Steroidogenic Acute Regulatory Protein mRNA Research Articles

Male testicular development is affected by estrogens but not altered in neonatal piglets receiving soy infant formula

Soy infant formula contains many phytochemicals, including phytoestrogens, which are structurally similar to estradiol (E2). As a result of their potentially estrogenic properties, soy infant formulas have been the subject of concern with regards to reproductive toxicity. To address these concerns regarding male reproductive development, we fed male piglets a soy‐based infant formula, a dairy milk‐based infant formula, milk formula supplemented with E2, or milk formula supplemented with putative estrogenic soy phytonutrient genistein (GEN), in comparison with piglets receiving sow's breast milk from postnatal day 2–21. In the E2 supplemented group, serum testosterone and testicular weight were significantly reduced compared to the milk formula‐fed and sow's milk controls. Testis tubule area, Sertoli cell and germ cell numbers were also increased in the E2 supplemented group compared to milk formula controls, P<0.05. When compared to the sow's milk control, RNAseq analysis in testes showed that E2 supplemented milk formula altered expression of 2012 testicular genes (±1.5 fold, P<0.100). Functional annotation analysis indicated up‐regulation of several pathways in the E2 supplemented group, including canonical Wnt signaling, cell differentiation and migration, angiogenesis and spermatogenesis. E2 also down‐regulated cholesterol and androgen synthesis pathways. Real‐time RT‐PCR confirmed the decrease in steroidogenic acute regulatory protein (Star) mRNA in the E2 supplemented group, suggesting a potential mechanism for the observed decrease in serum testosterone in this diet group. In contrast, GEN supplementation of milk formula altered 104 genes, soy formula 49 genes, and the pathways impacted had no clear links to testes function or sex organ development. These data show that early infant diet exposure to phytoestrogens in soy‐based infant formula are not estrogenic in the male reproductive system.Support or Funding InformationThis work was funded by USDA‐ARS Project 6026‐5100‐010‐05S.

Molecular identification of StAR and 3βHSD1 and characterization in response to GnIH stimulation in protogynous hermaphroditic grouper (Epinephelus coioides).

Gonadal steroids are critical factors in reproduction and sex reverse process. StAR (steroidogenic acute regulatory protein), transferring the cholesterol from the outer mitochondrial membrane to the inner membrane, is the rate-limiting factor of steroidogenesis. 3βHSD (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase), converting Δ5-steroids into Δ4-steroids, is an important oxidoreductase in steroidogenesis. In the present study, StAR and 3βHSD1 were cloned and characterized from protogynous orange-spotted grouper. StAR cDNA contains an 861bp open reading frame (ORF), encoding a predicted protein of 286 amino acids, and the ORF of 3βHSD1 was 1125bp, encoding a predicted protein of 374 amino acids. The transcript of StAR was mainly expressed in gonad, while 3βHSD1 mRNA was predominantly detected in brain and gonad. In the previous study, we found the expression of GnIH mRNA level in male, as well as in 17 alpha-methyltestosterone (MT)-induced male fish was significantly higher than in female fish, this indicating that GnIH/GnIHR signaling might be involved in the regulation of sex reversal and male maintenance. In order to figure out the function of GnIH in steroidogenesis, the expression of StAR and 3βHSD1 regulated by GnIH was examined. In vitro study showed that treatment of cultured ovary fragments with gGnIH peptides significantly stimulated the expression of StAR and 3βHSD1. In addition, the mRNA levels of StAR and 3βHSD1 were significantly increased after intraperitoneal injection (i.p.) with gGnIH peptides. Moreover, during MT-induced sex change from female to male, the levels of StAR mRNA significantly increased by 5.2, 24.8 and 353.5 folds, and that of 3βHSD1 mRNA by 3.5, 32.5 and 55.4 folds at the 2nd, 4th and 6th week after MT implantation, respectively. Collectively, our results indicate that GnIH may be involved in the regulation of sex reversal or male maintenance by stimulating the expression of StAR and 3βHSD1 in protogynous grouper.

Interleukin-6 increases the expression of key proteins associated with steroidogenesis in human NCI-H295R adrenocortical cells

Mechanisms of interleukin-6 (IL-6)-induced cortisol release (CR) were investigated by exposing H295R cells to IL-6 and determining mRNA/protein expression (PCR/western blots) for steroidogenic enzymes (SE), steroidogenic acute regulatory protein (StAR), steroidogenic factor-1 (SF-1) (enhances SE/StAR expression), activator protein 1 (AP-1) (regulates SE/StAR expression) and adrenal hypoplasia congenita-like protein (DAX-1) (inhibits SE/StAR expression). Promoter activity of StAR (SPA) was measured by a luciferase-coupled promoter. Cortisol release was increased by 10ng/mL IL-6 (24h P<0.01). Proteins/mRNAs (StAR, cholesterol side chain cleavage enzyme, SF-1, AP-1) and SPA were increased by IL-6 (60min 1–50ng/mL IL-6; 5ng/mL IL-6 30–120min P<0.05). Four other SE proteins/mRNAs were also increased by 10ng/mL IL-6 (60min P<0.01). Protein/mRNA for DAX-1 was decreased by IL-6 (60min 1–50ng/mL IL-6; 5ng/mL IL-6 30–120min P<0.01). Phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) was increased by IL-6 (JAK2 60min 1–50ng/mL IL-6; 10ng/mL IL-6 5–60min P<0.05; STAT1 and STAT3 60min 10ng/mL IL-6 P<0.01). Inhibition of JAK/STAT with AG490 (10μM) or piceatannol (50μM) blocked (P<0.01 10ng/mL IL-6vs. IL-6 plus AG490 or piceatannol) IL-6-induced increases in SPA and StAR mRNA. In summary, IL-6-induced CR may be facilitated by increased StAR and SE mediated by increased SF-1 and AP-1, decreased DAX-1, and increased phosphorylation of JAK/STAT.

Low glucose availability stimulates progesterone production by mouse ovariesin vitro

Steroid production by the ovary is primarily stimulated by gonadotropins but can also be affected by biological cues that provide information about energy status and environmental stress. To further understand which metabolic cues the ovary can respond to, we exposed gonadotropin-stimulated mouse ovaries in vitro to glucose metabolism inhibitors and measured steroid accumulation in media. Gonadotropin-stimulated ovaries exposed to 2-deoxy-d-glucose increased progesterone production and steroidogenic acute regulatory protein mRNA levels. However, oocytes and granulosa cells in antral follicles do not independently mediate this response because targeted treatment of these cell types with a different inhibitor of glucose metabolism (bromopyruvic acid) did not affect progesterone production. Elevated progesterone production is consistent with the homeostatic role of progesterone in glucose regulation in mammals. It also may regulate follicle growth and/or atresia within the ovary. These results suggest that ovaries can regulate glucose homeostasis in addition to their primary role in reproductive activity.

Effects of dietary soybean isoflavones (SI) on reproduction in the young breeder rooster

Soybean isoflavones (SIs) are phytoestrogens that competitive with estrogens in body. Although SIs play an important role in reproduction, their role in testicular development in roosters is unknown. This study was conducted to investigate the effect of SIs on testicular development and serum reproductive hormone profiles in young breeder roosters (70–133days old). Gene expression of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are related to testosterone synthesis, in rooster testis were also evaluated after treatment with different SI doses. Although SIs had no significant effect on body weight, 5mg/kg SIs significantly increased the testis index and serum levels of reproductive hormones (gonadotropin releasing hormone, follicle- stimulating hormone, luteinizing hormone, and testosterone).To further investigate whether SIs regulate hormone synthesis via StAR, p450scc, 3β-HSD, real time-PCR was performed to measure the mRNA levels of the corresponding genes. The results showed that 5mg/kg of SIs significantly increased StAR mRNA levels. However, there were no significant effects on p450scc or 3β-HSD mRNA levels. Moreover, the spermatogonial development and the number of germ cell layers were increased by treatment with 5mg/kg of SIs. These results suggest that SIs promote testicular growth by increasing reproductive hormone secretion, which is closely related to StAR expression, to positively regulate reproduction in young roosters.

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.

1149 Expression of genes involved in the initial steps of steroidogenesis in adipose tissue depots of dairy cows during the dry period and early lactation

In view of the significant changes in body fat content related to parturition and lactation in dairy cows, together with the role of adipose tissue (AT) not only as a store of steroid hormones but also as a potential site of steroidogenesis, mobilization of body reserves may also alter steroid hormone secretion and eventually reproduction. With this background, our research objectives were (1) to assess the expression of two rate-limiting factors of steroidogenesis, i.e., steroidogenic acute regulatory protein (StAR) and Cytochrome P450scc (CYP11A1) in bovine AT, (2) to characterize the time course of their mRNA abundance during late pregnancy and early lactation, and (3) to compare this time course in a subcutaneous versus a visceral fat depot. StAR triggers cholesterol delivery to the inner mitochondrial membrane (IMM) where CYP11A1 then initiates steroidogenesis by converting cholesterol to pregnenolone, the precursor of all other endogenous steroids. Biopsies were collected from 20 Holstein cows from the subcutaneous (sc) and the retroperitoneal fat depot (rp) on d −42 and d 1, 21, and 100 relative to calving. The mRNA abundance of StAR and CYP11A1 was assessed in the tissue samples by qPCR and normalized using the 4 most stable reference genes. Data were analyzed using the MIXED procedure of SAS. In scAT, the StAR mRNA abundance was lowest on d −42, increased on d 1 (P < 0.05) until d 21 to remain at this level at d 100 (∼3-fold greater than on d −42; P < 0.001). Expression of StAR mRNA in rpAT increased with time of lactation, but differences between sampling days were limited to d −42 versus d 100 (3.3-fold increase; P = 0.01). Expression of CYP11A1 mRNA was not detectable in both AT with the protocol used herein (suitability of the protocol was confirmed with ovary as positive control). The increased expression of StAR mRNA abundance after calving in rpAT and scAT indicate an increased capacity for cholesterol uptake to the IMM. Given the apparently absent expression of CYP11A1, de novo synthesis of steroid hormones in bovine AT seems implausible and AT steroid metabolism likely depends on the uptake of preformed steroid precursors. Cholesterol reaching the IMM may thus rather be metabolized to oxysterols which were suggested to have regulatory functions in adipocytes.

Retinoic acid enhances progesterone production via the cAMP/PKA signaling pathway in immature rat granulosa cells

Retinoic acid (RA) is a metabolite of vitamin A and has important roles in development, differentiation, and reproduction. Activin has been shown to regulate the RA pathway and affect granulosa cell (GC) proliferation, suggesting that RA is important for early follicle development. However, little is known about the effects of RA on GC functions, particularly steroidogenesis, during the early follicle stage. The aim of this study was to investigate the effects of all-trans-RA (atRA) on progesterone production in immature rat GCs cultured without gonadotropin. Our results demonstrated that atRA enhanced progesterone production by upregulating the levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450scc (Cyp11a1) mRNAs, but not 3β-hydroxysteroid dehydrogenase mRNA in immature rat GCs. Additionally, analysis of the mechanisms through which atRA upregulated StAR and Cyp11a1 mRNAs revealed that atRA enhanced intracellular cAMP accumulation and phosphorylation of cAMP response-element binding protein (CREB). In addition, H-89, an inhibitor of protein kinase A (PKA), abolished the stimulatory effects of atRA, indicating that atRA enhanced progesterone synthesis through cAMP/PKA signaling. In conclusion, our data demonstrated that atRA has a crucial role in progesterone synthesis in rat GCs during the early follicle stage.

Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3′- and 5′-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.

Atrazine activates multiple signaling pathways enhancing the rapid hCG-induced androgenesis in rat Leydig cells

Atrazine (ATR) is an endocrine disruptor that affects steroidogenic process, resulting in disruption of reproductive function of the male and female gonads. In this study, we used the primary culture of peripubertal Leydig cells to investigate the effect of ATR on the rapid androgen production stimulated by human chorionic gonadotropin (hCG). We demonstrated that ATR activated multiple signaling pathways enhancing the rapid hCG-stimulated androgen biosynthesis in Leydig cells. Low hCG concentration (0.25ng/mL) caused cAMP-independent, but ERK1/2-dependent increase in androgen production after 60min of incubation. Co-treatment with ATR for 60min enhanced the cAMP production in hCG-stimulated cells. Accumulation of androgens was prevented by addition of U0126, N-acetyl-l-cysteine and AG1478. Co-treatment with hCG and ATR for 60min did not alter steroidogenic acute regulatory protein (Star) mRNA level in Leydig cells. After 120min, hCG further increased androgenesis in Leydig cells that was sensitive to inhibition of the cAMP/PKA, ERK1/2 and ROS signaling pathways. Co-treatment with ATR for 120min further enhanced the hCG-induced androgen production, which was prevented by inhibition of the calcium, PKC and EGFR signaling cascades. After 120min, ATR enhanced the expression of Star mRNA in hCG-stimulated Leydig cells through activation of the PKA and PKC pathway. Collectively, these data suggest that exposure to ATR caused perturbations in multiple signaling pathways, thus enhancing the rapid hCG-dependent androgen biosynthesis in peripubertal Leydig cells.

Advanced glycation end products inhibit testosterone secretion by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products(AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs(25, 50, 100 and200µg/ml). Testosterone production induced by human chorionic gonadotropin(hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein(StAR), cholesterol side-chain cleavage enzyme(P450scc) and 3β-hydroxysteroid dehydrogenase(3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitativePCR and western blot analyssi, respectively. Reactive oxygen species(ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate(DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins[C/EBP homologous protein(CHOP)andglucose-regulated protein78(GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control(P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control(P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine(NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid(TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

Changes in gene expression of histone modification enzymes in rat granulosa cells undergoing luteinization during ovulation.

BackgroundThe ovulatory LH surge rapidly alters the expression of steroidogenesis-related genes such as steroidogenic acute regulatory protein (StAR) in granulosa cells (GCs) undergoing luteinization. We recently reported that histone modifications contribute to these changes. Histone modifications are regulated by a variety of histone modification enzymes. This study investigated the changes in gene expression of histone modification enzymes in rat GCs undergoing luteinization after the induction of ovulation. The extracellular regulated kinase (ERK)-1/2 is a mediator in the intracellular signaling pathway stimulated by the ovulatory LH surge and regulates the expression of a number of genes in GCs. We further investigated whether ERK-1/2 is involved in the regulation of the histone modification at the StAR promoter region in GCs undergoing luteinization.ResultsGCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h) CG injection. The expressions of 84 genes regulating histone modifications or DNA methylation were measured using a PCR array. Five genes (HDAC4, HDAC10, EZH2, SETDB2, and CIITA) were identified as histone acetylation- or histone methylation-related genes, and were significantly altered after hCG injection. None of the genes were related to DNA methylation. mRNA levels of EZH2, SETDB2, HDAC4, and HDAC10 decreased and CIITA mRNA levels increased 4 or 12 h after hCG injection.GCs isolated after eCG injection were incubated with hCG for 4 h to induce luteinization. StAR mRNA levels were significantly increased by hCG accompanied by the increase in H3K4me3 of the StAR promoter region. StAR mRNA expression was inhibited by the ERK inhibitor with the significant decrease of H3K4me3. These results suggest that hCG increases StAR gene expression through the ERK-1/2-mediated signaling which is also associated with histone modification of the promoter region.ConclusionsGene expressions of histone modification enzymes change in GCs undergoing luteinization after ovulation induction. This change may play important roles in regulating the expression of various genes during the early stage of luteinization, which may be critical for the subsequent corpus luteum formation.

Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in guinea pigs' testis.

A lot of studies have shown that fluoride and aluminum have toxic effect on male reproductive system, but the mechanism of which and the interaction between fluoride and aluminum is still unknown. This study investigated the effects of fluoride (NaF) or/and aluminum (AlCl3) on serum testosterone level, gene and protein expression levels of Steroidogenic Acute Regulatory Protein (StAR) and Cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in the testes of guinea pigs. Fifty-two guinea pigs were divided randomly into four groups (Control, HiF, HiAl and HiF + HiAl). Fluoride (150 mg NaF/L) or/and aluminum (300 mg AlCl3/L) were orally administrated to male guinea pigs for 13 weeks. The results showed that F and Al reduced number and elevated abnormal ratio of sperm. Meanwhile, the concentrations of serum testosterone in all experimental groups were decreased. P450scc protein expression was significantly reduced in all treatment groups, and StAR expression was decreased remarkably in HiF group and HiF + HiAl group. The levels of StAR mRNA in three groups were reduced by 53.9%, 21.4% and 33.4%, respectively, while the expressions of P450scc mRNA were reduced by 67.8%, 17.0% and 47.8%. Therefore, we concluded that F induced the reduction in testosterone and sperm amount, and thus in lower fertility, which might occur as a consequence of depressed StAR and P450scc mRNA expression. There were no synergistic effects between F and Al, instead, Al weakened the toxicity of F to some extents. The results indicated that Al had antagonism effects on F.

Bisphenol A stimulates steroidogenic acute regulatory protein mRNA expression and progesterone production in rat immature granulosa cells

Bisphenol A stimulates steroidogenic acute regulatory protein mRNA expression and progesterone production in rat immature granulosa cells

Effects of monobutyl phthalate on steroidogenesis through steroidogenic acute regulatory protein regulated by transcription factors in mouse Leydig tumor cells.

Dibutyl phthalate (DBP) is one of the most widely used phthalate esters, and it is ubiquitous in the environment. DBP and its major metabolite, monobutyl phthalate (MBP), change steroid biosynthesis and impair male reproductive function. However, the regulatory mechanism underlying the steroid biosynthesis disruption by MBP is still unclear. We analyzed the progesterone production, steroidogenic acute regulatory protein (StAR) mRNA, protein expression, and DNA-binding affinity of transcription factors (SF-1 and GATA-4). Our results reveal that MBP inhibited progesterone production. At the same time, StAR mRNA and protein were decreased after MBP exposure. Furthermore, electrophoretic mobility shift assay showed that DNA-binding affinity of transcription factors (SF-1 and GATA-4) was decreased in a dose-dependent manner after MBP treatments. Western blot tests next confirmed that protein of SF-1 was decreased, but GATA-4 protein was unchanged. However, phosphorylated GATA-4 protein was decreased with 800 μM of MBP. This study reveals an important and novel mechanism whereby SF-1 and GATA-4 may regulate StAR during MBP-induced steroidogenesis disruption.

Perfluorooctanoic Acid‐Induced Progesterone Synthesis Inhibition is Associated with Decreased Expression of Steroidogenic Acute Regulatory Protein (StAR) mRNA but Not p450 Side‐Chain Cleavage mRNA in hCG‐Stimulated mLTC‐1 Cells

Perfluorooctanoic acid (PFOA) is an environmentally persistent synthetic fluoropolymer that has been identified as an endocrine disruptor. Although its mechanism of action remains largely unknown, it has been suggested to decrease steroidogenesis. The aims of this study were to determine if PFOA decreases progesterone (P4) synthesis in stimulated Mouse Leydig Tumor (mLTC‐1) cells and which steroidogenic enzymes are involved. Cells were treated with PFOA (100nm ‐ 100μM) for 24h before stimulation for 4h with human chorionic gonadotropin (hCG) or forskolin. Cell viability was measured using the CellTiter‐Blue® Cell Viability Assay. The effects of PFOA on P4 production were assessed by ELISA. The expression of steroidogenic acute regulatory protein (StAR), p450 side‐chain cleavage (p450scc), and the luteinizing hormone receptor (LHR) were determined by real‐time PCR. Cells treated with 100μM PFOA for 24h showed an 86% reduction in hCG‐induced P4 synthesis compared to untreated cells. To ensure that this decline was not due to diminished LHR expression (mRNA levels were decreased in PFOA‐treated cells), cells were also stimulated with forskolin. Similar results were obtained. There was no significant decrease in cell viability. There was no significant difference in levels of p450scc mRNA between PFOA‐stimulated and unstimulated cells. However, the levels of StAR mRNA levels were markedly declined in cells treated with 100μM PFOA. These data show inhibition of steroidogenesis in PFOA‐treated mLTC‐1 cells and an associated decrease in the expression of LHR and StAR mRNA. &#x0D; Funding: Center for Undergraduate Research and Dept of Biological Sciences, GRU

Mechanism of Toona sinensis-stimulated adrenal steroidogenesis in primary rat adrenal cells

Toona sinensis is a well-known nutritious plant food widely distributed in Asia. The effects of aqueous leaf extracts of T. sinensis (TSL-1) on steroidogenesis was examined using primary rat adrenal cells. TSL-1 stimulated corticosterone production in a time- and concentration-dependent manner. Cells were then challenged with forskolin, dibutyryl cyclic AMP (dbcAMP), pregnenolone, progesterone or deoxycorticosterone in the presence or absence of TSL-1 for 6 h to determine the mechanism. TSL-1 could enhance forskolin- and dbcAMP-induced steroidogenesis and promote pregnenolone-, progesterone- and deoxycorticosterone-induced steroidogenesis. Moreover, TSL-1 induced steroidogenic acute regulatory protein (StAR) mRNA and protein expression in rat adrenal cells and increased StAR promoter activity in CHO-K1 cells. Thus, the stimulatory effect of TSL-1 was presumed to be mainly due to methyl and ethyl gallates. Therefore, TSL-1-stimulated corticosterone production requires the participation of cAMP-dependent PKA, steroidogenic enzymes and the expression of StAR, which regulates the rate-limiting step in steroidogenesis.

Analysis of endocrine disruption effect of Roundup® in adrenal gland of male rats

The effect of Roundup® on adrenal gland steroidogenesis and signaling pathway associated with steroid production was investigated. Doses of 10, 50, 100 and 250mg/kg bw/d Roundup® were administered for two weeks to adult male rats. The 10mg/kg bw/d dose which reduced circulatory corticosterone levels, but did not change food consumption and body weight, was selected for further study. The expression of cholesterol receptor (low density lipoprotein receptor), de novo cholesterol synthesis enzyme (3-hydroxy-3-methylglutaryl-coenzyme A synthase), hormone-sensitive lipase, steroidogenic acute regulatory protein (StAR) mRNA and phosphorylated form was decreased. Adrenocorticotropic hormone receptor (ACTH), melanocortin-2 receptor, expression was not changed but circulatory ACTH levels and adrenal cortex protein kinase A (PKA) activity were reduced. Surprisingly, exogenous ACTH treatment rescued steroidogenesis in Roundup®-treated animals. Apoptosis was evident at 250mg/kg bw/d, but not at 10mg/kg bw/d dose. These results suggest that Roundup® may be inhibitory to hypothalamic–pituitary axis leading to reduction in cyclic adenosine monophosphate (cAMP)/PKA pathway, StAR phosphorylation and corticosterone synthesis in the adrenal tissue.

Effect of in utero exposure to endocrine disruptors on fetal steroidogenesis governed by the pituitary-gonad axis: a study in rats using different ways of administration.

The effects of endocrine disruptors on testicular steroidogenesis in fetal rats were investigated in a study involving in utero exposure. In the major part of this study, pregnant rats at gestational day (GD)15 were given a single oral administration of the test substance, and then the expression of the following mRNAs in GD20 fetuses was determined: testicular steroidogenic acute-regulatory protein (StAR), a cholesterol transporter mediating the rate-limiting step of steroidogenesis, a ß-subunit of pituitary luteinizing hormone (LH), and a regulator of gonadal steroidogenesis. Among the substances tested, only di(2-ethylhexyl)phthalate (DEHP) reduced the expression of fetal testicular StAR. The others listed below exhibited little effect on fetal StAR: 2,2',4,4'-tetrabromodiphenylether, tributyltin chloride, atrazine, permethrin, cadmium chloride (Cd), lead acetate (Pb) and methylmercury (CH3HgOH). None of them, including DEHP, lacked the ability to reduce the expression of pituitary LHß mRNA. The present study also examined the potential of metals as modifiers of fetal steroidogenesis by giving them to pregnant dams in drinking water during GD1 and GD20. Under these conditions, Cd and Pb at a low concentration (0.01 ppm) significantly attenuated the fetal testicular expression of StAR mRNA without a concomitant reduction in LHß. No such effect was detected with CH3HgOH even at 1 ppm. These results suggest that: 1) DEHP, Cd and Pb attenuate the fetal production of sex steroids by directly acting on the testis, and 2) chronic treatment during the entire gestational period is more useful than a single administration for determining the hazardous effect of a suspected endocrine disruptor on fetal steroidogenesis.

Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells.

Exerting a broad range of biological effects in various tissues, activins are homo- or heterodimers of activin/inhibin β-subunits (βA, βB, βC, and βE in humans). Although activins A (βAβA), B (βBβB), AB (βAβB), and AC (βAβC) have been demonstrated in the female reproductive system, little is known about their individual functions in the ovary. To investigate the biological roles and activities of activins in regulating steroidogenesis in human granulosa cells. Human granulosa-lutein cells obtained from 32 patients undergoing in vitro fertilization were used to investigate the effects of activin A, B, AB, and AC on the expression of steroidogenic enzymes and steroid production. An academic research center. mRNA and protein levels were examined by reverse transcription quantitative real-time PCR and Western blot analysis, respectively. The production of estradiol and progesterone was measured by enzyme immunoassay. P450 aromatase, FSH receptor, and estradiol levels were increased, whereas steroidogenic acute regulatory protein (StAR), LH receptor, and progesterone levels were decreased after treatment with activin A, B, and AB, but not activin AC. FSH or LH induced the production of aromatase/estradiol and StAR/progesterone; however, pretreatment with activin A, B, or AB enhanced the effects of gonadotropins on aromatase/estradiol, but suppressed their effects on StAR/progesterone. Treatment with activin A, B, or AB induced the phosphorylation of SMA- and MAD-related proteins (SMAD2 and 3), whereas activin AC had no such effects. Furthermore, co-culture of activin AC (1-100 ng/mL) with activin A (25 ng/mL) did not alter the effects of activin A on P450 aromatase or StAR mRNA levels. Activin A, B, and AB have similar effects on steroidogenesis in human granulosa cells. In contrast, activin AC is not biologically active and does not act as a competitive antagonist.