Effects of Electro-Acupuncture on Ovarian P450arom,P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg⁻¹ of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS.     

Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries

The aim of the present study was to investigate the localization/immunohistochemical distribution of AGEs and RAGE, as well as their putative signalling mediator NF-κB in ovaries of women with polycystic ovary syndrome (PCOS) compared to normal. Archival ovarian-tissue samples from biopsies of six women with PCOS and from six healthy of similar age women, were examined immunohistochemically with monoclonal anti-AGEs, anti-RAGE and anti-NF-κB(p50/p65) specific antibodies. In healthy women, AGE immunoreactivity was observed in follicular cell layers (granulosa and theca) and luteinized cells, but not in endothelial cells. PCOS specimens displayed AGE immunoexpression in theca interna and granulosa cells as well as in endothelial cells, but staining of granulosa cells was stronger than in that of normal ovaries. RAGE was highly expressed in normal and PCOS tissues. Normal tissue exhibited no staining differences between granulosa cell layer and theca interna. However, in PCOS ovaries, granulosa cells displayed stronger RAGE expression compared to theca interna cells in comparison to controls. NF-κB(p50/p65) was expressed in the cytoplasm of theca interna and granulosa cells of both normal and PCOS ovaries; whereas the NF-κB p65 subunit was only observed in granulosa cells nuclei in PCOS tissue. In conclusion, these findings demonstrate for the first time that RAGE and AGE-modified proteins with activated NF-κB are expressed in human ovarian tissue. Furthermore, a differential qualitative distribution of AGE, RAGE and NF-κB p65 subunit was observed in women with PCOS compared to healthy controls, where a stronger localization of both AGE and RAGE was observed in the granulosa cell layer of PCOS ovaries.     

Systemic Gene Delivery Protects the Photoreceptors in the Retinal Degeneration Slow Mouse

The retinal degeneration slow (rds/rds) mouse was used to test photoreceptor protection by systemic gene delivery of non-erythropoietic forms of erythropoietin (EPO). Two Epo mutants were generated and packaged into recombinant adeno-associated virus (rAAV) serotype 2/5, controls included rAAV2/5.Epo and rAAV2/5.enhanced green fluorescent protein (eGFP). Mice were injected in the quadriceps at postnatal day seven and analyses were performed at postnatal day 90. Hematocrit, serum EPO levels, and outer nuclear layer (ONL) thickness were quantified. Hematocrit and serum EPO levels in rAAV2/5.eGFP, rAAV2/5.Epo, and rAAV2/5.EpoR103E treated mice were: 46%, 8 mU/ml; 63%, 117 mU/ml; and 52%, 332 mU/ml, respectively. The ONL from rds/rds mice treated with the Epo vectors were approximately twice as thick as the negative controls. This demonstrates that the photoreceptors can be protected without performing an intraocular injection and without increasing the hematocrit to unsafe levels. Intramuscular delivery of rAAV.EpoR103E is an attractive treatment for retinal degenerative diseases.     

Alterations in mitogen-activated protein kinase kinase and extracellular regulated kinase signaling in theca cells contribute to excessive androgen production in polycystic ovary syndrome

We have investigated the involvement of the MAPK signaling pathway in increased androgen biosynthesis and CYP17 gene expression in women with polycystic ovary syndrome (PCOS). A comparison of MAPK kinase (MEK1/2) and ERK1/2 phosphorylation in propagated normal and PCOS theca cells, revealed that MEK1/2 phosphorylation was decreased more than 70%, and ERK1/2 phosphorylation was reduced 50% in PCOS cells as compared with normal cells. Infection with dominant-negative MEK1 increased CYP17 mRNA and dehydroepiandrosterone (DHEA) abundance, whereas constitutively active MEK1 reduced DHEA production and CYP17 mRNA abundance. Similarly, the MEK inhibitor, PD98059, increased CYP17 mRNA accumulation and CYP17 promoter activity to levels observed in PCOS cells. Remarkably, in theca cells maintained in the complete absence of insulin, ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, and CYP17 mRNA and DHEA synthesis were increased in PCOS theca cells. These studies demonstrate that in PCOS cells reduced levels of activated MEK1/2 and ERK1/2 are correlated with increased androgen production, irrespective of the insulin concentration. These findings implicate alterations in the MAPK pathway in the pathogenesis of excessive ovarian androgen production in PCOS.     

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.     

Increased cytochrome P450 17alpha-hydroxylase promoter function in theca cells isolated from patients with polycystic ovary syndrome involves nuclear factor-1

Cytochrome P450 17alpha-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells.     

Improvement of the folliculogenesis by transplantation of bone marrow mesenchymal stromal cells in mice with induced polycystic ovary syndrome

Background

Many studies have reported that inflammation and oxidative stress are involved in the pathogenesis of polycystic ovary syndrome (PCOS). Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-oxidant and anti-inflammation properties. In this study, we investigate the beneficial effect of stem cell therapy on folliculogenesis in mice with induced PCOS

Evaluation of Follicular Synchronization Caused by Estrogen Administration and Its Reproductive Outcome

To evaluate multiple follicular development synchronization after estrogen stimulation in prepubertal mice, follicular responsiveness to gonadotropin superovulation, the prospective reproductive potential and ovarian polycystic ovary syndrome (PCOS)-like symptoms at adulthood, prepubertal mice were intraperitoneally injected with estrogen to establish an animal model with solvent as control. When synchronized tertiary follicles in ovaries, in vitro oocyte maturation and fertilization rates, blastocyst formation rate, developmental potential into offspring by embryo transfer, adult fertility and PCOS-like symptoms, and involved molecular mechanisms were focused, it was found that estrogen stimulation (10μg/gBW) leads to follicular development synchronization at the early tertiary stage in prepubertal mice; reproduction from oocytes to offspring could be realized by means of the artificial reproductive technology though the model mice lost their natural fertility when they were reared to adulthood; and typical symptoms of PCOS, except changes in inflammatory pathways, were not remained up to adulthood. So in conclusion, estrogen can lead to synchronization in follicular development in prepubertal mice, but does not affect reproductive outcome of oocytes, and no typical symptoms of PCOS remained at adulthood despite changes related to inflammation.     

Moose density and habitat productivity affects reproduction,growth and species composition in field layer vegetation

Question: What is the effect of a gradient in moose density on reproduction, growth and functional composition of the field layer vegetation in a boreal forest, and how is this effect modified by habitat productivity? Location: Northwest of Umeå, Västerbotten, northern Sweden. Methods: Field layer vegetation was surveyed in an experimental setup with simulation of three different moose densities and a control in eight study sites along a gradient of habitat productivity. Results: We found that increased moose density led to decreased cover and reproductive effort of a browsed dwarf shrub (bilberry, Vaccinium myrtillus L.) and increased cover and reproductive effort of a non‐browsed graminoid (wavy hair‐grass, Avenella flexuosa (L.) Drejer). Increased moose density led to increased light availability and probably reduced competition from V. myrtillus. Total reproductive effort in the field layer vegetation increased, height decreased and cover of light‐demanding species and graminoids increased with increasing moose density. The effects of moose density were modified by the productivity gradient, leading to a higher relative increase in light availability and reproductive effort in highly productive areas than in low productive areas. Conclusions: Increased light availability was an important indirect effect of moose density, leading to less competition for light and a shift towards early successional species. The effect of moose density on light availability was modified by habitat productivity, leading to stronger relative effects in highly productive areas than in low productive areas.     

Morphological changes in the oviductal endometrium during the reproductive cycle of the tortoise,Gopherus polyphemus

The oviducts of 24 tortoises (Gopherus polyphemus) were examined using histological techniques and scanning electron microscopy to determine endometrial morphology. Measurements of endometrial characteristics (epithelial cell height, cilia length, thickness of endometrial glandular layer, and glandular diameter) in the uterus and tube (tuba uterina) were obtained to determine changes during the reproductive cycle. Epithelial cell height increases in both the uterus and the tube during vitellogenesis and remains hypertrophied during gravidity. Cilia length increases in the uterus during late vitellogenesis and gravidity, but the length of tubal cilia does not change during the reproductive cycle. The ratio of secretory to ciliated epithelial cells in the oviduct increases from quiescence to gravidity. The thickness of the glandular endometrial layer increases in both the uterus and tube during vitellogenesis. In the uterus, the glandular layer decreases in thickness during gravidity. The diameter of the uterine glands increases throughout vitellogenesis and gravidity; however, following ovulation glandular cells become depleted of secretory granules and cell height diminishes. The diameter of the tubal glands is unchanged during the reproductive cycle. Oviductal hypertrophy during vitellogenesis coincides with elevated circulating estradiol, whereas during gravidity progesterone concentrations peak (Taylor, '82, PhD Dissertation, University of Florida, Gainesville) and may induce secretion of albumen and eggshell components.     

DAY-NIGHT VARIATION IN THE IN VITRO CONTRACTILITY OF AORTA AND MESENTERIC AND RENAL ARTERIES IN TRANSGENIC HYPERTENSIVE RATS*

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Adiponectin and Its Receptors in the Ovary: Further Evidence for a Link between Obesity and Hyperandrogenism in Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS), characterized by ovarian androgen excess, is the commonest endocrine disorder in women. Obesity increases androgen synthesis, a phenomenon attributed to the accompanying hyperinsulinemia. Our hypothesis was that adipokines, fat cell-derived hormones, play a direct role in modulating ovarian androgen secretion. Therefore, the aims of this study were to explore the effects of adipokines (in particular, adiponectin) on ovarian steroidogenesis and compare the expression of adiponectin receptors in ovaries from women with and without PCO. Sections of archived human ovaries (nine from women with normal ovaries and 16 with PCOS, classified histologically, with reference to menstrual history and ultrasound) were analysed by quantitative morphometry and the proportion of positive-labelling cells compared. In addition, studies of androgen production in relation to adipokine function in primary bovine theca cell culture were also performed. A significantly lower proportion of theca cells expressed adiponectin receptors 1 and 2 (AdipoR1, AdipoR2) in polycystic ovaries than in normal ovaries. In cultured theca cells, adiponectin suppressed androstenedione production and gene expression of LH receptor and key enzymes in the androgen synthesis pathway. Moreover, knockdown of genes for AdipoR1 and AdipoR2 was associated with increased androstenedione secretion by bovine theca cells. These results provide evidence for a direct link between fat cell metabolism and ovarian steroidogenesis, suggesting that disruption of adiponectin and/or its receptors plays a key role in pathogenesis of hyperandrogenism in PCOS.     

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.     

Reproductive toxicity in male mice caused by organic extracts in tap water from the Jialing River in Chongqing, China

BACKGROUND : Recently, male reproductive disturbances caused by organic pollutants have aroused particular public concern about the safety of drinking water. The aim of this study was to investigate the toxicity of organic extracts (OE) in tap water from the source of the Jialing River in China on the reproductive system of male mice. METHODS : Kunming male mice were randomly divided into four groups, which included a solvent control (dimethylsulfoxide), a low‐, mid‐, and high‐dose of OE (12.5, 25, and 50 l/kg bw/day, respectively) treated groups. Mice were administered intraperitoneal injections of OE at different doses for five consecutive days. On the 15th day, after treatments, the mice were sacrificed. RESULTS : The results showed that the number of epididymal sperm in the high OE group was decreased significantly (p<0.05); however, the frequency of sperm abnormalities in all treated groups were increased significantly (p<0.05). In addition, serum testosterone and follicle‐stimulating hormone levels in the treated groups were also decreased significantly (p<0.05), and mid‐ and high‐doses of OE resulted in a significant decrease in the activity of acid phosphatase and increased activity of γ‐glutamyl transpeptidase (p<0.05). Histological changes were observed in the mid‐ and high‐dose OE‐treated groups. CONCLUSIONS : The findings of this study suggest that mid and high doses of OE could disturb the male reproductive system in mice. The potential adverse effects of these compounds on the male reproductive system are worthy of further study. Birth Defects Res (Part B) 92:1–9, 2010. © 2011 Wiley‐Liss, Inc.     

Relationships of plasma C-peptide and gender to the urinary excretion of inositols in older people.

PURPOSE: The urinary excretions of myo-inositol and D-chiro-inositol are elevated in diabetes, and have been suggested as possible markers or effectors of insulin action. The aim of the present study was to measure the urinary excretion of these compounds, and to assess possible relationships with the metabolic control of glucose, in older, non-diabetic men and women. SUBJECTS: 32 older (age range 54-71 yrs), moderately overweight (body mass index 29.1 +/- 0.4 kg/m2, mean +/- SEM), non-diabetic men (n = 17) and women (n = 15). METHODS: 75 g oral glucose tolerance testing was done the day after all subjects had consumed nutrient-defined menus for five days. Plasma samples were analyzed for the concentrations of glucose, insulin, and C-peptide, and the 180-minute area under the curve (AUC) for each of these compounds was calculated. Samples from 24-hour urine collections were analyzed for the concentrations of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. RESULTS: The fasting glucose, insulin, and C-peptide, and the AUC for glucose and insulin, were not different between men and women. C-peptide AUC was greater in the men versus the women (p < 0.001). The median urinary excretions (micromol/g creatinine) of myo-inositol (p < 0.001), D-chiro-inositol (p < 0.001), L-chiro-inositol (p < 0.05), and pinitol (p < 0.001) were higher, and the myo-inositol:D-chiro-inositol ratio was lower (p < 0.001), in the men versus women. For all subjects combined, C-peptide AUC was positively correlated with the urinary excretion of each of the measured inositols, as well as the myo-inositol:D-chiro-inositol ratio. The correlations between C-peptide AUC and these inositols were strongly influenced by the co-linear relationship between C-peptide AUC and gender. CONCLUSIONS: Collectively, these data show that older, moderately overweight, non-diabetic men and women with gender-related differences in glucose-stimulated C-peptide AUC, an indirect indicator of insulin secretion, also display differences in the urinary excretion of myo-inositol, D-chiro-inositol, L-chiro-inositol, and pinitol. The gender-related difference in the myo-inositol:D-chiro-inositol ratio suggests that, while the urinary excretion of all of the inositols measured were higher in the men than the women, the difference was more pronounced for D-chiro-inositol.