Isolation and Characterization of Submitochondrial Reticulum Complex Associated with Midpiece of Bovine Cauda Epididymal Sperm

Abstract

The mitochondrial sheath of mammalian spermatozoa adheres to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). The objective of the present study is to characterize the structural components of the SMR. We have isolated SMR fractions from midpieces of bovine cauda sperm. SDS-PAGE of SMR fractions revealed four major polypeptides of 56kDa, 54kDa, 34kDa, and 31kDa. Proteomic analyses of the four polypeptides revealed that 56kDa and 54kDa polypeptides identified as glycerol kinase and 34kDa and 31kDa polypeptide identified as VDAC2 and VDAC3, respectively (Voltage-Dependent Anion-Selective Channel Protein 2/3). Western blots of midpiece stained with anti-56kDa, anti-54KDa, anti-34kDaVDAC2, and anti-31kDaVDAC3 antibodies demonstrated the presence of 56kDa, 54kDa, 34kDa, and 31kDa immunoreactive bands, respectively. Four polypeptides are localized to midpiece. Glycerol kinase activity was quantitively assayed in tails. We proposed that glycerol kinase and VDAC2/VDAC 3 polypeptides play an important role in sperm motility and in midpiece function.