Steroidogenic Acute Regulatory Protein Protein Expression Research Articles

Chronic Effects of Prepubertal Ethanol Administration on Steroidogenic Acute Regulatory Protein in the Rat Ovary

A mitochondrial protein, steroidogenic acute regulatory protein (StAR), plays an essential role in steroidogenesis by facilitating delivery of cholesterol across the mitochondrial membrane. Because ethanol (EtOH) causes suppressed estradiol (E2) secretion in prepubertal female rats and rhesus monkeys, we evaluated the effects of chronic EtOH administration on prepubertal ovarian StAR. Rats were implanted with a gastric cannula on day 24 and began receiving control or EtOH diets on day 28. At 0800 hr on day 33, the experimental groups were subdivided. Half of the EtOH-treated animals received a subcutaneous injection of pregnant mare serum gonadotropin (PMSG; 15 IU), and the other half received an injection of saline. The chow-fed and liquid-diet control groups were also subdivided, with half receiving the PMSG and the other half receiving saline. Eight hours after the respective injections, the animals were killed, and their ovaries and blood were collected. The ovaries from EtOH-treated rats showed decreased basal expression of both the 3.8-kb (p < 0.05) and 1.7-kb (p < 0.01) StAR transcripts. PMSG-stimulated animals not exposed to EtOH showed a more than 2-fold increase (p < 0.01) in the ovarian levels of both transcripts. Western blot analysis revealed that EtOH exposure decreased (p < 0.001) the basal expression of StAR protein, which paralleled the decrease in basal StAR messenger RNA. PMSG induced an increase (p < 0.001) in the levels of StAR protein, and this effect was blunted (p < 0.01) by EtOH. These changes observed in ovarian StAR protein were paralleled by decreases in serum pregnenolone and E2. These results demonstrate for the first time that ovarian StAR is a target for the chronic action of EtOH to alter prepubertal steroidogenesis, resulting in suppressed serum E2 secretion during a critical time of development.

Acute Effects of Ethanol on Steroidogenic Acute Regulatory Protein (StAR) in the Prepubertal Rat Ovary

Background: Steroidogenic acute regulatory protein (StAR) is a 30 kDa mitochondrial protein that plays an essential role in steroid hormone biosynthesis by facilitating delivery of cholesterol across the mitochondrial membrane, where side chain cleavage occurs to initiate ovarian steroidogenesis. Because ethanol (EtOH) suppresses estradiol secretion in prepubertal female rats, we evaluated the effects of EtOH on prepubertal ovarian StAR.Methods: At 0700 hr, 28‐day‐old female rats were gavaged with saline or a 3 g/kg dose of EtOH. At 0800 hr, half of each of these two groups was treated with 15 IU of pregnant mare serum gonadotropin (PMSG). At 1000 hr, a 2 g/kg dose was administered to maintain moderately elevated blood alcohol levels. At 1600 hr, all of the animals were killed by decapitation, and blood and ovaries were collected for measurement of serum pregnenolone and estradiol and for ovarian StAR gene and protein expression.Results: Northern blot analysis showed two major transcripts of 3.8 and 1.7 kb of ovarian StAR mRNA. The ovaries from EtOH‐treated rats showed decreased (p &lt; 0.01) basal expression of both 3.8 and 1.7 kb StAR transcripts. PMSG‐stimulated animals showed a more than 4‐fold increase (p &lt; 0.001) in the levels of both transcripts, when compared with ovaries from animals that received saline or EtOH only. Conversely, in EtOH‐treated animals, the PMSG‐stimulated expression of the 1.7 kb transcript was blocked, and the increase in the 3.8 kb StAR transcript was blunted (p &lt; 0.05 vs. PMSG). Western blot analysis revealed that EtOH exposure also depressed (p &lt; 0.01) the basal expression of StAR protein. PMSG‐stimulated animals showed an increase (p &lt; 0.001) in levels of StAR protein, and this was blocked (p &lt; 0.01) by EtOH. These changes observed in ovarian StAR mRNA and protein were paralleled by changes in serum pregnenolone and estradiol. Specifically, acute EtOH exposure suppressed (p &lt; 0.05) the basal levels of both steroids. Furthermore, PMSG‐stimulated animals showed an increase in the production of pregnenolone (p &lt; 0.05) as well as estradiol (p &lt; 0.01), and EtOH blocked this stimulatory action of PMSG on both steroids.Conclusion: These results demonstrate for the first time that EtOH is capable of altering ovarian StAR expression, which contributes to the detrimental effect this drug has on ovarian steroidogenesis during prepubertal development.

Acute effects of ethanol on steroidogenic acute regulatory protein (StAR) in the prepubertal rat ovary.

Steroidogenic acute regulatory protein (StAR) is a 30 kDa mitochondrial protein that plays an essential role in steroid hormone biosynthesis by facilitating delivery of cholesterol across the mitochondrial membrane, where side chain cleavage occurs to initiate ovarian steroidogenesis. Because ethanol (EtOH) suppresses estradiol secretion in prepubertal female rats, we evaluated the effects of EtOH on prepubertal ovarian StAR. At 0700 hr, 28-day-old female rats were gavaged with saline or a 3 g/kg dose of EtOH. At 0800 hr, half of each of these two groups was treated with 15 IU of pregnant mare serum gonadotropin (PMSG). At 1000 hr, a 2 g/kg dose was administered to maintain moderately elevated blood alcohol levels. At 1600 hr, all of the animals were killed by decapitation, and blood and ovaries were collected for measurement of serum pregnenolone and estradiol and for ovarian StAR gene and protein expression. Northern blot analysis showed two major transcripts of 3.8 and 1.7 kb of ovarian StAR mRNA. The ovaries from EtOH-treated rats showed decreased (p < 0.01) basal expression of both 3.8 and 1.7 kb StAR transcripts. PMSG-stimulated animals showed a more than 4-fold increase (p < 0.001) in the levels of both transcripts, when compared with ovaries from animals that received saline or EtOH only. Conversely, in EtOH-treated animals, the PMSG-stimulated expression of the 1.7 kb transcript was blocked, and the increase in the 3.8 kb StAR transcript was blunted (p < 0.05 vs. PMSG). Western blot analysis revealed that EtOH exposure also depressed (p < 0.01) the basal expression of StAR protein. PMSG-stimulated animals showed an increase (p < 0.001) in levels of StAR protein, and this was blocked (p < 0.01) by EtOH. These changes observed in ovarian StAR mRNA and protein were paralleled by changes in serum pregnenolone and estradiol. Specifically, acute EtOH exposure suppressed (p < 0.05) the basal levels of both steroids. Furthermore, PMSG-stimulated animals showed an increase in the production of pregnenolone (p < 0.05) as well as estradiol (p < 0.01), and EtOH blocked this stimulatory action of PMSG on both steroids. These results demonstrate for the first time that EtOH is capable of altering ovarian StAR expression, which contributes to the detrimental effect this drug has on ovarian steroidogenesis during prepubertal development.

Cordyceps sinensis and its fractions stimulate MA-10 mouse Leydig tumor cell steroidogenesis.

The effects of Cordyceps sinensis (CS) and its extracted fractions on steroidogenesis in MA-10 cells were determined. Different concentrations of CS and 3 fractions of CS (F1, a water-soluble polysaccharide; F2, a water-soluble protein; and F3, a poorly water-soluble polysaccharide and protein) were added to MA-10 mouse Leydig tumor cells with or without human chorionic gonadotropin (hCG), and the production of steroid and the expression of steroidogenic acute regulatory protein (StAR) were examined. The results showed that CS alone (2-10 mg/mL) stimulated MA-10 cell progesterone production in a dose-dependent relationship. Fractions F1 and F3 (2-10 mg/mL) also had significant (P < .05) stimulatory effects on MA-10 cell steroidogenesis with a dose-dependent relationship. However, fraction F2 did not have an effect on MA-10 cells. CS and F3, but not F1, significantly induced more steroid production in hCG-stimulated MA-10 cells (P < .05). As a temporal relationship, F1 and F3 (2 mg/mL) maximally stimulated progesterone production between 1 and 3 hours after stimulation in MA-10 cells. In addition, CS and F3 significantly enhanced MA-10 cell StAR protein expression, which indicates that CS and F3 may use a cyclic adenosine monophosphate signal transduction pathway to activate MA-10 Leydig cell steroidogenesis in a manner to that of luteinizing hormone.

Regulation of steroidogenic acute regulatory protein and luteinizing hormone receptor messenger ribonucleic acid in hen granulosa cells.

The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.

Regulation of Steroidogenic Acute Regulatory Protein and Luteinizing Hormone Receptor Messenger Ribonucleic Acid in Hen Granulosa Cells

The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3–6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.

Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells

The steroidogenic acute regulatory protein (StAR) is required for the movement of cholesterol from the outer to the inner mitochondrial membrane, the site of cholesterol side chain cleavage. Here we describe a novel form of regulation of StAR gene expression in steroidogenic cells. Treatment of Y-1 BS1 adrenocortical cells with either low density lipoprotein (LDL) or high density lipoprotein (HDL) increases expression of endogenous StAR mRNA and protein in a dose-dependent manner. Induction of StAR mRNA by lipoprotein requires basal cAMP-dependent protein kinase, since the inhibitor, R(p)-8-Br-cAMP, inhibited induction of StAR protein by LDL. Likewise, basal StAR expression or LDL induction of StAR protein was not detectable in Y-1 kin-8 cells which are deficient in cAMP-dependent protein kinase. Aminoglutethimide and ketoconazole were used to determine if side chain cleavage of lipoprotein-derived cholesterol is required for induction of StAR mRNA. Treatment with either drug alone induced StAR mRNA expression 1.5-3-fold, while induction of StAR in cells treated with either drug plus LDL, was equal to, or greater than, induction seen with either agent alone, suggesting that lipoprotein does not regulate StAR via generation of an oxysterol intermediate. Both LDL and HDL increased expression of a mouse -966 StAR promoter-reporter construct 1.5-2.5-fold, indicating that regulation occurs at the level of transcription. In contrast, neither lipoprotein was able to induce transcription from a -966 StAR promoter in which the steroidogenic factor-1 site at -135 was abolished, indicating that regulation of StAR transcription by lipoproteins requires steroidogenic factor-1. The regulation of StAR gene expression by lipoproteins may represent a positive feedback circuit which links cholesterol availability with steroidogenic output.

Differences in StAR Protein Expression From the Luteinizing-Granulosa Cells of IVF-ET Patients: Implications for Cycle Outcomes

Objectives: Previous research from this and other laboratories has suggested a correlation between the subtle rise in Progesterone (P) induced by the administration of hCG, and cycle outcome in patients undergoing in vitro fertilization with embryo transfer (IVF-FT). The data suggests patients who have <2-fold increase in P following hCG will not conceive on that IVF-ET attempt (Poor responders; PR). Further, if the luteinizing-granulosa cells (LGC) of PR are placed in culture, their steroid hormone production will be significantly less than the LGC of patients with a normal response to hCG (>3-fold; NR). Recent studies have demonstrated steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis. The objective of the present study was to determine if StAR expression differed in NR and PR patients undergoing IVF-ET.

Prostaglandin F(2alpha) induces a rapid decline in progesterone production and steroidogenic acute regulatory protein expression in isolated rat corpus luteum without altering messenger ribonucleic acid expression.

With interest in steroidogenic acute regulatory protein (StAR) involvement in the luteolytic process, we studied changes in serum progesterone levels and the concomitant expression of StAR mRNA and protein (37-, 32-, and 30-kDa forms) in postovulatory Day 7 corpora lutea (CL) isolated from rats 1 h after injection with prostaglandin F(2alpha) (PGF(2alpha), n = 6) or saline (n = 6). Serum progesterone levels were determined by RIA, StAR and beta-actin mRNA expression by Northern analysis, and StAR and beta-actin protein expression by Western analysis. Adrenal, brain, and spleen from control animals were used as positive and negative controls for StAR expression. Scanning optical densitometry measurements were standardized by dividing the signal strength from each StAR autoradiogram lane by that from the corresponding beta-actin autoradiogram lane. ANOVA was used for significance testing, with alpha set at 0.05. The 37-, 32-, and 30-kDa forms of StAR protein were expressed in all adrenal samples, whereas only the 37- and 30-kDa forms were found in CL. Serum progesterone levels and expression of the 30-kDa and 37-kDa forms of the StAR protein in CL were all found to be significantly lower in the PGF(2alpha)-treated than the saline-treated group. StAR mRNA expression was not significantly different in the saline- and PGF(2alpha)-treated rats. The rapid decline in StAR protein expression that accompanies PGF(2alpha) induced luteolysis, therefore, does not result from significant decline in mRNA expression.

Expression of the steroidogenic acute regulatory protein and luteinizing hormone receptor and their regulation by tumor necrosis factor alpha in rat corpora lutea.

Expression of both mRNA and protein of the steroidogenic acute regulatory protein (StAR), in correlation with progesterone (P) production and LH receptor (LHR) mRNA expression, was studied in the corpora lutea (CL) of gonadotropin-induced-pseudopregnant and pregnant rats at various stages of CL development. Immature female rats, 21-22 days old, were injected s.c. with 20 IU eCG to stimulate follicle growth and then with 20 IU hCG 48 h later to induce ovulation. The ovaries were removed at various stages of CL development; either CL were isolated and snap frozen for total RNA analysis, or whole ovaries were fixed in Bouin's fluid for paraffin sectioning. The results of in situ hybridization, immunohistochemistry, and Northern blotting showed that the increase in StAR mRNA and protein expression was well correlated with the increase in serum P concentration. StAR expression was restricted to the luteal cells or theca cells in antral follicles. Both StAR mRNA and protein in the CL of pseudopregnant rats increased steadily on Day 1 and Day 4, reached highest levels on Day 4, and then dropped sharply on Day 8 when luteolysis takes place. LHR mRNA content was high on Day 1 but dropped significantly on Day 2. LHR mRNA increased to high levels on Day 4 and 8 and then declined on Day 12. StAR mRNA and protein levels in the CL of pregnant rats were high during early luteal development (Day 2, 4), increased even further on Day 9, and decreased on Day 13 when luteolysis takes place. It is therefore suggested that the expression of StAR coincides well with the capacity of P production in the CL and that StAR expression can be used as a functional "marker" of CL development. To study the possible effect of cytokines on StAR expression, pseudopregnant rats on Day 5 were injected s.c. with 10 IU hCG plus 20 microg prolactin (PRL), with or without 500 IU tumor necrosis factor alpha (TNFalpha) 30 min later. TNFalpha significantly inhibited hCG/PRL-induced StAR and LHR mRNA expression at 1 and 3 h post-TNFalpha. It is suggested that the luteolytic effect of TNFalpha may be mediated by its direct inhibition on StAR expression or by an indirect decrease in LHR expression.

Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.