Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination.

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Endometrial stromal cells from women with repeated implantation failure display impaired invasion towards trophoblastic spheroids.

In brief: Implantation failure can occur even after the transfer of good-quality embryos. This study showed that the migration of human endometrial stromal cells towards embryonic trophoblasts is higher in women with live births in the first in vitro fertilization cycle than those with repeated implantation failure, suggesting that the chemotactic response of stroma cells is associated with successful pregnancy. Abstract: The success rate of in vitro fertilization (IVF) remains limited in some women despite transfers of good-quality embryos in repeated attempts. There is no reliable tool for assessing endometrial receptivity. This study aimed to assess the interaction between decidualized human primary endometrial stromal cells (1°-EnSC) and human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) and to compare the invasion ability of decidualized 1°-EnSC towards BAP-EB between women attaining live birth in the first IVF cycle and those with repeated implantation failure (RIF). The invasion of the decidualized human endometrial cell line (T-HESC) and 1°-EnSC towards BAP-EB was studied. Real-time quantitative PCR and immunocytochemistry were employed to determine the expression of decidualization markers at mRNA and protein levels, respectively. Trophoblast-like BAP-EB-96h, instead of early trophectoderm (TE)-like BAP-EB-48h, facilitated the invasion ability of decidualized T-HESC and decidualized 1°-EnSC. Human chorionic gonadotropin at supra-physiological levels promoted the invasiveness of decidualized 1°-EnSC. The extent of BAP-EB-96h-induced invasion was significantly stronger in decidualized 1°-EnSC from women who had a live birth in the first IVF cycle when compared to those with RIF. While no difference was found in the expression of decidualization markers, PRL and IGFBP1 among two groups of women, significantly lower HLA-B was detected in the non-decidualized and decidualized 1°-EnSC from women with RIF. Collectively, the findings suggested that the invasion of decidualized 1°-EnSC towards trophoblast-like BAP-EB-96h was higher in women who had a live birth in the first IVF cycle than those with RIF.

High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation.

Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not ß-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception.

DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation.

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin ß3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor ß and integrin ß3 in Ishikawa cells.

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 µg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 µg/mL and ETU at 300 µg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 µg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor ß and integrin ß3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor ß and integrin ß3 expression of endometrial epithelial cells.

Human embryonic stem cell-derived blastocyst-like spheroids resemble human trophectoderm during early implantation process.

OBJECTIVE: To study the use of human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) as human blastocyst surrogates for studying early implantation and trophoblast development. DESIGN: Laboratory study. SETTING: University research laboratory. PATIENT(S): Infertile in vitro fertilization patients donating endometrial aspirates and human embryonic stem cells (hESCs: VAL3 and H9/WA09). INTERVENTION(S): In BAP-EB derived from hESC, transcriptomes analyzed by next-generation RNA sequencing, effects of Hippo signaling pathway studied by a YAP inhibitor, comparison of attachment of BAP-EB onto primary endometrial epithelial cells (EEC) collected at prereceptive and receptive phases, and antibody blocking assay used to study the molecule(s) involved in BAP-EB attachment. MAIN OUTCOME MEASURE(S): Gene expression profiles and endometrial cell attachment rates. RESULT(S): The BAP-EB differentiation protocol for VAL3 could be used to induce trophoblast differentiation in another hESC line, H9. Transcriptomic analysis showed that the epiblast signature gene expression was reduced while that of the trophoblast was induced during BAP-EB differentiation. Specifically, trophectoderm signature genes were induced in BAP-EB at 48 hours and 72 hours after induction of differentiation. The Hippo signaling pathway was one of the pathways induced during BAP-EB differentiation, and YAP1 inhibitor statistically significantly reduced attachment, outgrowth, and trophoblast gene expressions of BAP-EB. A statistically significantly higher number of BAP-EB derived from both VAL3 and H9 attached onto receptive EEC than prereceptive EEC. The antibody blocking assay demonstrated that endometrial E-cadherin might be critical in early implantation. CONCLUSION(S): The data suggest that BAP-EB possesses a trophectoderm-like signature, which supports the use of BAP-EB as a blastocyst surrogate for the study of trophoblast development and endometrial receptivity.

Bisphenol compounds regulate decidualized stromal cells in modulating trophoblastic spheroid outgrowth and invasion in vitro†.

Bisphenol A (BPA) is commonly found in epoxy resins used in the manufacture of plastic coatings in food packaging and beverage cans. There is a growing concern about BPA as a weak estrogenic compound that can affect human endocrine function. Chemicals structurally similar to BPA, such as bisphenol F (BPF) and bisphenol S (BPS), have been developed as substitutes in the manufacturing industry. Whether these bisphenol substitutes have adverse effects on human endocrine and reproductive systems remains largely unknown. This study investigated the effects of BPA, BPF, and BPS on regulating the function of decidualized human primary endometrial stromal cells on trophoblast outgrowth and invasion by indirect and direct co-culture models. All three bisphenols did not affect the stromal cell decidualization process. However, BPA- and BPF-treated decidualized stromal cells stimulated trophoblastic spheroid invasion in the indirect coculture model. The BPA-treated decidualized stromal cells had upregulated expressions of several invasion-related molecules including leukemia inhibitory factor (LIF), whereas both BPA- and BPF-treated decidualized stromal cells had downregulated expressions of anti-invasion molecules including plasminogen activator inhibitor type 1 (PAI-1) and tumor necrosis factor (TNFα) . Taken together, BPA and BPF altered the expression of invasive and anti-invasive molecules in decidualized stromal cells modulating its function on trophoblast outgrowth and invasion, which could affect the implantation process and subsequent pregnancy outcome.

Establishment of a novel human embryonic stem cell-derived trophoblastic spheroid implantation model.

STUDY QUESTION: Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation? SUMMARY ANSWER: We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation. WHAT IS KNOWN ALREADY: Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research. STUDY DESIGN, SIZE, DURATION: Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast. PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-ß inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells. MAIN RESULTS AND THE ROLE OF CHANCE: After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture. LIMITATIONS, REASONS FOR CAUTION: The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles. WIDER IMPLICATIONS OF THE FINDINGS: BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells.

Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage.

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.

Sirtuin 1 facilitates generation of induced pluripotent stem cells from mouse embryonic fibroblasts through the miR-34a and p53 pathways.

Forced-expression of transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPSC). Recent studies show that the reprogramming efficiency can be improved by inclusion of small molecules that regulate chromatin modifying enzymes. We report here that sirtuin 1 (SIRT1), a member of the sirtuin family of NAD(+)-dependent protein deacetylases, is involved in iPSC formation. By using an efficient mouse secondary fibroblast reprogramming system with doxycycline (DOX) inducible Yamanaka's transcription factors delivered by piggyBac (PB) transposition (2°F/1B MEF), we show that SIRT1 knockdown decreased while resveratrol (RSV) increased the efficiency of iPSC formation. The treatments were associated with altered acetylated p53 and its downstream Nanog but not p21 expression. The stimulatory effect was also confirmed by SIRT1 over-expression, which stimulated the formation of colonies with induced Nanog and reduced p21 expression. Furthermore, the effects of RSV and SIRT1 knockdown on reprogramming were most pronounced during the initiation phase of reprogramming. MicroRNA-34a is a known regulator of SIRT1. Its inhibitor increased, while its mimics reduced iPSC formation. The stimulatory effect of SIRT1 during reprogramming was also confirmed in the primary MEF. RSV increased while tenovin-6, a small molecule that activates p53 through SIRT1 inhibition, suppressed reprogramming. In conclusion, SIRT1 enhances iPSC generation, in part, through deacetylation of p53, inhibition of p21 and enhancement of Nanog expression.

Up-regulation of endocrine gland-derived vascular endothelial growth factor but not vascular endothelial growth factor in human ectopic endometriotic tissue.

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF), endocrine gland-derived VEGF (EG-VEGF/PK1), and its receptors (PKR1 and PKR2) in eutopic and ectopic endometrial tissues. DESIGN: A case-control study. SETTING: University reproduction unit. PATIENT(S): Infertile women undergoing diagnostic laparoscopy for tubal patency. INTERVENTION(S): Endometrial and endometriotic tissue sampling from women with and without endometriosis. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction (PCR) analysis of genes in eutopic and ectopic endometrial tissues. The EG-VEGF protein was studied by immunohistochemistry. RESULT(S): In normal endometrium, EG-VEGF messenger RNA (mRNA) expression was 50-fold higher in the secretory than in the proliferative phase, but that of PKR1 was 6-fold higher in the latter than in the former. The PKR2 transcript was detected in the proliferative but not the secretory endometrium. In patients with endometriosis, eutopic endometrial PKR2 transcript level was 4-fold higher in the proliferative than in the secretory phase. No differences in EG-VEGF or PKR1 were found in proliferative versus secretory endometrium in these patients. There were no significant differences in the expression of EG-VEGF in eutopic endometrium of normal women and in those with endometriosis. In the paired laser-captured microdissected eutopic endometrial and ectopic endometriotic samples, a significantly higher EG-VEGF, but not VEGF, transcript level was detected in the ectopic when compared with eutopic samples; whereas the expressions of PKR1 and PKR2 were barely detectable. The H-scoring confirmed that the stroma of endometriotic samples had a significantly higher EG-VEGF protein expression than that in the paired eutopic endometrium. CONCLUSION(S): High levels of EG-VEGF expression may play an important role in angiogenesis in endometriotic tissues.

Increased fetal abortion rate in autoimmune thyroid disease is related to circulating TPO autoantibodies in an autoimmune thyroiditis animal model.

OBJECTIVE: To determine the fertility and abortion rates in a mouse model of autoimmune thyroiditis and its relationship with circulating anti-thyroid peroxidase (TPO) antibody. DESIGN: Experimental animal study. SETTING: University research laboratory. ANIMAL(S): C57bl/6 mice. INTERVENTION(S): Female C57bl/6 mice immunized with recombinant mouse TPO (rmTPO) in complete Freund adjuvant (CFA) or glutathione-S-transferase (GST-CFA) were allowed to mate. The pregnant mice were killed on day 14 of pregnancy for assessment of fetal development. The effects of TPO antibody on preimplantation embryo development and implantation rate were also studied. MAIN OUTCOME MEASURE(S): Litter size, resorption rate, preimplantation embryo development, and implantation rate. RESULT(S): All of the mice immunized with rmTPO-CFA possessed anti-TPO antibody. They had reduced litter size and increased incidence of resorbed fetus compared with the control. Higher serum TSH levels, but not T(4) levels, were demonstrated after rmTPO-CFA immunization. Anti-TPO antibody bound to preimplantation embryos. Treatment of the embryos with the antibody marginally decreased the formation of 3/4-cell embryos but had no effect on the subsequent development and implantation compared with the nonimmune control sera. CONCLUSION(S): Autoimmune thyroiditis is associated with reduced fertility and higher incidence of fetal loss. The anti-TPO antibody may affect post-implantation embryo development, leading to fetal loss.

Regulation of complement-3 protein expression in human and mouse oviducts.

The human oviduct derived embryotrophic factor-3 (ETF-3) contains complement protein-3 (C3) and its derivates. Although C3 is not embryotrophic, it is converted into the embryotrophic derivative, iC3b in the presence of embryos and oviductal cells. The regulation of C3 production in the oviduct is not known. The objectives of this study were to investigate the effects of presence of preimplantation embryos and hormones on C3 expression in the oviducts in vitro and in vivo. The expression of C3 in the oviduct of pregnant mice was compared to that of pseudo-pregnant mice. The hormonal action on C3 expression was studied in the ovariectomized mouse oviducts and human oviductal epithelial (OE) cells. The results showed that the level of C3 mRNA in the mouse oviduct was high on Day 1 and Day 2, but decreased to a minimum on Day 4 of pregnancy, whereas that of pseudo-pregnancy remained relatively stable within the same period. The protein levels of C3 and iC3b specific fragments, alpha-115 and alpha-40, respectively in the mouse oviductal luminal fluid were highest on Day 3 of pregnancy, when the embryos were expected to be most sensitive to the embryotrophic activity of ETF-3. Estrogen elevated C3 expression in the ovariectomized mouse oviduct and the OE cells. Progesterone suppressed estrogen-induced C3 expression in the mouse oviduct, but had no effect on OE cells. In conclusion, the presence of embryo and steroid hormones regulate the synthesis and secretion of oviductal C3.

Endocrine gland-derived vascular endothelial growth factor stimulates proliferation and tube formation in human uterine microvascular endothelial cell through the mitogen-activated protein kinase but not through the Akt pathway.

OBJECTIVE: To study the angiogenic functions of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) on a normal myometrial uterine microvascular endothelial cell line (UtMVEC-Myo) and the signaling pathways elicited by EG-VEGF in UtMVEC-Myo. DESIGN: Experimental laboratory study. SETTING: University gynecology unit. PATIENT(S): Infertile women undergoing diagnostic laparoscopy for assessment of tubal patency. INTERVENTION(S): Real-time polymerase chain reaction (PCR) analysis of mRNA of EG-VEGF and its receptors, PKR1 and PKR2, in UtMVEC-Myo and endometrial samples. The effects of EG-VEGF on the cell proliferation, tube formation, and cell signaling pathways of UtMVEC-Myo were studied. MAIN OUTCOME MEASURE(S): Cell proliferation, tube formation, and molecules of cell-signaling pathways in the treated UtMVEC-Myo. RESULT(S): UtMVEC-Myo cells had PKR1 and PKR2 but not EG-VEGF mRNA. EG-VEGF significantly stimulated cell proliferation and tube formation in UtMVEC-Myo cells. EG-VEGF activated p44/42 mitogen-activated protein kinase (MAPK) but not Akt signaling pathway. The effects of EG-VEGF on p44/42 MAPK phosphorylation and cell proliferation were nullified by the specific MAPK inhibitor, PD98059. CONCLUSION(S): EG-VEGF has a direct angiogenic effect on UtMVEC-Myo that expresses EG-VEGF receptors (PKR1 and PKR2) and modulates cell proliferation and sprouting of the endothelial cells. It is suggested that EG-VEGF enhanced cell proliferation through the activation of MAPK pathway but not through the Akt pathway.

Aberrant expression of angiopoietins-1 and -2 and vascular endothelial growth factor-A in peri-implantation endometrium after gonadotrophin stimulation.

BACKGROUND: Ovarian stimulation affects normal endometrial development. The expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A) and the vascular state in the peri-implantation endometrium in women with natural and gonadotrophin-stimulated cycles were compared. METHODS: The expression of these angiogenesis-associated molecules in endometrial biopsies, collected on Day 7 after human chorionic gonadotrophin injection or luteinizing hormone surge in stimulated or natural cycles respectively, or at mid-luteal phase of women undergoing diagnositic laparoscopy, were analysed. RESULTS: Women with gonadotrophin-stimulation had lower Ang-1, but higher Ang-2, mRNA and protein expression (P < 0.05), and increased concentrations of von Willebrand factor (vWF) and blood vessel density than those with natural cycles (P < 0.05). Although stimulated cycles had higher VEGF-A mRNA expression (P = 0.023), VEGF-A protein expression was similar between the groups. Lower Ang-1/Ang-2 but higher Ang-2/VEGF-A mRNA ratios (P = 0.025) were found after gonadotrophin-stimulation. The ratios were negatively (P < 0.001) and positively correlated (P < 0.001) with estradiol levels, respectively. Cyclical changes in Ang-1 and Ang-2, but not in VEGF-A expression were noted. CONCLUSIONS: The decreased Ang-1 concentration and Ang-1/Ang-2 ratio and the increased Ang-2 concentration, with the increased vWF concentration and blood vessel density, in stimulated cycles suggests advanced endometrial angiogenesis after gonadotrophin-stimulation.

Demilune cell and parotid protein from murine oviductal epithelium stimulates preimplantation embryo development.

In mammals, fertilization and early preimplantation embryo development occur in the oviduct. We hypothesized that interaction exists between the developing embryos and the maternal genital tract, such that the embryos modulate the physiology and gene expression of the oviduct so that it is conducive to their development. By comparing the gene expression patterns in mouse oviducts containing transferred preimplantation embryos with those of oviducts containing oocytes, we report here the characterization of demilune cell and parotid protein (Dcpp), which was up-regulated in the embryo-containing oviduct. Dcpp mRNA was highly expressed in the oviductal epithelium at the estrus stage. The Dcpp gene codes for a protein of 150 amino acids and contains a signal peptide suggestive of secretory function. The Dcpp mRNA level was maintained in the oviductal epithelium of pregnant females but decreased continuously in those of pseudopregnant mice. Exogenous estrogen stimulated the expression of Dcpp mRNA and protein in ovariectomized mice. The effect was abolished by an estrogen antagonist, ICI 182,780. Dcpp protein was present in mouse oviductal fluid but not in uterine fluid. More importantly, Dcpp immunoreactivity was found in embryos recovered from the oviduct but not in mature oocytes from the ovary. Supplementation of Dcpp to culture medium stimulated the development of mouse embryos to the blastocyst stage. Anti-Dcpp antibody decreased the beneficial effect of Dcpp on implantation of two-cell mouse embryos transferred to the oviducts of the foster mothers. In summary, our data demonstrated that Dcpp is highly expressed in the oviductal lumen in the presence of preimplantation embryos. It stimulates the growth of preimplantation embryos and may play an important role in embryo-maternal dialogue.

Expression of human oviductin in an immortalized human oviductal cell line.

OBJECTIVE: To determine whether OE-E6/E7, an immortalized human oviductal epithelial cell line, expresses oviductin messenger RNA (mRNA) and its translated protein. DESIGN: Transmission electron microscopy was employed to characterize the morphology of OE-E6/E7 cells followed by reverse-transcription polymerase chain reaction (PCR) analysis of oviductin mRNA and sequencing of the nested-PCR product. Confocal microscopy was used, using a polyclonal antibody against human oviductin and Con A as a marker for mannose residues, to reveal the colocalization of human oviduct-specific glycoprotein with the endoplasmic reticulum and Golgi compartments. SETTING: University-based anatomy and cell biology department. PATIENT(S): Women undergoing laparoscopy for tubal ligation or hysterectomy due to uterine fibroma. INTERVENTION(S): An immortalized OE-E6/E7 cell line was previously established using human oviductal epithelial cells. Electron microscopy, RT-PCR, sequencing, immunohistochemistry and confocal microscopy were performed. MAIN OUTCOME MEASURE(S): The presence of human oviductin mRNA and protein in OE-E6/E7 cells. RESULT(S): OE-E6/E7 cells retain morphological features characteristic of secretory cells and express human oviductin mRNA and its translated protein. CONCLUSION(S): OE-E6/E7 cells were characterized for the first time by electron microscopy and shown to exhibit histological features typical of secretory cells. Reverse-transcription PCR with sequencing and confocal microscopy showed, respectively, that human oviductin mRNA and protein are expressed in OE-E6/E7 cells. Our results suggest that OE-E6/E7 could be a useful tool for future studies of the function of human oviductin.