Maternal high-fat-diet exposure is associated with elevated blood pressure and sustained increased leptin levels through epigenetic memory in offspring.

Maternal metabolism dysregulation during pregnancy predisposes offspring to major diseases, including hypertension, in later life, but the mechanism involved remains to be fully elucidated. A high-fat-diet (HFD) pregnant rat model was used to investigate whether excessive intrauterine lipid exposure was associated with elevated blood pressure in offspring and increased levels of leptin, an important biomarker and mediator of vascular dysfunction and hypertension. We found that gestational hyperlipidemia predisposed offspring to blood pressure elevation and sustained increases in leptin levels with no difference in body weight in the rat model. Increased leptin expression and leptin promoter hypomethylation were found in adipose tissues of HFD-exposed offspring. The treatment of mesenchymal stem cells with free fatty acids during adipogenic differentiation resulted in increased leptin expression, accompanied by leptin promoter hypomethylation. In addition, we also followed up 121 children to evaluate the association between maternal triglyceride levels and offspring blood pressure. Consistent with the animal study results, we observed elevated serum leptin levels and blood pressure in the offspring born to women with gestational hypertriglyceridemia. Our findings provide new insights that maternal hyperlipidemia is associated with elevated blood pressure in offspring and is associated with increases in leptin levels through epigenetic memory.

LINC00673 exerts oncogenic function in cervical cancer by negatively regulating miR-126-5p expression and activates PTEN/PI3K/AKT signaling pathway.

BACKGROUND: Recent studies have indicated the crucial regulator roles of a long non-coding RNA (lncRNA) LINC00673 in cancer pathogenesis and development. However, the clinical significance and functional effects of LINC00673 in cervical cancer remains unknown. METHODS: LINC00673 mRNA expression in cervical cancer tissues was measured by quantitative Real-time PCR (qRT-PCR), and the association between LINC00673 expression and the overall survival (OS) time of patients was analyzed by Kaplan-Meier survival plot. Cell proliferation was assessed using CCK8 assay, Flow cytometry analysis and cell colony formation assay. The association between miR-126-5p and LINC00673 was clarified by Luciferase activity assay. Furthermore, xenografts model in mice in vivo were used to evaluate the effects of LINC00673 expression on tumor growth of cervical cancer. RESULTS: It was confirmed that the relative mRNA expression of LINC00673 was promoted in cervical cancer tissues and cancer cell lines compared with its corresponding normal tissues and cells (P < 0.05). Higher LINC00673 expression was associated with tumor size, lymph node metastasis, and International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.05). Survival analysis showed higher LINC00673 expression predicted poor OS of cervical cancer patients, and Multivariate Cox analysis demonstrated that higher LINC00673 expression was identified as an independent risk factor for OS. LINC00673 overexpression promoted cell proliferation and cell cycle progression, but LINC00673 knockdown inhibited cell proliferation and cell cycle progression significantly (P < 0.05). Besides, overexpression of LINC00673 was negatively correlated with lower miR-126-5p expression in cervical cancer tissues. In vivo xenograft tumor assay indicated that LINC00673 silencing reduced the tumor volume and weight. Bioinformatics analysis revealed that miR-126-5p targeted 3'-UTR of LINC00673, and LINC00673 promoted cell proliferation by sponging to miR-126-5p in cervical cancer cells. Additionally, it was demonstrated that LINC00673 significantly activated the PTEN/PI3K/AKT signaling pathway in cervical cancer cells. CONCLUSION: These results provide the evidence that LINC00673 overexpression promotes cervical cancer cells progression through regulating miR-126-5p and activating the PTEN/PI3K/AKT signaling pathway, indicating that LINC00673 may be a potential therapeutic target for cervical cancer treatment.

Oocyte exposure to supraphysiological estradiol during ovarian stimulation increased the risk of adverse perinatal outcomes after frozen-thawed embryo transfer: a retrospective cohort study.

Maternal supraphysiological estradiol (E2) environment during pregnancy leads to adverse perinatal outcomes. However, the influence of oocyte exposure to high E2 levels on perinatal outcomes remains unknown. Thus, a retrospective cohort study was conducted to explore the effect of high E2 level induced by controlled ovarian stimulation (COH) on further outcomes after frozen embryo transfer (FET). The study included all FET cycles (n = 10,581) between 2014 and 2017. All cycles were categorized into three groups according to the E2 level on the day of the human Chorionic Gonadotropin trigger. Odds ratios (ORs) and their confidence intervals (CIs) were calculated to evaluate the association between E2 level during COH and pregnancy outcomes and subsequent neonatal outcomes. From our findings, higher E2 level was associated with lower percentage of chemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth as well as increased frequency of early miscarriage. Preterm births were more common among singletons in women with higher E2 level during COH (aOR1 = 1.93, 95% CI: 1.22-3.06; aOR2 = 2.05, 95% CI: 1.33-3.06). Incidence of small for gestational age (SGA) was more common in both singletons (aOR1 = 2.01, 95% CI: 1.30-3.11; aOR2 = 2.51, 95% CI: 1.69-3.74) and multiples (aOR1 = 1.58, 95% CI: 1.03-2.45; aOR2 = 1.99, 95% CI: 1.05-3.84) among women with relatively higher E2 level. No association was found between high E2 level during COH and the percentage of macrosomia or large for gestational age. In summary, oocyte exposure to high E2 level during COH should be brought to our attention, since the pregnancy rate decreasing and the risk of preterm birth and SGA increasing following FET.

Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also 'rescued' the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

Proteomics of Uterosacral Ligament Connective Tissue from Women with and without Pelvic Organ Prolapse.

PURPOSE: Damage to the uterosacral ligaments is an important contributor to uterine and vaginal prolapse. The aim of this study is to identify differentially expressed proteins (DEPs) in the uterosacral ligaments of women with and without pelvic organ prolapse (POP) and analyze their relationships to cellular mechanisms involved in the pathogenesis of POP. EXPERIMENTAL DESIGN: Uterosacral ligament connective tissue from four patients with POP and four control women undergo iTRAQ analysis followed by ingenuity pathway analysis (IPA) of DEPs. DEPs are validated using Western blot analysis. RESULTS: A total of 1789 unique protein sequences are identified in the uterosacral ligament connective tissues. The expression levels of 88 proteins are significantly different between prolapse and control groups (≥1.2-fold, p < 0.05). IPA demonstrates the association of 14 DEPs with "Connective Tissue Function." Among them, fibromodulin, collagen alpha-1 (XIV) chain, calponin-1, tenascin, and galectin-1 appear most likely to play a role in the etiology of POP. CONCLUSIONS AND CLINICAL RELEVANCE: At least six proteins not previously associated with the pathogenesis of POP with biologic functions that suggest a plausible relationship to the disorder are identified. These results may be helpful for furthering the understanding of the pathophysiological mechanisms of POP.

DNA Methylation Inhibitor 5-Aza-2'-Deoxycytidine Modulates Endometrial Receptivity Through Upregulating HOXA10 Expression.

Endometrial receptivity is a critical factor for embryo implantation. A decrease in endometrial homeobox A10 (HOXA10) expression is associated with hypermethylation of its promoter and lower endometrial receptivity in animals and humans. 5-Aza-2'-deoxycytidine (AZA) is a DNA methyltransferase inhibitor. However, whether demethylation of the HOXA10 gene could increase the receptivity of the human endometrium remains unknown. Homeobox A10 promoter methylation was analyzed using bisulfite genomic sequencing polymerase chain reaction. Quantitative real time polymerase chain reaction and Western blotting were used to analyze the expression of HOXA10 and its downstream target genes (integrin subunit ß 3 [ITGB3] and insulin growth factor binding protein 1 [IGFBP1]) in Ishikawa cells treated with or without AZA for 24 hours. Their protein expression was analyzed with or without HOXA10 siRNA treatment. The effect of AZA on embryo implantation was examined using a Jeg-3 spheroid-endometrial cell attachment assay. The percentage of methylated CpG islands in the HOXA10 promoter was 72.0% without AZA treatment. However, it was 38% and 35% in the 1 and 10 µM AZA treatment groups, respectively. 5-Aza-2'-deoxycytidine strongly induced the expression of HOXA10, ITGB3, and IGFBP1 messenger RNA and their protein expression. Homeobox A10 knockdown led to decreased expression of HOXA10, ITGB3, and IGFBP1, with or without AZA treatment. The attachment rate of Jeg-3 spheroids increased significantly from 82% (control) to 95% (AZA 1 µM) and 96% (AZA 10 µM) after AZA treatment. 5-Aza-2'-deoxycytidine could upregulate the expression of ITGB3 and IGFBP1 via HOXA10 upregulation, and upregulation of ITGB3 and IGFBP1 plays an important role in endometrial receptivity during implantation. 5-Aza-2'-deoxycytidine may improve endometrial receptivity by upregulating the expression of HOXA10.

Effects of Levonorgestrel and progesterone on Oviductal physiology in mammals.

BACKGROUND: Our previous study indicated that emergency contraception, including levonorgestrel and progesterone, could lead to ectopic pregnancy following contraception failure. However, our understanding of the effects of levonorgestrel and progesterone on oviductal physiology is limited. METHODS: The receptivity of the fallopian tubal epithelium after levonorgestrel and progesterone treatment was examined through western blots for receptivity markers and JAr-spheroid-fallopian tubal epithelial cell attachment assays. The ciliary beat frequency was analyzed using an inverted bright-field microscope. Furthermore, an in vivo animal model of embryo-tubal transplantation was also studied to determine the effects of levonorgestrel- and progesterone-induced ciliary beat reduction. RESULTS: Our results showed that levonorgestrel and progesterone did not change the levels of fallopian tubal epithelial cell receptive markers, including LIF, STAT3, IGFBP1, ITGB3, MUC1, and ACVR1B, or affect JAr-spheroid implantation. However, levonorgestrel and progesterone reduced the ciliary beat frequency in fallopian tubes in a dose-dependent manner. An in vivo model also showed that levonorgestrel and progesterone could lead to embryo retention in the oviducts. CONCLUSIONS: These findings show that levonorgestrel and progesterone can reduce the ciliary beat frequency without altering receptivity, indicating a possible mechanism for progesterone- or levonorgestrel-induced tubal pregnancy.

Aberrant expression and DNA methylation of lipid metabolism genes in PCOS: a new insight into its pathogenesis.

Background: Polycystic ovary syndrome (PCOS), whose etiology remains uncertain, is a highly heterogenous and genetically complex endocrine disorder. The aim of this study was to identify differentially expressed genes (DEGs) in granulosa cells (GCs) from PCOS patients and make epigenetic insights into the pathogenesis of PCOS. Results: Included in this study were 110 women with PCOS and 119 women with normal ovulatory cycles undergoing in vitro fertilization acting as the control group. RNA-seq identified 92 DEGs unique to PCOS GCs in comparison with the control group. Bioinformatic analysis indicated that synthesis of lipids and steroids was activated in PCOS GCs. 5-Methylcytosine analysis demonstrated that there was an approximate 25% reduction in global DNA methylation of GCs in PCOS women (4.44 ± 0.65%) compared with the controls (6.07 ± 0.72%; P < 0.05). Using MassArray EpiTYPER quantitative DNA methylation analysis, we also found hypomethylation of several gene promoters related to lipid and steroid synthesis, which might result in the aberrant expression of these genes. Conclusions: Our results suggest that hypomethylated genes related to the synthesis of lipid and steroid may dysregulate expression of these genes and promote synthesis of steroid hormones including androgen, which could partially explain mechanisms of hyperandrogenism in PCOS.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients.

INTRODUCTION: Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). METHODS: To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. RESULTS: In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. DISCUSSION: This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.

Prevalence of Prediabetes Risk in Offspring Born to Mothers with Hyperandrogenism.

BACKGROUND: Excessive androgen exposure during pregnancy has been suggested to induce diabetic phenotypes in offspring in animal models. The aim of this study was to investigate whether pregestational maternal hyperandrogenism in human influenced the glucose metabolism in offspring via epigenetic memory from mother's oocyte to child's somatic cells. METHODS: Of 1782 reproductive-aged women detected pregestational serum androgen, 1406 were pregnant between 2005 and 2010. Of 1198 women who delivered, 1116 eligible mothers (147 with hyperandrogenism and 969 normal) were recruited. 1216 children (156 children born to mothers with hyperandrogenism and 1060 born to normal mother) were followed up their glycometabolism in mean age of 5years. Imprinting genes of oocyte from mothers and lymphocytes from children were examined. A pregestational hyperandrogenism rat model was also established. FINDINGS: Children born to women with hyperandrogenism showed increased serum fasting glucose and insulin levels, and were more prone to prediabetes (adjusted RR: 3.98 (95%CI 1.16-13.58)). Oocytes from women with hyperandrogenism showed increased insulin-like growth factor 2 (IGF2) expression. Lymphocytes from their children also showed increased IGF2 expression and decreased IGF2 methylation. Treatment of human oocytes with dihydrotestosterone upregulated IGF2 and downregulated DNMT3a levels. In rat, pregestational hyperandrogenism induced diabetic phenotypes and impaired insulin secretion in offspring. In consistent with the findings in human, hyperandrogenism also increased Igf2 expression and decreased DNMT3a in rat oocytes. Importantly, the same altered methylation signatures of Igf2 were identified in the offspring pancreatic islets. INTERPRETATION: Pregestational hyperandrogenism may predispose offspring to glucose metabolism disorder via epigenetic oocyte inheritance. Clinical trial registry no.: ChiCTR-OCC-14004537; www.chictr.org.

Altered DNA methylation in neonates born large-for-gestational-age is associated with cardiometabolic risk in children.

BACKGROUND: Infants being born Large-for-gestational-age (LGA) are prone to developing cardiometabolic disease. However, the underlying mechanisms remain unclear. RESULTS: Clinical investigation showed that children born LGA had significantly higher serum level of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-c), and insulin, ratio of TC/high-density lipoprotein-cholesterol (HDL-c) compared to children born appropriate for gestational age (AGA). Birth weight (BW) was positively correlated to TC, LDL-c, and the ratio of TC/HDL in serum. Genome-wide DNA methylation analyzed in umbilical cord blood of controls and macrosomia cases. We identified 3459 methylation variable positions (MVPs) achieving genome-wide significance (adjusted P-value < 0.05) with methylation differences of ≥ 5%. A total of 327 MVPs were filtered by methylation differences of ≥ 7% located within an island, which mapped to 213 genes. Function analysis using Ingenuity Pathway Analysis showed 16 genes enriched in "cardiovascular disease". Four genes included contributed to hyperlipidemia. MATERIALS AND METHODS: Fifty-eight children aged 3-6 years born LGA and 123 subjects born AGA were enrolled. Anthropometric parameters and blood pressure (BP) were measured, and metabolic assessment was performed in all subjects. Genome-wide DNA methylation in umbilical blood was assayed by the 450K BeadChip in six AGA and six macrosomia newborns. CONCLUSIONS: Our data indicate that excess birth weight may increase the risk of lipid dysfunction in children aged 3-6 years. It might through reprogramming a group of genes correlated to cardiovascular disease. The genes identified in this study might be potential biomarker for cardiometabolic disease.

The impaired myocardial ischemic tolerance in adult offspring of diabetic pregnancy is restored by maternal melatonin treatment.

Diabetic pregnancy, with ever increasing prevalence, adversely affects embryogenesis and increases vasculometabolic disorder risks in adult offspring. However, it remains poorly understood whether maternal diabetes increases the offspring's susceptibility to heart injuries in adulthood. In this study, we observed that cardiac function and structure were comparable between adult offspring born to diabetic mice and their counterparts born to nondiabetic mice at baseline. However, in response to myocardial ischemia/reperfusion (MIR), diabetic mother offspring exhibited augmented infarct size, cardiac dysfunction, and myocardial apoptosis compared with control, in association with exaggerated activation of mitochondria- and endoplasmic reticulum (ER) stress-mediated apoptosis pathways and oxidative stress. Molecular analysis showed that the impaired myocardial ischemic tolerance in diabetic mother offspring was mainly attributable to blunted cardiac insulin receptor substrate (IRS)-1/Akt signaling. Furthermore, the effect of maternal melatonin administration on offspring's response to MIR was determined, and the results indicated that melatonin treatment in diabetic dams during pregnancy significantly improved the tolerance to MIR injury in their offspring, via restoring cardiac IRS-1/Akt signaling. Taken together, these data suggest that maternal diabetes predisposes offspring to augmented MIR injury in adulthood, and maternal melatonin supplementation during diabetic pregnancy may hold promise for improving myocardial ischemic tolerance in the offspring.

Calcitriol attenuates cardiac remodeling and dysfunction in a murine model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex reproductive and metabolic disorder affecting 10 % of reproductive-aged women, and is well associated with an increased prevalence of cardiovascular risk factors. However, there are few data concerning the direct association of PCOS with cardiac pathologies. The present study aims to investigate the changes in cardiac structure, function, and cardiomyocyte survival in a PCOS model, and explore the possible effect of calcitriol administration on these changes. PCOS was induced in C57BL/6J female mice by chronic dihydrotestosterone administration, as evidenced by irregular estrous cycles, obesity and dyslipidemia. PCOS mice progressively developed cardiac abnormalities including cardiac hypertrophy, interstitial fibrosis, myocardial apoptosis, and cardiac dysfunction. Conversely, concomitant administration of calcitriol significantly attenuated cardiac remodeling and cardiomyocyte apoptosis, and improved cardiac function. Molecular analysis revealed that the beneficial effect of calcitriol was associated with normalized autophagy function by increasing phosphorylation levels of AMP-activated protein kinase and inhibiting phosphorylation levels of mammalian target of rapamycin complex. Our findings provide the first evidence for the presence of cardiac remodeling in a PCOS model, and vitamin D supplementation may be a potential therapeutic strategy for the prevention and treatment of PCOS-related cardiac remodeling.

Aquaporin7 plays a crucial role in tolerance to hyperosmotic stress and in the survival of oocytes during cryopreservation.

Hyperosmotic stress may induce apoptosis of different cells. However, oocytes show tolerance to osmotic stress during cryopreservation by vitrification, which is an assisted reproductive technique. The underlying mechanism is still not understood. Here, we demonstrated that hyperosmosis produced by high concentrations of cryoprotectants, including DMSO, ethylene glycol and sucrose, significantly upregulated the protein levels of aquaporin (AQP) 7, but not AQP3 and AQP9, in mouse oocytes. Knockdown of AQP7 expression by siRNA-injection significantly reduced the survival of oocytes after vitrification. In oocytes, AQP7 was shown to bind with F-actin, a protein involved in almost all biological events. Moreover, we found that hyperosmosis could upregulate the phosphorylation levels of CPE-binding protein (CPEB) and Aurora A. Inhibition of the PI3K and PKC pathways blocked the hyperosmosis-induced upregulation of AQP7 and the phosphorylation of CPEB and Aurora A in oocytes. In conclusion, hyperosmosis could upregulate the expression of AQP7 via Aurora A/CPEB phosphorylation mediated by the PI3K and PKC pathways, and upregulation of AQP7 plays an important role in improving of tolerance to hyperosmotic stress and survival of oocytes during cryopreservation by vitrification.

Differential proteomic analysis of umbilical artery tissue from preeclampsia patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS.

Epidemiological studies suggest that the impact of preeclampsia does not only affect the mother but also the children. We know that adverse events in utero may predispose individuals to premature cardiovascular disease in adulthood, but we do not know the mechanisms. To gain insights into the mechanisms of cardiovascular dysfunction in the offspring of preeclampsia, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2D-LC-MS/MS. We identified 1521 non-redundant proteins, and 1496 of these were quantified. Further analysis identified 53 differentially expressed proteins in umbilical artery; 22 proteins were up-regulated and 31 proteins were down-regulated. K-means clustering analysis showed that there was a specific protein expression profile in the umbilical artery which could distinguish between normal and preeclampsia patients. These 53 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the angiogenesis, vasculogenesis, and development of the cardiovascular system. In addition, the differential expression of three cardiovascular relative proteins (aldose reductase, fibronectin-1, fibrillin-1) was independently verified using western blot. These results may supply new insights into the mechanisms of vascular dysfunction in the offspring of preeclampsia patients. BIOLOGICAL SIGNIFICANCE: Increasing evidence suggests that the children who were exposed to preeclampsia in utero have an increased cardiovascular risk, and vascular dysfunction has been found in some children born of preeclampsia. However, the mechanism remains largely unknown. In this study, we identified 1521 non-redundant proteins, and 1496 of these were quantified. Further analysis identified 53 differentially expressed proteins in the umbilical artery from preeclampsia patients; 22 proteins were up-regulated and 31 proteins were down-regulated. Some of these differentially expressed proteins have been shown to play important roles in cardiovascular system development. Our results provide new insights into the potential mechanisms underlying the changed blood pressure of offspring of mothers with preeclampsia, and, the elevation of their risk of cardiovascular abnormality in later life.

Expression of aquaporins in human embryos and potential role of AQP3 and AQP7 in preimplantation mouse embryo development.

BACKGROUND/AIMS: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. METHODS: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. RESULTS: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. CONCLUSION: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

Aquaporin-1 plays a crucial role in estrogen-induced tubulogenesis of vascular endothelial cells.

CONTEXT: Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development. OBJECTIVE: Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells. SETTING: The study was conducted in a university hospital in eastern China. MAIN OUTCOME MEASURES: Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene. RESULTS: Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs. CONCLUSIONS: Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.

Cryoprotectants up-regulate expression of mouse oocyte AQP7, which facilitates water diffusion during cryopreservation.

OBJECTIVE: To investigate the effects of cryoprotectants on the expression of AQP7 in oocytes. DESIGN: Experimental animal study. SETTING: University-based research laboratory. ANIMAL(S): Adult female C57BL/6J mice. INTERVENTION(S): In metaphase II (MII) oocytes obtained from adult female C57BL/6J mice and from donations by fertile women, the mouse oocytes were treated with human tubal fluid medium containing 8% ethylene glycol (EG), 9.5% dimethylsulfoxide (DMSO), and 0.5 M sucrose, respectively; 293T cells transfected with GFP-hAQP7 expression vector were treated with the same solutions. MAIN OUTCOME MEASURE(S): AQP7 expression in oocytes examined by reverse-transcriptase-nested polymerase chain reaction and immunofluorescence, changes in the volume of mouse oocytes treated with different solutions calculated to determine their permeability to water, and survival rates of vitrified oocytes. RESULT(S): AQP7 is expressed in human and mouse oocytes. Cryoprotectants, including EG, DMSO, and sucrose, up-regulated AQP7 expression in mouse oocytes and 293T cells transfected with GFP-hAQP7 expression vector. Compared with other cryoprotectants, DMSO stimulated higher expression of AQP7, and this was associated with faster cell volume recovery and lower survival rates of vitrified oocytes. CONCLUSION(S): DMSO up-regulates AQP7 expression in mouse oocytes more than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance with the cryoprotectant solution during cryopreservation.

Functions of water channels in male and female reproductive systems.

Twelve water channels (aquaporins) are expressed in mammalian reproductive systems, and play very important roles in maintaining water homeostasis in reproductive cells. Impairment of their functions can result in attenuated male and female fertility. Alteration of AQPs expression is also found in reproductive tissues of the patients with polycystic ovarian syndrome, endometriosis or endometrium carcinoma. A lot of data have increased understanding of the functions and mechanisms of regulation of aquaporins at both the molecular and the clinical level. Researches have also focused on aquaporins as therapeutic targets. This review discusses recent advances in uncovering the physiological and pathophysiological roles of aquaporins in the reproductive systems.

Functional expression of large-conductance calcium-activated potassium channels in human endometrium: a novel mechanism involved in endometrial receptivity and embryo implantation.

BACKGROUND: Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. OBJECTIVE: The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. METHODS: BK(Ca) channel expression in human endometrium is described at different phases of the menstrual cycle using quantitative real time-PCR and Western blot techniques. Their effects on embryo implantation were examined using JAr spheroid attachment assays and in vivo mouse model. We examined their effects on endometrial receptivity factors, nuclear factor-κB (NF-κB) activity using quantitative real time-PCR, Western blot, and EMSA analyses. Changes in electrophysiological properties and cytosolic free Ca(2+) were measured in endometrial cells with or without specific BK(Ca) blocker or transfected with BK(Ca) small interfering RNA using patch-clamp and fluorescence analyses, respectively. RESULTS: BK(Ca) channels are expressed in human endometrial cells in a phase-related fashion during the menstrual cycle (proliferative, 0.20 ± 0.02, vs. mid-secretory, 0.72 ± 0.07; P < 0.01). Blocking BK(Ca) channel function or knockdown of endogenous BK(Ca) channel expression not only decreased JAr spheroid attachment rate and embryo implantation rate in mice but also significantly reduced the expression levels of endometrial receptive factors, including leukemia inhibitory factor, integrin ß3, claudin-4, and DKK-1, in human endometrial cells. Blocking BK(Ca) channels also reduced BK(Ca)-regulated NF-κB activity, cytosolic Ca(2+) concentrations, and membrane potentials in human endometrial cells. CONCLUSIONS: These observations demonstrate that BK(Ca) channels: 1) are expressed in endometrial cells; 2) affect embryo implantation by mediating endometrial receptive factors; and 3) alter the activity of NF-κB and homeostasis of Ca(2+) in the human endometrial cells.