Direct but Not Indirect Methods Correlate the Percentages of Sperm With Altered Chromatin to the Intensity of Chromatin Damage

Abstract

Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.

Sperm degradation after vasectomy follows a sperm chromatin fragmentation-dependent mechanism causing DNA breaks in the toroid linker regions

“…This leads to a highly condensed nucleus, which functions to protect the paternal genome from genotoxic agents during its transit toward the oocyte. This protection, however, is not totally effective since sperm with fragmented DNA are often observed after ejaculation, with different incidences among species ( Castro et al , 2018 ; Peris-Frau et al , 2019 ; Crespo et al , 2020 ; Ribas-Maynou et al , 2021 ; Esteves et al , 2022 ). In vitro experiments, which attempted to actively damage sperm DNA, have shown that sperm chromatin limits chromatin breaks caused by nucleases to the TLRs ( Sotolongo et al , 2003 ), while damage caused by reactive oxygen species may affect both toroidal regions and TLRs, thus leading to a more localized damage when double-strand breaks (DSBs) are produced by enzymes, and more extensive damage when DNA is oxidized ( Villani et al , 2010 ; Ribas-Maynou and Benet, 2019 ).…”

Is oxidative stress evaluated in viable human spermatozoa a marker of good semen quality?

“…To our knowledge, only one study evaluated the relationship between the two parameters considering viable oxidized spermatozoa ( 28 ), reporting a positive association. In this study, however, sDF was evaluated by sperm chromatin dispersion test, which detects the susceptibility of sperm chromatin to damage rather than the occurrence of real DNA breaks as done by TUNEL ( 29 ). In addition, the positive correlation was assessed only in 28 men, selected on the basis of unexplained infertility ( 28 ).…”

Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility

“…DNA fragmentation and parameters of sperm quality, fertilization, and embryo development are caused by extrinsic factors and their effector mechanisms that affect fertility levels [50]. Ribas-Maynou et al [51] have reported that increased DNA fragmentation in bovine sperm is associated with morphological abnormalities. However, Otero et al [52] reported a negative correlation between sperm motility parameters and several kinematic parameters with DNA fragmentation.…”