Yolk Progesterone Research Articles

Yolk corticosterone and progesterone levels in Greater Rhea (Rhea americana) eggs vary in a changing social environment.

Maternal hormones in avian egg yolks may signal and prepare offspring for the prevailing conditions. However, this adjustment requires some degree of flexibility in regulating yolk hormone deposition. The Greater Rhea (Rhea americana) has a particular mating system that combines mixed polygyny and polyandry, communal nesting, and exclusive paternal care of chicks. In this species, we previously found that yolk hormone deposition varies among eggs of different captive populations and could influence chicks' physiology and behavior. However, it is still unknown whether females can modify yolk hormone deposition in a changing social environment. Using a captive population of Greater Rheas, in this study, we quantified yolk hormone levels before and after a reduction in the number of females present in the population. We found that females deposited on average higher yolk corticosterone and lower yolk progesterone after the change in their social environment. Since corticosterone deposited into the yolk comes exclusively from the female's plasma, our results suggest that females had, on average, higher plasma corticosterone levels. The change in the number of females may increase the events of male-male competitions, courtships, and matings, leading to an increase of corticosterone in the females' plasma and then into their eggs. Since we previously found that higher yolk corticosterone and lower yolk progesterone were associated with the production of chicks that have an attenuated stress response, the present study results suggest that yolk hormone deposition is mediated by flexible mechanisms that could adjust development to the prevailing conditions.

Nest predation risk and deposition of yolk steroids in a cavity-nesting songbird: an experimental test.

Maternal hormones can shape offspring development and increase survival when predation risk is elevated. In songbirds, yolk androgens influence offspring growth and begging behaviors, which can help mitigate offspring predation risk in the nest. Other steroids may also be important for responding to nest predation risk, but non-androgen steroids have been poorly studied. We used a nest predator playback experiment and liquid chromatography with tandem mass spectrometry (LC-MS-MS) to assess whether nest predation risk influences deposition of 10 yolk steroids. We found no clear evidence that yolk androgen deposition changed when perception of nest predation risk was experimentally increased. However, elevated nest predation risk led to decreased yolk progesterone deposition. Overall, our results suggest yolk progesterone may be more important than yolk androgens in responses to offspring predation risk and highlight new avenues for research.

In ovo metabolism of progesterone to 5β-pregnanedione in chicken eggs: Implications for how yolk progesterone influences embryonic development

Progesterone has received substantial attention for the essential role it plays in establishing and maintaining pregnancy in placental vertebrates. Despite the prevalence of progesterone during development, relatively little is known about how embryos respond to progesterone. This is true of placental vertebrates as well as egg-laying vertebrates where levels of progesterone in the yolk tend to be higher than most other steroids in the yolk. Bird eggs provide an opportunity to investigate the effects of progesterone on embryonic development because progesterone can be easily manipulated without any confounding effects on maternal physiology. To understand how progesterone might influence embryonic development, it is important to characterize the metabolic fate of progesterone given its potential to be converted to a wide range of steroids. We investigated the metabolic fate of tritiated progesterone over the first four days of development using chicken eggs (Gallus gallus) and identified 5β-pregnanedione as the primary metabolite during this period. After only one day of development, 5β-pregnanedione could be detected within the yolk. Levels of 5β-pregnanedione in both the yolk and albumen tended to rise early in development but conjugated metabolites began to accumulate towards the end of our sampling period. Additionally, in vitro assays using embryo homogenates collected after 72 h of development demonstrated that embryos were capable of carrying out the conversion of progesterone to 5β-pregnanedione. Overall these results have important implications for deciphering the mechanisms through which yolk progesterone might influence embryonic development. Effects could arise via progesterone receptors or receptors capable of binding 5β-pregnanedione but we found no evidence that progesterone is serving as a precursor for androgen or estrogen production.

Human behaviour at the origin of maternal effects on offspring behaviour in laying hens (Gallus gallus domesticus)

Regular visual presence of humans is known to reduce chickens' human-generated stress responses. Here we questioned whether, more than mere visual presence, human behaviour affects laying hen behaviour and subsequently their offspring's behaviour. We hypothesized that human behaviour triggers maternal effects via variations in yolk hormone levels. For five consecutive weeks, two groups of hens were exposed to the same durations of human presence (30 min twice a day, five days a week) but the behaviour of the human differed between groups. The first group (H+) was exposed to predictable arrival of the experimenter, slow movements combined with static presence, stroking during handling and human voice. Whereas the second group of hens (H-) was exposed to unpredictable arrival of the experimenter which remained silent, in motion, and did not provide stroking during handling. At the end of the treatment, we evaluated egg quality and offspring behaviour. We found that avoidance of the experimenter by H+ hens but not by H– hens decreased significantly. Fertility rates and concentrations of yolk progesterone and estradiol in H+ hens' eggs were higher than in H– hens' eggs. Fear of humans, neophobia or the capacity to solve a detour task did not differ significantly between H+ and H– chicks. Social discrimination tests showed that H+ chicks but not H– chicks typically preferred a familiar conspecific to a stranger. These results show that, with the same duration in the presence of the birds, humans through their behaviour engender variations in fertility rates, yolk hormone levels and transgenerational effects on social skills. Rarely explored, our data suggest that maternal effects influence filial imprinting. These data have broad implications for laboratory, commercial systems and conservatory programs where the inevitable presence of humans could trigger maternal effects on offspring phenotype.

Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick.

We assessed whether the ratio of dietary n-6/n-3 polyunsaturated fatty acids (PUFA) during egg formation engenders transgenerational maternal effects in domestic chicks. We analyzed yolk lipid and hormone concentrations, and HPA-axis activity in hens fed a control diet (high n-6/n-3 ratio) or a diet enriched in n-3 PUFAs (low n-6/n-3 ratio) for 6 consecutive weeks. Their chicks were tested for neophobia during the first week of life. We found higher corticosterone metabolites in droppings of hens fed the diet enriched in n-3 and significantly higher concentrations of yolk progesterone, androstenedione, and estradiol in their eggs compared to controls. Chicks of hens fed the n-3 enriched diet showed a lower body mass at hatch than controls and expressed higher neophobia when exposed to a novel object. These results add support to the hypothesis that the nutritional state of female birds produces variation in yolk hormone levels and engender maternal effects.

Distribution and concentration of maternal progesterone in the yolk of Greater Rhea eggs (Rhea americana).

Progesterone is the most concentrated maternal yolk steroid characterized to date in birds; however, no information about it is available in ratite eggs. We collected freshly laid eggs from zoo-housed Greater Rhea females (Rhea americana) bred under similar rearing conditions during two breeding seasons to characterize concentration and distribution of maternal yolk progesterone. After high-performance liquid chromatography analysis, yolk hormone was measured using a commercial electrochemiluminescence immunoassay. Progesterone concentrations were found to vary significantly among the yolk layers, supporting a follicular origin for this steroid in Greater Rhea eggs. Additionally, highly similar mean absolute yolk progesterone concentrations were detected between 2013 and 2015 breeding seasons (1,332.98 ± 82.59 and 1,313.59 ± 85.19 ng/g, respectively). These values are also comparable to those found in some domestic carinate species. Findings suggest that at population level, when rearing conditions are similar, mean absolute yolk maternal progesterone concentrations also appear bounded. Future research on the factors and mechanisms that regulate progesterone deposition in Greater Rhea eggs is needed to better understand whether its levels depend on different rearing conditions.

Elevated yolk progesterone moderates prenatal heart rate and postnatal auditory learning in bobwhite quail (Colinus virginianus).

Previous studies have established that yolk hormones of maternal origin can influence physiology and behavior in birds. However, few studies have examined the effects of maternal gestagens, like progesterone, on chick behavior and physiology. We tested the effects of experimentally elevated egg yolk progesterone on embryonic heart rate and postnatal auditory learning in bobwhite quail hatchlings. Quail chicks were passively exposed to an individual maternal assembly call for 10 min/hr during the 24 hr following hatching. Preference for the familiarized call was tested at 48 hr following hatching in three experimental groups: chicks that received artificially elevated yolk progesterone (P) prior to incubation, vehicle-only controls (V), and non-manipulated controls (C). Resting heart rate of P, V, and C embryos were also measured on prenatal day 17. The resting heart rate of P embryos was significantly higher than both the V and C embryos. Chicks from the P group also showed an enhanced preference for the familiarized bobwhite maternal call when compared to chicks from the C and V groups. Our results indicate that elevated yolk progesterone in pre-incubated bobwhite quail eggs can influence arousal level in bobwhite embryos and postnatal perceptual learning in bobwhite neonates.

Artificially Increased Yolk Hormone Levels and Neophobia in Domestic Chicks.

In birds there is compelling evidence that the development and expression of behavior is affected by maternal factors, particularly via variation in yolk hormone concentrations of maternal origin. In the present study we tested whether variation in yolk hormone levels lead to variation in the expression of neophobia in young domestic chicks. Understanding how the prenatal environment could predispose chicks to express fear-related behaviors is essential in order to propose preventive actions and improve animal welfare. We simulated the consequences of a maternal stress by experimentally enhancing yolk progesterone, testosterone and estradiol concentrations in hen eggs prior to incubation. The chicks from these hormone-treated eggs (H) and from sham embryos (C) that received the vehicle-only were exposed to novel food, novel object and novel environment tests. H chicks approached a novel object significantly faster and were significantly more active in a novel environment than controls, suggesting less fearfulness. Conversely, no effect of the treatment was found in food neophobia tests. Our study highlights a developmental influence of yolk hormones on a specific aspect of neophobia. The results suggest that increased yolk hormone levels modulate specifically the probability of exploring novel environments or novel objects in the environment.

Yolk hormones influence in ovo chemosensory learning, growth, and feeding behavior in domestic chicks.

In this study, we assessed whether prenatal exposure to elevated yolk steroid hormones can influence in ovo chemosensory learning and the behavior of domestic chicks. We simulated a maternal environmental challenge by experimentally enhancing yolk progesterone, testosterone, and estradiol concentrations in hen eggs prior to incubation. The embryos from these hormones-treated eggs (HO) as well as sham embryos (O) that had received the vehicle-only were exposed to the odor of fish oil (menhaden) between embryonic Days 11 and 20. An additional group of control embryos (C) was not exposed to the odor. All chicks were tested following hatching for their feeding preferences between foods that were or were not odorized with the menhaden odor. In the 3-min choice tests, the behavior of O chicks differed significantly according to the type of food whereas C and HO chicks showed no preference between odorized and non-odorized food. Our result suggests weaker response in HO chicks. In addition, HO chicks showed impaired growth and reduced intake of an unfamiliar food on the 24-h time scale compared to controls. Our data suggest that embryonic exposure to increased yolk hormone levels can alter growth, chemosensory learning, and the development of feeding behaviors.

Increased yolk progesterone interferes with prenatal auditory learning and elevates emotional reactivity in bobwhite quail (Colinus virginianus) chicks.

Avian eggs contain maternally derived hormones, including testosterone and progesterone. Little is currently known about the effects of these hormones on early behavioral development. We assessed the effects of elevated levels of progesterone levels on prenatal perceptual learning and postnatal emotional reactivity in Northern bobwhite quail. Prior to incubation, eggs received an injection of either progesterone (P) or oil vehicle (V). In P eggs, levels of progesterone were elevated two standard deviations above the mean based on ELISA analysis of progesterone yolk concentrations from a previous study. A third group of eggs served as controls and received no injection (C). Chicks hatched from P eggs displayed elevated levels of emotional reactivity compared to V and C chicks in a tonic immobility task and a hole-in-the-wall emergence task. Chicks from P eggs also failed to demonstrate a preference for a familiarized bobwhite maternal call that had been presented prenatally. In contrast, the V and C chicks demonstrated a significant preference for the familiarized maternal call following hatching, indicating prenatal auditory learning. Our results are consistent with previous findings from precocial birds demonstrating that hormones of maternal origin can influence prenatal perceptual learning as well as emotional reactivity in the period following hatching..

Molecular cloning, expression analysis and developmental changes in ovarian follicles of goose 3β-hydroxysteroid dehydrogenase 1

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase1 (3βHSD1) can catalyse the conversion of pregnenolone to progesterone in the △4-3-ketosteroid metabolic pathway. The aim of the present study was to clone 3βHSD1 and to determine whether this enzyme in the follicular wall has an effect on yolk progesterone in geese (Anser cygnoides). A putative coding sequence of 3βHSD1, which was 1134 nucleotides in length, was successfully obtained by using reverse transcription polymerase chain reaction (RT–PCR). A comparison of the deduced amino acid sequence with chicken, quail, zebra finch, cattle, horse, pig, human and mouse 3βHSD1 showed 89.7%, 88.4%, 87.3%, 55.6%, 54.0%, 53.5%, 55.3% and 52.9% similarity, respectively. The detection of 3βHSD1 mRNA levels in several tissues by quantitative real-time PCR showed that the highest level of 3βHSD1 was in the adrenal gland, followed by the ovary, which indicated that the gene we obtained was the adrenal gland/gonad-specific one. We measured the level of 3βHSD1 mRNA in the follicular wall and determined the concentration of progesterone in the yolk of these ovarian follicles; the concentration of progesterone in the yolk had a pattern of expression similar to that of 3βHSD1 in the follicular wall during follicular development. This result suggests that the expression of 3βHSD1 in the follicular wall may be a main factor that contributes to the accumulation of yolk progesterone.

Moderate Heat Challenge Increased Yolk Steroid Hormones and Shaped Offspring Growth and Behavior in Chickens

BackgroundEnvironmental challenges might affect the maternal organism and indirectly affect the later ontogeny of the progeny. We investigated the cross-generation impact of a moderate heat challenge in chickens. We hypothesized that a warm temperature–within the thermotolerance range- would affect the hormonal environment provided to embryos by mothers, and in turn, affect the morphology and behavioral phenotype of offspring.Methodology/Principal FindingsLaying hens were raised under a standard thermal condition at 21°C (controls) or 30°C (experimental) for 5 consecutive weeks. A significant increase was observed in the internal temperature of hens exposed to the warm treatment; however plasma corticosterone levels remained unaffected. The laying rate was not affected, but experimental hens laid lighter eggs than the controls during the treatment. As expected, the maternal thermal environment affected yolk hormone contents. Eggs laid by the experimental hens showed significantly higher concentrations of yolk progesterone, testosterone, and estradiol. All chicks were raised under standard thermal conditions. The quality of hatchlings, growth, feeding behavior and emotional reactivity of chicks were analyzed. Offspring of experimental hens (C30 chicks) were lighter but obtained better morphological quality scores at hatching than the controls (C21 chicks). C30 chicks expressed lesser distress calls when exposed to a novel food. Unlike C21 chicks, C30 chicks expressed no preference for energetic food.Conclusion/SignificanceOur findings suggest that moderate heat challenge triggers maternal effects and modulate the developmental trajectory of offspring in a way that may be adaptive. This suggests that the impact of heat challenges on captive or wild populations might have a cross-generation effect.

The decline in yolk progesterone concentrations during incubation is dependent on embryonic development in the European starling

Oviparous amniotes, particularly birds, have become model systems in which to study how mothers may utilize steroids to adaptively adjust offspring development. Although there is now ample evidence that maternally derived steroids in the egg at oviposition can influence offspring phenotype, very little is known about how these steroids elicit such effects. Of the major avian steroid hormones found in yolk, progesterone is by far the most abundant at oviposition, but has received little research attention to date. In this study, we examine the metabolism of [3H]-progesterone injected into freshly laid European starling eggs throughout the first 5days of development by characterization of radioactivity within the egg homogenate. We also introduce a technique that utilizes a focal, freeze/thaw cycle to prevent embryonic development and allows us to assess the role of the embryo in metabolizing progesterone during early incubation. Two major findings result. First is that [3H]-progesterone is metabolized in eggs possessing a developing embryo, but not in eggs with disrupted embryonic development. Second is that the change in the distribution of radioactivity within eggs possessing an embryo is the result of metabolism of [3H]-progesterone to a more polar form that is subsequently conjugated. Together, these data suggest live embryos are necessary for metabolism of progesterone during early incubation, underscoring the potentially important contribution of embryos to functional modulation or mediation of maternal yolk steroid effects.

Intra-clutch ratio of yolk progesterone level changes with laying date in rockhopper penguins: a strategy to influence brood reduction?

Hatching asynchrony in avian species generally leads to a size hierarchy among siblings, favouring the first-hatched chicks. Maternally deposited hormones affect the embryo and chick's physiology and behaviour. It has been observed that progesterone, a hormone present at higher levels than other steroid hormones in egg yolks, is negatively related to body mass in embryos, chicks and adults. A differential within-clutch progesterone deposition could therefore be linked to the size hierarchy between siblings and to the resulting brood reduction. We tested whether yolk progesterone levels differed between eggs according to future parental ability to feed the entire clutch in wild rockhopper penguins Eudyptes chrysocome. This species presents a unique reversed egg-size dimorphism and hatching asynchrony, with the larger second-laid egg (B-egg) hatching before the smaller first-laid egg (A-egg). Yolk progesterone levels increased only slightly with female body mass at laying. However, intra-clutch ratios were not related to female body mass. On the other hand, yolk progesterone levels increased significantly with the date of laying onset for A-eggs while they decreased for B-eggs. Early clutches therefore had proportionally more progesterone in the B-egg compared to the A-egg while late clutches had proportionally less progesterone in the B-egg. We propose that females could strategically regulate yolk progesterone deposition within clutches according to the expected food availability during chick growth, an adaptive strategy to adjust brood reduction to conditions. We also discuss these results, relating to yolk progesterone, in the broader context of other yolk steroids.

Experimental Evidence for Genetic Heritability of Maternal Hormone Transfer to Offspring

In many animal species, embryos are exposed to maternal hormones that affect their development. Maternal hormone transfer varies with environmental conditions of the mother and is often interpreted as being shaped by natural selection to adjust the offspring to prevailing environmental conditions. Such hormone transfer requires genetic variability, which has not yet been experimentally demonstrated. Our study reports direct evidence for additive genetic variance of maternal androgens through a bidirectional selection on yolk testosterone (T) levels in Japanese quail. Lines selected for high egg T (HET) and low egg T (LET) concentration differed in yolk levels of this androgen, resulting in high realized heritability (h² = 0.42)Correlated responses to selection on other gonadal hormones indicated that selection specifically targeted biologically active androgens. Eggs of HET quail contained higher androstenedione and lower estradiol concentrations than did those of LET quail, with no line differences in yolk progesterone concentration. Plasma T concentrations in adult females were not affected by selection, seriously challenging the hypothesis that transfer of maternal hormones to offspring is constrained by hormone levels in a mother's circulation. Our results suggest that transfer of maternal T represents an indirect genetic effect that has important consequences for the evolution of traits in offspring.

THE DECLINE IN YOLK PROGESTERONE CONCENTRATIONS DURING DEVELOPMENT IS DEPENDENT ON THE PRESENCE OF A DEVELOPING EMBRYO IN THE EUROPEAN STARLING

Event Abstract Back to Event THE DECLINE IN YOLK PROGESTERONE CONCENTRATIONS DURING DEVELOPMENT IS DEPENDENT ON THE PRESENCE OF A DEVELOPING EMBRYO IN THE EUROPEAN STARLING Ryan Paitz1* and Joseph Casto1 1 Illinois State University, School of Biological Sciences, United States Oviparous amniotes, particularly birds, have become model systems in which to study how mothers may utilize steroids to adaptively adjust offspring development. Although there is now ample evidence that maternally derived steroids in the egg at oviposition can influence offspring phenotype, very little is known about how these steroids elicit such effects. Of the major avian steroid hormones found in yolk, progesterone is by far the most abundant at oviposition, but has received little research attention to date. In this study, we examine the metabolism of [3H] progesterone injected into freshly laid European starling eggs throughout the first five days of development by characterization of radioactivity within the egg homogenate. We also introduce a technique that utilizes a focal, freeze/thaw cycle to prevent embryonic development and allows us to assess the role of the embryo in metabolizing progesterone during early incubation. Two major findings result. First is that [3H] progesterone is metabolized in eggs possessing a developing embryo, but not in eggs with disrupted embryonic development. Second is that the change in the distribution of radioactivity within eggs possessing an embryo is the result of metabolism of [3H] progesterone to a more polar form that is subsequently conjugated. Together, these data suggest live embryos are necessary for metabolism of progesterone during early incubation, underscoring the potentially important contribution of embryos to functional modulation or mediation of maternal yolk steroid effects. Future research should investigate environmental and physiological conditions that might alter the metabolism of maternally derived steroids present in freshly laid eggs. Keywords: bird, embryo, Progesterone, steroid metabolism Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Oral Presentation Topic: Sex determination and differentiation Citation: Paitz R and Casto J (2011). THE DECLINE IN YOLK PROGESTERONE CONCENTRATIONS DURING DEVELOPMENT IS DEPENDENT ON THE PRESENCE OF A DEVELOPING EMBRYO IN THE EUROPEAN STARLING. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00081 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Ryan Paitz, Illinois State University, School of Biological Sciences, Normal, IL, 61790, United States, rpaitz@ilstu.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ryan Paitz Joseph Casto Google Ryan Paitz Joseph Casto Google Scholar Ryan Paitz Joseph Casto PubMed Ryan Paitz Joseph Casto Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Placental buffering of maternal steroid hormone effects on fetal and yolk hormone levels: a comparative study of a viviparous lizard, Sceloporus jarrovi, and an oviparous lizard, Sceloporus graciosus

We investigated maternal-fetal hormone transfer in the mountain spiny lizard, Sceloporus jarrovi, a viviparous species with a simple chorioallantoic placenta. In one experiment, we examined the effect of elevated maternal progesterone on fetal and yolk hormone levels. Progesterone implants increased maternal progesterone nearly 100-fold; however, the resulting increase in fetal and yolk progesterone was only about 2% of that seen in mothers, providing evidence that the placenta buffers hormone diffusion. In addition, some effects of progesterone treatment differed between male and female fetuses, suggesting that this buffering may differ between the sexes. In a second experiment, we examined the relationship between maternal and fetal hormone levels in viviparous versus oviparous species. We measured endogenous progesterone, testosterone, estradiol, and corticosterone levels in pregnant S. jarrovi and their fetuses and neonates, and in gravid S. graciosus (an oviparous congener) and their fetuses and hatchlings. No clear relationship was identified between maternal and fetal or hatchling S. graciosus hormone levels. However, the data for S. jarrovi suggest that maternal hormones may inhibit perinatal hormone secretion. These findings indicate that, despite the relatively recent evolutionary origin and simple structure of the S. jarrovi placenta, mechanisms for placental mediation of the maternal-fetal endocrine relationship have evolved. Although the placenta appears to buffer hormone transport, maternal hormones can affect fetal and yolk hormone levels, suggesting that disruption of endocrine regulation could be a physiological cost of the evolution of viviparity.

Yolk steroids and the development of the hatching muscle in nestling European Starlings

Hatching asynchrony in avian species often leads to the establishment of a size hierarchy among nestlings. Because the youngest nestlings in a brood are often at a significant disadvantage with respect to competition for parentally‐delivered food, one might expect to find that mechanisms have evolved to alleviate the detrimental effects of hatching asynchrony and enhance the survival of last‐hatched offspring. The musculus complexus, or hatching muscle, is responsible for breaking the shell during hatching and for dorsal flexion and extension of the neck during begging. An increase in its strength in last‐hatched nestlings could therefore reduce the duration of hatching or enhance the ability to beg for parentally‐delivered food, thereby enhancing the competitive abilities of those nestlings. I previously found in the Red‐winged Blackbird Agelaius phoeniceus that the mass of the complexus increases with laying order and that its development is mediated by the actions of yolk testosterone. I investigated the hypothesis that yolk steroids also affect the development of the complexus in the European Starling Sturnus vulgaris. I asked, first, whether the mass of the complexus increases with laying order and, if so, whether this increase is at least partially attributable to a corresponding increase in the concentration of yolk testosterone. Because exogenous progesterone increases the water content of the complexus in the domestic fowl Gallus gallus I also asked whether an increase in complexus mass, if present, could be partially attributable to an increase in progesterone concentration across the laying order. I found that both yolk testosterone concentration and the mass of the complexus increased with laying order; however, there was no variation in the concentration of either yolk progesterone or the proportion of water in the complexus across the laying order. There were no differences in the concentrations of either androstenedione or 5α‐dihydrotestosterone across the laying order.