Abstract
Globally, ∼1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.
Highlights
In excess of 80 million people suffer from infertility [1], with ϳ1 in every seven couples so affected [2]
A set of guidelines for evaluating semen quality was originally published by the World Health Organization (WHO) in 1980 [3]
We show that the sperm-specific protein, outer dense fiber 1 (ODF1), was dramatically underexpressed within the infertile donors
Summary
Materials—All chemicals and antibodies were purchased from Sigma-Aldrich at the highest research grade, with the exception of albumin and ammonium persulphate (Research Organics, Cleveland, OH), Percoll (GE Healthcare, Castle Hill, Sydney, NSW Australia), HEPES (Gibco, Invitrogen Australia, Melbourne, VIC, Australia), and 10 ϫ Ham’s F-10 (MP Biomedical, Seven Hills, Sydney, NSW Australia). The membrane was washed four times for 5 min with TBS containing 0.01% Tween-20 and incubated for 1 h at room temperature with goat anti-mouse immunoglobulin G horseradish peroxidase (HRP) conjugate, at a concentration of 1:3000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. In order to confirm equal loading of proteins, blots that had been probed were stripped and reprobed with an antibody against ␣-tubulin For this procedure, ϳ30 ml of stripping buffer, consisting of a 0.2 M NaOH solution was added to the membrane for 30 mins at room temperature. Cells were washed in PBS four times for 5 min and incubated for 1 h at 37 °C with FITC-conjugated, goat anti-mouse immunoglobulin G at the concentration of 1:400 in PBS containing 1% (w/v) BSA.
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