Investigation of Flotillin-1 in the Rat Testis during Development, Spermatogenesis, and Ischemia/Reperfusion Injury

Abstract

The level and cellular localization of flotillin-1, a lipid raft protein, was examined in the testis of rats during postnatal development and spermatogenesis in order to determine if flotillin-1 is involved in testicular development. The testes of rats were sampled on postnatal days 7, 14, 21, 40, and 60, and analyzed by Western blot and immunohistochemistry. Western blot analysis detected flotillin-1 in the testes at days 7 and 14 after birth but the level decreased significantly at postnatal days 21, 40 and 60. At postnatal days 7, 14, 21, and 40, flotillin-1 immunolocalisation was observed mainly in the Sertoli cells. However, there was little flotillin-1 immunolabeling in the spermatogenic cells from the seminiferous tubule of the testes. In the seminiferous tubule of the testes at postnatal day 60, flotillin-1 immunoreactivity in the Sertoli cells varied according to the stages of the spermatogenic cycle; intense immunoreactivity being observed in stages IX-III and less in stages IV-VIII. These results suggest that flotillin-1 participates in the developmental process of Sertoli cells and is involved in the regulation of spermatogenesis.

To investigate the involvement of flotillin-1 in acute experimental testicular torsion, we examined the expression and cellular localization of flotillin-1 and cathepsin D in the rat testis with ischemia/reperfusion (I/R) injury. Western blot analysis showed that the expression of flotillin-1 increased significantly 6 h after I/R and that the level remained elevated for 48 h. Immunohistochemically, flotillin-1 was constitutively localized in some Sertoli cells, peritubular myoid cells, and interstitial cells in the normal testis. After I/R injury, Sertoli cells in the damaged tubules were intensely immunostained for flotillin-1 at 24 and 48 h after I/R. Flotillin-1 was also detected in some inflammatory cells in the interstitial space around damaged tubules. Furthermore, flotillin-1 was colocalized with cathepsin D, a lysosomal marker, in normal testis (mainly in Sertoli cells), and the colocalization was greater in Sertoli cells and macrophages in I/R injured testes. Therefore, we postulate that flotillin-1 immunoreactivity is increased in some Sertoli and inflammatory cells (especially in ED1-positive activated macrophages) in testicular torsion and that flotillin-1 in the injured testis associates with lysosomes in Sertoli cells and macrophages, activating subsequent signals in inflammatory macrophages and Sertoli cells after I/R.