Steroidogenic Acute Regulatory Protein Protein Expression 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.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

Prevention of Testicular Damage by Indole Derivative MMINA via Upregulated StAR and CatSper Channels with Coincident Suppression of Oxidative Stress and Inflammation: In Silico and In Vivo Validation.

Cis-diamminedichloroplatinum (II) (CDDP) is a widely used antineoplastic agent with numerous associated side effects. We investigated the mechanisms of action of the indole derivative N’-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide (MMINA) to protect against CDDP-induced testicular damage. Five groups of rats (n = 7) were treated with saline, DMSO, CDDP, CDDP + MMINA, or MMINA. Reproductive hormones, antioxidant enzyme activity, histopathology, daily sperm production, and oxidative stress markers were examined. Western blot analysis was performed to access the expression of steroidogenic acute regulatory protein (StAR) and inflammatory biomarker expression in testis, while expression of calcium-dependent cation channel of sperm (CatSper) in epididymis was examined. The structural and dynamic molecular docking behavior of MMINA was analyzed using bioinformatics tools. The construction of molecular interactions was performed through KEGG, DAVID, and STRING databases. MMINA treatment reversed CDDP-induced nitric oxide (NO) and malondialdehyde (MDA) augmentation, while boosting the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymis and testicular tissues. CDDP treatment significantly lowered sperm count, sperm motility, and epididymis sperm count. Furthermore, CDDP reduced epithelial height and tubular diameter and increased luminal diameter with impaired spermatogenesis. MMINA rescued testicular damage caused by CDDP. MMINA rescued CDDP-induced reproductive dysfunctions by upregulating the expression of the CatSper protein, which plays an essential role in sperm motility, MMINA increased testosterone secretion and StAR protein expression. MMINA downregulated the expression of NF-κB, STAT-3, COX-2, and TNF-α. Hydrogen bonding and hydrophobic interactions were predicted between MMINA and 3β-HSD, CatSper, NF-κβ, and TNFα. Molecular interactome outcomes depicted the formation of one hydrogen bond and one hydrophobic interaction between 3β-HSD that contributed to its strong binding with MMINA. CatSper also made one hydrophobic interaction and one hydrogen bond with MMINA but with a lower binding affinity of -7.7 relative to 3β-HSD, whereas MMINA made one hydrogen bond with NF-κβ residue Lys37 and TNF-α reside His91 and two hydrogen bonds with Lys244 and Thr456 of STAT3. Our experimental and in silico results revealed that MMINA boosted the antioxidant defense mechanism, restored the levels of fertility hormones, and suppressed histomorphological alterations.

Intravenous Infusion of Nucleated Peripheral Blood Cells Restores Fertility in Mice with Chemotherapy-Induced Premature Ovarian Failure.

Cancer treatment with specific chemotherapeutic agents has been well documented to have an adverse impact on female fertility leading to premature ovarian failure (POF). The objective of this study is to investigate if chemotherapeutic induced POF can be reversed by the infusion of autologous nucleated peripheral blood cells (PBMC). To reach our goal, mice were treated with a single intraperitoneal injections of busulfan and cyclophosphamide to induce POF. This was followed by transfusion of PBMC. The ovarian morphology and functional parameters were monitored by radioimmunoassay, real-time PCR, immunofluorescence and immunohistochemistry analysis. Our study showed that chemotherapy (CTX) protracted estrous cycle period and repressed E2 production. In addition, CTX decreased the expressions of steroidogenesis markers, CYP-17 synthesis, StAR (steroidogenic acute regulatory protein), and Connexin-43 protein expression in the ovarian follicles. We also observed reduced numbers and sizes of the primordial and primary follicles in CTX-treated mice compared to untreated controls (p < 0.05). When both CTX and untreated control groups were stimulated with gonadotrophin, the control group produced ten times more ova than the CTX group. Finally, the treatment of premature ovarian failure induced by CTX with autologous PBMC transfusion resulted in over-expression and a statistically significant increase in several stem cell markers and restoration of fertility. Infusion with PBMC in CTX further decreased the estrous cycle length by 2.5 times (p < 0.01). We found that transfusion of autologous PBMC to mice with chemotherapy induced POF was very effective at restoring fertility. These results are similar to other studies using bone marrow derived mesenchymal stem cells.

Bisphenol A stimulates steroidogenic acute regulatory protein expression via an unknown mechanism in adrenal cortical cells.

Bisphenol A (BPA) is one of the most widespread endocrine disrupting chemicals in the environment. Exposure to BPA is known to be associated with disruption of steroidogenesis in reproductive tissues, but little is known about its effects on the adrenal gland. We previously showed that prenatal BPA exposure resulted in elevated plasma corticosterone levels concomitant with increased adrenal levels of steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis, in adult female mouse offspring. However, the molecular mechanisms underlying the BPA-induced StAR protein expression in the adrenal gland remain unknown. Therefore, the current study was designed to address this important question using the human cortical cell line, H295A cells, as an in vitro model system. We found that: (1) BPA increased StAR protein levels in a dose-dependent manner; (2) both estrogen receptor alpha (ERα)- and ERβ-specific agonists mimicked while the ER antagonist ICI abrogated the stimulatory effects of BPA on StAR protein levels; and (3) BPA did not alter StAR messenger RNA, 37kDa preprotein or protein half-life. Taken together, these findings demonstrate that BPA increases StAR protein levels through an unknown mechanism independent of StAR gene transcription, translation, and protein half-life. Furthermore, such effects are likely mediated by ERα and/or ERβ.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells.

Down Regulation of Acrolein on Corticosterone Secretion in Male Rats

Acrolein is a small unsaturated aldehyde and can be found in a wide range of resources including all types of smoke and exhaust gases from gasoline engines. Although the toxicity and damage of acrolein have been recognized, the action mechanisms of acrolein, especially that of acrolein on the response of stresshormones are still unclear. The present study hypothesized that administration of acrolein altered the secretion of both adrenocorticotropin (ACTH) and corticosterone via the regulation of steroid biosynthetic pathway in rat zona fasciculata-reticularis (ZFR) cells. Both in vivo and in vitro approaches were uased. In the in vivo study, intraperitonal injection of acrolein (2 mg/ml/kg) once daily for 1 or 3 days resulted in a reduction of plasma levels of ACTH and corticosterone as well as the intracellular cAMP and ACTH-induced secretion of corticosterone. The protein expression of ACTH receptor (ACTHR) in rat ZFR cells was also reduced by 40-60% after treatment of acrolein for 1 day and 3 days, respectively. In the in vitro study, rat ZFR cells were prepared and chanllenged with ACTH (10 -9 M), forskolin (an adenylyl cyclase activitior, 10 -5 M), 8-Br-cAMP (a permeable synthetic cAMP, 5x10 -5 M), 25-OH-cholesterol (10 -5 M) ± trilostane (an inhibitor of 3β-hydroxysteroid dehydrogenase, 3β-HSD, 10 -5 M). The evoked release of corticosterone by ACTH, forskolin, 8-Br-cAMP and the induced release of pregnenolone in response to 25-OH-cholesterol plus triolostane were decreased. Since the accumulation of pregnenolone after blocking 3β-HSD by trilostane represents the activity of P450 scc, therate-limiting step of steroid biosynthesis, we suggest that not only the cAMP pathway was inhibited, but also the enzyme activity of P450 scc was attenuated following administration of acrolein. Although insignificant, the protein expression of steroidogenic acute regulatory protein (StAR) was decreased by 40% in ZFR cells after treatment of acrolein in vivo . Incubation of ZFR cells with acrolein (10 -9 ~10 -7 M) also decreased the in vitro release of corticosterone. These results suggest that administration of acrolein inhibited corticosterone production via the attenuation of cAMP pathway, StAR protein expression, and the enzyme activity of P450 scc. The attenuation of protein expression of ACTHR (also named melanocortin 2 receptor, MC2R) and reduced secrection of ACTH indicated that the hypothalamus-pituitary-adrenal (H-P-A) axis was also down- regulated by the administration of acrolein.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

English

Exposure to phthalate esters is associated with changes in steroidogenesis, leading to the hypothesis that this is a primary mechanism of phthalate reproductive toxicity. However, some phthalate-induced male reproductive toxicity has been demonstrated in the absence of changes to testosterone production, suggesting additional mechanisms of action. There is evidence that phthalate exposure increases expression of the inflammatory enzyme cyclooxygenase 2 (cox-2). Furthermore, inhibition of cox-2 enhances expression of the steroidogenic acute regulatory protein (StAR), which mediates the rate-limiting step in steroidogenesis. This study hypothesized that phthalate-induced toxicity and testosterone perturbation are mediated in part by cox-2. A 3D in vitro rat testis co-culture to explore the role of cox-2 in phthalate toxicity was employed. Cells were treated with 100 µM dipentyl phthalate (DPP) with and without pre-treatment with the specific cox-2 inhibitor NS-398. Effects were evaluated after 8, 24, and 72 h. DPP exposure significantly increased cox-2 expression at 8 and 24 h (p<0.01) and resulted in significant, dose-dependent cytotoxicity. Pre-treatment with NS-398 significantly reduced the cytotoxicity of DPP at 8 and 24 h (p<0.01). NS-398 also mitigated the effects of DPP on testosterone regulation. Total testosterone concentrations in cell culture media were significantly increased following 8 and 24 hr of DPP exposure (p<0.001) and NS-398 reduced this effect (p<0.05). Simultaneously, DPP significantly decreased StAR protein expression after 8 h (p<0.01) and this effect was significantly attenuated by the presence of NS-398 (p<0.01). These results suggest that the DPP-induced changes in testosterone regulation observed in this experiment are mediated in part by an inflammatory response that is cox-2 dependent. Key words: dipentyl phthalate, testosterone, cyclooxygenase 2, in vitro toxicology

The contribution of serine 194 phosphorylation to steroidogenic acute regulatory protein function.

The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in human StAR) as an important residue for StAR activity. To explore the importance of S194 phosphorylation in StAR function in vivo, we developed a transgenic model using a bacterial artificial chromosome expressing either wild-type (WT) StAR or StAR mutation S194A to rescue StAR knockout (KO) mice. Despite StAR protein expression comparable to or higher than amounts seen with control animals or rescue with WT StAR, S194A StAR did not rescue the neonatal lethality and only partially rescued the sex reversal in male mice observed uniformly in StAR KO mice. Like the StAR KO mice, the adrenal cortex and testicular Leydig cells contained abundant lipid deposits when stained with oil red O. Adrenal StAR from S194A rescue animals lacks an acidic species, which appears upon corticotropin stimulation in animals rescued with WT StAR, consistent with defective StAR phosphorylation. These findings demonstrate that S194 is an essential residue for normal StAR function in the adrenal cortex and testes of mice.

Prostaglandin E2 functions as a luteotrophic factor in the dog

The luteal phase in dogs is governed by many poorly understood regulatory mechanisms. Functioning of the corpus luteum (CL) is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotrophic effect of prostaglandin E2 (PGE2) during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesis was tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the pre-implantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2 receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly up-regulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3βHSD (HSD3B2) and P450scc (CYP11A1) expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL during both pregnancy and dioestrus and generally localized to the luteal cells. After initial up-regulation during the earlier stages of the CL phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL.

Inhibitory Effects of Digoxin on Testosterone Production in Rat Leydig Cells

Digoxin has beneficial effects on heart failure due to a direct positive inotropic action as an inhibitor of Na^+- K^+-ATPase and endogenous digoxin-like immunoreactive factors (DLIFs) found in mammalian blood also correlates to the same issue. We have demonstrated that digoxin inhibits rat testosterone secretion but the mechanisms remain unclear. In the present study, the direct effects of digoxin on the expression of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) in rat Leydig cells were evaluated. Digoxin inhibited not only testosterone production after incubation of Leydig cells with or without human chorionic gonadotropin (hCG), forskolin, 8-Br-cAMP, but also pregnenolone production following stimulation of 25-hydroxy-cholesterol, suggesting that digoxin reduced adenylyl cyclase and P450scc activity. Digoxin significantly reduced the expression of StAR protein, but not that of P450scc. The stimulation of testosterone production induced by A23187 was decreased by digoxin indicating that digoxin might influence Ca^2+ utilization. The analog of digoxin, ouabain, did not affect testosterone production in rat Leydig cells, which indicated the drug specificity. These results suggested that, in rat Leydig cells, digoxin directly inhibited testosterone production partly by reducing P450scc activity, StAR protein expression, and Ca^2+ utilization. This report not only demonstrates the mechanism digoxin acts, but also implies a probable interaction between gonadal and adrenal axis linked by DLIFs (i.e. digoxin).

Administration of a gonadotropin-releasing hormone agonist affects corpus luteum vascular stability and development and induces luteal apoptosis in a rat model of ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS.

Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3β-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance.

Stimulatory Effects of Propylthiouracil on Pregnenolone Production through Upregulation of Steroidogenic Acute Regulatory Protein Expression in Rat Granulosa Cells

Propylthiouracil (PTU) is a common and effective clinical medicine for the treatment of hyperthyroidism. Our previous study demonstrated that short-term treatment with PTU inhibits progesterone production in rat granulosa cells. However, our present results indicate that a 16-h treatment with PTU was able to stimulate pregnenolone production in rat granulosa cells, although progesterone production was diminished by PTU through inhibition of 3β-hydroxysteroid dehydrogenase. Notably, we found that PTU treatment enhanced the conversion of cholesterol into pregnenolone, whereas the protein level of the cytochrome P450 side-chain cleavage enzyme (P450scc, which is the enzyme responding to this conversion) was not affected. Interestingly, the levels of steroidogenic acute regulatory protein (StAR) in both total cell lysate and the mitochondrial fraction were significantly increased by PTU treatment. Furthermore, the binding of steroidogenic factor-1 (SF-1) to the StAR promoter region was also enhanced by PTU treatment, which suggests that PTU could upregulate StAR gene expression. In addition to SF-1 regulation, we found that mitogen-activated protein (MAP) kinase kinase activation is an important regulator of PTU-stimulated StAR protein expression, based on the effects of the MEK inhibitor PD98059. In conclusion, these results indicate that PTU plays opposite roles in the production of progesterone and its precursor, pregnenolone. The regulation of negative feedback on speeding the cholesterol transportation and pregnenolone conversion after a 16-h PTU treatment may be the mechanism explaining PTU's inhibition of progesterone production in rat granulosa cells.

Growth Differentiation Factor 9 Reverses Activin A Suppression of Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Human Granulosa-Lutein Cells

Abstract Background: We have reported that growth differentiation factor (GDF) 9 can enhance activin A (βAβA)-induced inhibin B (αβB) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. Methods: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey’s test. Results: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of a-subunit siRNA, activin A level increased (P &amp;lt; 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P &amp;lt; 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. Conclusion: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Lipopolysaccharide directly stimulates aldosterone production via toll-like receptor 2 and toll-like receptor 4 related PI3K/Akt pathway in rat adrenal zona glomerulosa cells

The level of circulating endotoxin is related to the severity of cardiovascular disease. One of the indexes for the prognosis of cardiovascular disease is the plasma aldosterone level. Recently, the Toll-like receptors (TLRs), lipopolysaccharide (LPS)-regulated receptors, were found not only to mediate the inflammatory response but also to be important in the adrenal stress response. Whether LPS via TLRs induced aldosterone production in adrenal zona glomerulosa (ZG) cells was not clear. Our results suggest that LPS-induced aldosterone secretion in a time- and dose-dependent manner and via TLR2 and TLR4 signaling pathway. Administration of LPS can enhance steroidogenesis enzyme expression such as scavenger receptor-B1 (SR-B1), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage (P450scc) enzyme. LPS-induced SR-B1 and StAR protein expression are abolished by TLR2 blocker. Furthermore, we demonstrated that phosphorylation of Akt was elevated by LPS treatment and reduced by TLR2 blockers, TLR4 blockers, and LY294002 (PI(3)K inhibitor). Those inhibitors of PI(3)K/Akt pathways also abolish LPS-induced aldosterone secretion and SR-B1 protein level. In conclusion, LPS-induced aldosterone production and SR-B1 proteins expression are through the TLR2 and TLR4 related PI(3)K/Akt pathways in adrenal ZG cells.

Growth Differentiation Factor 9 Reverses Activin A Suppression of Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Human Granulosa-Lutein Cells

We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (β(A)β(A))-induced inhibin B (αβ(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Involvement of ERK Phosphorylation in the Prevention of Ischemia-Induced Ovarian Follicular Depletion by Stem Cells

Ovarian failure is commonly caused by aging, autoimmune disease, menopause and cancer therapy. We used an ischemic model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Three treatment regimes were employed: sham-operated control, ligation plus PBS, and ligation plus immortalized human bone marrow stromal cells (stem cells) groups. After ligation-induced ischemia, stem cells or PBS were injected into rat ovaries. Then, pregnant mare serum gonadotropin was given intra-peritoneally to initiate folliculogenesis. The animals were then sacrificed. The ovary gland was weighed, and ovarian folliculogenesis, stem cell differentiation and vascular neogenesis were evaluated. In order to study improvement of folliculogenesis after ovarian ischemia, steroidogenic acute regulatory protein (StAR), p44/p42 MAPK (T-ERK1/2), and phospho-p44/ p42 MAPK (P-ERK1/2) expression were specifically evaluated. Results indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in ovarian ischemic-reperfusion animals, but both recovered after stem cell treatment. The stem cells migrated into the ovary and differentiated into theca cells, granulosa cells, corona radiata cells and vascular endothelial cells. In addition, von Willebrand factor (vWF) expression was increased; 17beta-estradiol (E2), progesterone (P4), P-ERK1/2 and StAR protein expression was recovered by stem cells treatment in the ischemic ovaries. The serum LH was significantly increased in ovaries of ischemia-reperfusion animals, but the stem cell treatment restored the effects. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis through the MAPK pathway.

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by Zona fasciculata‐reticularis cells in male rats

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu-Chu-Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti-allergic, anti-nociceptive and anti-inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata-reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10(-9) M), forskolin (an adenylyl cyclase activator, 10(-5) M), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP, a permeable cAMP analog, 10(-4) M), or steroidogenic precursors including 25-hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10(-5) M each, in the presence or absence of EVO and RUT respectively (0-10(-3) M) at 37 degrees C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10(-4) M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH-, forskolin-, as well as 8-Br-cAMP-stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP-related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression.