Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To MidâPhase.

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 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.

ORGANISM(S): Bos taurus

SUBMITTER: MADHUSUDHAN GORAVANAHALLY

PROVIDER: E-GEOD-10662 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha.

Goravanahally Madhusudan P MP Salem Mohamed M Yao Jianbo J Inskeep E Keith EK Flores Jorge A JA

Biology of reproduction 20090121 5

Prostaglandin F2 alpha (PGF(2alpha)) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF(2alpha) is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is insensitivity of the early CL to luteolytic actions of PGF(2alpha). The mechanisms underlying this differential luteal sensitivity are poorly understood. The developing CL has a maximum number of PGF(2alpha) receptors; therefore, differences in signaling events may be respons ...[more]

PMID: 19164179

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Project description:The LH-like molecule chorionic gonadotropin (CG) is secreted by the macaque conceptus during and following implantation, “rescuing” the CL from impending regression and extending its functional lifespan in early pregnancy for approximately two weeks. CG binds to the same receptor as LH; i.e., LHCGR, and promotes production of steroids and other factors such as relaxin (RLN1). Our research group recently used Affymetrix™ rhesus macaque total genome arrays to compare the effects of CG on the luteal transcriptome from rhesus females during simulated early pregnancy (SEP) with changes during luteal regression in the non-fecund menstrual cycle. This analysis demonstrated that CG-rescue affected expression levels of 4,500 mRNA transcripts between days 10 and 15 of the luteal phase. Previous analyses indicated that a portion of the transcriptome in the macaque CL of the menstrual cycle was regulated indirectly by LH via the local actions of steroid hormones, including progesterone (P). Therefore, this study was designed to distinguish CG-regulated luteal genes that are dependent versus independent of local steroid (P) action. A protocol of increasing dosages of hCG (SEP) was begun on day 9 of the luteal phase in rhesus females combined with concurrent administration of the 3BHSD inhibitor trilostane (TRL) +/- the synthetic progestin (P) R5020. CL were collected on day 10 (no treatment) of the luteal phase to serve as a baseline comparison (n=8), 1 day of SEP (Day 10+hCG+/-TRL+/-R5020) and 6 days of SEP (Day 15+hCG+/-TRL+/-R5020); n=4/group. In the presence of CG, treatment with TRL reduced serum P levels to less than 1 ng/ ml after 1 day and all of the Day 15+h+TRL-treated females initiated menses before CL collection. The isolated CL were processed for total RNA and hybridized to microarrays. Compared to hCG treatment alone, 50 significantly altered mRNA transcripts were identified on day 10, rising to 95 on day 15 (P<0.05, ≥ 2-fold change in gene expression). The mRNA levels for several genes were validated in CL by real-time PCR. RNL1 levels increased with CG-treatment, but were not affected by steroid ablation, similar to previously reported relaxin protein expression. Steroid-sensitive genes included CDH11, IL1RN, INSL3, LDLR, OPA1, SERPINE1, SFRP4, and TNSF13B1; however differential sensitivity was observed and effects of steroid ablation and P replacement varied by day. Expression of some genes (e.g., 3BHSD2, ADAMTS1, ADAMTS5, MMP9, STAR, and VEGFA) previously identified as steroid regulated in the macaque CL during the menstrual cycle were not significantly altered by steroid ablation and P replacement during CG exposure in SEP. These data indicate that the majority of CG-regulated luteal transcripts are differentially expressed independently of local steroid actions. The proportion of steroid sensitive mRNA transcripts in the presence of CG is much smaller than in the presence of LH during the non-fecund cycle. Nevertheless, the steroid-regulated genes in the macaque CL may be essential during early pregnancy, based on the previous report that TRL treatment initiates premature structural regression of the CL during SEP. These data reinforce the concept that the structure, function, and regulation of the rescued CL in early pregnancy is different from the CL of the menstrual cycle.

Project description:The LH-like molecule chorionic gonadotropin (CG) is secreted by the macaque conceptus during and following implantation, M-bM-^@M-^\rescuingM-bM-^@M-^] the CL from impending regression and extending its functional lifespan in early pregnancy for approximately two weeks. CG binds to the same receptor as LH; i.e., LHCGR, and promotes production of steroids and other factors such as relaxin (RLN1). Our research group recently used AffymetrixM-bM-^DM-" rhesus macaque total genome arrays to compare the effects of CG on the luteal transcriptome from rhesus females during simulated early pregnancy (SEP) with changes during luteal regression in the non-fecund menstrual cycle. This analysis demonstrated that CG-rescue affected expression levels of 4,500 mRNA transcripts between days 10 and 15 of the luteal phase. Previous analyses indicated that a portion of the transcriptome in the macaque CL of the menstrual cycle was regulated indirectly by LH via the local actions of steroid hormones, including progesterone (P). Therefore, this study was designed to distinguish CG-regulated luteal genes that are dependent versus independent of local steroid (P) action. A protocol of increasing dosages of hCG (SEP) was begun on day 9 of the luteal phase in rhesus females combined with concurrent administration of the 3BHSD inhibitor trilostane (TRL) +/- the synthetic progestin (P) R5020. CL were collected on day 10 (no treatment) of the luteal phase to serve as a baseline comparison (n=8), 1 day of SEP (Day 10+hCG+/-TRL+/-R5020) and 6 days of SEP (Day 15+hCG+/-TRL+/-R5020); n=4/group. In the presence of CG, treatment with TRL reduced serum P levels to less than 1 ng/ ml after 1 day and all of the Day 15+h+TRL-treated females initiated menses before CL collection. The isolated CL were processed for total RNA and hybridized to microarrays. Compared to hCG treatment alone, 50 significantly altered mRNA transcripts were identified on day 10, rising to 95 on day 15 (P<0.05, M-bM-^IM-% 2-fold change in gene expression). The mRNA levels for several genes were validated in CL by real-time PCR. RNL1 levels increased with CG-treatment, but were not affected by steroid ablation, similar to previously reported relaxin protein expression. Steroid-sensitive genes included CDH11, IL1RN, INSL3, LDLR, OPA1, SERPINE1, SFRP4, and TNSF13B1; however differential sensitivity was observed and effects of steroid ablation and P replacement varied by day. Expression of some genes (e.g., 3BHSD2, ADAMTS1, ADAMTS5, MMP9, STAR, and VEGFA) previously identified as steroid regulated in the macaque CL during the menstrual cycle were not significantly altered by steroid ablation and P replacement during CG exposure in SEP. These data indicate that the majority of CG-regulated luteal transcripts are differentially expressed independently of local steroid actions. The proportion of steroid sensitive mRNA transcripts in the presence of CG is much smaller than in the presence of LH during the non-fecund cycle. Nevertheless, the steroid-regulated genes in the macaque CL may be essential during early pregnancy, based on the previous report that TRL treatment initiates premature structural regression of the CL during SEP. These data reinforce the concept that the structure, function, and regulation of the rescued CL in early pregnancy is different from the CL of the menstrual cycle. A protocol of increasing dosages of hCG (SEP) was begun on day 9 of the luteal phase in rhesus females combined with concurrent administration of the 3BHSD inhibitor trilostane (TRL) +/- the synthetic progestin (P) R5020. CL were collected on day 10 (no treatment) of the luteal phase to serve as a baseline comparison (n=8), 1 day of SEP (Day 10+hCG+/-TRL+/-R5020) and 6 days of SEP (Day 15+hCG+/-TRL+/-R5020); n=4/group.The isolated CL were processed for total RNA and hybridized to Affymetrix Rhesus Genome microarrays.

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.

Dataset Information

Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To MidâPhase.

Publications

Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To MidâPhase.

Publications

Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Identification of Differential Gene Expression During Transition of Bovine Corpus Luteum from Early To MidâPhase.