The comet assay in male reproductive toxicology

Abstract

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.

Evaluation of pH effects on genomic integrity in adipose-derived mesenchymal stem cells using the comet assay

“…This version is the most comprehensive, and in addition to detecting direct damage, it enables the evaluation of indirect damage such as lesions like methylation adducts, which are alkali-labile and expressed as simple breaks under high alkaline conditions (Singh, 2000;Tice et al, 2000). The other version (pH = 12) detects mainly single-and double-strand breaks (Tice et al, 2000;Baumgartner et al, 2009).…”

Impact of adrenalectomy and dexamethasone treatment on testicular morphology and sperm parameters in rats: insights into the adrenal control of male reproduction

“…The quality of spermatozoa is critical for successful fertilization and subsequent development of the embryo (O'Brien & Zini, 2005;Baumgartner et al, 2009). Thus, we evaluated qualitative parameters in spermatozoa collected from the cauda epididymis of 7-and 15-day ADX rats.…”

“…Suggested mechanisms by which sperm DNA fragmentation can occur include defective chromatin condensation during spermiogenesis, abortive apoptosis during spermatogenesis or sperm maturation, and/or oxidative stress from reactive oxygen species (ROS) during sperm maturation (Baumgartner et al, 2009). Although the role of the epididymis in facilitating sperm chromatin condensation is unclear, this organ possesses several antioxidant defense systems that may protect sperm DNA from the harmful effects of oxidative stress (Hinton et al, 1995).…”

“…Importantly, the short-and long-term reproductive outcomes of male patients who underwent adrenalectomy, especially those related to sperm quality, are yet to be established. This is a relevant topic, since experimental and clinical evidences have pointed out the possible association between sperm DNA fragmentation and adverse reproductive outcomes, such as male infertility and embryo implantation and development impairment (Baumgartner et al, 2009;De Iuliis et al, 2014;Evgeni et al, 2014). Thus, experimental models of bilateral adrenalectomy become important tools for the understanding of possible effects induced by this surgical procedure by itself or in association with different conditions of steroid hormone replacement therapy on sperm DNA integrity and male fertility.…”

Germ cell mutagens: Risk assessment challenges in the 21st century

“…It is worthy to consider whether these assays and other in vitro tools could perhaps be optimized for genotoxicity assays examining effects on specific stages of spermatogenesis. Numerous other studies have examined the induction of DNA strand breaks in spermatozoa treated with toxicants in vitro [reviewed in Baumgartner et al, 2009]. Although somatic and spermatozoal responses are generally similar, there are a few exceptions where cultured sperm are more responsive than lymphocytes (probably due to the lack of DNA repair in mature sperm).…”