Enhanced Inflammatory Transcriptome in the Granulosa Cells of Women With Polycystic Ovarian Syndrome.

CONTEXT: Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of reproductive-aged women, is associated with systemic low-grade inflammation. OBJECTIVE: We propose that increased or altered intrafollicular inflammatory reactions also occur in periovulatory follicles of PCOS patients. DESIGN: Gene profiling and quantitative PCR (qPCR) analyses in granulosa-lutein cells (GCs) collected from PCOS and non-PCOS women undergoing in vitro fertilization were compared with serum and follicular fluid (FF) levels of cytokines and chemokines. SETTING: This was a university-based study. PATIENTS: Twenty-one PCOS and 45 control patients were recruited: demographic, hormone, body mass index, and pregnancy outcomes were abstracted from patient data files. INTERVENTIONS: GC cytokine/chemokine mRNAs were identified and analyzed by gene-chip microarrays/qPCR before and after culture with human chorionic gonadotropin, DHT, IL-6, or IL-8; serum/FF cytokine levels were also analyzed. MAIN OUTCOME MEASURES: Relative serum/FF cytokine levels and GC cytokine expression before and after culture were compared and related to body mass index. RESULTS: The following results were found: 1) PCOS GCs express elevated transcripts encoding cytokines, chemokines, and immune cell markers, 2) based on gene profiling and qPCR analyses, obese PCOS patients define a distinct PCOS disease subtype with the most dramatic increases in proinflammatory and immune-related factors, and 3) human chorionic gonadotropin and DHT increased cytokine production in cultured GCs, whereas cytokines augmented cytokine and vascular genes, indicating that hyperandrogenism/elevated LH and obesity in PCOS women augment intrafollicular cytokine production. CONCLUSIONS: Intrafollicular androgens and cytokines likely comprise a local regulatory loop that impacts GC expression of cytokines and chemokines and the presence of immune cells; this loop is further enhanced in the obese PCOS subtype.

Effects of nonylphenol on aldosterone release from rat zona glomerulosa cells.

Alkylphenol ethoxylate, which consists of approximately 80% nonylphenol ethoxylate (NPE), is a major nonionic surfactant. Nonylphenol (NP), the primary degradation product of NPE, has been reported to interfere with reproduction in fish, reptiles, and mammals by inducing cell death in the gonads and by affecting other reproductive parameters. However, the effects of NP on rat adrenal zona glomerulosa cells (ZG) and the underlying mechanisms remain unclear. In this study, we explored the effects of NP on aldosterone release. ZG cells were incubated with NP in the presence or absence of the secretagogues angiotensin II (ANG II), potassium, 8-Br-cAMP, 25-OH-cholesterol, corticosterone or cyclopiazonic acid (CPA). After performing radioimmunoassay (RIA) and Western blot analysis, we found that (1) NP stimulated aldosterone release in cells induced by ANG II, KCl, 8-Br-cAMP, 25-OH-cholesterol, corticosterone, and CPA; (2) NP triggered the release of higher amounts of pregnenolone in cells treated with vehicle and 25-OH-cholesterol+trilostane than in cells treated with other compounds; and (3) the stimulatory effect of NP seemed to be mediated through steroidogenic acute regulatory protein (StAR) and aldosterone synthase activity. These observations suggest that the effects of NP are mediated via increased free Ca(2+) in the cytoplasm.

Effects and mechanisms of nonylphenol on corticosterone release in rat zona fasciculata-reticularis cells.

Alkylphenol ethoxylate, consisting of ∼80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactant. The primary degradation product of NPEO, nonylphenol (NP), interferes with reproduction, induces cell death in gonads, and leads to changes in other reproductive parameters. With such apparent stress, NP is believed to induce stress response mechanism, i.e., adrenal cortical hormone. However, the effects and action mechanisms of NP on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of NP on corticosterone release. ZFR cells were incubated with NP in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxyl cholesterol (25-OH-cholesterol), pregnenolone, progesterone, or deoxycorticosterone at 37°C for 1 h. The concentrations of corticosterone or pregnenolone in the spent media were measured by radioimmunoassay. The expressions of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc) protein, and 11ß-hydroxylase in the cells were measured by Western blot. The data demonstrated that (1) NP stimulated corticosterone release induced by ACTH, 8-Br-cAMP, FSK, 25-OH-cholesterol, pregnenolone, progesterone, or deoxycorticosterone; (2) NP significantly increased pregnenolone release in the control, 25-OH-cholesterol, trilostane, and 25-OH-cholesterol + trilostane groups; (3) NP-stimulated corticosterone release was estrogen receptor dependent, but mediated by nitric oxide and p38 mitogen-activated protein kinase pathway independent; and (4) NP did not affect StAR, 11ß-hydroxylase, or P450scc protein expression. These results suggest that NP acts directly on rat ZFR cells to stimulate corticosterone release and that the stimulation mechanism of NP mediates through post-cAMP corticosterone manufacture enzymes, i.e., P450scc and 11ß-hydroxylase.

Reprogramming of human somatic cells using human and animal oocytes.

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells.

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37 degrees C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca2+ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes.

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells.

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.

Effects of S-petasin on cyclic AMP production and enzyme activity of P450scc in rat zona fasciculata-reticularis cells.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of P. hybridus, which has been used to relieve gastrointestinal pain, lung disease, and spasms of urogenital tract. We have demonstrated that S-petasin inhibited corticosterone release from rat zona fasiculata-reticularis cells. However, the mechanism and molecular effects of S-petasin on zona fasiculata-reticularis cells are still unclear. This study explored the effects of S-petasin on cellular adenosine 3':5'-cyclic monophosphate (cAMP) production, the functions of steroidogenic enzymes including cytochrome P450 side-chain cleavage enzyme (P450scc), 11beta-hydroxylase, and the expression levels of steroidogenic acute regulatory protein or P450scc. In this experiment, zona fasciculata-reticularis cells were incubated with S-petasin in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), forskolin, 25-OH-cholesterol, deoxycorticosterone at 37 degrees C for 0.5, 1 or 3 h. The media were used to measure the concentration of corticosterone or pregnenolone by radioimmunoassay. The cells were used to measure the content of cAMP by radioimmunoassay and extracted protein for Western blot or messenger RNA (mRNA) for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Our data demonstrated that (1) S-petasin inhibits ACTH- or forskolin-stimulated cellular cAMP production, (2) S-petasin increased the Michaelis constants of P450scc and 11beta-hydroxylase and (3) S-petasin decreased the expression levels and mRNA of steroidogenic acute regulatory protein. In summary, the actions of S-petasin mediate the inhibition of cAMP formation, decrease the activities of key enzymes P450scc and 11beta-hydroxylase, and reduce mRNA of steroidogenic acute regulatory protein and expression of steroidogenic acute regulatory protein.

Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata-reticularis cells.

The decline of plasma dehydroepiandrosterone (DHEA) and maintenance of glucocorticoid levels with increasing age contribute to excess body fat accumulation, hyperglycaemia, hyperlipidaemia, hyperinsulinaemia and cancer. Although opposing actions of DHEA and corticosterone have been proposed in a rat model, the effects and action mechanisms of DHEA on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study addressed the effects of DHEA on corticosterone release, cellular cAMP production, the functions of steroidogenic enzymes and the expression levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc). ZFR cells were incubated with DHEA in the presence or absence of adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone or deoxycorticosterone at 37 degrees C for 30 min, 1 h or 5 h and the concentration of corticosterone or pregnenolone measured subsequently in the media by RIA. The cells were used to measure the content of cAMP by RIA and to extract protein for Western blot or mRNA for RT-PCR analysis. The data demonstrated that (1) DHEA inhibited ACTH-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA increased the K(m) of 11beta-hydroxylase but not P450scc; (4) DHEA affected the expression levels of StAR protein but not of P450scc. These results suggest that DHEA acts directly on rat ZFR cells to diminish corticosterone secretion by inhibition within the post-cAMP pathway, by inhibiting steroidogenic enzymes downstream from P450scc and by inhibiting StAR expression.

Effects of fasting on corticosterone production by zona fasciculata-reticularis cells in ovariectomized rats.

BACKGROUND: To investigate the function and mechanism of fasting on the production of corticosterone in vitro by zona fasciculata-reticularis (ZFR) cells from ovariectomized (OVX) rats. METHODS: Female rats were OVX for 4 days before decapitation. Rats were fed or fasted for 1 day before experiment. ZFR cells from fed and fasted rats were incubated with adrenocorticotropic hormone (ACTH), forskolin, 8-bromo-3',5'-cyclic adenosine monophosphate, SQ22536, nifedipine, chelerythrine chloride, trilostane or steroidogenic precursors at 37 degrees C for either 60 or 30 minutes. Corticosterone, pregnenolone concentrations in spent media, and the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration were determined by radioimmunoassay. The effects of fasting in response to ACTH on the protein expressions of steroidogenic acute regulatory protein (StAR) or cytochrome P450 side-chain cleavage enzyme (P450scc) in ZFR cells were determined by Western blot analysis. RESULTS: The concentration of plasma corticosterone in fasted rats was significantly higher than that in fed rats (P<0.01). One-day fasting significantly increased the responsiveness of ZFR cells to ACTH, forskolin, and precursor-stimulated corticosterone productions and to forskolin-stimulated cAMP accumulation. The corticosterone production was reduced in fasted group when adenylyl cyclase was inhibited by SQ22536. The fasting-enhanced level of corticosterone production in ZFR cells was decreased by the administration of nifedipine but not altered by that of chelerythrine chloride. Fasting significantly increased trilostane-stimulated production of pregnenolone in ZFR cells. The activities of enzymes which converting cholesterol, pregnenolone, progesterone, and deoxycorticosterone to corticosterone and the expressions of StAR in ZFR cells were greater in fasted rats than in fed rats. CONCLUSIONS: This study demonstrated that fasting increased the release of corticosterone and the accumulation of cAMP by rat ZFR cells. The action mediated through enhancing the responsiveness to ACTH stimulation, cAMP cascades and the activity of L-type calcium channels. The activities of steroidogenic enzymes including P450scc, 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11beta-hydroxylase were all enhanced by the fasting treatment.

Effects of S-petasin on corticosterone release in rats.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of Petasites hybridus. S-petasin has been used to relieve gastrointestinal pain, lung disease, and spasms of the urogenital tract. However, the side effect of S-petasin on endocrine systems are still not clear. This study explored the effects of S-petasin on the release of corticosterone in vivo and in vitro. An intravenous injection of S-petasin (10 microg/kg) decreased both basal and adrenocorticotropin (ACTH)-induced plasma corticosterone concentration in male rats. In vitro, S-petasin (3 x 10(-6) - 10(-4) M) caused a significant reduction of basal and ACTH-stimulated release of corticosterone from the enzymatically dispersed rat zona fasciculata-reticularis (ZFR) cells in a dose-dependent manner. In order to study possible mechanisms, ZFR cells were incubated with S-petasin (10(-5) M) in the presence or absence of forskolin (adenylate cyclase activator, 10(-6) - 10(-4) M), 8-Br-cAMP (a cAMP analogue, 10(-6) 10(-4) M), 25-OH-cholesterol (pregnenolone biosynthesis precursor, 10(-5) M) combined with trilostane (a blocker of 3beta-hydroxysteriod dehydrogenase, 3beta-HSD, 10(-6) M) and deoxycorticosterone (corticosterone biosynthesis precursor, 10(-9) - 10(-6) M) at 37 degrees C for 1h. The concentration of pregnenolone and corticosterone in media were measured by radioimmunoassay. The stimulatory effects of corticosterone secretion induced by forskolin (10(-5) - 10(-4) M), 8-Br-cAMP (10(-5) - 10(-4) M) and deoxycorticosterone (10(-7) - 10(-6) M) were reduced by S-petasin at 10(-5) M. The stimulatory effects of pregnenolone secretion induced by 25-OH-cholesterol combined with or without trilostane was reduced by S-petasin at 10(-5) M. These results suggest that S-petasin inhibits the production of corticosterone from rat ZFR cells in part through decreasing the activities of adenylyl cyclase, P450scc and 11beta-hydroxylase.