Vivian C. Goerlich

Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken.

Stress during early life can profoundly influence an individuals phenotype. Effects can manifest in the short-term as well as later in life and even in subsequent generations. Transgenerational effects of stress are potentially mediated via modulation of the hypothalamic-pituitary-adrenal axis (HPA) as well as epigenetic mechanisms causing heritable changes in gene expression. To investigate these pathways we subjected domestic chicken (Gallus gallus) to intermittent social isolation for the first three weeks of life. The early life stress resulted in a dampened corticosterone response to restraint stress in affected birds and in their male offspring. Stress-specific genes, such as early growth response 1 (EGR1) and corticotropin releasing hormone receptor 1 (CRHR1), were upregulated immediately after restraint stress, but not under baseline conditions. Treatment differences in gene expression were also correlated across generations which indicate transgenerational epigenetic inheritance. In an associative learning test early stressed birds made more correct choices suggesting a higher coping ability in stressful situations. This study is the first to show transgenerational effects of early life stress in a precocial species by combining behavioral, endocrinological, and transcriptomic measurements.

Testosterone has a long-term effect on primary sex ratio of first eggs in pigeons-in search of a mechanism

Despite accumulating evidence that birds, in which females are the heterogametic sex, are able to manipulate primary offspring sex ratio, the underlying mechanism remains elusive. Steroid hormones, which govern female reproduction and are also accumulated by the developing follicle could potentially affect primary sex ratio by differential follicle development in relation to future sex and meiotic drive, or by sex specific influence on oocyte abortion or fertilization. So far, experimental results on the involvement of maternal testosterone (T) in offspring sex manipulation are ambiguous. To investigate the effect of T on primary sex ratio and elucidate underlying mechanisms, we elevated circulating T levels in female homing pigeons (Columba livia). During the course of the experiment females produced three clutches--before and during T implantation, and one year after implant removal. Intriguingly, first eggs, but not second eggs of T females were significantly male biased relative to sham-implanted controls. One year after cessation of the treatment the male bias was still present, indicating long-term effects on female reproductive physiology. T treatment did not affect maternal body condition, nor was body condition correlated with offspring sex ratio. Our data on timing of oviposition, lack of infertile eggs, and yolk weight indicate a possible role for sex specific follicle abortion, perhaps in combination with meiotic drive. However, despite T treatment elevating maternal plasma levels, egg yolk T concentrations did not differ between treatment groups and did not vary with embryo sex, suggesting that yolk T is not involved in meiotic drive.

Differential effects of testosterone, dihydrotestosterone and estradiol on carotenoid deposition in an avian sexually selected signal

Recent studies have demonstrated that carotenoid-based traits are under the control of testosterone (T) by up-regulation of carotenoid carriers (lipoproteins) and/or tissue-specific uptake of carotenoids. T can be converted to dihydrotestosterone (DHT) and estradiol (E2), and variation in conversion rate may partly explain some contradictory findings in the literature. Moreover, most studies on the effect of T on sexual signals have focused on the male sex only, while in many species females show the same signal, albeit to a lesser extent. We studied the effects of T, DHT, and E2 treatment in male and female diamond doves Geopelia cuneata in which both sexes have an enlarged red eye ring, which is more pronounced in males. We first showed that this periorbital ring contains very high concentration of carotenoids, of which most are lutein esters. Both T and DHT were effective in enhancing hue, UV-chroma and size in both sexes, while E2 was ineffective. However, E2 dramatically increased the concentration of circulating lipoproteins. We conclude that in both sexes both color and size of the secondary sexual trait are androgen dependent. The action of androgens is independent of lipoproteins regulation. Potential mechanisms and their consequences for trade-off are discussed.

Food conditions affect yolk testosterone deposition but not incubation attendance

In many bird species with hatching asynchrony, yolk androgens increase across the laying sequence. This has been hypothesized to represent a compensatory mechanism for disadvantages of later-hatching chicks - via positive effects of yolk androgens on early competitiveness and growth. However, the costs and benefits of this compensatory strategy probably depend on environmental factors determining the survival chances of the chicks such as the food conditions, which should, therefore, influence maternal yolk androgen deposition. We studied the consequences of manipulated food conditions on the expected level of hatching asynchrony in canaries (Serinus canaria) assigning females to either a low (=LQ) or high quality (=HQ) diet. We measured the incubation behaviour (as incubation attendance) and the yolk androgen deposition in order to investigate whether and how females modulate hatching asynchrony in relation to the food conditions. Females on a HQ diet laid larger and heavier clutches, showed a stronger increase in yolk testosterone content towards the last-laid eggs, but did not alter their incubation attendance. Thus, females on a HQ diet seem to favour the survival of later hatching chicks, as indicated by their yolk testosterone deposition pattern. However, females on a HQ diet laid larger clutches and might need to compensate more in order to achieve a similar degree of hatching asynchrony than females on a LQ diet, given the lack of plasticity in incubation attendance. This suggests that canary females respond to food manipulations mainly via changes in clutch size rather than by altering the degree of hatching asynchrony.

Change in Body Mass Can Overrule the Effects of Maternal Testosterone on Primary Offspring Sex Ratio of First Eggs in Homing Pigeons

The phenomenon of primary offspring sex ratio adjustment is being extensively studied, yet knowledge of the underlying proximate mechanism is still mainly hypothetical. Female birds are the heterogametic sex, thus potentially controlling the sex of the gamete to be fertilized. In several bird species, independent studies showed effects of maternal plasma testosterone, corticosterone, or condition on primary offspring sex ratio. Our objective was to investigate the causal relation between these two maternal hormones, body condition, and offspring sex ratio in homing pigeons (Columba livia domestica). Following our earlier study, we again implanted females with testosterone and determined embryo sex of first eggs. To identify the pathway of sex ratio adjustment, we repeatedly measured not only maternal plasma testosterone and mass but also plasma corticosterone, cholesterol, and glucose, all indicators of body condition. We also calculated the temporal change in these parameters, which has been proposed to be a more accurate predictor of offspring sex ratio compared with the absolute values. Furthermore, we analyzed testosterone concentrations in outer yolk layers, which potentially influence the first meiotic division of the gamete. We found no relation between plasma parameter and embryo sex of first eggs; testosterone treatment did not affect any of the measured parameters. However, females that increased in mass produced more male embryos, irrespective of treatment group. Outer yolk layer testosterone concentrations did not differ between treatment groups or between male and female eggs. We propose that not only the absolute values but also the complex interactions between maternal hormones and body condition ultimately affect the mechanism of primary offspring sex manipulation.

Effects of In Vivo Testosterone Manipulation on Ovarian Morphology, Follicular Development, and Follicle Yolk Testosterone in the Homing Pigeon

To date, our understanding of the function of testosterone in female reproductive physiology is only marginal although there are indications that testosterone is involved in modulating follicular recruitment, growth, atresia, and ovulation. Studies elevating testosterone in breeding female birds have, in most instances, found detrimental effects, such as delayed clutch initiation or decreased clutch size. In our previous study, testosterone treatment of female homing pigeons delayed clutch initiation without diminishing fecundity. In this study, we explore whether the observed effect might have been caused by testosterone influencing follicle maturation or ovulation. We implanted mature female pigeons with testosterone prior to pairing, which resulted in constant elevation of circulating testosterone concentrations within the physiological range. We killed females after they had laid the first egg and measured ovarian and follicular development. Ovarian mass and pre-hierarchical yolky follicles were not affected by the treatment; however, testosterone females produced smaller and lighter preovulatory follicles. High plasma testosterone levels at oviposition or a strong temporal increase in testosterone were negatively related to mass and diameter of second follicles. We proposed that sustained elevation of testosterone delays follicular maturation, potentially via negative feedback on the hypothalamo-pituitary-gonadal axis. Furthermore, to gain better insight into the regulation of yolk hormone acquisition, we measured testosterone concentrations in the preovulatory follicles. We found no differences between treatment groups but follicle yolk contained much higher levels of testosterone than yolk of un-incubated eggs, suggesting that hormone measurements performed after oviposition do not correctly reflect maternal allocation.

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.

Sources of variation in yolk hormone deposition: Consistency, inheritance and developmental effects

Maternal effects occur when the phenotype of the mother affects the phenotype of their offspring. They are thought to have evolved to translate the environmental conditions experienced by the mother into adaptive phenotypic variation of the offspring. However, the integration of environmental cues allowing adaptive responses requires some form of plasticity that depends on the interaction of the maternal phenotype and her environment. In birds, maternal yolk hormones represent such a pathway for maternal effects, and their adaptive significance depends thus on the plasticity in maternal yolk hormone deposition. We studied sources of variation in yolk testosterone deposition, focusing on the often neglected contribution of the (partly heritable) maternal phenotype. We investigated consistency and heritability of yolk testosterone deposition in captive canaries of which the F(1) generation was raised in foster nests and analyzed the potential effects of the early developmental conditions. We found significant female consistency across years in egg mass, yolk mass and total amount of yolk testosterone but not in yolk testosterone concentrations. Females varied the yolk testosterone concentrations of their eggs across years mainly via changes in yolk mass. The heritable variation in egg mass, yolk mass and amount of yolk testosterone but not yolk testosterone concentrations was within the range of previous studies, but not significantly different from zero. Finally, the growth of the daughters as nestling had a significant effect on their yolk testosterone deposition at adulthood indicating the transgenerational potential for environmental effects - via the effects of yolk hormones on offspring development.

No evidence for selective follicle abortion underlying primary sex ratio adjustment in pigeons

Primary sex ratio adjustment in birds has been extensively studied, yet the underlying physiological mechanisms are far from understood. Avian females are the heterogametic sex (ZW), and the future sex of the offspring is determined at chromosome segregation during meiosis I, shortly before the oocyte is ovulated. Assuming that the mother can detect the sex of the developing oocyte before ovulation, it has been suggested that a follicle of the un-preferred sex could selectively be induced to become atretic and regress instead of being ovulated (selective follicle abortion). This potential mechanism has been proposed to underlie biased primary sex ratios in birds, including the homing pigeon (Columba livia domestica), which produces a modal clutch size of two eggs. However, without replacement by an additional, already mature follicle, abortion of a preovulatory follicle would most likely result in either reduced clutch sizes or laying gaps, since a not-yet-recruited follicle still needed to undergo the whole maturation phase. In the current study we killed female pigeons, which were adjusting embryo sex of first eggs according to change in body mass. We examined ovaries for signs of follicle abortion but did not find any supporting evidence. All females produced one or two mature follicles but only two out of the 56 experimental birds produced an additional third mature follicle. Therefore, our results do not corroborate the hypothesis that pigeon mothers manipulate primary offspring sex by selectively aborting follicles of the un-preferred sex.