Figure 5
Expression and characterization of LH/CGR, genes belonging to SFKs and PDE4D in the monkey CL following LH secretion inhibition and LH replacement. (A) Circulating P4 levels following different treatments. Each bar represents mean ± SEM values. For comparison among various treatment groups, one-way ANOVA analysis was performed and bars with different letters indicate statistical significance (p < 0.05). (B) The qPCR expression for LH/CGR mRNA in the CL following different treatments. Each bar represents mean ± SEM values (n = 3 animals/treatment). For comparison among various treatment groups, one-way ANOVA analysis was performed and bars with different letters indicate statistical significance (p < 0.05). (C) Levels of LH/CGR protein in the monkey CL post CET-induced luteolysis and rescue of CL function at 1 or 8 h post CET + rhLH treatment. Anti-β-actin antibody (the protein loading control) probed blot is presented to indicate equal loading of protein in each lane. (D) Semi-quantitative RT-PCR expression of genes belonging to SFKs (Fyn, Yes and Src) in the monkey CL obtained following different treatments. L-19 mRNA was used as internal control and the fold change in mRNA expression was calculated following densitometric analysis. Each bar represents mean ± SEM values (n = 3 animals/treatment). For comparison among various treatment groups, one-way ANOVA analysis was performed and bars with different letters indicate statistical significance (p < 0.05). (E) Immunoblot analysis was performed to examine functional activation of Src protein i.e., protein levels of active pSrc (Y-416) and total Src in the monkey CL collected 24 h post VEH/CET treatment and at 1 or 8 h post CET + rhLH treatments. Protein lysates (100-200 μg) prepared from CL were resolved on 10% SDS PAGE, transferred on to PVDF membrane and immunoblot analysis was performed using antibodies raised against pSrc (Y-416), total Src and total Erk. A representative immunoblot blot for each of the protein antibody probed is shown along with the size of the protein. Anti-Erk antibody (the protein loading control) probed blot is presented to indicate equal loading of protein in each lane. Densitometric analysis of immunoblots was determined and the values are indicated as mean ± SEM of relative amount of pSrc (Y-416) expressed as intensity of total Src bands in each group (n = 3 animals/treatment group). For comparison among various treatment groups, one-way ANOVA analysis was performed and bars with different letters indicate statistical significance (p < 0.05). (F) The qPCR expression of PDE4D mRNA, in the CL obtained from monkeys subjected to different treatments. Each bar represents mean ± SEM values (n = 3 animals/treatment). For comparison among various treatment groups, one-way ANOVA analysis was performed (represented by letter on each bar) and the data across treatment groups was not significantly different (p > 0.05). (G) Immunoblot analysis was performed to examine PDE4D protein levels in the CL collected from monkeys subjected to different treatments. A representative immunoblot probed along with anti-β-actin antibody is presented to indicate equal loading of protein in each lane.