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Expression and localization of NANOS1 in the spermatogenic cells of rat testis were studied by immunofluorescence and immunoelectron microscopy. Using immunofluorescence techniques, NANOS1 was localized in the cytoplasm and discrete granules of spermatocytes and spermatids. The staining intensity of NANOS1 signal varied depending upon the stage of the cycle of seminiferous epithelium. Double immunofluorescence staining with antibodies against NANOS1 and DDX4 showed that several DDX4-positive compartments of nuage were also stained for NONOS1. Immunoelectron microscopy revealed that the major subcellular localization sites for NANOS1 were the chromatoid body (CB) and satellite body (SB), and the minor sites were the rest of the nuage compartments, including the irregularly-shaped perinuclear granules (ISPG), and intermitochondrial cement (IMC). Non-nuage structures such as mitochondria-associated granules (MAG), granulated body (GB), and reticulated body (RB) were also labeled by theNANOS1 antibody. In addition, NANOS1 was found in the outer dense fibers of flagella of spermatids at steps 12-19, and in the head cap of late spermatids after step 15. These results suggest that NANOS1 is one of the nuage proteins and functions mainly in the CBs as well as in the cytoplasm. NANOS1 may also have additional functions in non-nuage structures such as MAGs, GBs and RBs.