PATERNAL STRESS PRIOR TO CONCEPTION ALTERS DNA METHYLATION AND BEHAVIOUR OF DEVELOPING RAT OFFSPRING R. MYCHASIUK, * A. HARKER, S. ILNYTSKYY AND R. GIBB University of Lethbridge, Lethbridge, AB, Canada Abstract—Although there has been an abundance of research focused on offspring outcomes associated with maternal experiences, there has been limited examination of the relationship between paternal experiences and off- spring brain development. As spermatogenesis is a contin- uous process, experiences that have the ability to alter epigenetic regulation in fathers may actually change devel- opmental trajectories of offspring. The purpose of this study was to examine the effects of paternal stress prior to con- ception on behaviour and the epigenome of both male and female developing rat offspring. Male Long-Evans rats were stressed for 27 consecutive days and then mated with control female rats. Early behaviour was tested in offspring using the negative geotaxis task and the open field. At P21 offspring were sacrificed and global DNA methylation levels in the hippocampus and frontal cortex were analysed. Pater- nal stress prior to conception altered behaviour of all off- spring on the negative geotaxis task, delaying acquisition of the task. In addition, male offspring demonstrated a reduction in stress reactivity in the open field paradigm spending more time than expected in the centre of the open field. Paternal stress also altered DNA methylation patterns in offspring at P21, global methylation was reduced in the frontal cortex of female offspring, but increased in the hippocampus of both male and female offspring. The results from this study clearly demonstrate that paternal stress dur- ing spermatogenesis can influence offspring behaviour and DNA methylation patterns, and these affects occur in a sex- dependent manner. Development takes place in the centre of a complex interaction between maternal, paternal, and environmental influences, which combine to produce the various phenotypes and individual differences that we perceive. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: epigenetics, frontal cortex, hippocampus, fathers. INTRODUCTION Although a relatively new field of investigation, researchers are beginning to examine indirect environmental influences capable of altering neurodevelopmental outcomes. As we have extended the macro-environment of the child to include fathers, paternal influences, as they pertain to maternal behaviour/depression and co-parenting expectations (Field et al., 2006; Outscharoff et al., 2006; Paulson and Bazemore, 2010; Seidel et al., 2011; Mashoodh et al., 2012), have come under scrutiny. Despite this expansion however, the underlying theory has not really changed; mothers are still considered the primary influencer of child neurodevelopment, with fathers influencing offspring only through modification of maternal characteristics. Hence, there has been limited examination of experiences whereby fathers directly influence the neurodevelopment of offspring. Despite emerging evidence that paternal age, nutrition, and drug use, may place offspring at risk of psychopathologies (Kaat et al., 2007; Curley et al., 2010), very few studies have been designed to tease out the mechanisms responsible for these associations. When examining the role of the father from a genetic perspective, the majority of this research has focused on possible paternal contributions to foetal alcohol syndrome (Randall et al., 1982; Abel, 1993, 2004) or genetic imprinting, with very limited analysis of epigenetic reprogramming of the sperm prior to conception (Franklin et al., 2010; Miller et al., 2010; Mychasiuk et al., 2012). Owing to the continuous nature of spermatogenesis, experiences that change DNA methylation patterns in sperm before fertilization have the potential to alter epigenetic programming of future offspring. DNA methylation is used by spermatozoa and is uniquely regulated to play an important role in the development of future gametes and embryos (Jenkins and Carrell, 2011). Robust paternal epigenetic contribution to embryogenesis requires that the DNA in spermatozoa contain layers of regulatory elements, including methyl groups that drive gene activation or silencing upon contact with the egg. Although critical to normal development, this abundance of regulatory control also leaves the DNA susceptible to damage from outside agents (Jenkins and Carrell, 2011). Damaging outside agents are commonly believed to be chemical toxins or drugs of abuse, however, prior research has demonstrated that stress impairs spermatogenesis in adult rats (Potemina, 2008), and alters methylation patterns in the germline of F2 male mice (Franklin et al., 2010). The purpose of this study was to examine the effects of paternal stress prior to copulation on behaviour and the 0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.03.025 * Corresponding author. Address: Canadian Centre for Behavioural Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada. E-mail address: r.mychasiuk@uleth.ca (R. Mychasiuk). Abbreviations: ANOVA, analysis of variance; PS, paternal stress. Neuroscience 241 (2013) 100–105 100