Effects Of Paternal Aerobic Treadmill Exercise On The Growth And Development Among Their Male Offsprings In C57BL/6Mice

Objective:Increasing evidence has shown that subtle factors in parents can influence long-term health in offspring. Although the impacts of exercise-induced maternal development on fetal size and metabolism in offspring are well established, the extent of any contribution of paternal physical activity is unclear, particularly the role of non-genetic factors in the causal pathway. In the present study, our goals were to determine the effects of paternal aerobic exercise on offspring growth and to investigate the mechanisms associated with such effects. Male C57BL/6mice were separated into sedentary or exercise cohorts with the exercise cohort having6weeks of exercise on a motor-driven rodent treadmill prior to mating. Offspring were weaned and analyses were performed on the pubescent offspring that did not have access to running treadmill during any portion of their lives. Male offspring from exercised dams showed increased body weight/quadriceps muscle mass percentage and decreased visceral fat mass compared to male offspring from sedentary mice. Paternal aerobic exercise significantly increased serum insulin-like growth factor1(IGF-1) and IGF-1mRNA expression levels in the liver of male offspring. The results of methylated DNA immunoprecipitation real-time PCR (MeDIP-qPCR) assays indicated that paternal aerobic exercise down-regulated DNA methylation of IGF-1in the liver of male offspring. These results suggested that paternal aerobic exercise may inhibit adipose growth and promote skeletal muscle growth through a mechanism that is associated with increased IGF-1.Methods:(1) Animal model:Sibling males were divided into two groups: the control (C) and the exercise group (E). The mice in the running group were exercised on a motor-driven rodent treadmill for5days a week for a total of6weeks. After6weeks of exercise, one male and one female were mated. One to two days later, male mice were removed, and pregnant females were left alone. After weaning, male pups underwent behavioral assessment.(2) Training protocol: Mice were randomized to C and E group, E group were given exercise training on motorized treadmill at12m/min (75%VO2max), training took place5days per week,60min per day, maintained for6weeks.(3) Morphological characteristics: the offspring of each group were weighed and measured before sacrificed under anesthesia. Livers, quadriceps muscle, visceral fat from the epididymal and perirenal fat depots of the male mice were quickly harvested, weights of the organs were also recorded.(4) Enzyme immunoassay: Serum IGF-1levels of the offspring were measured by using mice enzyme immunoassay (ELISA) kit following the manufacturer’s protocol.(5) Real-time PCR was used to detect IGF-1mRNA level in liver.(6) Western blot was used to detect IGF-1and IGF-1R protein level in quadriceps muscle and visceral fat.(7) MeDIP-qPCR was used to examine the changes in DNA methylation at the IGF-1promoters.Results:(1) Morphological characteristics:There was no difference in the body length (BL) of male offspring from control and exercise groups. Body weight (BW) and quadriceps muscle wet weight (QW) increased significantly in the male offspring born to exercised mice compared with offspring from sedentary mice (P<0.05). In addition, QW normalized to BW (QW/BW ratio) was enhanced in male offspring born to exercised mice (P＜.05). Finally, male offspring born to exercised mice had significantly reduced visceral fat compare with those born to sedentary mice.(2) IGF-1mRNA and serum levels:Paternal exercise significantly increased serum IGF-1levels in male mice vs. controls (P<0.01). Moreover, results from Real-time PCR also revealed that exercise male offspring showed significantly higher levels of IGF-1mRNA contents with411.81%increase relative to the control male offspring (P＜0.05).(3) IGF-1promoter1and promoter2DNA methylation:Promoter1(P1) and promoter2(P2) of IGF-1were analyzed for methylation status. In P1, the status of methylation decreased38.78%in male offspring from paternal exercise mice compared to control mice (P＜0.05). In P2, the IGF-1was significantly hypomethylated in male offspring from paternal exercise mice relative to control mice (P<0.01).(4) IGF-1and IGF-1R protein expression in quadriceps femoris muscle and visceral fat: when the protein levels in visceral fat were analyzed, we observed a significant increase in IGF-1(127.3%) and IGF-1R (84.3%) in male offspring with paternal exercise when compared to control male offspring. Paternal exercise increased quadriceps femoris muscle IGF-1and IGF-1R protein levels in male offspring with paternal exercise by84.26%and42.37%compared to control male offspring.Conclusions:The present study presents evidence that the paternal regular aerobic exercise can effect the grown and development on the male offspring, and may also extend the concept of grown and development to include a paternal role on long-term offspring health. Paternal exercise was associated with elevated offspring circulatory IGF-1levels and promotion of IGF-1expression levels in the liver, possibly via DNA methylation modifications and increase of IGF-1in growth plates. These changes in circulatory IGF-1levels may inhibit adipose growth and promote skeletal muscle growth. The underlying mechanisms seem to include epigenetic modifications, but further studies are needed to elucidate the underlying mechanisms by how IGF-1mediated the paternal exercise-induced alternation about the growth in the male offspring.