Steroidogenic Factor-1 Expression Research Articles

Effect of Notch Signal Pathway on Steroid Synthesis Enzymes in TM3 Cells.

Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Unilateral adrenalectomy for a drug-resistant bilateral primary aldosteronism with heart failure: pathophysiology and surgical indication

BackgroundPatients with bilateral primary aldosteronism (PA) generally are treated with antihypertensive drugs, but optimal treatment for patients with complications due to refractory hypertension has not been established. In this report, we present a case with bilateral PA who presented with persistent hypertension, despite treatment with 6 drugs, and left-dominant heart failure, which was improved after unilateral adrenalectomy.Case presentationA 61-year-old man was admitted to our hospital because of severe left-dominant heart failure. His heart rhythm was atrial fibrillation and the left ventricle was diffusely hypertrophic and hypokinetic. Coronary arteries were normal on coronary arteriogram. Primary aldosteronism was suspected based on severe hypokalemia (2.5 mEq/L) and plasma aldosterone concentration (PAC; 1,410 pg/mL). Although computed tomography (CT) showed a single left cortical nodule, adrenal vein sampling (AVS) indicated bilateral PA. Early in the case, heart failure and hyperkalemia in this patient were improved by treatment with a combination of 6 antihypertensive drugs (spironolactone 25 mg/day, eplerenone 100 mg/day, azosemide 60 mg/day, tolvaptan 7.5 mg/day, enalapril 5 mg/day, and bisoprolol fumarate 10 mg/day); however, heart failure relapsed after four months of treatment. We hypothesized that hypertension caused by excess aldosterone was inducing the patient’s heart failure. In order to reduce aldosterone secretory tissue, a laparoscopic adrenalectomy was performed for the left adrenal gland, given the higher level of aldosterone from the left gland compared to the right. Following surgery, the patient’s heart failure was successfully controlled despite the persistence of high PAC. Treatment with anti-hypertensive medications was reduced to two drugs (eplerenone 100 mg/day and bisoprolol fumarate 10 mg/day). In order to elucidate the mechanism of drug resistance, immunohistochemistry (IHC) and real time-polymerase chain reaction (RT-PCR) assays were performed to assess the expression of steroidogenic factor 1 (SF-1), a regulator of steroid synthesis in adrenal tissue. IHC and RT-PCR demonstrated that the expression of SF-1 in this patient (at both the protein and mRNA levels) was higher than that observed in unilateral PA cases that showed good responsivity to drug treatment.ConclusionsUnilateral adrenalectomy to reduce aldosterone secretory tissue may be useful for patients with drug-refractory, bilateral PA. Elevated expression of SF-1 may be involved in drug resistance in PA.

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, invitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

Steroidogenic factor 1 protects mice from obesity-induced glucose intolerance via improving glucose-stimulated insulin secretion by beta cells.

As a potential druggable nuclear receptor, steroidogenic factor 1 (SF1) regulates obesity and insulin resistance in the ventromedial hypothalamic nucleus. Herein, we sought to demonstrate its expression and functions in islets in the development of obesity-induced diabetes. SF1 was barely detected in the beta cells of lean mice but highly expressed in those of non-diabetic obese mice, while decreased in diabetic ones. Conditional deletion of SF1 in beta cells predisposed diet-induced obese (DIO) mice to glucose intolerance by perturbing glucose-stimulated insulin secretion (GSIS). Consistently, forced expression of SF1 restored favorable glucose homeostasis in DIO and db/db mice by improving GSIS. In isolated islets and MIN6, overexpression of SF1 also potentiated GSIS, mediated by improvement of mitochondrial ATP production. The underlying mechanisms may involve oxidative phosphorylation and lipid metabolism. Collectively, SF1 in beta cell preserves GSIS to promote beta-cell adaptation to obesity and hence is a potential therapeutic target for obesity-induced diabetes.

Endometriosis: Update of Pathophysiology, (Epi) Genetic and Environmental Involvement.

Endometriosis is a chronic disease caused by ectopic endometrial tissue. Endometriotic implants induce inflammation, leading to chronic pain and impaired fertility. Characterized by their dependence on estradiol (via estrogen receptor β (ESRβ)) and their resistance to progesterone, endometriotic implants produce their own source of estradiol through active aromatase. Steroidogenic factor-1 (SF1) is a key transcription factor that promotes aromatase synthesis. The expression of SF1 and ESRβ is enhanced by the demethylation of their promoter in progenitor cells of the female reproductive system. High local concentrations of estrogen are involved in the chronic inflammatory environment favoring the implantation and development of endometriotic implants. Similar local conditions can promote, directly and indirectly, the appearance and development of genital cancer. Recently, certain components of the microbiota have been identified as potentially promoting a high level of estrogen in the blood. Many environmental factors are also suspected of increasing the estrogen concentration, especially prenatal exposure to estrogen-like endocrine disruptors such as DES and bisphenol A. Phthalates are also suspected of promoting endometriosis but throughmeans other than binding to estradiol receptors. The impact of dioxin or tobacco seems to be more controversial.

Unveiling the identities of null cell tumours: Epigenomics corroborate subtle histological cues in pituitary neuroendocrine tumour/adenoma classification.

Pituitary neuroendocrine tumour (PitNET)/adenoma classification is based on cell lineage and requires immunopositivity for adenohypophysial hormones and/or transcription factors (TFs) SF1, TPIT, or PIT1. PitNET/adenomas lacking lineage affiliation are termed "null cell" tumours (NCTs). NCT diagnosis may be afflicted by methodological limitations and inconsistent diagnostic approaches. Previous studies have questioned the existence of true NCTs. In this study, we explore the epigenomic identities of PitNET/adenomas lacking clear TF-immunopositivity. 74 hormone-negative PitNET/adenomas were immunostained and scored for SF1, TPIT, and PIT1 expression. All tumours were classified as gonadotroph, corticotroph, PIT1-positive or "null cell". NCTs were subjected to global DNA methylation analysis. Epigenomic profiles of NCTs were compared to reference tumours using UMAP plotting and methylation-based classification. TF-immunostaining revealed definite lineage identity in 59 of 74 (79.7%) hormone-negative PitNET/adenomas. Of the remaining 15 NCTs, 13 demonstrated minimal and inconclusive nuclear SF1- or TPIT-expression (5 and 8, respectively). Two NCTs were entirely immunonegative. UMAP plotting and methylation-based classification demonstrated that the epigenomes of NCTs with minimal SF1- or TPIT-expression, were adequately affiliated with gonadotroph or corticotroph lineages, respectively. The two immunonegative NCTs were located near the corticotroph PitNET/adenomas via UMAP, while the methylation classifier could not match these two cases to predefined tumour classes. Epigenomic analyses substantiate lineage identification based on minimal TF-immunopositivity in PitNET/adenomas. This strategy dramatically decreases the incidence of NCTs and further challenges the legitimacy of NCTs as a distinct PitNET/adenoma subtype. Our study may be useful for guiding diagnostic efforts and future considerations of PitNET/adenoma classification.

RF07 | PSUN355 Downstream Targets of the Steroidogenic Factor-1 (SF-1) Antagonist OR-449 in PDX Models of ACC

Abstract Background Adrenocortical cancer (ACC) is a rare endocrine malignancy frequently diagnosed at an advanced stage when prognosis is dismal. Mitotane, the only approved therapy for locally advanced or metastatic disease, is minimally effective, and recent clinical trials with multi-kinase inhibitors or immunotherapies have been disappointing. OR-449 is a potent, selective, and orally bioavailable small molecule antagonist to steroidogenic factor-1 (SF-1 or NR5A1), a nuclear receptor and a transcription factor essential for the growth and development of the adrenal gland. SF-1 was recently described as a candidate "master transcription factor" in adult ACC (1). We previously reported that OR-449, dosed orally at 30 mg/kg for 28 days, completely blocked the growth of SJ-ACC3, a pediatric ACC patient-derived tumor xenograft (PDX), in immunocompromised mice. To help identify biomarkers for following the action of OR-449 in patient tumors, we evaluated a second pediatric ACC PDX, SW1939, which has high SF-1 expression and responded to OR-449 (10, 30 or 100 mg/kg) with inhibition of tumor growth and improved event-free survival over 24 days of dosing (an event is a 4-fold increase in tumor volume). Methods We first measured circulating steroids, steroid precursors and steroid sulfates in SW1939 tumor-bearing mice with or without OR-449 treatment using liquid chromatography/tandem mass spectrometry. Second, we investigated common pathways of gene regulation by OR-449 in SW1939 and SJ-ACC3 tumors by RNAseq after seven days of dosing (100 mg/kg or 30 mg/kg, respectively). Results Cortisol, DHEAS, steroid precursors (11-deoxycortisol, 17OH-progesterone, 17OH-pregnenolone) and sulfated Δ5 steroid precursors (pregnenolone sulfate, 17OH-pregnenolone sulfate, androstenediol-sulfate) were elevated in serum from SW1939 tumor-bearing mice compared to CD-1 non tumor-bearing mice, consistent with tumor-derived origin. Moreover, DHEAS, pregnenolone sulfate, 17OH-pregnenolone sulfate and androstenediol-sulfate were significantly suppressed (12-fold, 7.6-fold, 10.6-fold and 4.4-fold, respectively) following administration of OR-449 (100 mg/kg/day for 24 days) in SW1939 tumor-bearing mice. Final tumor weights were ∼2-fold lower than controls, indicating that circulating steroid reduction was disproportionately greater than the change in tumor volume. Global gene expression was significantly altered following 7-day exposure to OR-449 in both tumors. Significantly, PAPSS2 and SULT2A1 within the sulfation pathway were down-regulated in both SW1939 (2.3-fold and 1.2-fold, respectively) and SJ-ACC3 (1.6-fold and 5-fold, respectively). Conclusions OR-449 reduces steroid production, alters gene expression, and suppresses growth of two PDX models of ACC in mice. Tumor gene expression and steroid regulation by OR-449 are currently under investigation in other pediatric and adult ACC PDX models to evaluate these endpoints as potential biomarkers of SF-1 receptor engagement for initial clinical trials of OR-449 in ACC. Reference: (1) Corces et al., Science. 2018 Oct 26;362(6413): eaav1898; Reddy et al., Sci Adv. 2021 Nov 26;7(48): eabf6123. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Low H3K27 acetylation of SF1 in PBMC: a biomarker for prenatal dexamethasone exposure-caused adrenal insufficiency of steroid synthesis in male offspring.

Dexamethasone is widely used to treat pregnancy disorders related to premature delivery. However, lots of researches have confirmed that prenatal dexamethasone exposure (PDE) could increase the risk of offspring multiple diseases. This study was designed to elucidate the epigenetic mechanism of adrenal developmental programming and explore its early warning marker in peripheral blood mononuclear cells (PBMC). We found the adrenal morphological and functional changes of PDE male offspring rats before and after birth, which were mainly performed as the decreased serum corticosterone concentration, steroidogenic acute regulatory (StAR) protein expression, and histone 3 lysine 27 acetylation (H3K27ac) level of steroidogenic factor 1 (SF1) promoter region and its expression. Simultaneously, the expressions of glucocorticoid receptor (GR) and histone acetylation enzyme 5 (HDAC5) in the PDE male fetal rats were increased. In vitro, dexamethasone reduced the expression of SF1, StAR, and cortisol production and still increased the expression of GR and HDAC5, the binding between GR and SF1 promoter region, and protein interaction between GR and HDAC5. GR siRNA or HDAC5 siRNA was able to reverse the above roles of dexamethasone. Furthermore, in vivo, we confirmed that H3K27ac levels of SF1 promoter region and its expression in PBMC of the PDE group were decreased before and after birth, showing a positive correlation with the same indexes in adrenal. Meanwhile, in clinical trials, we confirmed that prenatal dexamethasone application decreased H3K27ac of SF1 promoter region and its expression in neonatal PBMC. In conclusion, PDE-caused adrenal insufficiency of male offspring rats was related to adrenal GR activated by dexamethasone in uterus. The activated GR, on the one hand, increased its direct binding to SF1 promoter region to inhibit its expression, on the other hand, upregulated and recruited HDAC5 to decrease H3K27ac level of SF1 promoter region, and strengthened the inhibition of SF1 and subsequent StAR expression.

Extracellular Vesicles Inhibit Proliferation and Invasion of Ovarian Endometrial Stromal Cells and Their Expression of SF-1, ERβ, and Aromatase.

ObjectiveEndometriosis is an estrogen-dependent chronic disease. The abnormal proliferation and invasion of ectopic stromal cells (ESCs) are important manifestations of endometriosis, and it is necessary to find safer and more effective treatments. Extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (UC-MSCs) have been shown to be promising for the treatment of many diseases, except endometriosis. The main purpose of this study was to explore the effect of EVs derived from UC-MSCs on ESCs and evaluate the therapeutic value of EVs on endometriosis.Study DesignFollowing the successful culture and identification of UC-MSCs, we collected the medium of UC-MSCs and extracted EVs by ultracentrifugation. Then, 120 μg/mL EVs were used to stimulate ESCs, which were collected to evaluate cell proliferation and invasion and expression of the estrogen-related proteins steroidogenic factor-1 (SF-1), estrogen receptors β (ERβ), and aromatase.ResultsCompared with the control group treated with isodose phosphate buffered saline (PBS), 120 μg/mL EVs exposure significantly decreased the expression of cyclin D1 (mRNA: n = 6, P = 0.02; protein: n = 6, P = 0.000) and matrix metalloproteinase (MMP) 9 (mRNA: n = 6, P = 0.04; protein: n = 6, P = 0.000) of ESCs, which were consistent with Cell Counting Kit-8(CCK-8) results (day 0: NC: 0.29 ± 0.04, 120 μg/mL EVs: 0.28 ± 0.04; day 1: NC: 0.42 ± 0.08, 120 μg/mL EVs: 0.32 ± 0.01; day 2: NC: 0.64 ± 0.07, 120 μg/mL EVs: 0.50 ± 0.05, P = 0.000; day 3: NC: 0.82 ± 0.09, 120 μg/mL EVs: 0.65 ± 0.07, P = 0.000; day 4: NC: 0.95 ± 0.11, 120 μg/mL EVs: 0.76 ± 0.07, P = 0.012; n = 6) and Transwell experiments (n = 6, P = 0.000). In addition, the expression of SF-1 (encoded by NR5A1; mRNA: n = 6, P = 0.000; protein: n = 6, P = 0.000), ERβ (encoded by ESR2; mRNA: n = 6, P = 0.000; protein: n = 6, P = 0.000), and aromatase (encoded by CYP19A1; mRNA: n = 6, P = 0.04; protein: n = 6, P = 0.000) in ESCs decreased significantly.ConclusionTaken together, the results show that 120 μg/mL EVs derived from UC-MSCs can effectively inhibit the proliferation and invasion of ESCs, as well as their expression of SF-1, ERβ and aromatase, and thus may lead to the alleviation of endometriosis.

Changes in the Expression of Steroidogenic Factor 1 (SF-1) in Neurons in the Ventromedial Nucleus of the Hypothalamus in Rats on Aging

Expression of steroidogenic factor 1 in neurons in the ventromedial nucleus of the hypothalamus in rats was studied using an immunohistochemical method and western blotting in young (2–3 months) and aged (2–2.5 years) male and female rats. Steroidogenic factor 1 was present in a majority of neurons in the ventromedial nucleus of the hypothalamus in young and aged rats, in the dorsomedial part of the nucleus. In aged rats, the proportion of neurons immunoreactive to steroidogenic factor 1 decreased significantly, from 88% to 68% in males and from 82 to 66% in females. There were no significant sex-related differences in the numbers of immunopositive neurons in either age group. Expression of steroidogenic factor 1 was also significantly lower in aged male and female rats in terms of western blot results. Thus, aging was associated with changes in the neurochemical composition of the ventromedial nucleus of the hypothalamus, which is involved in regulating energy metabolism and sexual behavior.

Establishment of an Immortalized Endometriotic Stromal Cell Line from Human Ovarian Endometrioma.

Endometrial-like stromal cells, one of the main components of endometriotic lesions, are an important in vitro model for studying cellular and molecular mechanisms associated with lesion development in endometriosis. However, the short life span of primary endometriotic stromal cells (Ec-ESCs) limits their use. Human telomerase reverse transcriptase (hTERT) plasmids can be used to develop immortalized cell lines. Here we aimed to establish an endometriotic stromal cell line by hTERT immortalization. Primary Ec-ESCs were obtained from a human ovarian endometriotic cyst. The purity was assessed by morphology and the expression of vimentin, cytokeratin, and human interferon-inducible transmembrane protein 1 (hIFITM1). Cells were infected with hTERT lentiviral vector and selected with hygromycin. hTERT mRNA levels were confirmed by RT-qPCR. Immortalized Ec-ESCs (iEc-ESCs) were characterized by examining the expression of morphological markers and key genes of interest, TP53, estrogen receptor β (ERβ), progesterone receptor (PR), and steroidogenic factor-1 (SF-1). Karyotyping and in vitro decidualization studies were also performed. Ec-ESCs were positive for vimentin and hIFITM1 and negative for cytokeratin, indicating that they were representative of Ec-ESC. The fibroblast-like morphology, expression of TP53, ERβ, PR, and SF-1 did not change before and after hTERT immortalization. iEc-ESCs showed an impaired decidualization response like primary Ec-ESCs when compared to normal eutopic stromal cells. Karyotyping showed that 15/19 cells had normal female karyotype, while 4/19 cells had partial trisomy 11q. Collectively, we successfully established and characterized an immortalized endometriotic stromal cell line. It is potentially useful as an in vitro experimental model to investigate endometriosis biology.

Evaluation of carvacrol on pituitary and sexual hormones and their receptors in the testicle of male diabetic rats.

Diabetes mellitus (DM) is a complex metabolic disease and it is also closely associated with a reduction in fertility in male patients. The purpose of the present study was to investigate the antidiabetic effect of carvacrol (CRV), as a potent antioxidant, on the numbers of germ cells and Sertoli cells in testicular tissue, and the messenger RNA (mRNA) and protein expression of some genes involved in spermatogenesis, including luteinizing hormone/choriogonadotropin receptor (LHCGR), follicle-stimulating hormone receptor (FSHR), and steroidogenic factor 1 (SF-1), as well as hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and insulin. Adult male Wistar rats (n = 32) were randomly divided into four groups (eight animals per group), including healthy control that received 0.2% Tween 80, diabetic control group, the diabetic group treated orally with CRV (75 mg/kg), and CRV group that received orally CRV (75 mg/kg). The duration of the treatment period lasted 8 weeks. In the diabetic group, the numbers of Sertoli cells and germ cells were significantly decreased, while the treatment with CRV prevented the degree of the damage to the cells mentioned earlier. CRV administration elevated the concentrations of insulin, T, FSH, and LH. Moreover, treatment with CRV significantly enhanced the levels of the mRNA and protein expression of SF-1, LHCGR, and FSHR. According to the obtained results, CRV administration could prevent the deleterious effects of DM on testicular germ cells, and it increases the levels of hormones and some essential genes, such as SF-1, LHCGR, and FSHR, involved in the process of spermatogenesis.

Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat.

Brain glycogen is a vital energy source during metabolic imbalance. Metabolic sensory neurons in the ventromedial hypothalamic nucleus (VMN) shape glucose counter-regulation. Insulin-induced hypoglycemic (IIH) male rats were infusedicv with the glycogen breakdown inhibitor CP-316,819 (CP) to investigate whether glycogen-derived fuel controls basal and/or hypoglycemic patterns of VMN gluco-regulatory neuron energy stability and transmitter signaling. CP caused dose-dependent amplification of basal VMN glycogen content and either mobilization (low dose) or augmentation (high dose) of this depot during IIH. Drug treatment also prevented hypoglycemic diminution of tissue glucose in multiple structures. Low CP dose caused IIH-reversible augmentation of AMPK activity and glutamate decarboxylase (GAD) protein levels in laser-microdissected VMN GABA neurons, while the higher dose abolished hypoglycemic adjustments in these profiles. VMN steroidogenic factor-1 (SF-1) neurons exhibited suppressed (low CP dose) or unchanged (high CP dose) basal SF-1 expression and AMPK refractoriness of hypoglycemia at each dose. CP caused dose-proportionate augmentation of neuronal nitric oxide synthase protein and enhancement (low dose) or diminution (high dose) of this profile during IIH; AMPK activity in these cells was decreased in high dose-pretreated IIH rats. CP exerted dose-dependent effects on basal and hypoglycemic patterns of glucagon, but not corticosterone secretion. Results verify that VMN GABA, SF-1, and nitrergic neurons are metabolic sensory in function and infer that these populations may screen unique aspects of neurometabolic instability. Correlation of VMN glycogen augmentation with attenuated hypoglycemic VMN gluco-regulatory neuron AMPK activity implies that expansion of this fuel reservoir preserves cellular energy stability during this metabolic threat.

Dog Steroidogenic Factor-1: Molecular cloning and analysis of epigenetic regulation.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that is important in steroid hormone production, and adrenal and gonad development. The SF-1 gene is highly conserved among most vertebrates. However, dog SF-1 registered in public databases, such as CanFam3.1, lacks the 5’ end compared to other mammals including mouse, human, bovine, and cat. Whether this defect is due to species differences or database error is unclear. Here, we determined the full-length dog SF-1 cDNA sequence and identified the missing 5’ end sequence in the databases. The coding region of the dog SF-1 gene has 1,386 base pairs, and the protein has 461 amino acid residues. Sequence alignment analysis among vertebrates revealed that the 5’ end sequence of dog SF-1 cDNA is highly conserved compared to other vertebrates. The genomic position of the first exon was determined, and its promoter region sequence was analyzed. The DNA methylation state at the basal promoter and the expression of dog SF-1 in steroidogenic tissues and non-steroidogenic cells were examined. CpG sites at the basal promoter displayed methylation kinetics inversely correlated with gene expression. The promoter was hypomethylated and hypermethylated in SF-1 expressing and non-SF-1 expressing tissues, respectively. In conclusion, we identified the true full sequence of dog SF-1 cDNA and determined the genome sequence around the first exon. The gene is under the control of epigenetic regulation, such as DNA methylation, at the promoter.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells.

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)-1[[(2 S)-1-(1-oxo-4-phenylbutyl)-2-pyrrolidinyl carbonyl]-2-pyrrolidinecarbonitrile (KYP-2047). We found that KYP-2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side-chain cleavage enzyme (CYP11A), 3β-hydroxysteroid dehydrogenase (3β-HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP-2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP-regulated progesterone synthesis, U0126-EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126-EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126-EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β-HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.

Expression and regulation of GnRHR2 gene and testosterone secretion mediated by GnRH2 and GnRHR2 within porcine testes

Gonadotropin-releasing hormone 2 receptor (GnRHR2) together with its cognate ligand involves in regulating reproductive behavior. However, little is known concerning the effect of transcription factor steroidogenic factor1 (SF-1) regulation on porcine GnRHR2 gene expression and GnRH2 regulation mechanism in testosterone secretion through GnRHR2. Our study demonstrated that GnRHR2 transcription levels were high in porcine testis. Immunohistochemistry analyses showed that GnRHR2 immunoreactivity was strong in the Leydig cells in boar testes. Two SF-1 binding sites were predicted in GnRHR2 promoter and the second site (-159/-149) was considered to be important for GnRHR2 promoter activity through site-directed mutagenesis. The binding of SF-1 to GnRHR2 promoter was confirmed by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Overexpression and knockdown experiments revealed that SF-1 could up-regulate porcine GnRHR2 expression. DNA methylation of GnRHR2 promoter CpG island also specifically regulated GnRHR2 expression. Meanwhile, our study also demonstrated GnRH2 treatment promoted the expression of SF-1 and steroidogenic acute regulatory protein (StAR), and that this treatment stimulated cAMP responsive element binding protein (CREB) phosphorylation, regulated the expression of GnRHR2, especially that of GnRHR2-X1, and promoted testosterone secretion in porcine Leydig cells. We speculated that testosterone secretion mediated by GnRH2 and GnRHR2 (mainly GnRHR2-X1) was regulated by phosphorylated CREB interacting with SF-1 to control StAR expression. Taken together, the present study indicates that SF-1 and GnRH2 are the essential regulatory factors for GnRHR2 expression. This study also explores the regulation mechanism of testosterone secretion mediated by GnRH2 and GnRHR2 in porcine Leydig cells.

Dietary supplementation with linseed oil improves semen quality, reproductive hormone, gene and protein expression related to testosterone synthesis in aging layer breeder roosters

Omega-3/n-3 polyunsaturated fatty acids (ω-3/n-3 PUFAs) play an important role in male reproductive function. The present study was designed to evaluate the effects of linseed oil (LO) as a source of α-linolenic acid (ALA, n-3 PUFA) on semen quality, plasma reproductive hormone and expression of key enzyme and protein related to steroidogenesis in aging layer breeder roosters. Ninety-six 57-wk-old Nongda No.3 layer breeder roosters were randomly assigned into one of four dietary treatments. All birds were fed a basal diet for 1wk and then assigned to a corn-soybean meal-based diet containing 0, 1, 2, 4% LO for 4 wk. After feeding trial, the roosters were slaughtered and investigated. The results showed that semen volume was dramatically increased relative to the other treatments in 2% LO group (P < 0.05). With an increase in dietary LO, semen concentration, sperm viability, sperm motility and total sperm count increased linearly (P < 0.05). Plasma follicle-stimulating hormone (FSH) level increased gradually and reached a maximum when 4% LO was fed (P < 0.01). Plasma luteinizing hormone (LH) levels in 1% and 2% LO group were improved significantly (P < 0.05) relative to the control group. Plasma testosterone (T) levels were remarkably improved compared with the control when birds were fed 2% and 4% LO (P < 0.05). A significant increase of steroidogenic acute regulatory protein (StAR) mRNA expression in 2% and 4% LO group was observed relative to the control group (P < 0.05). An increase in dietary LO supplementation from 1% to 4% markedly enhanced (P < 0.05) the mRNA expression of cholesterol side-chain cleavage enzyme (P450scc) compared to the control. A significant increase (P < 0.05) in the Steroidogenic Factor 1 (SF-1) mRNA levels was observed in the 2% and 4% LO-added groups. SF-1 protein expression was markedly increased by adding LO in diets (P < 0.05), and reached a maximum in 2% LO group.In conclusion, the results above suggest that dietary LO may improve semen quality by increasing the T hormone secretion, which may be related to higher StAR and P450scc mRNA expression and SF-1 expression. These findings provide a potential for using LO to attenuate the age-related sub-fertility in commercial layer breeder roosters.

SF-1 mediates reproductive toxicity induced by Cerium oxide nanoparticles in male mice

BackgroundCerium oxide nanoparticles (CeO2 NPs) have potential application for use in biomedical and in various consumer products. However, it is largely unclear whether CeO2 NPs have effects on male reproductive function.MethodsIn this study, male mice were examined for toxicity, if any, following chronic oral administration of CeO2 NPs for 32 days. In each animal, epididymides were examined for sperm motility and DNA integrity. Bloods were tested for testosterone levels. Testicular tissues were collected to determine the element Ce content, the daily sperm production (DSP), marker enzymes such as ACP, G6PD, γ-GT and SDH, mRNA expression levels of steroidogenesis genes Star, P450scc, P450c17, 3β-Hsd, and 17β-Hsd, as well as steroidogenic factor-1 (SF-1) gene/protein levels.ResultsThe results showed that CeO2 NPs (20 mg/kg and 40 mg/kg) increased the element Ce content in testis, the testis histopathological patterns and sperm DNA damage whereas decreased the testis weight, DSP and sperm motility. There were also remarkable reduction in testosterone levels and marker enzymes activities, down-regulated mRNA expression levels of several steroidogenesis genes such as Star, P450scc, P450c17, 3β-Hsd, and 17β-Hsd, as well as altered gene and protein expressions of SF-1.ConclusionThese results reveal the male reproductive toxicity of chronic exposure of CeO2 NPs in mice, hinting that the utilization of CeO2 NPs need to be carefully evaluated about their potential reproductive toxicity on the human health.

Corticosterone rather than ethanol epigenetic programmed testicular dysplasia caused by prenatal ethanol exposure in male offspring rats

ABSTRACTPrenatal ethanol exposure (PEE) could affect offspring’s testicular development. This study aimed to illuminate its intrauterine origin and the programming mechanism caused by PEE. Pregnant Wistar rats were given ethanol (4 g/kg.d) by gavage administration during gestational days (GD) 9–20. Serum samples and testes of male offspring rats were collected on GD20, postnatal week (PW) 6, and PW12. We found that PEE induced testicular morphological abnormality, low serum testosterone levels, expressive suppression of 3β-hydroxysteroid dehydrogenase (3β-HSD), and low acetylation levels of histone 3 lysine 14 (H3K14ac) of 3β-HSD before and after birth. In utero, when fetal rats were overexposed to corticosterone by PEE, the expression levels of testicular glucocorticoid receptor (GR) and histone deacetylase 2 (HDAC2) were increased, while that of steroidogenic factor 1 (SF1) was decreased. In vitro, corticosterone (rather than ethanol) at 500 to 2,000 nM concentration decreased testosterone production and 3β-HSD expression in a concentration-dependent manner. Moreover, corticosterone downregulated SF1 and upregulated HDAC2 via activating GR, accompanied by a low H3K14ac level of 3β-HSD; SF1 overexpression could reverse the increased HDAC2 expression, and knockdown of HDAC2 could partially reverse the inhibitory effects of corticosterone on H3K14ac level and 3β-HSD expression but not on SF1 expression. Taken together, PEE caused testicular dysplasia in male offspring rats, which was associated with corticosterone-induced low-functional programming of 3β-HSD through the GR/SF1/HDAC2/H3K14ac pathway. This study provides new academic perspectives to illuminate the theory of ‘Developmental Origins of Health and Disease.’