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Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To Mid–Phase.

ABSTRACT: Prostaglandin F2 alpha (PGF2alpha) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF2alpha is used in the beef and dairy cattle to synchronize estrus. A limitation of this protocol is an insensitivity of the early CL to luteolytic actions of PGF2alpha. The mechanisms underlying luteal sensitivity (LS) are poorly understood. The early CL has maximum number of PGF2alpha receptors; therefore differences in signaling events might be responsible for LS. Hence differential gene expression at two developmental stages, days 4 (D-4) and 10 (D-10) post estrus, might account for differences in signal transduction pathways associated with LS. This possibility was examined in these studies. Microarray analysis (n=3 per stage) identified 167 genes that were differentially expressed (p<0.05). These were categorized into genes involved in cell signaling, steroidogenesis and metabolism, protein degradation, transcription regulation and DNA biosynthesis, protein biosynthesis and modification, extracellular matrix and cytoskeletal proteins, antioxidant property, miscellaneous, and unknown functions. Real-time PCR confirmed the differential expression of 9 randomly selected genes, including protein kinase C inhibitor protein-1 (KCIP-1) and regulator of G-protein signaling 2 (RGS2), observed in microarray. Further, the in vivo effect of exogenous PGF2alpha (n=3 per stage) on selected genes that were found differentially expressed during this developmental transition was examined. PGF2alpha increased the expression of a guanine nucleotide binding protein beta polypeptide 1 (GNB1) in D-4 CL and Ca2+/calmodulin dependent kinase kinase 2 beta (Camkk2) in D-10 CL. Therefore, GNB1, Camkk2, KCIP-1, and RGS2 are candidate genes that might play significant role in acquisition of luteal sensitivity to PGF2alpha. Keywords: Direct comparison between Day-4 and Day-10 CL, Developmental type comparision

ORGANISM(S): Bos taurus

PROVIDER: GSE10662 | GEO | 2009/02/23

SECONDARY ACCESSION(S): PRJNA107629

REPOSITORIES: GEO

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Project description:Prostaglandin F2 alpha (PGF2alpha) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF2alpha is used in the beef and dairy cattle to synchronize estrus. A limitation of this protocol is an insensitivity of the early CL to luteolytic actions of PGF2alpha. The mechanisms underlying luteal sensitivity (LS) are poorly understood. The early CL has maximum number of PGF2alpha receptors; therefore differences in signaling events might be responsible for LS. Hence differential gene expression at two developmental stages, days 4 (D-4) and 10 (D-10) post estrus, might account for differences in signal transduction pathways associated with LS. This possibility was examined in these studies. Microarray analysis (n=3 per stage) identified 167 genes that were differentially expressed (p<0.05). These were categorized into genes involved in cell signaling, steroidogenesis and metabolism, protein degradation, transcription regulation and DNA biosynthesis, protein biosynthesis and modification, extracellular matrix and cytoskeletal proteins, antioxidant property, miscellaneous, and unknown functions. Real-time PCR confirmed the differential expression of 9 randomly selected genes, including protein kinase C inhibitor protein-1 (KCIP-1) and regulator of G-protein signaling 2 (RGS2), observed in microarray. Further, the in vivo effect of exogenous PGF2ï¡ (n=3 per stage) on selected genes that were found differentially expressed during this developmental transition was examined. PGF2alpha increased the expression of a guanine nucleotide binding protein beta polypeptide 1 (GNB1) in D-4 CL and Ca2+/calmodulin dependent kinase kinase 2 beta (Camkk2) in D-10 CL. Therefore, GNB1, Camkk2, KCIP-1, and RGS2 are candidate genes that might play significant role in acquisition of luteal sensitivity to PGF2alpha. Keywords: Direct comparison between Day-4 and Day-10 CL, Developmental type comparision The present experiment made a direct comparision of gene expression in early (Day-4) vs mid-phase CL (Day-10). Each experiment contained two technical relicates per slide. Total of three biological replicates were used ( 3 animals for Day-4 and 3 animals for Day-10). Two Day-4 samples were labelled Cy5 and one Day-10 with Cy5. Two Day-10 samples were labelled with Cy3 and one Day-4 with Cy3. The Day-4 sample was control for our experiments.

Project description:To explore chorionic gonadotropin (CG)-regulated gene expression in the primate corpus luteum (CL), adult female rhesus macaques were treated with a model of simulated early pregnancy (SEP). Total RNA was isolated from individual CL and hybridized to AffymetrixM-bM-^DM-" GeneChip Rhesus Macaque Genome Arrays The level of 1192 transcripts changed expression > 2-fold (one-way ANOVA, FDR correction; P<0.05) during SEP when compared to Day 10 untreated controls, and the majority of changes occurred between Days 10 and 12 of SEP. To compare transcript levels between SEP rescued and regressing CL, previously banked rhesus GeneChip array data from the mid- to late and very late luteal phase were analyzed with time-matched intervals in SEP. Comparing RMA-normalized transcripts from the natural cycle with those from luteal rescue revealed 7677 transcripts changing in expression pattern >2 fold (one-way ANOVA, FDR correction; P<0.05) between the two groups. Clustering of samples revealed that the SEP samples possessed the most related transcript expression profiles. Regressed CL (days 18-19, around menses) were the most unlike all other CL. The most affected KEGG pathway was Steroid Biosynthesis, and most significantly absent pathways following SEP treatment includes groups of genes whose products promote cell-death. By further comparing the genome-wide changes in luteal gene expression during rescue in SEP, with those in CL during luteolysis in the natural menstrual cycle, it is possible to identify key regulatory pathways promoting fertility. Simulated early pregnancy (SEP) treatment was begun on day 9 as Duffy and Stouffer (1997) by treatment of females with recombinant human chorionic gonadotropin (hCG; NovarelM-bM-^DM-", Ferring Pharmaceuticals Inc. Parsippany, NJ, USA) in increasing dosages (15, 30,45,90,180,360,720,1440, and 2880 IU) twice daily by intramuscular injection. CL were collected by laparotomy on days 10, 12, 15, and 18, representing 1, 3, 6 and 9 days of hCG treatment (n=4 CL/day). Additionally, luteal day 10 untreated CL were collected to serve as baseline controls for SEP CL. All CL were dissected away from luteal tissue, sectioned, and snap-frozen in liquid nitrogen and stored at -80M-BM-0C until RNA and protein isolation by TRIzolM-BM-. extraction (Invitrogen, Carlsbad, CA, USA) according to manufacturerM-bM-^@M-^Ys protocols.

Project description:In monoovulatory species like bovines, a striking diversity in regulation of the corpus luteum (CL) function exists within species during different stages of the luteal phase. The function and life span of CL appears to be regulated by the interplay of both luteotrophic and luteolytic factors, whose roles and actions are varied from one species to another. Although, studies continue to expand our current understanding of the cellular and molecular actions of prostaglandin F 2alpha (PGF2M-NM-1 - a physiological luteolysin)-induced luteolysis, knowledge of the mechanism whereby, PGF2M-NM-1 mediates its luteolytic actions remains poorly understood. The mechanism of PGF2M-NM-1-induced luteolysis is a complex process involving changes in expression of multiple genes encoding gene products that regulate steroidogenesis, stress related or apoptotic genes, factors involved in immune response and enzymes that stimulate PGF2M-NM-1 production. Thus, in the present study, efforts were made to identify the temporal changes in the global gene expression profile in the CL of buffalo cows in response to PGF2M-NM-1 treatment. The molecular signature of genome-wide transcriptional changes in the CL tissue subjected to PGF2M-NM-1-induced luteolysis will expand our knowledge on basic mechanism/s that regulates the luteolytic process. The results obtained in this study provide insight into the mechanisms underlying the PGF2M-NM-1- induced regression of CL.Keywords: CL, PGF2M-NM-1, gene expression The objective of this study was to identify the PGF2M-NM-1-induced temporal changes in transcriptome of bovine CL on day 11 of estrous cycle. In the experiments with buffalo cows, animals with history of normal estrous cyclicity were treated with Cloprostenol (an PGF2M-NM-1 analogue; 500 M-BM-5g i.m.) on day 11 of the estrous cycle to induce luteolysis and CL tissues were collected at (control; n=4 amimals/time point), 3 (n=2 animals/time point), 6 and 18 (n=3 animals/time point) h post PGF2M-NM-1 administration to examine gene expression changes associated with immediate early and delayed changes in the CL, respectively. Following retrieval of CL from the ovary, flash frozen in liquid nitrogen and stored at -70 M-BM-0C for the purpose of RNA isolation. The isolated RNA was labeled and hybridized to Affymetrix GeneChip Bovine Genome Arrays as per the manufacturerM-bM-^@M-^Ys instructions.

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Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To Mid–Phase.

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