Figure 1.
MALDI-TOF spectrum of trypsin digested peptides of FMSF.
Three concentrations of the same sample were spotted on the CHCA matrix for analysis, which produced qualitatively identical results. All the peaks on the spectrum were checked for homology in the online protein database; only those with a score >65 were considered significant for discussion in the result section.
Figure 2.
Dose response curve of forskolin on forward motility of mature caprine spermatozoa.
Primary X- axis denotes graduations for applied Forskolin concentrations; secondary X-axis denotes graduations for control curve where equivalent amounts of solvent of Forskolin that were applied to produce treatment points, were used. The data represent mean ± SEM for n = 3 samples. Asterisk (*) denotes statistically significant difference vs. control (p<0.05). Viable cell count throughout the different assay conditions remained ≥97%.
Table 1.
Study of kinetic properties of adenylyl cyclase activity of purified sperm membrane fraction.
Figure 3.
Role of transmembrane adenylyl cyclase in mediation of FMSF activity.
A. Dose-response curve of ddAdo on effect on forward motility of caudal spermatozoa; B. Results of Spectrophotometric vertical motility assay of caprine mature spermatozoa. Viable cell count throughout the different assay conditions remained ≥95%. C. Dose-dependency of tmAC activity in presence of FMSF (light bars). Involvement of Gα-subunit in manifestation of FMSF activity represented by dense bars where GTPγS was used at a concentration of 10 µM. Pre-treatment periods for both ddAdo and GTPγS were of 30 min. Data for figure A and B represent mean ± SEM for n = 5 samples, while that of figure C is of n = 3 samples. Asterisks (***) and hash marks (#, ##, ###) denote statistically significant difference vs. control (p<0.001) and vs. only FMSF-treated (p<0.05, p<0.01 and p<0.001), respectively.
Figure 4.
Involvement of cAMP in FMSF-initiated signaling.
Dose-response curve of FMSF for generation of intracellular cAMP and corresponding forward motility. Inset figure represents correlation between [cAMP]i and forward motility induced by FMSF (r = +0.995).
Figure 5.
Elucidation of role of tmAC in quality of sperm forward movement.
A. Effect of ddAdo (dideoxyadenosine) and db-cAMP (dibutyryl cAMP) in relation with FMSF-induced forward motility. B. Effect of ddAdo and KH7 in relation with FSK induced forward motility. Incubation periods with ddAdo, KH7, FSK and db-cAMP were of 30 min each. All the data represent mean ± SEM for n = 6 samples. Double asterisks (**) denote statistically significant difference vs. control (p<0.01), hash mark (#) indicates statistically significant inhibition vs. FMSF-treated (p<0.01), double hash mark (##) indicates statistically significant inhibition vs. control (p<0.01), asterisk (*) denotes statistically significant difference vs. (ddAdo+FMSF) (p<0.05), nsd denotes not significant difference vs. control. Viable cell count throughout the different assay conditions remained ≥95%. C. Effect of different doses of ddAdo on modulation of intracellular cAMP level in presence of FMSF (0.5 µM). Duration of ddAdo and FMSF incubation were 30 and 2 min, respectively. All the data represent mean ± SEM for n = 4 samples. Hash mark (##) indicates statistically significant inhibition vs. ddAdo-untreated (p<0.01).
Figure 6.
A. Effect of PKA inhibitor in forward motility of the FMSF activated sperm cells.
Duration of IP20 incubation was 30 min. B. Effect of tyrosine-kinase inhibitor on forward movement profile of FMSF stimulated spermatozoa. Duration of Genistein incubation was 30 min. Viable cell count throughout the different assay conditions remained ≥95%. C. Phospho-kinase stimulating potential of FMSF. Only absorbance values from ELISA results have been plotted, actual activity units have not been shown; anti-phospho-serine/threonine for Protein Kinase A and tyrosine antibodies for Tyrosine kinase were used in ELISA of the assayed treatments. All the data represent mean ± SEM for n = 3 samples. Hash mark (#) indicates statistically significant inhibition vs. only FMSF-treated (p<0.01), Asterisks (*, **) denote statistically significant difference vs. control (p<0.05 and p<0.01), respectively.
Figure 7.
Flow-cytometric analysis of the phosphorylation status of spermatozoa.
Panel I–IV: Ser/Thr phosphorylation status; I- Control, II- db-cAMP (0.5 mM), III- FMSF (0.5 µM), IV- Genistein (250 µM). Panel V–VI: Tyr phosphorylation status; V- Control, VI- FMSF (0.5 µM). The figure is representative and the data represent mean ± SEM for n = 3 samples. The R2 region signifies fluorescence of stained cells (R1, not indicated denotes the entire region); percentage in the figure is of gated cells in the R2 region and corresponding Mean Fluorescence Intensity (MFI) are given beside.
Figure 8.
Immuno-localization of Tyrosine-phosphorylation site, (A).
Panel a: Bright-field images, Panel b: Fluorescent images; I- Protein A-FITC only control, II- untreated, III- FMSF (0.5 µM; incubation time 10 min). White arrows indicate sperm head where enhanced phosphorylation was observed. The figure is representative of different such views and was taken at 600× magnification. B. Western blot profile of tyrosine-phosphorylated protein. Electrophoresis was done in 10% denaturing polyacrylamide gel. FMSF dose: 0.5 µM/incubation time 10 min. The MW markers used were beta-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), REase Bsp98I (25 kDa). The set of bands with differential profile has only been shown. The SDS-PAGE band is of whole cell lysate; panels (a) and (b) correspond to developed Western blot of whole cell lysate protein and membrane protein fraction, respectively. Arrow indicates the detected ≈ 50 kDa band.