α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells.

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 µmol/L) or pretreated with a p38 inhibitor (50 µmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 µmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 µmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 µmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression significantly increased (P < 0.05) in the 40 µmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 µmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 µmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 µmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). CONCLUSION: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.

Cadmium-induced preeclampsia-like phenotype in the rat is related to decreased progesterone synthesis in the placenta.

Cadmium (Cd) is a toxic element, which is associated with preeclampsia (PE).We treated pregnant rats with cadmium chloride from gestational days (GDs) 9-12 to introduce the PE-like animal model. Maternal systolic blood pressures (SBPs) and body weights were measured on GDs 0, 5, 10, 15, and 20. Foetuses were delivered by Caesarean section on GD20. Then, the dams were sacrificed and the specimens were obtained. The morphological analysis of the placentae was carried out by haematoxylin and eosin staining examination and immunohistochemistry assay.Our study showed that Cd-treated rats developed PE-like phenotypes, such as hypertension, albuminuria, and foetal growth restriction. Moreover, Cd-injected rats displayed abnormal placental angiogenesis and lower progesterone (P4) levels. We further demonstrated that Cd also inhibited the mRNA and protein expressions of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), which are involved in P4 synthesis in the rat placenta.Our study demonstrates that maternal Cd exposure disrupts the local synthesis of P4 in the placenta, which contributes to the onset of PE in pregnant rats. Supplementing P4 at the early gestational stage may be a promising therapeutic strategy to prevent PE, which requires further investigation.

Linum usitatissimum seeds oil down-regulates mRNA expression for the steroidogenic acute regulatory protein and Cyp11A1 genes, ameliorating letrezole-induced polycystic ovarian syndrome in a rat model.

The safety and effectiveness of nutricetics suggest that they may offer an alternative to pharmaceutical and surgical therapy for hormone-dependent disorders, such as polycystic ovarian syndrome (PCOS). We investigated the effects of Linum usitatissimum seed oil (LSO) on ovarian functionality, its molecular targets, and the oxidative response in hyperandrogenism-induced polycystic ovary. The composition of LSO has been analyzed using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS). A well-established PCOS rat model orally administered with letrozole daily for 21 days was used to investigate the effect of LSO at doses of 1 and 2 mL/kg body weight for 28 days. The effect on hormonal profile and antioxidant status, histopathology (cell proliferation), and the expression ratio of the steroidogenic acute regulatory protein (StAR) and Cyp11A1 gene were evaluated. LSO exerted beneficial effects on PCOS rat models via restoring glutathione (GSH), malondialdehyde (MDA), beta subunit subunit luteinizing hormone (LH), testosterone levels, and histopathological scoring. Furthermore, LSO reversed the elevated StAR and Cyp11A1 genes in the PCOS rat model. This study demonstrated the molecular and cellular mechanisms of the beneficial effect of LSO against the reproductive and metabolic disorders of PCOS.

Novel promising reproductive and metabolic effects of Cicer arietinum L. extract on letrozole induced polycystic ovary syndrome in rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Chickpea was used in both greek and indian traditional medicine for hormonal related conditions as menstrual induction, acceleration of parturation, treatment of retained placenta and stimulation of lactation. Chickpea (Cicer arietinum) sprout isoflavone isolates exhibited reasonable estrogenic activities. Isoflavones, a subtype of phytoestrogens, are plant derivatives with moderate estrogenic activity that tend to have protective effects on hormonal and metabolic abnormalities of women with polycystic ovary syndrome (PCOS). AIM OF THE STUDY: In this study, we investigated the effect of UPLC/ESI-MS characterized Cicer arietinum L. seeds ethanol extract (CSE) on ovarian hormones, oxidative response and ovarian histological changes on induced PCOS rat model. MATERIALS AND METHODS: Thirty-five rats were divided into five groups including negative control, PCOS, and treatment groups. PCOS was induced using letrozole (1 mg/kg) daily orally for 21 days. Each treatment group was treated with one of the following for 28 days after induction of PCOS: clomiphene citrate (1 mg/kg), and CSE at 250 and 500 mg/kg. Ovaries and uteri were excised, weighed and their sections were used for quantitative real-time reverse transcriptase polymerase chain reaction, antioxidant assays and histomorphometric study of the ovaries. The antioxidant assays, histopathological examination, hormonal and metabolic profiles, and Cyp11a1(steroidogenic enzyme) mRNA expression were measured. RESULTS: In all treatment groups, ovarian weight was significantly decreased despite having no significant effect on uterine weight. Histomorphometric study in the treatment groups revealed a significant decrease in the number and diameter of cystic follicles, a significant increase in granulosa cell thickness while, thickness of theca cells was significantly decreased when compared to PCOS. Hormone levels, metabolic profile and antioxidant status were improved in the treatment groups. Moreover, Cyp11a1 mRNA expression was significantly downregulated in the treatment groups compared to PCOS. CONCLUSIONS: In the current study, CSE enhanced the reproductive and metabolic disorders which were associated with PCOS induction. For the first time, we have highlighted the effect of CSE in treating PCOS and its associated manifestations.

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Effect of genistein on the gene expressions of androgen generating key enzymes StAR, P450scc and CYP19 in rat ovary.

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary.

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3ß-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

Paeoniflorin extract reverses dexamethasone-induced testosterone over-secretion through downregulation of cytochrome P450 17A1 expression in primary murine theca cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic Ovarian Syndrome (PCOS) is a complex endocrine and reproductive disorder. A main hallmark includes increased androgen production. The root of Paeonia lactiflora Pall. (Bai Shao) is used in Chinese herbal medicine for reproductive disorders, however its effects and mechanisms on ovarian theca cells has not yet been fully elucidated. AIM OF THE STUDY: The aim of this study was to evaluate effect of paeoniflorin extract (PFE), the main constituents of Bai Shao, on androgen production in ovarian theca cells. MATERIALS AND METHODS: Primary murine theca cells were treated with concentrations of PFE (1-100 µg/mL) in the presence of dexamethasone (10 µM) with media-only treated cells used as the control. After 24 h, culture media was collected for biochemistry assays of testosterone and progesterone. Expression of key steroidogenic enzymes, cholesterol side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17A1) was characterized using immunofluorescence staining, immunoblotting and qRT-PCR. RESULTS: Dexamethasone significantly enhanced testosterone secretion (P < 0.05 vs. the control cells). PFE reversed over-production of testosterone induced by dexamethasone in a dose-dependent manner. The treatment with PFE also normalized production of progesterone in dexamethasone-treated cells. Expression of CYP11A1 and CYP17A1 in the theca cells were visualised by immunofluorescence staining. All doses of PFE significantly inhibited CYP17A1 expression detected by immunoblotting, but only 100 µg/mL of PFE downregulated CYP11A1 expression and reduced CYP11A1 significantly in dexamethasone-treated theca cells. CONCLUSIONS: PFE may reduce over-secretion of testosterone in theca cells through downregulation of CYP17A1 and CYP11A1. These findings provide scientific evidence to treat ovarian hyperandrogenism with the root of Paeonia lactiflora Pall.

Exposure to a glyphosate-based herbicide formulation, but not glyphosate alone, has only minor effects on adult rat testis.

Glyphosate has been suggested to be an endocrine disrupting chemical capable of disrupting male reproduction. There are conflicting data, however, with studies reporting effects from exposure to either glyphosate alone or to herbicide formulations, making comparisons difficult. We assessed rat testis histopathology and androgen function following two weeks exposure to either glyphosate at 2.5 and 25 mg/kg bw/day (5x and 50x Acceptable Daily Intake, ADI, respectively), or equivalent high dose of glyphosate in a herbicide formulation; Glyfonova. We observed no significant effects on testes or testosterone synthesis in rats exposed to glyphosate. Limited effects were observed in rats exposed to Glyfonova, with a small upregulation of the steroidogenic genes Cyp11a1 and Cyp17a1. We conclude that glyphosate alone has no effect on adult rat testis at exposure levels up to 25 mg/kg bw/day. Glyfonova induced only minor effects on steroidogenic gene expression, likely caused by additives other than glyphosate.

Omega-3 fatty acids modulate the lipid profile, membrane architecture, and gene expression of leiomyoma cells.

Uterine leiomyomas (fibroids or myomas) are the most common benign tumors of premenopausal women and new medical treatments are needed. This study aimed to determine the effects of omega-3 fatty acids on the lipid profile, membrane architecture and gene expression patterns of extracellular matrix components (collagen1A1, fibronectin, versican, or activin A), mechanical signaling (integrin ß1, FAK, and AKAP13), sterol regulatory molecules (ABCG1, ABCA1, CAV1, and SREBF2), and mitochondrial enzyme (CYP11A1) in myometrial and leiomyoma cells. Myometrial tissues had a higher amount of arachidonic acid than leiomyoma tissues while leiomyoma tissues had a higher level of linoleic acid than myometrial tissues. Treatment of primary myometrial and leiomyoma cells with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) reduced the monounsaturated fatty acid (MUFA) content and increased the polyunsaturated fatty acid (PUFA) content in both cell types. Myometrial and leiomyoma cell membranes were in the liquid-crystalline phase, but EPA- and DHA-treated cells had decreased membrane fluidity. While we found no changes in the mRNA expression of ECM components, EPA and DHA treatment reduced levels of ABCG1, ABCA1, and AKAP13 in both cell types. EPA and DHA also reduced FAK and CYP11A1 expression in myometrial cells. The ability of omega-3 fatty acids to remodel membrane architecture and downregulate the expression of genes involved in mechanical signaling and lipid accumulation in leiomyoma cells offers to further investigate this compound as preventive and/or therapeutic option.

Continuous soy isoflavones exposure from weaning to maturity induces downregulation of ovarian steroidogenic factor 1 gene expression and corresponding changes in DNA methylation pattern.

Female Wistar rats were treated with orally administered soy isoflavones at concentrations of 0, 25, 50, or 100mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian steroidogenesis was evaluated. After soy isoflavones were administered, a significant (P<0.05) decrease (44%) in the serum estrodial levels of the high-dose (HD) group were observed. Cultured granulosa cells from the middle- (MD) and HD groups showed significantly (P<0.05) reduced (31%, 45%, respectively) in vitro estradiol secretion, and those from the HD group showed significantly (P<0.05) reduced progesterone (25%) secretion. Compared with the control group, the mRNA expression of the steroidogenic acute regulatory protein (Star), cytochromeP450 cholesterol side chain cleavage (Cyp11a1 and Cyp19a1), and hydroxysteroid dehydrogenase 3b (Hsd3b) genes also decreased. Real-time quantitative PCR and Western blotting revealed a significant (P<0.05) decrease in key transcription factor steroidogenic factor-1 (SF-1) expression in the HD group. The detection of DNA methylation using bisulfitesequencing PCR (BSP) suggested a significantly (P<0.05) increased total methylation rate in the proximal SF-1 promoter in the HD group. Further studies showed that treatment with soy isoflavones can significantly (P<0.05) increase the mRNA expression of DNA methyltransferase (DNMT) 1 and DNMT3a. This study proved that soy isoflavone administration from weaning until sexual maturity could inhibit ovarian steroidogenesis, suggesting that SF-1 might play an important role in this effect. In addition, DNA methylation might play a role in the downregulation of SF-1 gene expression induced by soy isoflavones.

Expression of Cholesterol Hydroxylase/Lyase System Proteins in Yeast S. cerevisiae Cells as a Self-Processing Polyprotein.

2A peptide discovered in Picornaviridae is capable of self-cleavage providing an opportunity to carry out synthesis of several proteins using one transcript. Dissociation in the 2A sequence during translation leads to the individual proteins formation. We constructed cDNA including genes of the bovine cholesterol hydroxylase/lyase (CHL) system proteins-cytochrome P450scc (CYP11A1), adrenodoxin (Adx) and adrenodoxin reductase (AdR), that are fused into a single ORF using FMDV 2A nucleotide sequences. The constructed vectors direct the expression of cDNA encoding polyprotein P450scc-2A-Adx-2A-AdR (CHL-2A) in Escherichia coli and Saccharomyces cerevisiae. The induced bacterial cells exhibit a high level of CHL-2A expression, but polyprotein is not cleaved at the FMDV sites. In yeast S. cerevisiae, the discrete proteins P450scc-2A, Adx-2A and AdR are expressed. Moreover, a significant proportion of AdR and Adx is present in a fusion Adx-2A-AdR. Thus, the first 2A linker provides an efficient cleavage of the polyprotein, while the second 2A linker demonstrates lower efficiency. Cholesterol hydroxylase/lyase activity registered in the recombinant yeast cell homogenate indicates that the catalytically active CHL system is present in these cells. Consequently, for the first time the mammalian system of cytochrome P450 has been successfully reconstructed in yeast cells through expressing the self-processing polyprotein.

Gossypol inhibits LH-induced steroidogenesis in bovine theca cells.

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0-25 µg/mL) for 24 h. Gossypol inhibited LH-stimulated theca cell A4 and P4 production in a dose-dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down-regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down-regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.

Suppressed Sex Hormone Biosynthesis by Alkylresorcinols: A Possible Link to Chemoprevention.

Alkylresorcinols (ARs, 5-n-alkylresorcinols) are amphiphilic phenolic lipids in whole grain rye and wheat, with a long odd-numbered carbon chain. A preventive effect of whole grain diet on sex hormone-dependent cancers has been recognized, but the active component(s) or mechanisms are not known. We have investigated the effects of the ARs C15:0, C19:0, and C21:0, individually and in combination, on steroid hormone production by using the human adrenocortical cell line H295R. Decreased synthesis of dehydroepiandrosterone (DHEA), testosterone, and estradiol was demonstrated at low concentrations of C15:0 and C19:0. There were no indications of additive effects on steroid secretion from the combined treatment with equimolar concentrations of the three ARs. Gene expressions of CYP21A2, HSD3B2, and CYP19A1 were downregulated and CYP11A1 was upregulated by the ARs. The results on gene expression could not explain the effects on steroidogenesis, which may be due to direct effects on enzyme activities, such as inhibition of CYP17A1. Our results demonstrate suppressed synthesis of testosterone and estradiol by ARs suggesting a novel mechanism for ARs in the chemoprevention of prostate and breast cancer.

CYP11A1 expression in bone is associated with aromatase inhibitor-related bone loss.

Aromatase inhibitors (AIs) used as adjuvant therapy in postmenopausal women with hormone receptor-positive breast cancer cause diverse musculoskeletal side effects that include bone loss and its associated fracture. About half of the 391 patients treated with AIs in the Barcelona-Aromatase induced bone loss in early breast cancer cohort suffered a significant bone loss at lumbar spine (LS) and/or femoral neck (FN) after 2 years on AI-treatment. In contrast, up to one-third (19.6% LS, 38.6% FN) showed no decline or even increased bone density. The present study aimed to determine the genetic basis for this variability. SNPs in candidate genes involved in vitamin D and estrogen hormone-response pathways (CYP11A1, CYP17A1, HSD3B2, HSD17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, DHCR7, GC, CYP2R1, CYP27B1, VDR and CYP24A1) were genotyped for association analysis with AI-related bone loss (AIBL). After multiple testing correction, 3 tag-SNPs (rs4077581, s11632698 and rs900798) located in the CYP11A1 gene were significantly associated (P<0.005) with FN AIBL at 2 years of treatment. Next, CYP11A1 expression in human fresh bone tissue and primary osteoblasts was demonstrated by RT-PCR. Both common isoforms of human cholesterol side-chain cleavage enzyme (encoded by CYP11A1 gene) were detected in osteoblasts by western blot. In conclusion, the genetic association of CYP11A1 gene with AIBL and its expression in bone tissue reveals a potential local function of this enzyme in bone metabolism regulation, offering a new vision of the steroidogenic ability of this tissue and new understanding of AI-induced bone loss.

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

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

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

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

Effect of azaline B on follicular development and functions in the hamster.

The usefulness of azaline B, a GnRH antagonist, in suppressing gonadotropin secretion in the golden hamster was examined by examining follicular development, steroidogenesis and expression of steroidogenic enzymes. Serum levels of P and E declined significantly, while FSH or LH was undetectable in azaline B-treated hamsters. FSH significantly increased serum E levels, whereas LH upregulated serum P levels. The formation of antral follicles ceased in azaline-treated hamsters, but was reversed by FSH with or without LH supplement. FSH also activated the primordial follicle pool resulting in increased formation of primary and preantral follicles. Further, an increasing trend in the formation of preantral follicles in response to E or E + P, and the formation of antral follicles in response to E + P treatment was evident. The level of Cyp11a1 mRNA increased markedly in LH- or LH + FSH-treated hamsters, whereas FSH with or without LH upregulated Cyp17a1, Cyp19a1 and Fshr mRNA expression. E without or with P also upregulated ovarian Cyp19a1 mRNA expression. The expression of enzyme protein corroborated the mRNA data. In summary, azaline B is an efficient GnRH antagonist in the hamster, and will be useful in studying the selective effect of gonadotropins on ovarian functions without disrupting the physiological functions of other hormones in ovarian cells.

Genistein and daidzein affect in vitro steroidogenesis but not gene expression of steroidogenic enzymes in adrenals of pigs.

Phytoestrogens (PEs), including genistein and daidzein, are plant-derived substances that mimic or antagonize estrogen action in animals. The majority of studies investigated the effects of PEs on reproduction in humans and laboratory animals. The mechanisms of phytoestrogen action on reproductive processes in domesticated animals, including pigs, are garnering increasing attention. However, very few in vivo and in vitro studies investigating the effects of PEs on adrenal glands have been carried out on models other than humans and rats. The aim of the present study was to determine whether the effects of genistein and daidzein on adrenal in vitro steroidogenesis are accompanied by changes in expression of genes encoding key steroidogenic enzymes in porcine adrenocortical cells. The following genes were analyzed: cholesterol side-chain cleavage enzyme (P450scc, CYP11A1 gene), 3ß-hydroxysteroid dehydrogenase (3ß-HSD, HSD3B1 gene), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17A1 gene) and 21-hydroxylase (P450c21, CYP21A2 gene). Porcine adrenocortical cells collected from both luteal- and follicular-phase gilts were exposed for eight hours to genistein (10 µM), or daidzein (10 µM), in the absence or presence of ACTH (5 nM). Genistein and daidzein inhibited basal and ACTH-stimulated secretion of cortisol and corticosterone and stimulated secretion of androstenedione. PEs did not affect the expression of CYP11A1, HSD3B1, CYP17A1 and CYP21A2 in the adrenocortical cells of luteal- and follicular-phase gilts. It can be concluded that the influence of PEs on steroid secretion in porcine adrenal glands is not mediated by changes in the expression of genes encoding major steroidogenic enzymes. More studies are needed to elucidate the intracellular mechanisms leading to the PE-induced changes in adrenal steroidogenesis in pigs.

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

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