Inosine-specific cleavage of RNA for microarray analysis of RNA A-to-I editing targets.

Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Although several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of adenosine deaminases that act on RNA (ADARs) remain unknown. Here we report a modified I-specific cleavage method that improves the quality of the RNA product. Preliminary microarray comparison of RNAs subjected to I-specific cleavage or mock digestion reported 165 genes that showed more than 0.2-fold reductions due to the cleavage. Six of the 165 genes were randomly selected for further verification, and three were verified to be targets of I-specific cleavage. This method may provide an alternative method of identifying novel RNA A-to-I editing sites using a microarray and facilitate the inquiry into the roles of RNA A-to-I editing in various biological processes.

A method to identify RNA A-to-I editing targets using I-specific cleavage and exon array analysis.

RNA A-to-I editing is the most common single-base editing in the animal kingdom. Dysregulations of RNA A-to-I editing are associated with developmental defects in mouse and human diseases. Mouse knockout models deficient in ADAR activities show lethal phenotypes associated with defects in nervous system, failure of hematopoiesis and reduced tolerance to stress. While several methods of identifying RNA A-to-I editing sites are currently available, most of the critical editing targets responsible for the important biological functions of ADARs remain unknown. Here we report a method to systematically analyze RNA A-to-I editing targets by combining I-specific cleavage and exon array analysis. Our results show that I-specific cleavage on editing sites causes more than twofold signal reductions in edited exons of known targets such as Gria2, Htr2c, Gabra3 and Cyfip2 in mice. This method provides an experimental approach for genome-wide analysis of RNA A-to-I editing targets with exon-level resolution. We believe this method will help expedite inquiry into the roles of RNA A-to-I editing in various biological processes and diseases.

Screening of a panel of steroid-related genes showed polymorphisms of aromatase genes confer susceptibility to advanced stage endometriosis in the Taiwanese Han population.

OBJECTIVE: To establish a multilocus model for studying the effect of steroid-related genes on advanced stage endometriosis. MATERIALS AND METHODS: A total of 121 patients with advanced stage endometriosis and 171 control women were included. Eighteen single-nucleotide polymorphisms (SNPs) from nine genes (HSD17B1, HSD17B2, HSD17B5, HSD17B6, CYP17, CYP19, ERα, ERß, and PGR) were genotyped using the TaqMan assays. Logistic regression models were used to evaluate the genetic effects, with adjustment for other covariates. RESULTS: Only the presence of the mutant CYP19 (aromatase gene) was associated with a significantly increased risk of endometriosis after adjusting for age, BMI, and parity (p = 0.002, OR = 2.69; 95% CI = 1.44-5.02). No association was ascertained between the other investigated SNPs and endometriosis. CONCLUSION: Polymorphisms of the aromatase gene confer susceptibility to advanced stage endometriosis in the Taiwanese Han population.

Polymorphisms of dioxin receptor complex components and detoxification-related genes jointly confer susceptibility to advanced-stage endometriosis in the taiwanese han population.

PROBLEM: To establish a multilocus model for studying the effect of dioxin receptor complex components and detoxification-related enzymes on advanced endometriosis. METHOD OF STUDY: Six single-nucleotide polymorphisms (SNPs) and two deletion polymorphisms from eight genes (CYP1A1, CYP1B1, GSTM1, GSTT1, GSTP1, AhR, ARNT, and AhRR) were genotyped. RESULTS: In the single SNP analysis, GSTM1 null type and AhRR variant type were associated with a significantly increased risk of endometriosis [odds ratio (OR)=2.38 and 2.45, respectively]. Using multiple SNPs in the logistic regression for covariates, wild-type AhR and mutant AhRR combination was significantly higher in patients (67.8%) than in controls (48.0%) (OR=2.76). On the other hand, mutant AhRR in combination with GSTM1 null genotype was significantly higher in patients (35.5%) than in controls (19.3%) (OR=6.12). CONCLUSION: Polymorphisms of dioxin receptor complex components and detoxification-related genes jointly confer susceptibility to advanced-stage endometriosis in the Taiwanese Han population.

Effects of sleep deprivation on serum testosterone concentrations in the rat.

Sleep deprivation (SD) leads to decreases in circulating levels of testosterone with unknown mechanisms. We tested the hypothesis that decreased testosterone levels associated with SD may be caused by serotonin-mediated inhibition of its production. Male rats were subjected to SD for 24 or 48 h using the dish-over-water-method with a Rechtschaffen apparatus. Serum testosterone, corticosterone and serotonin (5-HT) concentrations were assessed thereafter, as were testicular StAR and 5-HT 2 receptor levels. SD, regardless of duration led to significant decreases in serum testosterone levels and testicular steroid acute regulatory protein (StAR) protein expression, while 5-HT levels were significantly elevated (all P<0.05). Corticosterone concentrations were significantly increased in 48 h SD rats (P<0.05). In primary Leydig cell cultures, 5-HT decreased chorionic gonadotropin-induced testosterone secretion and StAR expression, which appeared to be dependent on 5-HT 2 receptor activation but independent of cyclic AMP signaling. These findings suggest that decreased serum testosterone levels in SD rats may be the result of 5-HT-related inhibition of testosterone production and decreased testicular expression of StAR protein.

Androgen excess down-regulates connexin43 in a human granulosa cell line.

By use of a cell model, we found that high levels of androgen reduce connexin43 expression and impair gap junction intercellular communication between human granulosa cells through the androgen receptors. High levels of androgen may impair folliculogenesis and in turn lead to ovulatory dysfunction in polycystic ovary syndrome patients.

Mitochondria DNA deletion and copy numbers of cumulus cells associated with in vitro fertilization outcomes.

OBJECTIVE: Mitochondria are important organelles in cell biology. We aimed to study the effects of mitochondrial DNA variations in cumulus cells (CCs) upon in vitro fertilization and embryo transfer (IVF-ET) outcomes. STUDY DESIGN: A total of 51 women undergoing IVF-ET were recruited for the study. The CCs were collected during oocyte retrievals. Mitochondria DNA 4977-bp deletion (dmtDNA-delta5Kb) and copy numbers (MCN) of CCs were analyzed by polymerase chain reaction. The relationships of dmtDNA-delta5Kb and MCN with patients' age, embryo qualities and pregnancy rates (PRs) were detected and compared. RESULTS: PRs were positively correlated with younger age, better transferred embryo qualities and lower dmtDNA-delta5Kb ratios in CCs. The dmtDNA-delta5Kb status was positively associated with older age and higher MCN but was not associated with embryo morphologic scoring. The dmtDNA-delta5Kb ratios of transferred embryos in pregnancy and nonpregnancy groups were 0% and 10.4%, respectively. The dmtDNA-delta5Kb in > or = 34-year-old and <34-year-old groups were 6.9% and 3.2%, respectively. CONCLUSION: The dmtDNA-delta5Kb and MCN statuses of CCs are negatively associated with PRs, which might be potential tools for oocyte evaluation and embryo selections during IVF-ET.

Serum anti-Müllerian hormone predicts ovarian response and cycle outcome in IVF patients.

PURPOSE: This prospective study was designed to investigate whether anti-Müllerian hormone (AMH) levels at basal and ovulation triggering day are associated with ovarian response and pregnancy outcome for in vitro fertilization (IVF). METHOD: 60 infertility women undergoing IVF were prospectively studied. On day 3 of the menstrual cycle (D3), measurements of AMH, inhibin B, FSH, LH, and E2 and ultrasound evaluation of antral follicle count (AFC) were performed. Serum AMH and inhibin B levels were remeasured on the day of hCG administration (DhCG). The outcome measures were the number of retrieved oocytes and clinical pregnancy. RESULTS: Number of retrieved oocytes was statistically significant and correlated with D3 AMH, AFC, DhCG AMH, DhCG inhibin B, FSH, and age (r=0.885, 0.874, 0.742, 0.732, -0.521, -0.385, respectively). Statistically significant differences were found between pregnant and non-pregnant women regarding D3 AMH and AFC. Multiple regression analysis for prediction of pregnancy showed D3 AMH to be a good predictor of clinical pregnancy. CONCLUSION: AMH correlates better than age, FSH, and inhibin B with the number of retrieved oocytes. Serum basal AMH may offer a better prognostic value for clinical pregnancy than other currently available markers of IVF outcome in our preliminary study.

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells.

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.

Effects of ingested fruiting bodies, submerged culture biomass, and acidic polysaccharide glucuronoxylomannan of Tremella mesenterica Retz.:Fr. on glycemic responses in normal and diabetic rats.

Mushroom polysaccharides have been shown to regulate glucose metabolism. Using male Wistar rats injected with saline (normal rats), streptozotocin (STZ-NT rats), or streptozotocin plus nicotinamide (STZ+NT rats), we investigated the hypoglycemic activity of orally ingested fruiting bodies (FB), submerged culture biomass (CM), or the acidic polysaccharide glucuronoxylomannan (GXM) of Tremella mesenterica, an edible jelly mushroom. Our results demonstrated that FB ingestion significantly attenuated the elevated blood glucose levels in an oral glucose tolerance test (OGTT) in STZ-NT rats. However, in STZ+NT rats, FB, CM, and GXM ingestion significantly attenuated the increases in food and water intake, 2-h postprandial blood glucose concentrations, and blood glucose levels in OGTT. Moreover, FB and GXM ingestion significantly decreased serum concentration of fructosamine in STZ+NT rats. Our results indicated that T. mesenterica might be developed as a potential oral hypoglycemic agent or functional food for diabetic patients and for persons with high risk for diabetes mellitus.

The effects of Tremella aurantia on testosterone and corticosterone productions in normal and diabetic rats.

Tremella aurantia (TA) has been traditionally used as food and crude medicine in Chinese society. The polysaccharide isolated from the fruiting bodies of TA exhibits significant hypoglycemic activity in diabetic mouse models of insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). Diabetes will cause sexual dysfunction in patients. In the present study, we examined if the treatment of TA on IDDM and NIDDM rats will restore steroidogenesis and then the reproductive function. The fruiting bodies (FB), mycelium (TM) and polysaccharide (GX) of TA were fed to the IDDM and NIDDM rats, and testosterone and corticosterone levels in plasma, the weight of steroidogenic organs, and the expression of steroidogenic acute regulatory (StAR) protein and P450scc enzyme were determined. Plasma testosterone productions were significantly suppressed with the feeding of FB or TM in normal rat (p < 0.05). Testosterone productions were also significantly suppressed in IDDM diabetes rats (p <0.05), and FB or TM could not restore the inhibitory effects (p > 0.05). There was no significant difference of the testosterone production between normal and NIDDM rats (p > 0.05). In plasma corticosterone production, there were no differences among control, FB- or TM-fed normal rats (p > 0.05). Corticosterone levels were reduced in IDDM rats compared to control, and FB or TM could restore its level. Corticosterone levels were induced in NIDDM rats compared to control (p <0.05), but FB, TM or GX significantly brought the corticosterone back (p < 0.05) to the control levels. Considering steroidogenic organs, IDDM rats with or without TA treatments had heavier testis and adrenal glands, but not epididymis, than normal rats with or without TA treatments. There were no effects of TA on the weight of steroidogenic organs among normal and NIDDM rats. However, GX feeding in NIDDM rat had lesser testis weight compared to NIDDM rats. The expression of StAR protein and P450scc enzyme were not different among groups in IDDM and NIDDM rats. Plasma testosterone productions were suppressed in normal rats with the feeding of TA (FB and TM). IDDM rats did have lower testosterone, but not in NIDDM, and FB or TM could not restore the inhibitory effects. The induction of IDDM or NIDDM rats did affect steroidogenesis and steroidogenic organ weights, and the feeding of TA had different effects on steroidogenesis in different types of diabetic rats.