Presence of Endogenous Nicks in DNA of Ejaculated Human Spermatozoa and its Relationship to Chromomycin A3 Accessibility1

Abstract

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.

Human Cervical Mucus Can Act in Vitro as a Selective Barrier Against Spermatozoa Carrying Fragmented Dna and Chromatin Structural Abnormalities

“…During the last decade, a number of studies have shown that variable but consistent fractions of human spermatozoa not always detectable on the basis of classic morphological parameters, contain serious chromatin structural abnormalities and DNA strand breaks (12)(13)(14)(15)(16)(17)(18). The existence of an association between sperm chromatin malformation and male infertility has been repeatedly observed in humans and structural chromatin abnormalities have been found to exert negative effects both on the percentage of fertilization and on embryonic development (17,(19)(20)(21).…”

Variation of the genome size estimate with environmental conditions in Drosophila melanogaster

“…A correlation between estimates obtained using different methods is not in itself sufficient, however, to eliminate the possibility of an artifact, because DNA accessibility depends on complex interactions between intercalating or nonintercalating fluorochromes and DNA and various proteins. Variation in DNA accessibility due to the state of condensation of the chromatin has been reported in plants (21,23), during spermatogenesis (24) and in frozen and thawed human spermatozoa (25), in mouse thymocyte nuclei (26), in ethanol-fixed cells (27), in dividing and stationary Euglena cells (28), during differentiation of Friend leukemia cells (29), and in DNAse I treated HeLa nuclei (30,31). The exact impact of external environmental conditions on the genome size estimate therefore needs to be determined before a protocol can be defined that would permit reliable and comparable genome size estimates between individuals and populations within a given experiment and between different experiments.…”

An Assessment of New Sperm Tests for Male Infertility

“…Stains proteins in loosely condensed chromatin 23 Chromomycin a (inexpensive, simple). Competes with protamines for association with DNA and staining relates to the degree of protamination in mature sperm 24 Toluidine blue (inexpensive, simple). Stains phosphate residues of loosely packed and fragmented sperm nuclear DNA 25 Figure 1.…”

“…This assay quantifies the incorporation of biotinylated deoxyuridine triphosphate (dUTP) at single-stranded DNA breaks (SSBs) within sperm DNA in a reaction catalyzed by DNA polymerase I. 24 Not widely used clinically, this assay lacks sensitivity and correlative studies with fertility outcomes are lacking. 24 Terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL).…”

“…24 Not widely used clinically, this assay lacks sensitivity and correlative studies with fertility outcomes are lacking. 24 Terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL). This assay accurately detects mainly double-stranded breaks (DSBs) in DNA through the incorporation of dUTP at DNA breaks catalyzed by terminal deoxynucleotidyl transferase.…”