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Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG. Lussier JG et al. (2017) Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles. Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened. Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, (774)KNSHNEL(780). Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The (774)KNSHNEL(780) sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression. We conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.//////////////////
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Changes in granulosa cells gene expression associated with increased oocyte competence in bovine. Nivet AL et al. One of the challenges in mammalian reproduction is to understand basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, a previous experiment from our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post FSH withdrawal (coasting). Using such protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation by real-time PCR. Results shows that the major changes in gene expression occurred from 20h to 44h of coasting, when oocyte quality rising. Secondly, among up-regulated genes (20h to 44h), 25%were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified 2 patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, 3 major functional changes were identified: 1) the end of follicle growth (BMPR1B, IGF2, RELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2, TF, VNN1) and 2) apoptosis (KCNJ8) followed by 3) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase of apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases.
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