Title: Identification of changes in bovine oviductal mRNA expression by RNAseq from animals with high and low circulating estradiol concentrations during timed artificial insemination Authors
Submitted to: International Symposium on Reproduction in Domestic Ruminants
Publication Type: Abstract Only
Publication Acceptance Date: April 16, 2014
Publication Date: N/A
Technical Abstract: Timed artificial insemination of beef cows with high concentrations of estradiol at time of insemination are known to impact pregnancy success by 27%±5% compared to animals with low estradiol. Therefore, understanding the mechanisms responsible for this biological variation is key to improving reproductive efficiency. To explore the role the oviductal environment plays in this reproductive success, we conducted RNA-seq on DNAse treated RNA isolated from the isthmus and ampullary-isthmic junction collected 24 hr after AI from six angus-cross beef cows with differential estradiol concentrations (High-E2, 10.12±0.62 pg/ml [n=3] vs Low-E2, 5.97±0.62 pg/ml [n=3], P=0.0093) at the time of fixed-time AI. Paired end libraries were constructed using the Truseq version 2.0 RNA-seq library prep kits and sequenced on a Hi-seq 2000. Reads were mapped and differential expression conducted using the Tuxedo suite of software. Transcript abundance is conveyed using the FPKM method and differences were considered significant when the FDR adjusted P-value was less than 0.05. Pathway analysis was conducted using Ingenuity Pathway Analysis software. Within the ampullary-isthmic junction, 49 genes were up-regulated in the High-E2 group and 2107 genes were up-regulated in the Low-E2 group. An additional 60 and 255 genes were uniquely expressed in the High-E2 and Low-E2 groups, respectively. Within the isthmus, 176 genes were up-regulated in the High-E2 group and 184 genes were up-regulated in the Low-E2 group. Twenty and 21 genes in the isthmus were also uniquely expressed in the High-E2 and Low-E2 groups, respectively. Pathway analysis was consistent with our classification of the animals and revealed that estrogen receptor signaling is predicted to be activated in the High-E2 group compared to the Low-E2 group (Junction z= 1.755, Isthmus: z= 2.573). In the isthmus, the top canonical pathways affected were glycolysis 1 and inhibition of matrix metalloproteases (P=2.09x10**-6 and 6.98x10**-6, respectively). For the ampullary-isthmic junction, the top canonical pathways affected were EIF2 and mTOR signaling (P=8.96x10**-22 and 2.91x10**-16, respectively). The biological functions most affected by changes in the transcriptome were protein metabolism in the ampullary-isthmic junction (P=6.12x10**-10, Z=2.494) and migration of cells in the isthmus (P=5.17x10**-29, Z=-4.028). In conclusion, selecting only cows that have exhibited estrus before timed artificial insemination can improve pregnancy success, and the pathways reported here give a starting point to investigate how preovulatory estradiol concentrations are impacting fertilization, early embryo development, and overall pregnancy success.