Protamine Levels Vary Between Individual Sperm Cells of Infertile Human Males and Correlate With Viability and DNA Integrity

Abstract

Sperm protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in protamine protein content. The objectives of this study were to evaluate variations in protamine-1 (P1) and protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with protamine immunostaining to assess the relationship between protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and protamine levels was assessed via viability staining concomitant with protamine staining. The protamine fluorescence data demonstrate significant variations in protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of protamine levels. The data also demonstrate individual sperm cells displaying the lowest protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.