Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Abstract

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

Polarized Light Microscopy in Mammalian Oocytes

“…As some research studies have shown that PLM does not have detrimental effects on oocyte developmental competence, researchers have been using PLM to enucleate oocytes from mice and hamsters (Liu et al 2000a), cattle (Lu et al 2005) and non-human primates (Chen et al 2007;Mitalipov et al 2007) with promising results. In addition, rhesus macaque embryos were produced for nuclear transfer of adult skin fibroblasts with the aid of PLM to enucleate the oocytes.…”

Effects of enucleation method on in vitro and in vivo development rate of cloned pig embryos

“…This approach results in an enucleation rate that varies widely among laboratories (40%-90%) depending on the skill and experience of the operators; it is usually difficult to reach a high enucleation rate due to "blind" localization of the nucleus [2][3][4][5] . To increase enucleation efficiency, spindle imaging system-assisted (SIS-A) enucleation has been used for oocyte enucleation in pig, cattle and other species [6][7][8][9][10][11][12][13] . This method employs a polarized light microscope to visualize the meiotic spindle-containing oocyte nucleus.…”

“…This method employs a polarized light microscope to visualize the meiotic spindle-containing oocyte nucleus. The SIS-A approach can achieve a high oocyte enucleation rate (88%-100%) in several mammalian species [8,10,12,13] and improve the in vitro development efficiency of cloned embryos in pigs and cattle [6,7] . Fluorescence dye staining has also been used to localize clearly the oocyte nucleus before enucleation [14,15] .…”