Mouse Leydig tumor cells produce C-19 steroids,including testosterone

The mouse Leydig tumor cells (MLTC-1) were derived from a transplantable Leydig cell tumor carried in C57BL/6 mice. The original cell line (M5480) produced testosterone and little progesterone. However, it was later shown that there were two subtypes of the cell line, one producing mainly progesterone and termed M5480P and the other which produced androgens and termed M5480A. MLTC-1 cells are reportedly derived from the former. We studied the production of testosterone by MLTC-1 cells using a specific and sensitive testosterone RIA, tandem mass spectrometry (TMS) and examined the expression of mRNA of some key enzymes involved in steroidogenesis. Although the molar yields were 1:20:60 for testosterone, androstenedione and progesterone, respectively, in response to human chorionic gonadotropin (hCG), testosterone measured by our RIA accounted for 94% of the testosterone immunoreactivity. Both MLTC-1 and Balb/c Leydig cells expressed Steroidogenic Acute Response (StAR) protein mRNA in response to hCG. Cytochrome P450 17alpha-hydroxylase/17,20-lyase mRNA was expressed constitutively in MLTC-1 and Balb/c Leydig cells. Whereas the latter expressed 17beta-hydroxydehydrogenase/17-ketoreductase isoform Type 3mRNA in response to hCG, MLTC-1 cells expressed isoform Type 7 constitutively. The absence of isoform Type 3 in MLTC-1 cells thus may account for the low conversion of androstenedione to testosterone in this cell line. However, with a very specific and sensitive RIA even the low production of testosterone becomes meaningful. In conclusion MLTC-1 cells produce testosterone.     

Evidence for nitrite reductase activity in intact mouse Leydig tumor cells

Nitric oxide (NO) supposedly derived via L-arginine-NO synthase (NOS) pathway has been implicated in inhibiting steroidogenesis by binding the heme moiety of steroidogenic enzymes. Previously, nitrite, and to a lesser extent nitrate ions inhibited steroidogenesis via NO by hitherto unknown reduction mechanism. Recently, a putative mammalian nitrite reductase activity ascribed to complex III of mitochondrial respiratory chain complexes (MRCC) has been reported, where MRCC inhibitors reduced NO production from nitrite variably. We thus studied the effects of MRCC inhibitors on testosterone production in mouse Leydig tumor cells (MLTC-1) without (basal) or with human chorionic gonadotropin (hCG) stimulation. In stimulated MLTC-1, MRCC inhibitors decreased testosterone production, order being: complex III (antimycin A and myxothiazol) > complex I (rotenone) > complex II (thenoyltrifluoroacetone), while cAMP production increased inversely. In unstimulated MLTC-1, MRCC inhibitors in same order, increased basal testosterone production, which correlated inversely with the percentage inhibition of NO production, with one exception; while antimycin A did not inhibit NO production in the nitrite reductase study mentioned above, it increased basal testosterone production in the present study. While MLTC-1 expressed mRNA for endothelial and neuronal, but not inducible NOS, various stimulators and inhibitors of L-arginine-NOS pathway had no effect on basal testosterone production in MLTC-1 or fresh Balb/c Leydig cells. Moreover, hCG increased nitrate uptake into MLTC-1, which suggests the gonadotropin aids nitrite and nitrate ions in their steroidogenesis inhibitory activity. In conclusion, this study supports the existence of a surrogate mammalian nitrite reductase and the dormancy of L-arginine-NOS pathway in MLTC-1.     

Brain-derived neurotrophic factor: A steroidogenic regulator of Leydig cells

The brain-derived neurotrophic factor (BDNF) was first recognized for its roles in the peripheral and central nervous systems, and its complex functions on mammalian organs have been extended constantly. However, to date, little is known about its effects on the male reproductive system, including the steroidogenesis of mammals. The purpose of this study was to elucidate the effects of BDNF on testosterone generation of Leydig cells and the underlying mechanisms. We found that BDNF-induced proliferation of TM3 Leydig cells via upregulation of proliferating cell nuclear antigen ( Pcna) and promoted testosterone generation as a result of upregulation of steroidogenic acute regulatory protein ( Star), 3b-hydroxysteroid dehydrogenase ( Hsd3b1), and cytochrome P450 side-chain cleavage enzyme ( Cyp11a1) both in primary Leydig cells and TM3 Leydig cells, which were all attenuated in Bdnf knockdown TM3 Leydig cells. Furthermore, the possible mechanism of testosterone synthesis was explored in TM3 Leydig cells. The results showed that BDNF enhanced extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, and the effect was disrupted by Bdnf deletion. Moreover, PD98059, a potent selective inhibitor of ERK1/2 activation, compromised BDNF-induced testosterone generation and upregulation of Star, Hsd3b1, and Cyp11a1. The Bdnf knockdown assay, on the other hand, indicated the autocrine effect of BDNF on steroidogenesis in TM3 Leydig cells. On the basis of these results, we concluded that BDNF, acting as an autocrine factor, induced testosterone generation as a result of the upregulation of Star, Hsd3b1, and Cyp11a1 via stimulation of the ERK1/2 pathway.     

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.     

Binding of all-trans-retinoic acid to MLTC-1 proteins

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 mean ± SD) fmoles/8 × 10⁴ cells at 37 °C. About 90% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.9 μM and 0.65 μM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 μM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 μM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [³H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [³H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells. (Mol Cell Biochem 276: 55–60, 2005)This paper is dedicated to the memory of Prof. E. Quagliariello.     

Age-related changes in responsiveness of rat Leydig cells to hCG

The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30-80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and "total 17beta-hydroxy androgen" (17beta-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17beta-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 10(6) cells. These differences probably reflect changes in the metabolism of testosterone to 5alpha-reduced products with increasing age because 80% of androgen secreted at 30 days is 3alpha-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.     

Establishment of gonadotropin-responsive murine leydig tumor cell line

Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-5'-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.     

Stimulation of leydig cell testoterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone

Injection of a luteinizing hormone-releasing hormone (LHRH) agonist into 55-day-old male rats which had been hypophysectomized 3 days earlier resulted in a 10- to 30-fold increase in the levels of testosterone in serum and testicular interstitial fluid (IF) in the 4h following injection. The levels achieved were within or above the normal range for intact untreated rats of this age. In similar animals, injection of LHRH agonist also enhanced the serum testosterone response to injected hCG at $\text{1}\text{1}\text{2}\text{h}$, but not at later times after injection, and by 24h reduced IF levels of testosterone suggested that LHRH agonist had begun to inhibit stimulation by hCG. $\text{In vitro}$, dispersed Leydig cells from untreated hypophysectomized rats showed a 2-fold increase in testosterone responsiveness to LHRH agonist when compared to cells from intact rats, and this change was associated with an 80% increase in the number of Leydig cell LHRH-receptors.     

Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro

Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post‐EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. It also decreased the levels of Leydig cell‐specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA‐Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell‐specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM‐mediated action was reversed by S3I‐201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.     

Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor

As a cis‐acting non‐depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC‐1 tumour cells. Rat Leydig cells and MLTC‐1 cells were treated with CAC (5, 10 and 50 μmol/L) or nAChR agonists (50 μmol/L nicotine or 50 μmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC‐1 cells at 5 μmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC‐1 cells by up‐regulating the expression of Lhcgr and Star. Up‐regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up‐regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.