Localization of BRUNOL2 in Rat Spermatogenic Cells as Revealed by Immunofluorescence and Immunoelectron Microscopic Techniques

Distribution and localization of a RNA-binding protein, BRUNOL2 in rat spermatogenic cells were studied by dot blotting of cell fractions, immunofluorescence (IF), and immunoelectron microscopy (IEM). BRUNOL2 distributed in nuclear (23%), mitochondrial (19%), microsomal (15%), and cytosol fractions (43%). BRUNOL2 was detected in all spermatogenic cells. In the cytoplasm and nucleoplasm of the spermatogonia, spermatocytes and spermatids, both diffuse and granular staining patterns were observed. Many cytoplasmic granules were stained also for DDX4 and DDX25. Large granules in the cytoplasm of elongated spermatids were stained for BRUNOL2 but not for the nuage proteins. IEM showed that gold signals for BRUNOL2 were concentrated in nuage components including loose aggregates of small particles, chromatoid body (CB), intermitochondrial cement (IMC), and satellite body (SB). In addition, many non-nuage structures such as ER-attached small granules, less dense material surrounding connecting piece of flagellum, reticulated body, mitochondria-associated granules (MAG), granulated body, ribosome aggregate, and manchette, were stained for BRUNOL2 with different staining intensities. In the nucleus, gold signals were concentrated in heterochromatin area and nucleolus. The results suggest that BRUNOL2 is one of the nuage proteins and also associated with the other non-nuage structures, suggesting multiple functions of this protein.