(1/365) Antibodies with the cell-type specificity to the morula cells of the solitary ascidians Styela rustica and Boltenia echinata.
The separation of the blood cells of Styela rustica (Styelidae, Stolidobranchiae) in discontinuous Percoll gradient showed 4 fractions. The 4th and bottom most fraction contained 90-100% of morula cells. The protein composition of the morula cell fraction revealed on SDS-PAGE showed two major proteins with m.w. 47 and 26 kDa. These proteins were heavily positively charged. Polyclonal antiserums against these proteins were raised. Each antiserum reacted with both proteins only in morula cells on the blot after SDS-PAGE and stained the proper protein without crossreaction on the blot after AU-PAGE. The only type of cells stained with antibodies in circulating blood, in the tunic and on the tunic wound surface in paraffin sections of another species Boltenia echinata (Pyuridae, Stolidobranchiae) were morula cells. The morula-type specific antibodies obtained recognized major positively charged proteins which were apparently structural substrates for the phenoloxidase tanning. (+info)
(2/365) Mammalian transgenesis by intracytoplasmic sperm injection.
Coinjection of unfertilized mouse oocytes with sperm heads and exogenous DNA encoding either a green fluorescent protein (GFP) or beta-galactosidase reporter produced 64 to 94 percent transgene-expressing embryos, reflecting DNA-sperm head association before coinjection. Nonselective transfer to surrogate mothers of embryos in the GFP series generated about 20 percent offspring expressing the integrated transgene. These data indicate that exogenous DNA can reproducibly be delivered into an oocyte by microinjected spermatozoa and suggest an adaptable method of transgenesis. (+info)
(3/365) Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.
Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis. (+info)
(4/365) Microelectrophoretic analysis of changes in protein expression patterns in mouse oocytes and preimplantation embryos.
One- and two-dimensional polyacrylamide microslab gel electrophoresis followed by silver staining was devised to visualize picogram to nanogram levels of proteins and was applied to the analysis of 1-20 mouse oocytes and embryos (approximately 16.5-330 ng of protein) during preimplantation development. Compared with values in embryos, more bands in the higher molecular weight range were found only for unfertilized oocytes in one-dimensional microelectrophoresis. A marked decrease in the number of protein spots occurred after fertilization in two-dimensional microelectrophoresis. Both findings indicate a decrease in maternal proteins caused by fertilization. Silver-staining densities were almost invariable for 8 major spots, but increased, decreased, or varied for 32 minor spots in developing embryos from the 1-cell to the morula stage, signifying spot-specific changes in the expression of zygotic proteins during development. The protein patterns in cumulus cells and blastocysts were different from those in oocytes and embryos. Even in a single 1-cell embryo, major spots and some minor spots were detectable by our two-dimensional microelectrophoretic technique, but many more minor spots were visualized in five 1-cell embryos, exemplifying the limit of our microelectrophoretic technique. As a preliminary result, a two-dimensional immunoblot pattern is shown for glucose transporter 1 expressed in morulae. (+info)
(5/365) Retinol administration to superovulated ewes improves in vitro embryonic viability.
Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development. (+info)
(6/365) Developmentally regulated loss and reappearance of immunoreactive somatic histone H1 on chromatin of bovine morula-stage nuclei following transplantation into oocytes.
One difference between chromatin of bovine oocytes and blastomeres is that somatic subtypes of histone H1 are undetectable in oocytes and are assembled onto embryonic chromatin during the fourth cell cycle. We investigated whether this chromatin modification is reversed when nuclei containing somatic H1 are transplanted into ooplasts. Donor nuclei obtained from morula-stage bovine embryos were fused to ooplasts at different times before and after parthenogenetic activation of the ooplasts. After fusion, immunoreactive H1 became undetectable, and the loss occurred more rapidly when fusion was performed near the time of ooplast activation compared with several hours after activation, when the host oocytes were at a stage corresponding to interphase. Although the loss of immunoreactive H1 occurred independently of DNA replication and transcription, exposure of reconstructed oocytes to cycloheximide or 6-dymethylaminopurine (6-DMAP) delayed the loss of immunoreactive H1 from transplanted nuclei. During further development of nuclear-transplant embryos, somatic H1 remained undetectable at the 2- and 4-cell stages, and it reappeared on the chromatin at the 8- to 16-cell stage, as previously observed in unmanipulated embryos. We conclude that factors in oocyte cytoplasm are able to modify morula chromatin so that somatic H1 becomes undetectable, and that the amount or activity of these factors declines over time in activated ooplasts. (+info)
(7/365) Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.
We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(-/-) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845-1854). (+info)
(8/365) Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos.
In hope of understanding possible roles of estrogen during early embryogenesis, we examined the expression of both estrogen receptor alpha (ER alpha) and ER beta, a recently cloned novel subtype, in mouse oocytes and preimplantation embryos by means of reverse transcription polymerase chain reaction (RT-PCR). To investigate whether estrogen actually exerts its action, we further determined the expression of efp (estrogen-responsive finger protein), a newly characterized estrogen responsive gene belonging to the RING finger family. ER alpha mRNA was detected in whole ovaries, cumulus-oocyte complexes, denuded oocytes, 2-cell and 4-cell embryos, whereas it was undetected in 8-cell embryos. Interestingly it reappeared in morulae and blastocysts. ER beta mRNA was detected similarly to ER alpha except for the absence of ER beta mRNA in morulae. The efp mRNA was detected in whole ovaries, cumulus-oocyte complexes, 4-cell embryos, morulae and blastocysts. The stage specific expression of ER alpha and ER beta along with detection of the product of the estrogen responsive gene in early preimplantation embryos may indicate the possible physiological roles of estrogen in early embryogenesis. (+info)