification of rat Sertoli cells.

    PubMed

    Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohamadreza Baghaban; Sedighi-Gilani, Mohammadali; Mokarizadeh, Aram

    2014-09-01

    Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p < 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.

  16. The Effect of Early Mosquito Insecticides Exposure on Spraque Dawley Rat Testis: A Histopathological Feature Towards Malignancy?

    NASA Astrophysics Data System (ADS)

    Indah Winarni, Tri; Auzan Aziman, Milzam; Abshar Andar, Anindyo; Pawitra, Ika

    2017-02-01

    The incidence of health problems associated with endocrine-disruption have increased. Many studies suggesting that endocrine disruptor chemicals (EDC) do contribute to cancer through estrogen-related receptors. Many chemicals have EDCs properties including insecticides. Early life exposure to EDCs can increased the risk of testicular cancer have been reported in the last decade. This study was aimed to determine the effect of insecticides exposure on histopathological tumor cell development of germ and Leydig cell. True experiment research design with posttest only control group design was applied. Sprague Dawley (SD) rat (n = 25) were randomly divided into 5 groups (control group, 25 mg β estradiol 3-benzoate, spiral mosquito coil repellent, 3 ml of liquid mosquito repellent, and 4 ml of liquid mosquito repellent). The exposure were administered for 20 days started at aged 3 days. At the age of 100 days (older adult), testis was stained using Hematoxyllin Eosin (HE) and histological features predicting malignancy were observed. The number of tumor cell development in both testicular germ cells and Leydig cells significantly increased in all treated group compared to those of control and the changes towards malignancy were also observed in all treated group. Exposure to mosquito insecticides causes significant changes in testicular germ and Leydig cell histological features that leads to malignancy.

  17. Neurofilament expression in cultured rat adenohypophysial cells.

    PubMed

    Quintanar, J L; Salinas, E

    2001-01-01

    The aim of the present work was to investigate in cultured rat adenohypophysial cells: a) the presence of neurofilaments of 200 kDa (NF-H), b) the effect of thyroid hormone (T(3)) and thyrotropin releasing hormone (TRH) on the expression of NF-H and c) the possible role of NF-H on thyrotropin (TSH) secretion. The presence of NF-H was observed by immunocytochemistry in cultured rat adenohypophysial cells. The exposure to T(3) for 12 h produced a significant increase in NF-H expression; whereas incubation with TRH or T(3)+TRH resulted in no change. The cells treated with T(3) or TRH or T(3)+TRH for 24 h showed no alteration. However, incubation for 48 h with TRH or T(3)+TRH caused significant decrease in NF-H expression. Incubation with NF-H antibodies produced a significant inhibition of calcium-induced TSH release in digitonin-permeabilized adenohypophysial cells. These results provide evidence that NF-H is present in cultured rat adenohypophysial cells, and that T(3) and TRH can modify NF-H expression. It can be suggested that in cultured adenohypophysial cells, NF-H may play a role in the secretory process.

  18. Valproate inhibits GnRH-induced gonadotropin release from anterior pituitary cells of male rat in vitro.

    PubMed

    Wasilewska-Dziubinska, Elzbieta; Gajewska, Alina; Wolinska-Witort, Ewa; Chmielowska, Magdalena; Martynska, Lidia; Elbanowski, Jerzy; Kochman, Kazimierz

    2011-01-01

    Valproate (VPA) a potent antiepileptic drug has been claimed to induce reproductive disturbances in men. Long-term VPA treatment can affect sperm morphology and induce testicular atrophy in non-epileptic rats. It has been reported that VPA reduced testosterone secretion stimulated by hCG in isolated rat Leydig cells. These results suggest direct effect of VPA on testes in rats. However centrally mediated effects at hypothalamo-pituitary level can therefore not be excluded. This study focused on the dose and time-dependent effects of VPA on basal and GnRH-induced LH and FSH release from the primary anterior pituitary cells culture of male rats. The dose-dependent effect of 10 nM-100 mM of VPA on basal LH release from anterior pituitary cells after 3h of incubation was examined. To determine the time-dependent effects on LH, FSH, TSH and PRL release short (3 h) and long-term (24 h) incubations in the presence of 10 nM, 100 nM and 1 μM of VPA were maintained.To assess whether VPA can affect GnRH-induced LH and FSH release, cells were incubated for 3 h with 10 nM, 100 nM and 1 μM of VPA in the presence of GnRH. The concentration of rLH, rFSH, rPRL and rTSH in incubation medium was determined by RIA method. VPA did not affect the basal LH, FSH, PRL and TSH release from the primary anterior pituitary cells culture of male rats. VPA in concentration 1µM significantly suppressed GnRH-induced LH secretion. However VPA at all tested doses diminished GnRH-induced FSH release. VPA may diminish gonadotropin release in vitro but this effect can only be achieved after GnRH-dependent specific receptor activation. Both gonadotropins differ in their pattern of response for increasing doses of VPA.

  19. Pancreas Transplantation Delays the Progression of Morphological, Morphometric and Ultrastructural Changes in Testes of Alloxan-Induced Diabetic Rats.

    PubMed

    Tadeu Spadella, César; Teixeira Trindade, Amélia Arcângela; Natália Lucchesi, Amanda; Sperandéo de Macedo, Célia

    2017-02-01

    Aim: The aim of this study was to assess the effects of pancreas transplantation on the progression of testicular lesions in diabetic rats. Methods: Diabetic rats were subjected to pancreas transplantation and sacrificed after 6, 14, 26 and 50 weeks of follow-up, using non-diabetic and untreated diabetic rats as controls. Results: Successful pancreas transplantation corrected all of the metabolic changes observed in diabetic rats, including low levels of testosterone. The testicular mass was decreased, and the relative weight of the testes was high in diabetic rats. The seminiferous tubules of diabetic rats showed progressive atrophy of the germinal epithelium, with cytoplasmic vacuolization, detachment of germ cells to the tubular lumen and the appearance of giant cells. Leydig cells were abnormally distributed, and hyperplasia of Sertoli cells was observed. Sperm were not detectable within the tubular lumen in late follow-up. The diameter, total area, lumen area, and germinal epithelium area of the seminiferous tubules were low, and tubular density was high in diabetic rats. Ultrastructural changes were also observed in these rats, compromising the cytoplasm, organelles and cellular nuclei of the germ, Sertoli, and Leydig cells. The most frequent changes consisted of accumulation of lipid droplets and electron-dense dark material in the cell cytoplasm, cellular degeneration and apoptosis. Similar to non-diabetic rats, pancreas-transplanted rats showed progressive testicular lesions, but they were much less severe and occurred much later than in the untreated diabetic controls. Conclusion: Diabetes causes morphological and ultrastructural changes in rat testes, but the progression of lesions can be significantly delayed by successful pancreas transplantation, which may have a positive impact on male infertility due to diabetes.

  20. Effects of arecoline on testosterone release in rats.

    PubMed

    Wang, Shyi-Wu; Hwang, Guey-Shyang; Chen, Te-Jung; Wang, Paulus S

    2008-08-01

    Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 microg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentrations of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34 degrees C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10(-8) to 10(-6) M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10(-6) M), or 8-Br-cAMP (10(-5) M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17beta-hydroxysteroid dehydrogenase and enhancing the expression of StAR.

  1. Carcinogenicity in rats of the SGLT2 inhibitor canagliflozin.

    PubMed

    De Jonghe, Sandra; Proctor, Jim; Vinken, Petra; Feyen, Bianca; Wynant, Inneke; Marien, Dirk; Geys, Helena; Mamidi, Rao N V S; Johnson, Mark D

    2014-12-05

    The carcinogenicity potential of canagliflozin, an inhibitor of SGLT2, was evaluated in a 2-year rat study (10, 30, and 100 mg/kg). Rats showed an increase in pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors. Systemic exposure multiples at the highest dose relative to the maximum clinical dose were 12- to 21-fold. Pheochromocytomas and renal tubular tumors were noted in both sexes at 100 mg/kg. Leydig cell tumors were observed in males in all dose groups and were associated with increased luteinizing hormone levels. Hyperplasia was increased in the adrenal medulla at 100 mg/kg, but only a limited increase in simple tubular hyperplasia was observed in the kidney of males at 100 mg/kg. Hyperostosis occurred and was accompanied by substantial effects on calcium metabolism, including increased urinary calcium excretion and decreased levels of calcium regulating hormones (1,25-dihydroxyvitamin D and parathyroid hormone). A separate study with radiolabeled calcium confirmed that increased urinary calcium excretion was mediated via increased calcium absorption from the gastrointestinal tract. It was hypothesized that, at high doses, canagliflozin might have inhibited glucose absorption in the intestine via SGLT1 inhibition that resulted in glucose malabsorption, which increased calcium absorption by stimulating colonic glucose fermentation and reducing intestinal pH. Pheochromocytomas and adrenal medullary hyperplasia were attributed to altered calcium homeostasis, which have a known relationship in the rat. In conclusion, Leydig cell tumors were associated with increased luteinizing hormone levels and pheochromocytomas were most likely related to glucose malabsorption and altered calcium homeostasis. Renal tubular tumors may also have been linked to glucose malabsorption. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Experiment K-7-16: Effects of Microgravity or Simulated Launch on Testicular Function in Rats

    NASA Technical Reports Server (NTRS)

    Amann, R. P.; Clemens, J. W.; Deaver, D.; Folmer, J.; Zirkin, B.; Veeramachaneni, D. N. R.; Grills, G. S.; Gruppi, C. M.; Wolgemuth, D.; Serova, L. V.; hide

    1994-01-01

    Fixed or frozen testicular tissues from five rats per group were analyzed by: subjective and quantitative evaluations of spermatogenesis; Northern-blot analysis for expression of selected genes; quantification of testosterone and receptors for LH; and morphometric analysis of Leydig cells. Based on observations of fixed tissue, it was evident that some rats in the flight and vivarium groups had testicular abnormalities unassociated with treatment, and probably existing when they were assigned randomly to the four treatment groups; the simulated-launch group contained no abnormal rat. Lesions induced in testes of caudal-elevation rats precluded discernment of any pre-existing abnormality. Considering rats without pre-existing abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (E less than 0.05) in flight rats than in simulated-launch or vivarium rats. However, ratios of germ cells to each other, or to Sertoli cells, and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. There was no effect of flight on normal expression of testis-specific hsp gene products, or evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rLH in testicular tissue, and surface densities of smooth endoplasmic reticulum and peroxisomes in Leydig cells, were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20 percent of values for simulated-launch or vivarium controls. Thus, spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Sequela of reduced androgen production on turnover of muscle and bone should be considered when interpreting data from mammals exposed to microgravity.

  3. Experiment K-7-16: Effects of Microgravity or Simulated Launch on Testicular Function in Rats

    NASA Technical Reports Server (NTRS)

    Amann, R. P.; Clemens, J. W.; Deaver, D.; Folmer, J.; Zirkin, B.; Veeramachaneni, D. N. R.; Grills, G. S.; Gruppi, C. M.; Wolgemuth, D.; Serova, L. V.; Sapp, W. J.; Williams, C. S.

    1994-01-01

    Fixed or frozen testicular tissues from five rats per group were analyzed by: subjective and quantitative evaluations of spermatogenesis; Northern-blot analysis for expression of selected genes; quantification of testosterone and receptors for LH; and morphometric analysis of Leydig cells. Based on observations of fixed tissue, it was evident that some rats in the flight and vivarium groups had testicular abnormalities unassociated with treatment, and probably existing when they were assigned randomly to the four treatment groups; the simulated-launch group contained no abnormal rat. Lesions induced in testes of caudal-elevation rats precluded discernment of any pre-existing abnormality. Considering rats without pre-existing abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (E less than 0.05) in flight rats than in simulated-launch or vivarium rats. However, ratios of germ cells to each other, or to Sertoli cells, and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. There was no effect of flight on normal expression of testis-specific hsp gene products, or evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rLH in testicular tissue, and surface densities of smooth endoplasmic reticulum and peroxisomes in Leydig cells, were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20 percent of values for simulated-launch or vivarium controls. Thus, spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Sequela of reduced androgen production on turnover of muscle and bone should be considered when interpreting data from mammals exposed to microgravity.

  4. Cell-surface marker analysis of rat thymic dendritic cells.

    PubMed Central

    Bañuls, M P; Alvarez, A; Ferrero, I; Zapata, A; Ardavin, C

    1993-01-01

    Rat thymic dendritic cells have been isolated by collagenase digestion, separation of the low-density cell fraction by centrifugation on metrizamide, and differential adherence. The resulting dendritic cell preparation had a purity of > 90%, and has been analysed by flow cytometry (FCM) using a large panel of monoclonal antibodies (mAb). Dendritic cells expressed major histocompatibility (MHC) class I and class II molecules, the leucocyte common antigen CD45, the rat leucocyte antigen OX44, the rat macrophage marker ED1, and the adhesion molecules Mac-1, LFA-1 and ICAM-1. They were negative for the T- and B-cell-specific forms of CD45, CD45R and B220, and the B-cell marker OX12. Concerning T-cell marker expression, they were negative for T-cell receptor (TcR) and OX40, but they expressed CD2, CD4 and CD8, and interestingly, 50% of DC were CD5+, 50% expressed the alpha-chain of interleukin-2 receptor (IL-2R), and 80% were positive for the T-cell activation antigen recognized by the mAb OX48. Moreover, 60% of DC expressed high levels of Thy-1, whereas 40% displayed intermediate levels of this T-cell marker. PMID:8102122

  5. Paraptosis-like cell death in Wistar rat granulosa cells.

    PubMed

    Torres-Ramírez, Nayeli; Escobar, María L; Vázquez-Nin, Gerardo H; Ortiz, Rosario; Echeverría, Olga M

    2016-10-01

    Follicular atresia, a common process present in all mammals, involves apoptotic and autophagic cell death. However, the participation of paraptosis, a type of caspase-independent cell death, during follicular atresia is unknown. This study found swollen endoplasmic reticulum in the granulosa cells of adult Wistar rats. Calnexin was used as a marker of the endoplasmic reticulum at the ultrastructural and optical levels. The cells with swelling of the endoplasmic reticulum were negative to the TUNEL assay and active caspase-3 immunodetection, indicating that this swelling is not part of any apoptotic or autophagic process. Additionally, immunodetection of the CHOP protein was used as a marker of endoplasmic reticulum stress, and this confirmed the presence of the paraptosis process. These data suggest that paraptosis-like cell death is associated with the death of granulosa cells during follicular atresia in adult Wistar rats.

  6. Effects of gamma rays on rat testis tissue according to the morphological parameters and immunohistochemistry: radioprotective role of silymarin

    PubMed Central

    Marzban, Mohsen; Anjamshoa, Maryam; Jafari, Parnia; Masoumi, Hossien; Ahadi, Reza; Fatehi, Daryoush

    2017-01-01

    Objective To determine the radioprotective effects of Silymarin in adult male Sprague-Dawley rats irradiated with γ-rays. Methods The present experimental study was performed in Tehran University of Medical Sciences, Tehran, Iran from December 2009 to March 2010. The study was performed on 40 rats, which were randomly and equally divided into four groups: 1) control group: neither received Silymarin nor irradiated with γ-rays; 2) γ-irradiation group: testis region exposed to 2Gy of γ-rays; 3) Silymarin & γ-irradiation: rats received 100 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays; 4) Silymarin & γ-irradiation: rats received 200 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays. After animal experiments and preparing the tissue sections, different histological and histomorphological parameters of seminiferous tubules and the biological characteristics of Leydig cells were evaluated applying quantitative assessment, Johnson scoring, and Leydig cell apoptosis assay by TUNEL method. The data were analyzed applying ANOVA and Tukey’s post hoc test, using SPSS software (V.19). Results Irradiation of 2 Gy γ-rays to the testis of the rats significantly affected the frequency of spermatogonia, primary spermatocyte, round spermatid, spermatozoa, seminiferous tube and lumen diameters, thickness of the epithelium, Leydig cell nuclear diameter and volume, epithelium height, and apoptotic cells (p<0.05). However, administration of Silymarin improved the mentioned parameters specifically in 200 mg/kg of dosage. Conclusion Silymarin could act as a potent radioprotector and it can be used in modulation as well as improvement to radiation therapy to prevent male reproductive function, specifically seminiferous tubules in an animal model; however, its molecular mechanism is still not clear and needs more molecular researches. PMID:28848626

  7. Effects of gamma rays on rat testis tissue according to the morphological parameters and immunohistochemistry: radioprotective role of silymarin.

    PubMed

    Marzban, Mohsen; Anjamshoa, Maryam; Jafari, Parnia; Masoumi, Hossien; Ahadi, Reza; Fatehi, Daryoush

    2017-06-01

    To determine the radioprotective effects of Silymarin in adult male Sprague-Dawley rats irradiated with γ-rays. The present experimental study was performed in Tehran University of Medical Sciences, Tehran, Iran from December 2009 to March 2010. The study was performed on 40 rats, which were randomly and equally divided into four groups: 1) control group: neither received Silymarin nor irradiated with γ-rays; 2) γ-irradiation group: testis region exposed to 2Gy of γ-rays; 3) Silymarin & γ-irradiation: rats received 100 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays; 4) Silymarin & γ-irradiation: rats received 200 mg/kg of Silymarin 24hrs before exposure to 2Gy of γ-rays. After animal experiments and preparing the tissue sections, different histological and histomorphological parameters of seminiferous tubules and the biological characteristics of Leydig cells were evaluated applying quantitative assessment, Johnson scoring, and Leydig cell apoptosis assay by TUNEL method. The data were analyzed applying ANOVA and Tukey's post hoc test, using SPSS software (V.19). Irradiation of 2 Gy γ-rays to the testis of the rats significantly affected the frequency of spermatogonia, primary spermatocyte, round spermatid, spermatozoa, seminiferous tube and lumen diameters, thickness of the epithelium, Leydig cell nuclear diameter and volume, epithelium height, and apoptotic cells (p<0.05). However, administration of Silymarin improved the mentioned parameters specifically in 200 mg/kg of dosage. Silymarin could act as a potent radioprotector and it can be used in modulation as well as improvement to radiation therapy to prevent male reproductive function, specifically seminiferous tubules in an animal model; however, its molecular mechanism is still not clear and needs more molecular researches.

  8. The influence of hollyhock extract administration on testicular function in rats.

    PubMed

    Papiez, Monika A

    2004-11-01

    It has been reported, recently that an aqueous extract from hollyhock flowers (Althaea rosea Cav. varietas nigra) induces weak metabolic changes in rat testes. In the present study, the in vivoinfluence of a methanolic extract was investigated on the metabolism and morphology of the rat testis. To this end, histochemical, morphometric and radioimmunological methods were used. The rats drank the extract at a dose of 100 mg/day for 7 weeks. The histochemical activities of glucose-6-phosphate dehydrogenase (G6PDH) and Delta(5)beta-hydroxysteroid dehydrogenase (Delta(5)betaHSD) increased significantly statistically in the Leydig cells of the experimental rats in comparison with controls. There were no significant changes in either the diameter of seminiferous tubules or the height of seminiferous epithelium after hollyhock administration. Further, only a small amount of hyperplasia of the interstitial tissue was observed. The morphological and histoenzymatic changes in the Leydig cells indicate that the methanolic hollyhock extract has a direct but small influence on rat testes. The insignificant changes in testicular testosterone and estradiol content suggest that the extract does not disturb steroidogenesis.

  9. Cell Therapy for Liver Disease Using Bioimaging Rats

    PubMed Central

    Haga, Junko; Enosawa, Shin; Kobayashi, Eiji

    2017-01-01

    Advances in stem cell research suggest that cell therapy is a potential alternative to liver transplantation. The use of individualized and minimally invasive cell therapy is desirable to avoid rejection and reduce patient burden. While allo-hepatocyte transplantation has been performed for metabolic hepatic disease, auto-bone marrow transplantation (BMT) has shifted toward mesenchymal stem cells (MSCs) transplantation for liver cirrhosis. In this article, an overview of cell transplantation research for liver disease is provided through our recent rat studies. We have developed various kinds of rat imaging models and have evaluated the effect of cell therapy for liver disease. Bone marrow cells (BMCs) of the Alb-DsRed2 rat were transplanted via the portal vein (PV) in acute and chronic liver damage models. The number of Alb-DsRed2+ albumin-producing cells increased, and the size of the cells increased in the chronic liver damage model as well as in the acute liver damage model. Luciferase transgenic (luc-Tg) rat hepatocytes were transplanted into the hepatectomized LEW rat via the PV. Luminescence intensity lasted for 2 months in the hepatectomized rat. BMCs obtained from green fluorescent protein (GFP) Tg rats were transplanted repeatedly via the PV using an implanted catheter with a port. Repeated BMT via the PV reduced the liver fibrosis. Adipocyte-derived MSCs from the luc-Tg rat were transplanted into the hepatectomized rat model via the PV after ischemic reperfusion. MSCs inhibited hepatocyte apoptosis and promoted liver regeneration. Transplanting the optimal number of cells by an effective and safe way is important for clinical application. Bioimaging rats are a powerful tool for cell transplantation research because it makes observation of the in vivo kinetics of transplanted cells possible. Cell transplantation research using bioimaging rats contributes greatly to evaluating effective methods of cell therapy. PMID:28174669

  10. Antioxidant protective effect of honey in cigarette smoke-induced testicular damage in rats.

    PubMed

    Mohamed, Mahaneem; Sulaiman, Siti Amrah; Jaafar, Hasnan; Sirajudeen, Kuttulebbai Nainamohamed Salam

    2011-01-01

    Cigarette smoke (CS) can cause testicular damage and we investigated the possible protective effect of honey against CS-induced testicular damage and oxidative stress in rats. CS exposure (8 min, 3 times daily) and honey supplementation (1.2 g/kg daily) were given for 13 weeks. Rats exposed to CS significantly had smaller seminiferous tubules diameter and epithelial height, lower Leydig cell count and increased percentage of tubules with germ cell loss. CS also produced increased lipid peroxidation (TBARS) and glutathione peroxidase (GPx) activity, as well as reduced total antioxidant status (TAS) and activities of superoxide dismutase (SOD) and catalase (CAT). However, supplementation of honey significantly reduced histological changes and TBARS level, increased TAS level, as well as significantly restored activities of GPx, SOD and CAT in rat testis. These findings may suggest that honey has a protective effect against damage and oxidative stress induced by CS in rat testis.

  11. Testicular germ-cell apoptosis in stressed rats following combined exposure to pyridostigmine bromide, N,N-diethyl m-toluamide (DEET), and permethrin.

    PubMed

    Abou-Donia, Mohamed B; Suliman, Hagir B; Khan, Wasiuddin A; Abdel-Rahman, Ali A

    2003-01-10

    This study reports and characterizes the testicular apoptosis following daily exposure of male Sprague-Dawley rats to subchronic combined doses of pyridostigmine bromide (PB, 1.3 mg/kg/d in water, oral), a drug used for treatment of myasthenia gravis and prophylactic treatment against nerve agents during the Persian Gulf War; the insect repellent N,N-diethyl m-toluamide (DEET, 40 mg/kg/d in ethanol, dermal); and the insecticide permethrin (0.13 mg/kg in ethanol, dermal), with and without stress for 28 d. Combined exposure to these chemicals was implicated in the development of illnesses including genitourinary disorders among many veterans of the Persian Gulf War. Previous studies from this laboratory have shown that exposure to combination of these chemicals produced greater toxicity compared to single components. Exposure to stress alone did not cause any significant histopathological alterations in the testes. Administration of combination of these chemicals induced apoptosis in rat testicular germ cells, Sertoli cells, and Leydig cells, as well as in the endothelial lining of the blood vessels. Testicular damage was significantly augmented when the animals were further exposed to a combination of chemicals and stress. Histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells and spermatocytes, indicating suppression of spermatogenesis. Increased apoptosis of testicular cells coincided, in timing and localization, with increased expression of the apoptosis-promoting proteins Bax and p53. Furthermore, significant increase of 3-nitrotyrosine immunostaining in the testis revealed oxidative and/or nitrosation induction of cell death. In conclusion, combined exposure to real-life doses of test compounds caused germ-cell apoptosis that was significantly enhanced by stress.

  12. Cell cycle of globose basal cells in rat olfactory epithelium.

    PubMed

    Huard, J M; Schwob, J E

    1995-05-01

    The olfactory epithelium of adult mammals has the unique property of generating olfactory sensory neurons throughout life. Cells of the basal compartment, which include horizontal and globose basal cells, are responsible for the ongoing process of neurogenesis in this system. We report here that the globose basal cells in olfactory epithelium of rats, as in mice, are the predominant type of proliferating cell, and account for 97.6% of the actively dividing cells in the basal compartment of the normal epithelium. Globose basal cells have not been fully characterized in terms of their proliferative properties, and the dynamic aspects of neurogenesis are not well understood. As a consequence, it is uncertain whether cell kinetic properties are under any regulation that could affect the rate of neurogenesis. To address this gap in our knowledge, we have determined the duration of both the synthesis phase (S-phase) and the full cell cycle of globose basal cells in adult rats. The duration of the S-phase was found to be 9 hr in experiments utilizing sequential injections of either IdU followed by BrdU or 3H-thy followed by BrdU. The duration of the cell cycle was determined by varying the time interval between the injections of 3H-thy and BrdU and tracking the set of cells that exit S shortly after the first injection. With this paradigm, the interval required for these cells to traverse G2, M, G1, and a second S-phase, is equivalent to the duration of one mitotic cycle and equals 17 hr. These observations serve as the foundation to assess whether the cell cycle duration is subject to regulation in response to experimental injury, and whether such regulation is partly responsible for changes in the rate of neurogenesis in such settings.

  13. Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Takemoto, N; Sanae, F; Koshiura, R

    1985-05-01

    The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

  14. Induction of antifertility with lupeol acetate in male albino rats.

    PubMed

    Gupta, R S; Bhatnager, A K; Joshi, Y C; Sharma, M C; Khushalani, Veena; Kachhawa, J B S

    2005-10-01

    The present study was undertaken to evaluate the antifertility activity of the active principle, i.e. lupeol acetate, isolated from benzene extract of Alstonia scholaris in male albino rats. The treatment with lupeol acetate at the dose level of 10 mg/rat/day did not cause any significant change in the body weights, but significant reduction in the weight of reproductive organs, i.e. testes, epididymides, seminal vesicle and ventral prostate, was observed. Testicular sperm count, epididymal sperm count and motility were found significantly declined when compared with controls, which resulted in reduction of male fertility by 100%. Arrest of spermatogenesis was noted at various stages with production of primary spermatocytes (preleptotene and pachytene), secondary spermatocytes and step-19 spermatids were decreased by 52.36, 54.91, 55.67 and 69.65%, respectively. The seminiferous tubules appeared reduced in size by 24.62%. Cross-sectional surface area of Sertoli cells as well as their counts were found to be significantly depleted. Leydig cell nuclear area and number of mature Leydig cells were decreased by 27.65 and 35.47%. Biochemical parameters of tissues i.e. protein, sialic acid, glycogen and cholesterol content of testes and seminal vesicular fructose also showed significant reduction. Copyright 2005 S. Karger AG, Basel.

  15. Effects of whole body CO60 irradiation on the male genital tract of gerbil (Meriones hurrianae), hedgehod (hemiechinus auratus Collaris), and house rat (Rattus rattus Rufescens).

    PubMed

    Dixit, V P

    1977-06-01

    1. The effects of 400 and 575 rads of whole body irradiation in gerbil (Meriones hurrianae), hedgehog (Hemiechinus auratus Collaris) and house rat have been studied with a view to compare the effects of irradiation on the desert mammalian species with those of domestic ones. 2. Typical signs and symptoms of radiation sickness, transient body loss and radiation mortality in gerbils and house-rats were apparent, whereas the hedgehogs were not affected at this dose level. 3. Extensive necrosis, exfoliation of seminiferous element, intertubular edema and shrinkage of the seminiferous tubule and Leydig cell nuclei in gerbils and house-rats were conspicuous in histological preparations. Mild regressive tubular lesions were also observed in hedgehogs treated with 575R. 4. Whole body irradiation depleted the RNA, protein and sialic acid contents of the testes. High cholesterol contens in the testes of irradiated gerbil, hedgehog and house rats identified this as the site of degenerating cells and fragments. 5. Reduced androgen production was confirmed in non active Leydig cells and decreased fructose contents in the seminal vesicles of gerbil and house rats. At 575 R dose level, hedgehog was comparatively radio-resistant. But at 875 R extensive damage to the reproductive organs could be noticed.

  16. Studies on gonadotropin receptor of rat ovary and testis

    SciTech Connect

    Zhang, Q.

    1989-01-01

    The subunit structure of the testicular LH/hCG receptor was studied by a chemical cross-linking technique. Leydig cells isolated from rat testis were incubated with {sup 125}I-hCG, following which the bound {sup 125}I-hCG was covalently cross-linked to the receptor on the cell surface with a cleavable or a non-cleavable cross-linking reagent. The hormone-receptor complex was extracted and then either subjected to gel permeation chromatography under nondenaturing conditions, or resolved by SDS-polyacrylamide gel electrophoresis, followed by autoradiographic analysis. The ovarian LH/hCG receptor was studied with luteal cells from pseudopregnant rats. Purification of the receptor was achieved by ligand affinity chromatography following detergent solubilization of the plasma membrane. The purified hCG receptor displayed properties identical to the membrane bound receptor with regard to binding specificity and affinity, and exhibited a molecular weight of approximately 130,000 dalton.

  17. Generation of transgenic rats through induced pluripotent stem cells.

    PubMed

    Jiang, Ming-Gui; Li, Tianda; Feng, Chunjing; Fu, Rui; Yuan, Yan; Zhou, Quan; Li, Xin; Wan, Haifeng; Wang, Liu; Li, Wei; Xiao, Yamei; Zhao, Xiao-Yang; Zhou, Qi

    2013-09-20

    The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.

  18. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  19. The toxic effects of sodium fluoride on the reproductive system of male rats.

    PubMed

    Gupta, R S; Khan, T I; Agrawal, D; Kachhawa, J B S

    2007-10-01

    The present study was undertaken to evaluate the effect of fluoride toxicity on the reproductive system of male rats. Sexually mature male Wistar rats were exposed to 2, 4, and 6 ppm sodium fluoride in their drinking water for 6 months ad libitum. Sperm motility and density in cauda epididymis were assessed. Biochemical and histological analysis were performed in reproductive organs. Fluoride treatment brought about a significant decrease in the weight of testis, epididymis, and ventral prostate. The sperm motility and density were significantly reduced. There was a marked reduction in the number of primary spermatocyte, secondary spermatocyte, and spermatids. The Sertoli cell counts and their cross sectional surface areas were significantly decreased. The Leydig cell nuclear area and the number of mature Leydig cells were also significantly decreased. The protein content of the testis and epididymis were significantly reduced. Fructose in the seminal vesicles and cholesterol in testes were increased significantly. In conclusion, sodium fluoride administrated in drinking water of 2, 4, and 6 ppm concentration for 6 months to male rats adversely affected their fertility and reproductive system.

  20. Bioengineered teeth from cultured rat tooth bud cells.

    PubMed

    Duailibi, M T; Duailibi, S E; Young, C S; Bartlett, J D; Vacanti, J P; Yelick, P C

    2004-07-01

    The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

  1. Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas.

    PubMed

    Iscache, Anne-Laure; Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Pedros, Christophe; Saoudi, Abdelhadi; Buelow, Roland; Anegon, Ignacio

    2011-10-01

    The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

  2. Localization of ANP-synthesizing cells in rat stomach

    PubMed Central

    Li, Chun-Hui; Pan, Li-Hui; Li, Chun-Yu; Zhu, Chang-Lin; Xu, Wen-Xie

    2006-01-01

    AIM: To study the morphological positive expression of antrial natriuretic peptide (ANP)-synthesizing cells and ultrastructural localization and the relationship between ANP-synthesizing cells and microvessel density in the stomach of rats and to analyze the distribution of the three histologically distinct regions of ANP-synthesizing cells. METHODS: Using immunohistochemical techniques, we studied positive expression of ANP-synthesizing cells in rat stomach. A postembedding immunogold microscopy technique was used for ultrastructural localization of ANP-synthesizing cells. Microvessel density in the rat stomach was estimated using tannic acid-ferric chloride (TAFC) method staining. Distribution of ANP-synthesizing cells were studied in different regions of rat stomach histochemically. RESULTS: Positive expression of ANP-synthesizing cells were localized in the gastric mucosa of rats. Localization of ANP-synthesizing cells identified them to be enterochrochromaffin cells (EC) by using a postembedding immunogold electron microscopy technique. EC cells were in the basal third of the cardiac mucosa region. ANP-synthesizing cells existed in different regions of rat stomach and its density was largest in the gastric cardiac region, and the distribution order of ANP-synthesizing cells in density was cardiac region, pyloric region and fundic region in mucosa layer. We have also found a close relationship between ANP-synthesizing cells and microvessel density in gastric mucosa of rats using TAFC staining. CONCLUSION: ANP-synthesizing cells are expressed in the gastric mucosa. EC synthesize ANP. There is a close relationship between ANP-synthesizing cells and microvessel density in gastric mucosa of rats.The distribution density of ANP-synthesizing cells is largest in the gastric cardiac region. PMID:17007021

  3. Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

    PubMed

    Lobo, M V T; Arenas, M I; Huerta, L; Sacristán, S; Pérez-Crespo, M; Gutiérrez-Adán, A; Díaz-Gil, J J; Lasunción, M A; Martín-Hidalgo, A

    2015-01-15

    The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3β-hydroxysteroid dehydrogenase (3β-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3β-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery. Copyright © 2015 the American Physiological Society.

  4. Moringa oleifera extract enhances sexual performance in stressed rats*

    PubMed Central

    Prabsattroo, Thawatchai; Wattanathorn, Jintanaporn; Iamsaard, Sitthichai; Somsapt, Pichet; Sritragool, Opass; Thukhummee, Wipawee; Muchimapura, Supaporn

    2015-01-01

    Aphrodisiacs are required to improve male sexual function under stressful conditions. Due to the effects of oxidative stress and dopamine on male sexual function, we hypothesized that Moringa oleifera leaves might improve male sexual dysfunction induced by stress. Therefore, the effects on various factors playing important roles in male sexual behavior, such as antioxidant effects, the suppression of monoamine and phosphodiesterase type 5 (PDE-5) activities, serum testosterone and corticosterone levels, and histomorphological changes in the testes, of a hydroethanolic extract of M. oleifera leaves were investigated. Various doses of extract including 10, 50, and 250 mg/kg body weight (BW) were given orally to male Wistar rats before e