Comparison of anterior-posterior development in the porcine versus chicken embryo, using goosecoid expression as a marker.

During early embryonic development, pig and chicken embryos share striking morphological similarities. In the present study, the timing and location of expression of mRNA for goosecoid (gsc), a gene classically expressed in the nodal region of developing embryos, was examined and compared in preprimitive streak and gastrulating pig and chicken embryos. The expression of gsc appeared first in the hypoblast and second in the hypoblast of pig and chicken embryos. Because gsc expression in these tissues was not symmetrical, gsc appears to be a useful marker for the onset of embryonic polarity. During gastrulation in both species, gsc expression became confined to cells in and around the node, in the epiblast and mesoderm layers. The only significant species-related difference in the distribution of gsc expression at these stages of development was the presence of gsc expression in the gut endoderm of chicken but not pig embryos. Certainly, our results suggest that the molecular mechanisms that control anterior-posterior development in different classes of vertebrates are remarkably similar. In addition, we were able to demonstrate that the pattern of gsc expression appears to provide a more sensitive and accurate means of determining the developmental stage of early porcine embryos than the more commonly used trophoblast or embryoblast size. Using gsc expression and accompanying embryo morphometric changes, we were able to develop a four-point scale that may offer a more accurate means of quantifying early embryo development in pigs.

Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation.

The mRNA expression patterns of activin beta(A) and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin beta(A) and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development. Mol. Reprod. Dev. 59: 390-399, 2001.

TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes.

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos.

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos.

The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

Blastoderm structure, cell migration and formation of the embryonic shield during gastrulation in the carp (Cyprinus Carpio); a scanning electron microscopic study.

This paper describes an ultrastructural study of the cell movements during the gastrulation of the common carp, Cyprinus carpio, using scanning electron microscopy. The morphology of the deep cells was studied in several consecutive stages ranging from 0-100% epiboly. Furthermore, the formation of the embryonic shield was followed from its earliest appearance at 50% epiboly onwards. This paper gives morphological evidence for the existence of two different pathways for involving and convergent movements. Firstly, cells may move along the inner surface of the not (yet) involuted cells. Secondly, a much smaller group may use the YSL as their substrate. These results are discussed in the light of the hypothesis that the two migrating cell populations may be differently induced, subsequently leading to the formation of mesoderm and endoderm.

The carp homeobox gene Ovx1 shows early expression during gastrulation and subsequently in the vagal lobe, the facial lobe and the ventral telencephalon.

The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections.

Blastomeres and cells with mesendodermal fates of carp embryos express cth1, a member of the TIS11 family of primary response genes.

The carp cth1 gene, related to the mammalian TIS11 family of primary response genes, encodes a novel fish protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during cleavage, blastula and gastrula stages of carp embryos. Cth1 mRNA is present in all cleavage stage blastomeres as a maternal message. After the late blastula stage, the maternal expression decreases, revealing a spot of higher expression at the margin of the blastoderm of the dome stage embryo. Further decrease of the maternal message reveals a ring of cth1 expressing cells at the blastoderm margin from the stage of 40% epiboly onwards. By alpha-amanitin treatment we established that this local cth1 expression is of zygotic origin. At the onset of gastrulation the cells of the cth1 ring involute, starting with those in the shield region, and at approximately 60% epiboly the ring is fully involuted and occupies the hypoblast layer. All cth1 transcripts have disappeared at completion of epiboly. We discuss a possible role for the putative cth1 protein during cleavage and gastrulation.

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Expression of Hoxb-1 during gastrulation and segmentation stages of carp (Cyprinus carpio).

This report describes the cDNA sequence and embryonic RNA expression pattern of carp Hoxb-1. Carp Hoxb-1 is a labial-like, homeobox-containing gene of the 3' end of the Hox gene cluster. The expression pattern in carp is compared to that of homologs in other vertebrates. As holds for other Hox genes, carp Hoxb-1 is expressed with highest intensity at a sharp anterior boundary, and expression fades out towards posterior. At later stages, gaps were found in the domain. The gene is expressed from late gastrulation onwards, first mainly in the hypoblast but later in all germ layers. Its most prominent expression area is rhombomere 4 (r4) of the hindbrain. Transcripts were also found in the neural tube, mesoderm (lateral, head and presomite), epidermis and neural crest. At 30 hours post fertilization, Hoxb-1 was still expressed in r4, in the anterior trunk neural tube and in the branchial arches posterior to r4. Hox genes are thought to be involved in the specification of positional values along the embryonic anterior-posterior axis, and Hoxb-1 expression in r4 is supposed to be important for specifying the unique identity of this hindbrain segment. The conserved expression in r4 suggests that this is also true for carp Hoxb-1.

Isolation of carp cDNA clones, representing developmentally-regulated genes, using a subtractive-hybridization strategy.

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.

Morphological indications of secretion by uterine epithelial cells and changes in the ratio between acidic and basic uterine proteins during early pregnancy in Chinese Meishan pig.

In Chinese Meishan pig embryonic mortality appears relatively low compared to European breeds. Most of embryonic loss in pig is believed to take place during early pregnancy. It is of interest to know possible specific features associated with low mortality. Therefore, the ultrastructure of the endometrial epithelium of Meishan pig was studied between Days 4 and 12 of pregnancy, and compared with earlier results on Yorkshire/Dutch Landrace interbreed (Y/DL). Furthermore, total protein and the relative amounts of acidic and basic proteins were determined in the uterine flushings, and compared with earlier results on Dutch Landrace (DL). As holds for European breeds, uterine glandular and luminal epithelium have to be considered as functionally different cell populations. Their morphology differs and suggests the synthesis of different secretory products. The periods of secretion are not the same: the luminal epithelium shows signs of product release during the whole period studied, the glands deliver their secretions from Day 12. This is correlated with a sudden increase in total protein in uterine flushings. Between Days 4 and 12, the relative amount of acidic proteins decreases from 92% to 47% in DL and from 88% to 38% in Meishan, resulting in a shift from acidic protein dominance to a mild dominance of basic proteins in both breeds, but most prominent in Meishan.

Differential susceptibility of early steps in carp (Cyrinus carpio) development to α-amanitin.

Carp embryos were dechorionated and their early development was studied in the presence or absence of a-amanitin. Cleavage and the formation of the enveloping layer and yolk syncytial layer were not influenced by the drug. However, a-amanitin largely blocked epiboly which started 6 h after fertilization in controls. Involution of deep cells, taking place during gastrulation movements, appeared to be blocked to a lesser degree. This might reflect differences in the degree to which maternal transcripts influence these developmental steps.

Morphological and radiochemical evidence for the metabolism of exogenous proteins by the preimplantation sheep blastocyst.

The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with 125I-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with [14C]leucine. A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.

Embryonic and uterine development during early pregnancy in pigs.

Comparison of the timing of pig preimplantation development, alterations in the ultrastructure of embryonic germ layers, and cytological changes of the uterine epithelial cells leads to the supposition that a close relationship exists between embryonic and uterine development during early pregnancy. The results of in-vitro studies of embryonic development and of experiments concerning asynchrony between embryos and uterine environment confirm this supposition, especially as far as the post-hatching period is concerned. It is suggested that successive steps in embryonic germ layer differentiation may induce specific developmental events and secretory activity of the embryos. A mutual influence of maternal and embryonic tissues appears to exist, but we can only speculate about the causes of many of the described phenomena.

Integrity of the preimplantation pig blastocyst during expansion and loss of polar trophectoderm (Rauber cells) and the morphology of the embryoblast as an indicator for developmental stage.

The embryonic ectoderm of the pig differentiated and became part of the outer barrier of the blastocyst (earlier formed by the trophectoderm alone) before shedding of the overlying polar trophectoderm around Day 10, thus securing the integrity of the rapidly expanding blastocyst. Ferritin, added to the medium of the blastocyst, was taken up rapidly by trophectoderm cells, but did not reach the blastocoele, and consequently no tracer was found within hypoblast cells. Embryonic ectoderm cells did not absorb the macromolecule, before or after loss of the polar trophectoderm. When ferritin was injected into the blastocoele, trophectoderm, hypoblast and embryoblast cells all absorbed the tracer. At Day 11, blastocyst diameter and embryoblast cell number varied widely and were hardly correlated. We suggest that embryoblast development may be a more reliable indicator for the developmental stage of a blastocyst than its diameter, which may merely be an indication of the viability of the trophoblast.

The ultrastructure of the uterine epithelium of the pig during the estrous cycle and early pregnancy.

The ultrastructure of the endometrial epithelium of the pig was studied during the estrous cycle and early pregnancy up to implantation. Special attention was given to the luminal epithelium and morphological indications of protein synthesis. Although the general morphology of the luminal and glandular epithelia is similar (both tissues consist of secretory cells and ciliated cells at all the stages studied), it appears that the two epithelia should be considered as two functionally different units in the pre-implantation period. Morphological evidence suggests the presence of at least three different secretory products within luminal epithelial cells; they are released at different times, i.e. at estrus, between day 8 and 10 and after day 11. The glandular epithelium shows release of secretory products from day 10-11. Increasing amounts of glycogen were found within epithelial cells, especially in pregnant gilts from day 12. The possible significance of secretory activity of the epithelium is discussed in relation to the development of the embryos.