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

ABSTRACT: 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.

ORGANISM(S): Bos taurus

PROVIDER: GSE121588 | GEO | 2018/10/23

REPOSITORIES: GEO

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Project description:In mammals, ovarian folliculogenesis leading to the ovulation of completely mature oocytes is a long and complex process that is regulated at different levels. The mechanisms that underlie the selection of one or several dominant follicles as well as the regulation of the number of ovulating follicles are largely unknown. In this project, we proposed to study the genetic determinism that underlies the difference of ovulation rate between species (cattle and pigs), by studying two processes that exist in ovary: follicular development and follicular atresia. Towards this purpose, we made a comparative transcriptomics study on granulosa cells. Pig and cattle comparison was achieved by a transcriptome analysis with a 9K nylon pig microarray (GPL3729) on granulosa cells from either small healthy antral follicles (SHF), small atretic follicles (SAF) or large healthy antral follicles (LHF). The images were quantified using AGscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Transcriptomic analysis on pig and cattle evidenced 997 differentially expressed genes (FDR1%) between the three follicle classes and/or the two species. This research project which implicated three laboratories from INRA: "Laboratoire de Genetique Cellulaire" (UMR444-LGC) , "Station d'Amelioration Genetique des Animaux" (UR 631-SAGA) and "Physiologie des Comportements et de la reproduction" (UMR 85-PRC) benefited from both European funding through SABRE project and French ANR funding through GenOvul project. Keywords: transcriptome analysis, pig, cattle, ovary, folliculogenesis, gene expression, cDNA microarray The data were obtained from 34 RNA samples: 10 small healthy follicles samples (6 for sows and 4 for cows), 13 small atretic follicles samples (7 for sows and 6 for cows) and 11 samples for large healthy follicles (6 for sows and 5 for cows). They were hybridized on a 9K pig nylon microarray (GPL3729).

Project description:Direct exposure to physiologically-relevant elevated temperatures during the first half of in vitro maturation heightens cumulus-derived progesterone production, suppresses matrix metallopeptidase 9 secretion, and alters cumulus transcriptome. We hypothesized that heat-induced perturbations in cumulus cells enveloping maturing oocyte may extend to the mural granulosa of the periovulatory follicle in the heat-stressed cow to subsequently follicular fluid proteome. Lactating Holsteins were pharmacologically stimulated to produce a dominant follicle then given GnRH to induce an LH surge. Following GnRH administration, cows were maintained at ~67 temperature humidity index (THI; thermoneutral conditions) or exposed to conditions simulating an acute heat stress event (71 to 86 THI; heat stress for ~12 h). Fluid was aspirated from periovulatory follicle ~16 h after GnRH. Follicular fluid proteome from thermoneutral (n = 5) and hyperthermic (n = 5) cows was evaluated by quantitative tandem mass spectrometry (nano LC-MS/MS). We identified 35 differentially-abundant proteins. Functional annotation revealed numerous immune-related proteins. Subsequent efforts revealed an increase in levels of the proinflammatory mediator bradykinin in follicular fluid (P = 0.0456) but not in serum (P = 0.9319) of hyperthermic cows. Intrafollicular increases in transferrin (negative acute phase protein) in hyperthermic cows (P = 0.0181) coincided with a tendency for levels to be increased in the circulation (P = 0.0683). Nine out of 15 cytokines evaluated were detected in follicular fluid. Heat stress increased intrafollicular IL6 levels (P = 0.0160). Whether hyperthermia-induced changes in the heat-stressed cow’s follicular fluid milieu reflect changes in mural granulosa, cumulus, other cell types secretions, and/or transudative changes from circulation remains unclear. Regardless of origin, heat stress/hyperthermia related changes in the follicular fluid milieu may have an impact on components important for ovulation and competence of the cumulus-oocyte complex contained within the periovulatory follicle

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

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