Mouse Leydig Cell Line Research Articles

The SET protein promotes androgen production in testicular Leydig cells.

SummaryApproximately 40% of middle‐aged men exhibit symptoms of late‐onset hypogonadism (LOH). However, the mechanism of androgen deficiency is still currently unclear. As shown in our previous studies, the SET protein is expressed in testicular Leydig cells and ovarian granule cells. This study was designed to investigate the effect of the SET protein on androgen production in Leydig cells. The AdCMV/SET and AdH1siRNA/SET adenoviruses were individually transduced into a cultured mouse Leydig cell line (mLTC‐1) with or without human chorionic gonadotropin (HCG) stimulation in vitro. The primary mouse Leydig cells were used to confirm the main data from mLTC‐1 cells. The SET protein was expressed in the cytoplasm and nucleus of mLTC‐1 cells. Testosterone production was significantly increased in mLTC‐1 cells overexpressing the SET protein compared with the control group (p < 0.05), whereas testosterone production was significantly decreased in the SET knockdown mLTC‐1 cells (p < 0.05). Consistent with the testosterone levels, the expression levels of the steroidogenic acute regulatory (StAR) and cytochrome P450c17α‐hydroxylase (CYP17a1) mRNAs and proteins synchronously changed according to the expression level of the SET protein. Interestingly, the expression of the SET protein was significantly increased in the mLTC‐1 cells stimulated with 0.04 and 0.1 U/mL hCG. In the mLTC‐1 cells transfected with AdH1siRNA/SET and concurrently stimulated with 0.1 U/mL hCG, both testosterone production and StAR expression were significantly lower than in the cells without SET knockdown (p < 0.05). In conclusion, the SET protein participates in regulating testosterone production by increasing the expression of StAR and CYP17a1, and it may be a downstream factor of the classic luteinizing hormone (LH)/luteinizing hormone receptor (LHR) signaling pathway. This study improves our understanding of the intracellular mechanism of testicular steroidogenesis and the pathophysiological mechanism of LOH in the aging male.

Perturbation of microRNA signalling by doxorubicin in spermatogonial, Leydig and Sertoli cell lines in vitro.

We have previously shown that in addition to its widely recognised cardiotoxicity, the chemotherapeutic doxorubicin (DOX) is able to induce transcriptional, microRNA (miRNA) and DNA methylation changes in the mouse testis. These changes perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. Here, we further investigated the differential miRNA expression induced by DOX in mouse spermatogonial (GC1), Leydig (TM3) and Sertoli (TM4) cell lines in vitro. We began by performing cell cycle analysis of the three mouse testicular cell lines to evaluate their sensitivity to DOX and thus select suitable doses for miRNA profiling. In keeping with our in vivo data, the spermatogonial cell line was the most sensitive, and the Sertoli cell line the most resistant to DOX-induced cell cycle arrest. We then further demonstrated that each cell line has a distinct miRNA profile, which is perturbed upon treatment with DOX. Pathway analysis identified changes in the miRNA-mediated regulation of specialised signalling at germ-Sertoli and Sertoli-Sertoli cell junctions following treatment with DOX. Amongst the most significant disease categories associated with DOX-induced miRNA expression were organismal injury and abnormalities, and reproductive system disease. This suggests that miRNAs play significant roles in both normal testicular function and DOX-induced testicular toxicity. Comparison of our in vitro and in vivo data highlights that in vitro cell models can provide valuable mechanistic information, which may also help facilitate the development of biomarkers of testicular toxicity and high-throughput in vitro screening methods to identify potential testicular toxicants.

Effects of enzymatic hydrolysate from seahorse Hippocampus abdominalis on testosterone secretion from TM3 Leydig cells and in male mice

Protein hydrolysates, the so-called bioactive peptides, are specific protein fragments that have positive effects on several body functions and may improve human health. Marine organism-derived protein hydrolysates and bioactive peptides have shown to possess many physiological functions. Seahorses, used in traditional medicine, are generally ground to powder form either for direct application or for application following dissolution in warm water. In this present study, we purified, hydrolyzed, and characterized two bioactive peptides (ALC and PEP) obtained from seahorse (Hippocampus abdominalis). The hydrolysates derived from seahorse significantly upregulated the expression of cyclin D and cyclin E and increased cell proliferation and testosterone level in the TM3 mouse Leydig cell line. These findings suggest that the hydrolysates stimulate the proliferation of TM3 cells via the AKT, ERK, and JNK pathways. The decline in the circulating testosterone levels in older men is associated with various adverse health effects. Following daily intake of hydrolysates for 12 weeks, the circulating level of testosterone and the sperm count in mice were measured. We found increased sperm motility (sperm count) as well as an increase in the testosterone level in male mice following a 12-week intake of hydrolysates derived from H. abdominalis. Hence, it can be suggested that seahorse-derived hydrolysates play an important role in improving male health by improving the serum testosterone level.

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

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

Di-(2-ethylhexyl) phthalate inhibits testosterone level through disturbed hypothalamic–pituitary–testis axis and ERK-mediated 5α-Reductase 2

Di-(2-ethylhexyl) phthalate (DEHP) has reproductive toxicity and can affect male reproductive development. In order to clarify adverse effects of DEHP on testicular physiology and testosterone production, Sprague-Dawley (SD) rats were dosed daily with DEHP by gavage for 30days; TM3 cells (mouse Leydig cell line) were treated with DEHP for 24h after pretreatment with vitamin C or U0126. Results indicated that the hypothalamic–pituitary–testis (HPT) axis was disturbed and serum testosterone, LH and FSH levels were decreased following DEHP exposure. Histomorphological changes of rat testes were also observed, such as deformed seminiferous tubules, aggregated chromatin, multiple vacuoles, swollen mitochondria, apoptotic germ cells and Sertoli cells, as well as increased Leydig cell numbers. Moreover, DEHP caused oxidative stress in vivo and in vitro and then induced the ERK pathway, which was required to mediate 5α-Reductase 2 and scavenger receptor class B-1 (SRB1) levels. However, levels of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), P450 17α-hydroxylase/17.20 lyase (P450c17), and P450 side-chain cleavage enzyme (P450scc) were not significantly altered after DEHP exposure. Taken together, DEHP-disturbed HPT axis and induced 5α-Reductase 2 contribute to the reduction of serum testosterone level. The activated ERK pathway is required to modulate expressions of 5α-Reductase 2 and SRB1.

The implication of pro-inflammatory cytokines in the impaired production of gonadal androgens by patients with pulmonary tuberculosis

The chronic nature of tuberculosis and the protracted immuno-inflammatory reactions are implied in a series of metabolic and immune-endocrine changes accompanying the disease. We explored components from the hypothalamous-pituitary-gonadal axis and their relationship with cytokines involved in disease immunopathology, in male TB patients. Plasma samples from 36 active untreated pulmonary TB male patients were used to determine TNF-α, IFN-γ, TGF-β, IL-6, cortisol, dehydroepiandrosterone, testosterone, progesterone, estradiol, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by ELISA. Healthy controls corresponded to 21 volunteers without contact with TB patients and similar age (40±16,8 years). Testicular histological samples from necropsies of patients dying from TB were immune-stained for IL-1β, TNF-α, IL-6 and IFN-γ. The TM3 mouse Leydig cell line was incubated with recombinants TNF-α, IFN-γ and TGF-β, supernatants were collected and used to measure testosterone by ELISA. Patients showed decreased levels of testosterone in presence of high amounts of LH, together with augmented IFN-γ, IL-6 and TGF-β levels. Testicular histological sections showed abundant presence of IL-1β, TNF-α, IL-6 and IFN-γ in interstitial macrophages, Sertoli cells and some spermatogonia. Invitro treatment of Leydig cells with these cytokines led to a remarkable reduction of testosterone production.

Uncarboxylated osteocalcin stimulates 25-hydroxy vitamin D production in Leydig cell line through a GPRC6a-dependent pathway.

Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells.

The mouse Leydig cell line MLTC-1 prefers L-thyroxine over 3,3',5-triiodo-L-thyronine in transport across plasma membrane and shows steroidogenesis response to thyroid hormone treatment

The mouse Leydig cell line MLTC-1 prefers L-thyroxine over 3,3',5-triiodo-L-thyronine in transport across plasma membrane and shows steroidogenesis response to thyroid hormone treatment

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

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

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

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

Microarray and gene ontology analyses reveal downregulation of DNA repair and apoptotic pathways in diethylstilbestrol-exposed testicular Leydig cells

This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17β-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories "DNA repair" and "apoptotic program" in DES-exposed cells. PAGE showed that "cell death," which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the "cell death" category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.

Leydig cells express the myelin proteolipid protein gene and incorporate a new alternatively spliced exon

Although the myelin proteolipid protein gene ( Plp1) is highly expressed in the central nervous system encoding the most abundant myelin protein in oligodendrocytes, it is also expressed in other tissues, including testis. Transgenic studies with mice that harbor Plp1–lacZ fusion genes suggest that Leydig cells are the source of Plp1 gene expression in testis. However, virtually nothing is known about Plp1 gene regulation in Leydig cells, which is the focus of this study. The first intron contains both positive and negative regulatory elements that are important in regulating Plp1 gene expression in oligodendrocytes. To test whether these elements are functional in Leydig cells, a battery of Plp1–lacZ fusion genes with partial deletion of Plp1 intron 1 sequence was transfected into the mouse Leydig cell line, TM3. Results presented here suggest that an enhancer, which is very potent in oligodendrocytes, is only nominally active in TM3 cells. The intron also contains several negative regulatory elements that are operative in TM3 cells. Moreover a new exon (exon 1.2) was identified within the first ‘intron’ resulting in novel splice variants in TM3 cells. Western blot analysis suggests that these splice variants, along with those containing another alternatively spliced exon (exon 1.1) derived from intron 1 sequence, give rise to multiple Plp1 gene products in the mouse testis.

Suppressive action of pituitary adenylate cyclase activating polypeptide (PACAP) on proliferation of immature mouse Leydig cell line TM3 cells

We aimed in the current study to understand the participation of PACAP in stage-specific Leydig and Sertoli cell functions. For this purpose, clonal cell lines TM3 (Leydig) and TM4 (Sertoli) cells, derived from the testis of immature BALB/c mice, were used. PACAP-specific receptors were detected in TM3 cells, but not in TM4 cells, which were characterized as PAC1 (type I) receptors. Stimulation of cAMP accumulation and testosterone secretion were observed in TM3 cells during 1-2 h treatment with PACAP38 (10(-10)-10(-7) M) or PACAP27 (10(-11)-10(-7) M). After around 10 h treatment with 10(-11)-10(-7) M PACAP38 or PACAP27, proliferation of TM3 cells was suppressed in time- and dose-dependent manners, which was confirmed by real-time cell electronic sensing (RT-CES) system and phase-contrast microscopy. At 6 h after the addition of PACAP38, the percent cell population in G(2)/M phases increased significantly, while that in S phase showed significant decrease with little change in G(0)/G(1) phases. The results revealed that PACAP exerts, in addition to early stimulatory effect on cAMP formation-steroidogenesis, sustained suppressive effect on cell proliferation in TM3 cells by controlling progression of the cell cycle. The suppressive action of PACAP on proliferation in TM3 cells supports the stage-specific participation of the peptide in differentiation of immature mouse Leydig cells.

Transforming Growth Factor-β Blocks Inhibin Binding to Different Target Cell Types in a Context-Dependent Manner through Dual Mechanisms Involving Betaglycan

Inhibin antagonizes activin and bone morphogenetic protein actions by sequestering their type II receptors in high-affinity complexes with betaglycan, a coreceptor that inhibin shares with TGF-beta. To clarify the nature and extent of interactions between inhibin and TGF-beta, we therefore examined 1) the mutual competition between these ligands for binding, 2) the regulation of endogenous betaglycan expression by inhibin and TGF-beta isoforms, and 3) the consequences of such betaglycan regulation for subsequent inhibin binding in mouse Leydig (TM3), Sertoli (TM4), adrenocortical cancer (AC), and gonadotroph (LbetaT2) cell lines, chosen to model cellular targets for local and endocrine actions of inhibin. Recognized inhibin, activin, and TGF-beta binding proteins and TGF-beta/activin signaling components were expressed by all four cell types, but AC and LbetaT2 cells notably lacked the type II receptor for TGF-beta, TbetaRII. Overnight treatment of TM3 and TM4 cells with TGF-beta1 suppressed the levels of betaglycan mRNA by 73 and 46% of control and subsequent [(125)I]inhibin A binding by 64 and 41% of control (IC(50) of 54 and 92 pm), respectively. TGF-beta2 acted similarly. TGF-beta pretreatments commensurately decreased the [(125)I]inhibin A affinity labeling of betaglycan on TM3 and TM4 cells. TGF-beta isoforms as direct competitors blocked up to 60% of specific inhibin A binding sites on TM3 and TM4 cells but with 9- to 17-fold lower potency than when acting indirectly via regulation of betaglycan. Only the competitive action of TGF-beta was observed with TbetaRII-deficient AC and LbetaT2 cells. Neither inhibin A nor inhibin B regulated betaglycan mRNA or competed for binding of [(125)I]TGF-beta1 or -beta2. Thus, inhibin binding to its target cell types is controlled by TGF-beta through dual mechanisms of antagonism, the operation of which vary with cell context and display different sensitivities to TGF-beta. In contrast, TGF-beta binding is relatively insensitive to the presence of either inhibin A or inhibin B.

Cross-Inhibitions of Human Chorionic Gonadotropin (hCG) and Ethynyl-Estradiol (EE2) at Pico-Molar Concentrations on Mouse Leydig Tumor Cells (mLTC) Progesterone Secretion

P-365 Introduction: The synthetic steroidal hormone ethynyl-estradiol is the most-abundantly used œstrogen component of oral contraceptives (about 0.2–0.3 ton per year worldwide) and its concentration in sewage treatment plant influents has been found to amount 3 to 66 ng/l (around 10−11 to 2x10−10M). These concentrations appear low compared to the doses in contraceptive pills (20–50 μg/day) but they are in the order of magnitude of the free active molecule in blood. In order to point out their eventual impact on human fecundity, we test these sub-nanomolar doses in various in vivo and in vitro assays related to reproduction to determine whether they can exhibit stimulatory or inhibitory effects. In the present paper, we present data concerning EE2 interaction with gonadotropin-stimulated steroidogenesis of a mouse Leydig cell line. Methods: Mouse Leydig Tumor cells (mLTC) were preincubated for 40h with Ethynyl-Estradiol EE2 (0 or 10−11-10−8 M) and subsequently incubated for 3h with hCG (0 or 10−11 M-2.4x10−10 M). Progesterone secretion in the medium was assessed by specific RIA. Results: Even at the lowest dose tested (10−11 M), EE2 induced an inhibition of the progesterone secretion by mLTCs during subsequent incubation with hCG. Interestingly, the inhibition was almost complete on the highest doses of hCG (1.2–2.4x10−10 M) but only marginal with lowest doses of hCG (1-4x10−11 M). In the absence of hCG during the incubation period, a stimulatory effect of EE2 present during the pre-incubation period, could be revealed. Interestingly, the presence of hCG even at the lowest stimulatory concentrations tested (10−11M), exerted an inhibitory effect on the progesterone response due to EE2 (10−10M) present during pre-incubation. This response was also much lower than that of the same dose of hCG (10−11M) in the absence of EE2 during the preincubation period. Discussion and conclusions: Both hCG and EE2 when alone were shown to exert stimulatory effects on the secretion of progesterone by mLTCs. Unexpectingly, not only they did not show any additive stimulations but, in the contrary, they exhibited strong cross-inhibition of their stimulatory effects. The inhibitory effect of pre-incubation of mLTCs with EE2 on hCG stimulation of progesterone secretion might be due either to interference with the hCG stimulatory pathway or to stimulation of progesterone-binding proteins. Although the precise molecular mechanisms of these effects are not clear, the observation that EE2 can act on mLTCs at pico-molar concentrations even over short periods of time might be of significance for possible interaction with human reproduction. Further in-vitro studies on testicular Leydig cells and in vivo assays will be necessary to assess the physiological and pharmacological relevance of these data.

Development of a self-proliferating Leydig cell line: a hyper-sensitive E-screening model

The mechanisms of estrogenic endocrine disruption on the male reproductive tract are poorly understood. In order to examine estrogenic properties of xenobiotic chemicals on male tissues, we have developed a mouse Leydig cell line (TM3-SF) that self-proliferates under serum-free conditions. This cell line was derived from ATCC's cell line, TM3. The development of TM3-SF was accomplished over a 4-month period by a progressive serum starvation of the original TM3 cells. The newly established cell line was maintained under serum-free conditions for 20 passages prior to testing. Sensitivity of the TM3-SF cells to estrogens was assayed by cell proliferation studies. A total of four compounds, diethylstilbestrol (DES), 17beta-estradiol, 17alpha-estradiol, and Bis-phenol A, were tested. Significant increases in cell proliferation occurred at various concentrations ranging from 1 pg/ml to 100 ng/ml for all four compounds. The order of potency observed was DES > Bis-phenol A > 17beta-estradiol and > 17alpha-estradiol. In addition, we investigated the mechanism for the self-proliferative properties of TM3-SF. The results of these trials indicate that either inhibin or activin is a primary growth factor for this cell line as a 50% inhibition of growth was noted when cell cultures were exposed to the anti-betaa subunit of inhibin/activin. Furthermore, the addition of the anti-betaa subunit of inhibin/activin blocked the DES-induced proliferation of TM3-SF. We conclude that the growth of TM3-SF cells is estrogen sensitive and that either inhibin or activin is involved in the self-regulation of growth.

Orphan Nuclear Receptor Nur77 Induces Zinc Finger Protein GIOT-1 Gene Expression, and GIOT-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor SF-1 via Recruitment of HDAC2

Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.

Cellular Distribution, Post-translational Modification, and Tumorigenic Potential of Human Group III Secreted Phospholipase A2

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.