FSCB phosphorylation during mouse spermatozoa capacitation

Objective To investigate the molecular mechanism underlying the phosphorylation of fibrous sheath CABYR binding protein (FSCB) during the process of mouse sperm capacitation. Methods Recombinant FSCB was phosphorylated with the presence of protein kinase A (PKA) in-vitro. The products were electrophoresed, followed by Western blot using anti-phospho-PKA substrate antibody and anti-phosphotyrosine antibody, respectively. Mouse spermatozoa before and at 2 h after capacitation were lysed and subjected to immunoprecipitation using anti-phospho-PKA substrate antibody, anti-phosphotyosine or anti-FSCB antibodies, respectively, followed by Western blot using anti-FSCB antibody. Immunofluorescence localization using FSCB antibody and phospho-PKA substrate antibody were performed. Results The recombinant FSCB could be phosphorylated in-vitro in the presence of PKA catalytic subunit. FSCB can be phosphorylated by PKA with simultaneous tyrosine phosphorylation during the process of sperm capacitation. Immunofluorescence analysis showed that FSCB and PKA phosphorylated substrate were co-localized to the spermatic principal piece. Conclusion FSCB is involved in the process of sperm capacitation as one of the PKA substrates. Key words: Sperm capacitation; Fibrous sheath; Protein kinase A; Phosphorylation