ERK1/2-dependent gene expression in the bovine ovulating follicle

Purpose: establishing a role for ERK1/2 in bovine ovulation by using RNA-Seq to compare granulosa cells collected from the dominant follicle at different times post GnRH treated with PD0325901 (ERK1/2 signaling inhibitor) Methods: The dominant follicle of cattle was subject to ultrasound-guided intrafollicular injection of either a Vehicle treatment or PD0325901 inhibitor treatment. Thirty minutes later we collected granulosa cells from Vehicle treated cows (Vehicle_0h_GnRH), while other cows were administered with an intramuscular injection of goonadotropin releasing hormone (GnRH) to simulate an LH surge. Six hours later granulosa cells were collected from Vehicle treated and PD0325901 treated cows (Vehicle_6h_GnRH & PD0325901_6h_GnRH). All granulosa cell RNA was subject to the Illumina HiSeqq 2500 PE 125. Quality check for reads was performed using FastQC v0.11.5 and adapters were removed by Trimmomatic v0.36. The data was aligned to the bovine Genome (Btau4.6.1) using Bowtie v2.2.1.0 and TopHatv2.0.11. Next, the rnumber of reads mapped to each gene were counted using HTSeq v0.6.1 Results: The counts from our 8 samples (Vehicle_0h_GnRH; n=2 , Vehicle_6h_GnRH; n=3, PD0325901_6h_GnRH; n=3) were subject to analysis to determine differentially expressed genes (DEGs) between our groups. Using The DESeq2 R package we observed 2 121 DEGs between Vehicle_0h_GnRH & Vehicle_6h_GnRH and 285 DEGs between Vehicle_6h_GnRH and PD0325901_6h_GnRH. We confirmed our RNA-seq analysis for a number of known genes in ovulation (ADAMTS1, TNFAIPs, EGR1, etc..) by qPCR. Conclusions: ERK1/2 is essential for ovulation in cattle as observed by the DEGs in granulosa cells in the pre-ovulatory follicle. To our knoweldge, we are the firs tto iinhbiti ERK1/2 signaling in vivo in cattle. Overall design: bovine granulosa cell mRNA profiles from the dominant follicle/pre-ovulatory follicle treated with PD0325901 (in vivo) were generated by deep sequencing using Illumina HiSeq 2500 PE125