Reproductive consequences of EDCs in birds

Neurotoxicology and Teratology 24 (2002) 17 – 28

Reproductive consequences of EDCs in birds What do laboratory effects mean in field species?$ Mary Ann Ottinger*, Mahmoud Abdelnabi, Michael Quinn, Nancy Golden, Julie Wu, Nichola Thompson Department of Animal and Avian Sciences, Room 3113, Animal Sciences Center, University of Maryland, College Park, MD 20742, USA Received 1 July 2001; received in revised form 25 August 2001; accepted 26 October 2001

Abstract The varied reproductive strategies of birds present a challenge in developing reliable indices for the assessment of effects of endocrine disrupting chemicals (EDCs). Precocial species, such as quail, appear to be most sensitive to EDC effects during embryonic development. Although the Japanese quail (Coturnix japonica) is a nonnative lab species, its reproductive strategy is similar to that of many free-ranging species. Because a great deal is known about the reproductive biology of this species and Japanese quail have a short generation time, this species is an ideal candidate for testing EDC effects. In this review, we present data collected in a two-generation design with embryonic exposure to estradiol benzoate (EB). This study was conducted to provide fundamental information for establishing reliable reproductive endpoints associated with estrogenic EDC exposure. Data were collected for a variety of endpoints, which were chosen as measures of reproductive capability and success. These reproductive fitness measures included fertility, hatching success, and offspring viability. Endocrine measures consisted of plasma hormone levels and gonad weight/condition. Neuroendocrine systems, such as the monoamine neurotransmitter systems, regulate hypothalamic gonadotropin releasing hormone (GnRH) and reproductive behavior. Therefore, these variables should potentially be very sensitive indicators. Behavioral measures included reproductive behavior. Results showed that embryonic estradiol exposure affected endocrine and behavioral responses in males and impacted productivity in females. Therefore, quails provide an excellent model to determine fundamental actions of EDCs. The laboratory trials then serve as a basis for the extrapolation of findings of controlled laboratory studies to effects that may be observable in free-ranging species. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Avian model; Endocrine disrupting chemicals; Endpoints; Biomarkers

1. Introduction Understanding and assessing the impact of endocrine disrupting chemicals (EDCs) is a difficult issue. Not only is there a vast array of compounds that are suspected EDCs, but also the mechanisms of action for these compounds often differ. Moreover, differences in response and sensitivity to specific compounds are expected across species and phyla. This is especially relevant when considering variations in reproductive processes and strategies, which also translate into species differences in developmental patterns. $ Supported in part by US EPA Grant R826134 and NSF #IBN 9817024 (MAO). * Corresponding author. Tel.: +1-301-405-1366; fax: +1-301-3149059. E-mail address: [email protected] (M.A. Ottinger).

Often, the phase of the life cycle that is most sensitive to effects of EDC exposure will also vary with the timing of these ontogenic patterns and events. As a result, it becomes extremely complex to develop clear indices of EDC impact that apply across a number of species, even in one phylum. Considerable data exist across phyla and the reader is referred to a number of review papers for more information [9,25,28,34,35,49,51,69,72,100]. The problem of environmental contaminant effects on avian reproduction first came to light with the recognition of DDT effects on eggshell thinning in eagles. More recently, both toxicological and endocrine disrupting effects have been described for a variety of chemicals, including pesticides, herbicides, industrial products, and plant phytoestrogens. Because there is tremendous variation in reproductive strategies and developmental processes across avian species, it is essential to develop reliable indices of exposure to

0892-0362/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 9 5 - 7

18

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

Fig. 1. Diagrammatic representation of the relationship between laboratory trials and application for avian species in the field.

EDCs to assess potential risk to birds in the wild. Although the models for this type of risk assessment are as yet unclear, it is critical to develop specific endocrine and behavioral measures, which provide some basis for regulatory decisions. In this review, we will consider avian species. The primary focus of this review will be on laboratory trials in which fundamental data have been collected on the effects of estrogen exposure in the Japanese quail model. Although these experiments have been conducted with treatment via egg injection, a low dose of estradiol has been used. In addition, a great deal of information exists in the poultry and other literature that describe differential porosity of the eggshell. Therefore, chemicals pass through the pores in the eggshell and in fact, early experiments by Glick and others used dipping the egg in a solution as a method for administration of hormone treatments. This mode of exposure is relevant for field species as pesticides and other chemicals, which are sprayed or dusted on crops, would be likely to contact the eggshell. We will overview the relevant literature for the Japanese quail and discuss the potential application of responsive endpoints as indices. Finally, a discussion of biomarkers that may be useful in the field is presented. The relationship of laboratory trials to field applications is presented in Fig. 1.

2. Avian reproductive characteristics There are dramatic differences, both in reproductive strategies and lifetime reproductive patterns across avian species. The greatest differences in developmental characteristics may be observed in precocial compared to altricial birds (for a comprehensive survey, see Ref. [104]). In the case of precocial birds, the hatchling is relatively well developed. Most of the ground dwelling birds are precocial species and include such species as quails, ducks, geese, and others. In contrast, passerine birds, collectively termed songbirds, are generally altricial, with hatchlings dependent on intensive parental care for some period of time often postfledging. Many species, especially the songbirds, exhibit a great deal of neural flexibility throughout their life. As a result, many studies have been conducted on the effects of steroid hormones on the organization and function of the song system. These studies indicate that sexual differentiation in songbirds may involve both steroid as

well as nonsteroidal signals [4,5,11,12,44,110]. Moreover, plasma testosterone, 5adihydrotestosterone, and estradiol levels change in a similar manner in male and female zebra finches in late embryonic and early posthatch birds [6]. Precocial species, such as Japanese quail, appear to have a much more limited critical period in which gonadal steroids affect sexual differentiation of the brain. Conversely, altricial species have a more gradual developmental period in terms of sexual behavior and song. As such, many of these birds appear to be affected by steroid hormones throughout their life. It should be noted, however, that gonadal differentiation may progress in a manner similar to the precocial species. Therefore, the response of altricial species to EDCs may be more difficult to characterize due to this flexibility, especially in the song system. It may ultimately be that passerines are less likely to be impacted by EDCs as embryos, but may be sensitive to EDC effects throughout life. This would mean that sporadic exposure to EDCs might temporarily impair reproductive function more in an adult passerine compared to a precocial species. Conversely, if a precocial species is EDC-exposed as an embryo or perinatally, these birds may suffer permanent reproductive impairment. Therefore, it is important to determine brain versus gonad effects and compare differential responses in precocial and altricial species; research is needed in this area. Japanese quail show evidence of a defined timing in the events that result in sexual differentiation of the reproductive system and behavior. That is, gonadal sex and sexspecific endocrine and behavioral responses are primarily organized during embryonic development. As a result, there is a window of time or critical period(s) in which gonadal sex, behavior, or endocrine responses would be the most sensitive to the hormone-like action of EDCs. Our purpose in this review is to identify specific endpoints that are likely to be meaningful in the assessment of EDC effects in an avian model. Many of the suspected EDCs are estrogenic in their action. Therefore, we will present and discuss studies in which embryonic steroid hormone exposure has been conducted in Japanese quail. These experimental results will be discussed in light of responsive endpoints that may be considered as indices for exposure to EDCs. Finally, we will discuss the applicability of these findings for field birds. 2.1. Sexual differentiation in galliformes Mammals and birds differ in that the avian female is the heterogametic sex, ZW (female) vs. ZZ (male). Because this appears to be the opposite of mammals, it has been suggested that sexual differentiation is also the reverse of the process in mammals. This means that the avian female would require estradiol exposure at appropriate stages in development in order to later exhibit endocrine and behavioral responses appropriate for the female. Conversely, the avian male should be the ‘‘neutral sex’’ and, as such, would

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

not need any hormonal influence for sexual differentiation. Unfortunately, this process does not appear to be quite as simple as originally hypothesized for birds. In songbirds, there may be two separate processes required for sexual differentiation of the song system, behavior, and the endocrine axis. The specific process and mechanisms involved in sexual differentiation in passerines is under intense investigation and there are a number of excellent reviews available in this area [5,11]. There are considerable data available for galliform species because of the importance of these animals in production agriculture. The Japanese quail has been utilized as a smaller galliform model for study of avian physiology, including reproductive, thyroid, and immune system function. This precocial species has a number of advantages as a model (Fig. 2). 2.1.1. The hypothalamic – pituitary– gonadal axis (HPG) in Japanese quail The avian reproductive axis functions in a similar manner to that in mammals and other phyla (Fig. 3). The hypothalamus, regulated by neurotransmitters and neuropeptides, synthesizes and secretes gonadotropin releasing hormone-I (GnRH-I). GnRH-I stimulates pituitary gland gonadotrophs to produce luteinizing hormone (LH) and follicle stimulating hormone (FSH), which in turn stimulate gonadal function and ovulation in the female. Gonadal steroids act in a feedback manner, primarily to the hypothalamic systems and also to the pituitary gland. During development, each level of the reproductive (HPG) axis develops and begins to function as early as embryonic day 5 (gonadal differentiation), embryonic day 12 – 14 (hypothalamus and pituitary gland), or later in embryonic development (accessory structures). The HPG axis initiates function midway in embryonic development in both chickens and quail. Observations by Woods et al. [117 – 121] provided a detailed profile of developmental events in the domestic chicken. They found plasma steroid hormones, including estradiol (E2) and androgen (A), follow sexually dimorphic patterns in the domestic chicken with circulating androgen levels peaking at 13.5 days of incubation in males, whereas estradiol levels slowly increase in females until hatching [117,119 – 121]. The relative concentration of E2 was higher in females, whereas males generally had higher androgen levels relative to estradiol [117,120]. Based on hormonal patterns, the HPG axis was functional between 11.5 and 14.5 days of embryonic age [118].

Fig. 2. Advantages of Japanese quail as a model system.

19

Fig. 3. The HPG axis.

Pituitary gland production of LH and FSH mirrored these hypothalamic responses [47,120]. In Japanese quail, similar patterns were observed during the 17-day incubation relative to plasma steroid hormones and initiation of the HPG axis [64]. Females have generally higher relative levels of E2/A, whereas males tend to have relatively low E2/A throughout embryonic development [1,74,80]. In quail, the developmental hormonal profiles also differ between males and females; in the female embryo, endogenous plasma E2 rose until hatch, and decreased posthatch [1,75,80]. In males, plasma androgen peaked at embryonic day 14 –17 and declined posthatch [76]. This observation was particularly interesting because the testes did not show microscopic evidence of steroidogenesis, meaning that lipid droplets and smooth ER were not apparent in the Leydig cells, similar to observations from other labs [97]. Conversely, the female has relatively high levels of gonadal steroids and the ovary appears steroidogenic at the microscopic level [1,75,80]. In subsequent experiments, both gonadal and adrenal steroid content was measured in an effort to determine the major source of these steroids. This was based on the observation that in the chick embryo there is significant steroid production by the adrenal gland and that there was a shift in steroid production with hatching [106,107]. The adrenal gland also contained substantial amounts of steroids throughout embryonic development, and levels remained relatively constant [1]. 2.2. Organization and regulation of sexual behavior The male Japanese quail has clear courtship and mating behaviors that are primarily modulated by specific areas of the central nervous system, primarily the preoptic area [114,115]. Because this area is known to be important in modulating courtship and mating behavior, a number of studies have focused on the neural systems contained in this area [82,83]. In addition, the preoptic –septal region also contains many of the gonadotropin releasing hormone (GnRH) cell bodies and the cells project to the median eminence of the hypothalamus, making this region critical in the regulation of both endocrine and behavioral components of reproduction over the entire life cycle [73]. Therefore, it

20

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

is important to study neural systems that become clearly sexually dimorphic because it is these systems that are likely to be affected by EDC exposure. Sexual behavior in the male Japanese quail is androgen dependent. That is, sufficient circulating concentrations of testosterone must be available to stimulate behavioral areas in the brain [77 –79]. In male Japanese quail, the preoptic area modulates sexual behavior. Metabolism of testosterone to estradiol by aromatase enzyme (AROM) in the brain is required for male behavior [83]. Male embryos and adults have higher hypothalamic AROM activity compared to females [13,41,58,83]. It is highly associated with reproductive status and the decline during aging is associated with a loss of sexual behavior and decreasing plasma androgen levels; exogenous testosterone restored both AROM and sexual behavior in old males [31,83]. Sexually dimorphic patterns in AROM are also observed in the gonads, which have high levels of p450 aromatase enzyme in the ovary, but not in the testes during embryonic development [32,101]. In addition, treatment with fadrozole or estradiol at embryonic day 3 resulted in altered expression of p450 17ahydroxylase and p450 aromatase in the gonads, thereby providing evidence for a hormonal regulation of these early enzyme activities in a sexually dimorphic manner [70]. Neurotransmitter systems are critical in the regulation of both endocrine and behavioral components of reproduction. In quail, the norepinephrine (NE) system stimulates GnRH-I as well as male sexual behavior [64 – 66,73]. Catecholamine levels, including NE and dopamine (DA) are sexually dimorphic in the embryonic brain during development; females have relatively higher NE and males have relatively higher DA concentrations [75]. Further, embryonic estradiol exposure resulted in long-term alterations in catecholamine levels in adult quail. Similarly, prenatal steroid treatment altered serotonergic systems in adult male and female quail (Abdelnabi and Ottinger, manuscript under revision). These data provide evidence for long-term effects of embryonic steroid exposure on later status of these neural systems. Moreover, males exposed to estradiol or testosterone as embryos are feminized; that is, they show diminished male sexual behavior [1,3,7]. Further, these studies have shown that the sensitive period for steroid exposure is between embryonic days 10– 12. Early steroid exposure or treatment with fadrozole or tamoxifen also affects gonadal development, producing defeminization of the ovary and accessory structures [32,85,92,101,105]. In summary, Japanese quail use relative concentrations of androgen and estradiol to provide the appropriate signals for sexual differentiation of both endocrine and behavioral responses. The timing of sensitivity to endogenous steroids also defines the time of greatest probable sensitivity to EDC impact. Altricial passerine species may be less sensitive to EDC effects during embryonic development, but are likely to be affected to some extent throughout life. Conversely, precocial species appear to be exquisitely

sensitive to consequences of EDC exposure during a critical period in embryonic development [2]. Therefore, the timing of exposure and the target(s) for EDCs determine the specific consequences of EDC exposure for an avian species.

3. Assessing the potential for EDC impact: laboratory studies Toxicologists have taken advantage of the accessibility and relative isolation of the avian embryo in testing a variety of compounds as well as exposure to the embryo by maternal deposition [54,57,88 – 90]. Studies have shown that diethylstilbestrol and ethinyl estradiol exposure result in the formation of an ovotestis or feminization of the testis along with long-term effects on reproductive processes [17,53]. In our laboratory, several studies were conducted to understand the potential mode and types of effects of EDCs on Japanese quail. One study was based on a previous experiment in which Japanese quail hens were given estradiol by daily injection or by implant; egg yolk estradiol content was then determined from eggs produced by these hens [6]. Results showed that estradiol transferred into the yolks of these eggs. As a result, we have recently measured endogenous steroid hormone in the yolk. This study revealed that both androgen and estradiol were measurable, even in the first week of incubation. During this week, the highest (19.93±8.60 ng/g) and the lowest (3.76±1.84 ng/g) levels of androgen were observed at embryonic days 3 and 7. The highest (8.08±3.85 ng/g) and the lowest (0.21±0.05 ng/g) concentrations of 17b-estradiol were observed at embryonic days 3 and 5 of the incubation period, respectively. Levels of yolk steroid hormones in the last half of incubation reflected circulating plasma levels in the embryo, suggesting that the yolk may serve as a depot for steroid hormones during embryonic development (Abdelnabi and Ottinger, unpublished data). 3.1. Effects of estradiol exposure during embryonic development In this section, original data will be presented that was collected in a two-generation design. Although a great deal is known about the behavioral and morphological effects of steroid exposure in quail, experiments have not aplproached these studies measuring endpoints of interest to toxicologists. Therefore, this study was conducted using estradiol benzoate (EB) as the chemical under examination with measurements that include endpoints of interest for EDC impact. 3.1.1. Experimental design and data collected This study assessed the response of endocrine and behavioral endpoints to EB, as a positive control for estro-

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

21

Fig. 4. Egg production and fertility in P1 pairs.

genic EDCs. In the parent generation (P1), birds were injected at Day 11 of embryonic development. These birds were raised, males were tested for sexual behavior and then paired with females from the same treatment, and then the pairs were monitored for egg production and fertility. Chicks from these pairs were also treated. However, some of the offspring (F1) from treated pairs were either EBinjected (same treatment as their parents) or not treated (given the vehicle only). Specifically, in the P1, fertile quail eggs (n = 50/treatment) were injected with EB (20 mg/egg; oil controls; uninjected controls) on Day 11 of incubation. As mentioned above, birds were monitored during sexual maturation; fertility was measured in pairs of treated or control birds. The number of animals monitored varied with the measure; most variables had n = 10/treatment/sex. In addition, 10 control and 10 treated males were photoregressed, testosterone implanted, and behaviorally tested. This allowed assessment of sexual behavior in males with the same circulating levels of testosterone, in case the treatment altered endogenous androgen levels. Also, as discussed above, in the F1 generation offspring from treated parents were either treated with EB or left as uninjected controls (20 mg/egg; n = 50 EB treated, n = 50 uninjected controls) on Day 11 of incubation. Sexual maturation, sexual behavior, fertility, egg production, and other parameters were monitored similar to the P1 birds.

3.1.2. Results Results showed reduced fertility and egg production in treated P1 pairs compared to both control groups (Fig. 4). EB impacted both males and females as shown in the figure. When birds were paired according to treatment, there were significant ( P < .05) decreases in both egg production and fertility. When treated females were paired with control males, they still showed lower egg production, as expected. These EB – control pairs also showed fertility that was intermediate between the treated and control P1 pairs, which suggests that the EB treatment impacted fertility through the females’ reproductive system. As shown below, control P1 females would not tolerate EB-treated males. Thus, fertility of treated P1 males alone was not assessed. A number of additional parameters were also determined in this study (Table 1). These results showed that the EB-treated P1 females were significantly ( P < .05) later in initiation of egg production. Similarly, the EB-treated F1 females were slower to begin egg production as a group. This may be seen at the ages when hens reached either 20% or 50% of their final laying rate. As shown below, control or oil-treated females reached 20% of their final egg production rate (#eggs/day/hen) at 56 and 52 days of age. However, EB-treated females did not reach their 20% of final laying rate until they were 67 days of age. A similar delay was also seen later when the age at

Table 1 Effects of embryonic estradiol benzoate on reproductive parameters Treatment Sex Females

Males

Trait Age at first egg (n = 15 – 30/trt) Age at which 20% lay (n = 15 – 34) Age at which 50% lay (n = 15 – 34) Lay rate (%; n = 14 – 34) Ovary weight (%; n = 14 – 16) Body weight (g; n = 14 – 16) Age at cloacal foam production (n = 23 – 33) Cloacal gland area (mm2; n = 6 – 9) Body weight (g)

Control

Oil injected a

53±0.72 56 60 81±2.5a 3.9±0.2a 152±2.6a 49±0.7a 177±16a 116±2

a

51±0.95 52 56 81±4.1a Not determined Not determined 48±1.0a 178±13a 115±6

n shown in the parenthesis. Significant differences in a row ( P < .05) denoted by different letters (no statistics conducted for age at which 20% or 50% lay achieved).

EB injected 61±1.7b 67 74 52±8.9b 4.0±0.5a 143±2.6b 49±2.0a 172±8a 114±2

22

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

Fig. 5. Sexual behavior in control and treated P1 male quail (all birds were photoregressed and testosterone implanted prior to testing for sexual behavior).

attaining 50% of their final egg production rate was calculated. Moreover, the overall laying rate (%/hen/treatment) differed ( P < .05) between EB-treated and control or oil-treated controls. The EB-treated P1 females were also lighter. In males, the accessory androgen-dependent cloacal gland did not differ with treatment. Similarly, the onset of cloacal gland foam was similar across groups. Therefore, accessory structures and peripheral gland function were not affected by EB when administered at Day 11 of embryonic development. This is expected because the gonads are sexually differentiated by embryonic day 5 and the accessory structures are likely to also be differentiated by the time that EB treatment occurred in this experimental design. In addition, EB-treated males showed drastically reduced mating behavior compared to controls in spite of no difference in plasma testosterone levels (Fig. 5). Therefore, there may be some compensation for embryonic exposure to exogenous hormones and similarly to EDCs in some of the steroid-sensitive neuroendocrine systems that are also sexually dimorphic. As such, these systems must be carefully evaluated for their usefulness as reliable indices for EDC exposure. The F1 offspring of treated P1 pairs that were also EB treated at embryonic day 11 showed decreased productivity and fertility similar to that observed in their parents (P1). This shows that embryonic EB effect was consistent across generations. There were also F1 offspring from treated P1 pairs that were not EB treated. In these pairs, there was a slight reduction in egg production and fertility (90% vs. 86% productivity and 90% vs. 83% fertility, respectively; Table 2). This suggests the possibility of some type of Table 2 EB injection: F1 productivity/fertility F1

n

% productivity

% fertility

Control EB injected Noninjected

8 8 14

90 57 86

83 32 72

and fertility were produced in EB injected F1 birds, similar
to Productivity the P1 birds. Noninjected offspring of P1 EB-treated pairs showed more subtle effects,
which may be an indication of carryover effects.

carryover effect from the EB-treated parents, in spite of no treatment to the F1 pairs.

4. Summary: determining relevant endpoints for EDC exposure in birds A variety of endpoints have been used in toxicology; however, it is not clear which of these measures may be useful evaluating EDCs in birds. Liver response has been examined, with measurement of cytochrome p450 activity and apolipoprotein II mRNA in chicken hepatocytes in response to EDCs [8,26,33,94]. Vitellogenin and estradiol receptor assays have been viewed as potential indices of EDC exposure as well [91,122]. Neurotoxic effects of dioxin were associated with altered symmetry in major brain areas in great blue herons and methyl parathione was associated with reduced eggshell weight in northern bobwhite quail [16,56]. Embryonic measures of enzyme activation, gonadal morphology, steroid hormone levels, and specific neurotransmitters and neuropeptides can provide indices of EDC exposure. It is interesting that more subtle effects of EDC exposure in parental generations may result in reduced plasma steroid concentrations, both in the parents and in the offspring in mammals and birds ([36], Ottinger et al., unpublished data). Growth parameters are often also affected by exposure to EDCs [48]. This would make sense due to the sexually dimorphic growth patterns that occur in many species of birds. Similarly, the timing of sexual maturation may be an important endpoint, which may be a more sensitive measure of EDC exposure than later egg production or fertility. Several types of behavioral endpoints should be assessed as well, because behavioral responses may prove to be reliable indicators of EDC exposure at critical stages in development. For example, motor tests in young chicks, such as those used in determining fearfulness and early social behaviors, may be excellent indicators of neuromotor consequences that may involve the dopaminergic neurotransmitter systems [43,59]. In addition, these types of tests may provide an indication of neurotoxicities, even those that may be delayed in clinical symptoms [111]. In the adults, earlier work has

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

Fig. 6. Potential endpoints for EDC exposure in birds.

established that exogenous estradiol exposure during midembryonic development greatly diminished male sexual behavior and reproductive success in adults ([75], Ottinger et al., unpublished). Similar results have been observed in bobwhite quail and also in mammals, indicating that this type of effect may be common across species and even phyla [29,67]. In addition, other estrogenic chemicals also impair sexual behavior, providing evidence that testing courtship and mating behavior in embryonically exposed male quail would offer a reliable indicator of estrogenic EDC exposure [53]. These potential endpoints have been grouped in the list below (Fig. 6).

5. Assessing the potential for EDC impact in field birds 5.1. Evidence from field studies Pesticides, including organophosphorus compounds may disrupt neuroendocrine regulatory mechanisms and impair reproductive function [18,30,60,88 – 90]. PCBs have also been associated with altered courtship and nesting behavior in some species, and a delay in onset of reproductive activity, stunted development and reduced hatching success [15,63]. In birds, additional studies have found EDCassociated effects including: abnormal growth, impaired behavior and reproduction, and measurable population declines [27,34,35,42,57,89]. Contaminant exposure has been associated with abnormal gonad development, especially in precocial species [42,45]. Estrogenic EDCs underlie egg shell thinning associated with o,p0-DDT exposure [9,25]. Other agents, including white phosphorous and DDE had endocrine disrupting activity in mallard ducks and cormorants [19,68,109]. 5.2. Bioindicators: use of plumage as an indicator of EDC exposure Although the use of feathers as biomarkers for heavy metal exposure has been validated and is in use in current ecotoxicological methodology, their use as biomarkers for EDC exposure has only recently begun to be explored. The development of plumage is dependent on an interplay of many different chemical and environmental influences, and is therefore a good record of an individual’s responses to

23

challenges encountered during development [87]. The effects of EDCs on feather production are important when considering the role of hormones on molt and pigment deposition. Some of the main hormones that are affected by EDCs and are involved with plumage development include thyroid hormones, estrogens, and androgens [46,84]. Thyroid hormones play a strong role in the initiation and maintenance of photo-refractoriness and molt [116]. Excesses and deficiencies in these hormones can cause molting schedules to begin or cease earlier or later than normal. Since plumage is often an important secondary sexual characteristic for many birds, disruption in the timing of molt could potentially affect their fitness [62]. Molt in birds often follows administration of thyroxine [84,99]. Feathers grown under hyperthyroid conditions often differ in structure, pigmentation, and pattern compared to feathers grown under normal conditions [113]. Hyperthyroidism causes a higher degree of melanization in feathers of some species and a lesser degree in others [84]. Hypothyroid effects can be as disruptive to the development of plumage as those observed during periods of hyperthyroidism. The structure of feathers that are produced during abnormally low levels of thyroid hormones is often irregular. Payne [84] described them as lanceolate in shape and lacking many barbules. The melanin content of these feathers was also altered. Feathers of dark-eyed juncos that were hormonally prevented from molting suffered noticeable wear and fading [71]. One individual that had its molt inhibited for a year suffered so much from abrasion that its wing was shortened by 7 mm. The feathers of hens that had completed molt under propylthiouracil, an antithyroid agent, treatment were old and rough appearing; many of these birds also had bare spots on their necks, wings, and abdomens [102]. When gonads are active, molt is inhibited in both sexes [84]. Estrogen, the primary steroid hormone involved with feather production, can cause molt in birds to be delayed or completely inhibited [50]. Showy male plumage is most often estrogen dependent in sexually dimorphic species [81]. In estrogen-dependent dichromatism, dull female plumage coloration develops in the presence of estrogen, and the brighter male coloration develops in the absence of estrogen [61]. Male coloration is therefore most often the default state of plumage for both sexes. Administration of estrogen can cause changes in melanin distribution, barbule structure, growth rate, and final length of developing feathers [112]. It has been suggested that testosterone plays a stronger role in the development of other secondary sexual characteristics such as combs, spurs, and wattles [22,93]. However, much research exists that supports the influence of testosterone on plumage characteristics. The main effect of testosterone on the development of plumage appears to deal with pigmentation. Testosterone treatments suppressed pigmentation of juvenile black-headed gull chicks [95]. Feathers of Japanese quail became darker after castration and lighter after injection of testosterone propionate [37]. The

24

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

effects of androgens on the deposition of specific melanin types in mallards are becoming clearer; testosterone reduced eumelanin content and increased pheomelanin content in feathers around the head and tail of castrates [52]. One argument against the possible effects of androgens on plumage coloration is that most studies have not tested whether the observed effects were due to the androgens themselves or due to possible metabolites of the androgens, such as estradiol. Whatever the exact mechanisms may be, it is clear that many aspects of the development of plumage development are modulated by hormone action. Disruption of the hormonal systems involved with the development of plumage could potentially change the appearance or structure of the resulting feathers, thus impacting sexual selection, thermoregulation, social interactions, and even flight. The collection of feathers as possible biomarkers for EDC exposure is noninvasive and relatively easy, and they can be stored for many years without significant changes being made to their colors and patterns [68]. One study has shown an association between polychlorinated biphenyl exposure and plumage color in tree swallows [68], but clearly more studies are needed to demonstrate causal relationships between EDCs and plumage characters before feathers can be confidently used as honest markers for EDC exposure in birds. The most extensive use of plumage in contaminant monitoring has been the measurement of heavy metal, metalloid, and trace element concentrations in feathers [23]. Concentrations of these elements in feathers reflect levels in the blood at the time of feather formation, either from current dietary exposure or mobilization from internal organs [21,96]. Once a feather is fully formed, the blood supply is terminated and internal metal concentrations in the feather remain constant. Feathers have become regarded as desirable tools for monitoring because they are easy to collect and store, can be sampled from the same bird in successive years, and exact minimal harm upon the individual [23,24]. The most commonly analyzed metal in feathers is mercury [23]. Controlled studies of mercury exposure in birds have revealed a number of reproductive effects including reduction in mating attempts, ova development and testes weight, reduced hatchability of eggs, misshapen and truncated eggs, decreased eggshell strength and eggshell absence, nerve tissue lesions, and early mortality of offspring [38,40,55,98,103,108]. While increased concentrations of mercury in wild bird eggs and tissues have been found in populations experiencing impaired reproduction [14,39], the inability to isolate the effects of mercury from those of other environmental contaminants or stressors in the field leaves the establishment of a causal relationship inconclusive. The possible contribution of mercury to these effects, whether alone or in combination with other stressors, calls for continued field study and monitoring of this element in wildlife. Feathers provide a reliable tool for this

purpose, in that mercury readily accumulates in the feather, accounting for up to 93% of the total body burden [21], and once deposited, remains unchanged in concentration in response to physical changes in the environment [10]. A great number of other elements can be sequestered in the feather, including the known toxicants cadmium, lead, and selenium [20,24,86]. Of these, lead, like mercury, can accumulate to a high degree into the feather, where deposition has been reported to account for up to 60% of the adult body burden [24]. While interpretation of feather concentrations is hindered for many elements by a lack of controlled studies, the growing body of literature reporting these values will provide important baseline data from which to detect and interpret changes in the future.

6. Summary For the purpose of applications in the field, both the timing and duration of EDC exposure must be considered. Based on this information, the potential risk to avian species, both precocial and altricial, can be made. In the case of precocial birds, exposure during embryonic development presents the greatest potential risk. The consequences of estrogenic EDC exposure may be estimated based on some of the endpoints that we examined in our study with estradiol exposure. In Fig. 7, we have attempted to group these endpoints in terms of duration of effects of EDC exposure during embryonic development. Therefore, with embryonic exposure, similar to the paradigm used in our estradiol study, it would be expected that fitness endpoints, such as viability and growth or metabolic measures, might show immediate or long-term response to the exposure. Endocrine endpoints, such as plasma hormones, would also show immediate as well as long-term responses. Conversely, behavioral endpoints would be affected at some time potentially much after exposure, especially in the case of effects of embryonic EDC exposure on sexual behavior. However, because the male quail is exquisitely sensitive to the effects of embryonic estradiol exposure, sexual behavior provides a very sensitive measure of embryonic EDC exposure. It should be mentioned that exposure in our study was limited to the time during which the brain differentiates and after gonadal differentiation has already occurred. Therefore, our experimental paradigm specifically tested for EDC effects

Fig. 7.

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

on the sexual differentiation of brain and behavior, not effects on gonadal differentiation.

Acknowledgments Research supported by the Maryland Agriculture Experiment Station, USGS-Patuxent Wildlife Research Center (PH), competitive grants from EPA #R826134010 (MAO) and NSF #IBN-9817024 (MAO). The authors thank Elizabeth Humphries, Kameka Henry, and Erin Quigley for their work in the research and to Edith Silvious for her help in the preparation of this manuscript.

References [1] M.A. Adelnabi, M. Bakst, J. Woods, M.A. Ottinger, Plasma 17 B-estradiol levels and ovarian interstitial cell structure in embryonic Japanese quail, Poult. Sci. 79 (2000) 564 – 567. [2] E.K. Adkins, Effects of embryonic treatment with estradiol or testosterone on sexual differentiation of the quail brain. Critical period and dose response relationships, Neuroendocrinology 29 (1979) 178 – 185. [3] E.K. Adkins, B.L. Nock, The effects of antiestrogen CI-628 on sexual behavior activated by androgen or estrogen in quail, Horm. Behav. 7 (1976) 417 – 429. [4] E.K. Adkins-Regan, M. Abdelnabi, M. Mobarak, M.A. Ottinger, Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata), Gen. Comp. Endocrinol. 78 (1990) 93 – 109. [5] E.K. Adkins-Regan, V. Mansukhani, C. Seiwert, R. Thompson, Sexual differentiation of brain and behavior in the zebra finch: Critical periods for effects of early estrogen treatment, J. Neurobiol. 25 (7) (1994) 865 – 877. [6] E.K. Adkins-Regan, M.A. Ottinger, J. Park, Maternal transfer of estradiol to egg yolks alters sexual differentiation of avian offspring, J. Exp. Zool. 271 (1995) 466 – 470. [7] E.K. Adkins-Regan, P. Pickett, D. Routnik, Sexual differentiation in quail: Conversion of androgen to estrogen mediates testosterone induced demasculinization of copulation but not other male characteristics, Horm. Behav. 16 (1982) 259 – 278. [8] P.L. Andersson, A.S.A.M. van der Burght, M. van den Berg, M. Tysklind, Multivariate modeling of polychlorinated biphenylinduced CYP1A activity in hepatocytes from three different species: Ranking scales and species differences, Environ. Toxicol. Chem. 19 (5) (2000) 1454 – 1463. [9] G. Ankley, E. Mihaich, R. Stahl, D. Tillitt, T. Colborn, S. McMaster, R. Miller, J. Bantle, P. Campbell, N. Denslow, R. Dickerson, L. Folmar, M. Fry, J. Giesy, E. Gray, P. Guiney, T. Hutchinson, S. Kennedy, V. Kramer, G. LeBlanc, M. Mayes, A. Nimrod, R. Patino, R. Peterson, R. Purdy, R. Ringer, P. Thomas, L. Touart, G. Van der Kraak, T. Zacharewski, Overview of a workshop on screening methods for detecting potential (anti-) estrogenic/androgenic chemicals in wildlife, Environ. Toxicol. Chem. 17 (1) (1997) 68 – 87. [10] H. Appelquist, S. Asbirk, I. Drabek, Mercury monitoring: Mercury stability in bird feathers, Mar. Pollut. Bull. 15 (1984) 22 – 24. [11] A.P. Arnold, B.A. Schlinger, The puzzle of sexual differentiation of the brain and behavior in zebra finches, Poult. Sci. Rev. 5 (1994) 3 – 13. [12] A.P. Arnold, J. Wade, W. Grisham, E.C. Jacobs, A.T. Campagnoni, Sexual differentiation of the brain in songbirds, Dev. Neurosci. 18 (1996) 124 – 136. [13] N. Aste, G.C. Panzica, P. Aimar, C. Viglietti-Panzica, N. Foidart, J. Balthazart, Morphometric studies demonstrate that aromatase-immu-

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

[27]

[28] [29]

[30]

[31]

[32] [33]

25

noreactive cells are the main target of androgens and estrogens in the quail medial preoptic nucleus, Exp. Brain Res. 101 (1996) 241 – 252. J.F. Barr, Population dynamics of the common loon (Gavia immer) associated with mercury-contaminated waters in northwestern Ontario, Can. Wildl. Serv. Occas. Pap. 56 (23 pp.). P.H. Becker, S. Schuhmann, C. Koepff, Hatching failure in common terns (Sterna hirundo) in relation to environmental chemicals, Environ. Pollut. 79 (1993) 207 – 213. R.S. Bennett, R. Bentley, T. Shiroyama, J.K. Bennett, Effects of the duration and timing of dietary methyl parathion exposure on bobwhite reproduction, Environ. Toxicol. Chem. 7 (1990) 1473 – 1480. C. Berg, K. Halldin, B. Brunstrom, I. Brandt, Methods for studying xenoestrogenic effects in birds, Toxicol. Lett. 102 – 103 (1998) 671 – 676. C.A. Bishop, N.A. Mahony, S. Trudeau, K.E. Pettit, Reproductive success and biochemical effects in tree swallows (Tachycineta bicolor) exposed to chlorinated hydrocarbon contaminants in wetlands of the great lakes and St. Lawrence River Basin, USA and Canada, Environ. Toxicol. Chem. 18 (1999) 263 – 271. A.T.C. Bosveld, M. Van den Berg, Effects of polychlorinated biphenyls, dibenzo-p-dioxins and dibenzofurans on fish-eating birds, Environ. Rev. 2 (1994) 147 – 166. W.W. Bowerman IV, E.D. Evans, J.P. Giesy, S. Postupalsky, Using feathers to assess risk of mercury and selenium to bald eagle reproduction in the Great Lakes Region, Arch. Environ. Contam. Toxicol. 27 (1994) 294 – 298. B.M. Braune, D.E. Gaskin, Mercury levels in Bonaparte’s gulls (Larus philadelphia) during autumn molt in the Quoddy region, New Brunswick, Canada, Arch. Environ. Contam. Toxicol. 16 (1987) 539 – 549. F. Briganti, A. Papeschi, T. Mugnai, F. Dessi-Fulgheri, Effect of testosterone on male traits and behaviour in juvenile pheasants, Ethol. Ecol. Evol. 11 (1999) 171 – 178. J. Burger, Metals in avian feathers: Bioindicators of environmental pollution, Rev. Environ. Toxicol. 5 (1993) 203 – 311. J. Burger, M. Gochfeld, Tissue levels of lead in experimentally exposed herring gull (Larus argentatus) chicks, J. Toxicol. Environ. Health 29 (1990) 219 – 233. A.O. Cheek, P.M. Vonier, E. Oberdorster, B.C. Burow, J.A. McLachlan, Environmental signaling: A biological context for endocrine disruption, Environ. Health Perspect. 106 (Suppl. 1) (1998) 5 – 10. S.-W. Chen, P. Dziuk, B.M. Francis, Effect of four environmental toxicants on plasma Ca and estradiol 179 and hepatic p450 in laying hens, Environ. Toxicol. Chem. 13 (5) (1994) 789 – 796. T. Colborn, F.S. vom Saal, A.M. Soto, Developmental effects of endocrine-disrupting chemicals in wildlife and humans, Environ. Health Perspect. 101 (5) (1993) 378 – 384. D. Crews, E. Willingham, J.K. Skipper, Endocrine disruptors: Present issues, future directions, Q. Rev. Biol. 75 (3) (2000) 243 – 260. G. Csaba, C. Karabelyos, J. Dallo, Fetal and neonatal action of a polycyclic hydrocarbon (benzpyrene) or a synthetic steroid hormone (allylestrenol) as reflected by the sexual behaviour of adult rats, Am. J. Dev. Physiol. 19 (1993) 67 – 70. T.W. Custer, C.M. Custer, R.K. Hines, S. Gutreuter, K.L. Stromborg, P.D. Allen, M.J. Melancon, Organochlorine contaminants and reproductive success of double-crested cormorants from Green Bay, Wisconsin, USA, Environ. Toxicol. Chem. 18 (6) (1999) 1209 – 1217. T.L. Dellovade, E.F. Rissman, N. Thompson, N. Harada, M.A. Ottinger, Co-localization of aromatase enzyme and estrogen receptor immunoreactivity in the preoptic area during reproductive aging, Brain Res. 674 (1995) 181 – 187. A. Elbrecht, R.G. Smith, Aromatase enzyme activity and sex determination in chickens, Science 255 (1992) 467 – 470. J.E. Elliott, S.W. Kennedy, A. Lorenzen, A comparative toxicity of polychlorinated biphenyls to Japanese quail (Coturnix c. japonica) and American kestrels, J. Toxicol. Environ. Health 51 (1997) 57 – 75.

26

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

[34] A. Fairbrother, Comparative aspects of estrogen functions and measurements in oviparous and viviparous vertebrates, Hum. Ecol. Risk Assess. 6 (1) (2000) 73 – 102. [35] A. Fairbrother, W.G. Landis, S. Dominquez, T. Shiroyama, P. Buchholz, M.J. Roze, G.B. Matthews, A novel nonmetric multivariate approach to the evaluation of biomarkers in terrestrial field studies, Ecotoxicology 7 (1998) 1 – 10. [36] A.S. Faqi, P.R. Dalsenter, H.-J. Merker, I. Chahoud, Reproductive toxicity and tissue concentrations of low doses of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation, Toxicol. Appl. Pharmacol. 150 (1998) 383 – 392. [37] I. Feuerbacher, R. Prinzinger, Einflub van testosteron auf gefiederfarbung unddepotfett bei der Japanwachtel Coturnix coturnix japonica, J. Ornithol. 123 (1982) 203 – 209. [38] N. Fimreite, Effects of dietary methylmercury on ring-necked pheasants with special reference to reproduction, Can. Wildl. Serv. Occas. Pap. 9 (39 pp.). [39] N. Fimreite, Mercury contamination of aquatic birds in Northwestern Ontario, J. Wildl. Manage. 38 (1974) 120 – 131. [40] M.T. Finley, R.C. Stendell, Survival and reproductive success of black ducks fed methylmercury, Bull. Environ. Contam. Toxicol. 21 (1978) 105 – 110. [41] A. Foidart, N. Harada, J. Balthazart, Effects of steroidal and non steroidal aromatase inhibitors on sexual behavior and aromatase-immunoreactive cells, Brain Res. 657 (1994) 105 – 123. [42] G.A. Fox, A.P. Gilman, D.B. Peakall, B. Anderka, Behavioral abnormalities of nesting Lake Ontario Herring gulls, J. Wildl. Manage. 42 (3) (1978) 477 – 483. [43] N. Francois, A.D. Mills, J.M. Faure, Inter-individual distances during open-field tests in Japanese quail (Coturnix japonica) selected for high or low levels of social reinstatement behaviour, Behav. Processes 47 (1999) 73 – 80. [44] F. Freking, T. Nazairians, B.A. Schlinger, The expression of the sex steroid-synthesizing enzymes CYP11A1, 39-HSD, CYP17, and CYP19 in gonads and adrenals of adult and developing zebra finches, Gen. Comp. Endocrinol. 119 (2000) 140 – 151. [45] M. Fry, Reproductive effects in birds exposed to pesticides and industrial chemicals, Environ. Health Perspect. 103 (Suppl. 7) (1995) 165 – 171. [46] F. Gill, Ornithology, W.H. Freeman & Co, New York, 1990, pp. 533 – 551. [47] C.B. Gonzalez, E.H. Charreau, E.H. Aragone´s, C.P. Lantos, B.K. Follett, The ontogenesis of reproductive hormones in the female embryo of the domestic fowl, Gen. Comp. Endocrinol. 68 (1987) 369 – 374. [48] J.C. Gould, K.R. Cooper, C.G. Scanes, Effects of polychlorinated biphenyl mixtures and three specific congeners on growth and circulating growth-related hormones, Gen. Comp. Endocrinol. 106 (1997) 221 – 230. [49] L.E. Gray Jr., J. Ostby, C. Wolf, C. Lambright, W. Kelce, The value of mechanistic studies in laboratory animals for the prediction of reproductive effects in wildlife: Endocrine effects on mammalian sexual differentiation, Environ. Toxicol. Chem. 17 (1998) 109 – 118. [50] E.D. Greij, Effects of sex hormones on plumages of the blue-winged teal, Auk 90 (1973) 533 – 551. [51] L.J. Guillette, D.A. Crain, M.P. Gunderson, S.A.E. Kools, M.R. Milnes, E.F. Orlando, A.A. Rooney, A.R. Woodward, Alligators and endocrine disrupting contaminants: A current perspective, Am. Zool. 40 (2000) 438 – 452. [52] E. Haase, S. Ito, K. Wakamatsu, Influences of sex, castration, and androgens on the eumelanin and pheomelanin contents of different feathers in wild mallards, Pigm. Cell Res. 8 (1995) 164 – 170. [53] K. Halldin, C. Berg, I. Brandt, B. Brunstrom, Sexual behavior in Japanese quail as a test end point for endocrine disruption: Effects of in ovo exposure to ethinylestradiol and diethylstilbestrol, Environ. Health Perspect. 107 (11) (1999) 861 – 866. [54] Y. Hatano, A. Hatano, Influence of polychlorinated biphenyls on the growth of chicken embryos, J. Toxicol. Environ. Health 42 (1994) 357.

[55] G. Heinz, Effects of low dietary levels of methyl mercury on mallard reproduction, Avian Dis. 20 (1974) 9 – 17. [56] D.S. Henshel, J.W. Martin, R. Norstrom, P. Whitehead, J.D. Steeves, K.M. Cheng, Morphometric abnormalities in brains of great blue heron hatchlings exposed in the wild to PCDDs, Environ. Health Perspect. 103 (4) (1995) 61 – 66. [57] D.J. Hoffman, Embryotoxicity and teratogenicity of environmental contaminants to bird eggs, Rev. Environ. Contam. Toxicol. 115 (1990) 39. [58] J.B. Hutchison, M. Schumacher, R.E. Hutchison, Developmental sex differences in brain aromatase activity are related to androgen level, Dev. Brain Res. 57 (1990) 198 – 205. [59] R.B. Jones, C.G. Satterlee, G.G. Cadd, Timidity in Japanese quail: Effects of vitamin C and divergent selection for adrenocortical response, Physiol. Behav. 67 (1) (1999) 117 – 120. [60] R.J. Kavlock, G.P. Daston, C. DeRosa, P. Fenner-Crisp, L.E. Gray, S. Kaatari, G. Lucier, M. Luster, M.J. Mac, C. Maczka, R. Miller, J. Moore, R. Rolland, G. Scott, D.M. Sheehan, T. Sinks, H. Tilson, Research needs for the risk assessment of health and environmental effects of endocrine disruptors: A report of the US EPA-sponsored workshop, Environ. Health Perspect. 104 (Suppl. 4) (1996) 715 – 740. [61] R.T. Kimball, Evolution of avian plumage dichromatism from a proximate perspective, Am. Nat. 154 (1999) 182 – 193. [62] M. Kirkpatrick, Sexual selection and the evolution of female choice, Evolution 36 (1982) 1 – 11. [63] T.J. Kubiak, H.J. Harris, L.M. Smith, T.R. Schwartz, D.L. Stalling, J.A. Trick, L. Sileo, D.E. Docherty, T.C. Erdman, Microcontaminants and reproductive impairment of the Forster’s tern on Green Bay, Lake Michigan—1983, Arch. Environ. Contam. Toxicol. 18 (1989) 706 – 727. [64] Q. Li, B. Alston-Mills, M.A. Ottinger, Avian LHRH during embryonic development: Measurement by competitive ELISA with a monoclonal antibody, Gen. Comp. Endocrinol. 82 (1991) 444 – 450. [65] Q. Li, G.F. Paciotti, L. Tamarkin, M.A. Ottinger, LHRH I release from hypothalamic slices measured by specific EIA, Gen. Comp. Endocrinol. 95 (1994) 13 – 24. [66] Q. Li, L. Tamarkin, P. Levantine, M.A. Ottinger, Estradiol and androgen modulate chicken luteinizing hormone-releasing hormone-I release in vitro, Biol. Reprod. 51 (1994) 896 – 903. [67] R.J. Lien, J.R. Cain, S.L. Beasom, Effects of dietary and parenteral estrogens on bobwhite reproduction, Poult. Sci. 66 (1987) 154 – 161. [68] J.P. McCarty, A.L. Secord, Possible effects of PCB contamination on female plumage color and reproductive success in Hudson River tree swallows, Auk 117 (2000) 987 – 995. [69] J.A. McLachlan, K.S. Korach, Symposium on estrogens in the environment: III, Environ. Health Perspect. 103 (Suppl. 7) (1995) 3 – 5. [70] H. Nishikimi, N. Kansaku, N. Saito, M. Usami, Y. Ohno, K. Shimada, Sex differentiation and mRNA expression of P450c17, P450arom and AMH in gonads of the chicken, Mol. Reprod. Dev. 55 (2000) 20 – 30. [71] V. Nolan Jr., E.D. Ketterson, Testosterone and avian life histories: Effects of experimentally elevated testosterone on prebasic molt and survival in male dark-eyed juncos, Condor 94 (1992) 364 – 370. [72] NRC, Hormonally Active Agents in the Environment, National Academy Press, Washington, DC, 1999. [73] M.A. Ottinger, Male reproduction: Testosterone, gonadotropins, and aging, in: C.V. Mobbs, P.R. Hof (Eds.), Functional Endocrinology of Aging, Interdiscipl Top Gerolotol, vol. 29, Karger, Basel, 1998, pp. 105 – 126. [74] M.A. Ottinger, M.A. Abdelnabi, Neuroendocrine systems and avian sexual differentiation, Am. Zool. 37 (1997) 514 – 523. [75] M.A. Ottinger, M.A. Abdelnabi, P.F.P. Henry, S. McGary, N. Thompson, J. Wu, Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail, Horm. Behav. 40 (2001) 234 – 247. [76] M.A. Ottinger, M.R. Bakst, Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail, Gen. Comp. Endocrinol. 43 (1981) 170 – 177. [77] M.A. Ottinger, H.J. Brinkley, The relationship of testosterone and

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

[78] [79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87] [88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

sexual behavior during maturation of the male Japanese quail, Horm. Behav. 11 (1978) 174 – 182. M.A. Ottinger, H.J. Brinkley, The ontogeny of crowing and copulatory behavior in Japanese quail, Behav. Processes 4 (1979) 43 – 47. M.A. Ottinger, H.J. Brinkley, Testosterone and sex-related morphology during maturation of the male Japanese quail, Biol. Reprod. 20 (1979) 905 – 909. M.A. Ottinger, S. Pitts, M.A. Abdelnabi, Steroid hormones during embryonic development in Japanese quail: Plasma, gonadal, and adrenal levels, Poult. Sci. 80 (2001) 795 – 799. I.P.F. Owens, R.V. Short, Hormonal basis of sexual dimorphism in birds: Implications for new theories of sexual selection, Tree 10 (1995) 44 – 47. G.C. Panzica, N. Aste, C. Viglietti-Panzica, A. Fasolo, Neuronal circuits controlling quail sexual behavior. Chemical neuroanatomy of the septo-preoptic region, Poult. Sci. Rev. 4 (1992) 249 – 259. G.C. Panzica, E. Garcia-Ojeda, C. Viglietti-Panzica, N. Thompson, M.A. Ottinger, Testosterone effects on vasotocinergic innervation of sexually dimorphic medial preoptic nucleus and lateral septum during aging in male quail, Brain Res. 712 (1996) 190 – 198. R.B.B. Payne, Hormonal control of feather loss and replacement, in: D.S. Farner, J.R. King (Eds.), Avian Biology, vol. II, Academic Press, New York, 1972 (Chapter 3). R.M.R. Perrin, S. Stacey, A.M.C. Burgess, U. Mittwoch, A quantitative investigation of gonadal feminization by diethylstilboestrol of genetically male embryos of the quail Coturnix coturnix japonica, J. Reprod. Fertil. 103 (1995) 223 – 226. A. Pilastro, L. Congiu, L. Tallandini, M. Turchetto, The use of bird feathers for the monitoring of cadmium pollution, Arch. Environ. Contam. Toxicol. 24 (1993) 355 – 358. C.L. Ralph, The control of color in birds, Am. Zool. 9 (1969) 521 – 530. B.A. Rattner, V.P. Eroschenko, G.A. Fox, D.M. Fry, J. Gorsline, Avian endocrine responses to environmental pollutants, J. Exp. Zool. 232 (1984) 683 – 689. B.A. Rattner, M.J. Melancon, T.W. Custer, R.L. Hothem, K.A. King, L.J. LeCaptain, J.W. Spann, B.R. Woodin, J.J. Stegeman, Biomonitoring environmental contamination with piping black-crowned night heron embryos: Induction of cytochrome P450, Environ. Toxicol. Chem. 12 (9) (1993) 1719. B.A. Rattner, L. Sileo, C.G. Scanes, Hormonal responses and tolerance to cold of female quail following parathion ingestion, Pestic. Biochem. Physiol. 18 (1982) 132. L. Ren, S.K. Lewis, J.J. Lech, Effects of estrogen and nonylphenol on the post-transcriptional regulation of vitellogenin gene expression, Chem.-Biol. Interact. 100 (1996) 67 – 76. E.F. Rissman, M. Ascenzi, P. Johnson, E. Adkins-Regan, Effect of embryonic treatment with oestradiol benzoate on reproductive morphology, ovulation, and oviposition and plasma LH concentrations in female quail (Coturnix coturnix japonica), J. Reprod. Fertil. 71 (1984) 411 – 417. P.T. Riutamaki, J. Hoglund, E. Karoonen, R.V. Alatalo, N. Lundberg, A. Lundberg, O. Ratti, J. Vouti, Combs and sexual selection in black grouse (Tetrao tetrix), Behav. Ecol. 11 (2000) 465 – 471. M.J. Ronis, M. Ingelman-Sundberg, T.M. Badger, Induction, suppression and inhibition of multiple hepatic cytochrome p450 isozymes in the male rat and bobwhite quail (Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs), Biochem. Pharmacol. 48 (10) (1994) 1953 – 1965. A.F.H. Ros, Effects of testosterone on growth, plumage pigmentation, and mortality in black-headed gull chicks, Ibis 141 (1999) 451 – 459. P.F. Scanlon, T.G. O’Brien, N.L. Schauer, J.L. Coggin, D.E. Steffen, Heavy metal levels in feathers of wild turkeys from Virginia, Bull. Environ. Contam. Toxicol. 21 (1979) 591 – 595. D. Scheib, A. Guichare, Th.-M. Mignoti, M. Reyss-Brion, Early sex differences in hormonal potentialities of gonads from quail embryos

[98]

[99]

[100] [101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112] [113] [114]

[115]

[116]

27

with a sex-linked pigmentation marker: An in vitro radioimmunoassay study, Comp. Endocrinol. 60 (1985) 266 – 272. M.L. Scott, J.R. Zimmerman, S. Marinsky, P.A. Mullenhoff, G.L. Rumsey, R.W. Rice, Effects of PCBs, DDT, and mercury compounds upon egg production, hatchability, and shell quality in chickens and Japanese quail, Poult. Sci. 54 (1975) 350 – 368. K. Sekimoto, K. Imai, M. Suzuki, H. Takikawa, Thyroxine-induced molting and gonadal function of laying hens, Poult. Sci. 66 (1987) 752 – 756. D.M. Sheehan, Isoflavone content of breast milk and soy formulas: Benefits and risks, Clin. Chem. 43 (1997) 850. K. Shimada, K. Yoshida, N. Saito, Effects of aromatase inhibitor on sex differentiation and levels of P450 (17 alpha) and P450arom messenger ribonucleic acid of gonads in chicken embryos, Gen. Comp. Endocrinol. 102 (1996) 241 – 246. T. Siopes, Transient hypothyroidism reinitiates egg laying in turkey breeder hens: Termination of photorefractoriness by propylthiouracil, Poult. Sci. 76 (1997) 1776 – 1782. J.W. Spann, R.G. Heath, J.F. Kreitzer, L.N. Locke, Ethyl mercury p-toluene sulfonanilide: Lethal and reproductive effects on pheasants, Science 175 (1972) 328 – 331. J.M. Starck, R.E. Ricklefs, Avian growth and development: Evolution within the altricial – precocial spectrum, Oxford Ornithol. Ser. 8 (1997) 1 – 12. R. Stoll, F. Ichas, N. Faucounau, R. Maraud, Action of estradiol and tamoxifen on the testis-inducing activity of the chick embryonic testis grafted to the female embryo, Anat. Embryol. 188 (1993) 587 – 593. Y. Tanabe, T. Nakamura, K. Fujioka, O. Doi, Production and secretion of sex steroid hormones by the testes, the ovary, and the adrenal glands of embryonic and young chickens (Gallus domesticus), Gen. Comp. Endocrinol. 39 (1979) 26 – 33. Y. Tanabe, N. Saito, T. Nakamura, Ontogenetic steroidogenesis by testes, ovary, and adrenals of embryonic and postembryonic chickens (Gallus domesticus), Gen. Comp. Endocrinol. 63 (1986) 456 – 563. J.P. Thaxton, R.P. Carmen, Abnormal mating behavior and reproductive dysfunction caused by mercury in Japanese quail, Proc. Soc. Exp. Biol. Med. 144 (1973) 252 – 255. S.L. Vann, D.W. Sparling, M.A. Ottinger, Effects of white phosphorus on mallard reproduction, Environ. Toxicol. Chem. 19 (10) (2000) 2525 – 2531. A. Vanson, A.P. Arnold, B.A. Schlinger, 3b-Hydroxysteroid dehydrogenase/isomerase and aromatase activity in primary cultures of developing zebra finch telencephalon: Dehydroepiandrosterone as substrate for synthesis of androstenedione and estrogens, Gen. Comp. Endocrinol. 102 (1996) 342 – 350. R.G. Varghese, S.J. Bursian, C. Tobias, D. Tanaka Jr., Organophosphorus-induced delayed neurotoxicity: A comparative study of the effects of tri-ortho-tolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail, Neurotoxicology 16 (1) (1995) 45 – 54. H.G. Vevers, The influence of the ovaries on secondary sexual characters, The Ovary, Academic Press, New York, 1962 (Chapter 17). A.A. Voitkevich, The Feathers and Plumage of Birds, October House, New York, 1966. J.T. Watson, E. Adkins-Regan, Activation of sexual behavior by implantation of testosterone propionate and estradiol enzoate into the preoptic area of the male Japanese quail (Coturnix japonica), Horm. Behav. 23 (1989) 251 – 268. J.T. Watson, E. Adkins-Regan, Testosterone implanted in the preoptic area of male Japanese quail must be aromatized to activate copulation, Horm. Behav. 23 (1989) 432 – 447. F.E. Wilson, B.D. Reinert, A one-time injection of thyroxine programmed seasonal reproduction and postnuptual molt in chronically thyroidectomized male American tree sparrows Spizella arborea exposed to long days, J. Avian Biol. 26 (1995) 225 – 233.

28

M.A. Ottinger et al. / Neurotoxicology and Teratology 24 (2002) 17–28

[117] J.E. Woods, D.M. Brazzill, Plasma 17b-estradiol levels in the chick embryo, Gen. Comp. Endocrinol. 44 (1981) 37 – 43. [118] J.E. Woods, M.P. Honan, R.C. Thommes, Hypothalamic regulation of the adenohypophyseal – testicular axis in the male chick embryo, Gen. Comp. Endocrinol. 4 (1989) 167 – 172. [119] J.E. Woods, L.M.G. Otten, R.C. Thommes, Ontogeny of 17b-estradiol (E2)- and estrogen receptor (ER)-immunostained cells in the hypothalamus and adenohypophyseal pars distalis of the chick embryo, Growth Dev. Aging 59 (1995) 93 – 105. [120] J.E. Woods, C.G. Scanes, M. Seeley, P. Cozzi, F. Onyeise, R.C.

Thommes, Plasma LH and gonadal LH-binding cells in normal and surgically decapitated chick embryos, Gen. Comp. Endocrinol. 74 (1989) 1 – 13. [121] J.E. Woods, R.C. Thommes, Ontogeny of hypothalamo – adenohypophyseal – gonadal (HAG) interrelationships in the chick embryo, J. Exp. Zool. 232 (1984) 435 – 441. [122] T. Zacharewski, Identification and assessment of endocrine disruptors: Limitations of in vivo and in vitro assays, Environ. Health Perspect. 106 (Suppl. 2) (1998) 577 – 582.