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Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co\u2010localize with the proton pumping V\u2010ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co\u2010localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and\/or the final fertilization process. J. Cell. 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