(A) AC assays of germ cells and sperm. The amount of cAMP produced in the presence of magnesium (Mg), magnesium + bicarbonate (Mg+B), and magnesium + forskolin (Mg+FSK) was assayed using soluble (S) and particulate (P) proteins prepared from pachytene spermatocytes, round spermatids, condensing spermatids, and sperm.
(B) Immunoblot (using biotinylated sAC monoclonal antibody R21) of immunoprecipitates (using sAC monoclonal antibody R5 recognizing a nonoverlapping epitope) from wild-type (+/+) and sAC null (−/−) testis cytosol. The first lane is a protein extract from sACt-overexpressing cells (sACt is the Mr ~50,000 isoform generated by alternative splicing).
(C) Immunofluorescent analysis of sAC in noncapacitated wild-type sperm using monoclonal antibody R21 (Zippin et al., 2003). Arrows refer to midplece region.
(D) Corresponding phase image of (C).
(E) Immunofluorescent analysis of sAC in capacitated wild-type sperm using the R21 antibody. Arrow refers to annular region.
(F) Corresponding phase image of (E).