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PubMed1997

Cryopreservation of mouse embryos affects later embryonic development possibly through reduced expression of the glucose transporter GLUT1.

In order to study the effects of cryopreservation on later embryonic development, two-cell mouse embryos were frozen, thawed, and then allowed to develop into blastocysts. The percentage of cryopreserved embryos which developed into blastocysts was significantly lower than that of fresh two-cell embryos. The amount of glucose incorporation in terms of 3H-2-deoxyglucose uptake in blastocysts developed in vivo, and in vitro from fresh or frozen-thawed two-cell embryos, was 473 +/- 108, 105 +/- 75, and 43.0 +/- 28.3 fmol per embryo per hour, respectively. Quantification of glucose transporter GLUT1 in these embryos by Western blotting was reflective of the degree of glucose incorporation. The implantation rate of blastocysts developed in vitro from frozen-thawed two-cell embryos (22.0%) was significantly lower than that developed in vivo (41.1%). These data suggest that cryopreservation may have later consequences on embryonic development through a mechanism that involves altered GLUT1 expression.

Animals↗
PubMed1999

Gene expression of vasoactive intestinal contractor/endothelin-2 in ovary, uterus and embryo: comprehensive gene expression profiles of the endothelin ligand-receptor system revealed by semi-quantitative reverse transcription-polymerase chain reaction analysis in adult mouse tissues and during late embryonic development.

Vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2) is a 21 amino acid intestinal peptide characterized as a potent vasoactive and intestinal smooth muscle-contracting compound. To investigate the physiological roles of VIC/ET-2 further, we characterized the specificity of VIC gene expression relative to that of other members of the endothelin (ET) ligand-receptor system in adult mouse tissues and during embryonic development. Gene expression of ET-1, ET-3, ETA and ETB was ubiquitous in almost all tissues we examined while gene expression of VIC was localized to certain tissues. A high level of VIC gene expression was observed in ovary and uterus. The gene expression of VIC, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was approximately 2.0%, 0.4%, and 2.3% in ovary, uterus, and intestine respectively, and was approximately 1.6 and 7. 1 times higher than that of ET-1 in ovary and intestine respectively. Thus, VIC may have some physiological role in adult ovary and uterus as well as intestine. In embryonic development, VIC gene expression sharply increased between 11 and 15 days post coitus and decreased after birth, suggesting an involvement in the later stages of embryonic development.

Animals↗
PubMed1989

[Total proteins levels and embryonic development of eggs of sea urchins (Lytechinus variegatus) treated with YbCl3].

Total proteins levels and the embryonic development of sea urchin (Lytechinus variegatus) eggs treated with 25 micrograms/ml of YbCl3 in sea water for one hour were studied. After the treatment, eggs were let to develop for 24 hours when the total proteins levels were determined and morphological changes as well as the synchronism of the embryonic development have been checked.

Animals↗
PubMed2001

CpG binding protein is crucial for early embryonic development.

Epigenetic modification of DNA via CpG methylation is essential for the proper regulation of gene expression during embryonic development. Methylation of CpG motifs results in gene repression, while CpG island-containing genes are maintained in an unmethylated state and are transcriptionally active. The molecular mechanisms involved in maintaining the hypomethylation of CpG islands remain unclear. The transcriptional activator CpG binding protein (CGBP) exhibits a unique binding specificity for DNA elements that contain unmethylated CpG motifs, which makes it a potential candidate for the regulation of CpG island-containing genes. In order to assess the global function of this protein, mice lacking CGBP were generated via homologous recombination. No viable mutant mice were identified, indicating that CGBP is required for murine development. Mutant embryos were also absent between 6.5 and 12.5 days postcoitum (dpc). Approximately, one-fourth of all implantation sites at 6.5 dpc appeared empty with no intact embryos present. However, histological examination of 6.5-dpc implantation sites revealed the presence of embryo remnants, indicating that CGBP mutant embryos die very early in development. In vitro blastocyst outgrowth assays revealed that CGBP-null blastocysts are viable and capable of hatching and forming both an inner cell mass and a trophectoderm. Therefore, CGBP plays a crucial role in embryo viability and peri-implantation development.

Animals↗
PubMed2002

Identification of known and novel genes whose expression is regulated by endogenous retinoic acid during early embryonic development of the mouse.

Retinoic acid (RA) derived from vitamin A is necessary for, among other things, mammalian embryonic development. Although the impact of RA-dependent gene-regulation on embryonic development has been examined through genetic disruption of the retinoid receptors, the understanding of the underlying molecular mechanism remain unclear, in part, due to the difficulty in identifying RA-regulated genes in an intact embryo. We report here that RA-regulated genes can be identified from total RA-deficient embryos created by retinol-binding protein antisense (RBP-AS) oligodeoxynucleotide treatment in conjunction with differential display. Of the 28 genes isolated, 15 genes matched known genes in the GenBank database and the others either represented EST sequences or encoded novel genes. Semi-quantitative reverse transcriptase-polymerase chain reaction verified that the mRNA levels of mouse DN 38, COL VI 3 alpha, cul-1, alpha-tropomyosin, and PP2A-C alpha were substantially increased, whereas mouse Msh 2, Ndufa2, Ribosomal protein S19, sFRP-1, GDAP-10 and mSmcD were significantly decreased in vitamin A deficient (VAD) embryos compared to the control embryos. The utility of the method is exemplified by our finding that several genes in the Wnt signaling pathway are vitamin A regulated in day 9.0 post coitum (p.c.) embryos.

Animals↗
PubMed2001

Relationships among salinity, egg size, embryonic development, and larval biomass in the estuarine crab Chasmagnathus granulata Dana, 1851.

We studied interrelationships between initial egg size and biomass, duration of embryogenesis at different salinities, and initial larval biomass in an estuarine crab, Chasmagnathus granulata. Ovigerous females were maintained at three different salinities (15 per thousand, 20 per thousand and 32 per thousand); initial egg size (mean diameter), biomass (dry weight, carbon and nitrogen) as well as changes in egg size, embryonic development duration, and initial larval biomass were measured.Initial egg size varied significantly among broods from different females maintained under identical environmental conditions. Eggs from females maintained at 15 per thousand had on average higher biomass and larger diameter. We hypothesise that this is a plastic response to salinity, which may have an adaptive value, i.e. it may increase the survivorship during postembryonic development. The degree of change in egg diameter during the embryonic development depended on salinity: eggs in a late developmental stage were at 15 per thousand significantly larger and had smaller increment than those incubated at higher salinities. Development duration was longer at 15 per thousand, but this was significant only for the intermediate embryonic stages. Initial larval biomass depended on initial egg size and on biomass loss during embryogenesis. Larvae with high initial biomass originated either from those eggs that had, already from egg laying, a high initial biomass (reflecting individual variability under identical conditions), or from those developing at a high salinity (32 per thousand), where embryonic biomass losses were generally minimum. Our results show that both individual variability in the provisioning of eggs with yolk and the salinity prevailing during the embryonic development are important factors causing variability in the initial larval biomass of C. granulata, and thus, in early larval survival and growth.

Journal Article↗
PubMed1999

Effects of heavy metals (Hg2+, cd2+, Pb2+) during the embryonic development of acridid grasshoppers (Insecta, caelifera)

Heavy metals present in the soil get differently accumulated in organisms and show different rates of toxicity at different stages of the life cycle of any organism living there. To see whether such toxicants get accumulated during the embryonic development and/or change the normal developmental processes of organisms exposed to heavy metals, freshly laid eggs and egg pods of two acridids, Aiolopus thalassinus and Eyprepocnemis plorans, were tested against different concentrations of Hg2+, Cd2+, and Pb2+. At increasing Hg2+ concentration in the substrate, an increased egg mortality and reduced nymphal hatching in A. thalassinus were observed, yet no change in the normal duration for embryonic development was noticed. A higher hatching rate of E. plorans nymphs than that of A. thalassinus could possibly be due to the higher tolerance, contributed by larger egg volume and thicker foam around the egg pods of the former. Treated concentrations of Hg2+ could be of sublethal (0.121 &mgr;g Hg2+/g substrate) to lethal (0.605 &mgr;g and more Hg2+/g substrate) doses. The mortality of eggs did not always increase with increasing substrate concentration of Cd2+ and Pb2+, and the hatching rates of both grasshopper species in many cases were even higher than that in the control. Still, lower accumulation factors of Cd2+ and Pb2+ than that of Hg2+ were found coupled with not increased mortality. The tested concentrations of Cd2+ and Pb2+ in the substrates, thus, could be of subacute doses. The embryonic development was found prolonged due to Cd2+ and Pb2+. During parallel egg and egg pod treatments, higher metal concentrations were found in not developed eggs than that in fresh nymphs hatching from the same substrates. This indicated the role of foam around the egg pods as "protective filter" against the toxic substances in the soil.

Journal Article↗
PubMed1997

Oviductal regulation of fertilization and early embryonic development.

During the period of late follicular development and the first four days of the oestrous cycle, the oviduct occupies a central role in the establishment of pregnancy. Oviductal function is regarded as being either 'passive' or biologically active, providing an environment that sustains and enhances fertilization and early cleavage-stage embryonic development. Recent reports have focused on this microenvironment and shown that ovarian steroids induce marked morphological, physiological and biochemical changes. Alterations include changes in the biosynthetic activity and release of macromolecules by the oviductal epithelium which become part of the luminal microenvironment. Furthermore, both regional and temporal differences in activity and protein production occur through hormonal changes during the oestrous cycle and early pregnancy. Studies on identification, characterization and regulation of several proteins synthesized de novo have indicated oocyte-oviduct and embryo-oviduct interactions. However, the identification of oviduct-derived proteins, their regulation and their potential function in vivo needs to be examined. Studies in other species also suggest roles for growth factors in early embryonic development, but little information is available for the pig. We propose that ovarian hormones control changes in synthetic activity, synthesis of some oviduct-derived proteins and the presence of specific factors in the luminal microenvironment which sustain and enhance fertilization and early cleavage-stage embryonic development.

Animals↗
PubMed1988

Cell proliferation and early differentiation during embryonic development and metamorphosis of Hydractinia echinata.

The early embryonic development of Hydractinia lasts about 2.5 days until the developing planula larva acquires competence for metamorphosis. Most embryonic cells stop cycling on reaching the larval stage. In older larvae of Hydractinia, cells that are still proliferating occur exclusively in the endoderm in a typical distribution along the longitudinal axis. During metamorphosis, proliferation activity begins again. The number of S-phase cells has increased by the 9th hour after induction of metamorphosis. Proliferative activity starts in the middle gastric region and in basal parts of primary polyps. Tentacles and stolon tips are always free of replicating cells.

Animals↗
PubMed1999

Regulation of somatotroph differentiation and growth hormone (GH) secretion by corticosterone and GH-releasing hormone during embryonic development.

The role of extracellular factors in the regulation of anterior pituitary cell differentiation and GH secretion during embryonic development was investigated. Previously, we reported that somatotrophs become a significant population by embryonic day (e-) 16 of the chick and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of e-16 serum. More recently, the influence of hormone interactions on somatotroph differentiation and GH secretion during mid- to late embryogenesis was evaluated. Anterior pituitary cells from e-12, -14, and -17 chicks were cultured for 2, 3, and 6 days with corticosterone (10(-9) M) and GH-releasing hormone (GHRH; 10(-10)-10(-7) M) alone and in combination. Medium samples were analyzed for GH concentrations, and recovered cells were subjected to GH reverse hemolytic plaque assay for determination of somatotroph percentages and the relative amount of GH secretion from individual somatotrophs. GHRH significantly (P < 0.05) increased GH secretion from e-17, but not e-12 and e-14, pituitary cells during 2 and 3 days of culture. Corticosterone alone failed to increase GH secretion from e-12, -14, and -17 pituitary cells; however, corticosterone in combination with GHRH increased GH secretion from cells of all three ages. Culture with GHRH decreased percentages of e-17 GH-secreting cells in a concentration-dependent manner (from basal levels of 12.3 +/- 2.4% to 3.2 +/- 0.7% by 2 days), but did not affect percentages of e-12 and e-14 somatotrophs. Conversely, corticosterone increased percentages of e-12 and e-14 GH-secreting cells (by as much as 14- and 3-fold above basal levels, respectively), but did not alter the proportions of e-17 GH cells. Corticosterone in combination with GHRH was more effective than either hormone alone for increasing percentages of e-12 GH-secreting cells (from 9.6 +/- 0.8% with corticosterone to 15.9 +/- 1.5% with corticosterone plus GHRH), but this synergistic effect was not apparent until after 3 days of culture. Exposure to corticosterone in culture for 2, 3, and 6 days increased subsequent GH release from e-12 and e-14 pituitary cells during reverse hemolytic plaque assay. Combined treatment with corticosterone and GHRH further increased subsequent GH release from e-12 and e-14 cells. We conclude that glucocorticoids induce GH cell differentiation and that corticosterone and GHRH can interact at specific stages of embryonic development to regulate somatotroph differentiation and GH secretion.

Animals↗
PubMed1998

Ontogeny of prolactin-secreting cells during chick embryonic development: effect of vasoactive intestinal peptide.

It was previously reported that prolactin (PRL)-containing cells differentiate by days 15 to 19 of embryonic development and that vasoactive intestinal peptide (VIP) increased plasma PRL concentrations and pituitary PRL protein and mRNA levels in vivo. In the present study, anterior pituitaries derived from day 15, 16, 17, 18, and 19 embryos were subjected to reverse hemolytic plaque assays (RHPA) for chicken PRL to determine the ontogeny of lactotrophs. We found that PRL secreting cells were first consistently detected on day 17 of embryonic development, indicating that lactotroph differentiation during normal development occurs by this age. However, extended treatment in the RHPA with VIP exposed lactotrophs as early as day 15, suggesting that a lactotroph precursor population was present earlier. Next, primary cultures of embryonic anterior pituitary cells from day 12, 13, 14, 15, 16, and 17 embryos were incubated for 0, 2, or 4 days in serum-free medium or medium supplemented with 10 nM VIP. After the culture periods, cells were subjected to RHPAs for chicken PRL. PRL-secreting cells differentiated spontaneously by 2 days in culture for day 15 and 16 cells and by 4 days in culture for all embryonic ages tested, except day 17. Culturing with VIP for 2 days did not increase PRL-secreting cells at any embryonic age tested, whereas VIP treatment for 4 days increased lactotroph numbers at all ages except day 12. Cells from days 15, 16, and 17 were responsive to VIP in the RHPA after 2 days in untreated cultures, but after 4 days only day 17 cells continued to respond to VIP in the RHPA. Treatment with VIP in culture for 4 days maintained VIP responsiveness in the RHPA for cells derived from embryos as early as day 14. We conclude that lactotroph differentiation during normal chicken development occurs by embryonic day 17. Moreover, our results indicate that responsiveness to VIP is an early event in PRL cell development and that lactotroph differentiation may be stimulated in vitro by VIP as early as embryonic day 13.

Animals↗
PubMed1986

Embryonic development of mevalonate metabolism by sterol and nonsterol pathways in chick brain and liver.

The embryonic development of the sterol and nonsterol mevalonate metabolism has been investigated in chick brain and liver. The shunt pathway of mevalonate was negligible in both tissues throughout 10-21 days of embryo development. Mevalonate incorporation into nonsaponifiable lipids was higher in liver than in brain. A pronounced peak was found in liver at 12 days of incubation, while only small differences were observed in brain. Lanosterol and cholesterol were the major sterols synthesized in brain, followed by desmosterol and squalene. Their relative percentages did not change significantly during 10-16 days and slightly decreased thereafter. In liver, cholesterol and squalene were the major sterols observed during the first days of incubation with a developmental pattern similar to that found in the mevalonate incorporation into nonsaponifiable lipids, while relative percentage of squalene oxides sharply increased between 12 and 16 days of embryonic development. The importance of cholesterol esters accumulation in the inhibition of cholesterogenic activity is discussed.

Animals↗
PubMed2002

Differential expression of iodothyronine deiodinases in chicken tissues during the last week of embryonic development.

In the current study, the authors examined the type 1 (D1), type 2 (D2), and type 3 deiodinase (D3) activity and mRNA expression patterns in thyroid, lung, brain, pituitary, heart, liver, spleen, gonads, skin, muscle, intestine, Fabricius' bursa, and kidney during the last week of chicken embryonic development and the first 2 days posthatch. The D3 was the most widely expressed, occurring in all examined tissues. Also, the D1 knows a widespread distribution, although no D1 activity or mRNA expression could be detected in the brain, the thyroid, the muscle, and the skin. In contrast, the D2 has a much more restricted expression pattern, since the brain is the only organ where, prior to hatching, both in vitro D2 activity and D2 mRNA expression can be detected. Taken together, these results demonstrate that during the last week of chicken embryonic development, the majority of tissues express D3, together with either D1 or D2, indicating that each tissue possesses the necessary tools to regulate local thyroid hormone levels at least partly independent from T(3) and T(4) levels in plasma. In addition, the deiodinase expression data could be correlated to certain thyroid hormone dependent tissue-specific developmental events. This strongly suggests that in birds, as in mammals and amphibians, the correct spatial and temporal expression of iodothyronine deiodinases are essential for normal embryonic development.

Animals↗
PubMed2004

A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice.

Inactivation of the murine tissue factor (TF) gene or tissue factor pathway inhibitor 1 (TFPI) gene results in embryonic lethality, indicating that both are required for embryonic development. We have shown that expression of low levels of TF from a transgene (hTF) rescues TF-null embryos. However, low-TF mice (mTF(-/-)/hTF+) have hemostatic defects in the uterus, placenta, heart, and lung. In this study, we hypothesized that the death of TFPI-/- embryos was due to unregulated TF/FVIIa activity and that the hemostatic defects in low-TF mice were due to insufficient TF expression. Therefore, we attempted to rescue TFPI-/- embryos by reducing TF expression, and to restore hemostasis in low-TF mice by abolishing TFPI expression. Intercrossing TFPI(+/-)/mTF(+/-)/hTF+/- mice generated close to the expected number of TFPI(-/-)/low-TF mice at weaning age from 128 offspring, indicating rescue of TFPI-/- embryos from embryonic lethality. Conversely, a decrease in TFPI levels dose-dependently prolonged the survival of low-TF mice and rescued the hemorrhagic defects in the lung and placenta but not in the heart or uterus. These results indicate that the correct balance between TF and TFPI in different organs is required to maintain hemostasis during embryonic development and in adult mice.

Age Factors↗
PubMed1997

Heat shock protein gene expression during embryonic development of the zebrafish.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.

Animals↗
PubMed2005

Release of ecdysteroid-phosphates from egg yolk granules and their dephosphorylation during early embryonic development in silkworm, Bombyx mori.

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

Animals↗
PubMed1981

Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development.

Monoclonal antibodies (mAbs) against a preparation of intermediate filaments from trophoblastoma cells were studied for their reactivity pattern during embryonic development and on adult tissue cells. Up to day 12 of embryonic development, epithelial cells of the three germ layers reacted with these mAbs. Later during development and in adult tissues, positive reactions could be observed only with epithelial cells derived from mesoderm and endoderm. Because of their tissue distribution, the proteins reacting with these mAbs might belong to the keratin family of intermediate filaments or they might represent a new group of intermediate filaments.

Animals↗
PubMed1995

Expression and localization of glucose transporter 1 (GLUT1) in the rat oviduct: a possible supplier of glucose to embryo during early embryonic development.

The oviduct fluid mainly derived from the oviduct epithelium is reported to provide the environment necessary for embryonic development. To elucidate the origin of glucose in the oviduct fluid, we examined the expression and localization of glucose transporter 1 (GLUT1) in the rat oviduct by Northern blot analysis, immunoblot analysis and immunohistochemistry using both light and electron microscopy. Northern blot and immunoblot analyses both showed the presence of the GLUT1 mRNA and protein. Specific staining for GLUT1 was observed in the ampulla and the isthmus, but only slightly in the fimbria and the utero-tubal junction. Staining was confined to the luminal surface of the epithelial mucosa. Immunoelectron microscopic analysis revealed that GLUT1 was observed only on the surface of the microvilli in non ciliated secretory cells, but not in ciliated cells. These findings suggest that GLUT1 plays an important role in the glucose transfer from the oviduct epithelium into the lumen and in maintaining the adequate glucose concentration of the oviduct fluid for embryonic development in rat oviduct.

Animals↗