Yin-Lau Lee

The embryotrophic activity of oviductal cell-derived complement C3b and iC3b, a novel function of complement protein in reproduction.

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.

Complement 3 Deficiency Impairs Early Pregnancy in Mice

Human oviductal cells produce complement‐3 (C3) and its derivative, iC3b. These molecules are important in immune responses. Our recent study suggested that iC3b also possessed embryotrophic activity and it stimulates the blastulation and hatching rates of in vitro cultured mouse embryos. The objective is to study the impact of C3 deficiency on early pregnancy in vivo using homozygous C3‐deficient (C3KO) and wild‐type (C3WT) mice. C3 protein was undetectable in the reproductive tissues of C3KO mice. Deficiency in C3 is associated with significantly longer estrous cycle (P = 0.037). No significant difference was found in the ovulation rate, total cell count in blastocysts and implantation rate between the wild‐type and the C3KO mice, though C3KO mice tended to have lower values in the latter two parameters. On day 15 of pregnancy, C3KO mice had fewer conceptus (P < 0.001) and higher resorption rate (P < 0.001) than that of C3WT mice. The fetal and placental weights (P < 0.001) were lower in the C3KO mice. The placenta of C3KO mice had smaller spongiotrophoblast (P = 0.001) and labyrinth (P = 0.037). Deficiency in C3 is associated with mild impairment in early pregnancy including longer estrous cycle and higher resorption rates after implantation. The impairment may be related to compromised placental development leading to under‐developed fetuses. Mol. Reprod. Dev. 76: 647–655, 2009.

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct.

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene‐expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo‐containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo‐pregnant mice when assayed by real‐time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.

Vero cells, but not oviductal cells, increase the hatching frequency and total cell count of mouse blastocysts partly by changing energy substrate concentrations in culture medium

AbstractPurpose: To investigate the embryotrophic mechanisms of Vero and oviductal cells coculture. Methods: Mouse embryos were cultured in Chatot, Ziomek, and Bavister medium (CZB), in modified CZB media (MM) with nutrient concentrations adjusted to that found in conditioned media after different periods of Vero cells or oviductal cells culture, in reconstituted medium (RM) containing the purified >100-kDa components of Vero cell conditioned medium that had been reconstituted with CZB medium, and cocultured with Vero cells with an interposing membrane. Results: The blastulation rate was not different among embryos cultured in different Vero-cell–derived MMs. Nine-hour Vero-cell-derived MM significantly increased the total cell number and hatching frequency of the embryos. There was no difference in these parameters with oviductal-cell–derived MMs. The RM of Vero cells did not possess embryotrophic activity. The presence of a porous membrane between Vero cells and embryos did not affect the embryotrophic activity of coculture. Conclusions: Vero cells, but not oviductal cells, improved mouse embryo development partly by modifying the energy substrate concentration in culture medium.

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.

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, α‐115 and α‐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. Mol. Reprod. Dev. 76: 301–308, 2009.

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.

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-β 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.

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.

Study of transforming growth factor alpha for the maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have self-regenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor α (TGFα) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFα in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFα maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFα treatment activated the p44/42 MAPK pathway but not the PI3K/Akt pathway. In addition, TGFα treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFα provides insights for the development of clinical grade hESCs for therapeutic applications.