New Challenges in the Analysis of Gene Transcription in Bovine Blastocysts
Pablo Bermejo-ÃlvarezEva PericuestaAlberto Miranda BedateC. de FrutosSerafín Pérez‐CerezalesAC LucioD. RizosAlfonso Gutiérrez‐Adán
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Contents In the last years, enormous progress has been made in the analysis of gene transcription at the blastocyst stage. The study of gene expression at this early stage of development is challenging because of the very small amount of starting material, which limits the use of traditional mRNA analysis approaches such as Northern blot. Another problem is the difficulty for data normalization, particularly the identification of the best housekeeping gene with the lowest fluctuation under different developmental conditions. Moreover, the transcriptional analysis of embryo biopsies or individual embryos needs to take into consideration that the blastocyst is a transitional stage of development, which is composed of three different types of cells (trophoblast, epiblast and primitive ectoderm) with different patterns of gene expression, and that there are large differences between male and female blastocysts. In this review, we analyse the different specific and sensitive tools available to compare mRNA expression levels of specific genes at the blastocyst stage, and how the protocol and the analytical method used can influence the results dramatically. Finally, we describe future research challenges to identify candidate genes related to developmental competence of bovine blastocysts, not only in terms of pregnancy rates but also in relation to adverse long‐term consequences in the adult animal.Keywords:
Housekeeping gene
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Epiblast
Northern blot
Implantation is a process whereby the blastocyst becomes embedded in the endometrium. Firstly, the blastocyst must contact the epithelium, then the trophoblast cells will start invading the endometrium. In species with delayed implantation, the blastocyst requires activation before its attachment. Thus there are three stages involved in delayed implantation: blastocyst activation, trophoblast attachment and trophoblast invasion. The following account indicates what characterizes these three stages at a cellular and subcellular level and what suggestions structural analysis can give as to the mechanisms controlling these processes. DELAYED IMPLANTATION The blastocysts of some mammalian species are normally kept in a state of delay before implantation (Enders, 1963; Lanman, 1970). In some other species, for instance the mouse, a delayed implantation also can be obtained experimentally (Yoshinaga & Adams, 1966; Humphrey, 1967; Smith & Biggers, 1968; McLaren, 1971). This offers a biological
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Summary. Twelve blastocysts, collected 7–12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to > 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cells, forming an irregular layer of cells capping the epiblast. With continued increase in blastocyst size, polar trophoblast cells became isolated but maintained their characteristic apical endocytic structures. By Days 10–12, the scattered trophoblast cells showed evidence of deterioration, and vacuoles containing cell debris were common within the epiblast. It is suggested that polar trophoblast cells become scattered, rather than withdrawing as a unit, because they become more adherent to subjacent epiblast cells than to adjacent trophoblast cells. It is further suggested that most of the isolated cells are eventually phagocytosed by epiblast cells. Keywords: horse; blastocyst; trophoblast; differentiation; inner cell mass
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Abstract One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid 1 . Here we show that naive human pluripotent stem cells cultured in PXGL medium 2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development 3,4 .
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Abstract The spatiotemporal pattern of DNA synthesis in the mouse embryo at the beginning of metabolic dormancy was examined. Embryos were recovered from females at intervals following ovariectomy at 1100 hours on day 4 of pregnancy, incubated in vitro for 1 h in the presence of [ 3 H]thymidine, and prepared for light microscopic autoradiography. The proportion of labeled cells in the embryo remained high (40–60%) for 18 h after ovariectomy and then declined gradually to 12% by 96 h. However, analysis of individual cell subpopulations showed that the decline was not uniform in all regions of the blastocyst. Labeling was high over the inner cell mass (ICM) during all time intervals in the study, while labeling over the mural trophoblast cells declined sharply by 24 h after ovariectomy. Labeling over the polar trophoblast also declined but had values that were intermediate between the ICM and mural trophoblast regions of the blastocyst. These regional differences in DNA synthesis during the arrest of development suggest that intermediate steps are involved in control of DNA synthesis in the embryo and that the ICM may play a role in the different responses of the trophoblast cell populations.
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Summary. A hatched human blastocyst obtained after in-vitro fertilization and culture was examined by transmission electron microscopy and the ultrastructural features compared with hatched mouse and bovine blastocysts. The human blastocyst contained a continuous layer of trophoblast cells with apical junctional complexes, an inner cell mass and the beginning of a primitive endoderm layer. Certain ultrastructural features were common to the blastocysts of all 3 species; these included characteristic junction regions between adjacent trophoblast cells, an abundance of microvilli on the external surfaces of the blastocysts and the presence of well developed mitochondria and numerous ribosomes in the trophoblast cells. The features that were dissimilar included the extent of development of the endoderm layer, the appearance of the inner cell mass and the nature and extent of vesicular inclusions in the trophoblast cells.
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Abstract The germinal area of the rabbit blastocyst between 108 h postcoitum (pc) and 168 h pc has been examined by scanning electron and transmission electron microscopy. At 108 h and 120 h pc the polar trophoblast (Rauber's layer) is an intact epithelium overlying the epiblast of the inner cell mass. By 132 h pc the polar trophoblast cells begin to separate at multiple foci, exposing the underlying epiblast. Most of the polar trophoblast cells have become individually separated at 144 h pc. The villous, electron‐dense polar trophoblast cells can be easily distinguished from cells for the epiblast, which have smooth apical surface. By 162 h pc the polar trophoblast cells have disappeared from the germinal area. Before the polar trophoblast breaks up, the underlying epiblast cells are only loosely attached to one another. Concurrent with the disintegration of the trophoblast epithelium, the epiblast cells change in shape so that their lateral borders become closely apposed, and juctions develop to form a new epithelium. The epiblast becomes contiguous with the mural trophoblast, and thus the blastocyst does not lose its turgidity as the permeability sea is maintained. There are two classical theories on the fate of the polar trophoblast: the cells die, or they become incorporated into the epiblast as living cells. In newly exposed epiblast the presence of very large phagosomes, which are not found when the polar trophoblast is still intact, favors the first hypothesis and indicates that in the rabbit the epiblast is involved in the phagocytosis of the polar trophoblast.
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Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.
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Mammalian preimplantation development, which is the period extending from fertilization to implantation, results in the formation of a blastocyst with three distinct cell lineages. Only one of these lineages, the epiblast, contributes to the embryo itself, while the other two lineages, the trophectoderm and the primitive endoderm, become extra-embryonic tissues. Significant gains have been made in our understanding of the major events of mouse preimplantation development, and recent discoveries have shed new light on the establishment of the three blastocyst lineages. What is less clear, however, is how closely human preimplantation development mimics that in the mouse. A greater understanding of the similarities and differences between mouse and human preimplantation development has implications for improving assisted reproductive technologies and for deriving human embryonic stem cells.
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