Additional file 3: Figure S1. of Analysis of 17β-estradiol (E2) role in the regulation of corpus luteum function in pregnant rats: Involvement of IGFBP5 in the E2-mediated actions

In vitro aromatisation of testosterone (T) and effects of AI on circulating E2 and P4 levels, weight of CL and Cyp19a1 mRNA expression during rat pregnancy. (A) Sliced pieces of CL tissue collected on day 7, 11, 12 and 16 of rat pregnancy was incubated without or with 20 ng T for 4 h. E2 content in the medium was estimated and represented as pg/mg tissue/4 h. Each bar represents mean±SEM, n = 4 to 12/time point, *** P <0.001, * P <0.05. (B-E) Pregnant rats received oral administration of AI (1 mg/kg BW) or VEH (2 % ethanol) for four days daily. Circulating mean±SEM serum E2 (B) and P4 (C) concentrations during different treatments (n = 5 to 10 animals/time point, B and n = 3 animals/time point, C, ** P <0.01, * P <0.05). (E) Weight of CL during different treatments with a representative photo for each treatment is shown on each bar (mean±SEM, n = 8 to 13 CL/time point) (F) qPCR expression of Cyp19a1 mRNA in CL post different treatments. The results are shown as fold changes of mRNA expression compared to day 7 CL. Individual bars represents mean±SEM fold change in mRNA expression value for qPCR analysis during different treatments (n = 4 animals/time point). (PPTX 338 kb

Additional file 2: Table S2. of Analysis of 17β-estradiol (E2) role in the regulation of corpus luteum function in pregnant rats: Involvement of IGFBP5 in the E2-mediated actions

List of primers used for qPCR analyses. The list of genes and details of the primers employed along with the expected amplicon size and annealing temperature are provided. (PPTX 63 kb

Schematic diagram illustrating a model for PGF_2α-induced luteolysis in the bovine corpus luteum.

<p>The model shows interaction amongst various intracellular signaling pathways activated by LH, PGF_2α and E₂. The luteolysis process induced by PGF_2α treatment appears to be a result of distinct molecules that involve hormone biosynthesis pathways (StAR, CYP19A1) which is downstream of PTGFR signaling leading to decrease in serum and luteal P₄ and E₂ levels. The model also shows involvement of Bcl-2 family members, Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) that cause changes in mitochondrial permeability to apoptogenic molecules. The model also shows inhibition of cell survival pathway as shown by LH/CGR and ER (both genomic and non-genomic signaling) down regulation and in turn inhibition of downstream molecules (CREB, FKHR). Few of the selected genes out of differentially expressed genes at different time points post PGF_2α treatment compared with 0 h PGF_2α treatment (StAR, CYP19A1, Bax, Bcl-2, CREB, FKHR) has been provided in the Figure. The information on gene names, general function, fold change in mRNA expression and implicated pathways are represented. The stimulation and inhibition of specific molecules downstream of different signaling pathways are represented by green (+) and red (−) signs, respectively.</p

Profiling of Luteal Transcriptome during Prostaglandin F2-Alpha Treatment in Buffalo Cows: Analysis of Signaling Pathways Associated with Luteolysis

<div><p>In several species including the buffalo cow, prostaglandin (PG) F_2α 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 PGF_2α in buffalo cows. Circulating progesterone levels decreased within 1 h of PGF_2α 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 PGF_2α 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 PGF_2α 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 PGF_2α-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 PGF_2α interferes with luteotrophic signaling, impairs intra-luteal E₂ levels and regulates various signaling pathways before the effects on structural luteolysis are manifest.</p></div

Effects of PGF_2α on expression of LH/CGR and downstream signaling molecules, Akt and FKHR.

<p>(A) Protein levels of LH/CGR in bovine CL. Protein lysate (100 µg) prepared from CL tissue collected before (0 h) and post (3, 6 and 18 h) PGF_2α treatment were resolved on 10% SDS PAGE, transferred onto PVDF membrane and immunoblot analysis was performed using anti-LHCGR and anti-β-actin antibody. A representative immunoblot for each of the antibody probed is shown. The immunoblot probed with β-actin antibody indicates loading control for each lane. Densitometric values were determined and indicated as mean±SEM (n = 3 animals/time point), relative to intensity of β-actin for each time point post PGF_2α treatment. The values of immunoblot analysis has been put on top of each lane and lane with different alphabets indicate statistical significance, p<0.05. (B) PKA activity in the bovine CL before (0 h) and after (3 and 18 h) PGF_2α administration. Values represent mean±SEM for each time point post PGF_2α administration (n = 3 animals/time point). Individual bars with different alphabets indicate statistical significance (p<0.05). (C, D and E) Protein levels of pCREB, CREB, pAkt, Akt, pFKHR and FKHR in the CL. Protein lysate (50 µg) of each CL tissue collected at different time points was subjected to immunoblot analysis employing anti-pCREB, anti-CREB, anti-pAkt, anti-Akt, anti-pFKHR, anti-FKHR antibodies. A representative immunoblot for each of the antibody probed is shown. Densitometric values were determined and represented as mean±SEM (n = 3 animals/time point), relative to intensity of total CREB (C), Akt (D) and FKHR (E) for each time point post PGF_2α treatment. The values of immunoblot analysis has been put on top of each lane and lanes with different alphabets indicate statistical significance (p<0.05).</p

Schematic representation of differentially expressed genes in the CL post PGF_2α treatment.

<p>(A and B) Venn diagrams representing the number of differentially expressed genes identified after microarray analysis of CL tissues collected before (0 h) and at different time points post PGF_2α treatment. Data analyzed by Bioconductor analysis tool employing ≥2 fold change cut-off and statistical filters with Benjamini and Hochberg correction factor for false discovery rate. The comparison of total number of differentially expressed up (A) and down (B) regulated genes found common between 0 vs. 3 h (blue circle) and 0 vs. 6 h (red circle), as well as between 0 vs.18 h (green circle) post PGF_2α treatment are presented. (C) Correlation analysis between expression ratios obtained from microarray and qPCR analyses of few genes post PGF_2α treatment (n = 15 genes/time point). Linear regression analysis was performed for selected differentially expressed genes using qPCR fold change in expression values (2^−ΔΔCT; Y-axis) with fold change expression values obtained by microarray analysis (X-axis). The r value, correlation coefficient, generated for the theoretical line of best fit (represented as solid line in each panel) and the p value indicate the significance of the correlation as determined by ‘F’ test.</p

Ingenuity Pathway Analysis (IPA) of differentially expressed genes.

<p>(A) The pathway analysis indicated that a large number of differentially expressed genes belong to canonical pathways such as IGF-1 signaling, steroidogenesis, chemokine signaling, prolactin signaling, cellular growth and proliferation, extracellular matrix modulation and apoptosis. The orange line represents score for the likelihood [-log (B-H p<0.05)] that genes belonging to a specific canonical pathway category affected at 3 h post PGF_2α administration. The stacked bars indicate the percentage of genes distributed according to regulation, i.e., green (down), red (up) and open bars (no overlap with dataset) in each canonical pathway. (B) Network 0 vs. 3 h: Ingenuity Pathway Analysis of the differentially regulated genes 3 h post PGF_2α administration shows a network of 21 focus molecules with a score of 48, with top biological functions of cell death, cellular growth and proliferation (immune cells), and cell-to-cell signaling. The network is displayed graphically as nodes (genes/gene products) and edges (biological relationship between nodes). The node color intensity indicates the fold change expression of genes; with red representing up-regulation, and green representing down-regulation of genes between 0 vs. 3 h post PGF_2α administration. The fold change value for individual gene is indicated under each node. The shapes of nodes indicate the functional class of the gene product and the lines indicate the type of interaction.</p

Validation of microarray data and its comparison with qPCR analysis.

<p>Log ratio of microarray fold change expression of the selected 20 up and down regulated genes associated with specific biological process at 3, 6 and 18 h post PGF_2α administration in the CL. The genes selected by microarray analysis were subjected to qPCR analysis and log ratio of fold expression changes at different time points post PGF_2α administration are represented as bar graphs. Individual bar for each gene represents mean±SEM log₂ (fold change) in mRNA expression value for microarray analysis and qPCR analysis at each time point (n = 3 animals/time point). For each gene, bars with different alphabets above them are significantly different (p<0.05).</p

Effect of PGF2α on expression of Bax, Bcl-2 and Akt genes during PGF2α treatment.

<p>(A) qPCR expression and analysis of Bax and Bcl-2 in the bovine CL. Mean (±SEM) fold expression changes before and after PGF_2α treatment is presented. Bars with different alphabets above them indicate statistical `significance (p<0.05). (B) Protein levels of Bax and Bcl-2 in the bovine CL. The CL tissue lysates were resolved on 10% SDS PAGE, transferred onto PVDF membrane and subjected to immunoblot analysis employing anti-Bax, anti-Bcl-2 and anti-β-actin antibodies. The immunoblot probe with β-actin antibody was used as a loading control. Densitometric values were determined and indicated as mean±SEM (n = 3 animals/time point), relative to intensity of β-actin for each time point post PGF_2α treatment. Bars with different alphabets indicate statistical significance (p<0.05). (C) Fold expression changes in mRNA and quantitative changes in protein as ratio of Bax/Bcl-2 levels in the luteal tissue during PGF_2α treatment. A representative immunoblot for each of the antibody probe is shown. Individual bars with different alphabets above them indicate statistical significance (p<0.05). Individual bars in solid box and open box represent mRNA expression and protein levels, respectively. (D) mRNA and protein levels of PI3k p85 in bovine CL. Mean (±SEM) fold expression changes in PI3k p85 mRNA examined by qPCR analysis. Quantitative changes in the ratio of pPI3k p85 and PI3k p85 protein levels were estimated. For the protein loading control, blots were probed with β-actin antibody. Densitometric values were determined and represented as mean±SEM (n =  3 animals/time point), relative to intensity of total PI3k p85 for each time point post PGF_2α treatment. Individual bars with different alphabets above them indicate statistical significance (p<0.05). Individual bars in solid box and open box represent mRNA expression and protein levels, respectively.</p

Expression of Creatine Biosynthetic and Transport Genes in Maternal and Neonatal Tissues Following Gestational Creatine Supplementation in a Non-Human Primate Model

Expression of Creatine Biosynthetic and Transport Genes in Maternal and Neonatal Tissues Following Gestational Creatine Supplementation in a Non-Human Primate Mode