Study question Does maternal circadian rhythm change during pregnancy and lactation cause transgenerational epigenetic changes on DNA methylation in the gonads of adult male offspring? Summary answer The expression of DNA methyltransferases (DNMTs) is altered in the testis of offspring rats due to maternal circadian rhythm changes during pregnancy and lactation periods. What is known already Epigenetic mechanisms are essential for normal development and maintenance of tissue specific expression in mammals. Key mechanisms involved in epigenetic gene regulation include specifically DNA methylation and histone modifications. DNA methylation is an important regulatory mechanism that plays a role in many cellular processes and it is catalyzed by specific DNMT enzymes. Regulation of the circadian clock is also subject to epigenetic influence. Current evidence increasingly indicates that environmental conditions during pregnancy may have lifelong effects on offspring in both humans and animal models. Study design, size, duration Male offspring from pregnant Wistar rats (n = 60) which were exposed to control (12 h light/12 h dark), short day (SD) (8 hours light/16 hours dark) or long day (LD) periods (16 hours light/8 hours dark) during only pregnancy, only lactation or both periods (n = 15/group) categorized as control (C), SD pregnancy (SD-P), SD lactation (SD-L), SD pregnancy and lactation (SD-P+L), LD pregnancy (LD-P), LD lactation (LD-L), LD pregnancy and lactation (LD-P+L) groups. Participants/materials, setting, methods Maternal body weights were recorded during pregnancy and lactation periods. The global methylation pattern was evaluated with 5-methylcytosine (5-mC) immunostaining in line with histopathological analyzes in the testis tissues. In addition, the expression of three DNMTs, DNMT1, DNMT3A and DNMT3B, were evaluated to analyze the maintenance of DNA methylation and de novo establishment of DNA methylation patterns, respectively. Main results and the role of chance DNMT1 immunolocalization showed a nuclear staining pattern, predominantly in spermatogonia, spermatocytes, round spermatids and Sertoli cells in all experimental groups. A significant increase in DNMT1 expression was determined in SD-P, SD-L, SD-P+L, LD-P and LD-L groups compared to the control group (p < 0.001). On the other hand, DNMT3A was especially expressed in spermatogonia, spermatocytes, and Leydig cells in the interstitial areas. Spermatogonia, round spermatids and the head of elongated spermatids and also in Leydig cells in the interstitial areas were immunopositively stained with DNMT3B and 5-mC. 5-mC expression was also present in spermatocytes. There was a significant increase in DNMT3A, DNMT3B and 5-mC expressions in SD-P, SD-L and SD-P+L groups compared to the control group (p < 0.001). DNMT3B and 5-mC expressions were statistically higher in LD-P than control group (p < 0.001). Limitations, reasons for caution Although using a rat model has several advantages, translating these results to the human setting is a limitation of this study. Wider implications of the findings The recent studies regarding circadian regulation in reproductive tissues suggests that it plays important role in several aspects of fertility. Our study indicates that expression of DNMT proteins alters in testis due to maternal circadian rhythm changes suggesting potential influences on transgenerational reproductive health of offspring. Trial registration number This work was supported by research grants from the Scientific and Technological Research Council of Turkey (TUBITAK) (SBAG 219S636) and Akdeniz University, The Scientific Research Projects Coordination Unit (TYL-2018-3580)

Study question Do maternal circadian rhythm changes during pregnancy and lactation periods cause transgenerational behavioral and cognitive alterations in male offspring? Summary answer Maternal circadian rhythm changes cause impaired behavioral alterations such as motor activity impairments, increased anxiety and weakening of spatial memory in male offspring. What is known already The circadian system regulates the daily temporal organization in behavior and physiology in most species. Recent publications in model systems have emphasized the negative effects of circadian rhythm disruption on both female and male reproduction and fertility. There is growing concern about the long-term effects of circadian rhythm disruptions during pregnancy and lactation periods on human offspring and their descendants as circadian regulation during these periods can potentially alter epigenetic programing in offspring. However, there is a lack of information about the possible transgenerational effects of circadian rhythm disorders for the health of offspring in the literature. Study design, size, duration Male offspring from pregnant Wistar rats (n = 60) which were exposed to control (12 hours light/12 hours dark), short day (SD) (8 hours light/16 hours dark) or long day (LD) periods (16 hours light/8 hours dark) during only pregnancy, only lactation or both periods (n = 15/group) categorized as control, SD pregnancy (SD-P), SD lactation (SD-L), SD pregnancy and lactation (SD-P+L), LD pregnancy (LD-P), LD lactation (LD-L), LD pregnancy and lactation (LD-P+L) groups. Participants/materials, setting, methods The length of gestation, litter size, sex ratio of pups (fraction male) and birth weights were recorded. The maternal locomotor activity and anxiety were analyzed by open field (OF) and elevated plus maze (EPM) tests, respectively (n = 15 mothers/group). Learning and memory, locomotor activity and anxiety related changes were studied in the male offspring in pre-pubertal, pubertal and adult periods with object localization (OL) and Morris water maze (MWM), OF and EPM tests, respectively. Main results and the role of chance Newborn weights was significantly increased in SD (5.01±0.07) and LD (4.47±0.29) groups compared to control group (3.73±0.03) (p < 0.001). Male sex ratio of pups born to mothers on SD schedule was unusually low (0.21±0.62) compared to mothers on normal light schedule (0.48±0.05) (p < 0.001). The gestation length was not altered. Locomotor activity was increased in mothers exposed to SD schedule (p < 0.01). A significant motor activity impairment in the prepubertal period was remarkable in SD-P+L, LD-P, LD-L and LD-P+L groups (p < 0.001). Compared to control group, EPM test showed that all the offspring in SD groups had significant increase in their anxiety in the prepubertal period. Motor functions that decreased before puberty seems gradually improved at adulthood. Weakened short-term spatial memory was observed at puberty, especially in SD-P (p < 0.001), SD-P+L (p < 0.05), LD-P (p < 0.05) groups. Time to find the platform was significantly higher than those measured for control group for SD-P-L (p < 0.001) in the prepubertal period and LD-P+L group (p < 0.001) in the pubertal period meaning decreased spatial memory and learning. A probe trial given on day 6 with MWM test showed that LD groups had no alterations in spatial memory whereas SD-P (p < 0.05) and SD-G+L (p < 0.001) groups showed impaired changes in long-term spatial memory. Limitations, reasons for caution Due to limitations of working with human subjects exposed to shift work schedules, most chronoregulation research including ours has used rodent models with limitations in translating to human studies. However, to truly know if such concerns apply to humans will require retrospective and prospective human studies. Wider implications of the findings Epigenetic transitions are crucial for transcriptional reprogramming triggered by environmental cues. Our study might provide valuable information regarding transgenerational effects of maternal chronodisruption on health later in life of the offspring, particularly at prepubertal and pubertal periods, create suggestions and guidelines for reproductive biology, shift work and possible infertility problems. Trial registration number This work was supported by research grants from the Scientific and Technological Research Council of Turkey (TUBITAK) (SBAG 219S636) and Akdeniz University, The Scientific Research Projects Coordination Unit (TYL-2018-3580).