30 SCRIPTAID TREATMENT IMPROVES POST-IMPLANTATION DEVELOPMENT OF SHEEP CLONED EMBRYOS

Abstract

Increased histone acetylation by exposure to inhibitors of deacetylase enzymes has been reported to improve development of embryos produced by somatic cell nuclear transfer (SCNT). However, the response to such treatment seems to vary according to the species, cell line, and type of inhibitor used. The main objective of this study was to evaluate if treatment with the histone deacetylase inhibitor Scriptaid could improve the development to term of sheep SCNT-embryos. The 2 fibroblast cell lines used in this study were obtained from skin biopsies collected from 2 adult rams of the Santa Ines breed. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 30 crossbred sheep that were hormonally stimulated as described previously (Baldassarre et al. 2002 Theriogenology 57, 275). Oocyte maturation, cell transfer, fusion and activation, culture and transfer to recipients were conducted following procedures previously described (Baldassarre et al. 2003 Cloning Stem Cells 5, 279). Briefly, oocytes were matured in vitro for 24 h in TCM 199 supplemented with hormones and 10% fetal bovine serum, at 38.5°C in 5% CO₂. Cells were transferred into enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6 kV cm^–1 for 70 μs. The reconstructed embryos were then activated using ionomycin (5 μM/5 min) followed by cycloheximide (10 μg mL^–1) and cytochalasin B (7.5 μg mL^–1) for 4 to 5 h and then cultured in mSOF media (control); while half of the reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Subsequent to culture in mSOF ± Scriptaid as above, selected embryos were finally transferred into the oviducts of synchronized recipients within 24 h from fusion. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. A total of 258 oocytes were recovered (8.6/donor), of which 203 resulted in fused embryos after micromanipulation (79%) and 178 (69%) were selected for transfer into the oviducts of 18 synchronized recipients (avg. 10 embryos/recipient). Initial pregnancy was significantly higher in the Scriptaid group (40 v. 12.5%; P < 0.01). Interestingly, pregnancy was maintained through gestation Day 90 in the Scriptaid group, while the pregnant recipient carrying the control embryos lost her pregnancy by Day 60. All 4 pregnant recipients are due in early August. Our results are consistent with a previous report from (Zhao et al. 2010 Cel. Reprog. 12, 75) working with pig embryos and suggest that Scriptaid treatment can improve post-implantation development of SCNT sheep embryos. The results above will be further evaluated when data from births becomes available.