Litter and sex effects on maternal behavior and DNA methylation of the Nr3c1 exon 17 promoter gene in hippocampus and cerebellum

• Sex of pup and altering the sex composition of the litter affects frequency of pup licking and mRNA and DNA methylation levels of the glucocorticoid gene in adolescent rat brains.
• Male rat pups raised in mixed-sex litters are licked more frequently by their dams than male pups raised in single-sex litters.
• Mixed-sex male adolescent rats have the lowest DNA methylation levels of the Nr3c1 exon 17 promoter gene in cerebellum.
• Mixed-sex female adolescent rats have the lowest mRNA levels of the Nr3c1 gene in hippocampus.

Early life events can alter gene expression through DNA methylation. The methylation status of the exon 17 promoter of the glucocorticoid receptor (Nr3c1 gene) in hippocampus associates with frequency of pup licking. Much of this work was conducted with male rats. Because dams more frequently lick male pups, this may contribute to sex differences in phenotypes through DNA methylation. Modifying litter gender composition (LGC), in which offspring of single-sex litters are compared to mixed-sex litters, alters maternal behavior. Previously, we demonstrated that LGC and sex affected pup licking times as well as anxiety and hippocampal DNA methylation of the Nr3c1 exon 17 promoter gene in adolescence. Now, we expand upon this work by examining effects in cerebellum and measuring mRNA levels. We also re-assessed DNA methylation in hippocampus using pyrosequencing and re-analyzed pup licking with the more commonly used frequency measure. Litters, culled to 8 pups on postnatal day 1 (PN1), were assigned to one of three conditions: all male (n = 10), all female (n = 12), or half of each sex (n = 20). Licking was rated on PN4, 7, and 10. On PN35, hippocampal and cerebellar samples were obtained. Single-sex males were licked the least and mixed-sex males, the most. Hippocampal Nr3c1 mRNA levels were lowest in mixed females with no LGC or Sex effects in DNA methylation. Cerebellar DNA methylation levels were lowest in mixed males with no effect on mRNA levels. Maternal pup licking associated with DNA methylation of the Nr3c1 exon 17 promoter gene in cerebellum and with hippocampal mRNA.