Guo-Lian Ding

Zhejiang University, Hangzhou, Zhejiang Sheng, China

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Publications (15)74.59 Total impact

  • Guo-Lian Ding, He-Feng Huang
    Asian Journal of Andrology 06/2013; · 2.14 Impact Factor
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    ABSTRACT: 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.
    The Journal of clinical endocrinology and metabolism 02/2013; · 6.50 Impact Factor
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    ABSTRACT: 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.
    Fertility and sterility 01/2013; · 3.97 Impact Factor
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    ABSTRACT: Background: Low expression levels of S100A11 proteins were demonstrated in the placental villous tissue of patients with early pregnancy loss, and S100A11 is a Ca(2+)-binding protein that interprets the calcium fluctuations and elicits various cellular responses. Objectives: The objective of the study was to determine S100A11 expression in human endometrium and its roles in endometrial receptivity and embryo implantation. Methods: S100A11 expression in human endometrium was analyzed using quantitative RT-PCR, Western blot, and immunohistochemical techniques. The effects of S100A11 on embryo implantation were examined using in vivo mouse model, and JAr (a human choriocarcinoma cell line) spheroid attachment assays. The effects of endometrial S100A11 on factors related to endometrial receptivity and immune responses were examined. Using a fluorescence method, we examined the changes in cytosolic Ca(2+) and Ca(2+) release from intracellular stores in epidermal growth factor (EGF)-treated endometrial cells transfected with or without S100A11 small interfering RNA. Results: S100A11 was expressed in human endometrium. S100A11 protein levels were significantly lower in endometrium of women with failed pregnancy than that in women with successful pregnancy outcomes. The knockdown of endometrial S100A11 not only reduced embryo implantation rate in mouse but also had adverse effects on the expression of factors related to endometrial receptivity and immune responses in human endometrial cells. Immunofluorescence analysis showed that S100A11 proteins were mainly localized in endoplasmic reticulum. The EGF up-regulated endometrial S100A11 expression and promoted the Ca(2+) uptake and release from Ca(2+) stores, which was inhibited by the knockdown of S100A11. Conclusions: Endometrial S100A11 is a crucial intermediator in EGF-stimulated embryo adhesion, endometrium receptivity, and immunotolerance via affecting Ca(2+) uptake and release from intracellular Ca(2+) stores. Down-regulation of S100A11 may cause reproductive failure.
    The Journal of clinical endocrinology and metabolism 08/2012; 97(10):3672-83. · 6.50 Impact Factor
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    ABSTRACT: Gestational diabetes mellitus (GDM) has been shown to be associated with high risk of diabetes in offspring. However, the mechanisms involved and the possibilities of transgenerational transmission are still unclear. We intercrossed male and female adult control and first-generation offspring of GDM (F1-GDM) mice to obtain the second-generation (F2) offspring in four groups: C♂-C♀, C♂-GDM♀, GDM♂-C♀, and GDM♂-GDM♀. We found that birth weight significantly increased in F2 offspring through the paternal line with impaired glucose tolerance (IGT). Regardless of birth from F1-GDM with or without IGT, high risk of IGT appeared as early as 3 weeks in F2 offspring and progressed through both parental lineages, especial the paternal line. IGT in male offspring was more obvious than that in females, with parental characteristics and sex-specific transmission. In both F1 and F2 offspring of GDM, the expression of imprinted genes Igf2 and H19 was downregulated in pancreatic islets, caused by abnormal methylation status of the differentially methylated region, which may be one of the mechanisms for impaired islet ultrastructure and function. Furthermore, altered Igf2 and H19 gene expression was found in sperm of adult F1-GDM, regardless of the presence of IGT, indicating that changes of epigenetics in germ cells contributed to transgenerational transmission.
    Diabetes 03/2012; 61(5):1133-42. · 7.90 Impact Factor
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    ABSTRACT: Early pregnancy loss (EPL) is a frustrating clinical problem, whose mechanisms are not completely understood. DNA methylation, which includes maintenance methylation and de novo methylation directed by DNA methyltransferases (DNMTs), is important for embryo development. Abnormal function of these DNMTs may have serious consequences for embryonic development. To evaluate the possible involvement of DNA methylation in human EPL, the expression of DNMT proteins and global methylation of DNA were assessed in villous or decidua from EPL patients. The association of maintenance methylation with embryo implantation and development was also examined. We found that DNMT1 and DNMT3A were both expressed in normal human villous and decidua. DNMT1 expression and DNA global methylation levels were significantly down-regulated in villous of EPL. DNMT3A expression was not significantly changed in the EPL group compared to controls in either villous or decidua. We also found that disturbance of maintenance methylation with a DNMT1 inhibitor may result in a decreased global DNA methylation level and impaired embryonic development in the mouse model, and inhibit in vitro embryo attachment to endometrial cells. Our results demonstrate that defects in DNA maintenance methylation in the embryo, not in the mother, are associated with abnormal embryonic implantation and development. The findings of the current study provide new insights into the etiology of EPL.
    BMC Medicine 03/2012; 10:26. · 6.68 Impact Factor
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    ABSTRACT: The present study was designed to investigate the expression of small-conductance calcium-activated K(+) channels 3 (SK3) in preimplantation embryos and to explore their role in the underlying mechanism of blastocyst hatching. Human preimplantation embryos were donated by patients who achieved successful pregnancy with in vitro fertilization. Mouse preimplantation embryos in different stages were collected and cultured with or without siRNA cell injection. The expression of SK3 was examined by RT-PCR, quantitative real-time PCR, western blot and immunofluorescence. Functional expression of SK3 was investigated using the patch-clamp technique. [Ca(2+)]i was measured by fluorescent imaging. Embryos were cultured in vitro to investigate the effect of SK3 knockdown or apamin, an SK3 inhibitor, on blastocyst hatching and F-actin formation. In human blastocysts, the level of SK3 expression was significantly lower in blastocysts that failed to hatch than in blastocysts that hatched successfully. In mouse embryos, SK3 mRNA and protein were not found in zygotes, but were detected from the 2-cell stage onward, with the highest levels observed in blastocysts. SK3 was predominately located in the trophectoderm cell membrane of expanded blastocysts. SK3 knockdown in trophectoderm cells not only suppressed the SK3 current, but also reduced [Ca(2+)]i elevation and membrane potential hyperpolarization induced by thapsigargin. Although the formation of expanded blastocysts was not affected, blastocyst hatching and F-actin formation were significantly inhibited after SK3 knockdown in trophectoderm cells. SK3-mediated [Ca(2+)]i elevation and membrane potential hyperpolarization in trophectoderm cells are important for blastocyst hatching, and defects in SK3 expression may contribute to infertility.
    Human Reproduction 03/2012; 27(5):1421-30. · 4.67 Impact Factor
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    ABSTRACT: The objective of this study was to explore whether hyperandrogenism induces epigenetic alterations of peroxisome proliferator-activated receptor gamma 1 (PPARG1), nuclear corepressor 1 (NCOR1), and histone deacetylase 3 (HDAC3) genes in granulosa cells (GCs) of polycystic ovary syndrome (PCOS) women and whether these alterations are involved in the ovarian dysfunction induced by hyperandrogenism. Thirty-two infertile PCOS women and 147 infertile women with tubal blockage were recruited. PCOS women were divided into the hyperandrogenism (HA) PCOS group (n = 13) and nonhyperandrogenism (N-HA) PCOS group (n = 19). Sixty female Sprague-Dawley rats were used for PCOS model establishment. In GCs of HA PCOS women, PPARG1 mRNA expression was lower, whereas NCOR1 and HDAC3 mRNA expression were higher than N-HA PCOS women and controls (P < 0.05). When all women were divided into successful and failed pregnancy subgroups according to the following clinical pregnancy outcome, we found lower PPARG1 mRNA levels and higher NCOR1 and HDAC3 mRNA levels in the failed subgroup of HA PCOS (P < 0.05). Two hypermethylated CpG sites in the PPARG1 promoter and five hypomethylated CpG sites in the NCOR1 promoter were observed only in HA PCOS women (P < 0.01 to P < 0.0005). The acetylation levels of histone H3 at lysine 9 and p21 mRNA expression were decreased in human GCs treated with dihydrotestosterone in vitro (P < 0.05). PCOS rat models also showed alterations of PPARG1, NCOR1, and HDAC3 mRNA expression and methylation changes of PPARG1 and NCOR1, consistent with the results from humans. Hyperandrogenism induces the epigenetic alterations of PPARG1, NCOR1, and HDAC3 in GCs, which are involved in the ovarian dysfunction of HA PCOS.
    Journal of Molecular Medicine 02/2012; 90(8):911-23. · 4.77 Impact Factor
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    ABSTRACT: Large-conductance calcium-activated potassium channels (BK(Ca) channels) mediate physiological processes in nonexcitable cells. The aim of the study was to determine BK(Ca) channel expression in human endometrium and its role in endometrial receptivity and embryo implantation. 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. 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 β3, 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. 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.
    The Journal of clinical endocrinology and metabolism 12/2011; 97(2):543-53. · 6.50 Impact Factor
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    ABSTRACT: Accumulating evidence suggests that aquaporins (AQP) can facilitate cell migration, invasion, and proliferation in tumor development in addition to water transport. The aim of this study was to examine AQP2 expression in the endometrial tissues from patients with endometrial carcinoma (EC) and determine the roles and mechanisms of AQP2 in estrogen-related cell migration, invasion, adhesion, and proliferation of Ishikawa (IK) cells. AQP2 expression levels were measured in human endometrial cells and estradiol (E(2))-treated IK cells, and the estrogen-response element was identified. After blocking down and up-regulating the endogenous expression of AQP2 in IK cells, cell morphology, capacity for invasion, migration and adhesion, and expression markers of membrane/cytoskeleton were analyzed. AQP2 was expressed in endometrial tissues from patients with EC and endometriosis, both of which are estrogen-dependent diseases. In IK cells, E(2) dose-dependently increased AQP2 expression, which was blocked by the estrogen receptor inhibitor ICI182780. An estrogen-response element was identified in the AQP2 promoter. E(2) significantly increased the migration, invasion, adhesion, and proliferation of IK cells. AQP2 knockdown attenuated E(2)-enhanced migration, invasion, and adhesion. AQP2 knockdown reduced not only the E(2)-enhanced expression of F-actin and annexin-2 but also the E(2)-induced alteration of cell morphology. Moreover, higher expression levels of F-actin and annexin-2 were detected in the endometrial tissues from patients with EC. AQP2 mediates E(2)-enhanced migration, invasion, and adhesion through alteration of F-actin and annexin-2 expression and reorganization of F-actin, and inhibition of AQP may be a potential method for antitumor therapy.
    The Journal of clinical endocrinology and metabolism 06/2011; 96(9):E1399-408. · 6.50 Impact Factor
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    ABSTRACT: To investigate whether the embryotoxic effect of peritoneal fluid (PF) from infertile women with mild endometriosis on mouse oocytes and embryos is associated with changes in embryonic epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and their receptors. Experimental animal study. University-based research laboratory. Adult ICR mice. Peritoneal fluid was obtained from fertile women with no endometriosis (PF-NE) and infertile women with mild endometriosis (PF-E). In vitro fertilization was performed, and mouse two-cell stage embryos were cultured in human tubal fluid medium with or without PF. Rates of fertilization, cleavage, and blastulation. The embryonic EGF and IGF-I levels in culture medium were analyzed by enzyme-linked immunosorbent assay, and EGF receptor, IGF-I receptor, and phosphorylated extracellular signal-regulated protein kinases (p-ERK) expression was determined by immunofluorescence and confocal microscopy. When oocytes and embryos were cultured in media with PF-E, the fertilization capability of oocytes and the development potential of embryos were decreased. The levels of embryonic EGF, IGF-I, and their receptors were increased. However, p-ERK of the postreceptor signal transduction pathway was down-regulated. Endometriotic PF may attenuate oocyte and embryo development by impairing embryonic growth factor/receptor/signal transduction, resulting in endometriotic infertility.
    Fertility and sterility 05/2010; 93(8):2538-44. · 3.97 Impact Factor
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    ABSTRACT: The present study was designed to evaluate whether the alteration of aquaporin-9 (AQP-9) expression in granulosa cells (GCs) of patients with polycystic ovary syndrome (PCOS) was associated with the hyperandrogenism in follicular fluid (FF). We recruited infertile women with PCOS (n = 14) and infertile women with tubal blockage (controls, n = 31) for this study. We examined total testosterone (TT), free androgen index (FAI), sex hormone-binding globulin (SHBG), FSH, LH and estradiol in FF. Real-time PCR and western blotting were performed to assess AQP-9 expression in GCs, including effects of dihydrotestosterone (DHT) in vitro. AQP-9 protein was localized in the nucleus, cytoplasm and cell membrane of the human GCs. The TT, FAI and LH levels were all higher, and SHBG levels lower, in the FF of women with PCOS versus controls (P = 0.0145, 0.0001, 0.0191, 0.0001, respectively). AQP-9 mRNA level in GCs of patients with PCOS was tightly correlated with the TT, SHBG levels and FAI in FF (P = 0.0020, 0.0001, 0.0020, respectively). In vitro, DHT (10(-9) mol/l) decreased AQP-9 mRNA (lowest at 12 h) and protein levels in control GCs (P = 0.0005, 0.0247, respectively). The inhibitory effect of DHT on AQP-9 mRNA was attenuated by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor (P = 0.0013). Fifty micromolar 4-(hydroxymercuri) benzoic acid sodium salt (PMB) and 10(-9) mol/l DHT blunted the swelling of GCs in hypotonic medium, respectively (P = 0.0350, 0.0027). Hyperandrogenism in FF of women with PCOS inhibited AQP-9 in GCs through the PI3K pathway.
    Human Reproduction 04/2010; 25(6):1441-50. · 4.67 Impact Factor
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    ABSTRACT: In vitro maturation (IVM) of oocyte is an effective procedure for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). To investigate the influences of IVM on epigenetic reprogramming and to search for the possible reasons for the lower rates of fertilization and cleavage in IVM oocytes, we examined the expression of two enzymes controlling histone acetylation, histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in mouse metaphase II (MII) oocytes and preimplantation embryos. Results showed that IVM downregulated the protein expression of GCN5 in MII oocytes and two-cell embryos and changed the distribution of GCN5 in two-cell embryos. Expression of HDAC1 mRNA in MII oocytes and two-cell embryos decreased in the IVM group. However, none of these changes persisted after two-cell embryos. Levels of Ac-H3 in both oocytes and embryos remained unchanged after IVM. Our studies indicated that IVM could affect the protein and gene expression related to histone acetylation in oocytes and early cleavage embryos. By function of selection, parts of the changes could be recovered in late embryo development.
    Obstetrics and Gynecology International 01/2010; 2010:989278.
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    ABSTRACT: To investigate the effects of elevated nitric oxide (NO) levels in peritoneal fluids (PF) on oocyte fertilization and pre-implantation embryo development, and the relation of those effects to endometriosis-associated infertility. PF from women undergoing laparoscopy for infertility of minor endometriosis, tubal blockage and operation for tubal ligation was aspired at the pouch of the cul-de-sac during surgery. Oocytes and embryos of adult ICR mice were cultured in vitro with or without endometriotic PF. The fertilization rate of oocyte and the cleavage rate of 2-cell embryos were examined. Also, the clinical indexes of IVF-ET of women with minor endometriosis and tubal infertility were analyzed. Oocyte fertilization rate of endometriotic women with IVF-ET treatment was significantly lower than that of tubal block women. The dose-related adverse effects of endometriotic PF and SNP (NO donor) in culture medium on oocyte fertilization and embryos development were confirmed. Increased NO levels in PF play an important role in mediating the effects of endometriotic PF on oocyte fertilization and embryo development. IVF might serve as an alternative treatment for endometriosis-associated infertility.
    Cellular Physiology and Biochemistry 01/2010; 26(6):1023-8. · 3.42 Impact Factor
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    ABSTRACT: Our objective was to evaluate whether polycystic ovarian syndrome (PCOS)-associated infertility is related to alterations of leptin, leptin receptor (Ob-R), and the phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/suppressor of cytokine signal 3 (SOCS3) system in the ovary. A case-control study was conducted in a university hospital. Thirty-one infertile PCOS women with oligoovulation plus polycystic ovarian morphology and 79 infertile women with tubal blockage (control) participated in the study. The subjects were stratified according to in vitro fertilization outcomes: successful and failed subgroups. Serum and follicular fluid (FF) leptin levels were measured with ELISA. RT-PCR and Western blotting were performed to assess expression of mRNA encoding leptin and Ob-R and proteins of p-STAT3 and SOCS3 in granulosa cells (GCs). Leptin levels in serum and FF of PCOS women were significantly higher than those of control (P < 0.01). There were no significant differences in expression of leptin mRNA and short and long Ob-Rs between PCOS and control (P > 0.05). The p-STAT3 level was decreased in PCOS compared with control (P < 0.01), whereas SOCS3 remained significantly unchanged (P > 0.05). Further analysis showed that serum and FF leptin levels were significantly higher, whereas p-STAT3 in GCs was lower in the failed subgroup of PCOS than those in the successful subgroup of PCOS (P < 0.05). Hyperleptinemia and high FF leptin are important pathologies of PCOS with infertility. Lower levels of p-STAT3 in GCs may be related to ovarian leptin resistance and fecundity in PCOS women. Relatively high serum and FF leptin and low p-STAT3 in GCs may account for decreased fertilization, implantation, and pregnancy rates of in vitro fertilization in PCOS women.
    Journal of Clinical Endocrinology &amp Metabolism 12/2007; 92(12):4771-6. · 6.43 Impact Factor