Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive and metabolic disorder characterized by high androgen levels. The aim of this study was to evaluate the effects of hyperandrogenism on the hypothalamus and subsequently on the food intake and obesity in females. METHODS: A dihydroxy testosterone (DHT)-induced rat model was established to recapitulate the hyperandrogenism features of PCOS patients. Body weight and food intake of the rats were recorded. The food intake of DHT-induced rats was restricted by pair feeding to exclude possible effects of weight gain on the hypothalamus. The expression levels of relevant proteins and mRNAs in the hypothalamus and primary hypothalamic neurons exposed to DHT were analyzed by Western blotting and RT-PCR, respectively. The leptin levels in the serum and cerebrospinal fluid (CSF) were measured, and leptin was injected via the intracerebroventricular (ICV) route to test the leptin sensitivity of the hypothalamus. RESULTS: The excessive prepuberty androgen levels in the DHT-induced rats markedly elevated food intake prior to weight gain. Consistent with this, the expression of neuropeptide Y and agouti-related peptide mRNAs was upregulated, which occurred prior to obesity and even with restricted food intake. In addition, the hypothalamic sensitivity to insulin and leptin was also impaired in the DHT-induced rats before obesity and with restricted food intake. DHT significantly reduced the leptin levels in the CSF, and ICV injection of leptin inhibited the DHT-induced increase in food intake. CONCLUSIONS: Androgen excess increased food intake in rats and promoted obesity by downregulating insulin and leptin signaling in the hypothalamus, most likely by suppressing leptin levels in the CSF.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

Aminopeptidase N expression in the endometrium could affect endometrial receptivity.

Aminopeptidase N (ANPEP) is a membrane-bound zinc-dependent peptidase. Although it is widely believed that ANPEP acts as an important angiogenesis regulatory factor, there are few studies about its function in the female reproductive system. In our previous research, we applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to analyze the influence of different controlled superovulation treatments for Assisted Reproductive Technology, In Vitro Fertilization and Embryo Transfer (IVF-ET)) patients from a global proteomic perspective to search for potential biomarkers associated with endometrium receptivity and embryo implantation. ANPEP is one of the proteins that demonstrated differential expression between different treatment groups and may be closely associated with endometrial receptivity. In this study, we assessed the expression of ANPEP in the endometrium of mice at different ages and found it to be highest in the mature period. We also detected ANPEP expression in the endometrium of IVF-ET patients in the proliferative, preimplantation and implantation stages, and the highest expression level of ANPEP was found in the last group. Human primary endometrial stromal cells were infected with an adenovirus expression vector containing the ANPEP gene and a green fluorescent protein (GFP) fusion protein; the mRNA levels of HOXA-10, LIF, and integrin ß3 were found to be increased. Therefore, we conclude that ANPEP could be involved in the regulation of endometrial receptivity.

Hypoxia-induced and HIF1α-VEGF-mediated tight junction dysfunction in choriocarcinoma cells: Implications for preeclampsia.

INTRODUCTION: Accumulated data indicate that placental hypoxia is implicated in the pathogenesis of preeclampsia (PE). Tight junction (TJ) is important structure that sustains normal placental barrier function, its dysregulation under hypoxia has been observed. This study was designed to explore hypoxia-induced TJ dysfunction in trophoblast cells and its possible involvement in PE pathophysiology. METHODS: Choriocarcinoma cells were grown in a monolayer and treated with cobalt chloride (CoCl2) to induce hypoxia. TJ architecture was assessed using transmission electron microscopy, and locations of TJ proteins were determined by immunofluorescence. TJ functions were assessed by transepithelial electrical resistance (TER) and increased cell paracellular permeability (CPP), and the expression of TJ-related proteins, HIF-1α and VEGF was measured. RESULTS: The TJ functions of trophoblast cells were significantly altered by hypoxia; TER decreased and CPP increased in a time- and concentration-dependent manner. Significant alterations in TJ protein expression and increases in HIF1α and VEGF expression were observed in hypoxic cells, and these effects were attenuated by pretreatment with YC-1. Moreover, corresponding changes in TJ protein expression were also detected in preeclamptic placentas. CONCLUSION: These data demonstrate that trophoblast cells undergo significant changes in TJ protein expression under hypoxic conditions and highlight the potential significance of the HIF1α-VEGF axis in the regulation of TJ structure and function in the preeclamptic placenta.

[Preimplantation genetic diagnosis for infertile males with autosomal dominant polycystic kidney disease].

OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic renal diseases, which may cause oligoasthenospermia and azoospermia and result in male infertility. This study aimed to analyze the outcomes of preimplantation genetic diagnosis (PGD) in male patients with ADPKD-induced infertility. METHODS: We retrospectively analyzed the clinical data on 7 male patients with ADPKD-induced infertility undergoing PGD from April 2015 to February 2017, including 6 cases of oligoasthenospermia and 1 case of obstructive azoospermia, all with the PKD1 gene heterozygous mutations. Following intracytoplasmic sperm injection (ICSI), we performed blastomere biopsy after 5 or 6 days of embryo culture and subjected the blastomeres to Sureplex whole-genome amplification, followed by haplotype linkage analysis, Sanger sequencing, array-based comparative genomic hybridization to assess the chromosomal ploidy of the unaffected embryos, and identification of the unaffected euploid embryos for transfer. RESULTS: One PGD cycle was completed for each of the 7 patients. Totally, 26 blastocysts were developed, of which 12 were unaffected and diploid. Clinical pregnancies were achieved in 6 cases following 7 cycles of frozen embryo transplantation, which included 5 live births and 1 spontaneous abortion. CONCLUSIONS: For males with ADPKD-induced infertility, PGD may contribute to high rates of clinical pregnancy and live birth and prevent ADPKD in the offspring as well. This finding is also meaningful for the ADPKD patients with normal fertility.

Abnormal CFTR Affects Glucagon Production by Islet α Cells in Cystic Fibrosis and Polycystic Ovarian Syndrome.

Glucagon, produced by islet α cells, functions to increase blood glucose. Abnormal glucose levels are often seen in cystic fibrosis (CF), a systematic disease caused by mutations of the CF transmembrane conductance regulator (CFTR), and in polycystic ovarian syndrome (PCOS), an endocrine disorder featured with hyperandrogenism affecting 5-10% women of reproductive age. Here, we explored the role of CFTR in glucagon production in α cells and its possible contribution to glucagon disturbance in CF and PCOS. We found elevated fasting glucagon levels in CFTR mutant (DF508) mice compared to the wildtypes. Glucagon and prohormone convertase 2 (PC2) were also upregulated in CFTR inhibitor-treated or DF508 islets, as compared to the controls or wildtypes, respectively. Dihydrotestosterone (DHT)-induced PCOS rats exhibited significantly lower fasting glucagon levels with higher CFTR expression in α cells compared to that of controls. Treatment of mouse islets or αTC1-9 cells with DHT enhanced CFTR expression and reduced the levels of glucagon and PC2. The inhibitory effect of DHT on glucagon production was blocked by CFTR inhibitors in mouse islets, and mimicked by overexpressing CFTR in αTC1-9 cells with reduced phosphorylation of the cAMP/Ca2+ response element binding protein (p-CREB), a key transcription factor for glucagon and PC2. These results revealed a previously undefined role of CFTR in suppressing glucagon production in α-cells, defects in which may contribute to glucose metabolic disorder seen in CF and PCOS.

Use of electroacupuncture and transcutaneous electrical acupoint stimulation in reproductive medicine: a group consensus.

With the rapid development of assisted reproductive technology, various reproductive disorders have been effectively addressed. Acupuncture-like therapies, including electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS), become more popular world-wide. Increasing evidence has demonstrated that EA and TEAS are effective in treating gynecological disorders, especially infertility. This present paper describes how to select acupoints for the treatment of infertility from the view of theories of traditional Chinese medicine and how to determine critical parameters of electric pulses of EA/TEAS based on results from animal and clinical studies. It summarizes the principles of clinical application of EA/TEAS in treating various kinds of reproductive disorders, such as polycystic ovary syndrome (PCOS), pain induced by oocyte retrieval, diminished ovarian reserve, embryo transfer, and oligospermia/ asthenospermia. The possible underlying mechanisms mediating the therapeutic effects of EA/TEAS in reproductive medicine are also examined.

Expression of the SET protein in testes of mice at different developmental stages.

SET is a multifunctional protein involved in regulating many biological processes of the cell cycle. It is also a regulator of steroidogenesis in the ovary. However, the expression of SET protein in testis, and its function, still remains ambiguous. In this study, we observed the expression of SET in the testes of mice at different developmental stages, and have discussed its potential function in regulating spermatogenesis and androgen production. Forty-eight male mice at different developmental stages (1 week old as the infancy group; 4 weeks old as the prepubertal group; 12 weeks old as the adult group; over 12 months old as the ageing group) were used. Cellular location of SET protein in the testes was observed by immuno-histochemistry. Expression levels of Set mRNA and SET protein were analyzed by quantitative polymerase chain reaction and Western blotting. SET protein was expressed in spermatogonial cells and spermatocytes; the highest level was mainly in haploid and tetraploid cells of the prepubertal and adult groups, and Leydig cells of the adult and ageing groups. There was a low expression in Sertoli cells. Expression of Set mRNA in the prepubertal group was significantly higher than that in the adult group (P < 0.05), while expression of SET protein was at the highest level in the adult group (P < 0.05). SET protein is mainly expressed in spermatogonial cells and spermatocytes, and poorly expressed in Sertoli cells, suggesting that it is involved in spermatogenesis. Expression of SET protein in Leydig cells suggests a possible role in steroidogenesis.

Insulin-like 3-induced rat preantral follicular growth is mediated by growth differentiation factor 9.

The communication of somatic cells and oocytes by intrafollicular paracrine factors is essential for follicular growth in the ovary. Insulin-like 3 (INSL3) is a theca cell-secreted paracrine factor. Androgens and growth differentiation factor 9 (GDF9), an oocyte-derived growth factor, are essential for follicular development. Using a rat preantral follicle culture model, we examined in the present study the influence of INSL3 on preantral follicular growth and the molecular mechanisms involved. We have observed that the receptor for INSL3, relaxin/insulin-like family peptide receptor 2 (RXFP2), was exclusively expressed in oocytes. Recombinant INSL3 stimulated Gdf9 expression, preantral follicular growth, and testosterone synthesis in vitro. Inhibition of the cAMP/protein kinase A signaling pathway (with cAMP antagonist, 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer) attenuated INSL3-induced Gdf9 expression and preantral follicular growth. Moreover, knocking down Gdf9 expression (with small interfering RNA) or inhibiting GDF9 signaling (with SB431542, an activin receptor-like kinase receptor 5 inhibitor, or specific inhibitor of mothers against decapentaplegic homolog 3) or androgen action (with flutamide, an androgen receptor antagonist) suppressed INSL3-induced preantral follicular growth. In addition, LH and DHT regulated the expression of Insl3 mRNA in preantral follicles. These observations suggest that INSL3 is a key theca cell-derived growth factor for preantral follicle and that its action is mediated by GDF9.

Effect of HSP10 on apoptosis induced by testosterone in cultured mouse ovarian granulosa cells.

OBJECTIVE: To investigate the effect of heat shock protein 10 (HSP10) on apoptosis induced by testosterone in granulosa cells (GCs) of mouse ovaries in order to define the possible roles of HSP10 in ovarian pathological development of polycystic ovarian syndrome (PCOS) and hyperandrogenic conditions. STUDY DESIGN: Cultured mouse ovarian GCs were treated with testosterone (10(-5) mol/l). Apoptosis was assessed using flow cytometry, and proliferation was assessed using the MTT assay. HSP10 expression in the treated GCs was detected by real-time polymerase chain reaction (PCR). HSP10 gene was downregulated in the cultured GCs by AdCMV-H1-SiRNA/HSP10 or overexpressed by AdCMV-HSP10. PD98059 [phosphorylated ERK (p-ERK) inhibitor] was used to treat GCs to induce a high apoptosis index. Critical apoptotic factors and proliferation factors, including P-ERK, Bcl-2, Bax, caspase 9, caspase 3 and Ki67, were monitored by real-time reverse transcriptase PCR (RT-PCR) and Western blot. RESULTS: Compared with the control group, the apoptosis index was higher (p<0.05) and HSP10 expression was lower (p<0.05) in the testosterone-treated groups. In the AdCMV-H1-SiRNA/HSP10-treated group, cell viability was decreased (p<0.05) and the cell cycle was arrested at G2. Expression of p-ERK, Bcl-2 and Ki67, and the Bcl-2:Bax ratio were lower, while expression of apoptotic factors, including Bax, caspase 9 and caspase 3, was higher (p<0.05). Compared with the control group, Bcl-2 expression in the GCs that overexpressed HSP10 was increased (p<0.05), while the reduction of p-ERK and Bcl-2 and the elevation of caspase 9 and caspase 3 induced by PD98059 were significantly suppressed (p<0.05). CONCLUSIONS: Hyperandrogenic conditions induced apoptosis of mouse GCs. Testosterone may have reduced HSP10 expression in GCs, leading to reduced Bcl-2 expression and increased Bax expression.

Downregulated expression of peroxiredoxin 4 in granulosa cells from polycystic ovary syndrome.

Peroxiredoxin 4 (PRDX4), a member of Peroxiredoxin (PRDX) family, is a typical 2-Cys PRDX. PRDX4 monitors the oxidative burden within cellular compartment and reduces hydrogen peroxide and alkyl hydroperoxide related to oxidative stress and apoptosis. Antioxidant, like PRDX4, may promote follicle development and participate in the pathophysiology of PCOS. In our previous study, we found that PRDX4 was expressed in mice oocyte cumulus oophorus complex, and that PRDX4 could be associated with follicle development. In this study, we explored the expression of PRDX4 in human follicles and possible role of PRDX4 in PCOS pathophysiology. Our data showed that PRDX4 was mainly expressed in granulosa cells in human ovaries. When compared to control group, both PRDX4 mRNA level and protein level decreased in PCOS group. The lowered levels of PRDX4 may relate to oxidative stress in the pathophysiologic progress of PCOS. Furthermore, expression of PRDX4 in the granulosa cells of in vivo or in vitro matured follicles was higher than that in immatured follicles, which suggested that PRDX4 may have a close relationship with follicular development. Altogether, our findings may provide new clues of the pathophysiologic mechanism of PCOS and potential therapeutic strategy using antioxidant, like PRDX4.

Chemerin suppresses ovarian follicular development and its potential involvement in follicular arrest in rats treated chronically with dihydrotestosterone.

In the present study, we have investigated the cellular mechanisms of androgen-induced antral follicular growth arrest and the possible involvement of chemerin and its receptor chemokine-like receptor 1 (CMKLR1) in this process, using a chronically androgenized rat model. We hypothesize that hyperandrogenism induces antral follicle growth arrest via the action of chemerin and ovarian structural changes, resulting from granulosa cell and oocyte apoptosis and theca cell survival. Dihydrotestosterone (DHT) treatment resulted in increased expression of chemerin and CMKLR1 in antral follicles, absence of corpus luteum, and increased atypical follicles. Addition of chemerin to follicle cultures induced granulosa cell apoptosis and suppressed basal, FSH- and growth differentiation factor-9-stimulated follicular growth. DHT down-regulated aromatase expression and increased active caspase-3 content and DNA fragmentation in granulosa cells in vivo. These changes were accompanied by higher phosphatase and tensin homolog and lower phospho-Akt (Ser473) content in antral follicles and higher calpain expression and down-regulation of cytoskeletal proteins in atypical follicles, which were constituted predominantly of theca cells. DHT also activated granulosa cell caspase-3, decreased X-linked inhibitor of apoptosis protein, poly(ADP-ribose) polymerase, and phospho-Akt contents and induced apoptosis in vitro, responses readily attenuated by forced X-linked inhibitor of apoptosis protein expression. These findings are consistent with our hypothesis that antral follicular growth arrest in DHT-treated rats results from increased chemerin expression and action, as well as changes in follicular cell fate and structure, which are a consequence of dysregulated interactions of pro-survival and pro-apoptotic modulators in a cell-specific manner. Our observations suggest that this chronically androgenized rat model may be useful for studies on the long-term effects of androgens on folliculogenesis and may have implications for the female reproductive disorders associated with hyperandrogenism.

SET/PP2A system regulates androgen production in ovarian follicles in vitro.

SET has multiple cell functions including nucleosome assembly, histone binding, transcription control, and cell apoptosis. In ovaries SET is predominantly expressed in theca cells and oocytes. In our study, SET overexpression in theca cells stimulated testosterone production whereas SET knockdown decreased testosterone production. Moreover, SET negatively regulated PP2A activity. Treatment with PP2A inhibitor okadaic acid (OA) led to increased testosterone synthesis, while treatment with PP2A activators resulted in the decreased testosterone synthesis. Furthermore, PP2A knockdown confirmed the key role of PP2A in the testosterone synthesis, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition of testosterone production. The central role of PP2A in SET-mediated regulation of testosterone production was confirmed by the finding that SET promoted the lyase activity of P450c17 and that PP2A inhibited its lyase activity. Taken together, these results reveal a specific, SET-initiated, PP2A-mediated, pathway that leads to the increased lyase activity of P450c17 and testosterone biosynthesis.

Promoter characterization and role of cAMP/PKA/CREB in the basal transcription of the mouse ORMDL3 gene.

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5' flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (-27/-20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.

[Study on the factors associated with clinical pregnancy rate of in-vitro fertilization in endometriosis related infertility].

OBJECTIVE: To evaluate the factors associated with clinical pregnancy rate of in-vitro fertilization (IVF) in endometriosis related infertility. METHODS: Total of 326 patients with endometriosis related infertility undergoing IVF between January 2007 and December 2011 were studied in Department of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, retrospectively, which were divided into 141 cases in clinical pregnancy group and 185 cases in non-pregnancy group. Those factors including age, body mass index (BMI), basic FSH, antral follicle count (AFC), CA125 and CA199, endometriotic stage and history of surgery, stimulation scheme were analyzed by bivariate analysis and multivariable logistic regression. RESULTS: (1) Pregnancy rate:total of 141 pregnant cases and 185 non-pregnant cases treated by IVF were observed, pregnancy rate was 43.2% (141/326). (2) Basic parameters: there was no statistical difference in age, BMI, basic FSH, AFC, CA125 and CA199 between clinical pregnancy group and non-pregnancy group (P > 0.05). (3) Bivariate analysis: clinical pregnancy rate of 50.0% (87/174) among patients with infertility year less than five years was significantly higher than 35.5% (54/152) in patients with more than five years. Pregnancy rate of 56.1% (46/82) in stage I-II was significantly higher than 42.5% (79/186) in stage III-IV. Pregnancy rate of 46.6% (125/268) with history of surgery was significantly higher than 27.6% (16/58) with no history of surgery (P < 0.05). Pregnancy rate of 48.2% (79/164) in long-term scheme was higher than 38.3% (62/162) in short-term scheme, but there was no significant difference (P = 0.075). (4) Multivariable logistic regression: clinical pregnancy rate of infertility year with less than 5 years, stage I-II, history of surgery proved stage I-II and stage III-IV was significantly higher compared with infertility year more than 5 years, stage III-IV and no history of surgery respectively (adjusted OR and 95%CI: 2.003, 1.263 - 3.175; 1.899, 1.110 - 3.248; 3.769, 1.802 - 7.887, P < 0.05). CONCLUSION: Factors affecting clinical pregnancy rate of IVF in endometriosis related infertility were infertility year, stage and surgery.

Chemerin, a novel regulator of follicular steroidogenesis and its potential involvement in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, minimal granulosa cell proliferation, dysregulated sex hormone profile, hyperthecosis, and insulin resistance. Using a 5α-dihydrotestosterone (DHT)-induced rat model that recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the steroidogenic capability of granulosa cells from DHT-treated rats. Gene expression of several key steroidogenic enzymes including p450 side-chain cleavage enzyme (p450scc), aromatase, steroidogenic acute regulatory protein, hydroxysteroid dehydrogenase-17ß, and hydroxysteroid dehydrogenase-3ß were markedly lower in DHT-treated rats than the controls, although the responsiveness of their granulosa cells to FSH was higher. Expression of the adipokine chemerin and its receptor, chemokine receptor-like 1, was evident in control and DHT-treated rats, with significantly higher ovarian mRNA abundances and protein contents of chemerin and its receptor. Recombinant chemerin decreases basal estradiol secretion in granulosa cells from DHT-treated rats. When the inhibitory role of chemerin on steroidogenesis was further examined in vitro, chemerin suppressed FSH-induced progesterone and estradiol secretion in cultured preantral follicles and granulosa cells. Chemerin also inhibits FSH-induced aromatase and p450scc expression in granulosa cells. Overexpression of nuclear receptors NR5a1 and NR5a2 promotes p450scc and aromatase expression, respectively, which is suppressed by chemerin. These findings suggest that chemerin is a novel negative regulator of FSH-induced follicular steroidogenesis and may contribute to the pathogenesis of PCOS.

[Protective effects of SUR2B/Kir6.1 potassium channel opener natakalim against RAVECs injuries induced by hypoxia].

OBJECTIVE: To investigate the protective effects of natakalim against rat aortic vascular endothelial cells (RAVECs) injuries induced by hypoxia and its mechanisms. METHODS: Selecting RAVECs as a cell model injured by hypoxia, these RAVECs were divided into 5 groups: i.e. control group, hypoxia group, natakalim low, medium and high group. The cell survival rate was determined by MTT assay, con was measured using Griess Assay, RT-PCR was used to examine t he expression of intercellular adhesion molecule-1 (ICAM-1), vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) mRNA in RAVEC. RESULTS: Natakalim could reverse hypoxia-induced changes in endothelial cell function, including increased endothelial cell survival rate and level of NO concentration, significantly inhibited the hypoxia-induced endothelial ICAM-1, ET-1, VEGF mRNA expression levels increased. CONCLUSION: Natakalim have protective effects on hypoxia-induced changes in endothelial cell function, increasing of permeation, excess expression of cell adhesion molecules.

Mechanisms elevating ORMDL3 expression in recurrent wheeze patients: role of Ets-1, p300 and CREB.

The first genetic factor identified for childhood asthma by genome-wide association study (GWAS) is the locus on chromosome 17q21, harboring the Orosomucoid 1-like 3 (ORMDL3) gene. ORMDL3 is implicated in facilitation of endoplasmic reticulum-mediated inflammatory responses, believed to underlie its association with asthma. In the present study, we demonstrated that mRNA expression of ORMDL3 is significantly increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects by real-time RT-PCR. To elucidate the molecular mechanisms involved in human ORMDL3 regulation, we cloned and characterized the promoter region of ORMDL3. Applying 5'-rapid amplification of cDNA end analysis (RACE), we revealed that ORMDL3 gene used multiple transcriptional start sites (TSSs). Using a series of 5' deletion promoter plasmids in luciferase reporter assays, we identified that the proximal minimal promoter of ORMDL3 was located within the region -84/+58 relative to the TSS. Mutational analysis, RNA interference experiments and sequential chromatin immunoprecipitation (ChIP) assay demonstrated that transcriptional activity of the ORMDL3 gene was cooperatively regulated by multiple transcription factors, including Ets-1, p300 and CREB. The expression levels of Ets-1, p300 and CREB were increased in the peripheral blood of recurrent wheeze patients compared with normal control subjects and showed a strong linear correlation with the expression of ORMDL3. Our findings indicate that Ets-1, p300 and CREB binding to the promoter region drive the ORMDL3 transcription.

Effect of estradiol on proliferation and differentiation of side population stem/progenitor cells from murine endometrium.

BACKGROUND: In our previous study, endometrium side population cells (SP cells) were isolated from postpartum murine uterus, and characterized by a heterogeneous population of stem/progenitor cells. In this study, we investigated the effect of estrogen on the proliferation and differentiation of SP cells. METHODS: SP and non-SP cells of postpartum murine endometrium were isolated by DNA dye Hoechst 33342. The expression of estrogen receptor 1 (ESR1) was measured by reverse transcription polymerase chain reaction (RT-PCR), Real-time PCR, Western blot, immunofluorescence and immunohistochemistry. The proliferation and differentiation of SP cells treated with different concentrations [10(-8) M-10(-6) M] of estradiol (E2) and E2+ ICI182780 (Faslodex, inhibitor of ESR1) were measured by 3-(4, 5-dimethylthiazoly1-2)-2,5-diphenyltetrazolium bromide(MTT) and clonogenic assays. RESULTS: (1) SP cells expressed ESR1 at a higher level than non-SP cells. (2) The level of E2 in the serum and the expression of ESR1 in the uterus of postpartum murine changed in the same manner with the ratio of SP cells to total uterus cells at a different postpartum time point. ESR1, as ABCG2 is also predominantly located in the stroma and the glandular epithelium of the uterus. (3) 10(-6) M E2 notably promoted the proliferation of SP cells after treatment for 24 h. This effect could be inhibited by ICI182780. E2 at the concentration of 10(-7) M or 10(-8) M was sent to impair the large cloning efficiency (CE) of SP cells. CONCLUSIONS: The effect of estrogen on the proliferation and differentiation of endometrium SP cells via ESR1 was observed and it was in a concentration dependent fashion. Clearly, more work is needed to understand the in vivo effect of E2 at the physiological concentration on the differentiation of SP cells.

Heat shock protein 10 regulated apoptosis of mouse ovarian granulosa cells.

OBJECTIVES: To study the roles of heat shock proteins10 (HSP10) in the regulation of mouse ovarian granulose cell (GC) apoptosis, and to further define the possible roles of HSP10 in the development of polycystic ovary syndrome (PCOS). METHODS: Mouse HSP10 small interfering RNA (siRNA) and recombinant adenoviruses overexpressing HSP10 were constructed and subsequently transfected into cultured mouse ovarian GCs. After an infection period of 48 h, the expression levels of the HSP10 gene in mouse GCs were confirmed by Western blot. The GCs were also assessed for apoptosis using flow cytometry and the TUNEL assay. Apoptosis of GCs overexpressing HSP10 was assessed by flow cytometry after cisplatin treatment. RESULTS: Compared with control group, the expression of HSP10 was decreased in mouse GCs infected with AdCMV-siRNA/HSP10, whereas mouse GCs infected with AdCMV-HSP10 showed increased HSP10 expression p < 0.05. Knock-down of HSP10 in mouse GCs significantly increased apoptosis (p < 0.05), whereas overexpression of HSP10 significantly suppressed apoptosis induced by cisplatin (p < 0.05). CONCLUSION: In the present primary study, we have successfully employed recombinant adenovirus technologies to modulate HSP10 gene expression in mouse GCs, and examined the effects on apoptosis. Our experiments have demonstrated that knock-down of HSP10 induces apoptosis of mouse ovarian GCs, whereas overexpression of HSP10 suppresses apoptosis. These findings suggested that HSP10 may play a role in the regulation of apoptosis of mouse ovarian GCs.