Characterization of flagellar cysteine‐rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis

Abstract

Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels

“…Free cysteine levels are an indirect marker of the number of disrupted disulphide bonds, as despite not all free cysteine radicals resulting from the breakage of disulphide bonds, a significant percentage has this origin (Yeste et al, 2013. Specifically, disulphide bonds are crucial for a protein associated with the outer dense fibre 1 (ODF1; Cabrillana et al, 2011). Another issue is the observed increase in free cysteine residues in sperm tail extracts during IVC and the melatonin-counteracting effect of that increase.…”

“…Another issue is the observed increase in free cysteine residues in sperm tail extracts during IVC and the melatonin-counteracting effect of that increase. Thus, it could be hypothesised that changes in the number of disrupted disulphide bonds along the flagellum, specifically in flagellum-bond-related proteins such as sperm hexokinase-1 (Cabrillana et al, 2011(Cabrillana et al, , 2016, could also be related to the changes in motion parameters observed during IVC. Specifically, disulphide bonds are crucial for a protein associated with the outer dense fibre 1 (ODF1; Cabrillana et al, 2011).…”

“…Disulphide bonds are important for the maintenance of a proper sperm flagellum structure (Ijiri et al, 2014). Specifically, disulphide bonds are crucial for a protein associated with the outer dense fibre 1 (ODF1; Cabrillana et al, 2011). Thus, one could suggest that changes in free cysteine residues of sperm tail would affect the structure of the 732 Andrology, 2018, 6, 720-736 flagellum which could, in turn, induce subtle changes in sperm motion.…”

Role of Posttranslational Protein Modifications in Epididymal Sperm Maturation and Extracellular Quality Control

“…Within spermatozoa, disulfi de bond formation occurs in both the head, particularly in nuclear protamines as part of sperm chromatin condensation, as well as within the sperm tail [ 95 ]. The sulfhydryl rich organelles in the sperm tail that undergo oxidation to disulfi des include the outer dense fi bers, including the protein outer dense fi ber 1, connecting piece, outer mitochondrial membranes, and the fi brous sheath [ 96 ]. Disulfi de bond formation is thought to be important for sperm motility maturation since incubation of spermatozoa with a sulfhydryl-specifi c membrane impermeant dye inhibited the motility of goat spermatozoa [ 97 ].…”

Proteomics of post-translational modifications of mammalian spermatozoa

“…By labelling epididymal spermatozoa with sulfhydryl compounds such as [N-3 H]ethylmaleimide (Miller and Masui 1982), N-(4-carboxy-3-hydroxy-phenyl) maleimide (which forms fluorescent adducts) (Mercado et al 1976) and [ 14 C]iodoacetamide, it is clear that nuclear protein thiol oxidation occurs during epididymal transit, which is likely to be a reflection of the redox status of protamines (Shalgi et al 1989). In addition, others have shown that cysteine oxidation occurs within the outer dense fiber 1 protein (ODF1) (Cabrillana et al 2011) and hexokinase (HK1) (Nakamura et al 2008) as sperm descend the epididymis. ODF1 is a chaperone and, as such, its redox status may help in the folding of various proteins as well as in the elastic recoil of the sperm tail (Morales et al 1994).…”