Sheng-Ping Chang

Induction of p38 Mitogen-Activated Protein Kinase-Mediated Apoptosis Is Involved in Outgrowth of Trophoblast Cells on Endometrial Epithelial Cells in a Model of Human Trophoblast-Endometrial Interactions

Abstract During embryo implantation in species with hemochorial placentation, such as the mouse and human, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Signs of apoptosis were demonstrated in luminal endometrial epithelial cells (EEC) adjacent to the trophoblast cells; however, the signaling mechanisms leading to apoptosis in EEC remain unclear. Because mitogen-activated protein kinases (MAPK) were shown to mediate apoptosis in several model systems and found to be activated in the uterus during decidualization, the possible involvement of MAPK during trophoblast-EEC interactions was studied. By coculturing BeWo human trophoblast spheroids with RL95-2 human EEC monolayers to mimic the blastocyst-endometrial interaction, we found that most spheroids rapidly attached to EEC monolayers and then progressively expanded, with marked dislodgment of EEC adjacent to the spreading trophoblast cells. Immunoblotting analysis showed that both p38 MAPK and extracellular signal-regulated kinase (ERK) were activated in EEC after coculture. However, only SB203580 (a p38 MAPK inhibitor), but not PD98059 (an ERK inhibitor), inhibited trophoblast outgrowth on EEC monolayers through the suppression of p38 MAPK activation in EEC. Furthermore, trophoblast expansion caused prominent EEC apoptosis at the spheroid-EEC interface, as detected by annexin V labeling and valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (which binds activated caspases) staining, and SB203580 significantly decreased the percentage of apoptotic cells. Our results, based on a model of human trophoblast-EEC interactions, establish that trophoblast cells cause activation of p38 MAPK in EEC and, consequently, induce apoptosis and displacement of EEC, a process that may facilitate implantation.

Collagen IV significantly enhances migration and transplantation of embryonic stem cells: involvement of α2β1 integrin-mediated actin remodeling.

Embryonic stem (ES) cell transplantation represents a potential means for the treatment of degenerative diseases and injuries. As appropriate distribution of transplanted ES cells in the host tissue is critical for successful transplantation, the exploration of efficient strategies to enhance ES cell migration is warranted. In this study we investigated ES cell migration under the influence of various extracellular matrix (ECM) proteins, which have been shown to stimulate cell migration in various cell models with unclear effects on ES cells. Using two mouse ES (mES) cell lines, ESC 26GJ9012-8-2 and ES-D3 GL, to generate embryoid bodies (EBs), we examined the migration of differentiating cells from EBs that were delivered onto culture surfaces coated with or without collagen I, collagen IV, Matrigel, fibronectin, and laminin. Among these ECM proteins, collagen IV exhibited maximal migration enhancing effect. mES cells expressed α2 and β1 integrin subunits and the migration enhancing effect of collagen IV was prevented by RGD peptides as well as antibodies against α2 and β1 integrins, indicating that the enhancing effect of collagen IV on cell migration was mediated by α2β1 integrin. Furthermore, staining of actin cytoskeleton that links to integrins revealed well-developed stress fibers and long filopodia in mES cells cultured on collagen IV, and the actin-disrupting cytochalasin D abolished the collagen IV-enhanced cell migration. In addition, pretreatment of undifferentiated or differentiated mES cells with collagen IV resulted in improved engraftment and growth after transplantation into the subcutaneous tissue of nude mice. Finally, collagen IV pretreatment of osteogenically differentiated mES cells increased osteogenic differentiation-like tissue and decreased undifferentiation-like tissue in the grafts grown after transplantation. Our results demonstrated that collagen IV significantly enhanced the migration of differentiating ES cells through α2β1 integrin-mediated actin remodeling and could promote ES cell transplantation efficiency, which may be imperative to stem cell therapy.

Roles of Estrogen and Progesterone in Endometrial Hemodynamics and Vascular Endothelial Growth Factor Production

Background: The endometrium becomes receptive to the embryo after sequential actions of estrogen and progesterone. The purpose of this study was to examine the effects of estrogen and progesterone on endometrial hemodynamics and on secretion of vascular endothelial growth factor (VEGF) from endometrial epithelial cells (EEC). Methods: Six early postmenopausal women taking sequential estrogen and progestin [days 1–11: estradiol valerate (estrogen) 2 mg daily; days 12–21: estradiol valerate 2 mg plus norethisterone acetate (progestin) 1 mg daily] were recruited. Three‐dimensional power Doppler angiography (3D‐PDA) was performed before hormone treatment (phase 0), on days 10–11 of hormone treatment (phase E), and on days 18–20 of hormone treatment (phase E + P). Ishikawa EEC were treated with or without 17‐β‐estradiol and progesterone for 24 hours, followed by determination of VEGF concentrations in the supernatants. Results: The endometrial volume was significantly increased in phase E and phase E + P as compared with that in phase 0. The vascularization index, flow index, and vascularization flow index in the subendometrial region, as measured by 3D‐PDA, were significantly higher in phase E + P than in phase 0, but there were no significant differences in these indices between phase 0 and phase E. While treatment of EEC with 17‐β‐estradiol had little enhancing effect on VEGF production, progesterone alone or in combination with 17‐β‐estradiol significantly increased VEGF secretion from EEC. Conclusion: Our data suggested that progesterone could stimulate VEGF secretion from EEC and subsequently increase subendometrial vascularity and blood flow.

ESTABLISHMENT OF AN EFFICIENT METHOD TO QUANTIFY EMBRYO ATTACHMENT TO ENDOMETRIAL EPITHELIAL CELL MONOLAYERS

SummaryDuring implantation, complex embryo-endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo-endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25×g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids wereaattached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200–1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst-EEC adhesion. Spheroid-EEC adhesion was inhibited by blocking Ca2+ influx with extracellular Ca2+ chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) or a Ca2+ channel blocker (verapamil) but not by interfering with Ca2+ release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.

Small supernumerary marker chromosome originating from chromosome 10 associated with an apparently normal phenotype

Small supernumerary marker chromosomes (sSMC) originating from chromosome 10 are rare. Only seven cases have been documented, and among those three cases were diagnosed prenatally. We reported on another prenatal diagnosis of a de novo mosaic sSMC in an apparently normal female fetus whose mother had conceived with assisted reproductive technology (ART) procedures. G‐banding analysis of amniotic cells was performed. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) studies with chromosome 10‐specific alphoid satellite DNA probe were used to identify the chromosome 10 origin of the sSMC. Further FISH study with telomeric sequence probes showed that the sSMC lacked a hybridization signal, suggesting that the marker could be a ring chromosome. FISH studies using BAC clone probes specific for the regions within 10p11.2, 10q11.1, and 10q11.2 showed that the short arm breakpoint was located between 29.8 and 30.7 Mb from the 10p telomere, and that the long arm breakpoint was located less than 43.6 Mb from the 10p telomere. The karyotype of the fetus was 47,XX,+mar. ish der(10)(SKY+ CEP 10+, CTD‐2130I7+, RP11‐89J23−)/46,XX. Oligonucleotide microarray‐based copy number variations (CNV) analysis was also performed and showed a 6.7 Mb duplication from 10p11.2 to 10q11.2 (36.2–42.9 Mb) with Affymetrix SNP‐array 6.0 genotype: arr cgh. 10p11.2q11.2(CN_519687 → CN_541524) X 3. At the 1‐year follow‐up, the baby did not have any findings of the trisomy 10p syndrome. This observation provided further credence to the concept that additional chromosome material of proximal 10p11.2 may not contribute to the trisomy 10p syndrome phenotype.

Anti-fas activating antibody enhances trophoblast outgrowth on endometrial epithelial cells by induction of P38 MAPK/JNK-mediated apoptosis.

In species with hemochorial placentation, such as the mouse and human, trophoblast cells of the implanting blastocyst induce apoptosis and displace endometrial epithelial cells (EEC) to cross the luminal epithelium of the endometrium. Since Fas and Fas ligand (FasL) are expressed in EEC and trophoblast cells respectively and mitogen-activated protein kinases (MAPKs) mediate Fas-induced apoptosis, the roles of Fas/FasL and MAPK signaling in trophoblast-EEC interactions were studied. By co-culturing BeWo trophoblast spheroids with RL95-2 EEC monolayers to mimic blastocyst-endometrial interactions, we found that trophoblast spheroid outgrowth on EEC was significantly enhanced by anti-Fas activating antibody. Since anti-Fas activating antibody had no effect on spheroid expansion on EEC-free culture surfaces, its enhancing effect on spheroid outgrowth on EEC may be mediated by acting on EEC to facilitate trophoblast-induced EEC apoptosis and displacement. Valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (VAD-FMK) staining showed that the percentage of apoptotic EEC at the spheroid-EEC interface was markedly increased by anti-Fas activating antibody. Moreover, the pancaspase inhibitor benzyloxycarbonyl-VAD-FMK was able to suppress the enhancing effect of anti-Fas activating antibody on spheroid expansion on EEC. Upon anti-Fas activating antibody stimulation, both p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) were activated. Furthermore, the anti-Fas activating antibody-enhanced EEC apoptosis and spheroid expansion on EEC were significantly inhibited by the p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125. Our results establish that anti-Fas activating antibody could activate p38 MAPK and JNK to induce EEC apoptosis, thereby promoting trophoblast outgrowth on EEC.

INTERLEUKIN-8 CAN STIMULATE PROGESTERONE SECRETION FROM A HUMAN TROPHOBLAST CELL LINE, BEWO

SummaryPrecise paracrine cross-talk between the embryo and the endometrium is essential for the establishment of a successful pregnancy. Previous studies have demonstrated that the expression of interleukin-8 (IL-8) in the endometrium is enhanced during the late-secretory phase and early pregnancy. Furthermore, IL-8 receptor (IL-8R) expression has been detected in trophoblast cells of the developing embryo. To clarify the roles of IL-8 in the endometrium-embryo interactions, the effects of IL-8 on hormone secretion by trophoblast cells were studied using the BeWo trophoblast cell line that retains hormone-secreting properties of normal trophoblast cells. Using reverse transcription-polymerase chain reaction, we found that IL-8R messenger ribonucleic acid (mRNA) was expressed in BeWo cells. The levels of IL-8R mRNA and protein expression in BeWo cells were similar to those in primary first-trimester trophoblast cells. Progesterone (P4) secretion of BeWo cells was comparable with that of first-trimester trophoblast cells but higher than that of third-trimester trophoblast cells. Treatment of BeWo cells with recombinant human IL-8 (rhIL-8) had no effect on cell proliferation, as determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Interestingly, secretion of P4, but not human chorionic gonadotropin, from cultured BeWo cells was significantly enhanced when the cells were incubated with rhIL-8. Our results demonstrate that IL-8 may play an important role in the endometrium-embryo interactions by stimulating trophoblast secretion of P4 for maintenance of a successful pregnancy.

The Effects of Sequentially Combined Hormone Replacement Therapy on Climacteric Symptoms and Lipid Profiles

Objectives: To compare the climacteric symptoms, bleeding patterns, and lipid metabolism in Taiwanese postmenopausal women using 2 different cyclic sequential hormone replacement regimens, containing either 1 to 2 mg 17βestradiol plus 1 mg norethisterone acetate (Sevina®) or 0.625 mg conjugated estrogen plus 5 mg medroxyprogesterone acetate (Premarin®/Provera®; PP). Material(s) and Method(s): Fifty-seven generally healthy, female, postmenopausal patients from 45 to 60 years of age were randomized into groups receiving either 1 tablet Sevina® daily or I tablet of 0.625 mg Premarin® daily with 1 tablet of 5 mg Provera® daily for the first 12 days (days 1 to 12) (PP group) for 6 months. Climacteric symptoms and bleeding patterns were assessed at the baseline and at 1, 3, and 6 months. The effects on lipid variables, hormone profiles, and liver function tests were measured at the baseline and at 6 months. Result(s): Both drugs produced a statistically significant reduction in most climacteric symptoms during the 6 months of treatment (p ＜ 0.05). After 6 months of treatment, total cholesterol showed no significant change in the PP group, whereas there was a significant decrease in the Sevina® group. Meanwhile, low-density lipoprotein cholesterol also revealed a significant reduction in both treatment groups, while high-density lipoprotein cholesterol remained unchanged in both groups. The triglyceride level decreased in the Sevina® group, but showed a significant increase in the PP group (p ＜ 0.05). There was also a significant difference between the 2 groups in changes in triglycerides. Conclusions: This study demonstrates that Sevina® is effective in decreasing low-density lipoprotein cholesterol and the total cholesterol level, without increasing the level of triglycerides, and can effectively alleviate climacteric symptoms during treatment. Sevina® is a highly effective and acceptable sequential hormone replacement regimen for the treatment of climacteric symptoms and lipid effects in postmenopausal women.

Buserelin treatment of endometriosis in Chinese women

Twenty five patients with endometriosis of varying degree had been treated with Buserelin (GnRH analogue) for 6 months. Among them, 83.4% reached castrated level by measuring the serum estradiol (E2) 2 months after therapy. Dysmenorrhea was alleviated or completely disappeared during therapy. Hot flush was the one mostly complained. Vaginal dryness was the second and decreased libido the third. Persisted periodic bleeding was noted in 3 patients. Ovulation was suppressed as evidenced by low serum progesterone throughout the whole course of treatment. Second-look laparoscopy was done at the end of 6-month therapy. Scoring assigned by the American Fertility Society (AFS) was reduced by 27.5%. The adrenal gland, liver and renal functions as well serum calcium and phosphate were retained at the end of treatment. Ovulation and menstruation also returned to normal within 2 months after cessation of therapy. There were 4 pregnancies during the 6-month follow period (4/15 = 26.6% pregnancy rate). 7 patients had improved symptoms whereas 7 patients sustained recurrent dysmenorrhea. The hormonal profile showed that dysmenorrhea improved group had better ovarian suppression than the dysmenorrhea recurrent group.

Transient hyperprolactinemia in gonadotropin-stimulated cycles for in vitro fertilization and its effect on conception

The significance of transiently increased serum prolactin (PRL) levels on pregnancy rates in vitro fertilization (IVF) is unknown. The aim of this study was to evaluate PRL levels in IVF patients who conceived and in matched controls who did not. Ten IVF cycles resulting in pregnancy and forty nonpregnant cycles were compared. Prolactin was measured before ovarian stimulation with gonadotropins and estradiol (E2), progesterone (P) and PRL were measured 36 hours, 12 hours, 10 minutes before and 12 hours after human chorionic gonadotropin (hCG) administration at mid-cycle. Serum PRL levels at various time were not significantly higher in the nonpregnant women than in the pregnant women. Twelve hours before hCG, P levels were significantly higher in the nonpregnant women than in the pregnant women (1.5 +/- 0.2 ng/ml [mean +/- standard error] and 0.9 +/- 0.3 ng/ml, respectively; P < 0.05). All women had transient hyperprolactinemia for one to three times during ovarian hyperstimulation. There was no correlation between PRL and E2 in either group. These results do not support the treatment of transient hyperprolactinemia with dopamine agonists in IVF patients.