Identification of a Hamster Sperm 26-Kilodalton Dehydrogenase/Reductase That Is Exclusively Localized to the Mitochondria of the Flagellum1

Abstract

Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.

Human DCXR – another ‘moonlighting protein’ involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

“…Immunocytochemistry experiments repeatedly showed that P26h‐specific staining was restricted to the acrosomal cap of mature hamster spermatozoa (Berube & Sullivan, ; Begin et al , ; Legare et al , 1999 a ). By contrast, Nagdas, Winfrey & Olson () showed that hamster P26h was exclusively localized to the midpiece of spermatozoa from the cauda epididymis, while no staining was observed on the head region. Moreover, P26h was detected in total cauda sperm lysate but was absent in both the plasma membrane fraction of spermatozoa and cauda epididymal fluid, also in contrast to previous findings.…”

“…It seems that both groups detected isoforms of hamster P26h, thus one possible explanation for their conflicting results might be the application of different fixation/permeabilization methods or different antibodies that were produced in‐house. The respective P26h proteins used for antibody production were either extracted from spermatozoa with detergent (Berube & Sullivan, ) or purified from isolated sperm tails (Nagdas et al , ). Interestingly, the antibodies from each group exclusively detected P26h in that particular region of the sperm from which the respective immunizing protein was extracted (sperm acrosome versus sperm flagellum).…”

Mitochondria in Mammalian Sperm Physiology and Pathology: A Review

“…2003). As well, glycolitic enzymes have been identified in the principal piece/flagellum in mammalian spermatozoa, including hexokinase, LDH and glyceraldehydes 3‐ phosphate dehydrogenase (GADP‐S) (Nagdas et al. 2005, 2006; Perl et al.…”

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis

“…The released sperm suspension was centrifuged at 100 g for 2 min at 4 8C. The pellet containing sedimented tissue fragments was discarded and supernatant centrifuged at 1500 g for 10 min at 4 8C to pellet spermatozoa (Nagdas et al 2006, Chakrabarty et al 2007. The pellets were washed twice in buffer containing 10 mM HEPES/NaOH and 100 mM NaCl and resuspended in the same buffer.…”

“…Mitochondria were isolated according to previously reported methods (Nagdas et al 2006). In brief, the washed pellets were suspended in buffer containing 10 mM HEPES/NaOH, 100 mM NaCl, and protease inhibitor cocktail (1:100 (v/v)).…”