P-114 Transgenerational effects of maternal circadian rhythm changes on DNA methylation pattern in the testis of offspring rats

Abstract

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)