In Vitro Developmental Potential of Nuclear Transfer Embryos Cloned with Enucleation Methods using Pre‐denuded Bovine Oocytes

Abstract

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.

Effects of the Timing of Cumulus Cell Removal from Bovine Oocytes on Enucleation Rate and Subsequent Development after Somatic Cell Nuclear Transfer

“…In blind enucleation, the removal of metaphase II (MII) chromosomes is usually carried out blindly by aspirating or pushing out the cytoplasm underlying the first polar body (PB1). The rate of successful enucleation by this method is reportedly lower than those of Hoechst staining and chemical treatment [2,3]. On the other hand, enucleation using UV irradiation after staining of the oocyte DNA with Hoechst dye is an efficient and reliable method, but exposure of the recipient oocytes to Hoechst dye and UV irradiation may have detrimental effects on their DNA and cytoplasmic organelles [1,4].…”

“…In this method, recipient oocytes are denuded of cumulus cells after 12 h of in vitro maturation (IVM) at the metaphase I stage, subsequently cultured for an additional 6 h until nuclear maturation and enucleated [3]. Malenko et al also demonstrated that removal of cumulus cells at 15 h after IVM leads to a high success rate of enucleation [6].…”

“…Recently, Jeon et al reported that blind enucleation of pre-denuded oocytes raises the enucleation success rate to the same level as that of chemically assisted enucleation [3]. In this method, recipient oocytes are denuded of cumulus cells after 12 h of in vitro maturation (IVM) at the metaphase I stage, subsequently cultured for an additional 6 h until nuclear maturation and enucleated [3].…”

“…These reports suggest that the removal of cumulus cells prior to PB1extrusion is effective for blind enucleation. The low efficiency of conventional blind enucleation is probably a result of displacement of the PB1 in the perivitelline space of oocytes by vortexing and pipetting after PB1 extrusion [3,6].…”

“…We compared the enucleation success rates of recipient oocytes from which cumulus cells were removed at 12 [3], 15 [6] or 18 h after IVM. In vitro developmental ability of somatic cell NT (SCNT) embryos derived from these enucleated oocytes was also examined.…”