Deficiency of the paternally-expressed imprinted Peg3 gene in mice has sexually dimorphic consequences for offspring communication and social behaviour

Abstract

Introduction: Imprinted genes are expressed from one parental allele as a consequence of epigenetic processes initiated in the germline. Consequently, their ability to influence phenotype depends on their parent-of-origin. Recent research suggests that the sex of the individual expressing the imprinted gene is also important. We have previously reported that genetically wildtype (WT) dams carrying and caring for pups mutant for PEG3 exhibit anxiety-like behaviours and their mutant pups show a reduction in ultrasonic vocalisation when separated from their mothers. Sex-specificity was not examined.

Methods: WT female mice were mated with WT, heterozygous Peg3^-/+ or homozygous Peg3^-/- studs to generate all WT (control), 50:50 mixed or 100% mutant litters, respectively, followed by behavioural assessment of both dams and their pups.

Results: We reproduced our original finding that WT dams carrying and caring for 100% mutant litters exhibit postpartum anxiety-like symptoms and delayed pup retrieval. Additionally, these WT dams were found to allocate less time to pup-directed care behaviours relative to controls. Male Peg3-deficient pups demonstrated significantly reduced vocalisation with a more subtle communication deficit in females. Postweaning, male mutants exhibited deficits across a number of key social behaviours as did WT males sharing their environment with mutants. Only modest variations in social behaviour were detected in experimental females.

Discussion: We have experimentally demonstrated that Peg3 deficiency confined to the offspring causes anxiety in mouse mothers and atypical behaviour including deficits in communication in their male offspring. A male-specific reduction in expression PEG3 in the fetally-derived placenta has previously been associated with maternal depression in human pregnancy. Maternal mood disorders such as depression and anxiety are associated with delays in language development and neuroatypical behaviour more common in sons. Peg3 deficiency could drive the association of maternal and offspring behavioural disorders reported in humans.

Keywords: Peg3; epigenetics; imprinted genes; sexual dimorphism; social behaviour; ultrasonic vocalisation.

Figure 1

Schematic of the breeding design used to generate experimental dams and offspring (A) Control group = 100% wild-type litter. Wild-type (WT) females mated with WT male to produce litters composed entirely of WT pups WT^{(WT Litter)}. (B) Mixed genotype litter: WT females mated with heterozygous Peg3^−/+ male to produce litters composing ~50% Peg3^+/+ [WT^{(Mixed Litter)}] pups and ~50% heterozygous mutant Peg3^+/− [Peg3KO^{(Mixed Litter)} pups]. (C) 100% mutant litter: WT females mated with homozygous Peg3^−/− male to produce litters composed entirely of mutant pups [Peg3KO^{(Mutant Litter)}]. Data used from litter sizes > 4 and, for mixed genotype litters, where the proportion of transgenic pups within the litter was between 45% and 60%.

Figure 2

Maternal anxiety-like behaviour. Analysed using ANCOVA controlling for litter size as a covariate. (A) Elevated zero maze, 4 days post-partum. Dam^{(Mutant Litter)} demonstrated significantly more stretch attend postures than Dam^{(WT Litter)} (p = 0.01). No further significant differences were detected. (B) Light Dark Box, 7 days post-partum. Dam^{(Mutant Litter)} demonstrated significantly more stretch attend postures than Dam^{(WT Litter)} (p = 0.01). Differences were observed for both time spent in, and time taken to enter the anxiogenic section of the LDB. Dam^{(Mixed Litter)} spent significantly less time in the light than Dam^{(WT Litter)} (p = 0.04) and Dam^{(Mutant Litter)} were slower to enter the anxiogenic section than Dam^{(WT Litter)} (p = 0.01). No further significant differences were detected. (C) Unified Anxiety Score. When all anxiety-like outcome measures were combined to generate a unified anxiety score, Dam^{(Mutant Litter)} demonstrated greater anxious-like behaviour than Dam^{(WT Litter)} (p = 0.02). Number of dams: Dam^{(WT Litter)} = 13, Dam^{(Mixed Litter)} = 12, and Dam^{(Mutant Litter)} = 12. Data are mean ±SEM. *p ≤ 05, **p ≤ 0.01.

Figure 3

Pup retrieval at P3. Analysed using ANCOVA controlling for litter size as a covariate. Average time taken to retrieve each pup by genotype and sex. A significant main effect of genotype was detected, with Peg3KO^{(Mutant Litter)} being retrieved slower than WT^{(WT Litter)} at P3. Number of offspring: WT^{(WT Litter)} M = 16, F = 20; WT^{(Mixed Litter)} M = 16, F = 15; Peg3KO^{(Mixed Litter)} M = 13, F = 13, and Peg3KO^{(Mutant Litter)} M = 14, F = 14. Data are mean ±SEM. **p ≤ 0.01.

Figure 4

Maternal behaviour during the pup retrieval task at P3. Analysed using ANCOVA controlling for litter size as a covariate. (A) Percentage time engaged in pup-directed behaviour. Dam^{(Mutant Litter)} spent a significantly smaller percentage of time engaged in pup-directed behaviour than both Dam^{(WT Litter)} and Dam^{(Mixed Litter)}, (B) Average number of failed retrieval attempts. (C) Number and latency to call for dam. (D) Combined number of calls from 4 pups. Number of dams: Dam^{(WT Litter)} = 13, Dam^{(Mixed Litter)} = 12 and Dam^{(Mutant Litter)} = 12. Data are mean ±SEM. **p ≤ 0.01, ***p < 0.001.

Figure 5

Neonatal isolation induced USVs of individual male and female pups aged P2 to P10. Analysed using ANCOVA controlling for body temperature as a covariate. (A) Number of calls at each age for male pups. (B) Number of calls at each age for female pups. Males: WT^{(WT Litter)} = 41, WT^{(Mixed Litter)} = 18, Peg3KO^{(Mixed Litter)} = 20, Peg3KO^{(Mutant Litter)} = 25; Females: WT^{(WT Litter)} = 39, WT^{(Mixed Litter)} = 17, Peg3KO^{(Mixed Litter)} = 20, Peg3KO^{(Mutant Litter)} = 28. Data are mean ±SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Direct social interaction test. Age = P28. Data is presented from the direct social interaction task for males (A,B) and females (C,D). Panels (A,C) show the average time spent engaged in social behaviour. A one-way ANOVA showed that mutant males from both single and mixed genotype litters spent significantly less time engaged in social behaviour than WT males from a single genotype litter. (B,D) Average time engaged in non-social behaviour. Mutant males from mixed genotype litters spent less time engaged in non-social behaviour than WT males from single genotype litters. No difference were observed between females. Number of offspring: WT^{(WT Litter)} M = 16, F = 20; WT^{(Mixed Litter)} M = 16, F = 15; Peg3KO^{(Mixed Litter)} M = 13, F = 13, and Peg3KO^{(Mutant Litter)} M = 14, F = 14. Data are mean +SEM. ^*p < 0.05, ^***p < 0.001.

Figure 7

Social propinquity test. Age = P35. Data is presented from the Social Propinquity test for males (A–C) and females (D–F) analysed using one-way ANOVAS. Panels (A,D) show the latency to share the tube. No significant effect of group was detected for males or females. Panels (B,E) show the percentage time mice spent sharing the tube. (B) Peg3KO^{(Mixed Litter)} male mice spent a significantly greater percentage of time sharing the tube than WT^{(WT Litter)}. Panels (C,F) show percentage of time the tube was vacant. (C) Both WT^{(Mixed Litter)} and Peg3KO^{(Mixed Litter)} littermate males spent a greater percentage of time in the aversive arena than males from single genotype litters. (F) Female Peg3KO^{(Mixed Litter)} spent a greater percentage of time in the aversive arena than WT^{(WT Litter)}, Number of pairs tested: WT^{(WT Litter)} M = 7, F = 10; WT^{(Mixed Litter)} M = 8, F = 8; Peg3KO^{(Mixed Litter)} M = 7, F = 7, and Peg3KO^{(Mutant Litter)} M = 7, F = 7. Data are mean ±SEM. ^*p < 0.05.

Figure 8

Three-chamber test and scent marking test. Age = P63. Data is presented for males (A–C,G) and females (D–F). All data were analysed using one-way ANOVAs. Panels (A,D) show the latency to enter the occupied chamber. Panels (B,E) show the total duration spent in the occupied chamber. Panels (C,F) show the number of entries made into the occupied chamber. WT^{(mixed litter)} males made a greater number of crosses into the occupied chamber than both Peg3KO^{(Mixed Litter)} (p = 0.006) and Peg3KO^{(Mutant Litter)} (p = 0.03) litter. Panel (G) shows the total number of 1 cm² squares containing scent markings made during the three-chamber trial (males only). All experimental groups scent marked less that WT^{(WT Litter)} males. Number of offspring: WT^{(WT Litter)} M = 16, F = 20; WT^{(Mixed Litter)} M = 16, F = 15; Peg3KO^{(Mixed Litter)} M = 13, F = 13, and Peg3KO^{(Mutant Litter)} M = 14, F = 14. Data are mean ±SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 9

Courtship USVs and behaviours. Age = P70. Male mice during interaction with a female WT mouse in oestrous—initial phase data (above) and reunion phase data (below). (A) Latency to call. (B) Total number of calls. (C) Mean duration of call. (D) Time spent engaged in social behaviour. (E) Time spent engaged in self-directed behaviour. No effect of genotype was detected for any of the USV parameters in either phase. Number of offspring: WT^{(WT Litter)} M = 16; WT^{(Mixed Litter)} M = 16; Peg3KO^{(Mixed Litter)} M = 13, and Peg3KO^{(Mutant Litter)} M = 14. Data are mean ±SEM.