IDEAL CULTURE CONDITIONS FOR ENHANCING FULL PREIMPLANTATION DEVELOPMENT OF EMBRYOS GENERATED THROUGH SOMATIC CELL HAPLOIDIZATION

Abstract

To identify the ideal culture conditions for promoting full preimplantation development of artificial embryos generated through somatic cell hapoloidization (SCH). Metaphase II (MII) oocytes from B6D2F1 mice were enucleated under Oosight™ visualization and exposure to cytochalasin B. Nuclear transfer of an individual FVB mouse cumulus cell was conducted by transfer into the perivitelline space of the resulting ooplast and fused using Sendai virus. Development of a pseudo-meiotic spindle was assessed in the reconstructed oocytes 2 hours after transfer, and the oocytes were then piezo-ICSI inseminated. Zygotes were cultured either in KSOM medium (KSOM), KSOM supplemented with Trichostatin A (TSA), or KSOM supplemented with fasudil, scriptaid, and RS-1 (cocktail). Trichostatin A and scriptaid are both histone-deacetylase inhibitors that may improve cellular reprogramming and embryo development after nuclear transfer. Fasudil, a Rho-associated kinase inhibitor, reduces apoptosis, while RS-1 aids in DNA damage repair. Zygotes were cultured in TSA and cocktail for 24 hrs only, before being changed to KSOM. Intact oocytes were ICSI inseminated as controls and cultured in KSOM (control). All four groups were monitored in a time-lapse imaging system for 96hr. A total of 527 MII oocytes were enucleated, resulting in 522 ooplasts (99.0%). Nuclear transfer of these ooplasts resulted in a survival rate of 99.6%, generating 520 reconstructed oocytes. A positive spindle was assessed in 360 artificial oocytes (69.2%), and piezo-ICSI of these spindle-positive oocytes resulted in a survival rate of 75.0%. ICSI insemination of 60 control oocytes had a comparable survival rate (83.3%). Fertilization rates were similar among all culture conditions, as well as to the control, with rates of 91.2% for the cocktail group, 93.8% for the KSOM group, 94.4% for the TSA group, and 94.0% for the control. The cocktail group exhibited significant delays at all stages (P<0.05) except the 2-cell stage when compared to the control. KSOM-cultured embryos had comparable morphokinetic development to the control up to the blastocyst stage, except for a delay in pronuclei appearance (P<0.002). Morphokinetic parameters of TSA embryos were comparable to the controls at all stages, beginning with the pseudo-second polar body extrusion (haploidization) up until the blastocyst stage. The intact oocyte control had an 82.3% blastulation rate compared to all experimental groups (P<0.00001). The cocktail cohort had a blastulation rate of only 9.6%, and the KSOM group had a blastulation rate of 14.5%; the highest blastulation rate was reached by the TSA group at 23.5%. Although the blastocyst development was still suboptimal, among the culture media tested, the addition of a histone-deacetylase inhibitor (TSA) yielded artificial embryos with morphokinetic parameters similar to the control.