Effects of maternal androgens and their metabolite etiocholanolone on prenatal development in birds

Offspring phenotypes can be affected by maternal testosterone and androstenedione (A4), which are considered a tool of mothers to adjust offspring to a fluctuating environment. Yet testosterone and A4 are very rapidly metabolized by developing avian embryos, suggesting that either the maternal testosterone and A4 have potent organizational effects on the embryos extremely early before being metabolized or it is the metabolites that evoke phenotypic variation in the offspring. One of the metabolites, etiocholanolone, increases substantially during early embryonic development and is a likely candidate for mediating maternal effects as it can promote erythropoiesis. To investigate and compare the effects of testosterone and A4 with the possible effects of etiocholanolone during prenatal embryonic development, we increased their levels in black-headed gull eggs (Larus ridibundus), and used sham-injected eggs as controls. This species usually has 3-egg clutches in which maternal androgen levels increase with the egg-laying sequence. We analysed embryonic heart rate, peri-hatching biometric traits, the ratio of white to red blood cells (W/R ratio) and bursa development. We found that testosterone and A4 treatment increased embryonic heart rate irrespective of egg-laying sequence and decreased bill length and W/R ratio, whereas etiocholanolone did not mimic these effects. Instead, etiocholanolone treatment decreased tarsus length and brain mass. Our finding that etiocholanolone does not mimic the effects induced by testosterone and A4 suggests that the embryonic metabolism of maternal testosterone and A4 can potentially diversify the function of these maternal androgens.