Identification of Drosophila-Based Endpoints for the Assessment and Understanding of Xenobiotic-Mediated Male Reproductive Adversities

Increased incidence of infertility in men, in part, as a consequence of exposure to the environmental chemicals (xenobiotics), is of global concern. In the last two decades, sperm counts in men have reduced well below World Health Organization threshold of average concentration of 55 million/ml and significant alterations in semen quality parameters critical for male fertility have been reported (Carlsen et al., 1992; Rolland et al., 2012). Environmental chemicals hamper male fertility by adversely affecting the testicular signaling and reducing sperm counts as well as quality of the semen of exposed males. These xenobiotics inflict detrimental effects on male fertility through disruption of endocrine function and/or causing oxidative stress (Aitken et al., 2014; Knez, 2013). The magnitude of observed reduction in male fertility, accordingly, prompted the assessment of reproductive toxicity potential of various environmental chemicals. Male reproductive toxicity assessment of xenobiotics primarily utilizes rodents or nonhuman primates, with a focus on human safety (Foster, 2006; Sharpe, 2001). Therefore, the assessment of innumerable chemicals on their ability to hamper male fertility would need a large number of these animals. However, the regulatory restrictions and ethical concerns along with limitations associated with using laboratory animals pose a greater challenge in taking in vivo reproductive toxicity assessment to high-throughput scale. In order to minimize these concerns and to promote the principle of three Rs (reduce, refine, and replace), European Centre for Validation of Alternate Methods (ECVAM) recommended the use of lower vertebrates and invertebrates as alternatives. Given the complexity of reproductive processes and their precise regulation through various feedback mechanisms, designing in vitro assays that can recapitulate the same is no trivial goal. Accordingly, existing in vitro male reproductive toxicity assays faithfully capture only certain aspects of male reproduction. In addition, the artificial nonphysiological conditions of the cultures, lack of homeostasis, impaired intracellular interactions, and dearth of defense mechanisms, which thrusts probably stronger impact on the precision of toxicity estimations (Hartung, 2007), exhort the pursuit of better substitutes. However, alternatives that facilitate the reproductive toxicity assessment in vivo are relatively scarce. In this context, Drosophila, with its well characterized development and reproduction, offers a potential alternative and a promising model for the male reproductive toxicity assessment. Interestingly, Drosophila is an OECD approved model for genotoxicity studies that quantifies DNA damage using sperm DNA (Siddique et al., 2005). However, despite the homology of genes associated with testicular development, spermatogenesis (Fischer et al., 2012; Mikhaylova et al., 2008), sperm maturation, and conservation of protein classes in seminal fluids from insects to mammals (Avila et al., 2011; LaFlamme et al., 2012), Drosophila remained under utilized for male reproductive toxicity assessment studies. A few studies have attempted to utilize Drosophila for reproductive toxicity assessment, but these have been mostly confined to the preliminary analysis of fertility. This is, in part, due to lack of predefined endpoints that can be attributed to male reproductive adversities in Drosophila. Therefore, in the present study, we carried out a systematic as well as comprehensive analysis of sperm as well as seminal fluid parameters at organismal, genetic, biochemical, and endocrine levels on male flies fed with a chemical to develop Drosophila melanogaster as an alternative model for the assessment of chemical-mediated male reproductive adversities. We show here that Drosophila recapitulates male reproductive toxicity phenotypes observed in mammals when exposed to dibutyl phthalate (DBP), a chemical belonging to a group of phthalate esters and known to hamper male fertility (Foster, 2006). We observed significant reduction in male fertility, as a consequence of altered semen quality and endocrine disruption, in response to DBP. Exposure to DBP resulted in a significant decline in the expression as well as activity of a Drosophila hormone receptor, Estrogen-Related Receptor (dERR), a sole Drosophila ortholog of the vertebrate ERR nuclear receptor subclass. In addition, we detected monobutyl phthalate (MBP), a major metabolite of DBP in higher organisms, in Drosophila males exposed to DBP. Our data suggest that even in Drosophila, MBP is more toxic than DBP to male fertility. Our findings reflect the potential of Drosophila as an alternative for prescreening of innumerable environmental chemicals on their ability to hamper male fertility. This, together with its genetic tractability makes Drosophila an excellent model to gain insights into chemical-mediated reproductive toxicity and endocrine disruption.