Steroidogenic Factor-1 mRNA Levels Research Articles

Increased circulating miR-370-3p regulates steroidogenic factor 1 in endometriosis.

Endometriosis is a gynecologic disease common among reproductive-aged women caused by the growth of endometrial tissue outside the uterus. Altered expression of numerous genes and microRNAs has been reported in endometriosis. Steroidogenic factor 1 (SF-1), an essential transcriptional regulator of multiple genes involved in estrogen biosynthesis, is aberrantly increased and plays an important role in the pathogenesis of endometriosis. Here, we show the expression of SF-1 in endometriosis is regulated by miR-370-3p. Sera and tissue were collected from 20 women surgically diagnosed with endometriosis and 26 women without endometriosis. We found that miR-370-3p levels were decreased in the serum of patients with endometriosis while SF-1 mRNA levels were inversely upregulated in endometriotic lesions compared with respective controls. Transfection of primary endometriotic cells with miR-370-3p mimic or inhibitor resulted in the altered expression of SF-1 and SF-1 downstream target genes steroidogenic acute regulatory protein (StAR) and CYP19A1. Overexpression of miR-370-3p inhibited cell proliferation and induced apoptosis in endometriotic cells. This study reveals that miR-370-3p functions as a negative regulator of SF-1 and cell proliferation in endometriotic cells. We suggest a novel therapeutic strategy for controlling SF-1 in endometriosis.

Methylation of a Novel CpG Island of Intron I Is Associated With Steroidogenic Factor I Expression in Endometriotic Stromal Cells

Steroidogenic factor 1 (SF-1), a transcriptional factor essential for estrogen biosynthesis, is undetectable in endometrial stromal cells and aberrantly expressed in endometriotic stromal cells. We tried to gain further insight into the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells. We had previously identified a novel CpG island in SF-1, which is located in the downstream intron 1 region. Here, we evaluated the methylation status of this CpG island. We obtained the eutopic endometrium from disease-free participants (n = 8) and the walls of cystic endometriosis lesions of the ovaries from another group of participants (n = 8). None of the patients had received any preoperative hormonal therapy. Stromal cells were isolated from these 2 types of tissues and subjected to DNA bisulfite treatment and sequence analysis. The SF-1 messenger RNA (mRNA) levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells. Bisulfite sequencing showed strikingly increased methylation of a 1-kbp region around the previously identified CpG island in endometriotic cells compared with endometrial cells (P < .001). A strong correlation between SF-1 mRNA levels and percentage methylation of the intron 1 region of the SF-1 gene was observed in endometriotic cells (Spearman correlation coefficient, .96; P < .001). Methylation of the intron 1 region of the SF-1 gene is associated with its expression in endometriotic cells. This CpG island therefore plays an important role in regulating SF-1 expression.

Decoding the Functional Role of Fast, a Long Non-Coding RNA Transcribed Antisense to Nr5a1.

Over the past decade, research on long non-coding RNAs implicates them in regulatory roles associated with gene expression and chromatin architecture. While searching for conserved regulatory elements, an expressed sequence tag lacking an identifiable open reading frame was identified associated with Ftz-f1 (Nr5a1), the gene encoding steroidogenic factor 1 (SF-1). The nuclear receptor SF-1 is a key determinant and regulator of the adrenal and reproductive axes. Mammals with SF-1 mutations display a range of phenotypes, including absence of adrenal glands and gonads, diminished pituitary gonadotropins, disruption of the ventromedial hypothalamus, ovarian failure, and XY-sex reversal. While there are numerous studies on the function and regulation of SF-1, none involved mechanisms that require a noncoding RNA (ncRNA). Identification of the Ftz-F1 associated transcript, or Fast, and its association with SF-1 suggested a role for the ncRNA in SF-1 expression and/or activity and prompted further investigation of Fast. Directional RT-PCR was used to determine transcriptional orientation of Fast and demonstrated it is transcribed in the opposite orientation of Ftz-F1. Fast full-length structure was delineated using 5' and 3' RACE and sequence analysis of amplified Fast cDNA. This showed Fast has three exons and is polyadenylated and derived from multiple transcriptional start sites that border and extend into exon 1g of Ftz-F1. In addition to identification of a third exon, sequence analysis revealed two amplified, possible spliced variants that lacked part of exon 3. RT-PCR analysis of RNA from multiple mouse tissues and endocrine cell lines showed Fast and SF-1 share the same expression profile. To help define the sub-cellular location of Fast, nuclear and cytoplasmic RNA was isolated from MA-10 cells, a Leydig cell line, and analyzed for the presence of Fast by RT-PCR. This revealed that Fast is predominantly located in the cytoplasm. The similar tissue expression pattern for SF-1 and Fast suggested they share regulatory mechanisms. Further investigation showed that P-19 embryonal-carcinoma cells treated with retinoic acid had decreased SF-1 and Fast transcript levels and that granulosa cells isolated from mice treated with pregnant mare's serum gonadotropin had elevated levels of both Fast and SF-1 mRNA. Together, these findings indicate that Fast is a long non-coding RNA that shares the same tissue-restricted expression profile and hormone regulation as SF-1, suggesting it acts within a similar biological pathway. We hypothesize that Fast acts either in conjunction with SF-1 and thus is required for proper development and function of adrenal glands and gonads.

Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles ( Rana catesbeiana)

The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog ( Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48 h exposure to 1 and 100 μg/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5 ng/l of the estrogenic chemical, 17α-ethynylestradiol (EE 2), and 673 ng/l of the thyroid hormone, 3,5,3′-triiodothyronine (T 3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48 h exposure to EE 2 but not T 3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development.

Elementary studies on elevated steroidogenic factor-1 expression in aldosterone-producing adenoma

ObjectivesThe expression of steroidogenic factor-1 (SF-1) was elevated in adrenal aldosterone-producing adenoma (APA). The influence of SF-1 on adrenal tumorigenesis by adrenocortical cell line H295R cells was investigated. Materials and methodsReal-time PCR and Western blotting were used to detect SF-1 expression in 16 APA samples and 12 normal adrenal samples. Specific SF-1-shRNA plasmid was transfected into H295R cells to inhibit SF-1 expression. Western blotting and real-time PCR were used to verify the effects of RNAi on SF-1 inhibition. Subsequently, WST-1 and cell count were applied to evaluate cell proliferation at different SF-1 levels. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to measure cell apoptosis, and proliferation marker Ki-67 was studied by immunohistochemistry. ResultsCompared with normal adrenal samples, SF-1 mRNA and protein levels in APA samples were significantly higher. It was 10.48:1 at SF-1 mRNA and 0.87 ± 0.05 vs. 0.39 ± 0.07 at protein levels, respectively (P < 0.01). A decreased SF-1 significantly inhibited cell proliferation in the experimental and control cells. These results were supported by weaker Ki-67 staining in SF-1-inhibited cells [(36.9% ± 4.17%) vs. (58.48% ± 7.16%) (P < 0.01)]. Moreover, SF-1 inhibition induced a 2.7-fold increase in the percentage of apoptotic H295R cells (P < 0.01). ConclusionsElevated SF-1 may play an important role in APA formation and primary aldosteronism. SF-1 acts as an oncogenic factor, and its inhibition provides new insight into the understanding and treatment of related adrenal diseases.

Transcriptional Activation of Steroidogenic Factor-1 by Hypomethylation of the 5′ CpG Island in Endometriosis

Endometriosis is an estrogen-dependent disease. Steroidogenic factor-1 (SF-1), a transcriptional factor essential for activation of multiple steroidogenic genes for estrogen biosynthesis, is undetectable in normal endometrial stromal cells and aberrantly expressed in endometriotic stromal cells. The objective of the study was to unravel the mechanism for differential SF-1 expression in endometrial and endometriotic stromal cells. We identified a CpG island flanking the SF-1 promoter and exon I region and determined its methylation patterns in endometrial and endometriotic cells. The study was conducted at Northwestern University. Eutopic endometrium from disease-free subjects (n = 8) and the walls of cystic endometriosis lesions of the ovaries (n = 8) were investigated. Stromal cells were isolated from these two types of tissues. Measures are mentioned in Results. SF-1 mRNA and protein levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells (P < 0.001). Bisulfite sequencing showed strikingly increased methylation in endometrial cells, compared with endometriotic cells (P < 0.001). Demethylation by 5-aza-2'-deoxycytidine increased SF-1 mRNA levels by up to 55.48-fold in endometrial cell (P < 0.05). Luciferase assays showed that the -85/+239 region bearing the CpG island regulated its activity (P < 0.01). Natural or in vitro methylation of this region strikingly reduced SF-1 promoter activity in both cell types (P < 0.01). Chromatin immunoprecipitation assay showed that methyl-CpG-binding domain protein 2 binds to the SF-1 promoter in endometrial but not endometriotic cells. This is the first demonstration of methylation-dependent regulation of SF-1 in any mammalian tissue. These findings point to a new mechanism for targeting local estrogen biosynthesis in endometriosis.

Effects of gonadotropin-releasing hormone analog (GnRHa) on steroidogenic factor-1 (SF-1) and estrogen receptor β (ERβ) gene expression in the black porgy ( Acanthopagrus schlegeli)

We examined effects of GnRHa on expression of steroidogenic factor-1 (SF-1) and estrogen receptor β (ERβ) in the pituitary and gonad of protandrous black porgy ( Acanthopagrus schlegeli). Fish were intraperitoneally injected with 0.2 μg GnRHa/g fish and then pituitary, gonad and plasma were sampled at 0, 6, 12, 24 and 48 h after injection. In gonad, the mRNA levels of the SF-1 were high at 6 h post injection, and then continuously decreased until 24 h; high expression of ERβ mRNA levels was only observed at 12 h. In contrast, pituitary SF-1 mRNA levels were very low during the experimental period. GnRHa stimulation caused a significant increase of plasma testosterone (T) and estradiol-17β (E 2) after 24 h. We suggest that SF-1 and ERβ play an important role in the development of gonad and these genes are involved with sex change in fish.

Regulation of genes encoding steroidogenic factor-1 (SF-1) and gonadotropin subunits in the ovine pituitary gland

Steroidogenic factor-1 (SF-1) is a transcription factor originally characterized as a mediator of gene expression in steroidogenic tissues. Studies in SF-1 knockout mice revealed that SF-1 has additional roles at multiple levels of the hypothalamic–pituitary–gonadal axis, including regulation of gene expression in pituitary gonadotropes. Specific binding sites for SF-1 have been demonstrated in several pituitary genes with essential roles in gonadotropin synthesis, including α subunit, LHβ subunit, and GnRH receptor. In studies aimed at identifying physiological factors controlling pituitary expression of SF-1, GnRH has been implicated as a co-regulator of SF-1 and gonadotropin subunit genes. In both rats and ewes, elevated endogenous secretion of GnRH following ovariectomy was associated with increased amounts of SF-1 mRNA in the anterior pituitary gland. Conversely, removal of GnRH input to the pituitary gland by hypothalamic–pituitary disconnection (HPD) in ovariectomized (OVX) ewes reduced SF-1 expression. Despite these changes, however, treatment of OVX ewes with GnRH following HPD only partially restored levels of SF-1 mRNA in the pituitary gland. Therefore, it is possible that regulation of SF-1 gene expression by GnRH during the estrous cycle may involve ovarian hormones or other hypothalamic factors. Additional studies are required to further define the physiological roles of SF-1 in regulation of the hypothalamic–pituitary–gonadal axis in domestic ruminants.

Expression of the mouse gonadotropin-releasing hormone receptor gene in alpha T3-1 gonadotrope cells is stimulated by cyclic 3',5'-adenosine monophosphate and protein kinase A, and is modulated by Steroidogenic factor-1 and Nur77.

Regulation of GnRH receptor (GnRHR) expression levels in the pituitary is a crucial control point in reproduction. The promoter of the mouse GnRHR gene contains nuclear receptor half-sites (NRS) at -244/-236 and -15/-7 relative to the translation start site. Although binding of steroidogenic factor-1 (SF-1) to the -244/-236NRS is implicated in mediating basal and gonadotrope-specific expression, no function or protein-DNA interactions have previously been described for the -15/-7NRS. We report that levels of the endogenous GnRHR mRNA in alpha T3-1 cells are stimulated by forskolin and 8-bromo-cAMP. We also show that the orphan nuclear receptor Nur77 is expressed in alpha T3-1 cells, and that both SF-1 and Nur77 bind to the -15/-7NRS and -244/-236NRS in vitro. We show that the activity of the proximal (-579/+1) mouse GnRHR promoter is up-regulated by protein kinase A, via a mechanism that is modulated by SF-1, both positively and negatively, through binding to the -244/-236NRS or the -15/-7NRS, respectively. Nur77 appears to be capable of acting as a negative regulator of this response, via the -15/-7NRS. Furthermore, we show that forskolin up-regulates SF-1 mRNA levels in alpha T3-1 cells, indicating that the levels of SF-1 play a role in modulating the protein kinase A response.

Activation of cAMP-dependent protein kinase increases the protein level of steroidogenic factor-1.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is an essential regulator of endocrine organogenesis, sexual differentiation, and steroidogenesis. SF-1 is a transcriptional regulator of cAMP responsive genes, but the exact mechanisms by which cAMP-dependent PKA modulates SF-1 dependent transcription leading to increased steroidogenic output have not been determined. In this report the effects of PKA activation on SF-1 in living cells have been examined by the use of full-length SF-1 cDNA fused to the cDNA encoding green fluorescent protein (GFP). The GFP-SF-1 fusion protein localized to the nucleus of both steroidogenic Y1 cells and nonsteroidogenic COS-1 cells, and the functional properties of wild-type SF-1 were conserved. When the catalytic subunit of PKA was coexpressed with GFP-SF-1, we observed that the fluorescence emission was markedly elevated. These findings were confirmed by Western blot analysis, showing that stimulation of PKA increased SF-1 protein levels. The PKA- induced expression of SF-1 protein was not accompanied by an increase in SF-1 mRNA levels. However, pulse-chase studies showed a decrease in SF-1 degradation rate in response to activation of PKA, indicating that PKA elevates the level of SF-1 by increasing the stability of SF-1 protein.

The role of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins essential for adrenal steroidogenesis in the late-gestation ovine fetus.

To further understand the relative roles of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins that are essential for adrenal development, we investigated the effects of, first, an ACTH infusion and labour in intact fetuses and, secondly, the effect of an ACTH infusion to fetuses with and without a pituitary gland, on the relative abundance of the mRNA encoding for the ACTH receptor (MC2R), steroidogenic factor 1 (SF-1), cholesterol side-chain cleavage enzyme (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase (P450(C17)) in the fetal adrenal gland. ACTH(1-24) infusion (14.7 pmol/kg per h) to intact fetuses was without effect on the abundance of mRNA encoding MC2R and SF-1, irrespective of whether the infusion was given for 18 (115-132 days of gestation) or 32 days (115 days to term (147 days of gestation)). Hypophysectomy (HX) did not alter the expression of MC2R mRNA; however, the abundance of SF-1 mRNA fell by approximately 50% following the removal of the pituitary gland. ACTH(1-24) infusion to HX fetuses failed to restore levels of SF-1 mRNA to that seen in intact animals. P450(scc) and 3betaHSD mRNAs were increased by ACTH(1-24) infusion for 18 days in intact animals, although no effects of the infusion were seen on P450(C17) mRNA levels. For all three of these mRNAs, there was a significant increase in their abundance between 132 days of gestation and term in intact fetuses. By term, ACTH(1-24) infusion was without any additional effect on their abundance. HX decreased the expression of P450(scc), 3betaHSD and P450(C17) mRNAs, while ACTH(1-24) infusion to HX fetuses increased the expression of these mRNAs to levels seen in intact animals. There were significant correlations between the abundance of the mRNA for P450(scc), 3betaHSD and P450(C17), but not MC2R and SF-1, and premortem plasma cortisol concentrations. These results emphasise the importance of the pituitary gland and ACTH in the regulation of the enzymes involved in adrenal steroidogenesis. Factors in addition to ACTH may also play some role, as the infusion was not always effective in increasing the abundance of the mRNAs. Surprisingly, the mRNA for MC2R and SF-1 did not appear to be regulated by ACTH in the late-gestation ovine fetus, though a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance.

The promoter of murine follicle-stimulating hormone receptor: functional characterization and regulation by transcription factor steroidogenic factor 1.

The promoter of the FSH receptor (R) gene has been cloned from several species. Although some of its regulatory elements have been identified, its function still remains poorly characterized. Using transient transfections of luciferase reporter constructs, driven by various fragments of the murine (m) FSHR promoter, we identified a cell-specific promoter region. This domain is located in the distal part of the mFSHR promoter, -1,110 to -1,548 bp upstream of the translation initiation site, and it contains two steroidogenic factor 1 (SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and protein closely correlated in various steroidogenic cell lines with activity of the transfected mFSHR promoter/luciferase reporter construct carrying the distal activator domain. A dose-dependent increase in FSHR promoter activity was shown in nonsteroidogenic HEK 293 cells transiently transfected with SF-1 cDNA. SF-1 was found to bind to a nonconsensus 5'-CAAGGACT-3' SLBS-3 motif in the distal part of the promoter; formation of the SF-1/SLBS-3 complex could be reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a significant loss of SF-1-mediated mFSHR promoter activity. The second SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In conclusion, we have identified a regulatory domain in the mFSHR promoter participating in the cell-specific regulation of FSHR expression. We demonstrated for the first time that the mFSHR promoter possesses functional SF-1 binding sites and thus belongs to the group of SF-1-regulated genes. These findings provide further evidence for the key role of SF-1 in the regulation of genes involved in gonadal differentiation and endocrine functions.

The Promoter of Murine Follicle-Stimulating Hormone Receptor: Functional Characterization and Regulation by Transcription Factor Steroidogenic Factor 1

The promoter of the FSH receptor (R) gene has been cloned from several species. Although some of its regulatory elements have been identified, its function still remains poorly characterized. Using transient transfections of luciferase reporter constructs, driven by various fragments of the murine (m) FSHR promoter, we identified a cell-specific promoter region. This domain is located in the distal part of the mFSHR promoter, −1,110 to −1,548 bp upstream of the translation initiation site, and it contains two steroidogenic factor 1 (SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and protein closely correlated in various steroidogenic cell lines with activity of the transfected mFSHR promoter/luciferase reporter construct carrying the distal activator domain. A dose-dependent increase in FSHR promoter activity was shown in nonsteroidogenic HEK 293 cells transiently transfected with SF-1 cDNA. SF-1 was found to bind to a nonconsensus 5′-CAAGGACT-3′ SLBS-3 motif in the distal part of the promoter; formation of the SF-1/SLBS-3 complex could be reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a significant loss of SF-1-mediated mFSHR promoter activity. The second SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In conclusion, we have identified a regulatory domain in the mFSHR promoter participating in the cell-specific regulation of FSHR expression. We demonstrated for the first time that the mFSHR promoter possesses functional SF-1 binding sites and thus belongs to the group of SF-1-regulated genes. These findings provide further evidence for the key role of SF-1 in the regulation of genes involved in gonadal differentiation and endocrine functions.

Steroidogenic factor-1 regulates the rate of proliferation of normal and neoplastic rat ovarian surface epithelial cells in vitro.

Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropin-primed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G0/G1 stage than in the S-, G2/M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector. Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P < 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with serum-induced proliferation and leads to apoptosis.

Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cells in Vitro

Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropin-primed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G0/G1 stage than in the S-, G2/M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector. Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P < 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with serum-induced proliferation and leads to apoptosis.

Steroidogenic factor-1 (SF-1) and the regulation of expression of luteinising hormone and follicle stimulating hormone b-subunits in the sheep anterior pituitary in vivo

Luteinising hormone (LH) and follicle stimulating hormone (FSH) comprise a common a-subunit and hormone-specific β-subunit, are expressed in gonadotroph cells of the anterior pituitary and during the sheep oestrous cycle, are regulated by gonadotrophin releasing hormone (GnRH), ovarian peptides and steroids. Transcription factor steroidogenic factor-1 (SF-1) transactivates the common a-subunit and LHβ subunit in other species. We investigated whether SF-1 regulates β-subunit expression and the regulation of SF-I expression in vivo in sheep. Immunocytochemistry co-localised SF-1, LH and FSH to sheep pituitary gonadotrophs and electrophoretic mobility shift assays (EMSA) demonstrated that SF-1 bound to the LHβ promoter in vitro. No SF-1 DNA binding site was found in the FSHβ promoter. No difference in mean levels of SF-1, FSHβ, LHβ and GnRHr mRNA was measured between the luteal and follicular phases of the oestrous cycle. However SF-1 mRNA levels were correlated to those of LHβ in individual luteal phase animals ( r = 0.88, p < 0.05), when the transcription rate of LHβ was significantly higher ( p < 0.01). GnRH antagonist treatment of luteal animals did not produce a significant reduction in mRNA levels of LHβ, SF-1 and FSHβ. Administering oestradiol benzoate with GnRH antagonist reduced levels of SF-I and FSHβ mRNA from luteal values ( p < 0.005). We conclude that: (1) expression of SF-1 is not solely dependent on GnRH and is downregulated by oestradiol; (2) the LHβ transcription rate is increased during the luteal phase but is not related to levels of LHβ or SF-1 mRNA; and (3) SF-1 does not appear to directly regulate expression of FSHβ, but may regulate expression indirectly.

Expression of the orphan receptor steroidogenic factor-1 mRNA in the rat medial basal hypothalamus.

Steroidogenic factor-1 (SF-1), an orphan receptor of the nuclear hormone receptor family, binds to the AAGGTCA motif in the promoter elements of several diverse target genes, including some that mediate steroidogenesis and sexual differentiation. In addition, SF-1 is expressed in embryonic forebrain, suggesting that it plays a role in neural development. This study was undertaken to study the distribution and regulation of SF-1 mRNA expression in the rat brain. SF-1 mRNA levels were measured in tissue dissections by ribonuclease protection assay. A 452 nt 32P-labeled cRNA probe, complementary to the putative ligand-binding domain of the rat SF-1 mRNA, was synthesized from the rat SF-1 cDNA inserted into pBluescript II KS, using a Sty 1 fragment and T3 polymerase. The probe protected a single 390 nt transcript in the medial basal hypothalamus (MBH) and peripheral steroidogenic tissues of the male rat. The size of this protected band corresponded to that of the protected sense RNA standard (HindIII fragment of the SF-1 cDNA transcribed with T7 polymerase). No SF-1 mRNA was detected in the preoptic area, amygdala or cingulate cortex. The levels of SF-1 mRNA in MBH were not affected by gonadectomy or androgen treatment, nor was there a sex difference in its expression in adults. In situ hybridization histochemistry revealed that SF-1 was localized to the ventromedial nucleus of the adult hypothalamus. The levels of SF-1 mRNA were high on gestational day 18 after which they fell by approximately 30% and remained constant throughout gestation, the first week of neonatal life, and into adulthood. These results demonstrate that the gene encoding SF-1 is expressed in a discrete region of the rat hypothalamus and appears to be developmentally regulated, but not affected by gonadal hormones in adults.

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period.

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.

GnRH regulates steroidogenic factor-1 (SF-1) gene expression in the rat pituitary.

Recent studies have demonstrated that the nuclear receptor, steroidogenic factor 1 (SF-1) plays a role in the regulation of pituitary gonadotropin gene expression. As GnRH is critical to stimulating LH and FSH gene expression, the present study was conducted to determine whether GnRH also regulates pituitary SF-1 mRNA. Pituitary SF-1 mRNA levels were measured in individual animals by RNase protection assay. In the first study, adult male and female rats were gonadectomized (GDX) for 7 days and some received testosterone (T) to prevent the post-GDX rise in GnRH, and compared to intact animals. Pituitary SF-1 mRNA levels increased significantly (3 fold in males, 2 fold in females; p < 0.05 vs intacts) after gonadectomy, which was blocked by exogenous T. Similar changes were observed in serum LH. To directly test whether GnRH stimulates SF-1 mRNA, we used a GnRH-deficient rat model (phenoxybenzamine-treated, ovariectomized females) and administered GnRH pulses for 6h (5ng at 30 min intervals; saline pulses to controls). Pulsatile GnRH stimulated a 51-64% increase in SF-1 mRNA levels (p < 0.05 vs controls). These results show that GnRH stimulates SF-1 gene expression, which may be a critical component in GnRH stimulation of gonadotropin subunit transcription.

Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene.

Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.