Fig 1: Expression and characterization of genes belonging to SFKs and their functional activation in the monkey CL throughout the luteal phase. (A) Diagrammatic representation of various signalling cascades and molecular players involved downstream of LH/CGR activation regulating various genes associated with steroidogenesis. Based on the review of literature the canonical pathway Gs/cAMP/protein kinase A (PKA)/pCREB and alternate signalling pathways like p38 MAPK or Erk have been illustrated in the diagram. Furthermore, recent studies describing involvement of SFKs and cAMP-PDE in modulation of hCG responsiveness and transactivation of tyrosine kinase receptors in mouse Leydig tumor cells are also represented for their possible similar role in the monkey luteal cells. (B) Semi-quantitative RT-PCR expression of genes belonging to SFKs (Fyn, Yes and Src) in the monkey CL obtained from different stages of the luteal phase. Each bar represents mean ± SEM values (n = 3 CL/stage). L-19 mRNA was used as internal control and relative expression was calculated following densitometric analysis. Bars with different letters indicate statistical significance (p < 0.05). (C) Levels of Src family of kinase protein in the monkey CL throughout the luteal phase. Immunoblot analysis was performed to determine functional activation of Src protein i.e., protein levels of active pSrc (Y-416), inactive pSrc (Y-527) and total Src in the monkey CL collected during different stages of the luteal phase. Anti-β-actin antibody (the protein loading control) probed blot is presented to indicate equal amount of protein being loaded in each lane. Densitometric analysis of immunoblots was determined and is indicated as mean ± SEM of relative amount of pSrc (Y-416/Y-527) expressed as intensity of total Src/β-actin bands for each stage of luteal phase (n = 3 CL/stage). Bars with different letters indicate statistical significance (p < 0.05).
Fig 2: Expression and characterization of LH/CGR and genes belonging to SFKs in the monkey CL during simulated early pregnancy condition. (A) The graph illustrates circulating mean P4 levels during different days of the luteal phase without (solid circles) and with hCG (solid triangle) treatment. Each point represents mean ± SEM values (n = 3 animals/control or hCG treatment). Bars with different letters indicate statistical significance (p < 0.05). (B) The qPCR expression for LH/CGR mRNA (upper panel) and the levels of LH/CGR protein (lower panel) in the CL from monkeys collected on day 14 without treatment i.e., late CL (L), on day 1 of menses (D1M) and on day 14 following hCG treatment from day 9-13 of luteal phase (hCG). Each bar in the upper panel represents mean ± SEM values (n = 3 animals/treatment). For comparison among various groups, one-way ANOVA analysis was performed and the data across groups was not significantly different (p > 0.05). The representative immunoblots probed with anti-LH/CGR antibody and anti-β-actin antibody are shown in the lower panel. Densitometric analysis (mean ± SEM for n = 3) of immunoblots was performed and the protein levels are indicated below respective bands. L19 mRNA was used as internal control for qPCR, while β-actin protein level was used as loading control. (C) Semi-quantitative RT-PCR expression of genes belonging to SFKs (Fyn, Yes and Src) in the CL collected from monkeys on day 14 of luteal phase that received hCG treatment to stimulate early pregnancy (hCG) or without treatment Late (L). Each bar represents mean ± SEM values (n = 3 animals/control or hCG treatment). L-19 mRNA was used as internal control and fold change in mRNA expression was calculated following densitometric analysis. (D) Immunoblot analysis was performed to determine functional activation of Src protein i.e., protein levels of active pSrc (Y-416) and total Src in the CL collected from monkeys on late luteal phase (L), on day 1 of menses (D1M) and on day 14 of luteal phase following hCG treatment on day 9-13 of luteal phase (hCG). A representative immunoblot for each of the protein antibody probed is shown along with the size of the protein. Anti-β-actin 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 level of pSrc (Y-416) is expressed as mean ± SEM relative to the intensity of total Src/β-actin. Individual bars with different letters are significantly different (p < 0.05).
Fig 3: Expression and characterization of LH/CGR and genes belonging to SFKs in the monkey CL during PGF2α-induced luteolysis. (A) Circulating P4 levels 24 h after VEH/PGF2α treatment. Each bar represents mean ± SEM values (n = 3 animals/treatment). (B) The fold change in qPCR expression for LH/CGR mRNA in the CL 24 h post VEH/PGF2α treatment. Each bar represents mean ± SEM values (n = 3 animals/treatment). (C) Semi-quantitative RT-PCR expression of genes belonging to SFKs (Fyn, Yes and Src) in the monkey CL obtained 24 h post VEH/PGF2α treatment. L-19 mRNA was used as internal control and the relative expression was calculated following densitometric analysis. Each bar represents mean ± SEM values (n = 3 animals/treatment). (D) Levels of LH/CGR, active pSrc (Y-416) and PDE4D protein in the monkey CL obtained 24 h post VEH/PGF2α treatment. Anti-β-actin antibody (the protein loading control) probed blot is presented to indicate equal loading of protein in each lane.
Fig 4: 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.
from Cell Signaling Technology for Src Antibody Sampler Kit