Parental and First Generation Effects of Exogenous 17β-Estradiol on Reproductive Performance of Female Zebra Finches (Taeniopygia guttata)

Hormones and Behavior 35, 135–143 (1999) Article ID hbeh.1998.1506, available online at http://www.idealibrary.com on

Parental and First Generation Effects of Exogenous 17b-Estradiol on Reproductive Performance of Female Zebra Finches (Taeniopygia guttata) Tony D. Williams Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada Received September 3, 1998; revised December 8, 1998; accepted December 10, 1998

Steroids hormones have numerous “activational” effects in adult birds, regulating sexual behavior, and more recently maternal androgens have been shown to have potentially important “organizational” effects in ovo, influencing offspring growth, development, and behavior. In this study I investigated parental and first-generation effects of exogenous estrogens on female reproduction in zebra finches (Taeniopygia guttata). 17b-Estradiol (E 2; 1.2 mg/g, 4 daily injections i.m.) elevated plasma levels of the yolk precursors, vitellogenin (VTG) and very lowdensity lipoprotein (VLDL), in nonbreeding females to levels similar to those of breeding females. However, E 2-treatment of breeding females caused no significant change in plasma VTG or VLDL levels compared to control birds (measured at the 1-egg stage), and there was no difference in reproductive performance between groups (egg size, clutch size, timing of laying). E 2treated females produced significantly more daughters than sons (21F:8M) at fledging, compared to control females (18F:19M). Nestling mortality was significantly higher in broods of E 2-treated females, suggesting that the skewed sex ratio may have resulted from differential mortality of male chicks. The pattern of chick mortality in E 2-broods was not consistent with this being caused by estrogen-mediated changes in parental behavior (e.g., provisoning). Mean egg mass of daughters of E 2-treated females was typical of experienced, adult breeders, and larger than normal, first-time breeders or control offspring (0.947 vs 0.850 g). There was no treatment effect on offspring clutch size or laying interval. These results suggest that early exposure to maternal estrogens in ovo might be involved in establishing intraindividual variation in female-specific phenotypic traits, as has previously been demonstrated for androgens and male behavioral traits (e.g., aggression). © 1999 Academic Press Key Words: 17b-estradiol; female reproductive effort; intergenerational effects. 0018-506X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

Estrogens have numerous “activational” roles in adult birds regulating both female sexual behavior (Balthazart, 1983; Nelson, 1995) and maturation and functioning of the reproduction system (stimulating ovarian development, yolk precursor synthesis by the liver, and, together with progesterone, oviduct growth; Burley and Vadhera, 1989; Williams, in press). As a consequence, several studies have shown that circulating levels of estrogens are highest coincident with the period of courtship, pair formation, egg formation, and onset of lay in a wide range of avian species (review, Wingfield and Farner, 1993). However, although species-specific patterns of plasma estrogens have been characterized in relation to temporal aspects of female reproduction (i.e., breeding chronology) it is unclear whether, or how, circulating estradiol levels are related to quantitative variation in female reproduction, such as variation in egg or clutch size, or timing of laying. There is large-scale intraspecific or interindividual variability in all of these traits in birds (Williams, in press), and this variation is repeatable within individual females and is maintained when birds are brought into captivity and bred in constant or controlled conditions on ad lib food (Williams, 1996a). Furthermore, there appear to have been no studies which have manipulated estradiol levels to assess subsequent effects on female reproductive physiology or performance (cf. behavior, see Discussion). In addition to their activational role in adult reproduction it is clear that estrogens have important “organizational” functions in genotypic sex determination and sexual differentiation during early development in birds, e.g., in expression of sex-spe-

135

136

cific morphological and behavioral phenotypes (Adkins, 1979; Balthazart, DeClerck, and Foidart, 1992; Elbrecht and Smith, 1992). Recent studies have demonstrated that maternal steroids, transfered via the yolk, can influence embryonic development and adult phenotype (androgens, Schwabl, 1993; estrogens, Adkins-Regan, Ottinger, and Parks, 1995; see also Riddle and Dunham, 1942) in addition to endogenous embryonic steroid secretion. Although it has been suggested that variation in yolk estrogen levels might serve adaptive functions (Winkler, 1993; McNabb, Scanes, and Zeman, 1998; cf. pathological effects of experimentally elevated estrogen levels, Adkins-Regan et al., 1995), no studies have demonstrated this to date. In this paper, I investigate the effects of exogenous 17b-estradiol (E 2) on female reproduction both withinand between generations in the zebra finch (Taeniopygia guttata). In particular, I tested the hypothesis that exogenous estradiol would increase measures of (a) primary reproduction (time of laying, egg and clutch size) through an increase in circulating levels of yolk precursors (vitellogenin and very low density lipoprotein or VLDL), and (b) secondary reproduction (survival and growth of offspring) and sex ratio in E 2treated adult females compared with control females. Second, I compared differences in primary reproductive effort between daughters of E 2-treated and control females to determine possible intergenerational effects of exogenous estradiol. A detailed understanding of activational/organizational role of estrogens in female reproduction is particularly relevant in light of current concerns over effects of xenoestrogens or estrogenmimics (e.g., Campbell and Hutchinson, 1998; Feyk and Giesy, 1998). Most interest to date has focused on feminizing effects of estradiol in male embryos, and there has been little or no consideration of possible “additive” effects of exogenous estradiol influencing the normal role of endogenous estradiol in female birds.

METHODS Animal Care and Breeding Protocol Zebra finches were maintained in controlled environmental conditions (temperature 19 –23°C; humidity 35–55%; constant light schedule, 14L:10D, lights on at 07.00). All birds were provided with a mixed seed diet (Panicum and white millet, 1:3, 11.7% protein, 0.6% lipid, and 84.3% carbohydrate by dry mass), water, grit, and cuttlefish bone (calcium) ad lib, and

Tony D. Williams

received a multivitamin supplement in the drinking water once per week. Breeding pairs were also provided with an egg food supplement (20.3% protein: 6.6% lipid) daily between pairing and clutch completion, and again during the chick-rearing period. Birds were assigned to experimental groups and pairs at random. All birds used in this experiment were of the “wild type” plumage morph, they had bred (laid eggs) at least once previously, and were $6 months old. Experiments and animal husbandry were carried out under a Simon Fraser University Animal Care Committee permit (No. 399B), in accordance with guidelines from the Canadian Committee on Animal Care (CCAC). Breeding pairs were housed individually in cages (61 3 46 3 41 cm) each with an external nest box (11.5 3 11.5 3 11.5 cm). Pairs were assigned to either the control (vehicle-injected) group (n 5 16) or estradiol-treated group (n 5 18) at random. Females were weighed (6 0.1 g, initial mass) at the time of pairing, at the 1-egg stage, and again at clutch completion. Nest boxes were checked daily between 09.00 and 11.00 and all new eggs were weighed (to 0.001 g) and numbered, to obtain data on egg size, clutch size, and laying interval (the time between pairing and laying of the first egg). All females were blood sampled between 10.00 and 12.00 on the day of laying of the first egg (1-egg stage) for measurement of plasma yolk precursor levels. If no new eggs were laid over 2 days the clutch was considered to be complete and birds were left undisturbed until hatching. For most of the E 2-treated and control adult females data were available on their previous reproductive history (egg size and clutch size for 1– 4 previous breeding attempts). Just prior to hatching, nest boxes were again checked daily to determine hatching success per clutch (brood size at hatching). All chicks were banded at 8 days of age. Chicks generally “fledged,” i.e., they left the nest box, around 18 days of age, and all chicks were weighed and measured (tarsus length, 6 0.01 mm) at 21 days of age, and brood size at fledging was determined. Once chicks were feeding independently (generally around 28 days of age) they were separated from the adult pair and maintained in large nonbreeding groups. As chicks developed their sexually dimorphic plumage (Zann, 1996) they were separated into single-sex groups until females (daughters) were bred at 90 days of age (as described above). Female offspring were all paired with experienced, unrelated adult males. Genetic sex of female offspring was confirmed prior to breeding (90 days of age) using

17b-Estradiol and Female Reproduction

a molecular DNA probe (Griffiths, Daan, and Dijkstra, 1996; B. Vanderkist, unpublished data). Estradiol Treatment To validate the protocol for estradiol treatment, and to determine the effect of exogenous estradiol on plasma yolk precursor levels, a pilot experiment was carried out using nonbreeding zebra finches. Adult females were given four daily injections i.m. of 20 mg 17b-estradiol (Sigma) in 30 ml of 1,2-propanediol (1.2 mg/g body weight; after Mathews, Brenowitz, and Arnold, 1988). Control birds received 30 ml of 1,2propandiol vehicle only. Birds were blood sampled 24 h after the final injection, killed with an overdose of anesthetic (Rompun/Ketamine 1:1 v/v), and their oviducts removed and immediately weighed. Breeding females received the same estradiol treatment and were then paired at random with males immediately after the fourth daily injection. Mean interval between pairing and laying is 6 days in zebra finches (Williams, 1996a), so to confirm that E 2-treatment elevated yolk precursor levels for the duration of this period, a further group of nonbreeding females was treated with estradiol following the same protocol. These birds were blood sampled 6 days after the last injection and circulating vitellogenin and VLDL levels were measured. All blood samples were centrifuged at 5000 rpm for 10 min and the plasma separated and frozen at 220°C until assayed for yolk precursors. Yolk Precursor Assays Plasma vitellogenin (VTG) was assayed using the zinc method developed for the domestic hen (Mitchell and Carlisle, 1991), which we have validated for passerines (Williams and Christians, 1997). This method measures total plasma zinc (Zn - Wako Chemicals, VA) then partitions zinc into the albumin-bound component and the VTG-bound component by depletion of VTG and VLDL from the plasma sample (following precipitation with dextran sulfate; VLDL accounts for only 2% of total zinc, Mitchell and Carlisle, 1991). Zinc is assayed in the depleted plasma sample and (total zinc - depleted zinc) provides an index of VTG-zinc. For some samples plasma volume was too small to allow measurement of depleted zinc, so only total zinc was assayed. However, total zinc and vitellogenin zinc are highly correlated (r 44 5 0.947, P , 0.001) confirming that total zinc can be used as a reliable measure of plasma vitellogenin (e.g., where plasma volumes are too small to allow the depletion step).

137

Intraassay CV (%) determined for a laying hen plasma pool were 3.0% and 2.6% (n 5 8) for total and depleted zinc, respectively. Interassay CV (%) was 8.4% using Control I serum (Sigma) and 13.0% for vitellogenin zinc using a laying hen plasma pool (n 5 27 assays). Plasma very low-density lipoprotein (VLDL) was assayed using a triglyceride kit (Wako Chemicals), following Mitchell and Carlisle (1991). Intra- and interassay CV (%) were 4.0% (n 5 10) and 6.7% (n 5 14 assays), respectively, using a 16-week hen plasma pool. All assays were run using 96-well microplates, and measured using a Biotek 340i microplate reader. Statistical Analysis Plasma yolk precursor levels were analyzed using a nonparametric Wilcoxon two-sample test, but all other data were analyzed using general linear models or ANCOVA (proc GLM; SAS Institute, 1989), controlling for significant covariates. Female body mass (at pairing) was included as a covariate in analyses of egg mass, and laying interval was included as a covariate in analyses of clutch size (Williams, 1996a). All statistical analyses were carried out using SAS (SAS Institute, 1989). Values are given as means 6 SE unless otherwise stated.

RESULTS Validation of 17b-Estradiol Treatment in Nonbreeding Females Estradiol treatment of nonbreeding females significantly elevated plasma levels of the yolk precursors vitellogenin (Wilcoxon two-sample test; total zinc, Z 5 3.73, P , 0.001: VTG-Zn, Z 5 3.72, P , 0.001) and VLDL (Z 5 2.98, P , 0.01), compared to control birds (Fig. 1). Circulating levels of yolk precursors in E 2treated nonbreeders were similar to (VLDL), or slightly higher than (VTG-Zn), those of breeding females (see Table 1). E 2-treatment also stimulated a fourfold increase in oviduct mass in nonbreeding females compared to controls (Z 5 3.17, P , 0.01; Fig.1). Plasma vitellogenin levels were still significantly higher (total zinc, 2.9 6 0.6 mg/ml) at 6 days postinjection in nonbreeders compared to controls (1.1 6 0.1 mg/ml; Z 5 3.69, P , 0.001). Effect of 17b-Estradiol Treatment on Reproductive Performance of Adult Females There was no difference in body mass at the 1-egg stage between E 2-treated and control females (15.3 6

138

Tony D. Williams

Estradiol-treated females laid a total of 94 eggs of which 39 hatched successfully (41%; 11/17 pairs hatched 1 or more chicks), whereas control birds laid a total of 71 eggs of which 38 hatched successfully (54%; 9/14 pairs hatched 1 or more chicks; test for difference in hatching success, x 2 5 2.35, df 5 1, P . 0.10). Fledging success was 74% for E 2-treated females (29/39 chicks surviving from hatching to fledging) and 92% for control females (35/38 chicks; x 2 5 4.32, df 5 1, P , 0.05), i.e., nestling mortality was higher in broods of E 2-treated females. Four chicks in E 2-broods died within 48 h of hatching and all but one chick (n 5 10) died within 1 week of hatching. In control broods, two of three chicks also died within 1 week of hatching (one unknown). Overall breeding success was therefore lower in E 2-treated females (31% or 29 chicks/94 eggs) compared to control females (49% or 35/71; x 2 5 5.80, df 5 1, P , 0.025). There was no difference in either body mass or size (tarsus length) of chicks at 21 days of age in broods of E 2-treated or control female (Table 1). Reproductive Performance of Female Offspring FIG. 1. Effect of administration of exogenous 17b-estradiol (1.2 mg/g body weight) on plasma yolk precursor levels (VTG and VLDL) and oviduct mass in nonbreeding adult female zebra finches. Open bars, control females; hatched bars, E 2-treated females.

0.4 g vs 15.0 6 0.4 g, F 5 0.40, df 5 1,29, P . 0.50). Similarly, there was no difference in reproductive performance (mean egg mass, F 5 0.04, df 5 1,28, P . 0.80; mean clutch size, F 5 1.99, df 5 1,27, P . 0.15) between treatment groups, based on data from 1– 4 previous non-manipulated breeding attempts per female. Plasma levels of yolk precursors did not differ between E 2-treated and control females sampled at the 1-egg stage, either for total zinc (Z 5 0.88, P . 0.30) VTG-Zn (Z 5 0.68, P . 0.40) or VLDL (Z 5 1.19, P . 0.20; Table 1). E 2-Treatment had no effect on mean egg mass, clutch size, or laying interval, compared to the control group (P . 0.50 in all cases; Table 1). However, overall E 2-treated females produced more daughters than sons (21F:8M; test against expected 1:1 sex-ratio, x 2 5 5.82, df 5 1, P , 0.025) surviving to fledging, that is the sex ratio of surviving offspring of E 2-treated females was significantly female-biased. In contrast, the overall sex ratio of offspring of control females did not differ from unity (19M:18F; P . 0.5). Only E 2treated females produced female-only broods (4 of 9 broods).

At 3 months of age, 18 of 21 daughters from E 2treated females successfully bred (laid eggs) when paired (86%, 9 broods), compared to 9 of 14 daughters from control females (64%, 5 broods; Fisher’s Exact x 2 5 2.19, df 5 1, P . 0.20). Body mass at 3 months of age did not differ among treatments (P . 0.50). Daughters of E 2-treated females laid larger eggs compared with

TABLE 1 Comparison of Plasma Yolk Precursor Levels, Reproductive Output, and Chick Growth for E 2-Treated (n 5 17) and Control (n 5 14) Female Zebra Finches Parameter Total zinc (mg/ml) Vitellogenin (VTG-Zn, mg/ml) VLDL (mg/ml) Mean egg mass (g) Clutch size Laying interval (days) Chick mass (g) Chick tarsus length (mm)

E 2-treated females

Control females

Z/F

P

4.34 6 0.24 2.05 6 0.29

4.01 6 0.27 1.78 6 0.37

0.88 0.68

ns ns

20.8 6 2.5 1.089 6 0.017 5.42 6 0.19 6.5 6 0.6

14.9 6 3.0 1.097 6 0.019 5.63 6 0.21 6.9 6 0.7

1.19 0.11 0.53 0.17

ns ns ns ns

12.9 6 1.1 16.55 6 0.39

12.4 6 0.9 16.48 6 0.22

0.86 0.37

ns ns

Note. Values are means or least-square means 6 SE. For chick growth, values are calculated from the mean per brood.

17b-Estradiol and Female Reproduction

139

TABLE 2 Comparison of Reproductive Output at 3-Months of Age for Daughters of E 2-Treated Females (n 5 18 Birds, 9 Broods) and Control Females (n 5 9 Birds, 5 Broods), and Their Mothers Egg and Clutch Size from Previous Breeding Attempts (See Text)

Parameter

Daughters of E 2-treated females

Daughters of control females

Mean egg mass (g) Clutch size Laying interval (days) Mother’s egg mass (g) a Mother’s clutch size a

0.946 6 0.030 3.08 6 0.45 7.3 6 0.8 0.985 6 0.036 4.25 6 0.51

0.853 6 0.024 3.13 6 0.64 8.1 6 0.6 0.962 6 0.045 4.40 6 0.64

Note. Values are means for siblings in each brood 6 SE. a Based on data from 1– 4 previous breeding attempts.

daughters of control females, either considering data from all female offspring (F 5 5.77, df 5 1,23, P , 0.025) or using mean egg mass of siblings for each brood (Z 5 2.00, P , 0.05; Table 2). However, there was no difference in clutch size (P . 0.80) or laying interval (P . 0.15; Table 2) among daughters of E 2treated or control females. Again, there was no difference in the mean egg mass or clutch size of adult females (i.e., mothers) that produced offspring surviving and laying eggs at 3 months of age, based on data from 1– 4 previous breeding attempts (P . 0.50 in both cases; Table 2).

DISCUSSION Treatment of adult nonbreeding female zebra finches with exogenous 17b-estradiol stimulated yolk precursor production and oviduct growth as has been reported in previous studies using male or immature birds (e.g., Sutherland, Mester, and Baulieu, 1977; Robinson and Gibbins, 1984; Mathews et al., 1988). Estradiol treatment at a dose rate of 1.2 mg/g body weight increased oviduct mass to only 30% of mature size (170 mg vs 570 mg; T. D. Williams, unpublished data; see also Mathews et al., 1988) probably reflecting the synergistic requirements for progesterone in oviduct development (e.g., Brant and Nalbandov, 1956). However, yolk precursor concentrations were elevated to levels similar to (VLDL), or slightly higher than (vitellogenin), those typical of breeding females. This suggests that the estradiol dose used in this experiment induced physiological, or at most supraphysiological, effects on these components of the female reproductive system, and not pharmacological

effects (cf. many other studies which have used doses of 10 –50 mg/g body weight, e.g., Rosebrough, McMurty, and Steele, 1982; Robinson and Gibbins, 1984; Mansukhani, Adkins-Regan, and Yang, 1996). Treatment of nonbreeding female zebra finches with 5 mg/g body weight estradiol induced pharmological levels of both yolk precursors three- to fivefold higher than those in breeding females (T.D. Williams, unpublished data). Effect of 17b-Estradiol Treatment on Adult Females Due to the small plasma volumes obtained from zebra finches (and the specific interest in circulating yolk precursor levels) it was not possible to assay samples directly for estradiol in this study. Nevertheless, effects of exogenous estradiol on mature nonbreeding females in this study, and other studies (Mathews et al., 1988) strongly suggest that intramuscular injections of estradiol elevate circulating estradiol levels. Furthermore, the effect of elevation of plasma estradiol is maintained for days (this study) or even weeks (Robinson and Gibbins, 1984) after injections are stopped, as determined from measurements of yolk precursor levels. Egg formation takes on average 4 days per egg in zebra finches (3 days for rapid yolk development and 1 day postovulation for albumen and shell formation; Haywood, 1993) and first eggs are laid 6 –7 days after pairing. Rapid yolk development (RYD) therefore commenced in most bird 2–3 days after the last daily estradiol injection and all eggs should have developed while circulating estradiol levels were elevated. Despite this, there was no effect of exogenous estradiol on circulating levels of yolk precursors or on the level of primary reproductive output (egg size or clutch size) in breeding female zebra finches. Plasma vitellogenin levels vary markedly among individual females (10-fold variation; Williams and Christians, 1997), but it is not known how variable vitellogenin levels are within individuals, either through a laying cycle or in successive breeding attempts. If yolk precursor levels are tightly regulated within individuals then negative feedback systems may “buffer” precursor synthesis against changes in plasma estradiol levels. In breeding female European starlings (Sturnus vulgaris), however, estradiol treatment using silastic implants did increase plasma vitellogenin levels (by 30%) but there was still no effect on reproductive output (Williams and Christians, 1997). This suggests that there are multiple levels of regula-

140

tion in estrogen-dependent control of reproductive performance (e.g., at the liver regulating yolk precursor production, but also at the follicle regulating uptake independent of the circulating pool of yolk precursors). Few previous studies have investigated effects of exogenous estradiol on reproductive physiology or performance (cf. reproductive behavior; Balthazart, 1983) in mature, laying females. Several studies have involved exposure of mature, nonbreeding females to exogenous estradiol but have focused on the behavioral response of untreated males to E 2-treated females (Runfeldt and Wingfield, 1985; Wingfield, 1994). Adkins-Regan et al. (1995) treated adult, laying female Japanese quail (Coturnix coturnix japonica) with estradiol benzoate (EB) and reported that egg-laying rates were decreased to values somewhat lower than laboratory norms, although there was no effect on hatchability, survival, and sex ratio of chicks from EBtreated females. However, even effects on egg-laying were most apparent at relatively high dose rates in this study (injections of 50 mg/g or in birds with two versus one silastic implants). Even though there was no effect of estradiol treatment on yolk precursor levels or primary reproductive output, there were signicant differences in secondary reproductive output (sex ratio and chick survival) between E 2-treated females and controls. Specifically, E 2-treated females produced more daughters than sons surviving to fledging (21 days). Free-living populations of zebra finches have sex ratios not significantly different from 1:1 at fledging (reviewed in Zann, 1996), but several studies of laboratory populations have reported skewed secondary sex ratios. In general, however, these appear to result from experimental manipulations, e.g., food restrictions (Kilner, 1998), brood size manipulation (de Kogel, 1997). Furthermore, recent studies have all reported significant male-biased sex ratios (Clotfelter, 1996; de Kogel, 1997; Bradbury and Blakey, 1998; Kilner, 1998), the opposite of the present study. In another (unrelated) experiment in our population, a large sample of nonmanipulated female zebra finches maintained on ad libitum seed plus egg food produced offspring with a sex ratio of 1:1 at independence (33M:34F). This strongly suggests that the female-biased sex ratio in E 2-treated females was not an artefact of rearing, or nutritional, conditions in our population but was specifically related to the estradiol treatment. In chickens, exposure of embryos to estrogens in ovo causes phenotypic sex reversal, all chicks hatching as phenotypic females (Etches and Kagami, 1997). However, this result is

Tony D. Williams

transient, genotypic sex is unaffected, and all genotypic males revert to phenotypic males by 18 weeks of age (Etches and Kagami, 1997). This was not the case in the present study because genotypic sex of female offspring from E 2-treated females was confirmed using the molecular sexing technique (Griffiths et al., 1996): all phenotypic female offspring were genotypic females. Estradiol-treated females had significantly lower fledging success, i.e., higher chick mortality during the nestling phase, compared to control females. Although the sex of dead chicks was not known, this suggests that the skewed fledging sex ratio might have been caused by differential mortality of male chicks in broods of E 2-treated females, rather than through effects of maternal estrogens on sex determination or primary sex ratio. Most chicks died within 7 days of hatching, in both E 2- and control broods, but the cause of death was unknown (dead chicks were quickly trampled in the nest, desiccated and sometimes eaten by adults). Chick mortality could have been induced directly via estrogenic effects on embryo or chick development, or indirectly via effects of estradiol on maternal parental behavior, e.g., chick provisioning. Late-hatched zebra finch chicks in large broods often have higher mortality, through inadequate provisioning and starvation (e.g., Skagen 1988). However, there was no evidence that mortality in E 2-broods was biased toward large broods or late-hatched chicks, and brood size at hatching did not differ with treatment. Even if the higher mortality in E 2-broods were related to parental care there is no evidence in this species for sex-biased provisioning which would have lead to differential male mortality (Clotfelter, 1996). Wingfield et al. (1989) treated adult female song sparrows (Melospiza melodia) with E 2-implants during egg-laying, which significantly elevated plasma estradiol levels, but they found no effect on parental behavior (incubation, chick feeding) or endogenous production of hormones related to parental behavior (e.g., prolactin). Reproductive Performance of Female Offspring Female offspring of estradiol-treated adult females laid significantly larger eggs than daughters of control females when paired at 3 months of age, although there was no detectable difference in other components of primary reproductive effort (propensity to lay, laying interval, or clutch size). Although eggs of estradiol-exposed daughters were on average only 11% heavier than “control” daughters, this difference

17b-Estradiol and Female Reproduction

is of similar magnitude to maximum variation reported among treatment groups for adult females in relation to, e.g., diet quality (Williams, 1996b). Zebra finches breeding for the first time (usually at 90 days of age) lay smaller eggs than during subsequent breeding attempts (T.D. Williams, unpublished data). Thus, daughters of E 2-treated females in this study laid eggs of a size typical of experienced, adult breeders, and larger than normal, first-time breeders. Whether this “enhanced” reproductive output is a positive or negative effect therefore depends on the fitness consequences of egg size, and whether there is an age-specific “optimum” egg size, something which is very poorly understood in birds (Bernardo, 1996). Larger eggs may increase offspring fitness (but see Williams, 1994), whereas increased (early) parental expenditure could decrease adult life-span (Burley, 1995). In most nonpasserine birds (e.g., quail, ducks, chicken) estrogens appear to play an important role in sexual differentiation and are required for female phenotypic development (Adkins-Regan, 1987; Etches and Kagami, 1997; but see Grisham, Wade, and Arnold, 1997). However, in zebra finches (review, Grisham et al., 1997) and some other passerines (Casto and Ball, 1996) estradiol treatment of nestling females has a masculinizing effect on the sexually dimorphic song control system. Casto and Ball (1996) suggested that as the oscine song system is a recently derived adaptation in avian evolution the mechanism of sexual differentiation could differ from that for other forms of reproductive behavior. However, female zebra finches treated with estrogen posthatching also show masculinized sexual partner preference, i.e., females were more likely to pair with other females (Mansukhani et al., 1996). These data appear to contrast with the “feminizing” effect of estradiol (increasing egg size) reported in the present study. However, direct comparisons are complicated by the fact that (a) previous studies have often used very high doses of estradiol (10 –20 mg/g body weight cf. 1.2 mg/g body weight in this study, see above), and (b) they involved different timing and different routes of estradiol exposure (typically posthatching and direct injection or implantation of nestlings). Furthermore, while egg production is clearly a sexually dimorphic trait in birds it is less clear whether the mechanisms of regulation of quantitative variation among females is analogous to the endocrine control of sexual differentiation. The role of estrogens in differentiation and development of neuroendocrine or endocrine systems controlling follicle development and ovulation in gen-

141

eral is poorly understood. Rissman et al. (1984) failed to demonstrate any effect of embryonic treatment with estradiol on ovulation, oviposition, or plasma luteinizing hormone levels in Japanese quail that initiated laying, although in general treated birds were less likely to lay. Adkins-Regan et al. (1995) reported that a small number of offspring from E 2-treated female Japanese quail retained a right oviduct and they suggested this might be associated with reduced fertility (Rissman et al., 1984). In chickens, hens exposed to high levels of estradiol in ovo (by dipping eggs in 10 mg/ml estradiol solution) showed delayed onset of laying (greater than 30 weeks posthatching), but hens exposed at lower concentration (0.1 and 1.0 mg/ml) matured normally with an average onset of lay of 21 weeks (Etches and Kagami, 1997). The present study did not explicitly investigate the mechanism by which maternal estrogens might have long-term effect on future reproductive performance of offspring. However, it seems most likely that estrogens in maternal circulation are transferred to egg yolk during rapid yolk development leading to exposure of embryos in ovo. Adkins-Regan et al. (1995) have demonstrated this pathway in quail: treatment of laying females with estradiol benzoate resulted in elevated yolk estradiol and permanent morphological alteration of female offspring (retention of right oviduct). This might represent a priming or “memory” effect, with initial exposure of the reproductive system to estrogens in ovo enhancing the response to subsequent estrogen stimulation, similar to the primary and secondary stimulation effects reported in Xenopus and chicks (Tata, 1976; Jost et al., 1986). Recent studies by Schwabl (1993, 1996a,b) have clearly shown that androgens from the maternal circulation are transferred to the egg and affect postnatal growth, development, and behavior. In particular, there is a positive correlation between social rank of offspring and yolk testosterone concentrations, and Schwabl (1993) suggested this might explain the large interindividual variation in aggressive behavior (typically, though not exclusively, a male phenotypic trait). The results of the present study therefore extend those of Schwabl (1993, a,b) and strongly suggest that maternal estrogens might also influence embryos in ovo and determine quantitative aspects of intraspecific variation in female-specific phenotypic traits (reproductive output). This supports the hypothesis that female birds can enhance their reproductive fitness through hormonal influences on sibling growth and reproduction (Winkler, 1993; Schwabl, 1996b), as has been demonstrated in mammals (review, Clark and Galef, 1995). Finally,

142

the present study has relevance to the issue of xenoestrogens as endocrine disruptors (e.g., Campbell and Hutchinson, 1998; Feyk and Giesy, 1998) as it confirms that exogenous estradiol can have significant additive effects on mature, laying females, not just on males or immatures.

ACKNOWLEDGMENTS This work was supported by an NSERC Operating Grant to T.D.W. Julian Christians provided helpful comments on an earlier draft of the paper.

REFERENCES Adkins, E. K. (1979). Effect of embryonic treatment with estradiol or testosterone on sexual differentiation of the quail brain: Critical period and dose–response relationships. Neuroendocrinology 29, 178 –195. Adkins-Regan, E. (1987). Sexual differentiation in birds. Trends Neurosci. 10, 517–522. Adkins-Regan, E., Ottinger, M. A., and Park, J. (1995). Maternal transfer of estradiol to egg yolks alters sexual differentiation of avian offspring. J. Exp. Zool. 271, 466 – 470. Balthazart, J. (1983). Hormonal correlates of behavior. In D. S. Farner, J. R. King, and K. C. Parkes (Eds.), Avian Biology, Vol.7, pp. 221–365. Academic Press, New York. Balthazart, J. A., De Clerck, A., and Foidart, A. (1992). Behavioual demasculinization of female quail is induced by estrogens: Studies with the new aromatase inhibitor, R76713. Horm. Behav. 26, 179 –203. Bernardo, J. (1996). The particular maternal effect of propagule size, especially egg size: Patterns, models, quality of evidence and interpretations. Am. Zool. 36, 216 –236. Bradbury, R. B., and Blakey, J. K. (1998). Diet, maternal condition, and offspring sex ratio in the zebra finch, Poephila guttata. Proc. R. Soc. Lond. B 265, 895– 899. Brant, J. W. A., and Nalbandov, A. V. (1956). Role of sex hormones in albumen secretion by the oviduct of chickens. Poult. Sci. 52, 692–700. Burley, N. (1985). Leg band color and mortality patterns in captive breeding populations of zebra finches. Auk 102, 647– 651. Burley, R. W., and Vadehra, D. V. (1989). The Avian Egg: Chemistry and Biology. Wiley, New York. Campbell, P. M., and Hutchinson, T. H. (1998). Wildlife and endocrine disruptors: Requirements for hazard identification. Environ. Tox. Chem. 17, 127–135. Casto, J. M., and Ball, G. F. (1996). Early administration of 17bestradiol partially masculinizes song control regions and a 2-adrenergic receptor distribution in European starlings (Sturnus vulgaris). Horm. Behav. 30, 387– 406. Clark, M. M., and Galef, B. G. (1995). Prenatal influences on reproductive life history strategies. Trends Ecol. Evol. 10, 151–153. Clotfelter, E. D. (1996). Mechanisms of facultative sex-ratio variation in zebra finches (Taeniopygia guttata). Auk 113, 441– 449. de Kogel, C. H. (1997). Long-term effects of brood size manipulation

Tony D. Williams

on morphological development and sex-specific mortality of offspring. J. Anim. Ecol. 66, 167–178. Elbrecht, A., and Smith, R. G. (1992). Aromatase enzyme activity and sex determination in chickens. Science 255, 467– 470. Etches, R. J., and Kagami, H. (1997). Genotypic and phenotypic sex reversal. In R. J. Etches, and H. Kagami (Eds.), Perspectives in Avian Endocrinology, pp. 57– 67. J. Endocrinol., Bristol. Feyk, L. A., and Giesy, J. P. (1998). Xenobiotic modulation of endocrine function in birds. In R. Kendall, R. Dickerson, and W. Suk (Eds.), Principles and Processes for Evaluating Endocrine Disruption in Wildlife, pp. 121–140. SETAC Press, Pensacola. Griffiths, R., Daan, S., and Dijkstra, C. (1996). Sex identification in birds using two CHD genes. Proc. Roy. Soc. Lond. B 263, 1251–1256. Grisham, W., Wade, J., and Arnold, A. (1997). Sexual differentiation of the songbird brain: Evidence for hormonal and non-hormonal mechanisms. In R. J. Etches, and H. Kagami (Eds.), Perspectives in Avian Endocrinology, pp. 37– 46. J. Endocrinol., Bristol. Haywood, S. (1993). Sensory control of clutch size in the zebra finch (Taeniopygia guttata). Auk 110, 778 –786. Jost, J.-P., Moncharmont, B., Jiricny, J., Saluz, H., and Hertner, T. (1986). In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei. Proc. Natl. Acad. Sci. USA 83, 43– 47. Kilner, R. (1998). Primary and secondary sex ratio manipulation in zebra finches. Anim. Behav. 56, 155–164. Mansukhani, V., Adkins-Regan, E., and Yang, S. (1996). Sexual preference in female zebra finches: The role of early hormones and social environment. Horm. Behav. 30, 506 –513. Mathews, G. A., Brenowitz, E. A., and Arnold, A. P. (1988). Paradoxical hypermasculinisation of the zebra finch song system by an antiestrogen. Horm. Behav. 22, 540 –551. McNabb, F. M. A., Scanes, C. G., and Zeman, M. (1998). The endocrine system. In J. M. Starck, and R. E. Ricklefs (Eds.) Avian Growth and Development, pp. 174 –202. Oxford Univ. Press, New York. Mitchell, M. A., and Carlisle, A. J. (1991). Plasma zinc as an index of vitellogenin production and reproductive status in the domestic fowl. Comp. Biochem. Physiol. 100A, 719 –724. Nelson, R. J. (1995). An Introduction to Behavioral Endocrinology. Sinauer, Massachusetts. Riddle, O., and Dunham, H. H. (1942). Transformation of males to intersexes by estrogen passed from blood of Ring Doves to their ovarian eggs. Endocrinology 30, 959 –968. Rissman, E. F., Ascenzi, M., Johnson, P., and Adkins-Regan, E. (1984). 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. Fert. 71, 411– 417. Robinson, G. A., and Gibbins, A. M. V. (1984). Induction of vitellogenesis in Japanese quail as a sensitive indicator of the estrogenmimetic effects of a variety of environmental contaminants. Poult. Sci. 63, 1529 –1536. Rosebrough, R. W., McMurty, J. P., and Steele, N. C. (1982). Effect of estradiol on the lipid metabolism of young turkey hens. Nutr. Rep. Int. 26, 373–376. Runfeldt, S., and Wingfield, J. C. (1985). Experimentally prolonged sexual activity in female sparrows delays termination of reproductive activity in their untreated mates. Anim. Behav. 33, 403– 410. SAS Institute Inc. (1989). SAS/STAT Users Guide, Version 6.0. SAS Institute, Cary, North Carolina. Schwabl, H. (1993). Yolk is source of maternal testosterone for developing birds. Proc. Natl. Acad. Sci. USA 90, 11446 –11450.

17b-Estradiol and Female Reproduction

Schwabl, H. (1996a). Maternal testosterone in the avian egg enhances postnatal growth. Comp. Biochem. Physiol. 114A, 271–276. Schwabl, H. (1996b). Environment modifies the testosterone levels of a female bird and its eggs. J. Exp. Zool. 276, 157–163. Skagen, S. K. (1988). Asynchronous hatching and food limitation: A test of Lack’s hypothesis. Auk 105, 78 – 88. Sutherland, R., Mester, J., and Baulieu, E.-E. (1977). Tamoxifen is a potent ‘pure’ anti-oestrogen in chick oviduct. Nature 267, 434 – 435. Tata, J. R. (1976). The expression of the vitellogenin gene. Cell 9, 1–14. Williams, T. D. (1996a). Intra- and inter-individual variation in reproductive effort in captive-breeding zebra finches (Taeniopygia guttata). Can. J. Zool. 74, 85–91. Williams, T. D. (1996b). Variation in reproductive effort in female zebra finches (Taeniopygia guttata) in relation to nutrient-specific dietary supplements during egg laying. Physiol. Zool. 69, 1255– 1275. Williams, T. D. (1999). Avian Reproduction—Overview. In E.

143

Knobil and J. D. Neill (Eds.), Encyclopedia of Reproduction. Academic Press, San Diego. Williams, T. D., and Christians, J. K. (1997). Female reproductive effort and individual variation: Neglected topics in environmental endocrinology. In S. Kawashima and S. Kikuyama (Eds), Proceedings of the XIIIth International Congress of Comparative Endocrinology, pp. 1669 –1675. Monduzzi Editore, Rome. Wingfield, J. C. (1994). Behavioral and hormonal responses of male song sparrows to estradiol-treated females during the non-breeding season. Horm. Behav. 28, 146 –154. Wingfield, J. C., and Farner, D. S. (1993). Endocrinology of reproduction in wild species. In D. S. Farner, J. R. King, and K. C. Parkes (Eds.), Vol. 9, pp. 163–327. Academic Press, New York. Wingfield, J. C., Ronchi, E., Goldsmith, A. R., and Marier, C. (1989). Interactions of sex steroid hormones and prolactin in male and female song sparrows, Melospiza melodia. Physiol. Zool. 62, 11–24. Winkler, D. W. (1993). Testosterone in egg yolks: an ornithologist5s perspective. Proc. Natl. Acad. Sci. USA 90, 11439 –11441. Zann, R. F. (1996). The Zebra Finch. Oxford Univ. Press, Oxford.