The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia.     

The cytoplasmic expression of E-cadherin and beta-catenin in bovine trophoblasts during binucleate cell differentiation

Binucleate cells are endocrine cells generated by the acytokinesis and endoreduplication of the trophectoderm in the ruminant placenta. These cells are migratory and secrete hormones into the maternal circulation after fusing with uterine epithelial cells. In this study, we performed immunohistochemistry for E-cadherin and beta-catenin in bovine placenta and a bovine trophoblast cell line (BT-1). We found that E-cadherin and beta-catenin were distributed not only at the cell to cell boundary but throughout the cytoplasm in binucleate cells, although they were concentrated at the cell to cell boundary in epithelial cells in bovine placenta. Moreover, beta-catenin was detected in the nuclei of binucleate cells. Binucleate cells after fusion with uterine epithelial cells (feto-maternal hybrid cells) in the maternal side showed no intracellular expression of E-cadherin and beta-catenin. The transformation into binucleate cells in the BT-1 cell line was also accompanied by the cytoplasmic accumulation of E-cadherin and beta-catenin. We further demonstrated that levels of cytoplasmic beta-catenin were well correlated with the DNA content of binucleate cells in BT-1. The dynamic changes in the distribution of E-cadherin and beta-catenin suggest an important role in binucleate cells, including the rearrangement of cadherin-mediated cell adhesions during cell migration and the onset of endoreduplication probably via the nuclear transfer of beta-catenin.     

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.     

Placental polyploidization: a comparative study in the mouse and the guinea pig

Initially diploid, pure trophectodermal derivatives were dissociated and grown in culture. Overthe 72-hour time course in vitro, uninucleate, binucleate and a small number of multinucleate cells appeared. Moreover, the pattern of binucleation found in these trophoblast cultures resembled that seen during the development of the mouse liver. Thus, the binucleates displayed a progressive increase in nuclear DNA content and the increased DNA values ranged from 2c to 32c. Furthermore, the proportion of uninucleate and binucleate cells changed systematically with growth in vitro and the final binucleate cell population became, on average, 10 to 15 per cent of the total. These results, together with those of other studies, suggest that mouse trophoblast can initially become giant through a binucleate phase.     

Mechanisms of growth in hydatidiform moles

Nuclear morphology and DNA synthesis were analyzed to determine the mechanism through which hydatidiform moles proliferate. Hydatidiform moles are characterized by a great variation in nuclear morphology and size. There are cells with small nuclei of variable size that have chromocenters and Barr bodies which do not undergo DNA synthesis or mitosis, as well as cells in the diploid range that have evenly stained nuclei that display numerous mitoses and a high proportion of interphase nuclei in the process of DNA synthesis. Nuclei in the medium range show classical endomitotic stages. Endomitotic nuclei in endometaphase do not label with tritiated thymidine; endoanaphase nuclei may have one or a few chromosomes labeled, and endotelophase nuclei are heavily labeled. Nuclei that are evenly stained and are in the medium- to giant-size range label differently, depending upon their size. Many of the medium-sized nuclei are labeled, indicating DNA synthesis; the large nuclei are rarely labeled, and the giant nuclei are never labeled. The growth of a hydatidiform mole appears to be the result of normal mitosis and cytokinesis, as well as polyploidization and accompanying cell enlargement achieved through andomitosis and endoreduplication.     

Control of trophoblastic growth

The investigations described herein have been aimed at elucidating the ways in which trophoblastic proliferation, endoreduplication, and multinucleation are controlled. The early post-implantation, trophectodermal derivatives of the mouse remain diploid and continue to divide even after they move away from the inner cell mass. This is due, in part, to the maintenance of close cell contacts within these tissues which suppress the giant cell change and keep the early post-implantation trophoblast in a diploid, non-giant state. However, such close cell contacts are not able to promote continued trophoblastic cell division. This suggests that other factors, such as contact with inner cell mass (ICM) derivatives, are needed to sustain trophoblastic proliferation. Conversely, the loss of close cell contacts and an appropriate tissue shape as well as the absence of ICM derivatives promote giant cell transformation. In the mouse, giant cell transformation may begin through a binucleate phase and subsequently proceed via endomitosis and/or endoreduplication. However, in species other than the mouse, trophoblastic giant cells may not arise through a binucleate phase but may, instead, become multinucleate and syncytial. Furthermore, in some species (e.g. ruminants), trophoblastic multinucleation may be consequential upon a multipolar mitosis without concomitant cytokinesis whilst in others (e.g., man) it may depend upon a cell fusion event with secondary syncytialization.The growth of guinea pig trophectoderm and its derivatives appears to be under ICM control. Thus the multilayered, abembryonic structure found within the guinea pig blastocyst, called the attachment cone, and the early post-implantation trophectodermal derivatives, presumed to be homologous to mouse extra-embryonic ectoderm, both stop dividing and become giant when isolated from the ICM and ICM-derived tissues.The development of primitive endoderm proceeds in a manner similar to the growth of trophectoderm. Thus the giant cell change appears to be a general feature of trophectodermal and primitive endodermal growth during mouse and guinea pig embryogenesis. Moreover, the giant cell change appears to begin in endoderm after trophectoderm has begun to degenerate. Endodermal polyploidization may occur during the growth of primitive, ICM-like, embryonal carcinoma cells as they form tissues with the biochemical and morphological features of normal primitive endoderm. Tumour-derived and normal primitive endoderm may also grow in a manner similar to trophectoderm since they both contain binucleate giant cells, possess polyploid mitotic figures, and display growth patterns potentially attributable to changes in homotypic and/or heterotypic cellular interactions.     

Quantitative analysis of the spreading of the mouse trophoblastin vitro: a model for early invasion

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study weanalysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear: cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40–46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 × C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 × C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96–120 h and no ‘catch-up’ was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.     

Characteristics of trophoblastic tissue derived from in vitro culture of preimplantation embryos of the common marmoset monkey

The features of trophoblastic tissue derived from the in vitro culture of marmoset monkey embryos have been described. Long-term trophoblast cultures (in excess of three years in one case) were established from the primary trophoblast monolayer of four of 38 embryos; division of one of these embryos produced two long-term cultures. The trophoblast cells retained their ability to synthesize and secrete chorionic gonadotrophin (CG) during maintenance in vitro and were capable of prolonging the luteal phase when transferred to the uterus of marmosets. A characteristic feature of the cultures was the formation of multiple fluid-filled vesicles enclosed by a single layer of cytotrophoblast cells and attached to the culture dish by a small monolayer of syncytiotrophoblast cells. The tissue was propagated by cutting vesicles into small pieces and placing into a fresh culture dish; attempts to subculture using single-cell suspensions were unsuccessful. These cultures provide a convenient source of marmoset CG for purification as well as an in vitro system for studying other secretory products of primate trophoblast.     

Establishment of mouse embryo culture system on collagen gel layer

In order to study the morphological features of mouse embryos in the early developmental stage, we first established an in vitro culture system applying a collagen gel layer, and then observed the morphology of the embryos cultured in this system. Embryos after hatching were attached to the collagen gel layer and grew to the egg cylinder stage. By means of morphological analysis of embryos cultured for 3 or 4 days, some interesting characteristics, such as processes and villi of the mural trophoblast, lacunae formation in the mural trophoblast, steroid synthesis of the mural and polar trophoblasts and desmosome or intermediate junctions between the mural and the polar trophoblasts, were revealed. Moreover, no morphological difference was observed between cells derived from the inner cell mass. From those findings, it has been concluded that the established culture system is useful in observing the morphology of early embryonal development and also in maintaining the viability of the embryo.     

Evaluation of Chang's culture medium for mouse in vitro fertilization and embryonic development

Chang's medium [with and without human serum (HS)] was compared with T6 medium [with and without bovine serum albumin (BSA)] for in vitro fertilization (IVF) and development of two-cell mouse embryos to the blastocyst stage. Chang's medium without any supplementation gave significantly better fertilization rates (83.3%) than Chang's with 10% HS (76.4%) or T6 and BSA (76.6%) (P <0.01). In a separate experiment 87.7% of the two-cell mouse embryos developed to the blastocyst stage in Chang's medium supplemented with 10% HS, 76.6% of the embryos developed to the blastocyst stage and 17.2% stopped development after the morula stage. After 72 hr in vitro hatched trophoblast and inner-cell-mass cells from 26.5 and 30.8% of the embryos grown in Chang's medium (with and without HS) attached to the plastic culture dishes and grew to form a mixed monolayer of epithelioid and fibroblastic cells. Chang's medium can thus be successfully used for IVF and growth of mammalian embryos. Further, inner cell mass and trophoblast cell lines could be established for various reproductive studies using this medium.     

The killing of mouse trophoblast cells by granulated metrial gland cells in vitro

Mouse placental cell preparations have been maintained in culture, and the types of cell that attached to the culture dish were classified according to morphological criteria. However, these morphological criteria were insufficient to determine from which trophoblast layer in the placenta all of the types of cell found in the cultures originated. Some placental cell preparations were co-cultured with granulated metrial gland (GMG) cells and these cultures were studied using time-lapse video. Various responses to contacts between GMG cells and trophoblast cells were observed. These responses included the killing of trophoblast cells by GMG cells.     

Isolation and characterization of trophoblast from murine placenta

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.     

From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: a study of nuclear number using the disector.

A method is presented for obtaining assumption-free estimates of the number of nuclei in the trophoblast of the human placenta and for defining the size of the trophoblast proliferative unit (TPU). The method relies on the disector, a stereological device for counting arbitrary particles in 3-dimensional space using pairs of parallel sections separated by a known distance. It is applied to investigate factors which contribute to trophoblast growth from 13 weeks of gestation to term. Physical disectors were sampled systematically using adjacent 4-4.6 microns thick paraffin sections. Nuclei in the trophoblast (syncytial and cellular) were counted if they appeared in an unbiased counting frame on one section but were absent from the adjacent section. Nuclear packing densities were converted to absolute numbers of nuclei by using placental volume as the reference space. At 37-39 weeks, the average placenta contained 6.4 x 10(10) trophoblast nuclei of which 90 per cent were located within the syncytium and the remainder in the cytotrophoblast. From a knowledge of total trophoblast volume, it was found that each nucleus is associated with 970 microns3 of trophoblast and each cytotrophoblast cell with 11,000 microns3. The latter may be regarded as the volume of a TPU. From 13 weeks of gestation to term, there was a ninefold increase in nuclear number but the trophoblast volumes associated with nuclei, including the size of the TPU, remained constant. Growth of trophoblast is purely hyperplastic and occurs by recruitment of new TPUs.     

Oxysterols inhibit differentiation and fusion of term primary trophoblasts by activating liver X receptors

Oxygenated cholesterol metabolites known as oxysterols display potent biological activities ranging from regulation of lipid homeostasis to cytotoxicity. Oxysterols have previously been shown to inhibit the invasion of first trimester trophoblasts, an effect which involves activation of the nuclear liver X receptors (LXRs). In the present study, we investigated the effects of several oxysterols on syncytialisation (differentiation and fusion) in term placental trophoblasts. Treatment of cultured term primary trophoblast cells with oxysterols [25-hydroxycholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol] and the synthetic LXR agonist T0901317 at non-toxic doses decreased expression of GCM-1 and HERV-W mRNA and reduced hCG secretion and placental alkaline phosphatase activity, indicative of diminished trophoblast differentiation. Furthermore, treatment with these compounds also decreased cell fusion measured by E-cadherin immunostaining and quantification of syncytialised nuclei. Treatment with an LXR antagonist (geranylgeranyl diphosphate) abrogated the inhibitory effects of oxysterols and T0901317 on trophoblast syncytialisation indicating that these effects are mediated by LXR. These findings suggest that oxysterols impair differentiation and fusion of term trophoblast cells via an LXR-dependent mechanism.     

Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome

OBJECTIVES: The epithelium of the human placenta comprises an inner cytotrophoblast (CT) which proliferates and fuses with the outer differentiated syncytiotrophoblast (ST). Turnover has been studied focussing on second and third trimester placentas but with a paucity of data describing the normal first trimester trophoblast. The aim of this study was to compare the nuclear CT:ST ratio in normal and pathological pregnancy and thus establish the relationship between cytotrophoblast and syncytiotrophoblast nuclear number during early gestation. METHODS: Archival first trimester material from placentas from healthy pregnancy and recurrent miscarriage (anti-phospholipid syndrome) was stained with H&E, cytokeratin-7 and Mib-1. The area of trophoblast as a fraction of total villous area was calculated and the number of sectioned cytotrophoblast and syncytiotrophoblast nuclei as well as the number of proliferating cytotrophoblast was evaluated. RESULTS: Normal features of trophoblast development during the first trimester (rise in trophoblast area, increase in number of syncytiotrophoblast nuclei, increase in number of proliferating cytotrophoblast, decrease in the nuclear CT:ST ratio) are absent/reversed in tissues from recurrent miscarriage (decreasing trophoblast area, constant number of syncytiotrophoblast nuclei, decreasing number of proliferating trophoblast, constant nuclear CT:ST ratio). CONCLUSIONS: Proliferation of cytotrophoblast in early gestation provides a pool of trophoblast stem cells critical for ongoing placental development. Premature cytotrophoblast differentiation in favour of syncytial fusion results in deficiencies of cytotrophoblast and rarification of villous trophoblast. Abnormal trophoblast differentiation in early gestation may be due to a premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation such as recurrent miscarriage.     

Identification of the white blood cell populations responsible for Th1 immunity to trophoblast and the timing of the response in women with recurrent pregnancy loss.

AIM: The purpose of this study was to identify the white blood cell populations responsible for Th1 immunity to trophoblast as evidenced in our in vitro assays following trophoblast activation and the timing of this response. STUDY DESIGN: Peripheral blood mononuclear cells (PBMC) were isolated from 32 nonpregnant women with a history of at least three prior first trimester spontaneous abortions of unknown etiology except their PBMC secreted Th1 embryotoxic cytokines in response to trophoblast stimulation. White blood cell populations were separated from PBMC by magnetic immunobeads and cultured with and without a trophoblast antigen extract. Supernatants from these cultures were added to two cell mouse embryos and after four days of culture assessment of blastocyst development was made to determine the white blood cell population responsible for embryotoxicity. In separate experiments trophoblast-activated PBMC culture supernatants were prepared over seven time points and individual Th1 cytokines (IL-2, INF-gamma, TNF-alpha) were measured by ELISA to determine the timing of the response to trophoblast stimulation. Results: The white blood cell (CD45) populations responsible for embryotoxicity in response to trophoblast were T cells (CD3) and NK (CD56) cells. Levels of IL-2 peaked in the first 24 h of culture followed by TNF-alpha and IFN-gamma levels which peaked at 96 h of culture. CONCLUSIONS: Our data demonstrated that the white blood cell populations responsible for embryotoxicity in our in vitro assays, were both T and NK cells. The kinetics of the cytokine response to trophoblast found in our study parallels the time course of a typical Th1 cytokine response. The profile of secreted cytokines support our hypothesis that trophoblast can produce Th1 immunity in some women with recurrent pregnancy loss that have embryotoxic effects in vitro.     

Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion

Fetal placental tissue from 11 days pregnant mice was dissociated in collagenase and DNase solution and then separated on a 40 per cent Percoll gradient. Trophoblast cells banded at a density of 1.05 g/ml. When cultured on rat tail collagen, these cells formed colonies of mono- and binucleate cells varying in size from 40 to 70 microns. At the time of plating, about 13 per cent of the trophoblast cells secreted mouse placental lactogen II (mPL-II) as determined by reverse haemolytic plaque assay. The ratio of mPL-II-producing cells increased significantly in culture and reached 63 per cent after 48 h. The secretion of mPL-II increased continuously during six days of culture, whereas the total protein release was highest after the first day, declined the second day and then remained relatively constant for the last four days of culture. The DNA content of the cells did not change significantly during the six-day period. When the trophoblast cells were incubated with insulin (1 ng/ml to 5 micrograms/ml), a modest but significant reduction in mPL-II secretion was observed. No change in the mPL-II secretion was seen when epidermal growth factor was administered to the culture in concentrations from 1 ng/ml to 10 micrograms/ml. It is concluded that this in vitro culture system is suitable for studying both mPL-II secretion and the differentiation of mPL-II-producing cells.     

卵裂期发育延缓胚来源的人类胚胎干细胞系的建立

目的观察体外受精（IVF）周期中24h内不发生卵裂的第3天（D3）发育延缓胚的体外发育潜能,为建立人胚胎干细胞库提供新的材料来源。方法通过囊胚序贯培养法将卵裂期发育延缓胚培养至囊胚,观察囊胚形成率及囊胚质量。用逐步logistic回归分析囊胚形成率与卵裂球数、卵裂球对称性及卵裂球碎片比率的相关性。用免疫外科法去除滋养细胞,将得到的原代克隆接种于小鼠胚胎成纤维细胞上,体外传代并鉴定。结果120枚卵裂期发育延缓胚培养出囊胚22枚（囊胚形成率18．7％）。第6天早期囊胚、囊胚、满囊胚、扩张胚、孵化胚及孵化后囊胚的比率分别为5．9％、23．5％、35．3％、23．5％、5．9％及5．9％。内细胞团分级及滋养外胚层分级均仅有1个为A级,其余均为B或C级。发育延缓胚的囊胚形成率与卵裂球数、卵裂球对称性相关,而与卵裂球碎片比率无关。获得内细胞团7个,原代克隆3个,成功培养出2株人类胚胎干细胞系（hESC）,并具有hESC的生物学特性：碱性磷酸酶（AKP）、阶段特异性胚胎抗原（SSEA）4、肿瘤排斥抗原（TRA）1-60、TRA-1-81呈阳性;悬浮培养可形成拟胚体;在严重免疫缺陷（SCID）小鼠体内可形成畸胎瘤;分别传45及35代后核型正常。结论部分发育延缓胚可以发育为囊胚,从而可为建立人胚胎干细胞系提供新的材料来源。