ODORANT RECEPTORS AND DESENSITIZATION PROTEINS COLOCALIZE IN MAMMALIAN SPERM

Background: The identification of transcripts encoding putative olfact ory receptors in mammalian germ cells (1) has generated the hypothesis that olfactory receptors may serve a chemosensory role in sperm chemo taxis during fertilization. We have sought to identify and localize th ese receptors and their regulatory machinery in rat sperm in order to gain further insight into mammalian sperm chemotaxis and odorant recep tor physiology. Materials and Methods: We conducted reverse transcript ion-polymerase chain reaction (RT-PCR) using degenerate primers direct ed against sequences conserved across members of the known odorant rec eptor family to identify transcripts from testis and round spermatids. Western analysis and immunohistochemistry were performed using antibo dies raised against two peptide sequences conserved among odorant rece ptors and using fusion protein antibodies to G-protein receptor kinase 3 (GRK3/beta ARK2) and beta-arrestin2. Results: We detected transcrip ts encoding putative odorant receptors in both testis and round sperma tids of the adult rat. Restriction digests of the PCR products demonst rated the existence of multiple gene products. Two anti-odorant recept or antibodies specifically recognized a 64 kD band in rat sperm prepar ations by Western blot. The proteins GRK3 and beta-arrestin2, implicat ed in olfactory desensitization, were detected in sperm cytosolic extr acts using Western analysis. Immunohistochemistry colocalized putative odorant receptors, GRK3 and beta-arrestin2 to elongating spermatids i n the testis and to the midpiece of mature sperm. Conclusions: The spe cific localization of odorant receptors to the respiratory center of m ature sperm is consistent with a role for these proteins in transducin g chemotactic signals. Based on the colocalization, it is plausible th at GRK3 and beta-arrestin2 function in sperm to regulate putative chem oreceptor responses.