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A putative non-genomic progesterone receptor was identified 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, reinforcing 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 influenced by progesterone via non-genomic progesterone receptor.

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