Role of karyoplasm in the emergence of capacity of egg cytoplasm to induce DNA synthesis in transplanted sperm nuclei.
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The behaviour of sperm nuclei was studied both in the cytoplasm of intact toad oocytes undergoing maturation and the cytoplasm of oocytes matured without germinal vesicles. The behaviour of the nuclei of pronase-treated sperm injected in the mature egg cytoplasm was shown to be exactly similar to that of the sperm nucleus after fertilization, i.e. they swelled, synthesized DNA, and divided. No changes in such sperm nuclei could be detected in the cytoplasm of the oocytes matured without germinal vesicles. (+info)
Development of parthenogenetic and fertilized mouse embryos in the uterus and in extra-uterine sites.
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Mouse eggs were activated with hyaluronidase in vitro and subsequently transferred to the oviduct. In the female reproductive tract they formed morulae and blastocysts which died soon after implantation. Haploid blastocysts were transferred beneath the kidney capsule and here some formed disorganized egg-cylinder structures in a week. Morulae and blastocysts from haploid and diploid parthenogenones were also transferred beneath the testis capsule. Two to four months later the growths which had formed were sectioned. They contained neural tissue, pigment, keratinized epithelium, glandular epithelium, ciliated epithelium, cartilage, bone, muscle, adipose tissue, and haemopoietic tissue. The range of cell types was similar to that produced by fertilized control blastocysts except that the parthenogenones did not form identifiable yolk-sac carcinoma or embryonal carcinoma cells. The growths from haploid and diploid parthenogenones in the testis were stained with Feulgen and their DNA content measured. Growths from diploid embryos contained the normal diploid amount of DNA while growths from haploid embryos contained less than this amount. Cell cultures were prepared from the growths. The cells which were investigated contained no Y chromosome, suggesting that they were derived from the embryonic cells rather than the cells of the male host. These cells contained a near diploid chromosome number, although some of them were originally derived from haploid embryos. (+info)
Preimplantational ectogenesis. Science and speculation concerning in vitro fertilization and related procedures.
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In recent years, technical advances have made preimplantational ectogenesis (in vitro maturation, fertilization and early embryonic development) more than a theoretical concept. Such procedures hold great promise in medical research. However, despite our newly-acquired skills in tissue culture and microsurgical manipulation, and contrary to many sensational articles in the lay press, it is not likely that preimplantational ectogenesis will soon attain wide clinical use in humans. Adverse societal attitudes, based largely upon moral and ethical dilemmas, will probably combine with still-unresolved technical difficulties to restrict the clinical applications. (+info)
Complete preimplantation development in culture of parthenogenetic mouse embryos.
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The present experiments were undertaken to determine whether, in parthenogenesis, heterozygous embryos develop better than homozygous embryos. Such experiments may provide an approach to elucidating whether fertilized embryos develop better than parthenogenetic ones because of heterozygosity, or if the sperm provides another contribution necessary for complete embryonic development. The parthenogenetic embryos studied included uniform haploids after extrusion of the second polar body, mosaic haploids in which each blastomere contained a genetically different haploid nucleus, and heterozygous diploid mouse embryos. Eggs were activated and cultured in a chemically defined medium. About three times as many mosaic haploid or heterozygous diploid eggs developed beyond the 4-cell stage after 98-100 h and to the blastocyst stage after 120 h in culture, than uniform haploid eggs. This indicates that the development of parthenogenetic embryos is probably under genetic control and that there was a better development of the heterozygous embryos. Mosaic haploid embryos showed the same high frequency of development as heterozygous diploids. The results therefore indicate that heterozygosity provided a developmental advantage even when distributed between two genetically different clones of cells in the same embryo. (+info)
Effect of reproductive status on in vitro developmental competence of bovine oocytes.
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The objectives of the present study were to compare the in vitro maturation (IVM), fertilization and early embryonic development of bovine oocytes recovered from ovaries during the follicular, metestrus and diestrus stages of the estrous cycle and at anestrus and pregnancy after maturation in a serum free culture medium. Cumulus oocyte complexes (COCs) collected from ovaries at different reproductive statuses were matured in medium 199 supplemented with 10 g/ml FSH, 10 g/ml LH, 1.5 g/m estradiol, 75 g/ml streptomycin, 100 IU/ml penicillin and 10 mM HEPES. COCs were incubated in 200 microl droplets of maturation medium 199 under oil for 24 h at 39 90 degree angle c and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim up, heparin capacitated sperm from two bulls separately in each replicate (20 h, 39C, 5% CO2). After fertilization, the presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin-G, 75 g/ml streptomycin and 10 mM HEPES for 144 h at 39C and 5% CO2 without medium freshening or change. Oocytes/embryos were fixed, stained with DAPI and evaluated under fluorescent microscope. The IVM rates were almost similar among oocytes from all reproductive statuses (range: 89.8 to 95.4%). However, IVM rates for oocytes from the metestrus (90.6%) and pregnant (89.9%) phases were lower than the other groups. The fertilization rates were lower (p<0.05) for oocytes from the diestrus phase (72.4%) than from the other phases (range: 81.1 to 86.6%). Oocytes, recovered during the metestrus phase of the estrous cycle, resulted in the highest cleavage rate (60.0%), while oocytes from the diestrus phase had the poorest embryonic development (39.8%: p<0.05). Majority of the embryos from all reproductive phases showed a developmental arrest around 8-cell stage. Although the developmental competence of oocytes from pregnant and anestrus animals was lower than that from the other reproductive stages, they could be potentially used as oocyte donors. Long term, in vitro embryo culture without medium freshening or change was hypothesized to have caused the failure to overcome the 8-cell block to development. (+info)
Effects of protein source and energy substrates on the in vitro development of bovine embryos in a two-step culture system.
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In this study, we examined the effects of a two-step culture system, which involves the use of different culture media for early cleavage and later stage embryos, on the in vitro development of bovine embryos. We also investigated the effect of glucose, phosphate and citrate on the in vitro early developmental period of bovine embryos in a two-step culture system. Moreover, the supplementation of different protein sources (BSA-V, BSA-FAF and FBS) during IVC did not affect the frequency of blastocyst development. Using two-step culture, embryos were cultured in protein-free media for an initial 5 days. This was then followed by the same culture media or an FBS supplemented media. The developmental rates of blastocysts in the FBS containing group were significantly higher than in the replaced with no serum containing group. Embryos cultured in mSOF supplemented with 1.5 mM glucose plus 1.2 mM phosphate were significantly inhibited. The inhibition of developmental competence by glucose plus phosphate was consistent with the existence of 0.5 mM sodium citrate. This study indicates that a two-step culture system, which applies different conditions for early cleavage embryos, i.e., serum-free media, vs. later stage embryos, with serum containing media, may be effective for in vitro production systems. In addition, the developmental competence of bovine embryos was depressed in the presence of glucose plus phosphate as compared to either alone or the absence of both. Therefore, the avoidance of this negative effect should allow more optimal conditions to be developed for in vitro production. (+info)
Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro.
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Among its many activities, leukemia inhibitory factor (LIF) can maintain embryonic stem cell monolayers in a pluripotent undifferentiated state. Presuming that this might reflect its physiologic role during embryogenesis, we have examined LIF expression in the embryonic environment by RNase protection assays and have determined its in vitro effect on differentiating embryonic stem cell embryoid bodies. Of all adult tissues analyzed, LIF transcripts appear only in the uterus, where their level fluctuates with the estrous cycle, peaking after ovulation. LIF expression continues in the uteri of pregnant and pseudopregnant females, with a relative peak when blastocysts are normally present. As for its effects on in vitro differentiation, we have found that LIF blocks embryoid body differentiation only partially, yet in a precise manner. Using molecular markers to follow the differentiation of defined cell types, we demonstrate that LIF selectively inhibits the formation of primitive ectoderm, while permitting the differentiation of primitive endoderm. These results suggest a specific role for LIF in preimplantation mouse development. (+info)
The effect of maternal cytomegalovirus infection on preimplantation development in the mouse.
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Mice were inoculated with murine cytomegalovirus at 14 and 7 days before and 1 and 4 days after mating. The effects of maternal infection on early pregnancy were investigated. Inoculation 7 days before and 1 day after mating, i.e. around ovulation and implantation, significantly reduced pregnancy rate. Embryos in these females were developmentally retarded, perhaps because of the inflammatory effect of the infection on the genital tract. Retarded embryos developed normally when cultured. (+info)