421 PRODUCTION AND CHARACTERIZATION OF TRANSGENIC BOVINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION-MEDIATED GENE TRANSFER ASSISTED BY DIFFERENT CHEMICAL ACTIVATION TREATMENTS

Abstract

Intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) is a powerful technique in the production of transgenic mice. However, it has not yet been exploited in cattle because of ICSI fertilization problems. The objective of this study was to evaluate fertilization rates, transgenesis, blastocysts rates, and enhanced green fluorescent protein (egfp) blastocyst quality parameters (mean cell number, Oct-4 expression, and day of formation) of ICSI-MGT derived bovine embryos treated with 5 different chemical activation protocols. COCs were aspirated from ovaries obtained from local slaughterhouses and IVM was performed using conventional protocols. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construct (pCX-EGFP) was carried out in 2.8% Na citrate, with 0.5 μg of plasmid per million spermatozoa for 5 min at 0°C. Then, spermatozoa were injected into metaphase II (MII) oocytes. Injected oocytes were treated with 5 μM ionomycin (Io) for 4 min, and after 3 h, incubated in 1.9 mM DMAP for 3 h (Io-DMAP); second Io followed by DMAP for 3 h (2Io-DMAP), second Io alone (2Io); 7% ethanol for 5 min (Io-EtOH); or 20 mM SrCl2 for 5 h (Io-SrCl2) immediately after first Io. Embryos were cultured in SOFaa medium. Fertilization efficiency (2 pronuclei and 2 polar bodies) at 16 h after ICSI and condensed sperm head presence at Day 4 were evaluated by Hoechst 33342 staining of ICSI-MGT embryos. Expression of EGFP was observed under the fluorescence microscope (488 nm). Oct-4 expression was detected by immunocytochemistry and confocal laser scanning microscopy. Each experiment was replicated 3 times. Differences between percentages (Fisher test) or means (ANOVA) were significant at P < 0.05. Fertilization rates did not differ between treatments. Transgenic and nontransgenic embryos were obtained by ICSI-MGT. All transgenic embryos were the product of ICSI fertilization (no condensed sperm head present at Day 4). On the other hand, at least 60% of nontransgenic embryos (>4 cells) were the result of parthenogenesis (condensed sperm present). Blastocyst rates after 2Io-DMAP (21/81; 25.9%) were not statistically different from Io-DMAP (15/80; 18.7%) or Io-EtOH (15/102; 14.7%) but they were higher than 2Io (8/85; 9.4%) or Io-SrCl2 (13/119; 10.9%). Transgenesis rates were higher for Io-DMAP (42/80; 52.3%), 2Io-DMAP (43/81; 53%) and Io-SrCl2 (51/119; 42.8%) than for 2Io (24/85; 28.2%) and Io-EtOH (30/102; 29.4%). Over 80% of the blastocysts were transgenic.An explant was derived from an egfp-blastocyst and it proliferated and expressed the transgene for 17 days. Mean cell numbers, Oct-4 expression, and day of formation (≤ Day 7 for all treatments except for 2Io) of the ICSI-MGT egfp-blastocysts were not statistically different neither between ICSI assistance treatments nor to IVF or parthenogenetic (Io-DMAP) controls, indicating similar embryo quality according to these parameters. All treatments proved to be highly efficient to assist ICSI-MGT and to produce transgenic bovine embryos. Moreover, ICSI-MGT with an early expressed marker can help to evaluate ICSI fertilization real efficiency.