Identification of a novel sperm motility–stimulating protein from caprine serum: its characterization and functional significance  Sudipta Saha, Ph.D., Sujoy Das, M.Sc., Arpita Bhoumik, M.Sc., Prasanta Ghosh, M.Sc., Gopal Chandra Majumder, Ph.D., Sandhya Rekha Dungdung, Ph.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 269-279.e5 (July 2013) DOI: 10.1016/j.fertnstert.2013.03.012 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Purification and physical characteristics of MSP. (A) Sephacryl S-200 gel filtration column chromatography showing the active MSP peak of the protein fraction (panel a). MSP activity obtained after ammonium sulfate fractionation and CM cellulose ion exchange chromatography was subjected to sephacryl S-200 gel filtration chromatography by the procedure described in Materials and Methods. Nondenaturing PAGE of the active gel filtration elute (panel b). The inset shows two prominent protein bands of Rf values 0.3 and 0.6. (B) Determination of molecular weight and subunit composition of goat blood serum MSP by 10% SDS-PAGE. Markers used were β-galactosidase (116 kDa), phosphorylase-b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), pepsin (35 kDa), carbonic anhydrase, (29 kDa), trypsinogen (24 kDa), and trypsin inhibitor (20 kDa). (C) HPLC gel filtration profile of purified goat blood serum MSP showing its homogeneity. The inset shows determination of molecular weight of goat blood serum MSP by HPLC. The molecular weight markers used as trypsinogen (24 kDa), ovalbumin (44.6 kDa), BSA (66 kDa), and anti-diuretic hormone-yeast (141 kDa). Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Biochemical characteristics of MSP. (A) Effect of goat serum MSP at different concentrations on sperm motility under the standard assay conditions. (-○-): FM %; (-▵-): activity units. The values indicate the mean ± SEM of four experiments. A statistically significant difference was found with respect to the control (P<.001). (B) Effect of MSP on horizontal and vertical velocities of goat cauda sperm. The values indicate the mean ± SEM of three experiments. There are statistically significant differences in horizontal (P<.005) and vertical (P<.05) velocities on MSP treatment with respect to their control values. (C) Effect of goat MSP (-●-, red), theophylline (-■-, orange), bicarbonate (-▲-, green), theophylline + bicarbonate (-◆-, blue), and MSP + theophylline + bicarbonate (-×-, black) at different concentrations on sperm motility under the standard assay conditions. The values indicate the mean ± SEM of four experiments. At all the data points on the X-axis, MSP was very significant compared with the other motility initiators. At the optimum concentration of MSP (0.9 μM), the significance level of MSP versus theophylline (P<.01), MSP versus bicarbonate (P<.001), and MSP versus theophylline + bicarbonate (P<.001) is worth mentioning. As expected MSP versus theophylline + bicarbonate + MSP was not significant at any data point. (D) Effect of goat MSP, 0.9 μM (-●-, red), theophylline 5 mM (-■-, orange), bicarbonate 20 mM (-▲-, green), theophylline + bicarbonate (-◆-, blue), and MSP + theophylline + bicarbonate (-×-, black) on sperm motility with respect to the control (-●-, black). At different times up to 5 minutes MSP-induced motility was found to be more significant with respect to other initiators (panel a). At 30 seconds to the 1 minute time point the statistical significance level is P<.001. At storage up to 3 hours, even at the end of 2 hours, there are significant differences in motility in MSP versus theophylline (P<.05), MSP versus bicarbonate (P<.01), and MSP versus theophylline + bicarbonate (P<.05; panel b). As anticipated, there were no significant differences in motility found between MSP versus theophylline + bicarbonate + MSP at any of the time points. The values indicate the mean ± SEM of four experiments. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunodetection of MSP and effect of its antibody on sperm motility. (A) Determination of antibody specificity by Western blotting as described in the Materials and Methods section. Lane 1: casein; lane 2: ovalbumin; lane 3: fetuin; lane 4: MSP. Polyacrylamide gel electrophoresis of proteins (panel a) and immunoblot on nitrocellulose paper (panel b). (B) Sperm FM inhibition by application of different dilution of MSP antibody. (-□-): 1:1,000; (-▵-): 1:500; (-⋄-): 1:100; (-○-): 1:10; and (-●-): control (preimmune serum 1:500). The values indicate the mean ± SEM of four experiments. At all time intervals, statistically significant differences were found with respect to the control values (P<.001). (C) Determination of the inhibitory effect of papain-digested monovalent MSP antibody on goat cauda sperm motility. (-●-): control; (-■-): 1:100; (-▲-): 1:50; (-◆-): 1:25; (-×-): 1:10. Data shown are mean ± SEM of three experiments. With respect to the control value, the significance level at all time intervals is same and P<.001. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Localization of MSP on goat spermatozoa and its distribution in different tissues and fluids. (A) Distribution of MSP on sperm cell surface as shown by FITC-tagged indirect immunofluorescence. (B) MSP antibody-mediated agglutination (panel b) of goat cauda epididymal spermatozoa when compared with the control (panel a). (C) Immunodetection of MSP in different goat tissues determined by ELISA as described in the Materials and Methods section (panel a). MSP distribution in cell-free epididymal tissue, sperm membrane, sperm cytosol, and EP of different regions of the epididymis as determined by ELISA (panel b). The values indicate the mean ± SEM of three experiments. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Determination of molecular weight of goat blood serum MSP by sephacryl S-200 gel filtration. Standard proteins and blue dextran were detected by absorption at 280 and 660 nm, respectively. The activity of MSP was estimated under standard assay conditions. The molecular weight markers used as standard were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa). Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Analysis with EXPASY protein sequence homology search (BLAST) with the N-terminal of goat blood serum MSP, which was found to be DTHKSEIAHRFNDLGEE. The diagram shows the matching areas in the sequence of different proteins. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Effect of different pH on the activity of goat MSP. Standard assay conditions were used except for variation in pH of the RPS medium. Amount of MSP used was (0.9 μM). (-○-): FM; (-●-): control; (-▵-): MSP activity. The values indicate the mean ± SEM of four experiments. Differences were found significant at all the pH points with P<.0001 at optimum pH 7–7.5. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Determination of intrasperm cAMP level of goat cauda spermatozoa after MSP antibody treatment. The data shown are mean ± SEM of three experiments. No significant difference was found with respect to the control value. Fertility and Sterility 2013 100, 269-279.e5DOI: (10.1016/j.fertnstert.2013.03.012) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions