Habituation to humans affects yolk steroid levels and offspring phenotype in quail

In the field as well as in the laboratory, human-generated stress responses are reduced in adult animals previously habituated to humans in comparison to non-habituated individuals. In birds, yolk steroid levels vary with maternal environment and condition. We tested the hypothesis that the experience of female birds with humans could affect yolk steroids levels and offspring phenotype. Two groups of Japanese quail, one habituated to humans (H) and a second non-habituated (NH), were exposed daily to brief human disturbances. We analysed egg quality, offspring growth, and offspring emotional reactivity. NH females produced eggs with less androgens (testosterone and androstenedione) and more immunoreactive progesterone compared to birds habituated to humans. NH females produced eggs with less yolk, heavier shell and chicks hatching later and being smaller as compared to habituated individuals. A lower emotional reactivity was found in young of NH females compared to young of H females. Thus, human disturbance of the mother triggered different effects on chick phenotype depending on previous experience of mother birds with humans. In addition, we describe for the first time the influence of environmental stimuli on yolk immunoreactive progesterone levels. Our results show that a relatively minor difference in behavioral habituation may have substantial effects on eggs and offspring. This has obvious implications for keeping and handling laboratory animals, for conservation biology and for animal welfare.     

Enhanced yolk testosterone influences behavioral phenotype independent of sex in Japanese quail chicks Coturnix japonica

Studies have demonstrated an effect of yolk testosterone levels on the physiology and behavior of nestling birds. In order to investigate this phenomenon experimentally in a precocial bird, we enhanced yolk testosterone, but within the physiological range, by injecting 50 ng testosterone in ethanol into Japanese quail Coturnix japonica eggs prior to incubation. The chicks hatching from these as well as from control eggs that had received the carrier-only or were left unmanipulated were subject to a number of behavioral tests from hatching to the age of 3 weeks. In addition, fecal samples were taken during a 90-min isolation period to determine a physiological response to a stressor. Experimental chicks performed a detour task faster and approached novel objects sooner than did the controls. Chicks from treated eggs took a longer time to start distress vocalizing and also produced less distress calls during open-field trials, took on average a larger number of trials for them to show tonic immobility and also excreted lower levels of corticosterone metabolites (BM) than did the controls. In response to a stressor, excreted BM was initially higher in the control chicks, as compared to the experimental birds. Induced behavioral effects were independent of sex with no sex treatment interactions found. In sum, experimentally enhanced testosterone levels in the eggs shifted individual behavioral phenotype towards "bold" or "proactive", irrespective of sex. We conclude that testosterone in the yolk influences the coping style of hatchlings and may be a potential means of maternal influence on offspring phenotype.     

Daily temporal organization of laying in Japanese quail: variability and heritability

In birds, many behavioral and physiological processes that occur during reproduction show daily rhythms in response to environmental temporal constraints. In this study, the individual daily organization of laying and its genetic determinant in Japanese quail (Coturnix coturnix japonica) were analyzed. For this purpose, the oviposition time of 102 randomly chosen females, maintained in long-day photoperiodic conditions (LD 14h:10h) for 1 mon was observed and extreme phenotypes selected. Laying is characterized by two parameters: oviposition interval and laying hour.

The birds showed a specific time of laying during 24 h. All eggs were laid in the afternoon between 6.5 and 14 h after lights on (HALO). Two laying profiles were determined: 20% of females with an oviposition interval greater than 24 h (24.7±0.2 h) (the “delayed” profile) laid progressively later each day until a pause day. The remaining 80% of the females laid at the same time each day, with few pause days and an oviposition interval close to 24 h (24.0±0.2 h) (the “stable” profile). Among the females, showing this last profile, an intra-individual stability and an inter-individual variability of laying hour was established. Two extreme laying phenotypes were then determined: the “early” phenotype (E) for females laying on average between 7.5 and 9.5 HALO and the “late” phenotype (L) for females laying between 12.5 and 14 HALO.

In order to study the genetic basis of the laying hour, three females of each extreme phenotype were selected and crossed with two different males. The E and L females produced 57 F1E and 42 F1L daughters, respectively. F1 females displayed both laying profiles. However, the proportion of females displaying a “delayed” profile was higher in the L line (50%) than in the E line (29.8%). For the “stable” daughters, artificial selection induced an advance in laying hour of 4.7% for the E line and a delay of 4.7% for the L line. Realized heritability was estimated at 0.5. Moreover, the laying hour of the daughters was correlated positively to that of the mothers (N=61;r=0.45). These results support the notion of heritability of oviposition time in Japanese quail.     

Vitellogenin transport and yolk formation in the quail ovary

Morphological and biochemical investigations were made on the yolk formation in ovaries of the quail Coturnix japonica. Morphologically, two ways of nutrient uptake were observed in follicles. In small oocytes of white follicles, vitellogenin (VTG) was taken up through fluid-phase endocytosis which was assisted by follicular lining bodies. The lining bodies were produced in follicle cells. They adhered to the lateral cell membrane, moved along the membrane in the direction of the enclosed oocyte and were posted to the tips of the microvilli. These tips, now with lining bodies, were pinched off from the main cell body, engulfed by indented cell membranes of the oocyte, and transported to yolk spheres. In large oocytes of yellow follicles, VTG and very-low-density lipoproteins (VLDL) were taken up through receptor-mediated endocytosis. The VTG and VLDL particles diffused through the huge interspaces between follicle cells, and once in oocytes were transported to yolk spheres via coated vesicles. Immunohistochemistry showed that the VTG resides on or near the surface of the follicle cell membrane at the zona radiata whereas the cathepsin D resides at or near the oocytic cell membranes. Tubular and round vesicles in the cortical cytoplasm of oocytes were also stained with both antisera, suggesting that these vesicles are the sites where the VTG is enzymatically processed by cathepsin D. Upon analysis by SDS-PAGE, a profile similar to that of yolk-granule proteins was produced by incubating VTG with a quail cathepsin D of 40 kD.     

Daily temporal organization of laying in Japanese quail: variability and heritability

In birds, many behavioral and physiological processes that occur during reproduction show daily rhythms in response to environmental temporal constraints. In this study, the individual daily organization of laying and its genetic determinant in Japanese quail (Coturnix coturnix japonica) were analyzed. For this purpose, the oviposition time of 102 randomly chosen females, maintained in long-day photoperiodic conditions (LD 14h:10h) for 1 mon was observed and extreme phenotypes selected. Laying is characterized by two parameters: oviposition interval and laying hour.

The birds showed a specific time of laying during 24 h. All eggs were laid in the afternoon between 6.5 and 14 h after lights on (HALO). Two laying profiles were determined: 20% of females with an oviposition interval greater than 24 h (24.7±0.2 h) (the “delayed” profile) laid progressively later each day until a pause day. The remaining 80% of the females laid at the same time each day, with few pause days and an oviposition interval close to 24 h (24.0±0.2 h) (the “stable” profile). Among the females, showing this last profile, an intra-individual stability and an inter-individual variability of laying hour was established. Two extreme laying phenotypes were then determined: the “early” phenotype (E) for females laying on average between 7.5 and 9.5 HALO and the “late” phenotype (L) for females laying between 12.5 and 14 HALO.

In order to study the genetic basis of the laying hour, three females of each extreme phenotype were selected and crossed with two different males. The E and L females produced 57 F1E and 42 F1L daughters, respectively. F1 females displayed both laying profiles. However, the proportion of females displaying a “delayed” profile was higher in the L line (50%) than in the E line (29.8%). For the “stable” daughters, artificial selection induced an advance in laying hour of 4.7% for the E line and a delay of 4.7% for the L line. Realized heritability was estimated at 0.5. Moreover, the laying hour of the daughters was correlated positively to that of the mothers (N=61;r=0.45). These results support the notion of heritability of oviposition time in Japanese quail.     

Daily organization of laying in Japanese and European quail: effect of domestication

In the present study, we aimed to examine the possible effects of domestication on the daily organization of laying in female quail. To achieve this we compared laying patterns in a laboratory strain of Japanese quail with those in European quail originating from a wild population. Under LD 14:10, the same specific laying window was observed, with ovipositions occurring mainly between 7 h and 15 h after lights on. Thus, domestication did not modify the specific daily organization of laying in females laying in the afternoon. Research about the functional value of this temporal organization in the field thus seems justified. At an individual level, two distinct laying profiles were recorded, as in a previous experiment on an industrial Japanese quail strain selected for meat and egg production. However, different frequencies were noted. Whereas stable profile females (laying at the same time each day) were predominant (80.4%, N=102) in the industrial Japanese quail strain, delayed profile females (laying successively later each day) appeared dominant in the European quail (81%, N=22). The Japanese quail from the laboratory strain constituted an intermediate group with a slight dominance of the stable profile (58.8%, N=17). Thus, domestication has changed the frequencies of the laying profiles, favoring stable females, which are the most productive birds. A model involving interaction between one circadian rhythm and one ultradian rhythm can explain the observed laying profiles.     

Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Effect of a cranium-directed daily illumination cycle on the laying rhythm in Japanese quail

This study tests the effect on the laying rhythm of a light cycle reaching directly the encephala via a diode in Japanese quail maintained in constant darkness. In DD, all the birds expressed their free-running laying rhythm (period close to 25 h). When the diode is switched on 14 h per 24 h cycle, females showed the same organization as in LD with the same laying time. Thus, a photoperiodic cycle, where light was only perceived through the skull of the female quail, could synchronize its laying rhythm. This device with a LED is an interesting alternative solution to eye-patching or blinding of birds.     

No sex difference in yolk steroid concentrations of avian eggs at laying

Yolk steroids of maternal origin have been proposed to influence genetic sex determination in birds, based on sex differences in yolk steroid concentrations of peafowl eggs incubated for 10 days. More recent reports dispute this proposal, as yolk steroids in eggs incubated for 3 days do not show such sex differences. To date, research examining this phenomenon has only analysed incubated eggs, although sex in avian species is determined before incubation begins. This may be a serious methodological flaw because incubation probably affects yolk steroid concentrations. Therefore, we investigated sex differences in yolk steroid concentrations of unincubated avian eggs. We withdrew yolk for steroid analysis from fresh, unincubated Japanese quail (Coturnix japonica) eggs by biopsy, and then incubated those eggs for 10 days, after which we harvested the embryonic material for genetic sexing and the incubated yolk for further steroid analysis. We found no sex differences in fresh Japanese quail eggs; however, sex differences were apparent in yolk steroids by day 10 of incubation, when female eggs had significantly more oestrogen in relation to androgen than male eggs. Concentrations of all yolk androgens decreased dramatically between laying and day 10 of incubation, whereas oestradiol (E2) concentrations increased marginally. Thus, yolk concentrations of androgens and E2 do not appear critical for avian sex determination.     

Maternal allocation strategies and differential effects of yolk carotenoids on the phenotype and viability of yellow-legged gull (Larus michahellis) chicks in relation to sex and laying order

Egg quality may mediate maternal allocation strategies according to progeny sex. In vertebrates, carotenoids have important physiological roles during embryonic and post-natal life, but the consequences of variation in yolk carotenoids for offspring phenotype in oviparous species are largely unknown. In yellow-legged gulls, yolk carotenoids did not vary with embryo sex in combination with egg laying date, order and mass. Yolk lutein supplementation enhanced the growth of sons from first eggs but depressed that of sons from last eggs, enhanced survival of daughters late in the season, and promoted immunity of male chicks and chicks from small eggs. Lack of variation in egg carotenoids in relation to sex and egg features, and the contrasting effects of lutein on sons and daughters, do not support the hypothesis of optimal sex-related egg carotenoid allocation. Carotenoids transferred to the eggs may rather result from a trade-off between opposing effects on sons or daughters.     

Pigmentation phenotype instability in Myxococcus xanthus.

Cells of Myxococcus xanthus FB2 produce tan or yellow colonies. Subcultures of tan colonies yielded tan and yellow colonies and subcultures of most yellow colonies yielded only yellow colonies. Strain FB2 variants in which the color type is more stable were obtained. Yellow cells were distinguishable from tan by the presence of pigment(s) with an absorption maximum at 379 nm. Fluctuation Test experiments and the presence of this pigment(s) in liquid cultures of FB2 indicated that tan phenotype cells spontaneously became or segregated yellow cells in liquid culture. The frequency of appearance of yellow cells was increased in low density cultures (less than 10(6)/ml). The increase cannot be explained by differences in growth rates of the two phenotypes. No evidence that cell-cell contact or culture medium constituents affect the appearance of the yellow phenotype was found. Ultraviolet irradiation of FB2 resulted in an increased proportion of cells producing yellow colonies among the survivors. Greater UV resistance of yellow cells and UV-induced conversion of tan to yellow accounts for this increase. Low level photoreactivation of viability and of the tan phenotype occurred. Incubation of FB2 in medium containing mitomycin C, nalidixic acid, phenethyl alcohol, or at 36.5 degrees C also resulted in conversion of tan to yellow cells.     

[Ciliogenesis in the mucous cells of the quail oviduct. I. Ultrastructural study in the laying quail]

The luminal epithelium of the oviduct (magnum) of laying quails is composed of ciliated cells and mucous cells. Ciliogenesis was observed in some of the mucous cells. Both centrioles of the diplosome migrate to the top of the cell, and one of them induces the formation of a rudimentary cilium. In some of the other cells, that are filled with mucous granules, the formation of basal bodies by an acentriolar pathway was observed. In these cells, numerous, dense fibrous masses are associated with the forming face of the Golgi apparatus. In the Golgi zone, generative complexes composed of a deuterosome and some forming procentrioles were found. Cilia develop from completed basal bodies. During ciliogenesis, the Golgi apparatus is disorganized, and generally the production of mucous granules is arrested. The nucleus is also modified: it becomes larger and the chromatin is dispersed. It is assumed that mucous cells are able to be transformed into ciliated cells in the oviduct of laying quails.     

Reproductive tradeoffs and yolk steroids in female leopard geckos, Eublepharis macularius

Life history theory predicts tradeoffs among reproductive traits, but the physiological mechanisms underlying such tradeoffs remain unclear. Here we examine reproductive tradeoffs and their association with yolk steroids in an oviparous lizard. Female leopard geckos lay two eggs in a clutch, produce multiple clutches in a breeding season, and reproduce for several years. We detected a significant tradeoff between egg size and the number of clutches laid by females during their first two breeding seasons. Total reproductive effort was strongly condition-dependent in the first season, but much less so in the second season. Although these and other tradeoffs were unmistakable, they were not associated with levels of androstenedione, oestradiol, or testosterone in egg yolk. Female condition and egg size, however, were inversely related to dihydrotestosterone (DHT) levels in egg yolk. Finally, steroid levels in egg yolk were not directly related to steroid levels in the maternal circulation when follicles were developing, indicating that steroid transfer to eggs is regulated. These findings suggest that maternal allocation of DHT could mitigate tradeoffs that lead to poor offspring quality (i.e. poor female condition) and small offspring size (i.e. small egg size).     

The effects of diet on plasma and yolk steroids in lizards (Anolis carolinensis)

Steroids present in egg yolk have been shown to vary as a resultof numerous social and environmental influences and to produceboth positive and negative phenotypic outcomes in offspring.In the present study, we examined how quality of the diet affectsplasma and yolk steroids in the green anole (Anolis carolinensis),a lizard species with genotypic sex determination. We documentedthe effects of body condition on plasma testosterone (T) andcorticosterone (CORT)—steroids with frequently opposingeffects—in breeding females and on the T and CORT contentof their eggs. We chose to manipulate body condition via dietbecause resource availability is a relevant, fluctuating variablein the environment to which females can be expected to respond.Field-collected females were housed in the laboratory and kepton either a reduced, standard, or enhanced diet (differing innutritional quality and/or quantity) for ten weeks. Althoughfemales did not differ in body condition at the beginning ofthe study, we found these diet regimes effective in producingfemales that differed in condition by the end of the study.Females on diets of enhanced quality were in better condition,produced more, but not heavier, eggs, and had higher plasmaT concentrations than did females on a standard diet or oneof reduced quality. There was also a significant positive relationshipbetween laying sequence of eggs and yolk T for females on dietsof enhanced quality, but not for the females on diets of standardor reduced quality. There were no effects of quality of dieton CORT in plasma or yolk, but yolk T and yolk CORT exhibiteda strong positive correlation irrespective of treatment. Femaleson diets of reduced quality did not differ from females on standarddiets either with respect to reproductive output or to endocrineprofiles, in spite of being in worse body condition. These resultsdemonstrate that females’ body condition, physiology,and reproductive output can be manipulated by quality of diet,and that changes in deposition of yolk steroids in responseto diet may be minimal.     

Toward a dynamic model of deposition and utilization of yolk steroids

The discovery by Schwabl that maternal steroid hormones aretransferred to the egg yolk and have effects on the phenotypeof offspring revealed a new pathway for non-genetic maternaleffects. The initial model relied on passive transfer. The thinkingwas that steroids passively entered the lipophillic yolk duringyolk deposition and then were deposited in the yolk until theywere passively delivered to the embryo as the yolk was used.Subsequent studies revealed that the system is much more dynamicthan that. Here, we explore questions about how dynamic thesystem really is and look at questions like: Is transfer ofmaternal steroids to the yolk passive or is it actively regulated?At what stages of the maternal reproductive cycle are steroidstransferred? During reproduction, how dynamic are the levelsof yolk steroids? Especially in the case of potentially deleterioussteroids (e.g., androgens in female offspring; glucocorticoids),once deposited can they come out of the yolk over time? Canthey be metabolized by the yolk or by the embryo? During incubation,how much do steroid levels in the yolk change? Can steroidsdiffuse from the yolk to the embryo prior to yolk utilization?Does the embryo contribute to yolk steroid levels as it develops?We believe that comprehensive answers to questions like thesewill eventually allow us to generate a much more accurate andcomplete model of the transfer and utilization of yolk steroidsand that this model will be much more dynamic and active thanthe one initially proposed.     

Effects of divergent selection for yolk testosterone content on growth characteristics of Japanese quail

Effects of yolk androgens on postnatal growth of offspring have been widely studied but their physiological role in the growth control is not fully understood due to an inconsistency in obtained results. We investigated androgen-mediated maternal effects on postnatal growth in relation to endocrine control mechanisms using two lines of Japanese quail divergently selected for high (HET) and low (LET) egg testosterone (T) content. Embryonic growth did not differ between the lines. During the growth period HET quail were heavier and displayed longer tarsi as compared with LET quail, with more pronounced line differences in males than females. HET males were heavier than LET males from the age of 2 weeks, reached the age of maximum growth rate earlier, and displayed higher asymptotic body weight than LET males. Accelerated growth in HET males was not accompanied by increased postembryonic plasma T concentrations. Plasma triiodothyronine levels did not differ between lines while plasma thyroxine levels were decreased in HET as compared with LET female chicks. Line differences in body weight disappeared in adult quail suggesting that yolk androgens, increased in a physiological way, resulted in stimulation of juvenile growth rate in precocial Japanese quail under stable social and environmental conditions.     

Genome instability: a mechanistic view of its causes and consequences

Genomic instability in the form of mutations and chromosome rearrangements is usually associated with pathological disorders, and yet it is also crucial for evolution. Two types of elements have a key role in instability leading to rearrangements: those that act in trans to prevent instability--among them are replication, repair and S-phase checkpoint factors--and those that act in cis--chromosomal hotspots of instability such as fragile sites and highly transcribed DNA sequences. Taking these elements as a guide, we review the causes and consequences of instability with the aim of providing a mechanistic perspective on the origin of genomic instability.     

Effects of adrenocortical steroids on long-term potentiation in the limbic system: Basic mechanisms and behavioral consequences

Summary Hippocampal structures are a major target for adrenal steroid hormones, and hence these neural regions are some of the most likely mediators of the effects of adrenocortical steroids on behavior. Memory disturbance, in particular biasing toward negative contents, are part of the symptomatology presented by depressive patients. In turn, a sizeable subset of depression also presents with hypercortisolemia. Adrenocortical hormones are also known to affect memory processes. Hippocampal formatio is essential for declarative memory. We thought it appropriate then to study the effects of adrenal steroids on long-term potentiation, a putative memory mechanism in the hippocampus. Two clearly distinguished components of the evoked response to perforant path stimulation can be studied in the hippocampus: the excitatory postsynaptic potential (EPSP) which denotes the graded depolarization of the somadendritic region of the neuron and the population spike (PS), a manifestation of the all-or-none-discharge of the cell action potential. Corticosterone had a significant depressant effect on the EPSP component of the evoked response immediately and 15 min after injection. Thereafter EPSP amplitudes were within normal values. Corticosterone significantly decreased the PS immediately after the train, the component remaining low 30 min after the train. 5-Dihydrocorticosterone (a ring A-reduced metabolite of corticosterone) significantly reduced the PS component of the response at all times after injection. 18-Hydroxydeoxycorticosterone and deoxycorticosterone significantly decreased both EPSP and PS components of the evoked response from the time of infusion. Contrary to expectation, tetrahydrodeoxycorticosterone was ineffective in decreasing and if anything, enhanced the development of long-term potentiation. 18-Hydroxydeoxycorticosterone 21-acetate behaved like vehicle, except for the first 30 min after injection when the EPSP was decreased. Allotetrahydroprogesterone decreased all EPSP's values and had no effect in the PS development in comparison with vehicle. The suggestion is made that the study of steroidal effects on hippocampal LTP can serve as a preclinical model of some aspects of depression in a specific subset of the disease.