Hormone-mediated Maternal Effects Research Articles

Female red-winged blackbirds (Agelaius phoeniceus) do not alter nest site selection, maternal programming, or hormone-mediated maternal effects in response to perceived nest predation or brood parasitism risk

Predation or brood parasitism risks can change the behaviors and reproductive decisions in many parental animals. For oviparous species, mothers can mitigate their reproductive success in at least three ways: (1) by avoiding nest sites with high predation or parasitism risks, (2) through hormonal maternal effects that developmentally prime offspring for survival in risky environments, or (3) by investing less in reproduction when predation or parasitism risks are high. Here, we tested if perceived predation and parasitism risks can induce any of these behavioral or physiological responses by exposing female red-winged blackbirds (Agelaius phoeniceus) to playbacks of two major nest threats, a predator (Cooper’s hawk, Accipiter cooperii) and an obligate brood parasite (brown-headed cowbird; Molothrus ater), as well as two controls (harmless Eastern meadowlark, Sturnella magna; and silence). We found that female blackbirds did not avoid nesting at sites treated with predator or brood parasite playbacks, nor were females more likely to abandon nesting attempts at these sites. Egg size and yolk hormone profiles, which are common proxies for maternal investment in oviparous species, were statistically similar across treatment sites. Instead, we found intraclutch variation in yolk steroid hormone profiles: concentrations of three progestogens (pregnanedione, 17α-hydroxypregnenolone, and deoxycorticosterone) and two androgens (testosterone and androstenedione) were higher in third-laid than first-laid eggs. Our study largely confirms previous findings of consistent intraclutch yolk hormone variation in this species, in birds in general, and in other oviparous lineages, but uniquely reports on several yolk steroid hormones largely overlooked in the literature on hormone-mediated maternal effects.

Urbanization and maternal hormone transfer: Endocrine and morphological phenotypes across ontogenetic stages

The phenotypes observed in urban and rural environments are often distinct; however, it remains unclear how these novel urban phenotypes arise. Hormone-mediated maternal effects likely play a key role in shaping developmental trajectories of offspring in different environments. Thus, we measured corticosterone (Cort) and testosterone (T) concentrations in eggs across the laying sequence in addition to Cort concentrations in nestling and adult female house wrens (Troglodytes aedon) at one urban and one rural site. We found that egg T concentrations were not different between birds from urban and rural sites. However, across all life stages (egg, nestling, and adult female), Cort concentrations were higher at the urban site. Additionally, urban nestling Cort concentrations, but not rural, correlated with fine-scale urban density scores. Furthermore, rural egg volume increased over the laying sequence, but urban egg volume leveled off mid-sequence, suggesting either that urban mothers are resource limited or that they are employing a different brood development strategy than rural mothers. Our study is one of the first to show that egg hormone concentrations differ in an urban environment with differences persisting in chick development and adult life stages. We suggest that maternal endocrine programing may shape offspring phenotypes in urban environments and are an overlooked yet important aspect underlying mechanisms of urban evolution.

Prenatal testosterone triggers long-term behavioral changes in male zebra finches: unravelling the neurogenomic mechanisms

BackgroundMaternal hormones, like testosterone, can strongly influence developing offspring, even generating long-term organizational effects on adult behavior; yet, the mechanisms facilitating these effects are still unclear. Here, we experimentally elevated prenatal testosterone in the eggs of zebra finches (Taeniopygia guttata) and measured male aggression in adulthood along with patterns of neural gene expression (RNA-seq) and DNA methylation (MethylC-Seq) in two socially relevant brain regions (hypothalamus and nucleus taenia of the amygdala). We used enrichment analyses and protein-protein interaction networks to find candidate processes and hub genes potentially affected by the treatment. We additionally identified differentially expressed genes that contained differentially methylated regions.ResultsWe found that males from testosterone-injected eggs displayed more aggressive behaviors compared to males from control eggs. Hundreds of genes were differentially expressed, particularly in the hypothalamus, including potential aggression-related hub genes (e.g., brain derived neurotrophic factor). There were also enriched processes with well-established links to aggressive phenotypes (e.g., somatostatin and glutamate signaling). Furthermore, several highly connected genes identified in protein-protein interaction networks also showed differential methylation, including adenylate cyclase 2 and proprotein convertase 2.ConclusionsThese results highlight genes and processes that may play an important role in mediating the effects of prenatal testosterone on long-term phenotypic outcomes, thereby providing insights into the molecular mechanisms that facilitate hormone-mediated maternal effects.

Maternally derived hormones, neurosteroids and the development of behaviour.

In a wide range of taxa, there is evidence that mothers adaptively shape the development of offspring behaviour by exposing them to steroids. These maternal effects have major implications for fitness because, by shaping early development, they can permanently alter how offspring interact with their environment. However, theory on parent-offspring conflict and recent physiological studies showing that embryos rapidly metabolize maternal steroids have placed doubt on the adaptive significance of these hormone-mediated maternal effects. Reconciling these disparate perspectives requires a mechanistic understanding of the pathways by which maternal steroids can influence neural development. Here, we highlight recent advances in developmental neurobiology and psychiatric pharmacology to show that maternal steroid metabolites can have direct neuro-modulatory effects potentially shaping the development of neural circuitry underlying ecologically relevant behavioural traits. The recognition that maternal steroids can act through a neurosteroid pathway has critical implications for our understanding of the ecology and evolution of steroid-based maternal effects. Overall, compared to the classic view, a neurosteroid mechanism may reduce the evolutionary lability of hormone-mediated maternal effects owing to increased pleiotropic constraints and frequently influence long-term behavioural phenotypes in offspring.

Manipulation of Prenatal Thyroid Hormones Does Not Affect Growth or Physiology in Nestling Pied Flycatchers

Hormones transferred from mothers to their offspring are thought to be a tool for mothers to prepare their progeny for expected environmental conditions, thus increasing fitness. Thyroid hormones (THs) are crucial across vertebrates for embryonic and postnatal development and metabolism. Yet yolk THs have mostly been ignored in the context of hormone-mediated maternal effects. In addition, the few studies on maternal THs have yielded contrasting results that could be attributed to either species or environmental differences. In this study, we experimentally elevated yolk THs (within the natural range) in a wild population of a migratory passerine, the European pied flycatcher (Ficedula hypoleuca), and assessed the effects on hatching success, nestling survival, growth, and oxidative status (lipid peroxidation, antioxidant enzyme activity, and oxidative balance). We also sought to compare our results with those of a closely related species, the collared flycatcher (Ficedula albicolis), that has strong ecological and life-history similarities with our species. We found no effects of yolk THs on any of the responses measured. We could detect only a weak trend on growth: elevated yolk THs tended to increase growth during the second week after hatching. Our results contradict the findings of previous studies, including those of the collared flycatcher. However, differences in fledging success and nestling growth between both species in the same year suggest a context-dependent influence of the treatment. This study should stimulate more research on maternal effects mediated by THs and their potential context-dependent effects.

Revisiting mechanisms and functions of prenatal hormone-mediated maternal effects using avian species as a model.

Maternal effects can adaptively modulate offspring developmental trajectories in variable but predictable environments. Hormone synthesis is sensitive to environmental factors, and maternal hormones are thus a powerful mechanism to transfer environmental cues to the next generation. Birds have become a key model for the study of hormone-mediated maternal effects because the embryo develops outside the mother's body, facilitating the measurement and manipulation of prenatal hormone exposure. At the same time, birds are excellent models for the integration of both proximate and ultimate approaches, which is key to a better understanding of the evolution of hormone-mediated maternal effects. Over the past two decades, a surge of studies on hormone-mediated maternal effects has revealed an increasing number of discrepancies. In this review, we discuss the role of the environment, genetic factors and social interactions in causing these discrepancies and provide a framework to resolve them. We also explore the largely neglected role of the embryo in modulating the maternal signal, as well as costs and benefits of hormone transfer and expression for the different family members. We conclude by highlighting fruitful avenues for future research that have opened up thanks to new theoretical insights and technical advances in the field. This article is part of the theme issue 'Developing differences: early-life effects and evolutionary medicine'.

Gonadal steroid levels in rock pigeon eggs do not represent adequately maternal allocation

Maternal hormones deposited in the egg can provide a powerful model for the study of maternal effects. The differential amount of maternal hormones in the yolk of freshly laid eggs is assumed to represent differential maternal allocation. However, some evidence suggests that these amounts do not reflect maternal allocation that in fact takes place before ovulation. We compared the amounts of a wide array of gonadal steroids and their metabolites in the yolk of pre-ovulatory follicles with those of freshly laid eggs of rock pigeons using mass spectrometry. We found that between the follicle and egg stages the levels of progesterone increase whereas androstenedione and testosterone decrease in which the strength of decrease was dependent on the laying order of the egg. For conjugated estrone the change between follicle and egg differed in direction for first and second laying position yielding a significant interaction effect. For conjugated testosterone the interaction did not reach but was close to significance. This extremely early steroid metabolism was not due to maternal enzymes in the yolk as indicated by incubation of pre-ovulatory yolks treated with proteinase-K, a protein digesting enzyme. The results have significant consequences for the functional and evolutionary interpretation as well as experimental manipulation of hormone-mediated maternal effects.

High heritability of egg testosterone and consequences on performance of offspring in Japanese quail. A review

DOI: 10.15414/afz.2014.17.04.130–135 Received 4. September 2014 ǀ Accepted 2. October 2014 ǀ Available online 24. November 2014 Individual phenotype is a result of an interacting genotype and environment with the most sensitive periods occurring within the late embryonic and early postnatal development. In this time, most of the signals come from the mother and such influences are termed maternal effects. In our review, we focus on hormone-mediated maternal effects in birds because the avian embryo develops without interactions with neuroendocrine milieu of the mother and the egg yolk contains biologically active substances including hormones. Thus, it is expected that the mother can adjust phenotypic development of her progeny to actual environmental conditions through differential deposition of hormones in the egg. The adaptive value of such hormone mediated maternal effects can have significant consequences for poultry performance but is still generally neglected. In the first part of our review, we summarise the data from wild living birds and urge the need to study maternal egg hormones also in poultry species. In the second part of this review, we present our experimental approach that is based on two genetic lines of Japanese quail divergently selected for yolk testosterone concentrations. We estimated relatively high heritability of yolk testosterone concentrations and proved that natural selection can shape hormone-mediated maternal effects. Our selected lines provided us with a unique model to explore mechanisms of maternal hormone transfer into the egg and epigenetic effects of maternal hormones on offspring. We expect that this approach will provide relevant data in the field of trans-generational maternal effects with potential application for poultry breeding and welfare. Keywords : maternal effects, hormones, androgens, genetic lines

Consequences of experimentally elevated yolk testosterone levels for intra- and inter-sexual selection in canaries

Hormones of maternal origin transferred to the eggs of oviparous species have been shown to significantly affect offspring development. Furthermore, there is now increasing evidence that these effects may last into adulthood. This underlines the persistence of yolk hormone-mediated maternal effects as well as their trans-generational potential as these changes may involve fitness-related traits such as mate choice behaviour, reproductive traits and longevity. Here, we tested the potential of yolk testosterone to affect sexual selection by experimentally increasing the yolk testosterone levels via egg injections. We focused on two central axes of sexual selection, male–male competition for access to a female (intra-sexual selection) and female mate choice behaviour (inter-sexual selection), using canaries (Serinus canaria) as a model species. Neither male agonistic behaviour nor access to the opposite sex, as measured in staged male–male encounters in the presence of a female, were affected by experimentally elevating yolk testosterone levels. We did not find any evidence for effects on female mate choice behaviour either, given the lack of significant effects on mate choice activity, consistency in female mate choice or choosiness. In conclusion, our results indicate that the consequences of yolk testosterone for sexual selection through changes in behavioural traits, which are expressed during pair formation or male–male competition, are probably limited.

Evaluation of a stress model induced by dietary corticosterone supplementation in broiler breeders: effects on egg yolk corticosterone concentration and biochemical blood parameters

1. This study aimed to evaluate a stress model induced by corticosterone (CORT) supplementation in the diet of broiler breeder hens.2. A total of 60 Ross broiler breeder hens at 29 weeks of age were randomly divided into 4 groups with 15 hens each. The first group served as the control. The rest of the hens were given 1, 1.5 or 2 mg of CORT/hen/d (CORT1, CORT1.5 and CORT2, respectively) for 7 d. Concentrations of yolk CORT, plasma uric acid, glucose, cholesterol, creatine kinase, heterophil (H):lymphocyte (L) ratio and duration of tonic immobility (TI) were measured at d 3, 5 and 7 of CORT supplementation. The same measurements were repeated at 3, 5 and 7 d after CORT was withdrawn from the diet.3. There were no significant CORT dose effect on yolk CORT and plasma glucose concentrations. Higher plasma uric acid and H:L ratio was obtained for CORT1.5 and CORT2 than for CORT1. From 3 to 7 d of dietary CORT supplementation, yolk CORT and plasma uric acid concentrations and H:L ratio increased whereas plasma glucose concentration decreased. After CORT was withdrawn from the diet, the H:L ratio remained elevated. The duration of TI and plasma creatine kinase concentration did not change during and after CORT supplementation.4. Yolk CORT concentration was correlated with plasma uric acid concentration during CORT supplementation.5. The results suggest that dietary CORT supplementation could be used as a stress model and to evaluate hormone-mediated maternal effects in broiler breeder hens.

Testosterone influences song behaviour and social dominance – But independent of prenatal yolk testosterone exposure

In the last two decades, maternally derived yolk androgens have been shown to significantly alter offspring development, and a number of these effects persist into adulthood. However, little is known about their underlying mechanisms. Mechanisms that have been suggested are changes in the endogenous androgen production post-hatching or changes in the sensitivity towards circulating androgens.We tested the effects of yolk testosterone on the plasma testosterone levels and the sensitivity to testosterone in 5months old male canaries that hatched from eggs that were either injected with testosterone (yT-males) or with a control solution (yC-males). Changes in sensitivity were investigated via the behavioural response to an experimental elevation of the plasma testosterone levels. We performed the experiment in fall (low endogenous testosterone production), focusing on testosterone dependent response traits (aggression and song).Before implantation, there was a non-significant trend that the plasma testosterone levels were lower in yT-males than in yC-males. Elevating the plasma testosterone concentrations increased aggressiveness, song bout length and similarity of repeated song elements (=consistency), with the latter likely being a consequence of testosterone-driven song crystallization. However, these effects were not different among yT- or yC-males in any of the parameters. Thus, our findings render it unlikely that changes in the sensitivity to testosterone post-hatching would form the main underlying mechanism of hormone-mediated maternal effects in birds. Further experiments are urgently needed in order to understand the nature of the phenotypic effects resulting from embryonic exposure to maternal yolk testosterone.

Increased exposure to yolk testosterone has feminizing effects in chickens, Gallus gallus domesticus

Competing for food by altricial and semiprecocial bird nestlings is a behaviour well known for its sensitivity to maternal androgens during prenatal development. Whether a similar effect is present in precocial species that do not beg is less well known. We therefore increased yolk testosterone levels within the physiological range at the onset of incubation to study its effects on food competition behaviour in the domestic chicken, Gallus gallus domesticus. We found an increase in competitiveness in testosterone-treated male domestic chicks, raising their level to that of the females. This is in line with the decrease in circulating plasma levels of males in the direction of the levels in females, and the overall decrease in androgen receptor densities after prenatal treatment as found previously. Hormones are known to have long-lasting organizing effects on behaviour and to affect sexual differentiation in vertebrates. Although research into hormone-mediated maternal effects has been productive, only a few studies describe (the ambiguous) effects into adulthood. Therefore we followed our animals into adulthood and recorded androgen-dependent social behaviour and secondary sexual characteristics, body mass and circulating plasma testosterone levels and checked whether these variables were treatment dependent. Treatment had a near significant effect on comb colour (both brightness and chroma). Again treatment caused a shift towards a more female-like phenotype. This suggests that, in contrast to earlier suggestions, maternal androgens may interact with (but not disrupt) sexual differentiation of brain and behaviour and the development of secondary sexual characteristics.

Hormonally mediated maternal effects, individual strategy and global change

A challenge to ecologists and evolutionary biologists is predicting organismal responses to the anticipated changes to global ecosystems through climate change. Most evidence suggests that short-term global change may involve increasing occurrences of extreme events, therefore the immediate response of individuals will be determined by physiological capacities and life-history adaptations to cope with extreme environmental conditions. Here, we consider the role of hormones and maternal effects in determining the persistence of species in altered environments. Hormones, specifically steroids, are critical for patterning the behaviour and morphology of parents and their offspring. Hence, steroids have a pervasive influence on multiple aspects of the offspring phenotype over its lifespan. Stress hormones, e.g. glucocorticoids, modulate and perturb phenotypes both early in development and later into adulthood. Females exposed to abiotic stressors during reproduction may alter the phenotypes by manipulation of hormones to the embryos. Thus, hormone-mediated maternal effects, which generate phenotypic plasticity, may be one avenue for coping with global change. Variation in exposure to hormones during development influences both the propensity to disperse, which alters metapopulation dynamics, and population dynamics, by affecting either recruitment to the population or subsequent life-history characteristics of the offspring. We suggest that hormones may be an informative index to the potential for populations to adapt to changing environments.

Negative effects of yolk testosterone and ticks on growth in canaries

Maternal yolk hormones in bird eggs are thought to adjust the offspring to the post-hatching environment. This implies that the effects of maternal yolk hormones should vary with the post-hatching environment, but to date such context-dependency has largely been ignored. We experimentally increased yolk testosterone concentrations in canary eggs and simultaneously manipulated the post-hatching context via an experimental tick-infestation of the chicks. This allows us to evaluate the context-dependency of hormone-mediated maternal effects, as it has previously been shown that ectoparasites alter the maternal yolk androgen deposition. The experimental tick infestation reduced growth in chicks from sham-treated eggs, indicating harmful effects of this ectoparasite in canaries. Chicks from testosterone-treated eggs were not affected in their development by ticks, suggesting lower ectoparasite vulnerability. But this may also be due to the fact that experimentally elevated yolk testosterone levels impaired growth even under parasite-free conditions. This contrasts previous studies, but these studies often manipulated first laid eggs, while we used eggs of subsequent laying positions. Later laid eggs are presumably of lower quality and contain higher yolk testosterone concentrations. Thus, the effects of elevated yolk testosterone on growth may be dose-dependent or vary with the egg quality, suggesting prenatal context-dependency.

Elevated Plasma Corticosterone Decreases Yolk Testosterone and Progesterone in Chickens: Linking Maternal Stress and Hormone-Mediated Maternal Effects

Despite considerable research on hormone-mediated maternal effects in birds, the underlying physiology remains poorly understood. This study investigated a potential regulation mechanism for differential accumulation of gonadal hormones in bird eggs. Across vertebrates, glucocorticoids can suppress reproduction by downregulating gonadal hormones. Using the chicken as a model species, we therefore tested whether elevated levels of plasma corticosterone in female birds influence the production of gonadal steroids by the ovarian follicles and thus the amount of reproductive hormones in the egg yolk. Adult laying hens of two different strains (ISA brown and white Leghorn) were implanted subcutaneously with corticosterone pellets that elevated plasma corticosterone concentrations over a period of nine days. Steroid hormones were subsequently quantified in plasma and yolk. Corticosterone-implanted hens of both strains had lower plasma progesterone and testosterone levels and their yolks contained less progesterone and testosterone. The treatment also reduced egg and yolk mass. Plasma estrogen concentrations decreased in white Leghorns only whereas in both strains yolk estrogens were unaffected. Our results demonstrate for the first time that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk. Maternal corticosterone could therefore mediate environmentally induced changes in yolk gonadal hormone concentrations. In addition, stressful situations experienced by the bird mother might affect the offspring via reduced amounts of reproductive hormones present in the egg as well as available nutrients for the embryo.

GnRH - a Missing Link between Testosterone Concentrations in Yolk and Plasma and Its Intergenerational Effects

Despite the strong interest in hormone-mediated maternal effects two key questions concerning their mechanisms are as yet unanswered: First, whether the deposition of hormones in the egg yolk is coupled with the levels of these hormones in the maternal circulation, and second, whether epigenetic changes as induced by embryonic exposure to maternal yolk hormones impinge on yolk hormone deposition at adulthood. We investigated the responsiveness to gonadotropin-releasing hormone (GnRH) in female canaries whose embryonic exposure to yolk testosterone had been manipulated. This enabled us to study to what extent GnRH interlinks testosterone concentrations in female circulation and egg yolk as well as the intergenerational potential of hormone-mediated maternal effects. As expected, canary females responded to GnRH with a rise in plasma testosterone. The GnRH-responsiveness was positively correlated with the yolk testosterone content. Factors stimulating the release of GnRH will, therefore, lead to an increase of testosterone in both plasma and egg, posing a potential constraint on the yolk hormone deposition due to testosterone related trade-offs within the laying female. Exposure to elevated yolk testosterone levels as embryo reduced the GnRH-responsiveness in adulthood, potentially limiting environmental influences on yolk testosterone deposition, but the concentrations of yolk testosterone itself were not affected.

Adaptive hormone-mediated maternal effects in free-ranging red squirrels

Adaptive hormone-mediated maternal effects in free-ranging red squirrels

Short- and long-term consequences of prenatal testosterone for immune function: an experimental study in the zebra finch

Hormone-mediated maternal effects play an important role in the formation of a differentiated phenotype. They have been shown to influence a wide array of offspring traits, both early in life and in adulthood. One important offspring trait that is under the influence of maternal androgens is the immune system. In birds, a growing number of studies show that yolk androgens modulate immune function during the chick stage. However, there is a lack of knowledge regarding long-term effects of prenatal androgens on offspring immunity. In this study, we therefore investigated the influence of prenatal testosterone (T) on several measures of immunity in fledgling and adult zebra finches (Taeniopygia guttata). Cell-mediated immune response (towards phytohaemagglutinin, PHA) of fledglings hatching from control eggs was negatively related to brood size, whereas there was no such association for fledglings hatching from eggs with experimentally elevated T levels (T fledglings). Male control fledglings showed reduced mass gain compared to female control fledglings within 24 h after the PHA injection. This pattern was reversed in T fledglings. Total antibody levels in fledglings were not affected by egg treatment. Neither cell-mediated immunity nor total antibody levels in sexually mature zebra finches were influenced by egg treatment. However, there was an immuno-enhancing effect of elevated egg T on both primary and secondary humoral immune responses toward diphtheria and tetanus antigens in ca 5 and 7 month old zebra finches. In addition, the covariation between different immune components differed between T and control offspring, suggesting that egg treatment may have altered the potential trade-offs between different parts of the immune system. Our results suggest that prenatal androgens could be an important factor contributing to individual variation in immune function even in adulthood.

Heritable Variation in Maternal Yolk Hormone Transfer in a Wild Bird Population

Differential reproductive investment by the mother can critically influence offspring development and phenotype, and strong selection is therefore expected to act on such maternal effects. Although a genetic basis is a prerequisite for phenotypic traits to respond to selection and thus to evolve, we still know very little about the extent of heritable variation in maternal effects in natural populations. Here, we present the first estimates of intrafemale repeatability across breeding seasons and estimates of heritability of hormone-mediated maternal effects in a wild population of collared flycatchers (Ficedula albicollis). We found that maternal yolk testosterone (T) concentrations, yolk mass, and egg mass were moderately to highly repeatable within females across years, whereas intrafemale consistency of maternal yolk androstenedione (A4) deposition was low yet statistically significant. Furthermore, maternal yolk T transfer, yolk mass, and egg mass were significantly heritable, whereas yolk A4 transfer was not. These results strongly suggest that two major maternal yolk androgens are differentially regulated by genes and the environment. Selection on heritable variation in maternal yolk T deposition has the potential to shape the rate and direction of phenotypic change in offspring traits and can thereby accelerate or impede the response to selection in natural populations.

Steroids in chicken egg yolk: Metabolism and uptake during early embryonic development

Effects of maternal hormones may adaptively adjust offspring development to prevailing conditions. However, Darwinian fitness of parents is maximized by investing in more than one offspring while each individual offspring benefits from receiving maximal investment. The control of mother and offspring over hormone-mediated maternal effects is thought to play a key role in the outcome of parent–offspring conflict, but these control mechanisms have hardly been studied. We investigated the potential embryonic control by analysing the changes in distribution and metabolism of steroid hormones in the egg during the first 6 days of incubation using injections of radiolabelled testosterone and corticosterone in freshly laid eggs. After 1 day of incubation the highest amount of radioactivity was concentrated in a small area at the top of the yolk. This challenges the use of hormones in oil as mimicking natural exposure. During incubation radioactivity spread within the egg with highest concentrations in yolk and yolk sac and lower concentrations in albumen, embryo, allantois, and amnion. Steroids were metabolised to other unconjugated and conjugated steroids, perhaps facilitating embryonic steroid uptake. Our study shows that the injected radiolabel is metabolised in the egg and taken up by the embryo, giving the embryo potential control over the effects of maternal hormones and thereby limiting maternal control over the outcome of hormone-mediated maternal effects.