Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate.
Sperm protamine deficiency is observed in a subset of infertile men, suggesting that the relative histone to protamine ratio may be altered in the spermatozoa of these men. We measured the ratio of nuclear histones to protamines in the spermatozoa of fertile (n = 10) and infertile men (n = 20). Sperm nuclear proteins were extracted and subsequently separated by acid-urea (AU) polyacrylamide gel electrophoresis. The relative histone (H2B) to protamine (PRM1 + PRM2) and PRM1 to PRM2 ratios were estimated by densitometric analysis of the AU gels. Immunoblotting experiments (using H2B, PRM1, and PRM2 antibodies) were conducted to confirm the specificity of the bands. The pattern and intensity of H2B staining in human spermatozoa was assessed by immunocytochemistry. Sperm samples from the infertile men in this study had a significantly higher proportion of histone H2B to protamine (PRM1 + PRM2) than did samples from the fertile men in this study (0.38 vs 0.08, P < .001). Immunocytochemistry experiments demonstrated a punctuated staining pattern (with strong, intermediate, or weak H2B staining intensity) throughout the sperm head. Infertile men had a higher proportion of spermatozoa exhibiting strong and intermediate staining than did samples from fertile men. These findings suggest that infertile men possess a higher proportion of spermatozoa with an increased histone to protamine ratio than fertile controls.