Steroidogenic Acute Regulatory Protein mRNA Research Articles

The expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during pre-spawning and pre-hibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in pre-hibernation rather than in pre-spawning, which is one of the physiological phenomena that occur in preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during pre-spawning and pre-hibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During pre-hibernation, oviductal pipe diameter and weight were significantly larger than during pre-spawning. 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), Low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in pre-hibernation and glandular cells during pre-spawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in pre-hibernation than during pre-spawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in pre-hibernation compared to pre-spawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during pre-hibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.

TGF-β1 and TGF-β3, but not TGF-β2, are upregulated in the ovaries of ovarian hyperstimulation syndrome†.

Ovarian hyperstimulation syndrome (OHSS) is a life-threatening and potentially fatal complication during in vitro fertilization treatment. The levels of transforming growth factor-β1 (TGF-β1) are upregulated in human follicular fluid and granulosa-lutein cells (hGL) of OHSS patients and could contribute to the development of OHSS by downregulating steroidogenic acute regulatory protein (StAR) expression. However, whether the same is true for the other two members of the TGF-β family, TGF-β2 and -β3, remains unknown. We showed that all three TGF-β isoforms were expressed in human follicular fluid. In comparison, TGF-β1 was expressed at the highest level, followed by TGF-β2 and TGF-β3. Compared to non-OHSS patients, follicular fluid levels of TGF-β1 and TGF-β3 were significantly upregulated in OHSS patients. The same results were observed in mRNA levels of TGF-β isoforms in hGL cells and ovaries of OHSS rats. In addition, StAR mRNA levels were upregulated in hGL cells of OHSS patients and the ovaries of OHSS rats. Treatment cells with TGF-β isoforms downregulated the StAR expression with a comparable effect. Moreover, activations of SMAD3 signaling were required for TGF-β isoforms-induced downregulation of StAR expression. This study indicates that follicular fluid TGF-β1 and TGF-β3 levels could be used as biomarkers and therapeutic targets for the OHSS.

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 3β-hydroxysteroid dehydrogenase (3β-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 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-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, 3β-HSD, and P450c17 expression in primary rooster Leydig cells.

Effect of maternal periconceptional undernutrition in sheep on cortisol regulation in offspring from mid-late gestation, through to adulthood.

Maternal periconceptional undernutrition (PCUN) alters fetal hypothalamic-pituitary-adrenal axis (HPAA) function and placental glucocorticoid metabolism in sheep. The effects of PCUN on HPAA function in adult life are not known. We investigated the effects of PCUN on fetal adrenal development across gestation and on cortisol regulation in adult offspring. Ewes were undernourished from 61 days before to 30 days after conception ('PCUN') or fed ad libitum ('N'). mRNA expression in the fetal adrenal gland of ACTH receptor (ACTHR), steroidogenic acute regulatory protein (STAR), cytochrome P450 17A1 (CYP17A1), 11beta-hydroxysteroid-dehydrogenase type 2 (11βHSD2), insulin-like growth factor-2 (IGF2), and in the fetal hippocampus of 11βHSD1, 11βHSD2, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) was determined at 50 (adrenal only), 85, 120 and 131 days of gestation (term=148 days). In adult offspring (≥ 3 years, N; 10 female, 5 male, PCUN; 10 female, 10 male) a combined arginine vasopressin (AVP, 0.1 μg/kg) and corticotropin-releasing hormone (CRH, 0.5 μg/kg) challenge and a metyrapone (40 mg/kg) challenge were undertaken. mRNA expression of ACTHR, STAR and CYP17A1 were determined in adult adrenals. Fetal adrenal STAR, CYP17A1 and IGF2 mRNA expression were not different between groups in early gestation but were higher in PCUN than N at 131 days' gestation (all p<0.01). PCUN reduced fetal hippocampal MR and GR mRNA expression by 50% at 85 day, but not in later gestation. Adult offspring plasma cortisol responses to AVP+CRH or metyrapone were not different between groups. Plasma ACTH response to AVP+CRH was lower in PCUN males but ACTH response to metyrapone was not different between groups. Adult adrenal ACTHR, STAR, and CYP17A1 mRNA expression were not affected by PCUN. We conclude that the effects of PCUN on fetal HPAA function that became apparent in late gestation, are not reflected in adrenal cortisol secretion in mid-adulthood.

Korean Ginseng Berry Extract Enhances the Male Steroidogenesis Enzymes In Vitro and In Vivo.

Testosterone hormonal replacement is the most commonly prescribed solution for men with reproductive issues; however, this treatment has various drawbacks. Hence, the identification of a natural product that promotes steroidogenesis is urgently needed. Ginseng is a popular traditional medicine. This study aimed to investigate steroidogenic effects of Korean ginseng berry extract (GBE; Panax ginseng C.A. Meyer) in vitro and in vivo. In vitro model, mouse Leydig cells were treated with varying concentrations of GBE, and the levels of steroidogenesis-related genes and proteins and testosterone were measured using western blotting, qRT-PCR, and enzyme-linked immunosorbent assay (ELISA). Similarly, in an in vivo model using lipopolysaccharide-injected C57BL/6J mice, expression of steroidogenesis-related genes and proteins and testosterone levels were analyzed. Additionally, sleep deprivation was used to simulate common life stressors related to late-onset hypogonadism (LOH) and the natural effects of aging. Mice were fed sham or GBE before being subjected to paradoxical sleep deprivation. In vitro, GBE induced steroidogenic effects by increasing the levels of enzymes associated with steroidogenesis, steroidogenic acute regulatory protein (STAR), CYP11A1, and CYP17A1. In vivo, GBE significantly increased mRNA and protein levels of steroidogenic enzymes. Furthermore, the synthetic testosterone levels in mouse Leydig cell supernatants and blood sera were increased. In the sleep deprivation study, mice fed GBE showed increased testosterone production and survival under such stressful conditions. GBE increased mRNA and protein levels of steroidogenesis-related enzymes STAR, CYP11A1, and CYP17A1. These key enzymes induced the increased production of testosterone both in vivo and in vitro. Thus, GBE might be a promising therapeutic or additive nutritional agent for improving men's health by increasing steroidogenesis or improving LOH.

AREG upregulates secreted protein acidic and rich in cysteine expression in human granulosa cells

The secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein and the expression of ovarian SPARC peaks during ovulation and luteinization. Besides, SPARC expression was induced by human chorionic gonadotropin (hCG) in rat granulosa cells. Amphiregulin (AREG) is the most abundant epidermal growth factor receptor (EGFR) ligand expressed in human granulosa cells and follicular fluid. AREG mediates the physiological functions of luteinizing hormone (LH)/hCG in the ovary. However, to date, the biological function of SPARC in the human ovary remains undetermined, and whether AREG regulates SPARC expression in human granulosa cells is unknown. In this study, we show that AREG upregulated SPARC expression via EGFR in a human granulosa-like tumor cell line, KGN. Treatment of AREG activated ERK1/2, JNK, p38 MAPK, and PI3K/AKT signaling pathways and all of them were required for the AREG-induced SPARC expression. Using RNA-sequencing, we identified that steroidogenic acute regulatory protein (StAR) was a downstream target gene of SPARC. In addition, we demonstrated that SPARC mRNA levels were positively correlated with the levels of StAR mRNA in the primary culture of human granulosa cells. Moreover, SPARC protein levels were positively correlated with progesterone levels in follicular fluid of in vitro fertilization patients. This study provides the regulatory role of AREG on the expression of SPARC and reveals the novel function of SPARC in progesterone production in granulosa cells.

A Short Promoter Region Containing Conserved Regulatory Motifs Is Required for Steroidogenic Acute Regulatory Protein (Star) Gene Expression in the Mouse Testis.

In the testis, Leydig cells produce steroid hormones that are needed to masculinize typical genetic males during fetal development and to initiate and maintain spermatogenesis at puberty and adulthood, respectively. Steroidogenesis is initiated by the transfer of cholesterol from the outer to the inner mitochondrial membrane through the action of steroidogenic acute regulatory protein (STAR). Given its importance for the steroidogenic process, the regulation of STAR gene expression has been the subject of numerous studies. These studies have involved the characterization of key promoter sequences through the identification of relevant transcription factors and the nucleotide motifs (regulatory elements) that they bind. This work has traditionally relied on in vitro studies carried out in cell cultures along with reconstructed promoter sequences. While this approach has been useful for developing models of how a gene might be transcriptionally regulated, one must ultimately validate that these modes of regulation occur in an endogenous context. We have used CRISPR/Cas9 genome editing to modify a short region of the mouse Star promoter (containing a subset of regulatory elements, including conserved CRE, C/EBP, AP1, and GATA motifs) that has been proposed to be critical for Star transcription. Analysis of the resultant mutant mice showed that this short promoter region is indeed required for maximal STAR mRNA and protein levels in the testis. Analysis also showed that both basal and hormone-activated testosterone production in mature mice was unaffected despite significant changes in Star expression. Our results therefore provide the first in vivo validation of regulatory sequences required for Star gene expression.

Gene expression and latitudinal variation in the stress response in Fundulus heteroclitus

Atlantic killifish, Fundulus heteroclitus, are intertidal marsh fish found along the east coast of North America. Associated with the thermal gradient along this coast, northern and southern killifish populations are known to differ in morphology, behavior, and physiology, including in their cortisol stress response. Our goal was to explore population differences in the stress response and identify underlying molecular mechanisms. We measured responses to both acute and repeated stress in plasma cortisol, stress axis mRNA expression, and body condition in northern and southern killifish. Following an acute stressor, the southern population had higher cortisol levels than the northern population but there was no difference between populations following repeated stress. In the brain, both corticotropin releasing factor and its binding protein had higher expression in the southern than the northern population, but the northern population showed more changes in mRNA levels following a stressor. In the head kidney, Melanocortin 2 Receptor and steroidogenic acute regulatory protein mRNA levels were higher in the southern population suggesting a larger capacity for cortisol synthesis than in the northern fish. Lastly, the glucocorticoid receptor GR1 mRNA levels were greater in the liver of southern fish, suggesting a greater capacity to respond to cortisol, and GR2 had differential expression in the head kidney, suggesting an interpopulation difference in stress axis negative feedback loops. Southern, but not northern, fish were able to maintain body condition following stress, suggesting that these differences in the stress response may be important for adaptation across latitudes.

Kisspeptin-10 Promotes Progesterone Synthesis in Bovine Ovarian Granulosa Cells via Downregulation of microRNA-1246.

The objective of this study was to clarify the effect of kisspeptin-10 (kp-10) on the synthesis of progesterone (P4) in bovine granulosa cells (BGCs) and its mechanisms via microRNA 1246 (miR-1246). According to the results, we found that treating with kp-10 for 24 h could increase P4 level, the mRNA expression of the steroidogenesis-related gene steroidogenic acute regulatory protein (StAR), free cholesterol content, and decrease miR-1246 expression in BGCs. Overexpression of miR-1246 significantly inhibited P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs, whereas underexpression of miR-1246 significantly reversed this effect in BGCs. Additionally, overexpression of miR-1246 counteracted the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Conversely, underexpression of miR-1246 enhanced the accelerative effect of kp-10 on P4 synthesis, StAR mRNA expression, and free cholesterol content in BGCs. Meanwhile, results of dual-luciferase reporter assays indicated that miR-1246 targeted the 3′UTR of StAR in BGCs. These results demonstrated that kp-10 induced P4 synthesis in BGCs by promoting free cholesterol transport via regulating expression of miR-1246/StAR.

Characterizing seasonal transitions: Breeding-like morphology and behavior during the late non-breeding season in green anole lizards

Seasonally breeding animals, such as green anole lizards (Anolis carolinensis), allow for the examination of the control of reproduction during different reproductive states. During the breeding season, the gonads are large and reproductively active. Following the breeding season, gonads regress and become less active, and the lizards enter a refractory period where breeding is inhibited. After this stage, a post-refractory period occurs during which the lizards are still in a non-breeding state, but environmental changes can trigger the onset of breeding. However, it is unclear what causes these changes in reproductive state and we hypothesized that this may be due to alterations in gonadotropin-releasing hormone (GnRH) signaling. The present study aimed to identify morphological and behavioral differences in GnRH- and saline-injected refractory and post-refractory male anoles when housed under the same non-breeding environmental conditions. We found that post-refractory anoles had increased testicular weight, recrudescence, sperm presence, and reproductive behavior, with no impact of GnRH injection. Renal sex segment size and steroidogenic acute regulatory protein (StAR) mRNA levels did not differ among groups, indicating that testosterone levels likely had not increased in post-refractory lizards. Post-refractory anoles in this study were beginning to transition towards a breeding state without exposure to changing environmental conditions, and GnRH was not necessary for these changes. These data reveal a complex interaction between the activation of breeding, changing environmental conditions, and the underlying physiology regulating reproduction in seasonally breeding lizards. Future studies are needed to further elucidate the mechanisms that regulate this relationship.

The functions of Patchouli and Elsholtzia in the repair of hen follicular granular cells after heat stress

The objective of this experimental study was to examine the effects of the Chinese herbal medicines Patchouli and Elsholtzia on the follicular granulosa cells of hens undergoing heat stress conditions. In the current investigation, hen follicular granulosa cells were isolated from the prehierarchical follicles of layer hens and then cultured in-vitro. The cells were randomly divided into the 6 groups. Following the completion of this study's experiments using different heat stress and medicinal treatments, the cell activities of each group were measured using an MTT method. The levels of the heat shock protein 70 (HSP70) were detected using ELISA. The expressions of the steroidogenic acute regulatory protein (StAR) mRNA; cytochrome P450 family 11, subfamily A, member 1 (CYP11A1) mRNA; proliferating cell nuclear antigen (PCNA) mRNA; and the follicle stimulating hormone receptor (FSHR) were detected using the real-time quantitative polymerase chain reactions. The concentration levels of estrogen and progesterone in the cell supernatant of each group were measured using ELISA. The results showed that cell activity had significantly decreased following the heat stress treatments at 43℃, 44℃, and 45℃ (P < 0.01), respectively. Meanwhile, cell activities observed in Patchouli and Elsholtzia were found to be much better than those of heat stress group (P < 0.05). In addition, the expression levels of HSP70 in the follicular granulosa cells of Patchouli and Elsholtzia groups were lower than those of heat stress group. Patchouli and Elsholtzia can maintain expressions of the receptor at 43℃. This study determined that the estrogen and progesterone in the supernatant fluid of Patchouli and Elsholtzia were higher than those observed in heat stress. Therefore, the results obtained in this study indicated that the Patchouli and Elsholtzia treatments administered prior the heat stress experiments had successfully protected the follicular granulosa cells from heat damages while maintaining the normal secretory functions of the granulosa cells.

Developmental expression of genes involved in progesterone synthesis, metabolism and action during the post-natal cerebellar myelination

Progesterone is involved in dendritogenesis, synaptogenesis and maturation of cerebellar Purkinge cells, major sites of steroid synthesis in the brain. To study a possible time-relationship between myelination, neurosteroidogenesis and steroid receptors during development of the postnatal mouse cerebellum, we determined at postnatal days 5 (P5),18 (P18) and 35 (P35) the expression of myelin basic protein (MBP), components of the steroidogenic pathway, levels of endogenous steroids and progesteroneʾs classical and non-classical receptors. In parallel with myelin increased expression during development, P18 and P35 mice showed higher levels of cerebellar progesterone and its reduced derivatives, higher expression of steroidogenic acute regulatory protein (StAR) mRNA, cholesterol side chain cleavage enzyme (P450scc) and 5α-reductase mRNA vs. P5 mice. Other steroids such as corticosterone and its reduced derivatives and 3β-androstanodiol (ADIOL) showed a peak increase at P18 compared to P5. Progesterone membrane receptors and binding proteins (PGRMC1, mPRα, mPRβ, mPRγ, and Sigma1 receptors) mRNAs levels increased during development while that of classical progesterone receptors (PR) remained invariable. PRKO mice showed similar MBP levels than wild type. Thus, these data suggests that progesterone and its neuroactive metabolites may play a role in postnatal cerebellar myelination.

The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Regulation and role of Acyl-CoA synthetase 4 in glial cells

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.

OR24-03 GRK2 Mediates Beta-Adrenergic Receptor Crosstalk to Enhanced Adrenocortical AngII-Dependent Aldosterone Production

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to hyperkalemia or angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone synthesis and secretion via both G proteins and the GPCR adapter proteins βarrestins. AZG cells express all three subtypes of β-adrenergic receptor (AR) and respond to catecholamines by producing aldosterone. Being GPCRs, both activated βARs and AT1Rs are phosphorylated by GPCR-kinases (GRKs), followed by βarrestin binding to initiate G protein-independent signaling. Herein, we investigated whether the major adrenal GRKs, GRK2 and GRK5, are involved in catecholaminergic regulation of AngII-dependent aldosterone production. We used the human AZG cell line H295R, in which we measured aldosterone secretion via ELISA and synthesis via real-time PCR for steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA levels. Isoproterenol (Iso, a βAR full agonist) treatment significantly augmented AngII-dependent aldosterone synthesis (2.2+0.8-fold CYP11B2 & 1.6+0.5-fold StAR mRNA inductions over AngII alone; p<0.05, n=4), as well as secretion (2.3+0.8-fold of vehicle with Iso; 3.2+1.1-fold of vehicle with AngII; 7.4+1.1-fold of vehicle with Iso+AngII, p<0.05 vs. either agent alone; n=5) in H295R cells. Importantly, GRK2, but not the other major GRK isoform expressed in human adrenals GRK5, was indispensable for the catecholamine-mediated enhancement of aldosterone production in response to AngII in H295R cells. Specifically, GRK2 inhibition with the small molecule Cmpd101 abolished Iso effects on AngII-induced aldosterone synthesis and secretion (Iso+AngII-induced aldosterone secretion: 8.1+2.3-fold of vehicle without Cmpd101; 2.8+0.8-fold of vehicle with Cmpd101; p<0.05, n=5). In contrast, GRK5 knockout via CRISPR/Cas9 did not affect the synergism between isoproterenol and AngII in stimulating aldosterone production. Mechanistically, βAR-activated GRK2, but not GRK5, phosphorylated and activated the Ca2+-activated chloride channel anoctamine-1 (ANO1), also known as transmembrane member (TMEM)16A, ultimately increasing aldosterone production in H295R cells (Iso+10–6 M [Ca2+]-induced ANO1 activity of Cmpd101-pretreated cells: 55+15 % of non-Cmpd101-pretreated cells; p<0.05, n=5). AngII alone failed to stimulate GRK2 in H295R cells. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk at the level of ANO1 activation, which results in enhanced aldosterone production in H295R cells. This finding suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems in the adrenal cortex and that GRK2 inhibition might be therapeutically advantageous for aldosterone suppression.

Effects of Butylparaben Supplementation on In Vitro Development of Mouse Preantral Follicle.

We investigated whether butylparaben supplementation to the culture media negatively effects on in vitro development of mouse preantral follicle. The preantral follicles were isolated from the ovaries of 7-8-week-old mice and cultured in growth medium for 10 days and then in maturation medium for 2 days. During in vitro culture, butylparaben (0, 0.01, 0.1, 1.0, or 10μM) was supplemented to the culture media. In the final spent media, the levels of 17ß-estradiol and anti-Müllerian hormone (AMH) were measured via enzyme-linked immunosorbent assay. In the final luteinized follicular cells, the mRNA levels of steroidogenic acute regulatory protein (StAR), superoxide dismutase 1 (Sod1), caspase 3 (Casp3), and extracellular signal-regulated kinase 1 (Erk1) were quantified via real-time reverse transcription-polymerase chain reaction. The metaphase II oocyte acquisition (per total oocyte) tended to decrease in the four butylparaben-supplemented groups, but not significant (26.8%, 23.2%, 21.4%, 15.1%, and 16.8%, respectively). The level of 17ß-estradiol and AMH tended to decrease in all butylparaben-supplemented groups, but statistically not significant. The expression level of StAR and Erk1 mRNA was significantly higher in all four butylparaben-supplemented groups, and a dose-dependent increment tendency was observed. Our findings suggest that butylparaben supplementation has largely no impact on in vitro development of mouse preantral follicle as well as 17ß-estradiol and AMH production. However, StAR, Sod1, Casp3, and Erk1 genes could be overexpressed in a certain concentration of butylparaben.

GRK2-Mediated Crosstalk Between β-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein βarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. β-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the βAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the βAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol’s effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the β-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Steroidogenic Effects of Korean Rice Bran Extract (Oryza sativa L.) on Mouse Normal Leydig Cells via Modulation of Steroidogenesis-Related Enzymes

Background and objective Many plant extracts have various effects on diseases. In particular, some medicinal plants have been shown to have steroidogenic effects in men. However, the effects of Korean rice bran extract (RBE) on steroidogenesis in men have not been reported previously. Accordingly, in this study, we investigated the effects of RBE (Oryza sativa L.) on mouse normal Leydig cells (TM3), which produce testosterone with increasing levels of key enzymes (e.g., StAR, CYP11A1, and CYP17A1) for male steroidogenesis. Material and methods Using mouse normal Leydig cells (TM3), we determined whether RBE had steroidogenic effects by eval-uating changes in the levels of steroidogenesis-related enzymes, such as StAR (steroidogenic acute regu-latory protein), CYP11A1, and CYP17A1, using quantitative real-time PCR, western blotting analysis, and ELISA method. Results RBE significantly enhanced mRNA and protein levels of StAR, CYP11A1, and CYP17A1, thereby enhanc-ing synthetic testosterone levels in mouse Leydig TM3 cell supernatants. These findings indicated that RBE increased the levels of steroidogenic enzymes to modulate steroidogenesis. Conclusion RBE enhanced mRNA and protein levels of key enzymes including StAR, CYP11A1, and CYP17A1 for male steroidogenesis. These changes could induce testosterone production by TM3 cells in vitro. Thus, RBE may be used as a food additive or medicinal plant for the treatment of diseases characterized by insuf-ficient testosterone (e.g., late-onset hypogonadism).

Steroidogenic acute regulatory protein transcription is regulated by estrogen receptor signaling in largemouth bass ovary

Estrogenic contaminants in the environment are linked to the occurrence of reproductive abnormalities in many aquatic species, including largemouth bass (Micropterus salmoides; LMB). Previous work has shown that many different types of xenoestrogens regulate expression of the Steroidogenic Acute Regulatory protein (StAR), a cholesterol-transporting protein vital to steroid hormone biosynthesis; however, the regulatory mechanisms of StAR are incompletely characterized in fish. To learn more about endogenous expression patterns of StAR in the ovary, LMB were collected from the St. John’s River (Florida, USA) over an entire breeding season to investigate StAR expression. Plasma 17β-estradiol (E2) and StAR mRNA levels were positively correlated in females, and StAR mRNA levels displayed ~ 100-fold increase between primary oocyte growth stages and final maturation. To further study the regulation of StAR, female LMB in the laboratory were fed at ≃2% of their weight on a diet laden with 17α-ethinylestradiol (EE2, 70 or 200 ng EE2 per gram feed). Diets were designed to achieve a physiologically-relevant exposure to EE2, and StAR expression was assessed in vivo. We observed a dose-dependent suppression of StAR mRNA levels, however both diets led to high, pharmacological levels in the blood and do not represent normal physiological ranges of estrogens. In the 200 ng EE2/gm feed group, ovarian StAR mRNA levels were suppressed to approximately 5% of that of the LMB control group. These investigations suggest that LMB StAR increases in expression during oocyte maturation and that it is suppressed by E2 feedback when estrogen levels are high, through the HPG axis. A 2.9 kb segment of the LMB StAR promoter was examined for putative E2 response elements using in silico software, and a putative estrogen receptor binding element (ERE/-1745) was predicted in the promoter. The functionality of the ERE was examined using MA-10 mouse Leydig cells transfected with the LMB StAR promoter. Estrogen receptor (ER) interaction with ERE/-1745 was evaluated under basal and human chorionic gonadotropin (hCG)-treated conditions in the presence and absence of E2. Chromatin immunoprecipitation (ChIP) experiments revealed that ESR1 binding to the promoter was enriched under basal conditions and E2 exposure elicited an increase in enrichment (4-fold) above that observed under basal conditions. ESR2 was not strongly enriched at the ERE/-1745 site, suggesting that StAR may be preferentially regulated by LMB estrogen receptor 1 (esr1). Taken together, these different experiments provide evidence that LMB StAR is under the control of estrogens and that ESR1 binds directly to the LMB StAR promoter in an E2-responsive manner.