Early embryonic modification of maternal hormones differs systematically among embryos of different laying order: A study in birds
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.
Section snippets
Animal housing
All animal procedures were approved by the animal welfare committee of the University of Groningen under license 6835B. The procedures were carried out at the animal facility of the University of Groningen according to the guidelines and regulations of the committee. Rock pigeons (80 pairs) were housed in outdoor aviaries (45 m long × 9.6 m wide x 3.75 m high) under natural light and temperature conditions, and ad libitum access to food and fresh water. All eggs (whether unfertilized or
Differential maternal deposition: Day 0 eggs
At the time of oviposition (Fig. 2: day 0, Table 1), second eggs had higher levels of 17-hydroxyprogesterone (p = 0.032), androstenedione (p < 0.001), and testosterone (p < 0.001), confirming earlier studies on androgens (Hsu et al., 2016). The levels did not differ between first and second eggs for progesterone, estradiol, and estrone. There was some conjugated estrone and conjugated testosterone already present at oviposition which also did not differ between the egg laying order.
Discussion
We analysed for the androgenic pathway the role of the embryo in the decline of androgens during the first days of incubation and to what extent this may be different between embryos of eggs that differ in laying position. As the decline was measured in the entire fertilized egg, including the embryo, the changes in amounts of steroids over incubation cannot be attributed to mixing of yolk-albumen or water influx into yolk or embryonic uptake over the course of incubation, as suggested in the
Conclusions
In conclusion, we experimentally demonstrate that the absolute levels as well as the relative differences in maternal hormones at oviposition tend to diminish very early during incubation due to embryonic metabolism, with the rate of androgen metabolism being higher in latter laid eggs. This creates a paradox as it is well known that initial differences in these hormones can have substantial effects on the chick, whereas we show that at 4.5 days of incubation these differences are hardly
Data availability
The dataset supporting this article can be accessed as Supplementary data set file.
Acknowledgements
We thank Bin-Yan Hsu, Gerard Overkamp, and the animal care takers (Saskia Helder, Diane ten Have, Martijn Salomons) for animal related work, Bernd Riedstra for conceptual discussions, and Alle Pranger for mass spectrometry related work.
Funding
This research was supported by Ubbo Emmius research grant by the University of Groningen to TG in collaboration with the Max Planck Institute for Ornithology.
Animal ethics
All the animal research was conducted according to the established guidelines and regulations of the animal welfare committee of the University of Groningen, and all relevant procedures were approved by the committee under the license 6835B.
Author contributions
NK and TG designed the details of the experiments. NK, MG, and TG discussed and interpreted the results. NK performed the experiments, analysed the data, and prepared the manuscript on which TG and MG provided feedback. MvF and IK provided the mass spectrometry data and wrote their part of the method section. All authors gave final approval for publication.
Competing interests
The authors declare that they have no competing interests.
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2020, Current Opinion in Behavioral SciencesCitation Excerpt :There is overwhelming evidence that injecting hormones in the avian egg before incubation affect a diversity of traits both prenatally and postnatally [7]. Interestingly, in the very first days of incubation, enzymes to metabolize maternal hormones and the relevant receptors are already present before the embryo produces its own hormones [16–19,20•,21,22•]. During this period, maternal hormones act as transcription factors that can influence the expression of several genes, including their own receptors (e.g. androgens [22•,23], glucocorticoids [24], and transmembrane transporters [25]).
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2020, General and Comparative EndocrinologyCitation Excerpt :In the European starling (Sturnus vulgaris), yolk testosterone (Paitz et al., 2011) and progesterone (Paitz and Casto, 2012) are metabolized in the first few days of development. In the rock pigeon (Columba livia), yolk steroids such as progesterone, androstenedione, and testosterone are all metabolized in the first 4.5 days of incubation (Kumar et al., 2018). Yolk corticosterone has been shown to be metabolized in ovo in both the chicken (Gallus gallus) (von Engelhardt et al., 2009) and the Japenese quail (Coturnix japonica) (Vassallo et al., 2014; Vassallo et al., 2019).
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2019, General and Comparative EndocrinologyCitation Excerpt :In the European starling (Sturnus vulgaris), AKR1D1 was shown to be present in the first five days of embryonic development and capable of metabolizing yolk testosterone during this period (Paitz et al., 2011). More recently, work in the rock pigeon (Columbia livia) has shown that 5β-reduced metabolites of both testosterone (i.e. ethiocholanolone) and progesterone (i.e. pregnanolone) accumulate in the egg during the first 4.5 days of development (Kumar et al., 2018). These results indicate that the 5β-reduction of yolk steroids occurs very early in development for several bird species.