(M427) DEFAULT PROTEIN SORTING AND DEGRADATION PATHWAYS IN MAMMALIAN TESTIS AND EPIDIDYMIS REVEALED IN THE UBC-GFP TRANSGENIC MOUSE.
Sutovsky, Miriam1, Manandhar, Gaurishankar1, Feng, Dawn1, Sutovsky, Peter 1, 1 University of Missouri-Columbia, Columbia, MO, 65211
ABSTRACT- Mammalian spermatogenesis and epididymal sperm maturation rely on unique pathways for protein sorting and degradation, including the docking of the pro-acrosomal Golgi vesicles to spermatid nucleus, rejection of spermatid cytoplasm, ubiquitin tagging of sperm organelles and apocrine secretion of otherwise non-secretory cytosolic proteins via epididymal apical blebs. We hypothesized that the above events are accompanied by the increased activity of the proteolytic ubiquitin system. We used transgenic mice with enhanced green fluorescent protein (eGFP) gene construct at the downstream of ubiquitin C (UBC) promoter to trace the expression of UBC gene and to map the protein sorting-pathways in the male reproductive system. As a result of UBC promoter activity, we observed high levels of GFP accumulation in spermatids belonging to various developmental stages, and in the principal and clear cells of the initial segment and caput epididymis. Testicular GFP was sorted preferentially into the acrosomal granules of step 1-5 round spermatids and into the cytoplasmic lobes of elongating spermatids. We also observed massive accumulation of GFP in the cytoplasmic droplets of normal testicular and epididymal spermatozoa, and in the redundant perinuclear and periaxonemal cytoplasm carried by defective spermatozoa. Consistent with the proposed apocrine secretion of ubiquitin and related enzymes into epididymal fluid, we observed increased GFP accumulation in the secretory sites on the apical surface of epididymal epithelium and in the endocytotic clear cells of caput epididymal epithelium. Semiquantitative RT-PCR demonstrated higher eGFP gene expression in testis as compared to other reproductive (epididymis, non-pregnant uterus, vagina, oviduct and ovary) and non-reproductive tissues (heart, lung, liver, kidney, striated muscle, brain and adipose tissue). These observations underscore the role of the ubiquitin system in spermatogenesis and epididymal function, indicating a unique pathway of protein sorting and recycling in the male reproductive system. The UBC-GFP mouse might serve as a useful experimental model to study spermatogenesis, germ cell transplantation, epididymal sperm maturation and sperm cytoplasmic droplet function. Supported by F21C UM-C and USDA-NRI award #2002-02069 to PS.
KEY WORDS: spermatogenesis, ubiquitin, green fluorescent protein, epididymis