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Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells
Abstract
Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L⁻¹ after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSD gene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.
... At present, studies are underway into intracellular signal pathways and additional mechanisms of testicular steroidogenesis; however, such inquiries are difficult due to low sensitivity and specificity of existing methods [28,[30][31][32]. Given a similar chemical structure of key steroid hormones, their precursors and their metabolites, and the difficulties of differentiating between them through standard immunoenzymometric methods of diagnostics [11][12][13], HPLC-MS/MS is currently the optimal method of analysing the components of steroidogenesis, as it provides virtually 100% selectivity, an ample sensitivity and repeatability [13][14][15]. ...
BACKGROUND: Type 2 diabetes mellitus (DM2) in men is associated with a high incidence of hypogonadism. Testosterone is a steroid hormone and one of the final metabolites of steroidogenesis, which causes interest in assessing the content of key steroid hormones, their precursors and metabolites in hypogonadal and eugonadal men with T2DM.
AIMS: Assessment of the features of steroidogenesis in men with hypogonadism in T2DM using tandem mass spectrometry.
MATERIALS AND METHODS: A full-design, cross-sectional, screening, single-center, non-interventional study included men with T2DM, who were he was treated in Endocrinology Research Centre, Moscow. The study was conducted from October 2021 to January 2022. Medical history assessment, physical examination with determination of body mass index (BMI), measurement of key steroid hormones, their precursors and metabolites by isotope dilution liquid chromatography/tandem mass spectrometry, glycated hemoglobin (HbA1c) were performed. The groups were compared using the Mann-Whitney U-test for quantitative indicators and χ² with Yates’ correction for qualitative ones. Correlation analysis was performed by the Spearman correlation method. When determining the criterion of statistical significance, the Bonferroni correction was applied.
RESULTS: Patients with hypogonadism had statistically significantly more pronounced obesity compared with eugonadal men. In a comparative analysis of patients, depending on the presence of hypogonadism, there were statistically significantly lower levels of androgen precursors 17-hydroxypregnenolone and 17-hydroxyprogesterone in hypogonadal men. At the same time, a positive statistically significant correlation was found between total testosterone and 17-hydroxyprogesterone. In addition, 17-hydroxyprogesterone, although to a lesser extent, but positively correlated with other androgens - androstenedione (r=0,328; p<0,001) and dehydroepiandrosterone (r=0,183; p=0,004). >< 0,001) and dehydroepiandrosterone (r=0,183; p=0,004).
CONCLUSIONS: In this investigation the prevalence of male hypogonadism in type 2 diabetes, determined by high-precision tandem mass spectrometry, was 69,5%. There was no effect of the disease on the mineralocorticoid and glucocorticoid links of adrenal steroidogenesis. Hypogonadism was associated with decreased levels of a number of testosterone precursors. The most significant of them was 17-hydroxyprogesterone, which can be considered as a marker of testicular steroidogenesis.
... Dapl1 deletion significantly increased the mRNA expression of APAK1 in the mouse testis (Fig. 6C). Another important upstream factor of the CREB/CREM pathway is MAPK3/1 (also known as ERK1/2) [20][21][22] . Activated MAPK3/1 and PKA activate the transcription factor CREB by increasing its expression and phosphorylation 23 . ...
Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.
... A defect in the pulsatile secretion of GnRH and a subsequent elevation of LH circulating concentrations, which, in turn, enhance ovarian androgen secretion, seems to be involved in the pathophysiology of this pathologic entity [48]. Due to their anatomical position, GnRH neurons interact with a range of neuroendocrine and metabolic inputs [49]. Kisspeptin-neurokinin B and dynorphin neurons, known as KNDγ neurons, represent major regulators of GnRH pulsatility [17]. ...
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age. It is a heterogeneous condition characterized by reproductive, endocrine, metabolic, and psychiatric abnormalities. More than one pathogenic mechanism is involved in its development. On the other hand, the hypothalamus plays a crucial role in many important functions of the body, including weight balance, food intake, and reproduction. A high-fat diet with a large amount of long-chain saturated fatty acids can induce inflammation in the hypothalamus. Hypothalamic neurons can sense extracellular glucose concentrations and participate, with a feedback mechanism, in the regulation of whole-body glucose homeostasis. When consumed nutrients are rich in fat and sugar, and these regulatory mechanisms can trigger inflammatory pathways resulting in hypothalamic inflammation. The latter has been correlated with metabolic diseases, obesity, and depression. In this review, we explore whether the pattern and the expansion of hypothalamic inflammation, as a result of a high-fat and -sugar diet, may contribute to the heterogeneity of the clinical, hormonal, and metabolic presentation in PCOS via pathophysiologic mechanisms affecting specific areas of the hypothalamus. These mechanisms could be potential targets for the development of effective therapies for the treatment of PCOS.
... Low testosterone levels in humans have been related to developmental defects of the male reproductive system 6,7 and disorders of male reproductive function, such as spermatogenesis failure, oligozoospermia, and male infertility 8 . Our previous study found that there are many important local regulatory mechanisms that accelerate testosterone synthesis in Leydig cells, including gonadotropin-releasing hormone (GnRH) and annexin A5 [9][10][11] . However, the cellular and molecular mechanisms underlying the steroidogenic impairment of Leydig cells are not clear. ...
Abnormal lipid/lipoprotein metabolism induced by obesity may affect spermatogenesis by inhibiting testosterone synthesis in Leydig cells. It is crucial to determine which components of lipoproteins inhibit testosterone synthesis. Circulating oxidized low-density lipoprotein (oxLDL), the oxidized form of LDL, has been reported to be an independent risk factor for decreased serum testosterone levels. However, whether oxLDL has a damaging effect on Leydig cell function and the detailed mechanisms have been rarely studied. This study first showed the specific localization of oxLDL and mitochondrial structural damage in testicular Leydig cells of high-fat diet-fed mice in vivo. We also found that oxLDL reduced the mitochondrial membrane potential (MMP) by disrupting electron transport chain and inhibited testosterone synthesis-related proteins and enzymes (StAR, P450scc, and 3β‑HSD), which ultimately led to mitochondrial dysfunction and decreased testosterone synthesis in Leydig cells. Further experiments demonstrated that oxLDL promoted lipid uptake and mitochondrial dysfunction by inducing CD36 transcription. Meanwhile, oxLDL facilitated COX2 expression through the p38 MAPK signaling pathway in Leydig cells. Blockade of COX-2 attenuated the oxLDL-induced decrease in StAR and P450scc. Our clinical results clarified that the increased serum oxLDL level was associated with a decline in circulating testosterone levels. Our findings amplify the damaging effects of oxLDL and provide the first evidence that oxLDL is a novel metabolic biomarker of male-acquired hypogonadism caused by abnormal lipid metabolism.
... It has been demonstrated that MAPK pathways might participate in the regulation of testosterone synthesis in various steroid-producing cells, while effects appear to be contradictory, some stimulatory and others inhibitory effects (Manna et al., 2007;Otis et al., 2005;Renlund et al., 2006;Yao et al., 2011). Interleukin-1α upregulated StAR protein expression via activating the ERK1/2, thereby promoting translocation of cholesterol into the mitochondria in immature Leydig cells (Renlund et al., 2006). ...
Nickel (Ni) can disorder testosterone synthesis in rat Leydig cells, whereas the mechanisms remain unclear. The aim of this study was to investigate the role of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) in Ni-induced disturbance of testosterone synthesis in rat Leydig cells. The testosterone production and ROS levels were detected in Leydig cells. The mRNA and protein levels of testosterone synthetase, including StAR, CYP11A1, 3β-HSD, CYP17A1 and 17β-HSD, were determined. Effects of Ni on the ERK1/2, p38 and JNK MAPKs were also investigated. The results showed that Ni triggered ROS generation, consequently resulted in the decrease of testosterone synthetase expression and testosterone production in Leydig cells, which were then attenuated by ROS scavengers of N-acetylcysteine (NAC) and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), indicating that ROS are involved in the Ni-induced testosterone biosynthesis disturbance. Meanwhile Ni activated the ERK1/2, p38 and JNK MAPKs. Furthermore, Ni-inhibited testosterone synthetase expression levels and testosterone secretion were all alleviated by co-treatment with MAPK specific inhibitors (U0126 and SB203580, respectively), implying that Ni inhibited testosterone synthesis through activating ERK1/2 and p38 MAPK signal pathways in Leydig cells. In conclusion, these findings suggest that Ni causes testosterone synthesis disorder, partly, via ROS and MAPK signal pathways.
... The anterior lobe is considered in this study. GnRH-I is the classic hypothalamic hormone responsible for the regulation, synthesis, and secretion of the pituitary gonadotropins FSH and LH [18]. GnRH-I acts on the anterior pituitary leading to the synthesis and storage of gonadotropins, movement of the gonadotropins from the reserve pool to a readily released point, and finally the secretion of gonadotropins. ...
... We found stimulatory effects of nesfatin-1 on GnRH, Kiss1R, and LHβ, and upregulation of NUCB2/nesfatin-1 by T in cells. Luteinizing hormone stimulation of T production in Leydig cells is augmented by GnRH, and is blocked by a GnRH antagonist [50]. Nesfatin-1 increased hCG-stimulated testosterone secretion in rat testicular explants ex vivo [31]. ...
Biology of Reproduction, 2017, 96(3), 635-651doi:10.1095/biolreprod.116.146621Research ArticleAdvance Access Publication Date: 28 January 2017The legend for Figure 7 was incorrectly placed under Figure 8.The legend for Figure 8 was incorrectly placed under Figure 7. Thishas now been corrected and the correct legends are under Figures 7and 8. The author regrets the error.
... We found stimulatory effects of nesfatin-1 on GnRH, Kiss1R, and LHβ, and upregulation of NUCB2/nesfatin-1 by T in cells. Luteinizing hormone stimulation of T production in Leydig cells is augmented by GnRH, and is blocked by a GnRH antagonist [50]. Nesfatin-1 increased hCG-stimulated testosterone secretion in rat testicular explants ex vivo [31]. ...
Neuroendocrine regulation of metabolism and reproduction are tightly interlinked. Nesfatin-1 is an 82 amino acid metabolic peptide derived from nucleobindin-2 (NUCB2). NUCB2 mRNA and protein significantly increase in the hypothalamus of rats during puberty-to-adult transition. Administration of nesfatin-1 modulates circulating LH and testosterone in male rats. However, whether nesfatin-1 acts directly on neurons and gonadotropes remain unknown. In addition, whether reproductive hormones of the hypothalamo-pituitary gonadal axis modulate NUCB2/nesfatin-1 is unclear. To address these, we employed murine hypothalamic (GT1-7) and pituitary (LβT2) cells in vitro. Nucb2 expression, and NUCB2/nesfatin-1 immunoreactivity were observed in both GT1-7 and LβT2 cells, and in the hypothalamus of mice. Nesfatin-1 co-localized GnRH in GT1-7 cells, and in the hypothalamic perikarya of mice. Cells were treated with kisspeptin, GnRH, and estradiol and testosterone, as well as nesfatin-1 for 2, 6 or 24 hours. Synthetic nesfatin-1 increased Kiss1r and Gnrh expression in GT1-7 cells and Lhβ in LβT2. Nesfatin-1 increased GnRH and LHβ protein expression in GT1-7 and LβT2 at 6-hour post incubation respectively. Both NUCB2 mRNA and protein were increased in GT1-7 cells treated with kisspeptin. Testosterone increased NUCB2 mRNA and protein expression in GT1-7 and LβT2. 17β-estradiol increased NUCB2 mRNA and protein expression in LβT2. Nesfatin-1 acts directly on hypothalamic neurons and gonadotropes to elicit a generally positive influence on the endocrine milieu regulating reproduction in mice. Reproductive hormones, in turn, modulate brain and pituitary NUCB2/nesfatin-1. In conclusion, we provide additional information to designate nesfatin-1 as a novel, additional factor that helps reproductive success.
... This may be attributed to same found influence LH and both LH-GnRH as promoting formation Leydig cells cluster enrich cell in hyperchromic granules of the testosterone formation. This finding approved the ketorolac effect exerted on cell viability for production testosterone at the level of biosynthesis more than cell proliferation dynamic influenced by GnRH craft, but they ketorolac diminished sterodiogenic actively diminished by reduction of viable cells and directly in the blocker activity particulate element presence of testosterone production [23,24]. ...
In the present thesis was conducted to clarify the effect of ketorolac (NSAID) on the Leydig cells of goats to achieve the ketorolac event, by the evaluation steroidoginic activity of ketorolac series on Leydig cell concentrations in vitro. Mitochondrial defects reduction of functional processes of sterodoginc activity reduction of testosterone levels via mitotracker probe indicator, ketorolac may be affected on the steroidogenic acute regulatory (STAR) protein and translocator protein (TSPO). Ketorolac might mutilation and impair the cells were via the uncoupling of oxidative phosphorylation in the cells followed by uncouple mitochondrial respiratory chain as well as ATP depletion. Testosterone level displays in all treated maneuvers LH, LH-GnRH and GnRH in ketorolac challenge decrease testosterone levels at a concentration of ketorolac from 0.6-0.15 mM with the exception in GNRH – ketorolac maneuvers result showed an increase in testosterone level in 0.03-0.06 mM, then fall up down other maneuvers of treatment. The testosterone synthesis in Leydig cells of mammalian is achieved almost motivated by the binding of luteinizing hormone (LH) to the plasma membrane of Leydig cell receptors efficaciously, that lead to creation of c.AMP Inhibition of LTB4 that due to direct inhibition of lipoxygenase action, and had a direct effect to reduce LTB4 and may be various eicosanoids also 12 (S) - hydroxyeicosatetraenoic acid and 15 (S) - hydroxyeicosatetraenoic acid binds to and activates LTB.
... The results of our recent study on the expression of GnRH I in the testis of P. obesus [32], suggest that b-endorphin and GnRH I act in P. obesus in an opposite way: GnRH I was present during sexual activity in the tubular as well as the interstitial compartment [32] whereas we observed no b-endorphin immunoreactivity in the period of sexual activity. In mammals, GnRH I seemed to exert a stimulatory action on androgen synthesis [33] inducing 3b-hydroxysteroid dehydrogenase expression [34]. ...
Introduction:
Testicular function in the sand rodent Psammomys obesus is subjected to seasonal alternations with a trigger of spermatogenesis in winter and a total quiescence which extends from late spring to summer. The aim of this study was to investigate the distribution of β-endorphin in the testis at the period of winter sexual activity and at its summer regression, and assess the effect of 17β-estradiol treatment on testicular morphology and β-endorphin expression.
Material and methods:
The adult males were grouped into 4 groups (rest group, sexually active group, rest treated with 17β-estradiol group and controls at sexual rest injected with olive oil, n = 5 in each group). Using anti-serum against β-endorphin, we studied its testicular expression by Western blot and cellular location by immunohistochemical (IHC) method, respectively.
Results:
We detected by Western blot a peptide of 3.5 kDa molecular weight corresponding to β-endorphin only in sexually resting and control males. The 17β-estradiol treatment induced a clear reduction in the β-endorphin band expression compared with the latter. These results were confirmed by the IHC analysis since β-endorphin was only observed in the testis at sexual rest and in controls, in majority of seminiferous tubules at the level of germ cells. The intensity of IHC labeling was significantly different between spermatogonia and spermatocytes I or round spermatids which revealed the strongest labeling. The intense immunoreactivity was also located in Leydig cells and highly significantly varied compared to the germ cells. The 17β-estradiol treatment decreased significantly the β-endorphin signal in germ cells but not in Leydig cells.
Conclusion:
The 17β-estradiol treatment induces a repressive effect on seasonal testicular endorphinergic system in P. obesus and this action targets exclusively the germ cells.
... 29 Our previous study demonstrated that the synthesis of annexin A5 is stimulated by GnRH and hCG, and GnRH positively regulates steroidogenesis by activating ERK in rat Leydig cells. 18,30 ERK1/2 but not JNKs or p38-MAPKs was activated by GnRH in our previous study, 30 and ERK1/2 is also activated by annexin A5 in the current study. Interestingly, JNKs or p38-MAPKs were not activated by annexin A5 in our study. ...
This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.
... It was later reported that activation of LHCGR leads to a transient cAMP-dependent activation of the EGFR and downstream mitogen-activated protein kinase (MAPK) cascade [75]. The involvement of the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) downstream of PKA have long been known to be essential for proper LH-induced steroidogenesis in Leydig cells [76][77][78][79][80][81][82] (Fig. 1). ...
Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. Consequently, the process of steroid hormone biosynthesis is finely regulated. In the testis, the main steroidogenic cells are the Leydig cells. There are two distinct populations of Leydig cells that arise during development: fetal and adult Leydig cells. Fetal Leydig cells are responsible for masculinizing the male urogenital tract and inducing testis descent. These cells atrophy shortly after birth and do not contribute to the adult Leydig cell population. Adult Leydig cells derive from undifferentiated precursors present after birth and become fully steroidogenic at puberty. The differentiation of both Leydig cell populations is controlled by locally produced paracrine factors and by endocrine hormones. In fully differentially and steroidogenically active Leydig cells, androgen production and hormone-responsiveness involve various signaling pathways and downstream transcription factors. This review article focuses on recent developments regarding the origin and function of Leydig cells, the regulation of their differentiation by signaling molecules, hormones, and structural changes, the signaling pathways, kinases, and transcription factors involved in their differentiation and in mediating LH-responsiveness, as well as the fine-tuning mechanisms that ensure adequate production steroid hormones.
Copyright © 2015. Published by Elsevier Inc.
... Accordingly, in human, the expression levels of GnRH-1, GnRH-2, GnRH-Rs, cytochrome P450 side-chain cleavage (CYP11A1), 3β-HSD type 2 enzyme, and the intra-testicular testosterone (T) levels are significantly increased in patients with spermatogenic failure (26). At molecular level, the transduction pathway involving the GnRH agonist-dependent activation of ERK1/2 has been reported (27). Interestingly, in mouse testis, GnRH-R activity well correlates with the increased steroidogenic activity observed during pubertal and adult stages and its decline parallels the decreased steroidogenic activity observed during the senescence (28). ...
Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic-pituitary control, being regulated by the secretion of pituitary gonadotropins, follicle stimulating hormone, and luteinizing hormone, in response to stimulation exerted by gonadotropin releasing hormone from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intra-gonadal signals sustains the production of high quality mature spermatozoa. In this review, we focus on the recent advances in the area of the intra-gonadal signals supporting sperm development.
... Studies have demonstrated that steroidogenesis is regulated by many signaling pathways, such as the cAMP/PKA, PI3K, and MAPK pathways [13,26]. It has been shown that gonadotropin releasing hormone (GnRH) activates ERK1/2, rather than JNK or p38, to increase transcription/translation of the gene encoding 3b-HSD and subsequent testosterone production in rat primary Leydig cells [29]. Furthermore, 8-Br-cAMP induces transcription/ translation of steroidogenic acute regulatory (StAR) protein and progesterone production through ERK1/2 activation in MA-10 Leydig cells [15]. ...
Fibroblast growth factor 9 (FGF9) is a multifunctional polypeptide belonging to the FGF family and has functions related to bone formation, lens-fiber differentiation, nerve development, gap-junction formation and sex determination. In a previous study, we demonstrated that FGF9 stimulates the production of testosterone in mouse Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse Leydig tumor cells to further investigate the molecular mechanism of FGF9-stimulated steroidogenesis. Results showed that FGF9 significantly activated steroidogenesis in both mouse primary and tumor Leydig cells (p<0.05). Furthermore, FGF9 significantly induced the expression of phospho-Akt at 0.5 and 24 hr, phospho-JNK at 0.25, 0.5, and 24 hr, phospho-p38 at 0.5 hr, and phospho-ERK1/2 from 0.25 to 24 hr in primary Leydig cells (p<0.05). Also, FGF9 significantly up-regulated the expression of phospho-Akt at 3 hr, phospho-JNK at 0.25 hr, and phospho-ERK1/2 at 1 and 3 hr in MA-10 cells (p<0.05). Using specific inhibitors of Akt, JNK, p38, and ERK1/2, we further demonstrated that the inhibitors of Akt and ERK1/2 significantly suppressed the stimulatory effect of FGF9 on steroidogenesis in mouse Leydig cells. In conclusion, FGF9 specifically activated the Akt and ERK1/2 in normal mouse Leydig cells and the Akt, JNK and ERK1/2 in MA-10 mouse Leydig tumor cells to stimulate steroidogenesis.
... Concurrently, some key steroidogenic enzymes were assessed in CTN-treated cells. It is well known that cholesterol conversion to biologically active steroids is a multi-step enzymatic process, including important enzymes such as P450scc (Payne and Hales, 2004;Luisa et al., 2004;Lin et al., 2008), 3b-HSD (Prisco et al., 2008;Yao et al., 2011), and StAR protein (Miller, 2007;Stocco et al., 2005;Lin et al., 1995). StAR protein appears to transfer cholesterol from cellular stores to the inner mitochondrial membrane, where P450scc converts cholesterol to pregnenolone and then 3b-HSD converts this product to progesterone. ...
A previous study has shown that CTN (Citrinin) inhibits mouse testosterone production. In this study, the mechanism by which testosterone production is inhibited by CTN in rat Leydig cells was investigated, and the morphological evidence of apoptosis, including nuclei fragmentation and phosphatidylserine (PS) exposure on cell surfaces, was clearly observed 36h after CTN exposure. The results showed that citrinin at 50 and 100μM significantly suppressed testosterone secretion by human chorionic gonadotropin (hCG) at 10IU/ml. Western blotting results showed that CTN induced formation of processed p53, caspase-9, and caspase-3 proteins in a dose-dependent manner; CTN also induced a dose-dependent increase in caspase-3 catalytic activity. Western blot assays also showed that CTN decreased expression of three key enzymes (P450scc, 3β-HSD-1, and StAR) of testosterone production. Taken together, these results suggested that CTN reduced testosterone secretion by inducing apoptosis in rat Leydig cells, a mechanism that might account for CTN stimulation of p53 expression followed by activation of multiple caspases.
... In order to further investigate the action of fenvalerate on the downstream pathway of IGF-I and the exact site(s) the pesticide interfere along the ERK1/2 downstream pathway, we examined the expression of ERK1/2 and p-ERK1/2 considering the important role ERK1/2 play in steroidogenesis (Martinat et al., 2005;Yao et al., 2011). Our data showed that fenvalerate did not alter the level of total ERK1/2 but markedly attenuated the expression of p-ERK1/2, which are in agreement with the description that decreased phosphorylation of ERK1/2 can result in decreased steroidogenesis (Renlund et al., 2006). ...
Exposure to fenvalerate has been shown to be associated with decreased steroid hormone production by mouse Leydig tumor cells (MLTC-1) in our previous study and the interference with cAMP-PKA pathway cannot explain this inhibitory effect completely. In this study, the same cell line was used to investigate the potential involvement of insulin-like growth factor I (IGF-I) signaling pathway in the downregulation of steroidogenesis by fenvalerate. Results showed that fenvalerate treatment decreased IGF-I secretion significantly which was consistent with the reduced expression of IGF-I mRNA. Then inhibitors of the two downstream pathways of IGF-I were added to the medium. The addition of LY294002 (inhibitor of phosphatidylinositol (PI)-3-kinase) did not alter the declining trend of progesterone production with increasing dosages of fenvalerate treatment while the addition of UO126 (inhibitor of extracellular signal-regulated kinases 1/2 (ERK1/2)) markedly attenuated this trend, which strongly indicated the possible involvement of pathway ERK1/2. In addition, phosphorylation of ERK1/2 was also suppressed by fenvalerate. The results suggest that the mechanism by which fenvalerate decreased steroid hormone production might involve the impairment of IGF-I signal pathway by attenuating the IGF-I production and ERK1/2 phosphorylation.
... Testicular GnRH receptors have been reported in several species, including humans (87,88). In rats, GnRH affects Ca 2+ mobilization (89) as well as steroidogenesis in Leydig cells (90). This reported direct effect of GnRH on steroidogenesis is especially noteworthy for the current study, as although testosterone implants returned testosterone concentrations to the normal range in castrated rats, the testosterone elevation was lower in deslorelin-treated intact rats. ...
Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHβ and LHβ, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHβ-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHβ-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHβ-ir cell population, although the loss of FSHβ in bi-hormonal cells increased the proportion of mono-hormonal LHβ-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHβ is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.
Female hypogonadism is a state characterized by absent or decreased ovarian function. It results from a gonadal (primary hypogonadism) or an extragonadal (secondary hypogonadism) princeps defect. In secondary hypogonadism, hypothalamic gonadotropin-releasing hormone or/and pituitary-secreted gonadotropins (follicle-stimulating hormone, luteinizing hormone) are either deficient or inactive leading to decreased secretion of gonadal steroids and subsequent amenorrhea. In certain conditions, both hypothalamic and pituitary dysfunctions are present. The genetic causes of secondary hypogonadism manifest mainly as congenital genetic syndromes (i.e., Kallmann syndrome) while some of them have been attributed to recognized single gene mutations and others have been characterized as idiopathic forms. Acquired causes of secondary hypogonadism include reversible causes such as functional hypothalamic amenorrhea, drugs, chronic illnesses, and irreversible causes such as central nervous system insults (trauma, irradiation, and intracranial tumors). Diagnosis should take in consideration the age at the clinical presentation (prepubertal or postpubertal), the physical findings as well as biochemical and imaging findings. Genetic investigation can be employed for more precise diagnosis. Finally, treatment should focus upon the treatment of the causal factor wherever possible and the hormone replacement therapy. The latter is adapted to the age of diagnosis of secondary female hypogonadism (prepubertal vs. postpubertal).
Menstruation is the cyclic and orderly sloughing of the endometrium. In order for normal menstruation to take place, a complex interaction between the brain, the ovaries, and the uterus has to take place. This interaction involves fluctuating levels of hormones produced by the brain as well as the ovaries, while the uterus responds to the changing hormone levels. This process of hormone production is initiated in the hypothalamus through the production and release of GnRH, which leads to FSH and LH release from the anterior pituitary gland. As a result, oocyte development takes place in the ovaries, and estradiol is produced from the growing ovarian follicle. As the growing follicle matures and estradiol rises further, LH from the pituitary surges, leading to the process of ovulation. After ovulation, the remnant of the follicle becomes the corpus luteum, which is responsible for progesterone production. If the cycle does not result in a pregnancy, progesterone levels decline and menstruation takes place. Throughout this process, there are many other hormones from the central nervous system as well as from the ovary that play a role in the control of the menstrual cycle. We will examine the role of the central nervous system and how it interacts with the ovaries to control the menstrual cycle.
Menstruation is the cyclic and orderly sloughing of the endometrium. In order for normal menstruation to take place, a complex interaction between the brain, the ovaries, and the uterus has to take place. This interaction involves fluctuating levels of hormones produced by the brain as well as the ovaries, while the uterus responds to the changing hormone levels. This process of hormone production is initiated in the hypothalamus through the production and release of GnRH, which leads to FSH and LH release from the anterior pituitary gland. As a result, oocyte development takes place in the ovaries, and estradiol is produced from the growing ovarian follicle. As the growing follicle matures and estradiol rises further, LH from the pituitary surges, leading to the process of ovulation. After ovulation, the remnant of the follicle becomes the corpus luteum, which is responsible for progesterone production. If the cycle does not result in a pregnancy, progesterone levels decline and menstruation takes place. Throughout this process, there are many other hormones from the central nervous system as well as from the ovary that play a role in the control of the menstrual cycle. We will examine the role of the central nervous system and how it interacts with the ovaries to control the menstrual cycle.
Abstract
In this study, investigated whether captopril inhibited steroidogenesis and components of the Leukotriene B4 pathways are involved in GnRH agonist (GnRH)-induced testis steroidogenesis in mice Leydig cells. Primary cultures of mice Leydig cells were established. Purified Leydig cells from adult albino mice were incubated with gradual various concentrations of GnRH with and without Captopril, Luteinizing hormone (LH); LTB4, steroidogenic (testosterone) activity and LTB4 concentration were measured after various time intervals and Leydig cell viability. The maneuvers of Leydig cells treated media was covered the singular and dual actions of antisteroidogenic of captopril and the reversible activity by GnRH-LTB4 as well as contribution of LTB4 in Leydig cells testosterone production endpoint. The different treatment media are Medium alone; Medium plus captopril 60 μM, 65 μM, 70 μM, 75 μM and 80 μM 100 μM; Medium plus 2.5mU/ml leukotriene B4; Medium plus 0.1 mM LH; Medium plus 0.1 μM GnRH; Medium plus 65 μM captopril plus 2.5 mU/ml leukotriene B4;Medium plus 65 μM captopril plus 0.1 mM LH; Medium plus 65 μM captopril plus 0.1 mM GnRH; Medium plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4 and Medium plus 65 μM captopril plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4. Basal testosterone levels were maximal at 0.1 μM GnRH concentration and superior testosterone yield in Leydig cells incubated 0.1 μM GnRH media than without GnRH media, and the activity profile LTB4 flow up. That comparable result led to highly correlated approved the contribution of LTB4 in GnRH stimulated Leydig cell steroidogenic end point. Furthermore captopril had an abolishment effect partially of testosterone yield and recovered and improved by GnRH and LTB4. The Leydig cells viability results suggest that the major effect of GnRH is probably beyond the LTB4. The entire key; GnRH induced testosterone production and upregulated LTB4 Levels at both the captopril inhibitory LTB4-testesteron Leydig cells culture media and captopril abolished LTB4 levels; it also activated endogenous LTB4, but not LH motivated testosterone pathway. Our data show that GnRH positively regulates steroidogenesis via LTB4 signaling in mice Leydig cells. LTB4 activation by GnRH may be responsible for the induction of Ca++ signaling indirect. Possibility improve the captopril steroidogenic disruption in Leydig cells via LTB4 and/or GnRH induction of endogenous LTB4, likewise the positive maintenance of Leydig cells viability matched induce testosterone synthesis. The LTB4 production, which may ultimately modulate steroidogenesis in mice Leydig cells, and promise new antidotal and preventative of captopril adverse effects.
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.
Orexin A (ORA) regulates food intake, energy metabolism, gastrointestinal and reproductive functions.
The purpose of this study was to demonstrate whether the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and testosterone was stimulated by ORA and mediated through mitogen-activated protein kinases (MAPK) in rat Leydig cells.
Primary Leydig cells were isolated from male rat testes, cultured, and treated with ORA under various conditions.
Orexin receptor 1 (OX 1 R) mRNA, but not orexin receptor 2 mRNA, was detected in primary Leydig cells. ORA up-regulated the expression of OX 1 R mRNA and protein in a dose-responsive manner and increased the phosphorylation of extracellular receptor kinase 1/2 (ERK1/2) and p38 MAPK levels, but did not affect the phosphorylation of the JNK MAPK. Phosphorylation of ERK1/2 and p38 MAPKs by ORA was blocked with U0126 and SB203580 inhibitors, respectively. An OX1R-specific inhibitor, SB334867, also blocked the phosphorylation of ERK1/2 and p38 by ORA. Inhibitor treatment also blocked 3β-HSD expression and testosterone production.
These results demonstrate that ORA activation of OX1R up-regulates 3β-HSD expression and testosterone production via the ERK1/2 and p38 MAPKs signaling pathways in primary rat Leydig cells.
Potential roles of gonadotropin-releasing hormone (GnRH) antagonists on GnRH/GnRH receptor systems and their effects on the extrapituitary tissues are largely elusive. In this narrated review, we summarized the systemic effects of GnRH antagonists on ovary, endometrium, embryo implantation, placental development, fetal teratogenicity, reproductive tissue cancer cells, and heart while briefly reviewing the GnRH and GnRH receptor system. GnRH antagonists may have direct effects on ovarian granulosa cells. Data are conflicting regarding their effects on endometrial receptivity. The GnRH antagonists may potentially have detrimental effect on early placentation by decreasing the invasive ability of cytotrophoblasts if the exposure to them occurs during early pregnancy. The GnRH antagonists were not found to increase the rates of congenital malformations. Comparative clinical data are required to explore their systemic effects on various extrapituitary tissues such as on cardiac function in the long term as well as their potential use in other human cancers that express GnRH receptors.
We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1α (IL-1α)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1α-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1α stimulated both androgen production and StAR protein expression in a dose-and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1α were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1α signaling in the immature Leydig cell. The present results suggest that IL-1α, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1α contributes to the differentiation of immature Leydig cells into adult cells.
Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.
GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Galpha(q/11), Galpha(i/o), and Galpha(s), their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Galpha(q/11)-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Galpha(i) or chimeric Galpha(qi5), transfection of Galpha(q) cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPgammaS binding assays confirmed that the GnRH receptor was unable to directly couple to Galpha(i) but could directly interact with Galpha(q/11). Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Galpha(q), and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Galpha(q/11), but not to Galpha(i/o) or Galpha(s), and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.
Activator protein 1 (JUN) transcription factors (JUN and FOS) play critical roles in a wide variety of signaling processes including those in the protein kinase C (PRKCC) pathway, a pathway that is instrumental in the expression of the steroidogenic acute regulatory (STAR) protein. In the present study, we determined the functional involvement of one of the key JUN family members, JUN, in the regulation of PRKCC-dependent STAR expression and steroidogenesis. MA-10 mouse Leydig tumor cells treated with an activator of PRKCC, phorbol 12-myristate 13-acetate (PMA), demonstrated increases in the expression of the STAR and CYP11A1 proteins and progesterone synthesis, which coincided with the expression and phosphorylation of JUN (P-JUN). PMA was also capable of enhancing the cAMP analog, (Bu)(2)cAMP, which stimulated JUN, STAR, P-STAR and progesterone levels. The induction of Jun mRNA expression and steroid synthesis by PMA requires de novo protein synthesis. Chromatin immunoprecipitation studies revealed the association of P-JUN with the STAR proximal promoter and that PMA specifically enhanced in vivo P-JUN-DNA interaction. Electrophoretic mobility shift assays and reporter gene analyses demonstrated that JUN binds to the JUN motif (-81/-75 bp) in the STAR promoter, and that JUN-DNA-binding activity was highly correlated with the induction of JUN by PRKCC signaling. Overexpression of JUN increased the PMA-mediated transcription of the Star gene, an event markedly decreased by TAM-67, a dominant negative mutant of JUN. Targeted silencing of endogenous JUN, by small interfering RNA, was correlated with the repression of basal- and PMA-mediated STAR expression and progesterone synthesis. These findings describe the mechanisms by which JUN influences PRKCC signaling and provide additional and novel insight into the regulation of the steroidogenic machinery in mouse Leydig cells.
Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.
In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.
Agonist analogs of gonadotropin-releasing hormone (GnRH) have been shown to exert antigonadal effects in male and female animals. In hypophysectomized male rats treated with follicle-stimulating hormone, administration of a potent GnRH agonist caused depletion of luteinizing hormone and prolactin receptors and marked suppression of serum testosterone levels. The possibility that such direct effects of GnRH agonists on testicular function could be expressed through specific receptors located in the interstitial cells of the testis was supported by the selective concentration of a 125I-labeled GnRH agonist by the testis in vivo. Specific receptors for the releasing hormone were demonstrated in testis particles and dispersed interstitial cells by direct binding analysis with the 125I-labeled GnRH agonist. The binding affinity (Ka = G X 10(9) M-1) and peptide specificity of the testicular GnRH binding sites were similar to those of anterior pituitary and ovarian GnRH receptors. The presence of GnRH receptors in the testis indicates that these sites mediate the direct inhibitory actions of GnRH agonists upon testicular endocrine function.
Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.
The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.
Different isoforms of testicular interleukin-1 (IL-1) were analysed to determine whether there were differences in the ability to modulate rat Leydig cell steroidogenesis in vitro. Rat 17K IL-1alpha and IL-1beta, 32K IL-1alpha precursor (32proIL-1alpha) and a 24K splice variant (24proIL-1alpha) stimulated testosterone production by Leydig cells from 40- but not 80-day-old rats. The potency of the isoforms was IL-1alpha>IL-1beta>32proIL-1alpha>24proIL-1alpha, IL-1alpha being 50-fold more potent than IL-1beta. IL-1 receptor antagonist reversed the effects and IL-1 receptor type I mRNA was expressed by the responding Leydig cells, indicating a receptor mediated action. Inhibition of PKA and Ca(2+) channels abolished IL-1-induced steroidogenesis, while inhibition of PKC had no significant effect. Except for 24proIL-1alpha which was stimulatory, all IL-1 isoforms suppressed hCG-driven testosterone production. This inhibitory effect was abolished by androstendione, suggesting that P450c17 was suppressed by IL-1. Our results indicate that IL-1 plays a paracrine role in the regulation of Leydig cell steroidogenesis.
We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.
The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca2+ influx, but not Ca2+ mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca2+ ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca2+, inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca2+, are involved in GnRH induction of the ovine FSHbeta gene.
Recent results indicate that a novel second form of GnRH, GnRH-II, has an antiproliferative effect on ovarian and endometrial cancer cells and might be considered as a possible therapy for gynecological tumors. However, the mechanism of the GnRH-II-induced antiproliferative effect is not known. The p38 MAPK, one of the stress-activated protein kinases, is activated by diverse cellular stress and proinflammatory cytokines. In this study, the effect of GnRH-II on the activation of p38 MAPK was investigated, and its possible role in the regulation of cell proliferation and apoptosis was further examined in the human ovarian cancer cell line, OVCAR-3. Treatment with GnRH-II (100 nM) resulted in an activation of p38 MAPK in a time-dependent manner. A significant activation of p38 MAPK was observed at 2, 5, 10, and 15 min after GnRH-II treatment. The activation of p38 MAPK by GnRH-II was reversed in the presence of a specific inhibitor of p38 MAPK, SB203580 (1 microM). The transcription factor, activator protein-1, was activated (1.5-fold) by GnRH-II and attenuated in the presence of SB203580 (1 microM). Treatment with GnRH-II (1 nM, 100 nM, 10 microM) for 2, 4, and 6 d resulted in an inhibition of cell growth in OVCAR-3 cells as determined by thymidine incorporation assay. The effect of GnRH-II (100 nM) on cell proliferation was blocked by pretreatment with SB203580 (1 microM). Furthermore, a significant increase of apoptosis (1.6-fold) was observed after GnRH-II treatment, which was also reversed by pretreatment with SB203580 (1 microM). Taken together, these results indicate that p38 MAPK is involved in the GnRH-II-induced inhibition of cell growth through activator protein-1 activation, which may be related to induction of apoptosis in ovarian cancer cells.
We studied the involvement of the ERK cascade in human chorionic gonadotropin (hCG)-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase A and protein kinase C function as upstream kinases in connection with transduction of the signal from the gonadotropin receptor to the ERK cascade. These MAPKs enhance the stimulatory effects of hCG on the de novo synthesis of the steroidogenic acute regulatory protein and the activity of protein phosphatase 2A, which are associated with increased androgen production by the Leydig cell. Specific inhibition of ERK1/2 by Uo126 suppressed all of these cellular responses to hCG. In contrast, steroidogenesis from 22OHC (a cell-permeable form of cholesterol) is not inhibited by Uo126, suggesting that cholesterol delivery to mitochondria is being affected by this compound. We propose that the ERK cascade is an important part of the signal transduction pathway involved in the rapid hormonal responses of Leydig cells to trophic hormones. In hCG-activated Leydig cells, these MAPKs may play a role in controlling the biosynthesis of the steroidogenic acute regulatory protein as well as regulating protein phosphatase 2A activity, thereby governing cholesterol transport across the mitochondrial membrane.
The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.
The luteinizing hormone (LH) plays a critical role in steroidogenesis, by stimulating cAMP-dependent protein kinase A (PKA) and phospholipase A2 activity, and by mobilizing calcium and chloride ions. In contrast, whether the ERK 1, 2 mitogen-activated protein (MAP) kinases are involved in LH-induced steroidogenesis is less obvious. Here, we sought to clarify this point in rat primary Leydig cells, naturally bearing the LH receptor (LH-R) in male, and in the mouse tumoral Leydig cell line (MLTC 1). Pre-incubation of both cell types with the mitogen-activated protein kinase kinase (MEK) inhibitors U0126 and PD98059 reduced LH-induced steroidogenesis, and tonically enhanced the expression of the StAR protein. Furthermore, ERK1, 2 were inducibly phosphorylated following LH exposure of MLTC 1 cells. Altogether, our results indicate that in primary as well as in tumoral Leydig cells, inhibiting MEK dampened LH-induced steroidogenesis but enhanced basal as well as LH-induced StAR expression, suggesting that ERK1,2 could be involved in these responses.
Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser(133)Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.
Interleukin-1alpha (IL-1alpha) plays an important role in the regulation of immune responses as well as in non-inflammatory events in different types of cells. Here we have investigated the involvement of the extracellular signal-regulated kinase (ERK) cascade in IL-1alpha-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase C functions as an upstream component of signal transduction from the IL-1 receptor type I (IL-1RI) to the ERK cascade. It was observed that IL-1alpha upregulated both steroidogenic acute regulatory (StAR) protein expression and its phosphorylation when compared with controls. Selective inhibition of these mitogen-activated protein kinases (MAPKs) by UO126 enhanced both the expression and phosphorylation of the StAR protein, but suppressed androgen production by the immature Leydig cells as well as dissipating the mitochondrial electrochemical potential (Psim) in these cells. The evidence that water-soluble cholesterol but not 22R-hydroxycholesterol-stimulated steroidogenesis was inhibited by UO126 suggested that an intact Psim across the inner mitochondrial membrane is required for cholesterol translocation and is positively regulated by the ERK cascade. We propose that activation of ERKs by IL-1alpha plays a dual role in the regulation of steroidogenesis in immature Leydig cells: these MAPKs downregulate StAR expression and phosphorylation, while at the same time they support an intact Psim across the inner mitochondrial membrane, thereby promoting translocation of cholesterol into the mitochondria of the Leydig cell.
It is well established that surfactants can elicit cytotoxic effects at threshold concentrations by changing the permeability and solubilizing components of cell membranes. The purpose of this study was to characterize the relationship between perturbation of the mitochondrial membrane resulting from treatment with representative cationic, nonionic, and anionic surfactants and the extent to which this perturbation affects steroid formation and StAR protein expression and activity in MA-10 Leydig cells. The StAR protein is synthesized as an active 37 kDa extramitochondrial form, which is processed into a 30 kDa intramitochondrial form after cholesterol transfer and mitochondrial import and processing. It has been shown in several in vitro studies that the mitochondrial electrochemical gradient is required for the StAR protein to transfer cholesterol to the inner mitochondrial membrane. Each substance that was tested produced a concentration-dependent decrease in steroid formation in hCG-stimulated MA-10 cells. Decreases in progesterone production were accompanied by loss of mitochondrial membrane potential and by a decrease in the levels of the 30 kDa form of the StAR protein. However, levels of the 37 kDa form of the StAR protein did not decrease, indicating no effect on StAR protein expression. These results demonstrate how perturbation of the mitochondrial membrane by surfactants inhibits import, processing, and cholesterol transfer activity and underscore the importance of including sensitive assays that evaluate mitochondrial function when screening for potential effects on steroidogenesis with in vitro test systems.
To investigate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated Leydig cell steroidogenesis, the effects of GnRH agonist [des-Gly10, (D-Ala6) GnRH] on phospholipid turnover were studied. GnRH agonist in concentrations of 10−9 to 10−7 M increased phosphatidic acid labeling 292 ± 16% (mean ± SE), and phosphatidylinositol labeling 258 ± 13.2%. GnRH agonist-stimulated phospholipid labeling was detectable as early as 2 minutes. GnRH antagonist completely blocked GnRH agonist-induced testosterone formation and phosphatidic acid and phosphatidylinosital labeling. Nifedipine in concentrations of 1 and 10 μg/ml inhibited GnRH agonist-stimulated testosterone formation but had no effect on 32P incorporation into phospholipids. Our results suggest that GnRH agonist-stimulated Leydig cell steroidogenesis is calcium dependent and correlated with increased phospholipid turnover.
The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHβ-promoter activity was examined in the gonadotroph cell line LβT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHβ-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nm) to LβT-2 cells resulted in a 6-fold increase in LHβ-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca²⁺ ionophore ionomycin, stimulated LHβ-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca²⁺, abolished the GnRH-A and the TPA response. Cotransfection of the LHβ-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHβ-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHβ-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHβ-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHβ-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHβ-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHβ-CAT activity. c-Src participates also in LHβ-promoter activation by a mechanism which might be linked to ERK and JNK activation.
Gonadotropin-releasing hormone (GnRH), the first key hormone of reproduction, is synthesized in the hypothalamus and is released in a pulsatile manner to stimulate pituitary gonadotrope-luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and release. Gonadotropes represent only about 10% of pituitary cells and are divided into monohormonal cells (18% LH and 22% FSH cells) and 60% multihormonal (LH+FSH) cells. GnRH binds to a specific seven transmembrane domain receptor which is coupled to Gq and activates sequentially different phospholipases to provide Ca2+and lipid-derived messenger molecules. Initially, phospholipase C is activated, followed by activation of both phospholipase A2(PLA2) and phospholipase D (PLD). Generation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG) lead to mobilization of intracellular pools of Ca2+and activation of protein kinase C (PKC). Early DAG and Ca2+, derived via enhanced phosphoinositide turnover, might be involved in rapid activation of selective Ca2+-dependent, conventional PKC isoforms (cPKC). On the other hand, late DAG, derived from phosphatidic acid (PA) via PLD, may activate Ca2+-independent novel PKC isoforms (nPKC). In addition, arachidonic acid (AA) which is liberated by activated PLA2, might also support selective activation of PKC isoforms (PKCs) with or without other cofactors. Differential cross-talk of Ca2+, AA, and selective PKCs might generate a compartmentalized signal transduction cascade to downstream elements which are activated during the neurohormone action. Among those elements is the mitogen-activated protein kinase (MAPK) cascade which is activated by GnRH in a PKC-, Ca2+-, and protein tyrosine kinase (PTK)-dependent fashion. Transcriptional regulation can be mediated by the activation of transcription factors such as c-fos by MAPK. Indeed, GnRH activates the expression of both c-jun and c-fos which might participate in gene regulation via the formation of AP-1. The signaling cascade leading to gonadotropin (LH and FSH) gene regulation by GnRH is still not known and might involve the above-mentioned cascades. AA and selective lipoxygenase products such as leukotriene C4also participate in GnRH action, possibly by cross-talk with PKCs, or by an autocrine/paracrine amplification cycle. A complex combinatorial, spatial and temporal cross-talk of the above messenger molecules seems to mediate the diverse effects elicited by GnRH, the first key hormone of the reproductive cycle.
Intracellular cAMP and Ca(2+) are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone (LH) and human chorionic gonadotropin (hCG). However, the identification of Ca(2+) entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca(2+) channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil (a putative T-type Ca(2+) channel blocker) on steroidogenesis were assessed using reverse transcription (RT)-polymerase chain reaction analysis for the steroidogenic acute regulatory protein (StAR) mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L(-1) extracellular Ca(2+), hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion (P < 0.05), and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner (P < 0.05). Moreover, the hCG-induced increase in testosterone production was completely removed when external Ca(2+) was omitted, implying that Ca(2+) entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca(2+) currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca(2+) entry carried out by the T-type Ca(2+) channel in the Leydig cells of mice.
To study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis.
Purified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined.
GnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05).
GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.
We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.
The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.
Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.
Gonadotropin-releasing hormone (GnRH) has specific receptor sites in rat Leydig cells and has direct effects on their steroidogenesis. The purpose of the present study was to examine whether activation of the calcium- and phospholipid-dependent protein kinase C (PK-C) is involved in GnRH effects on rat Leydig cells, as has been shown in pituitary gonadotrophs. Testosterone production of Percoll-purified Leydig cells was similarly stimulated (about 50-100%) by a GnRH agonist (buserelin, maximum effect at concentration of 10(-9) mol/l and above) and a tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, maximum effect at 10(-8) mol/l), which is known to activate PK-C. In contrast, a GnRH antagonist (10(-5) mol/l) and an inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate (10(-6) mol/l), were without effect on testosterone. None of these substances had clear effects on cAMP production. The maximum steroidogenic effects of GnRH agonist and TPA were the same whether used separately or together, suggesting that they share a common mechanism of action. TPA translocated the cytosolic proportion of Leydig cell PK-C activity to a membrane-associated form almost instantaneously, within 0.5-1 min. A similar translocation, though less complete, was observed in the presence of buserelin in 1-4 min. Inclusion of a 100-fold excess of a potent GnRH antagonist completely prevented the translocation of PK-C. These results provide evidence that GnRH agonist activates PK-C also in the testis tissue, and this may be the mechanism whereby it affects Leydig cell endocrine function.
Gonadotropin-releasing hormone agonist (GnRHa) markedly increased testosterone formation from 2.35 +/- 0.13 ng/ml of the controls to 14.92 +/- 0.33 ng/ml (mean +/- SE) in isolated and purified rat Leydig cells. GnRHa-induced testosterone formation was completely blocked by phospholipase A2 inhibitor (chloroquin, 10(-4) M), but was potentiated by the addition of either cyclo-oxygenase inhibitor (indomethacin) or lipoxygenase inhibitor (nordihydroguaiaretic acid, NDGA). Arachidonic acid also directly stimulated Leydig cell steroidogenesis and activated Ca/phospholipid dependent protein kinase. Steroidogenic effects of arachidonic acid were also potentiated by the addition of either indomethacin or NDGA. These results suggest that arachidonic acid may be important in mediating direct stimulatory effects of GnRH on Leydig cell steroidogenesis, and the conversion of arachidonic acid to either prostaglandins or leukotrienes is not required for its steroidogenic effect.
Leydig cells isolated from adult rat testes bound 125I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (KA=1.2 × 109M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with 125I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10−4M) concentrations but was inactive at 10−6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of 125I-LHRH agonist to Leydig cells by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.
Dispersed cells from whole testes or from isolated interstitial tissue of mature rats, yielded two distinct populations of Leydig cells when subjected to centrifugation in a 0-40% metrizamide gradient. One population (I) was found in a fractions with a density of 1.085-1.117 g/cm3, and the other population (II) was found in fractions with a density of 1.128-1.145 g/cm3. Binding of 125I-labeled hCG by each population of cells indicated a single class of binding sites with the same high binding affinity and similar concentrations of binding sites per Leydig cell. Testosterone production per fmol gonadotropin receptor site in the absence of gonadotropin stimulation was similar for cells of each population. However, when cells from each population were incubated with increasing concentrations of hCG or dibutyryl cAMP, only Leydig cells from population II exhibited a marked increase in testosterone production. The low responsiveness of Leydig cells in population I did not appear to be a result of either damage to these cells or inhibition by non-Leydig interstitial cells in population I.
To investigate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated Leydig cell steroidogenesis, the effects of GnRH agonist [des-Gly10, (D-Ala6) GnRH] on phospholipid turnover were studied. GnRH agonist in concentrations of 10(-9) to 10(-7)M increased phosphatidic acid labeling 292 +/- 16% (mean +/- SE), and phosphatidylinositol labeling 258 +/- 13.2%. GnRH agonist-stimulated phospholipid labeling was detectable as early as 2 minutes. GnRH antagonist completely blocked GnRH agonist-induced testosterone formation and phosphatidic acid and phosphatidylinosital labeling. Nifedipine in concentrations of 1 and 10 micrograms/ml inhibited GnRH agonist-stimulated testosterone formation but had no effect on 32P incorporation into phospholipids. Our results suggest that GnRH agonist-stimulated Leydig cell steroidogenesis is calcium dependent and correlated with increased phospholipid turnover.
Unlabelled:
The present study was designed to elucidate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated testosterone formation. Purified Leydig cells from adult Sprague-Dawley rats were incubated with varying concentrations of GnRH agonist (des-Gly10, (D-Ala6) GnRH N-ethylamide), hCG, 8-bromo cAMP or pregnenolone; testosterone, cAMP, cyclic GMP (cGMP) and cAMP-dependent protein kinase activity were measured after various time periods. Basal testosterone levels were 2.54 +/- 0.13 ng/10(5) cells, increasing to 3.18 +/- 0.14, 4.32 +/- 0.08, and 4.63 +/- 0.12 ng within 1 hour after the addition of 10(-9), 10(-8), and 10(-7) M GnRH agonist, respectively. After a 3-hour incubation a 10(-7) M dose of GnRH agonist increased testosterone production four-fold above control. GnRH agonist potentiated hCG-stimulated testosterone formation, but had no significant effects on cGMP levels and cAMP-dependent protein kinase activity. Cyclic AMP levels in the incubation medium increased slightly. GnRH agonist also enhanced 8-bromo-cAMP and pregnenolone-induced testosterone formation. Furthermore, GnRH agonist increased testosterone formation both in the absence and presence of phosphodiesterase inhibitor. These results suggest that the major effect of GnRH agonist is probably beyond the cAMP step. When purified Leydig cells were incubated in a calcium-free medium, the stimulatory effects of GnRH agonist on testosterone formation were completely abolished, but could be restored by the addition of calcium to the incubation medium. GnRH agonist-induced testosterone formation was also blocked by the addition of nifedipine (a calcium channel blocking agent, 0.1 to 10 micrograms/ml). Finally, GnRH antagonist in a concentration of 10 micrograms/ml completely inhibited GnRH agonist-stimulated testosterone formation.
In conclusion:
GnRH agonist stimulated Leydig cell testosterone formation in short-term incubations. The stimulatory effect is calcium dependent and not mediated by cyclic nucleotides.
THAT the testes are related to the sexual characteristics of the individual and have reproductive power was observed at an early time in history. One of the earliest descriptions of this relationship in men and in animals was made by Aristotle, three centuries before Christ. He described how the castration of immature male birds prevented the development of sexual characteristics, such as the coloring of the crest and the attraction to females. He linked these changes in the bird to those observed in castrated boys who experienced the persistence of a high-pitched voice of childhood into adulthood and the lack of sexual hair development. The first convincing evidence of the role of the testis in the maintenance of male sexual characteristics was given in the middle of the nineteenth century by Berthold (1) who showed that atrophy of the cock's comb observed after castration was prevented by implantation of the testis into the abdominal cavity.
The role of mitogen-activated protein kinase (MAPK, also known as extracellular signal regulated kinase; ERK) stimulation in gonadotropin-releasing hormone (GnRH) signaling was investigated in cultured pituitary cells of tilapia hybrids (Oreochromis niloticus x O. aureus). Exposure of the cells to salmon GnRH (sGnRH) resulted in a dose- and time-dependent elevation in ERK levels. The PKC activator, 1-O-tetradecanoyl phorbol-13-acetate (TPA) increased kinase levels, while addition of GnRH had no further effect. However, chronic exposure to TPA resulted in reduction of basal and GnRH-induced ERK elevation. When PKC was inhibited by GF109203X, the GnRH-elevated ERK levels were totally abolished. The role of MAPK activation on GPalpha, FSHbeta and LHbeta gene expression was determined by administration of MAPK-kinase (MEK) inhibitor (PD98059; PD). This inhibitor completely blocked GnRH-induced increases in ERK activity. Furthermore, it suppressed GPalpha and LHbeta mRNA responses to GnRH, but had no effect on FSHbeta transcript levels. PD also decreased basal LHbeta mRNA levels. These results indicate that in tilapia pituitary cells, GnRH activates MAPK cascade in a PKC-dependent manner. ERK is involved in GnRH elevation of GPalpha and LHbeta, but not in FSHbeta genes transcription.
The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHbeta-promoter activity was examined in the gonadotroph cell line LbetaT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHbeta-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nM) to LbetaT-2 cells resulted in a 6-fold increase in LHbeta-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca(2+) ionophore ionomycin, stimulated LHbeta-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca(2+), abolished the GnRH-A and the TPA response. Cotransfection of the LHbeta-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHbeta-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHbeta-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHbeta-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHbeta-CAT activity. c-Src participates also in LHbeta-promoter activation by a mechanism which might be linked to ERK and JNK activation.
The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[14C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[14C]citrulline accumulation. Increased intracellular Ca2+ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.
We have previously shown that type IV collagen (alpha1 (IV) and alpha2 (IV) collagen chains) (Col-IV) inhibits testosterone (T) production by Leydig cells (LC). The aim of this study was to analyze mechanism/s by which Col-IV exerts this effect. No significant differences in the specific binding of hCG to LH/hCG receptors in LC cultured on uncoated or Col-IV coated plates were observed. An inhibition of cAMP production in hCG-stimulated LC cultured on Col-IV was detected. The inhibition exerted by Col-IV on T production in response to hCG was also observed when cells were stimulated with 8Bromo-cAMP. In addition, conversion of steroid precursors to T in LC cultured on uncoated and Col-IV coated plates was similar. On the other hand, we detected an increase of ERK1/2 phosphorylation in hCG-stimulated LC cultured on Col-IV. Genistein added to LC cultures reduced the ability of Col-IV to increase ERK1/2 phosphorylation and reverted the inhibitory effect of Col-IV on T production. An inhibitor of MEK, PD98059 added to LC cultures also reverted the inhibitory effect of Col-IV on T production. A decrease of steroidogenic acute regulatory protein (StAR) expression in hCG-stimulated LC cultured on Col-IV coated plates that could be reverted by addition of PD98059 to the cultures was also demonstrated. All together these results suggest that Col-IV inhibits T production in LC by binding to integrins, activating ERK1/2, decreasing cAMP production and decreasing StAR expression.
GnRH controls the synthesis and release of the pituitary gonadotropic hormones. MAP kinase (MAPK) cascades, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, are crucial for GnRH-induced gene activation. In the present study, we investigated the function of GnRH-induced MAPK phosphatases (MKPs) using an in vivo mouse model as well as the alphaT3-1 cell line. Following GnRH agonist stimulation, in vivo gene profiling demonstrated that both MKP-1 and MKP-2 are induced with distinct temporal profiles, suggesting differential roles of these MKPs in the regulation of MAPK activation. Elevated activity of MKP-2 in alphaT3-1 cells, through either overexpression or activation of the endogenous MKP-2 gene, was correlated with inhibition of GnRH-induced activation of ERK and JNK, as well as the expression of ERK- and JNK-dependent proto-oncogenes. These data supported the conclusion that GnRH-induced MKPs likely serve as negative feedback regulators that modulate MAPK activity and function in the GnRH signaling pathway.
Geoffrey Wingfield Harris' demonstration of hypothalamic hormones regulating pituitary function led to their structural identification and therapeutic utilization in a wide spectrum of diseases. Amongst these, Gonadotropin Releasing Hormone (GnRH) and its analogs are widely employed in modulating gonadotropin and sex steroid secretion to treat infertility, precocious puberty and many hormone-dependent diseases including endometriosis, uterine fibroids and prostatic cancer. While these effects are all mediated via modulation of the pituitary gonadotrope GnRH receptor and the G(q) signaling pathway, it has become increasingly apparent that GnRH regulates many extrapituitary cells in the nervous system and periphery. This review focuses on two such examples, namely GnRH analog effects on reproductive behaviors and GnRH analog effects on the inhibition of cancer cell growth. For both effects the relative activities of a range of GnRH analogs is distinctly different from their effects on the pituitary gonadotrope and different signaling pathways are utilized. As there is only a single functional GnRH receptor type in man we have proposed that the GnRH receptor can assume different conformations which have different selectivity for GnRH analogs and intracellular signaling proteins complexes. This ligand-induced selective-signaling recruits certain pathways while by-passing others and has implications in developing more selective GnRH analogs for highly specific therapeutic intervention.
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