Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos.
In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.
Bauer
Bethany K
BK
Division of Animal Science, University of Missouri, Columbia, Missouri 65211, USA.
Isom
S Clay
SC
Spate
Lee D
LD
Whitworth
Kristin M
KM
Spollen
William G
WG
Blake
Sean M
SM
Springer
Gordon K
GK
Murphy
Clifton N
CN
Prather
Randall S
RS
D000822:Animal Husbandry / Q000379:methods; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D053624:Blastocyst Inner Cell Mass / Q000166:cytology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism; D002452:Cell Count / Q000662:veterinary; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D030561:Databases, Nucleic Acid; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D020869:Gene Expression Profiling / Q000379:methods / Q000662:veterinary; D018507:Gene Expression Regulation, Developmental; D008832:Microchemistry / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods / Q000662:veterinary; D034421:Sus scrofa / Q000196:embryology* / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology
D027201:Cationic Amino Acid Transporter 1; D018076:DNA, Complementary; D012333:RNA, Messenger; D001120:Arginine