GKP was supported by grants AR-047314, AR-051372, and AR-052730 from the National Institutes of Health and the Muscular Dystrophy Association. suggest a larger role for ORs during tissue repair. Keywords:fusion, migration, MOR23, Olfr16, myofiber branching, myofiber splitting, regeneration, adhesion, olfactory receptor == Introduction == Odorant receptors (ORs) are G-protein-coupled receptors (GPCR) expressed within the olfactory epithelium of the nose where they function as chemosensors to detect small molecules we perceive as smell. ORs are the largest receptor family in mammals comprising 3-5% of all genes (Young and Trask, 2002), with approximately 913 potential OR genes in the mouse genome, and 390 in the human genome (Olender et al., 2008;Zhang and Firestein, 8-Hydroxyguanine 2002). Microarray analyses suggest ORs are 8-Hydroxyguanine expressed in many tissues (Feldmesser et al., 2006), but functions of these receptors are known for only three: chemosensing in olfactory epithelium, proliferation in prostate cancer and chemotaxis in sperm of humans and mice (Fukuda et al., 2004;Neuhaus et al., 2009;Spehr et al., 2003). One of the ORs that regulates sperm chemotaxis is usually mouse OR23 (MOR23, Olfr16) (Fukuda et al., 2004). We unexpectedly observed an increase in MOR23 expression in a microarray analysis performed during myogenesis of primary cultured mouse muscle cells (unpublished data). This increase occurred when muscle cells were undergoing extensive cell migration, adhesion and fusion to form multinucleated cells. In adult skeletal muscle, myogenesis occurs to 8-Hydroxyguanine regenerate muscle after injury. This process is dependent on satellite cells, which are normally quiescent, but in response to injury proliferate, and their progeny myoblasts differentiate and fuse with each other or with existing myofibers to restore normal tissue architecture. Migration and cell-cell adhesion are necessary for muscle cell differentiation (Kang et al., 2004) and fusionin vitro(Jansen and Pavlath, 2006). Therefore, identifying the molecules that control muscle cell adhesion and migration may reveal potential molecular targets for improving muscle regeneration. As MOR23 regulates migration of sperm, we hypothesized that ORs may affect comparable processes during myogenesis. Here, we demonstrate that MOR23 is an important regulator of myogenesisin vitroandin vivothrough affects on cell migration and adhesion, influencing downstream fusion events. In addition, our data suggest a MOR23 8-Hydroxyguanine ligand is usually secreted by muscle cellsin vitroand is also present in muscle tissue. Furthermore, a number of ORs were also expressed by regenerating muscle, which may suggest a larger role for ORs in tissue repair. == Results == == Multiple ORs, with distinct patterns, are expressed during myogenesis == Multinucleated myofibers form through fusion of muscle cells, which requires cell migration and adhesion factors (Jansen and Pavlath, 2008). Myogenesisin vitrooccurs in distinct phases (Fig. 1A): proliferating myoblasts Dynorphin A (1-13) Acetate terminally differentiate to become fusion-competent myocytes in differentiation media (DM). By 24 hrs in DM, myocytes fuse with one another to form small, nascent myotubes with few nuclei. Further rounds of fusion produce mature myotubes with many nuclei, by 48 hrs in DM. By microarray analysis, MOR23 mRNA was upregulated at 24 hrs of myogenesis (unpublished data). To validate the expression of MOR23 and also to determine whether other ORs are expressed during myogenesisin vitroandin vivo, we performed Real Time RT-PCR. Given the large numbers of mouse ORs, we choose 18 ORs previously identified in microarray screens of skeletal muscle (Beggs et al., 2004;Melcon et al., 2006;Tseng et al., 2002;Zhao et al., 2002). Olfactory bulb RNA was used as a positive control for OR mRNA (Supp. Table 1). Thirteen ORs were expressed during myogenesis (Supp. Table 2 and 3,Fig. 1). The majority of ORs expressedin vitrowere most highly expressed at 0 hrs, when the muscle cells are proliferating myoblasts (Supp. Table 2,Fig. 1B, C). A smaller number of ORs were increased with terminal differentiation, and most highly expressed at 24 hrs in DM, when myocytes are fusing. Interestingly, no ORs were upregulated or exclusively expressed at 48 hrs in DM, when most cells have already fused. == Physique 1. Multiple ORs are expressed duringin vitromyogenesis and muscle regeneration. == Real Time RT-PCR was used to analyze the time-course of expression bothin vitroandin vivofor 19 ORs. A) During myotube formation the majority of myoblasts (red) differentiate into myocytes (blue). Myocytes fuse with one another to form small nascent myotubes with few nuclei. Subsequently, nascent myotubes fuse with myocytes to form large myotubes with many nuclei. B) Two patterns of expression were observedin vitro: peak expression either during proliferation or after terminal differentiation. Olfr15 and Olfr16 (MOR23) are.