Avian offspring prenatal DNA methylation response to maternal corticosterone dosing during reproduction

Avian embryos develop in an egg composition which reflects both maternal condition and the recent environment of their mother. In birds, yolk corticosterone (CORT) influences development by impacting pre- and postnatal growth, as well as nestling stress responses and development. One possible mechanism through which maternal CORT may affect offspring development is via changes to offspring DNA methylation. We sought to investigate this, for the first time in birds, by quantifying the impact of manipulations to maternal CORT on offspring DNA methylation. We non-invasively manipulated plasma CORT concentrations of egg-laying female zebra finches (Taeniopygia castanotis) with an acute dose of CORT administered around the time of ovulation and collected their eggs. We then assessed DNA methylation in the resulting embryonic tissue and in their associated vitelline membrane blood vessels, during early development (5 days after lay), using two established methods - liquid chromatography–mass spectrometry (LC-MS) and methylation-sensitive amplification fragment length polymorphism (MS-AFLP). LC-MS analysis showed that global DNA methylation was lower in embryos from CORT-treated mothers, compared to control embryos. In contrast, blood vessel DNA from eggs from CORT-treated mothers showed global methylation increases, compared to control samples. There was a higher proportion of global DNA methylation in the embryonic DNA of second clutches, compared to first clutches. Locus-specific analyses using MS-AFLP did not reveal a treatment effect. Our results indicate that an acute elevation of maternal CORT around ovulation impacts DNA methylation patterns in their offspring. This could provide a mechanistic understanding of how a mother’s experience can affect her offspring’s phenotype.

Methods
Australian Zebra finches (Taeniopygia castanotis) were bred in a laboratory setting. Treatment group females were orally dosed with corticosterone throughout breeding and reproduction (control females instead orally dosed with the oil vehicle, alone). Females were bred twice (with the same mate), once under treatment, once under control (half treatment first, half control first). Eggs were collected on the day of lay and artificially incubated for 5 days of development, then collected and immediately frozen. DNA from the egg was purified from both embryos and vitelline membrane blood vessels. The extracted DNA was hydrolysed and processed via mass spectrometry (LC-MS). Separately, the extracted DNA from the egg (vitelline) blood vessels was processed via enzymatic DNA profiling (MS-AFLP) and sent to AGRF for analysis (single base resolution of DNA fragments). The data was analysed with R (via RStudio) using packages nlme (LC-MS data) and msap (MS-AFLP data).