The high rates of embryonic mortalities which follow in vitro production of ruminant embryos have emphasized the need for increased knowledge of early development. It is likely that early failures in embryonic development and placenta formation involve abnormal differentiation of mesoderm. The aim of this study was to investigate the pattern of expression of two T-box genes known to control the gastrulation process, Brachyury and Eomesodermin, by whole-mount in situ hybridization. To allow a more precise comparison of both expression patterns between embryos, we describe a new staging of pre-implanted ovine embryos by gross morphology and histology from pre-gastrulation stages to the beginning of neurulation. In pre-streak embryos primitive mesoderm cells delaminated in between the primitive endoderm and the epiblast. At that stage, no expression of Brachyury or Eomesodermin could be detected in the embryos. Early expression of both T-genes was observed by the early-streak stages in epiblast cells located close to the presumptive posterior pole of the embryos. Later on, during gastrulation both genes followed a pattern of expression similar to the ones described in other mammals. These observations suggest that other genes, which remain to be identified, are responsible for extra-embryonic mesoderm differentiation in ruminant embryos.
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Michel Guillomot, Annick Turbe, Isabelle Hue and Jean-Paul Renard
Bernd Fischer, Pascale Chavatte-Palmer, Christoph Viebahn, Anne Navarrete Santos and Veronique Duranthon
The renaissance of the laboratory rabbit as a reproductive model for human health is closely related to the growing evidence of periconceptional metabolic programming and its determining effects on offspring and adult health. Advantages of rabbit reproduction are the exact timing of fertilization and pregnancy stages, high cell numbers and yield in blastocysts, relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structured similarly to the human placenta. To understand, for example, the mechanisms of periconceptional programming and its effects on metabolic health in adulthood, these advantages help to elucidate even subtle changes in metabolism and development during the pre- and peri-implantation period and during gastrulation in individual embryos. Gastrulation represents a central turning point in ontogenesis in which a limited number of cells program the development of the three germ layers and, hence, the embryo proper. Newly developed transgenic and molecular tools offer promising chances for further scientific progress to be attained with this reproductive model species.
LeAnn Blomberg, Kazuyoshi Hashizume and Christoph Viebahn
The molecular basis of ungulate and non-rodent conceptus elongation and gastrulation remains poorly understood; however, use of state-of-the-art genomic technologies is beginning to elucidate the mechanisms regulating these complicated processes. For instance, transcriptome analysis of elongating porcine concepti indicates that protein synthesis and trafficking, cell growth and proliferation, and cellular morphology are major regulated processes. Furthermore, potential autocrine roles of estrogen and interleukin-1-β in regulating porcine conceptus growth and remodeling and metabolism have become evident. The importance of estrogen in pig is emphasized by the altered expression of essential steroidogenic and trophoblast factors in lagging ovoid concepti. In ruminants, the characteristic mononucleate trophoblast cells differentiate into a second lineage important for implantation, the binucleate trophoblast, and transcriptome profiling of bovine concepti has revealed a gene cluster associated with rapid trophoblast proliferation and differentiation. Gene cluster analysis has also provided evidence of correlated spatiotemporal expression and emphasized the significance of the bovine trophoblast cell lineage and the regulatory mechanism of trophoblast function. As a part of the gastrulation process in the mammalian conceptus, specification of the germ layers and hence definitive body axes occur in advance of primitive streak formation. Processing of the transforming growth factor-β-signaling molecules nodal and BMP4 by specific proteases is emerging as a decisive step in the initial patterning of the pre-gastrulation embryo. The topography of expression of these and other secreted molecules with reference to embryonic and extraembryonic tissues determines their local interaction potential. Their ensuing signaling leads to the specification of axial epiblast and hypoblast compartments through cellular migration and differentiation and, in particular, the specification of the early germ layer tissues in the epiblast via gene expression characteristic of endoderm and mesoderm precursor cells.
Séverine A Degrelle, Kim-Anh Lê Cao, Yvan Heyman, Robin E Everts, Evelyne Campion, Christophe Richard, Céline Ducroix-Crépy, X Cindy Tian, Harris A Lewin, Jean-Paul Renard, Christèle Robert-Granié and Isabelle Hue
Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as ‘patterning’ the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.
Rebecca L Jones, Tu’uhevaha J Kaitu’u-Lino, Guiying Nie, L Gabriel Sanchez-Partida, Jock K Findlay and Lois A Salamonsen
Maternal–fetal communications are critical for the establishment of pregnancy. Embryonic growth and differentiation factors produced by the oviduct and uterus play essential roles during the pre- and early post-implantation phases. Although several studies indicate roles for activin in embryonic development, gene-knockout studies have failed to identify a critical role in mammalian embryogenesis. We hypothesized that activin is produced by maternal tissues during the establishment of pregnancy, and thus maternally derived activin could compensate for the absence of embryonic activin in null homozygotes during critical developmental stages. We investigated the expression of inhibin α, activin βA, and βB subunits in the mouse oviduct and uterus during the estrous cycle and early pregnancy, and in the early conceptus. Inhibin α subunit was weakly expressed, while activin βA and βB subunits were strongly expressed in oviduct and uterus at estrous, and dramatically upregulated in the uterus on each day of pregnancy between days 3.5 and 8.5 post coitum. Prior to implantation, activin βA and βB subunits were immunolocalized to oviductal and uterine epithelial cells; following implantation they were expressed in the stroma, in a wave preceding decidualization. Later in pregnancy, activin βA and βB subunits were present in decidua basalis, trophoblast giant cells, and labyrinth zone of the developing placenta. Expression of activin βA subunit was also detected in blastocysts and early post-implantation embryos. These data are consistent with a role for maternally derived activins in the support of the pre-implantation embryo, and during gastrulation and embryogenesis.
Yue Li, Ru Zheng, Rui Wang, Xiaoyin Lu, Cheng Zhu, Hai-Yan Lin, Hongmei Wang, Xiaoguang Yu and Jiejun Fu
The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.
D Hickford and L Selwood
This study describes for the first time the spatial and temporal distribution of a growth factor and its receptors in uteri and conceptuses of a marsupial species during the peri-gastrulation period. Uteri (gravid and non-gravid) and blastocysts from 40 female stripe-faced dunnarts (Sminthopsis macroura) were collected over the peri-gastrula period (days 6.0-8.5) and stained immunohistochemically for transforming growth factor beta2 (TGFbeta2) and its receptors, TbetaRI and TbetaRII, to determine possible roles for TGFbeta2 in marsupial embryonic development. The events that occurred during the period examined included the appearance and proliferation of hypoblast and mesoderm, primitive streak and node formation, and early neurulation. Differences in TGFbeta2 quantities between gravid and non-gravid uteri reflect differences in uterine morphology, indicating a role for TGFbeta2 in endometrial remodelling. In blastocysts, large quantities of all three proteins in the trophectoderm during the node stage coincide with both blastocyst expansion before implantation and the appearance of multinucleated cells, indicating that TGFbeta2 plays a role in conceptus elongation and trophectoderm differentiation. In contrast, lack of TbetaRII in blastocysts during hypoblast formation and proliferation negates any role for TGFbeta2 in these processes, as both receptors are required for a response to TGFbeta2. High concentrations of TGFbeta2 but low concentrations of TbetaRII in blastocysts during early primitive streak formation indicate that paracrinal embryo-maternal signalling may be occurring, as blastocysts cannot respond to TGFbeta2 at this stage, but uteri could. A similar situation, but reversed, also occurs during primitive node formation.
Natalie I Alexopoulos, Poul Maddox-Hyttel, Pernille Tveden-Nyborg, Nancy T D'Cruz, Tayfur R Tecirlioglu, Melissa A Cooney, Kirsten Schauser, Michael K Holland and Andrew J French
In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.
M. L. Norris
Summary. The efficacy of several exteroceptive factors including exposure to a 'strange' male, were tested to induce pregnancy failure in newly mated Mongolian gerbils. Only physical separation of monogamous pairs consistently induced pregnancy failure. This phenomenon was restricted to the immediate period after mating and is believed to be due to disruption of pair bonding, with associated luteal and implantation failure. Concurrent lactation annulled this effect.
S. Long, S. K. Dey and D. C. Johnson
Summary. An intravenous injection of 2-fluoro-oestradiol simultaneously with an implantation-inducing dose of oestradiol reduced the number of implantation sites in delayed implanting hypophysectomized rats maintained with progesterone. Administration of 2-fluoro-oestradiol 1 h before or after oestradiol had no effect. Furthermore, injection of as much as 500 ng 2-fluoro-oestradiol 48 h before administration of oestradiol failed to have any effect upon implantation, i.e. failure to block implantation was correlated with failure to induce the uterine refractory state. These results suggest that conversion of primary oestrogens to catechol oestrogens could be important for implantation as well as for the induction of the oestrogen refractory state in the uterus.