Abstract
Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.
Highlights
The mechanism underlying sperm flagellar development is unknown
Data base searches indicated that the membrane-associated RING-CH 10 (March10) gene is limited to mammals, whereas the March7 gene is present in vertebrates ranging from fish to humans
When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, and this pattern of distribution was dispersed by microtubule depolymerization
Summary
The mechanism underlying sperm flagellar development is unknown. Results: Mammalian MARCH10 is a microtubule-associated E3 ubiquitin ligase expressed in the tail of developing spermatids. MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids. The identification and characterization of the flagellar Ub-proteasome system should provide novel insight into molecular mechanisms regulating the assembly and function of the mammalian sperm flagella. We show that MARCH10 is localized to the principal piece of elongating spermatids and is associated with microtubules, which is important for its E3 activity. These results suggest that MARCH10 may play a role in the formation of the sperm flagellum
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