Early Placental Trophoblast Research Articles

ELAC2 Functions as a Key Gene in the Early Development of Placental Formation Based on WGCNA.

The placenta plays a crucial role in mammalian fetal growth. The most important cell type in the placenta is the trophoblast cell. Many genes have been reported to play important functions in the differentiation of early placental trophoblast cells. Weighted gene co-expression network analysis (WGCNA) is a systematic biological method for describing the correlation patterns among genes across microarray samples. We used WGCNA to screen placental trophoblast development-related genes, and through experimental confirmation, we showed that, among these genes, ELAC2 may play an important regulatory role in the early development of mammalian placental formation. ELAC2 regulates early placental trophoblast differentiation by affecting cell migration and cell proliferation. In addition, ELAC2 may be involved in regulating cell migration processes in a manner that affects epithelial mesenchymal transition (EMT).

In vitro fertilization-embryo transfer affects focal adhension kinase signaling pathway in early placenta

To study the effects of in vitro fertilization-embryo transfer (IVF-ET) technique on gene expression of focal adhension kinase (FAK) signaling pathway in early placental trophoblast cells, and to explore the effects of IVF-ET technology on the development and function of early placenta. We collected 7-8 weeks of gestation placenta tissue as a study group by ultrasound guided reduction of fetal from double embryo transfer under IVF-ET technology. In the control group, placenta tissues were obtained from the spontaneous abortion of natural pregnancy twin 7-8 weeks. Microarray hybridization analysis was performed on the placenta tissue of the two groups using the Affymetrix HG-U133 Plus 2.0 gene chip. Eight differentially expressed genes were identified by real-time quantitative polymerase chain reaction (qRT-PCR), and unsupervised clustering analysis and functional bioinformatics analysis were performed for the differentially expressed genes. Twenty-eight cases of IVF-ET reduced fetal villi and 8 cases of spontaneous abortion villi were collected. A total of 8 placental villi were detected by the gene chip. Compared with the natural pregnancy control group, 32 differentially expressed genes in the placental FAK signaling pathway were expressed in IVF-ET. The differential expression was greater than or equal to 2 times, of which 12 genes were up-regulated and 20 were down-regulated. The qRT-PCR showed that the expression of the 8 genes in FAK signaling pathways of IVF-ET was significantly different from that in the placenta of natural pregnancy, which was consistent with the result of the gene chip detection. The FAK signal pathway gene localization showed that the FAK gene was mainly located in the upstream of the signal pathway in the placenta of IVF-ET. The placental trophoblast cells maintained the FAK signaling pathway function through gene expression compensation. There are gene expression differences in the FAK signaling pathway between the IVF-ET derived early placenta and the natural pregnancy placenta. The differentially expressed genes are involved in many key functions of the FAK signaling pathway and affect the early development and function of the IVF-ET placenta, while the placental trophoblast cells change gene expression for interference to compensate for IVF-ET technology itself, maintain normal function of the FAK signaling pathway, and satisfy the need for placental and fetal development.

Matrix metalloproteinase-2 and -9 secretion by the human JAR choriocarcinoma cell line is stimulated by TNF-<i>α</i>

The JAR choriocarcinoma cell line share many of the characteristics of early placental trophoblast cells including the invasion properties. Matrix metallo-proteinases (MMPs), the main actors of matrix proteolysis, are involved in normal invasion as well as in the invasive character of tumor cells and the metastase formation. Tumor necrosis factor-α (TNF-α) is present in the placental environment and TNF-α levels are elevated in some placental pathologies. In the present work, we addressed whether TNF-α is a modulator of JAR cell MMP secretion. Following TNF-α stimulation, zymographic analysis showed the increased secretion of the active form of MMP-2 and to a lesser extent proMMP-2 and MMP-9. In addition, MMP-2 gene expression only increased slightly whereas MMP-9 and TIMP-1 transcripts were undetectable. This suggests that TNF-α may modulate the secretion of MMPs independently of MMP gene expression control.

IGF2, IGF binding protein 1, and matrix metalloproteinases 2 and 9 in implantation-stage endometrium following immunoneutralization of vascular endothelial growth factor in the rhesus monkey

Blastocyst implantation in the rhesus monkey is inhibited by administration of antibody against vascular endothelial growth factor (VEGF) A during peri-implantation period with no change in the circulatory concentrations of estradiol, progesterone, and VEGF. In this study, we have investigated the effect of administration of a MAB to VEGFA on days 5 and 10 after ovulation upon the mRNA expression, immunopositive protein expression, and immunohistological localization of IGF2, IGF binding protein 1 (IGFBP1) and matrix metalloproteinases (MMPs) 2 and 9 in the implantation-stage endometrium collected on day 13 after ovulation from fecund cycles of rhesus monkeys. The comparison between isotype-matched IgG (control; n=8)- and VEGF antibody (VEGF Mab; n=8)-treated animals revealed higher (P<0.05) IGF2 in lacunar and villous syncytiotrophoblasts, trophoblast cell columns, migrating extravillous trophoblast cells, and endovascular trophoblast cells in control animals, but with no change in the various cell types of maternal endometrium between the two groups. No change in IGFBP1 expression in the endometrium was observed between the two groups. MMPs 2 and 9 were detected in syncytiotrophoblast in lacunae and villi, trophoblast cell columns, and extravillous trophoblast cells in control samples. MMP9 transcript expression in maternal endometrium and its immunopositivity in endometrial stroma and trophoblast cells were lower (P<0.05) with no change in MMP2 level in VEGF Mab-exposed samples compared with those in control samples. A functional network involving VEGF, IGF2, and MMP9 in early placental trophoblast cells and maternal endometrium appears to be important for normal placentation.

Regulation of early trophoblast differentiation – Lessons from the mouse

The earliest stages of trophoblast differentiation are of tremendous importance to mediate implantation and to lay the anatomical foundations for normal placental development and function throughout gestation. Yet our molecular insights into these early developmental processes in humans have been limited by the inaccessibility of material and the unavailability of trophoblast cell lines that fully recapitulate the behaviour of early placental trophoblast. In this review we highlight recent advances that have come from the study of distinct stem cell types representative of the embryonic and extraembryonic lineages in the mouse, and from the study of mouse mutants. These models have revealed the presence of intricate transcriptional networks that are set up by signalling pathways, translating extracellular growth factor and cell positional information into distinct lineage-specific transcriptional programmes. The trophoblast specificity of these networks is ensured by epigenetic mechanisms including DNA methylation and histone modifications that complement each other to define trophoblast cell fate and differentiation. Despite the anatomical differences between mouse and human placentas, it seems that important aspects of early trophoblast specification are conserved between both species. Thus we may be able to build on our insights from the mouse to better understand early trophoblast differentiation in the human conceptus which is important for improving assisted reproductive technologies and may enable us in the future to derive human trophoblast stem cell lines. These advances will facilitate the investigation of genetic, epigenetic and environmental influences on early trophoblast differentiation in normal as well as in pathological conditions.

ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

The first definitive cell fate decision in development occurs at the blastocyst stage with establishment of the trophoblast and embryonic cell lineages. In the mouse, lineage commitment is achieved by epigenetic regulation of a critical gatekeeper gene, the transcription factor Elf5, that reinforces placental cell fate and is necessary for trophoblast stem (TS) cell self-renewal. In humans, however, the epigenetic lineage boundary seems to be less stringent since human embryonic stem (ES) cells, unlike their murine counterparts, harbour some potential to differentiate into trophoblast derivatives. Here, we show that ELF5 is expressed in the human placenta in villous cytotrophoblast cells but not in post-mitotic syncytiotrophoblast and invasive extravillous cytotrophoblast cells. ELF5 establishes a circuit of mutually interacting transcription factors with CDX2 and EOMES, and the highly proliferative ELF5(+)/CDX2(+) double-positive subset of cytotrophoblast cells demarcates a putative TS cell compartment in the early human placenta. In contrast to placental trophoblast, however, ELF5 is hypermethylated and largely repressed in human ES cells and derived trophoblast cell lines, as well as in induced pluripotent stem cells and murine epiblast stem cells. Thus, these cells exhibit an embryonic lineage-specific epigenetic signature and do not undergo an epigenetic reprogramming to reflect the trophoblast lineage at key loci such as ELF5. Our identification of the trophoblast-specific transcriptional circuit established by ELF5 will be instrumental to derive human TS cell lines that truly reflect early placental trophoblast and that will be most beneficial to gain insights into the aetiology of common pregnancy complications, including intra-uterine growth restriction and pre-eclampsia.

Effects of recombinant H2 relaxin on the expression of matrix metalloproteinases and tissue inhibitor metalloproteinase in cultured early placental extravillous trophoblasts

Relaxin promotes softening of the uterine cervix and inhibits uterine contractility in rats, mice and pigs. Little information, however, is available about the role of relaxin in humans. In 2002, LGR7 and LGR8 were discovered to be receptors for relaxin and those receptors were identified in the human placenta. Thus, in this study, effects of recombinant H2 (rH2) relaxin on human early placental extravillous trophoblasts (EVTs) were examined. Isolation of EVTs from early placental trophoblasts was performed using the procedures established in our laboratory. After 48-h subculture, the presence of relaxin receptors in cultured EVTs was characterized by RT-PCR and immunoblotting. The cultured EVTs were treated with different doses (0.3-3 ng/ml) of rH2 relaxin for 24 h. The effects of rH2 relaxin on MMP-2, -3, -9 and TIMP-1 mRNAs levels were examined by real-time RT-PCR. RT-PCR and immunoblotting revealed that relaxin receptors are present in early placental EVTs. Treatment with rH2 relaxin increased MMP-2 and -9 mRNAs levels and decreased TIMP-1 mRNA levels in cultured EVTs, whereas rH2 relaxin did not affect MMP-3 mRNA levels. These results suggest that relaxin may promote the invasive potential of early placental EVTs through up-regulating MMP-2, -9 mRNAs and down-regulating TIMP-1 mRNA in EVTs.

SAPKγ/JNK1 and SAPKα/JNK2 mRNA transcripts are expressed in early gestation human placenta and mouse eggs, preimplantation embryos, and trophoblast stem cells

To test early-gestation human placenta, a human trophoblast cell line, mouse eggs, preimplantation embryos, and a mouse trophoblast cell line for the expression of mRNA transcripts for stress-activated protein kinase/c-Jun N-terminal kinase (SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3). Whole RNA was isolated from the tissue sources listed above and control tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to assay for the qualitative and semiquantitative presence of SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3. None. None. None. The presence and magnitude of amplimer amounts in gels or gene hybridization on Affymetrix cDNA arrays of RT-PCR products of reactions for SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3. SAPKgamma/JNK1 and SAPKalpha/JNK2 mRNA transcripts are present in early-gestation human placenta, a human trophoblast cell line, mouse eggs, preimplantation embryos, and a mouse trophoblast cell line at levels similar to positive control levels. SAPKalpha/JNK2 is expressed at the highest level of the three transcripts in the family. SAPKbeta/JNK3 is present at levels that are 1/100-1/1,000 those of the positive control and in some cases at the apparent level of the negative control (previously measured by the less-sensitive Northern blot analysis). Analysis with an Affymetrix cDNA array suggested that SAPKalpha/JNK2 and 38 kDa mitogen-activated protein kinase had the highest mRNA expression measured for each of three family members. Mitotic placental trophoblast cell lines and primary conceptus/embryo samples containing early placental trophoblasts express SAPKalpha/JNK2 at higher levels than SAPKgamma/JNK1, but not (only low background levels of) SAPKbeta/JNK3 mRNA transcripts. This suggests that SAPKgamma/JNK1 and SAPKalpha/JNK2 may be important mediators of stress-induced responses in early implanting conceptuses that could mediate embryo loss.

Increase in the Expression of c-erb A and c-erb B mRNAs in the Human Placenta in Early Gestation: Their Roles in Trophoblast Proliferation and Differentiation

Previous studies have shown that human placental trophoblast is the site of the reception of thyroid hormone and epidermal growth factor (EGF) and that cellular levels of thyroid hormone receptor and EGF receptor are notably higher in early placenta than those in term placenta. On the other hand, it is now evident that a close similarity exists between thyroid hormone receptor and erb A protein and between EGF receptor and erb B protein. In the present study, we have therefore analyzed staged human placentas by Northern blot and In situ hybridization with c-erb A and c-erb B cDNA probes.In situ hybridization revealed that c-erb B mRNA is localized in the syncytiotrophoblast, consistent with our previous results, which showed the cytological localization of EGF receptor in developing human placenta. Northern blot analysis demonstrated that placental RNA contains c-erb A transcripts of 5.7-kb and c-erb B transcripts of 10.0-kb and 5.6-kb and that the amounts of the c-erb A and c-erb B transcripts vary remarkably during the course of gestation, being most abundant in early placenta, more abundant in midterm placenta, and least abundant in term placenta. It is therefore likely that the increased expression of c-erb A and c-erb B gene in early placental cells indicates an increase in cellular levels of thyroid hormone receptor and EGF receptor in early placental trophoblasts. These findings suggest an important role for the c-erb A and c-erb B oncogenes in the induction of trophoblast proliferation and differentiation in early gestation.

Human early placental trophoblasts produce an epidermal growth factor-like substance in synergy with thyroid hormone

Previous studies have shown that human trophoblast is the site of epidermal growth factor (EGF) localization, reception and action and that thyroid hormone exerts similar effects on trophoblasts endocrine function as observed with EGF. Thus, the present study was designed to examine local production of an EGF-like substance in synergy with thyroid hormone by early placenta. Explants of normal early (7-8 weeks) placentas were cultured in a serum-free condition in the presence or absence of L-triiodothyronine (T3), with or without cycloheximide for 4 days. The conditioned media were dialyzed, lyophilized, acidified and chromatographed over a Sephadex G-75 column equilibrated with 1 mol/l acetic acid. EGF was measured by a specific RIA for human EGF. Fractionation of the serum-free conditioned media resulted in the elution of immunoreactive EGF with an apparent molecular weight of 9,000 which is larger than [125I] human EGF. The addition of T3 (10(-8) mol/l) resulted in increased secretion of immunoreactive EGF by placental explants. By contrast, the addition of cycloheximide (5 x 10(-5) mol/l) dramatically reduced the secretion of immunoreactive EGF. The similarity of the immunoreactive EGF material to authentic human EGF was supported by parallel displacement in human EGF-RIA. These results suggest that human early placental trophoblast is capable of producing an EGF-like substance and that thyroid hormone enhances the local production of the EGF-like substance. This suggests that an autocrine/paracrine control system, wherein EGF serves as the signal in regulating placental growth and function in synergy with thyroid hormone, exists in human early placenta.

Control mechanisms for gonadotrophic hormone production in vitro.

The BeWo human trophoblastic cell line produces high levels of multiple pregnancy hormones comparable to the early placental trophoblast. This trophoblastic cell synthesizes large quanties of HCG (Human Chorionic Goadotropin) and small quantities of HPL (Human Placental Lactogen). The glycoprotein hormone synthesis was inhibited by incubation of gonadotropin-secreting cells with endogenous precursors of progesterone in the trophoblastic cell. Complete cessation of HCG production occurred after 72 hours of precursor incubation. Electron microscopy performed at the time of cessation of HCG production revealed almost total glycogen depletion.