Early life experiences reorganize the physiology [1, 2] immunity[3–6], and behavior [1, 4–6]of the exposed individual, with lifelong consequences. Our data extend previous studies that have demonstrated that the experience of NR in macaques is associated with reduced immune cell counts, dysregulated plasma cortisol [2, 30, 36]and greater emotion-related behavior. We now demonstrate that some of these effects are also observed in offspring of NR males. Male and female offspring of NR males exhibited greater trait emotionality and higher plasma cortisol compared with descendants of CONTROL males. Notably, these differences were observed in the absence of extended social exposure to fathers, mothers, or extended families and social groups, as our infant subjects were all themselves NR. It is unlikely that genetic influences explain these results, because parental NR was administered randomly, our population pairwise relatedness was low(less than 2%), and we controlled for pedigree in our analysis. In sum, our work suggests that vestiges of early stress may not be limited to exposed individuals in primates, but may influence subsequent generations through the paternal line via apparently non-social mechanisms.
In the present study, paternal NR predicted greater emotionality and higher plasma cortisol in NR offspring. Maternal effects of NR were not observed in this study. These findings are consistent with several studies that have demonstrated paternal line effects of adversity on anxiety-and health-related traits in offspring, even in the absence of social contact with fathers [18–23], but are somewhat inconsistent with rodent studies that have demonstrated equivalent transgenerational effects of both paternal and maternal exposures. This does not necessarily mean that maternal NR has no effect on offspring development, but its effects may be more complex to detect. Traits inherited via the maternal line arise from interactions among maternal genetics, prenatal factors, and postnatal investment. In the present study, the impact of postnatal investment would have been minimal: mothers and infant subjects remained together usually less than one full day (but no more than four days) after infant birth. But it is possible that maternal NR interacted with maternal genetics and prenatal factors in a way that obscured the effects of maternal rearing history on infant development in this analysis. Another possible source of heterogeneity is that, while all fathers were NR on Day 1 of life, as were the majority of NR mothers, a small subset (4/27) mothers had been separated from their own mothers later in development (two by Day 3 of life, but one on Day 23 and the other on day 85). We controlled for maternal postnatal day NR in our analysis. This variable did not affect out results, suggesting that the number of days that female infants remained with mothers before being NR did not change the effects on offspring. However, due to the small sample size of our NR mothers, it is possible that this heterogeneity in NR practices affected our results. To address this potential issue, we re-analyzed the data without these four females, and the results were identical. Thus, we believe that the main effects of maternal NR are, in fact, weaker than that of paternal NR, but future studies must corroborate this finding.
Because the effects we observed were specific to the paternal line, we considered the possibility that parental NR may be sex-specific, and thus, affect males and females differently. While other studies have demonstrated sex differences in sensitivity to experiences[37, 38], and more recently it has been discovered that there may be sex differences in the effects of parental environmental exposure as well [18, 23–27], this was not the case in our population. Males and females were similarly affected by paternal line NR in our study, but it remains to be determined whether affected males and females will equally transmit the effects of paternal NR to their own offspring.
The mechanisms for the effects of paternal early stress on offspring trait emotionality and plasma cortisol are not yet known. The potential for social or environmental mechanisms to explain the effects of paternal NR in this study is limited. Thus, the effects of paternal NR may arise from germ-line mechanisms. If germ line elements were changed in males following early experiences and were stably inherited by progeny, this would help explain the effects of paternal NR. Presumably substantial changes to the DNA sequence itself are not widespread in sperm following stress, but epigenetic signals, such as histone modifications, micro RNAs, hormones/transcription factors, and DNA methylation patterns are all present in paternal germ cells and may be inherited by the developing infant [18–20].While several recent studies in rodents have demonstrated such transgenerational inheritance of exposure-induced DNA methylation patterns and micro RNA expression via sperm[18–23], it is not yet known if such acquired germ-line changes are inherited in primates, including humans.
Why would paternal, but not maternal, NR effects be inherited through germ line mechanisms? Because most mammalian fathers, including rhesus macaques, contribute little more than germ cells to progeny (monogamous, biparental species represent an exception among mammals[39, 40]), it has been theorized that we might expect such germ-line mediated mechanisms to evolve, to allow for intergenerational transmission of acquired information between males and their descendants in the species-typical absence of social contact [41–43]. We would not necessarily expect such germ line mechanisms for transmission to arise between mother and offspring in mammals, because there are other opportunities for such mother-infant transmission, including prenatal and postnatal signaling.
An alternate explanation for our findings is that mothers may adjust their prenatal investment in infants based on NR-related qualities of the father. Avian and mammalian mothers have been shown to adjust postnatal investment in infants based on paternal characteristics. In one recent study, female mice that were mated with males from impoverished early life conditions negatively adjusted their postnatal maternal investment toward offspring, which corresponded in a linear fashion with the rate of anxiety-related behaviors exhibited by the father. This reduction in maternal care, in turn, predicted offspring growth. This study was consistent with the ethological literature that suggests that females may invest differently in offspring based on perceived characteristics of the male mate, for example, in mallards. In these previously described conditions, however, the female mated with only one male and therefore the identity of the father was likely known to the female. Macaques are a polygynous species, and females mate with multiple males throughout the breeding season. While evidence is scarce, there are few data available to suggest that females are aware of the identities of their offspring's fathers in natural group-living conditions. Similarly, in the present study, over 60% of our subjects’ mothers lived in conditions with multiple males, meaning that mothers most likely did not know the identity of offspring's fathers. Future studies must investigate this possibility more systematically, however.
These findings have important evolutionary implications. When adverse early experience in a previous generation is associated with an emotionally reactive temperament and heightened cortisol in subsequent generations, this parental programming may influence the fitness and reproductive success of future generations. Initially, it may seem that the transgenerational programming of such traits may be disadvantageous to descendants’ survival and reproductive success, especially if it leads to poorer mental and physical health. One might expect that a compensatory effect of parental environmental exposures on the health of their descendants would be a more adaptive strategy in an evolutionary context. Such a compensatory effect has been observed in humans [26, 27]. We provide some evidence for a compensatory effect as well. A previous study in this population demonstrated that NR predicts dampened cortisol output in 3-4 month olds. Our NR infants with NR fathers still exhibit dampened cortisol compared with CONTROL reared infants, but their cortisol was merely higher than other NR infants of CONTROL fathers. These results may suggest that the effects of NR on cortisol are somewhat, although not completely, reversed, in the next generation. It is also possible that challenges in a previous generation shape adaptive coping strategies in the next generation. For this to be an advantageous strategy, one might expect that transgenerational programming should optimize fitness under parental environmental conditions. To our knowledge, this has not yet been investigated, but at least two studies have demonstrated that early adversity may actually optimize survival skills and reproductive strategies in the same generation. Rats that received poor maternal care as infants reach puberty earlier and engage in higher rates of sexual behavior as adults, perhaps ensuring reproductive success in uncertain conditions . Additionally, while rats that received lower maternal care in infancy perform worse on a learning task than infants that received better care, these low care infants perform better than high care rats under stressful conditions, suggesting that early stress may have optimized performance for stress.Thus, transgenerational inheritance of stress coping strategies may actually optimize stress adaptation in the next generation – particularly if the offspring's environment matches the stressed parent's environment. Future studies will examine whether transgenerational stress programming may confer an advantage in subsequent generations in primates.
There were several important limitations of this study. The first limitation is that conditions similar to NR are not likely to occur in the wild, as infants that survive after being separated from parents have likely received care from other adult group members rather than peers. Thus, the ecological relevance of this paradigm is somewhat limited. However, we suggest that shifts in immunity, physiology, and behavior following variation in early experiences are likely facilitated by genomic, neural, and physiological shifts. Thus, the specific characteristics of the early experience may not be as important to the ecological relevance of our results as is the demonstration that these systems show shifts in response to different types of experiences. NR has been linked with similar biobehavioral shifts compared with other types of early experiences in humans and other animals [1–3, 5–7, 9], supporting its generalizability. Certainly, other types of early experiences with similar effects on behavior and physiology may have been transmitted across generations, as others and we have previously demonstrated [9–14]. Therefore, while we expect that other types of more ecologically relevant experiences would be subject to transgenerational inheritance, just as we have demonstrated with NR, more research is required. Similarly, another limitation of this study is that we restricted our analysis to offspring that were themselves NR. This approach was taken to eliminate the impact of social mechanisms involved with transgenerational stress effects, but we still do not know whether the effects would be as strong in infants raised in species-typical environments with their parents and social groups. Future studies must bear this out.
A methodological limitation of this study is that, because we assessed physiology and behavior at one time point [3-4 months of age), the lifetime effects of NR ancestry on behavior, physiology, and health, and the relevance to fitness outcomes, are not yet known. We believe that these measures are indicators of future outcomes, however, as previous work has linked these biobehavioral measures with critical health and social outcomes later in life. For example, macaques with biobehavioral profiles suggesting greater anxiety display airway hyper-responsiveness, a predictor of asthma, in adolescence[47, 48]. There are also social consequences associated with these measures: in adolescence, macaques tend to prefer to spend time with individuals with similar behavioral traits [49, 50], indicating that these traits measured early in life may influence social relationships and even mating preferences. These studies hint that the transgenerational inheritance of anxiety-related traits may impact important aspects of macaque social life and health, but future studies will investigate the continuity of effects into adulthood and ultimate effects on longevity and reproductive success.
In summary, we demonstrate that some of the effects of NR are transmitted to the second generation through non-social mechanisms via the paternal line in rhesus macaques. Stress related traits and diseases, such as cardiovascular disease, metabolic disease, inflammation, and psychiatric illness, have long been known to run in families. This phenomenon has historically been attributed to genetic causes. Our finding that an early experience influences subsequent generations through the paternal line in primates contributes to a growing literature that suggests that some acquired experience-related traits may also be “inherited” in mammals, including primates. Future studies will focus on understanding the likely complex social and germ line mechanisms of the effects of paternal-line NR in primates, and their consequences for health, reproductive success, and natural selection.