155 Elevated temperature on the phenotypic plasticity of female mice across five generations

Elevated temperatures disturb oocyte physiology at both cellular and molecular levels. Recently, we developed an experimental model in which mice undergo exposure to high temperatures during the first wave of oocyte growth. This environmental stress led to widespread alterations in DNA methylation patterns of fully grown oocytes, despite little effect on oocyte developmental competence. Here, we explored whether this environmental challenge leads to reproductive plasticity without impacting oocyte developmental competence and whether it persists for multiple generations. Parental females (Swiss strain; n = 23) with litters underwent random allocation to heat stress (HS): exposure to 35°C for 12 h light/21°C for 12 h dark cycle, or control ([CTL]/21°C for 24 h) at postnatal Day 10 (P10) for eleven days. HS and CTL female offspring (F0 generation) were kept under CTL conditions until puberty at P35 for in vivo embryo production or mated to age-matched CTL males to generate F1 progeny (one female pup per litter). After weaning, randomly chosen F1 generation females were mated to CTL males for generating F2, F3, and F4 generations, respectively (F0 was the sole generation exposed to HS). In vivo embryo production for selected generations (F0: n = 26, F1: n = 87, F2: n = 73, and F4: n = 46 female mice) relied on 5 IU PMSG and 5 IU human chorionic gonadotrophin (hCG) injections 44–48 h apart followed by embryo collection in M2 medium at 94 h post-hCG. Viable embryos were at the morula and blastocyst stages, while non-viable embryos were arrested in development, degenerated, or fragmented. Considering that the data did not attend to the assumptions of ANOVA, nonparametric analysis was conducted using the Wilcoxon test (SAS software). The statistical significance level was 0.05. The coefficient of variation (CV) was the ratio of the standard deviation to the mean. Survival of lactating females and offspring was similar between HS and CTL across generations. Litter size was indistinguishable between HS and CTL (P = 0.63) and remained unaffected until F4 generation. In vivo embryo production was not affected by HS (P = 0.35) and remained similar among generations. Neither the percentage of viable embryos nor their non-viable counterparts differed between groups (P = 0.77 and P = 0.98, respectively). To address whether there was increased variability despite no visible phenotype, we calculated dispersion measures of these results. The CV for litter size varied in the CTL from 26.4% on the F3 to 55.4% on F4. In turn, the CV for litter size in the HS varied from 23.2% on the F2 to 60.6% on F4. The CV for in vivo embryo production varied from 67.7% on F2 to 96.3% on F0 in the CTL. HS ranged from 52.3% on F4 to 97.4% on F0. Hence, our HS model leads to widespread DNA methylation perturbation but does not cause phenotypic plasticity of reproductive traits across generations.

This work was supported by the São Paulo Research Foundation (FAPESP; grant 2017/20125-3 F.F.P.-L.) and Coordination for the Improvement of Higher Level Education—Personnel (CAPES)—Finance Code 001 (M.T.M.).