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.

Background Rabbits are sensitive to dietary cholesterol and rapidly develop hypercholesterolemia, leading to prominent subfertility. Sterol regulatory element‐binding protein isoform 2 drives the intracellular cholesterol pathway in many tissues, including the testicles. Its abnormal regulation could be the mainly responsible for the failure of suppressing cholesterol synthesis in a cholesterol‐enriched environment, ultimately leading to testicular and seminal alterations. However, extra‐virgin olive oil consumption has beneficial properties that promote lowering of cholesterol levels, including the recovery of seminal parameters altered under a high‐fat diet. Objectives Our goal was to investigate the effects of high‐fat diet supplementation with extra‐virgin olive oil at the molecular level on rabbit testes, by analyzing sterol regulatory element‐binding protein isoform 2 protein and its corresponding downstream effectors. Materials and methods During 12 months, male rabbits were fed a control diet, high‐fat diet, or 6‐month high‐fat diet followed by 6‐month high‐fat diet plus extra‐virgin olive oil. Serum lipids, testosterone levels, bodyweight, and seminal parameters were tested. The mRNA and protein levels of sterol regulatory element‐binding protein isoform 2, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase, and low‐density lipoprotein receptor were determined by semi‐quantitative polymerase chain reaction and Western blotting techniques. The expression pattern of sterol regulatory element‐binding protein isoform 2 protein in the rabbit testicles was studied by indirect immunofluorescence. In addition, testicular cholesterol was detected and quantified by filipin staining and gas chromatography. Results The data showed that the addition of extra‐virgin olive oil to high‐fat diet reduced testicular cholesterol levels and recovered the expression of sterol regulatory element‐binding protein isoform 2, 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase, and low‐density lipoprotein receptor initially altered by the high‐fat diet. Discussion and conclusions The combination of high‐fat diet with extra‐virgin olive oil encourages testicular recovery by modifying the expression of the enzymes related to intracellular cholesterol management.