Heparin binding-epidermal growth factor improves blastocyst hatching and trophoblast outgrowth in the golden hamster

The effect of heparin binding-epidermal growth factor (HB-EGF) on the in-vitro development of hamster 8-cell embryos was investigated. Supplementation of HB-EGF to culture medium accelerated zona escape of blastocysts (63 +/- 9% compared with 33 +/- 9% after 36 h; P < 0.05). Complete zona escape of blastocysts persisted even after 48 h (61 +/- 11% versus 30 +/- 4%) and 60 h (75 +/- 6% versus 42 +/- 8%). Addition of anti-HB-EGF antibody drastically reduced the percentage of zona escaped-blastocysts (30.0 +/- 5.0% versus 92.3 +/- 2.8%; P < 0.05). Interestingly, a significant increase in the area of trophoblast outgrowth occurred in the presence of HB-EGF (116 x 10(3) +/- 8 x 10(3) microm(2) versus 74 x 10(3) +/- 8 x 10(3) microm(2) at 48 h; P < 0.05). This, however, was not due to an increased number of trophectodermal cells in HB-EGF-treated blastocysts. Immunoreactive HB-EGF was localized in blastocysts and uterine sections, visible by intense immunostaining in the luminal epithelium, particularly on the apical surface. Moreover, the expression of HB-EGF in the uterus was maximum on day 4 of pregnancy, coinciding with the timing of zona escape and implantation. The receptor of HB-EGF, viz. EGF receptor was also detected in blastocysts and the luminal epithelium of day 4 pregnant uterus. These results show that HB-EGF improves blastocyst hatching and trophoblast outgrowth in hamsters.     

Trophoblast growth in the lungs of mice.

Trophoblast giant cells grow from ectoplacental cones of 7 1/2 days' gestation that are implanted in the lung from the surface, intrabronchially, or via the circulation. When blastocysts of 3 1/2 days' gestation are implanted in lung, their behavior varies according to the route of implantation and the ages of the recipient mice. Blastocysts produced trophoblast giant cells when implanted in the bronchial tree. Trophoblast giant cells were not seen in adult lungs when blastocysts were introduced via the circulation. However, they were seen in 12-day-old mice injected in the same way 10 days previously. Something is apparently liberated from adult lung vessels which interacts with the blastocyst or zona pellucida and causes rapid degeneration of trophoblast. Experiments designed to extract this agent are in progress in the hope that the agent may be useful in the treatment of human trophoblastic disease.     

体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

Effect of curcumin on in vitro early post-implantation stages of mouse embryo development

Objectives

This study was designed to examine the embryotoxic potential of the curcumin at the blastocyst stage and during early post-implantation development of mouse embryos in vitro.

Study design

Curcumin was administered to ICR mice embryos at a dose of 0, 6, 12, 24 μM throughout in vitro culture. A total of 1015 embryos were randomly assigned to the different dosage groups. The embryotoxic effects were studied by the exposure of curcumin at the blastocyst, implanted blastocyst and early egg cylinder stages, respectively. For assessment of implantation in vitro and further embryonic differentiation, blastocysts were cultured for 8 days. The cell proliferation of outgrowth blastocysts was analysed by Giemsa staining.

Results

Exposure to 24 μM of curcumin at the implanted blastocyst stage or early egg stage cause adverse effects on development. The percentage of embryos in the later stages of development was changed depending upon the dose of curcumin used. Furthermore, exposure to 24 μM of curcumin at the blastocyst stage was lethal to all embryos. The number of nuclei per outgrowth of the blastocyst decreased significantly after curcumin pre-treatment. The percentage of trophoblastic giant cells per outgrowth increased significantly after curcumin pre-treatment.

Conclusions

These findings demonstrate that curcumin exerts an adverse effect on mouse embryos during the early post-implantation stages of development, equivalent to day 3–day 8 of gestation in vivo. Curcumin treatment or administration should be used carefully at the early post-implantation stage of gestation.     

Immunoregulatory activity in supernatants from cultures of normal human trophoblast cells of the first trimester

Supernatants from isolated trophoblast cell cultures (trophoblastic fluid) derived from first-trimester human placentas were assessed for immunoregulatory activity. Trophoblastic fluid at different days of culture consistently inhibited the blast transformation of allogenic lymphocytes. This suppressor effect had no apparent correlation with biosynthesis of human chorionic gonadotropin by trophoblast cells, since this hormone was secreted into the culture fluid only for the initial 3 days. However, the culture fluids of such purified trophoblast cells contained an immunosuppressive factor, pregnancy-associated plasma protein A, which was measurable throughout the culture period of 8 days. The presence of pregnancy-associated plasma protein A in significant amounts in trophoblastic fluid collected at daily intervals indicated a continuous secretion ability of pregnancy-associated plasma protein A by trophoblast cells in culture parallel to the suppressive immunoregulatory effect of the fluid. Such immunosuppressive effect was absent in the culture fluids of control BeWo malignant trophoblast cells; the BeWo cell culture fluids had markedly reduced levels of pregnancy-associated plasma protein A. The culture supernatant of normal trophoblast cells of placentas from first-trimester pregnancy activated in vitro the generation of a population of suppressor lymphocytes. This effect is generally considered responsible for immunologic tolerance. Therefore demonstration of immunosuppressive effects and the presence of relatively high levels of pregnancy-associated plasma protein A in trophoblastic fluid indicate that such proteins secreted by the trophoblast cells may be important in the local immunoregulatory processes of the fetal allograft.     

Transferrin receptor expression in early postimplantation mouse trophoblast and associated tissues

The expression of the transferrin receptor (TR) was examined on murine trophoblast cells on days 6, 8 and 10 of gestation, using a monoclonal antibody visualized by indirect immunofluorescence on cryostat sections of the implant site. In the day 6 tissues, TR were observed on both the ectoplacental cone (EPC) and mural giant cell trophoblast populations, as well as on the embryonic ectoderm, anti-mesometrial decidual cells, uterine glandular epithelium and myometrium. By the 8th day of gestation, TR expression was weak, or undetectable on trophoblast giant cells (TGC), but remained strong on the proliferating cells of the EPC and embryo. In the definitive placenta (day 10), TR are expressed primarily on the differentiated labyrinthine trophoblast cells involved in the maternal-fetal transfer of iron.     

A trophoblast specific antigen, recognized by monoclonal antibody MA21, locates a unique trophoblast cell population in the murine placenta

Monoclonal antibody MA21 recognized a 44kDa plasma membrane protein on F9 teratocarcinoma cells, trophectoderm of mouse peri-implantation-stage blastocyst and ectoplacental cone cells of 5 day postcoitum implanted blastocyst (Vernon, Linnemeyer and Hamilton, 1989). We show here that this antigen is expressed by trophoblast cells of the maturing placenta. Immunohistochemical assays of early and mature placental tissue sections, indirect immunofluorescence labelling of placental cultures and blastocyst outgrowths in vitro, and immunoprecipitation of 35S-labelled NP-40 extracts of placental cultures indicate the presence of a plasma membrane-associated antigen with the same characteristics as MA21 antigen of peri-implantation embryos and F9 teratocarcinoma cells. In sections of placentae, antigen-positive cells are always situated in a thin layer between trophoblastic giant cells and maternal tissue. In cultures of postimplantation stage embryos, attached trophoblast cells express MA21 antigen initially, but following transformation to the giant cell state, antigen is no longer expressed. These results indicate the presence of a plasma membrane protein antigen associated with a distinct population of cells believed to be trophoblast. We believe that these cells are the foremost trophoblast cells opposing maternal decidua and that they may give rise to secondary trophoblastic giant cells.     

Trophoblast alterations in the placental bed in relation to physiological changes in spiral arteries

Summary. Ninety-three placental bed biopsies containing a segment of a spiral artery at the level of the decidual–myometrial junction (53 with and 40 without physiological changes) were histologically investigated for depth of trophoblastic penetration of the uterine wall, formation of trophoblastic multinucleated giant cells and the enzyme histochemical characteristics of the interstitial (stromal) and vascular (intramural) trophoblast. The depth of trophoblastic penetration was not related to the presence or absence of the physiological changes. Conversely, in the absence of physiological changes, a significant accumulation of multi-nucleated giant cells at the decidual–myometrial junction was found before 36 weeks of gestation. The enzyme histochemical characteristics of the placental bed trophoblast suggest a stromal migration of trophoblast to the proximal (decidual–myometrial) part of the spiral artery whereas the distal part might be invaded by intraluminal (upstream) invasion. The hypothesis is put forward that in the absence of physiological changes a disturbed stromal migration is caused by intrinsic (trophoblastic) or extrinsic (interstitial of vascular) factors expressed by the augmentation of the multinucleated cells at the decidual–myometrial junction.     

The role of leukemia inhibitory factor in tubal ectopic pregnancy

Introduction

Ectopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy.

Methods

We immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined.

Results

LIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth.

Discussion

Our data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion.

Conclusion

LIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.     

Screening test for mouse blastocysts as an index of the vitality of embryos

Summary We attempted to investigate the viability of mouse blastocysts in vitro, comparing with embryo transfer to recipient mice. Despite normal appearances of the blastocysts, the rate of trophoblastic outgrowth of blastocysts fertilized in vitro was lower than that of blastocysts fertilized in vivo. However, the rate of transferred blastocysts developing into fetuses did not differ significantly between in vivo and in vitro fertilized blastocysts. It is considered that blastocyst hatching, attachment, and trophoblastic outgrowth in vitro can serve as indices for the vitality of individual embryos.     

Villous explant culture: characterization and evaluation of a model to study trophoblast invasion.

The mechanisms that control invasion of cytotrophoblast (CTB) cells into the maternal decidua and myometrium with transformation of the maternal spiral arteries are not fully understood, but oxygen is thought to be a key factor. We carried out a semiquantitative evaluation of an explant culture model for use in the study of trophoblast proliferation and invasion. Explants of human villous tissue (6-9 weeks of gestation) cultured on Matrigel in both standard culture conditions (18% O2) and in a low oxygen environment (2% O2) produced regions of outgrowth, of cytotrophoblast cells from villous tips and migration of cells into the Matrigel. The number of sites of outgrowth and migration, area of outgrowth, and extent of migration of cells into the Matrigel tended to increase throughout the culture period (144 h) but varied between explants from the same placenta and those from different placentas. There were no significant differences in the number of sites of outgrowth or migration scores in explants cultured in a low oxygen environment compared to those cultured in standard conditions. This study highlights the importance of careful validation, design and interpretation of experiments using in vitro culture systems, particularly those investigating the regulatory role of oxygen.     

Sera from women with histories of repeated pregnancy losses cause abnormalities in mouse peri-implantation blastocysts

Incubation of peri-implantation mouse blastocysts in the presence of untreated human sera resulted in destruction of the blastocysts. Heating the serum resulted in deactivation of the non-specific toxic factor. Whereas heat-treated serum from women with normal obstetrical histories, and men, supported normal trophoblast attachment and outgrowth, sera from women with reproductive dysfunction resulted in inhibition of attachment or disruption of the trophoblast cells. The inner cell masses were not adversely affected by the sera which were toxic to trophoblast. Fractionation of a serum sample by affinity chromatography resulted in removal of the toxic factor with the IgG fraction. Absorption of the toxic serum with human trophoblast membranes resulted in serum that supported trophoblast outgrowth indicating that the toxic factor was an antibody directed against trophoblast antigens.     

Does hypercapillarization influence the branching pattern of terminal villi in the human placenta at high altitude?

This study tests the hypothesis that exposure of the placenta to hypobaric hypoxia at altitude results in an altered branchingpattern of the villous tree. Histological material from 20 term placentae delivered at altitudes over 3000 m was compared with matched controls from 500 m. Estimates of the mean star volume of intermediate and terminal villous domains were 1.40 × 10⁶ μm³ (s.d. 0.63) in the high altitude group and 1.90 × 10⁶ μm³ (s.d. 0.34) in the controls (F=9.07, P<0.005 The volume fraction of the villous tree occupied by trophoblastic bridges and syncytial knots was 8.1 per cent (s.d. 3.5) in the high altitude group and 3.2 per cent (s.d. 1.6) in the controls (F=29.45, P<0.0001). Previous studies have shown that the majority (80 per cent) of bridges are artefacts caused by the plane of section passing tangentially through the trophoblast layer at points of villous bending or branching. The results are, therefore, consistent with the hypothesis that peripheral villi are shorter, knob-like protrusions at high altitude, clustered more closely together. This modified branching pattern was confirmed by scanning electron microscopy. The change in architecture may be due to enhanced angiogenesis stimulated by the lower partial pressure of oxygen prevailing at high altitude.     

The rat placenta expresses paternal class I major histocompatibility antigens

The expression of paternally inherited class I MHC antigens on the placental trophoblast of the rat has been investigated using a mouse anti-rat monoclonal antibody (MN4-91-6) in an indirect immunoperoxidase labelling assay on cryostat sections. Strong specific staining was obtained on the spongy zone trophoblast of the mature placenta from DA male (RT1a) X PVG female (RT1c) matings. In marked contrast, no staining was observed on the labyrinthine trophoblast nor on the trophoblastic giant cells at any stage of gestation from 8 to 19 days post-coitum. None of the trophoblastic cell populations at any stage of gestation were reactive with an anti-class II monoclonal antibody. Class I positive endovascular cytotrophoblast cells were present in the maternal arterial sinusoids of the decidua. These findings imply that maternal immunoregulatory mechanisms must be essential for the survival of the placenta and fetus.     

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro. II. The role of gamma interferon in the responses of primary and secondary giant cells

The induction of paternal Class I and II MHC antigens by crude lymphokine preparations or purified recombinant gamma interferon was investigated on (C57BL/6J X CBA/H)F1 primary and secondary trophoblast giant cell outgrowths from 3.5-day post-coital (pc) blastocyst and 7.5-day pc ectoplacental cone preparations, respectively, using sensitive immunogold labelling techniques and electron microscopy. Class I MHC (but not Class II) antigens could readily be induced on secondary trophoblast giant cells, by incubation in vitro with gamma interferon for 40 h. However, repeated attempts to induce detectable MHC antigens on primary trophoblast giant cells failed. Mock-treated (C57BL/6J X CBA/H)F1 secondary trophoblast giant cell control preparations failed to express detectable MHC antigens. These findings suggest that, at the time of implantation, there is a time window during which MHC antigens are neither expressed constitutively nor are inducible by soluble factors which normally modulate cell surface MHC antigen concentration.     

Phagocytic properties of cultured murine trophoblast

The phagocytic potential of cultured trophoblast from early (ectoplacental cone (EPC); day 7.5 post coitum) and mid-term (placenta; day 12 to 14 post coitum) pregnancy in the mouse has been examined using a variety of test particles and culture conditions. In suspension, small numbers (less than 1 per cent) of large placental trophoblast cells showed limited phagocytic uptake of Staphylococcus aureus but not of opsonized sheep red blood cells (RBCs). In contrast, trophoblast phagocytosis was never seen in monolayer placental cell culture. Placental macrophages consistently exhibited phagocytic uptake of both opsonized sheep RBCs and S. aureus under these conditions. In monolayer culture, EPC trophoblast phagocytosed S. aureus, but there was only limited uptake of RBCs (mouse or sheep) or spermatozoa. When cultured in a three-dimensional matrix (blood and plasma clots), however, EPC trophoblast demonstrated extensive phagocytosis of both RBCs and sperm. These results are discussed with reference to the use of in vitro systems for examining developmental processes, and a possible role for trophoblast phagocytosis in early gestation is proposed.     

Treatment with gonadotropins impaired implantation and fetal development in mice

Purpose The effect of gonadotropins on implantation and fetal development in mice was investigated by superovulation with pregnant mare serum gonadotropin and human chorionic gonadotropin. In a previous study fetal growth was found to be highly retarded.Results Assessment of implantation in vivo revealed that late implantation did occur. Gestational length was highly extended, the mean number of live fetuses per pregnant mouse was lower and their mean weight significantly reduced. In vitro experiments revealed no significant difference in the rate of blastocyst adhesion and trophoblast outgrowth development. Immunohistochemical staining, however, showed that blastocysts from superovulated mice had smaller trophoblastic outgrowths than control embryos. Staging embryonic development at the time of flushing, however, revealed retarded embryo development in vivo in hormone-treated mice. After correlation with embryonic stage at the beginning of the culture, there was no difference in the size of trophoblastic outgrowths.Conclusion Treatment with gonadotropins impaired implantation and embryonic/fetal development. Changes in maternal milieu, rather than in embryo quality, may be responsible for the adverse effects observed.     

Mechanics of human blastocyst hatching in vitro

Critical examination of 30 blastocysts by transmission electron microscopy at various stages of blastulation and hatching, has revealed the presence of specialized, plump, trophoblastic cells at the points of hatching, which seem to aid in initial breaking of the zona pellucida (ZP) and then widen its opening to permit the progressive emergence of the embryo in amoeboid fashion, when it acquires a characteristic dumb-bell shape. These cells are named 'zona-breaker' cells and their characteristics are described. Normally, trophoblast cells in expanding blastocysts are flattened (squamous), forming a continuous robust epithelium with specialized cell junctions. Bundles of tonofilaments anchor onto desmosomes, forming a terminal web. Proper expansion of blastocysts by intake of fluid into the blastocoele causes an increase in internal hydrostatic pressure that stretches the trophoblast epithelium leading to an enlargement of its volume two- to three-fold, consequently thinning the zona prior to hatching. This is an important prerequisite to normal hatching. The blastocysts usually hatch out at the pole opposite the inner cell mass (ICM), though a few hatch out at the embryonal pole or elsewhere. In all cases zona-breakers seem to play a vital role in the hatching process.     

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-μn pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of a5 and a6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl CAMP (db cAMP) and the CAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell (JEG-3) invasion. The collagenolytic activity of trophoblastic cells (IEG-3) was increased by hCG stimulation. No changes were shown in the expression of α5 and α6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.