Identification of a 71 kDa protein as a putative non-genomic membrane progesterone receptor in boar spermatozoa

A putative non-genomic progesterone receptor was ident-ified by Western blot analysis from the membrane fraction but not the cytosolic fraction of boar spermatozoa using monoclonal antibody (mAb) C-262. When the membrane and the cytosolic fractions of boar liver, kidney, uterus and spermatozoa were analyzed with mAb C-262, protein bands with molecular masses of 86 and 120 kDa were detected from the cytosolic fraction of the uterus, whereas a 71 kDa protein was detected from the membrane fraction of spermatozoa. Apparently, while the 86 and 120 kDa proteins from the uterus correspond to the genomic progesterone receptor isoforms A and B in boar, the 71 kDa protein of the sperm membrane fraction seems to be a novel membrane-associated progesterone receptor. Ligand blot assay of the membrane and the cytosolic fractions of boar spermatozoa performed with peroxidase-conjugated progesterone revealed that only the 71 kDa membrane protein binds specifically to progesterone, re-inforcing the results obtained from the Western blot analysis. Also ligand blot assays performed in the presence of mAb C-262 demonstrated that mAb C-262 inhibited progesterone binding to the 71 kDa protein in a dose-dependent manner. Ligand blot assays performed in the presence of free progesterone, RU486 or estrogen revealed that binding of peroxidase-conjugated progesterone to the 71 kDa protein was inhibited by free progesterone and RU486 in a dose-dependent manner but not by estrogen, which further confirms that progesterone binds to the 71 kDa protein specifically. Furthermore, the progesterone-induced acrosome reaction was inhibited by mAb C-262 in a dose-dependent manner. These results strongly imply that spermatozoa possess a progesterone receptor in a membrane-bound form and can be influ-enced by progesterone via non-genomic progesterone receptor