Reproduction impairment following paternal genotoxin exposure in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus)

Abstract

This work describes some consequences of paternal germ cell DNA damage on the reproduction success in two fish species. Male brown trout (n = 31) and male Arctic charr (n = 28) were exposed to the model genotoxicant MMS at the end of spermatogenesis to generate a significant DNA damage level in mature spermatozoa (28% and 25% tail DNA in trout and charr sperm, respectively, evaluated through the comet assay). Sperm from each MMS exposed and control fish was then used to fertilize in vitro an aliquot of a single pool of eggs collected from 4 unexposed females for each species. Each batch of fertilized eggs was monitored individually in the hatchery to follow embryonic and larval abnormalities during the fry development. Paternal exposure did not influence fertilization rate or survival rate at hatching in either species. However, MMS paternal treatment resulted in a large array of morphological abnormalities during embryonic and larval development. At the eyed stage, malformations exhibited a 8 fold increase in trout and a 2 fold increase in charr for larvae stemming from MMS treated males as compared with controls. At the end of yolk sac resorption, an increase in the gross morphological abnormality incidence was found in trout larvae originating from MMS exposed males (2.10% vs. 0.93% in control, p < 0.05). When looking more in detail at bony structures after Alizarin red S staining, a 20% incidence of skeletal defects was recorded at the swimming stage. A positive correlation was found between the paternal sperm DNA damage level and the skeletal abnormality incidence of its progeny. During the next 2 months of development, mortality in trout originating from DNA damaged sperm was 3 times higher than in control. After one year, no effect of paternal treatment was found on growth traits (length and weight) but the gross morphological abnormality incidence was still very high in the treated group (27% malformation incidence vs. 0.5% in control). These results demonstrate ecologically relevant consequences of fish spermatozoa DNA damage and stress the value of using this parameter as a biomarker signaling potential long term effects of environmental genotoxins in aquatic systems.

Introduction

Aquatic ecosystems are known to be the ultimate recipient of an increasing amount and range of anthropogenic chemical compounds. However, the global threat posed by the sublethal effects of pollutants is not yet adequately addressed although attention has been historically drawn since the Rio de Janeiro World Summit in 1992 to the reduction in biodiversity taking place world-wide (Depledge, 1996). A significant part of the man-made contaminants (up to one-third) has been described as being potentially genotoxic for the living organisms and possibly implicated as important causal factors in biodiversity loss (Claxton et al., 1998). Genotoxicants have been standing in the limelight because of their potential to lead to a cascade of adverse changes from the cellular to the population level (Würgler and Kramers, 1992, Bickham et al., 2000, Belfiore and Anderson, 2001). Thus, the interest of genotoxicity assessment in aquatic ecotoxicology has been highlighted both in vertebrates and invertebrates (Depledge, 1998, Jha, 2004). However, if some progress has been made in understanding the consequences of genotoxin exposure on human health, a huge gap remains concerning aquatic species. In aquatic ecotoxicology, DNA damage has been used for a long time as an exposure biomarker but the further biological consequences of this damage are still poorly understood or limited to the individual response. For example, in somatic cells, DNA strand breaks are known to give rise to chromosomal aberrations if they are un- or mis-repaired (Palitti, 1998). Such chromosomal aberrations can lead to detrimental effects, ranging from cell death up to physiological dysfunction of key organs, but such effects remain circumscribed to the individual. A specificity of ecotoxicological studies is that they should assess pollutant effects at the population level since environmental policies attempt to take into account drastic and population long term effects of pollutant pressure on aquatic ecosystems (Newman and Clements, 2008). However, so far, the majority of studies are individual based and there is still a clear need to construe the genotoxic exposure consequences at the population level. However, this level is difficult to work on. Because of its probable consequences on population parameters, reproductive impairment of fish can be considered as one of the most significant effects of aquatic pollution (Kime, 1995). To study this, one possibility is to pay particular attention to the consequences of germ cell DNA damage that could impact negatively progeny outcomes, since genotoxic damage in such cells can be passed on to future generations if not or misrepaired. A recent study carried out on fish has suggested a possible link between the level of DNA damage in spermatozoa of mature male exposed to a genotoxicant and reproduction impairment, but limited to an effect on hatching rate (Zhou et al., 2006). In the current work, we studied the reproductive consequences of a paternal genotoxin exposure by following the link between the sperm DNA integrity level and the occurrence of developmental abnormalities at early and late developmental stages in fish as well as the subsequent effects on juvenile survival and growth. The comet assay was chosen to measure DNA damage in sperm after male exposure to methyl methane sulfonate (MMS) during late spermatogenesis. MMS was used as a model alkylating genotoxicant because alkylating compounds are thought to be the most potent and abundant genotoxic contaminants in aquatic ecosystems (Kuehl et al., 1994). MMS binds covalently with nucleophilic centers of DNA bases. It primarily alkylates ring nitrogens and depurinates DNA, further leading to strand breaks. Among the different assays available for genotoxicity assessment, the comet assay (also called single cell gel electrophoresis assay) is a rapid, sensitive, relatively inexpensive method, requiring only a small number of cells and providing the opportunity to study DNA damage and repair in individual cells of aquatic organisms (Devaux et al., 1997, Devaux et al., 1998, Lee and Steinert, 2003, Jha, 2008). The alkaline version of this technique used in the present study allows the evaluation of a large array of DNA damages including single and double strand breaks, DNA cross-links, alkali-labile sites and incomplete repair sites (Singh et al., 1988). Development of fish progeny was assessed at different stages during 5 months from the fertilization, at embryonic and larval stages, respectively, by measuring fertilization rate, morphological abnormalities, hatching rate and survival. Two freshwater fish species were chosen, based on both ecological and experimental criteria. Arctic charr (Salvelinus alpinus) lives in the deep and oligotrophic lakes in the Alps and its spawning grounds are located in the deeper parts of the lakes. Brown trout (Salmo trutta) occupies many habitats (rivers, lakes, sea shores) in all the northern and middle Europe but it reproduces only in cold and little polluted brooks.