Significance Physical exercise is well known for its positive effects on general health (specifically, on brain function and health), and some mediating mechanisms are also known. A few reports have addressed intergenerational inheritance of some of these positive effects from exercised mothers or fathers to the progeny, but with scarce results in cognition. We report here the inheritance of moderate exercise-induced paternal traits in offspring’s cognition, neurogenesis, and enhanced mitochondrial activity. These changes were accompanied by specific gene expression changes, including gene sets regulated by microRNAs, as potential mediating mechanisms. We have also demonstrated a direct transmission of the exercise-induced effects through the fathers’ sperm, thus showing that paternal physical activity is a direct factor driving offspring’s brain physiology and cognitive behavior.

Abstract Physical exercise has positive effects on cognition, but very little is known about the inheritance of these effects to sedentary offspring and the mechanisms involved. Here, we use a patrilineal design in mice to test the transmission of effects from the same father (before or after training) and from different fathers to compare sedentary- and runner-father progenies. Behavioral, stereological, and whole-genome sequence analyses reveal that paternal cognition improvement is inherited by the offspring, along with increased adult neurogenesis, greater mitochondrial citrate synthase activity, and modulation of the adult hippocampal gene expression profile. These results demonstrate the inheritance of exercise-induced cognition enhancement through the germline, pointing to paternal physical activity as a direct factor driving offspring’s brain physiology and cognitive behavior.

The beneficial effects of exercise for health are largely well known (1, 2), as well as its basic action profile—a hormetic, biphasic curve (3). Specifically, its anxiolytic, antidepressant, and procognitive effects have been described in detail in past decades (for a recent review, both in laboratory rodents and humans, see ref. 4). Moreover, physical exercise has also been linked to brain function and to specific behavior-related phenomena such as adult hippocampal neurogenesis (5). However, very little data exist on whether these exercise-mediated effects are inheritable. Because many of the positive effects of exercise on the brain are induced through epigenetic changes (for a recent review, see ref. 6) and some epigenetic changes have also been reported in runners’ sperm (7⇓–9), we investigate whether the procognitive effects of exercise training in mice might be inherited by the (otherwise sedentary) progeny of runner fathers.

Several reports have revealed either no effects or positive effects of physical activity on the sedentary progeny of exercised pregnant female rodents and humans, due to its direct actions on fetuses through the placenta (10⇓–12). Intergenerational transference of exercise-induced effects on mood and conditioned fear has been reported (13⇓–15), and a recent paper (16) demonstrated the inheritance of an enriched environment-induced improvement in hippocampal long-term potentiation (LTP) through male sperm microRNAs. Despite the importance of knowing the basic biological mechanisms by which an activity-dependent improvement in cognition might be inherited by a nontrained progeny, to our knowledge, there are no reports addressing either the specific neuronal populations related to these improved functions or the cellular mechanisms mediating these effects. Furthermore, the inheritance of the effects on both nonspatial and spatial tasks and on cognitive processes, like object recognition memory and pattern separation (crucial to information processing), remain to be described. Taking into account that adult neurogenesis is a key player in hippocampal information processing and pattern separation (17) and the relevant role that mitochondrial function has for exercise-induced improvements in cognition (reviewed in ref. 18), we have designed hypothesis-driven patrilineal intergenerational inheritance experiments focusing on pattern separation, adult hippocampal neurogenesis, and nuclear-encoded mitochondrial protein effects. Considering this background, our working hypotheses are (i) the effects of exercise on some aspects of fathers’ cognition are inherited by the adult male offspring; (ii) the main features of the adult hippocampal neurogenesis subpopulations (cell proliferation, short- and long-term cell survival, and maturation) affected by the paternal exercise are inherited by the progeny; (iii) the changes in the pattern of gene expression in the hippocampus of exercised fathers might also be inherited by the offspring; and (iv) the main characteristics of mitochondrial functioning (either number or organelle activation or both) are transmitted intergenerationally.

To achieve these goals, we performed a triple approach to patrilineal intergenerational inheritance to test the strength of the biological process at hand. First, we compared litters from sedentary males with litters from the same males after training (running). This approach was used to minimize interfather variability (the progenitor effect). Second, we compared litters from sedentary males with litters from different, exercised males to study intergeneration effects of exercise in nonrelated offspring. This approach was used to compare animals from experimental groups processed at the same time and without the potentially confounding factor of the order of the litter. Third, to study whether these intergenerational exercise-driven effects were germline dependent, we designed an experiment in which interactions between male and female progenitors were eliminated by generating the progeny through in vitro fertilization (IVF) and embryo transfer. Our experimental design followed main gold-standard guidelines (19). Because we were interested in the cognitive effects of physical activity, we tested the pure effects of physical exercise, separating these effects from the cognitive influence of an environmental enrichment. To minimize intersubject variability, we employed moderately forced activity on a treadmill. Specific behavioral tests were used to analyze a possible enhancement of object recognition memory and spatial pattern separation. The novel object recognition (NOR) test is a common method used to assess the rodents’ ability to recognize a novel object in an environment without external cues and reinforcements. It is based on the rodent’s natural preference for novelty. When the animals are exposed to a familiar and a novel object, they spend more time exploring the novel object (20). There are several underlying neural circuits and brain structures involved in the NOR test (in which the hippocampal formation plays a key role) that support learning and memory processes, such as encoding, consolidation, and memory retrieval (21). On the other hand, pattern separation is a cognitive process that allows the formation of distinct representations out of similar inputs. A pattern separation task based on a novel object location test can be used to study spatial pattern separation, which is greatly supported by the dentate gyrus and adult hippocampal neurogenesis (22). Both the NOR test and the pattern separation task can be evaluated by a discrimination index (DI), which expresses the difference in the exploration times of the novel and familiar objects (the moving and fixed objects in the pattern separation task), divided by the total exploration time.

To relate the exercise effects with the heritability of the changes in specific hippocampal neuronal populations (including neurogenic populations), we used ad hoc-designed stereological protocols. Exercise-induced changes in gene expression in the brains of fathers and offspring were also analyzed, as well as the changes in induced methylation in the parents’ sperm. Finally, exercise-induced changes in mitochondrial physiology and cellular energetics in the liver, cerebellum, and hippocampus of fathers and offspring were also analyzed.

Discussion Previous studies have addressed inter- or transgenerational inheritance of activity-induced effects on behavior (13⇓⇓–16). In these studies, limited results were found. No changes were observed in anxiety or depression-like behaviors of the filial (F)1 generation after using environmental enrichment (14). Exercise alone only suppressed reinstatement of juvenile fear memory in the study by Short et al. (13), and isolated animals were tested only at juvenile stages after weaning (15). Benito et al. (16) reported an enhancement of synaptic plasticity after environmental enrichment, with limited results in cognition. The present study shows a significant, large effect of fathers’ pure physical activity on both the NOR memory and the spatial pattern separation of their adult offspring as compared with the offspring of sedentary fathers or the offspring of the same fathers before exercising. We have also found that offspring significantly replicated the exercise effects on the immature neuron subpopulation found in the fathers’ hippocampus. However, differences in cell proliferation were not replicated. This is not surprising, as pH3+ and 24-h survival BrdU+ cells are different subpopulations usually under compensating regulation. Our results indicate that specific subpopulations of cycling progenitors and immature, differentiating neurons in the dentate gyrus GCL are changed in sedentary brains because of the exercise program performed by their fathers. Despite this replication of cognitive advantage and effects in the immature neuron subpopulation, we did not find a similar gene expression profile in both generations. There were no matches between sDEGs from the fathers’ comparison and from the litters’ comparison. DAVID analysis of RNA-seq data showed that different annotation terms were enriched for each sDEG list, whereas GSEA analysis showed that some relevant biological processes were affected in the same direction in both generations (i.e., mitochondrial processes), but others in opposite directions (i.e., cell cycle, cell proliferation). These results indicate that different gene expression profiles are mediating the same cognitive, cellular, and molecular outcomes in fathers and their offspring. Moreover, as for the neurogenesis-related sets, an exercise-induced increase in the proliferation of neural progenitors can be the final outcome of the intervention both in fathers and offspring, even though the gene expression patterns are different, due to different compensatory mechanisms in parents and offspring. Our work provides an extensive and detailed functional analysis of the gene expression changes induced by physical exercise done in two generations: the exercised one and their offspring. Extensive descriptive analyses were previously made by other groups (30), but they were restricted to the exercised animals and not their offspring. The findings on mitochondrial proteins suggest that paternal exercise produces a specific reprogramming of hippocampal mitochondria in the offspring. In particular, we found that citrate synthase activity was enhanced, while mtDNA copy number per cell was unaffected. These data are suggestive of increased mitochondrial function and/or content in this specific area of the brain, which may have beneficial effects for the offspring. This finding is reinforced by our GSEA analysis, in which we found several gene sets related to mitochondria, enriched both in the fathers’ comparison and the litters’ comparison, including mitochondrial matrix set (where citrate synthase is located). The enhanced mitochondrial activity in the offspring might contribute to the cellular and behavioral changes observed in the present study. This is supported by recent works reporting that mitochondrial integrity is crucial for cell differentiation and dendritogenesis of newborn neurons (31) and for efficient lineage progression of adult NSCs in adult and aged hippocampus (32), as well as the finding that acute activation or deactivation of certain mitochondrial receptors is sufficient to modify memory abilities in adult mice (33). We believe that the fact that the mtDNA/nDNA ratio is not altered in the hippocampus of the offspring but that differences are found in the same samples in citrate synthase activity is remarkable and might reflect differences in mitochondrial functionality rather than differences in the number of mitochondria. Therefore, our data demonstrate that the specific brain effects of a physical activity program can be intergenerationally inherited. These transmitted effects include (i) enhancing the performance of nonspatial and spatial cognitive tasks; (ii) increasing the number of specific cell populations of adult hippocampal neurogenesis, inducing changes in hippocampal gene expression; and lastly (iii) increasing hippocampal mitochondrial citrate synthase activity. We found no exercise-induced changes in methylation of male sperm DNA, suggesting that intergenerational effects were not mediated by altered DNA methylation in spermatozoa. Our GSEA suggests a possible mechanism of epigenetic inheritance, since we found a huge number of enriched sets related to microRNA activity. This indicates that many of the genes that are microRNA targets show a tendency to be found overexpressed or underexpressed in the hippocampus of exercised fathers and their offspring compared with sedentary groups. It has been reported that paternal sperm microRNAs drive the changes in the progeny of stressed fathers (34, 35). Some of the gene sets that were enriched in our study are the target of microRNAs that have been proved as key in the epigenetic inheritance of an LTP improvement [e.g., the key role of microRNA 212/132 reported by Benito et al. (16)]. Therefore, the paternal sperm microRNAs of exercised fathers could well be originating the changes we observed here in mitochondria, neurogenesis, and behavior. We cannot discard other epigenetic marks such as histone methylation (36) or H3 retention sites (37) that may have mediated phenotype transmission. Our data suggest that the intergenerational transmission of these exercise effects is pleiotropic. Multiple mechanisms involved at different levels of the hippocampus mediate these effects. First, we found that specific gene sets were modified in exercised fathers and their sedentary offspring; second, at an organelle level, an increased mitochondrial function in the hippocampus of sedentary offspring of runner fathers was found; finally, at a tissue level, we found increased proliferation of hippocampal cells in both generations. Our gene expression analysis suggests mitochondrial and cell cycle-related genes as potential mechanisms mediating these effects in the hippocampus, whereas some of the microRNAs that were differentially regulated in the hippocampus of fathers and offspring are involved in the germline transmission of these changes (16). Further experiments would be worth carrying out to demonstrate whether transgenerational effects are also inherited (by examining the F2 generation). Most importantly, we have shown that the cognitive effects are germline dependent, because the main behavioral results were robust after IVF and embryo transfer in a patrilineal design. These findings demonstrate a patrilineal intergenerational inheritance of improved cognitive abilities in adult progeny, pointing to the physical activity levels of fathers as an unexpected, relevant factor in the brain physiology and cognitive performance of their descendants.

Materials and Methods Subjects. C57/BL6J mice (Harlan Laboratories) were housed under standard laboratory conditions, with ad libitum access to food and water, in accordance with European Union Directive 2010/63/EU. All experiments were performed according to the European Community Guidelines (Directive 2010/05/2016) and Spanish Guidelines (Real Decreto 53/2013), and have been approved by the Committee of Ethics and Animal Experimentation of the Cajal Institute (20/05/2016), Ethics Committee (Subcommittee of Ethics) of the Spanish Research Council (07/27/2016) and the Animal Protection Area of the Ministry of Environment of the Community of Madrid (10/26/2016). Male progenitors (F0). In both experiments (A and B), animals were randomly assigned to the experimental conditions. Ten subjects were used in experiment A [referred to as group sedentary (SED) A and runner (RUN) A] and 15 subjects in experiment B (referred to as group SED B and RUN B). They shared a home cage with two dams during mating periods and were housed individually immediately afterward. Animals were 3.5 mo old at the start of the preexercise behavioral assessment and 5.5 mo old at the beginning of the behavioral battery, and they were killed at 7 mo of age. All comparisons were made on subjects of the same age for each comparison. Male offspring (F1). Only male offspring were used. To prevent litter effects, each dam was considered the experimental unit, and all siblings from a given litter were considered as one sample. In experiment A, litters from sedentary fathers (L. SED A) and litters from the same fathers after exercising (L. RUN A) resulted in a sample size of eight subjects in each group. In experiment B, litters from sedentary fathers (L. SED B) resulted in a sample size of eight, while litters from different exercised males (L. RUN B) resulted in a sample size of four. After weaning, subjects were housed with their respective siblings. Animals were 3 mo old at the start of the behavioral assessment and were killed at 5.5 mo of age. All comparisons were made on subjects of the same age for each comparison. Experiment Design (Experiments A and B). A polygamous trio was selected as a breeding strategy, always housing one male with two females per cage during a whole week (different females were selected for the following trios). Dams were separated when visibly pregnant to prevent overcrowding and to keep a correct record of which pups belong to which female. A cross-fostering strategy was implemented to minimize the impact of the mother on the offspring. Siblings from a given litter remained together and were culled to generate as balanced numbers as possible within and between experimental groups. Experiment C Design. Experiment C was implemented to test the transmission of the positive effects of exercise in cognition through the germline, eliminating interactions between male and female progenitors by IVF and embryo transfer. In adulthood, litters underwent the same behavioral protocols as litters in experiments A and B. IVF and embryo transfer were conducted at the Mouse Embryo Cryopreservation Facility of the National Centre for Biotechnology, Spanish National Research Council and using described methods (38⇓–40). C57BL/6JOlaHsd females were superovulated (41). The IVF protocols used are available through the Center for Animal Resources and Development web page (card.medic.kumamoto-u.ac.jp/card/english/sigen/index.html). The process produced 10 males from sedentary fathers (referred to as L. SED C) and 13 males from runner fathers (referred to as L. RUN C). Animals were 3 mo old at the start of the behavioral assessment and were killed at 4.5 mo of age. Control Experiment Design. A control experiment was carried out to test whether continuous exposure to complex testing had an effect on the animals’ performance in easy behavioral protocols. Two groups of six adult males (sedentary and runner) exclusively underwent easy protocols of NOR and pattern separation. Exercise Protocol (All Experiments). The exercise protocol that was used was modified from Trejo et al. (42). Mice ran at 1,200 cm/min for 40 min, 5 d a week. Sedentary mice remained in the same room without running throughout the duration of the protocol. Behavioral Assessment. Activity assessment. To study the spontaneous locomotor activity in an open field arena, a VersaMax Legacy Open Field activity box (Omnitech Electronics) was used. Animals underwent a two-day protocol (5 min in the activity cage per day). NOR protocols. To assess memory enhancement, difficult and easy protocols were designed by modifying [from the original description (20) and recent modifications of the test (21, 43)] the time spent during the training phase (SI Appendix, Figs. S11 A and B and S12). Pattern separation. A modified version of the pattern separation test was used to study pattern separation performance enhancement. To do this, animals underwent two different protocols (SI Appendix, Fig. S11 C and D), referred to as low separation and high separation. BrdU Injections. All male experimental animals in experiments A and B received one i.p. injection of BrdU (50 mg/kg body weight; Sigma-Aldrich) 24 h before being killed. Tissue Collection. All male experimental animals were deeply anesthetized with pentobarbital (Euta-Lender). Each animal was transcardially perfused with 0.9% saline. The right hemisphere was used to store frozen tissue. The left hemisphere was fixed by immersion in 4% paraformaldehyde for histology. Histology. Coronal sections (50-μm width) were obtained on a Leica VT1000S vibratome. One random series was chosen for each immunohistochemistry as described previously (44). Slices were incubated for single or double staining (SI Appendix, Table S3). The Cavalieri method was used as described previously (45). Stereology. BrdU- and pH3-labeled cells were counted by the optical fractionator method. The physical-dissector method, adapted to confocal microscopy as previously described (46), was used to estimate the total number of SOX2+/GFAP+ cells, DCX+, and CLR+ cells. GFAP expression was analyzed in the dentate gyrus. RNA-seq. Total RNA extraction from hippocampal tissue. The QuickGene RNA Tissue Kit SII (RT-S2) and the QG-Mini80 (Kurabo) was used to extract total RNA from hippocampal tissue of a random selection of fathers (n = 10) and of eight animals representing each litter per group (n = 16) in experiment B. The final number of useful samples for analysis was n = 9 fathers (sedentary fathers, n = 5; exercised fathers, n = 4) and n = 15 offspring (animals from sedentary fathers, n = 8; animals from exercised fathers, n = 7). Stranded mRNA library preparation and sequencing. RNA-seq libraries were made with the TruSeq Stranded mRNA LT Sample Prep Kit (cat. no. 15031047 Rev. E, October 2013; Illumina). The libraries were sequenced on HiSeq2000 (Illumina) using TruSeq SBS Kit v4. Image analysis, base calling, and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.18.66.3) and followed by generation of FASTQ sequence files by CASAVA. RNA-seq data processing and analysis. RNA-seq reads were mapped with STAR version 2.5.2a (ENCODE parameters for long RNA), and genes were quantified with RSEM version 1.2.28 (with default parameters). Normalization and differential expression were performed with DESeq2 version 1.10. We considered significant genes with a false discovery rate (FDR) of <5%. Bioinformatic analysis of RNA-seq results of hippocampal tissue. Only animals of experiment B were used in this analysis. DAVID v6.8 was used for the functional description of the sDEGs of each comparison (exercised fathers vs. sedentary fathers and litters from exercised fathers vs. litters from sedentary ones). sDEGs have adjusted P values associated with their log2FoldChange of <0.05. Four databases were chosen for the extraction of terms: GOTERM_BP_DIRECT, GOTERM_CC_DIRECT, KEGG_PATHWAY, and GOTERM_MF_DIRECT, and an EASE Score of 0.05 was set as a threshold. GSEA of RNA-seq data was performed with GSEA (Broad Institute, v3.0). A preranked analysis was performed using log2FoldChange as a ranking metric. Only gene sets with an FDR of <25% were considered for descriptive analysis following the guidelines set by the Broad Institute (https://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html). Mitochondrial Assessment in Liver, Cerebellum, and Hippocampus. mtDNA/nDNA ratio analysis. Total DNA was extracted with the DNeasy Blood and Tissue Kit (QIAGEN). mtDNA was amplified using primers specific for the mitochondrial NADH dehydrogenase (ND1) gene. Primer sequences can be found in SI Appendix, Table S5. The RT-PCR was performed on individual DNAs by using iTAQ universal SYBR Green (Bio-Rad Laboratories). The relative DNA content was calculated by the 2−ΔΔCT method. Citrate synthase activity. Citrate synthase activity was determined in ∼50 µg of protein lysates following the method described by Spinazzi et al. (29). Citrate synthase was determined by spectrophotometric methods. Statistical Analysis. Depending on the type of comparison and the parameter analyzed, we used either the t test, the Mann–Whitney U test, the paired-sample t test, the Wilcoxon signed-ranked test, a repeated-measures ANOVA, a mixed ANOVA, a Friedman test followed by a post hoc Wilcoxon signed-ranked test, or the Chi-square test (detailed description of the statistical analysis can be found in the SI Appendix, Supplementary Materials and Methods). All data were analyzed using SPSS Statistics (IBM, v.24.0.0). For the dependent variables measured on a continuous scale, data are shown as mean ± SEM. To test normality, the Shapiro–Wilk test was applied. For each test, extreme values were removed from the analysis. For comparisons between independent groups (intergroup differences), *P < 0.05, **P < 0.01, ***P < 0.001; trends 0.05 ≥ #P < 0.09. For comparisons between dependent groups (intragroup differences), +P < 0.05, ++P < 0.01, +++P < 0.001; trends 0.05 ≥ #′P < 0.09. All graphs were created in GraphPad Prism 5. Effect size estimates are described as g (Hedges’ g), r2, and partial eta-squared (η p 2).

Acknowledgments We thank Cesar Cobaleda [Centre of Molecular Biology Severo Ochoa (CBMSO), Spanish National Research Council/Autonomous University of Madrid (CSIC/UAM), Madrid, Spain] and Alberto González-de la Vega (MegaLab, Madrid, Spain) for expert assistance and advice of the RNA-seq, DAVID, and GSEA analysis; María Llorens-Martín (CBMSO, CSIC/UAM, Madrid, Spain) for useful discussions; Silvia Fernández (Cellular and Molecular Biology Unit, Cajal Institute, Madrid, Spain) and Laude Garmendia (Animal House, Cajal Institute, Madrid, Spain) for volunteer help and advice; the Image Analysis Unit of the Cajal Institute; Carmen Sandi (Brain Mind Institute, Lausanne, Switzerland) for helpful and useful advice and assistance; and all members of the National Centre for Biotechnology Mouse Embryo Cryopreservation Facility—María Jesús del Hierro, Marta Castrillo, and Lluís Montoliu—for their huge efforts and impressive involvement in the IVF experiments. This work was supported by the Spanish Ministry of Economy and Competitiveness Project Grants BFU2013-48907-R and BFU2016-77162-R (to J.L.T.), SAF2016-78845-R (to S.R.F.), RYC-2012-10193 and AGL2014-85739-R (to P.B.Á.), CP14/00105 and PI15/00134 (to A.M.-M.); by the Instituto de Salud Carlos III of the Spanish Ministry of Economy and Competitiveness; and by the European Regional Development Fund Grant PT17/0009/0019 (to A.E.-C). Á.F.-L. was funded by a CSIC JAE-Doc Programme grant and VPlan Propio US-Acceso Grant, I.L.-T. was funded by a predoctoral fellowship (FPI) grant, and K.R.M. was funded by a contract associated with the above-mentioned project grants awarded to J.L.T.

Footnotes Author contributions: Á.F.-L. and J.L.T. designed research; K.R.M., P.T., I.F.-V., A.P., M.M.-S., A.E.-C., I.L.-T., P.B.-Á., J.F.-P., A.M.-M., R.M., S.R.F., E.J.R., Á.F.-L., and J.L.T. performed research; K.R.M., P.T., I.F.-V., A.P., M.M.-S., A.E.-C., I.L.-T., P.B.-Á., J.F.-P., A.M.-M., R.M., S.R.F., E.J.R., Á.F.-L., and J.L.T. analyzed data; and K.R.M., P.T., A.P., P.B.-Á., J.F.-P., A.M.-M., S.R.F., E.J.R., Á.F.-L., and J.L.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://ncbi.nlm.nih.gov/geo (accession no. GSE123582).

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