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Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

ABSTRACT: In several species including the buffalo cow, prostaglandin (PG) F2? is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF2? in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF2? treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression changes in several of immediate early genes and transcription factors within 3 h of treatment. Also, changes in expression of genes associated with cell to cell signaling, cytokine signaling, steroidogenesis, PG synthesis and apoptosis were observed. Analysis of various components of LH/CGR signaling in CL tissues indicated decreased LH/CGR protein expression, pCREB levels and PKA activity post PGF2? treatment. The novel finding of this study is the down regulation of CYP19A1 gene expression accompanied by decrease in expression of E2 receptors and circulating and intra luteal E2 post PGF2? treatment. Mining of microarray data revealed several differentially expressed E2 responsive genes. Since CYP19A1 gene expression is low in the bovine CL, mining of microarray data of PGF2?-treated macaques, the species with high luteal CYP19A1 expression, showed good correlation between differentially expressed E2 responsive genes between both the species. Taken together, the results of this study suggest that PGF2? interferes with luteotrophic signaling, impairs intra-luteal E2 levels and regulates various signaling pathways before the effects on structural luteolysis are manifest.

SUBMITTER: Shah KB

PROVIDER: S-EPMC4125180 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

Shah Kunal B KB Tripathy Sudeshna S Suganthi Hepziba H Rudraiah Medhamurthy M

PloS one 20140807 8

In several species including the buffalo cow, prostaglandin (PG) F2α is the key molecule responsible for regression of corpus luteum (CL). Experiments were carried out to characterize gene expression changes in the CL tissue at various time points after administration of luteolytic dose of PGF2α in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF2α treatment and evidence of apoptosis was demonstrable at 18 h post treatment. Microarray analysis indicated expression change ...[more]

PMID: 25102061

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Project description:BackgroundPGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFβ1, which plays an important role during luteal regression.MethodsThe current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFβ1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3βHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFβ1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9.ResultThe present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFβ1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFβ1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFβ1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells.ConclusionThese results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFβ1 signaling for luteolysis.

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.

Project description:BackgroundDuring female reproductive cycles, a rapid fall in circulating progesterone (P4) levels is one of the earliest events that occur during induced luteolysis in mammals. In rodents, it is well recognized that during luteolysis, P4 is catabolized to its inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP) by the action of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) enzyme and involves transcription factor, Nur77. Studies have been carried out to examine expression of 20alpha-HSD and its activity in the corpus luteum (CL) of buffalo cow.MethodsThe expression of 20alpha-HSD across different bovine tissues along with CL was examined by qPCR analysis. Circulating P4 levels were monitored before and during PGF2alpha treatment. Expression of 20alpha-HSD and Nur77 mRNA was determined in CL at different time points post PGF2alpha treatment in buffalo cows. The chromatographic separation of P4 and its metabolite, 20alpha-OHP, in rat and buffalo cow serum samples were performed on reverse phase HPLC system. To further support the findings, 20alpha-HSD enzyme activity was quantitated in cytosolic fraction of CL of both rat and buffalo cow.ResultsCirculating P4 concentration declined rapidly in response to PGF2alpha treatment. HPLC analysis of serum samples did not reveal changes in circulating 20alpha-OHP levels in buffalo cows but serum from pseudo pregnant rats receiving PGF2alpha treatment showed an increased 20alpha-OHP level at 24 h post treatment with accompanying decrease in P4 concentration. qPCR expression of 20alpha-HSD in CL from control and PGF2alpha-treated buffalo cows showed higher expression at 3 and 18 h post treatment, but its specific activity was not altered at different time points post PGF2alpha treatment. The Nur77 expression increased several fold 3 h post PGF2alpha treatment similar to the increased expression observed in the PGF2alpha-treated pseudo pregnant rats which perhaps suggest initiation of activation of apoptotic pathways in response to PGF2alpha treatment.ConclusionsThe results taken together suggest that synthesis of P4 appears to be primarily affected by PGF2alpha treatment in buffalo cows in contrast to increased metabolism of P4 in rodents.

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Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

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Profiling of luteal transcriptome during prostaglandin F2-alpha treatment in buffalo cows: analysis of signaling pathways associated with luteolysis.

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