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PubMed2001

Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei.

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

Animals↗
PubMed1982

Nonhuman primate in vitro fertilization: seasonality, cumulus cells, cyclic nucleotides, ribonucleic acid, and viability assays.

Since establishing our squirrel monkey (Saimiri sciureus) in vitro fertilization (IVF) system in 1972, 1788 oocytes have been laparoscopically recovered from follicles with 669 matured and 332 fertilized. Oocyte maturation was higher (P less than 0.05) from March to June. Among the oocytes, 50% showed first polar body extrusion by 21 hours after recovery. Maturation was significantly higher (P less than 0.05) in oocytes with corona radiata cells, compared with denuded oocytes. Sperm motility studies indicated that 2.6 and 5.2 mM caffeine enhanced motility by 10% to 30% over controls for the first 5 hours. Addition of 1 and 10 microM dibutyryl cyclic adenosine 3':5'-monophosphate (dbcAMP) enhanced fertilization by 50.3% and 50.0%, respectively. Preliminary studies of 3H-urindine incorporation and uptake by unfertilized oocytes showed levels of 3.43 +/- 0.21 and 55.01 +/- 7.16 fmole/embryo/3 hours, respectively. Exclusion of the vital dye trypan blue by oocytes and embryos gave a high correlation with morphology and survival. Autoradiographic analysis indicated nuclear grain densities in oocytes cultured with 3H-uridine. The results of the studies indicated that oocyte maturation was dependent on season and corona radiata cells, and that addition of dbcAMP enhanced IVF. Trypan blue was found be a useful embryo viability test.

Animals↗
PubMed1995

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes.

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.

Journal Article↗