Specific Activation Requirements of Zona-Free Sheep Oocytes before and after Somatic Cell Nuclear Transfer

Abstract

In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 μM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 μM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 μM ionomycin for 5 min rather than 2.5 μM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.

Comparison of efficiency of in vitro cloned sheep embryo production by conventional somatic cell nuclear transfer and handmade cloning technique

“…The cleavage percentage obtained in HMC was 66.92 ± 3.76% which was significantly higher than that obtained in conventional SCNT ( p < .05). Similar cleavage percentage has been observed with HMC in cattle and pig (Hajian et al., ; Lagutina et al., ). However, higher cleavage rates with similar technique have been obtained in goat (Khan et al., ) and buffalo (Shah et al., ).…”

Mitochondrial transfer: Implications for assisted reproductive technologies

“…In cases of reproductive cloning in other mammalian species (Bordignon et al, 2013, Hajian et al, 2013, Kamimura et al, 2013), although the techniques have been perfected, manipulated embryos undergo developmental failure and the resulting offspring are found to succumb to early death, which is presumed to be coupled to unanticipated epigenetic events. Regarding somatic cell nuclear transfer (SCNT), a genome-wide study revealed that although an oocyte has the ability to epigenetically reprogram transplanted DNA, it cannot fully replicate the DNA demethylation which occurs during natural fertilisation.…”