Determination of Optimal Conditions for Parthenogenetic Activation and Subsequent Development of Rat Oocytes In Vitro

Abstract

Abstract. The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.

Optimal Conditions for Successful In Vitro Fertilization and Subsequent Embryonic Development in Sprague-Dawley Rats1

“…Oocytes from the SD strain were more resistant to the strontium treatment than were those from the Wistar strain, and the overall activation efficiency was higher for SD oocytes [8]. However, when oocytes were electrically stimulated and then treated with 6-dimethylaminopurine, more activated oocytes from Wistar-Imamichi strain developed to the 2-cell and blastocyst stages than did those from the SD rats [9]. In addition, fertilized rat eggs of the SD outbred strain had a high capability of developing from the 1-cell to the blastocyst stage, whereas those of the SD inbred strains performed much more poorly [31].…”

“…It has been demonstrated that genetic background is an important determinant in the in vitro response of oocytes [7][8][9] and spermatozoa [10][11][12][13], as observed in different species, including mice and rats, as well as in different strains of the same species. These factors may be responsible for the different environmental requirements for successful IVF in different strains and species.…”

“…Recent investigations have indicated that oocytes from Wistar and SD rats respond differently to pathenogenetic activation [8,9], suggesting that different strains of rats may have different optimal conditions for successful IVF. Therefore, in the present study, we compared the IVF requirements for two rat strains (SD and Wistar), and we defined the optimal conditions for successful fertilization in vitro of oocytes from the SD rat.…”

Dynamic Oxygen Enhances Oocyte Maturation in Long-Term Follicle Culture

“…28 This suggested that the parameters for this culture condition needed to be altered to develop a successful long-term culture technique that could produce mature oocytes with the same potential as those matured in vivo. We hypothesized that if the oxygen tension in the incubator were decreased to in vivo levels for developing preantral follicles, the result would be an increase in the proportion of mature functioning oocytes obtained from the culture.…”

Efficient Activation of Reconstructed Rat Embryos by Cyclin-Dependent Kinase Inhibitors

“…However, increased space and cost requirements often limit the production of genetically modified rats in this manner [16]. Also, standard SCNT has proven to be very difficult to implement in the rat [17], [18], [19], [20], [21], [22], [23]. To date, only a single report [24] exists describing the successful generation of a rat by this method.…”