The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro.
BACKGROUND
Excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on in vitro fertilized bovine embryos.


RESULTS
Approximately 12,000-24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.


CONCLUSIONS
These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada. lfavetta@uoguelph.ca
Laura A
LA
Favetta
Pavneesh
P
Madan
Gabriela F
GF
Mastromonaco
Elizabeth J
EJ
St John
W Allan
WA
King
Dean H
DH
Betts
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D002417:Cattle; D002970:Cleavage Stage, Ovum / Q000378:metabolism; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D005307:Fertilization in Vitro; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D008845:Microinjections; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055765:Shc Signaling Adaptor Proteins
D048868:Adaptor Proteins, Signal Transducing; D012330:RNA, Double-Stranded; D017382:Reactive Oxygen Species; D055765:Shc Signaling Adaptor Proteins