Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos.
Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.
Purpera
M N
MN
Louisiana State University Agricultural Center, Baton Rouge, 70803, USA.
Giraldo
A M
AM
Ballard
C B
CB
Hylan
D
D
Godke
R A
RA
Bondioli
K R
KR
D000818:Animals; D002417:Cattle / Q000196:embryology*; D002452:Cell Count; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics; D003470:Culture Media; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development / Q000187:drug effects; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D051272:Glucose Transporter Type 1 / Q000096:biosynthesis / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation
D018031:Connexin 43; D003470:Culture Media; D051272:Glucose Transporter Type 1; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger