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Sperm Chromatin Condensation Level: Depending on Fixatives

Tulay Irez, Tugce Durmus

Sperm chromatin undergoes structural changes during spermatogenesis causing high condensation. This nuclear exchange is considered to be a predictor of sperm-fertilization capacity and pregnancy outcome. Various studies have shown a relationship between male infertility and sperm protamine deficiency, and it has been suggested that it may be an important determinant of sperm DNA integrity. Acidic aniline blue staining is often used with glutaraldehyde fixation and stains histones due to lysine’s in the sperm nucleus. Research has shown that the level of histones in sperm nuclei is closely related to DNA damage and infertility. Sperm chromatin condensation is expressed by the presence of protamine in sperm nucleus, that is, the absence of histone. In this study, we investigated how the aniline blue staining method can correctly determine sperm chromatin condensation when different fixation methods are applied. Four different fixatives were used in this study. The fixatives were applied with glutaraldehyde (2.5%), methanol/acetic acid Carney’s, (3/1), ethyl alcohol (98%), and Formaldehyde (10%) aniline blue dye as applied at conventional concentrations in cytological preparations. Of these fixatives, glutaraldehyde is widely used to display sperm histones. Students -T-test and repeated measures analysis of variance were performed to calculate differences in chromatin condensation (aniline blue negative) rates among different fixatives. When the chromatin condensation values obtained with glutaraldehyde fixation were compared with those obtained with methanol and alcohol fixation, statistically significant differences (p=0.014,p=0.0001) were observed. A positive correlation between Glutaraldehyde and Formaldehyde (p=0.51), and a negative correlation between Glutaraldehyde and Formaldehyde, methanol/ acetic acid and alcohol fixation (p=0.005,p=0.0005 Ve p=0.0005,p=0.007) were observed. The positive correlation of chromatin condensation with sperm concentration and motility was determined (p=0.012,p<0,001). According to the results obtained in this study, 2.5% glutaraldehyde and 10% Formaldehyde fixations were appropriate for determining chromatin condensation with aniline blue, but methanol/acetic acid and ethyl alcohol is not suitable for this purpose. It was observed that the alcohol-based fixatives we tested in our study did not specifically show sperm lysine’s and caused the entire nucleus to be stained with aniline blue.