Early embryonic modification of maternal hormones differs systematically among embryos of different laying order: A study in birds

Highlights

• Maternal steroids are substantially and rapidly metabolized only in fertilized eggs.

• The rate of metabolism differs systematically with egg laying order.

• The resulting metabolites are uncharacterized in the context of maternal effects.

• Effects of maternal steroids may take place either very early in development.

• Or via unconjugated metabolites directly or after deconjugation by the embryo.

Abstract

Vertebrate embryos are exposed to maternal hormones that can profoundly affect their later phenotype. Although it is known that the embryo can metabolize these maternal hormones, the metabolic outcomes, their quantitative dynamics and timing are poorly understood. Moreover, it is unknown whether embryos can adjust their metabolic activity to, for example, hormones or other maternal signals. We studied the dynamics of maternal steroids in fertilized and unfertilized rock pigeon eggs during early incubation. Embryos of this species are naturally exposed to different amounts of maternal steroids in the egg according to their laying position, which provides a natural context to study differential embryonic regulation of the maternal signals. We used mass spectrometric analyses to map changes in the androgen and estrogen pathways of conversion. We show that the active hormones are heavily metabolized only in fertilized eggs, with a corresponding increase in supposedly less potent metabolites already within one-fourth of total incubation period. Interestingly, the rate of androgen metabolism was different between embryos in different laying positions. The results also warrant a re-interpretation of the timing of hormone mediated maternal effects and the role of the supposedly biologically inactive metabolites. Furthermore, the results also provide a potential solution as to how the embryo can prevent maternal steroids in the egg from interfering with its sexual differentiation processes as we show that the embryo can metabolize most of the maternal steroids before sexual differentiation starts.

Introduction

Over the last decades there is a growing interest in the exposure of the vertebrate embryo to maternal hormones as a potential pathway for adaptive maternal effects. Egg laying species, especially birds (Gil, 2008, Groothuis et al., 2005, von Engelhardt and Groothuis, 2011), but also fish (Brown et al., 1988) and reptiles (Paitz and Bowden, 2011, Paitz and Bowden, 2008, Radder, 2007) have been used extensively to study the effects of maternal hormones, especially steroids, in the egg yolk since in oviparous species the embryo develops outside the body of the mother facilitating such manipulations. This has revealed a wide array of effects on the offspring phenotype, ranging from morphology to physiology and behaviour (Groothuis et al., 2005, Schwabl, 1993, von Engelhardt and Groothuis, 2011). Furthermore, systematic variation is found in egg steroid levels associated with the laying order as well as environmental variation surrounding the mother (Eising et al., 2001, Schwabl, 1997, Schwabl, 1993, von Engelhardt and Groothuis, 2011), including biotic and abiotic factors (Gil, 2008, Hahn, 2011, Müller et al., 2002, Welty et al., 2012). However, how, when and which hormones reach the embryo is as yet unclear.

In the course of egg incubation the hormone concentrations in the yolk decline rapidly (birds (Eising et al., 2003, Elf and Fivizzani, 2002, Wilson and McNabb, 1997), reptiles (Bowden et al., 2002, Paitz and Bowden, 2009), fish (Feist and Schreck, 1996)). One study showed that hormone levels decline in yolk-albumen homogenates (Paitz et al., 2011) suggesting that the decrease in yolk hormone concentrations is not entirely due to yolk dilution by mixing and/or water influx with albumen, and a few pioneering studies indicate metabolism of maternal yolk steroids by the embryo by conjugation (Paitz et al., 2011, Paitz and Casto, 2012, Vassallo et al., 2014, von Engelhardt et al., 2009). As suggested by Paitz and Bowden, 2008, Paitz and Bowden, 2013, von Engelhardt et al., 2009), this opens the possibility that embryos of oviparous species have in fact active control over their endocrine environment like in mammalian species (Braun et al., 2013, Cottrell and Seckl, 2009, Del Giudice, 2012) which would be favoured by natural selection (Del Giudice, 2012, Mock and Forbes, 1994, Müller et al., 2007, Wilson et al., 2005, Winkler, 1993). This is because of potential parent-offspring conflicts in which the endocrine environment created by the mother might be primarily in the interest of the mother but not always be in the best interest of the offspring as they share only half of their genes. For example, by distributing maternal androgens over the laying order mothers may favour certain offspring over others, creating a conflict with the latter. However, the detailed scope for such role of the embryo in translating maternal hormones is not well understood, especially in bird species, the most widely used species in this field. This includes the timing and quantitative dynamics of embryonic metabolism, metabolic differences based on embryo’s laying order in the clutch, the overall metabolic outcomes concerning detailed steroid metabolic pathway such as conversion of less potent metabolites to more potent ones or vice-versa, and their uptake and utilization by the embryo.

The aims of this study were (i) To verify which of the steroids of the androgenic and estrogenic pathway differ in maternal deposition between first and second eggs including the conjugated forms; (ii) To investigate to what extent the decline in yolk hormone levels during the first days of incubation is due to hormone conversion by analysing the decline in hormone amounts of the entire egg between oviposition and 4.5 days of incubation in the unfertilized eggs; (iii) To compare the metabolic profile of incubated fertilized and unfertilized eggs to discern the maternal and embryonic contribution to the steroid metabolism; (iv) To compare the metabolic outcomes of maternal steroid hormones between fertilized first and second eggs that would indicate scope for differential embryonic activity. To this end, we used rock pigeon species (Columba livia) because it provides an appropriate natural context to test whether the embryos of different laying order can utilize or metabolize maternal hormonal signals differently as the first and second embryos of a clutch are exposed to different levels of maternal androgens (Hsu et al., 2016). We analysed a wide spectrum of hormone profiles and their metabolites (Fig. 1) over the first 4.5 days of incubation to identify patterns of conversion to biologically active or inactive compounds, including conjugated forms.