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Maternal Thyroid Hormones in Japanese Quail Eggs and Their Influence on Embryonic Development
General and Comparative Endocrinology 107, 153–165 (1997) Article No. GC976906
Maternal Thyroid Hormones in Japanese Quail Eggs and Their Influence on Embryonic Development C. Morgan Wilson and F. M. Anne McNabb1 Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406 Accepted February 26, 1997
We addressed the relationship between the thyroid status of hens and the thyroid hormone content of their eggs, as well as the influences of egg hormones on embryonic development. Methods for measuring thyroid hormones in egg yolk were verified by demonstrating consistency in the recovery of yolk thyroid hormones following a methanol/chloroform extraction and in the measurement of thyroid hormones by RIA for a range of hormone concentrations in yolk extracts. Untreated hens produced eggs with yolk thyroxine (T4) concentrations that were low relative to plasma T4, but yolk triiodothyronine (T3) concentrations comparable to those of plasma. Hens dosed twice daily with T4 (1 or 33 the daily thyroid secretion rate, TSR, of T4 per dose) had significantly higher plasma and egg yolk T4 concentrations than did control hens dosed with saline. In general, the T4 concentration of egg yolk varied with the thyroid status of the hen. When the relationship between each hen’s plasma T4 and the yolk T4 concentration of her eggs was examined, hens appeared to regulate T4 deposition into yolk at ‘‘levels’’ characteristic of the ‘‘levels’’ of thyroid status produced by the different doses of T4. Embryonic pelvic cartilage, a thyroid hormone-responsive tissue, showed enhanced growth and differentiation in embryos from eggs of hens given the highest dose of T4. Specifically, alkaline phosphatase activity (a marker of differentiation) and pelvic cartilage wet and dry weights were significantly greater in embryos from high T4 eggs (hens on the 33 TSR dose) than those in controls. However, 1
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embryos from high T4 eggs did not differ in general body growth (body weight, length, and general morphology) or hatchability compared to controls. In a single T3 experiment, hens were dosed twice daily with 1 mg T3. The embryos from eggs of these hens had accelerated differentiation/maturation of pelvic cartilages (sampled at Day 12) compared to those from control eggs; body growth did not differ from that of controls. r 1997 Academic Press
Thyroid hormones are essential for the control of development in all vertebrate classes (Gorbman et al., 1983; McNabb, 1992; McNabb and King, 1993). However, some early mammalian studies suggested that maternal thyroid hormones did not cross the placenta, and therefore they were presumed not to play a role in embryonic development (Fisher et al., 1977; Schwartz, 1983). It is now known that maternal thyroid hormones are available to embryos of many vertebrates. Maternal thyroid hormones are transferred into fish eggs (Brown et al., 1987; Kobuke et al., 1987; Tagawa and Hirano, 1987) and cross the placenta in rats (Morreale de Escobar et al., 1985). In striped bass, treatment of prespawning females with T3 is associated with accelerated larval development, swimbladder inflation, and enhanced larval survival (Brown et al., 1988). Maternal thyroid hormones influence fetal development in rats prior to the time of appreciable fetal thyroid gland function (Morreale de Escobar et al., 1985, 1988). Although thyroid hormones are known to affect the
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development of avian embryos and chicks (review, McNabb and King, 1993), few studies have investigated maternal thyroid hormones in avian eggs. The first reports of thyroid hormones in avian eggs (chickens; Hilfer and Searls, 1980; Sechman and Bobeck, 1987) are difficult to evaluate because they provided no validation showing that the methods used effectively extracted or accurately measured yolk thyroid hormones. However, the data from these studies, as well as those from a more recent study of chicken eggs using validated techniques (Prati et al., 1992), indicated that maternal thyroid hormones are present in avian egg yolk. If utilized by the embryo, maternal thyroid hormones in egg yolk could influence development, especially before appreciable synthesis and release of hormones by the thyroid gland. During the first one-third to one-half of incubation, the thyroid gland appears to possess a very limited capability to synthesize hormones (chickens: Thommes, 1987; Japanese quail: McNichols and McNabb, 1988). Thommes and Hylka (1978) found detectable T4 in embryonic chicken plasma by Day 6.5 of the 21-day incubation period, and Prati et al. (1992) found extremely small amounts of T4 and T3 in pooled chicken embryos on Days 4 and 6. It is not clear whether these small amounts of thyroid hormones in early embryos are of maternal or embryonic origin or both. It is clear, however, that after linking of the hypothalamic–pituitary–thyroid (HPT) axis (Days 10.5–11.5 in chicken embryos) thyroid hormones from the embryonic thyroid gland predominate (Thommes et al., 1977). The objectives of this study were: (1) to validate methods for the extraction of thyroid hormones from avian egg yolk, (2) to determine the relationship between the thyroid status of the hen and thyroid hormones in the yolk of her eggs, and (3) to determine if thyroid hormones in the egg yolk affect thyroid hormone-responsive development of embryos.
MATERIALS AND METHODS Animals Adult Japanese quail (Coturnix japonica), ages 8 to 12 months, from a random-bred colony, were housed in
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Wilson and McNabb
pairs and maintained under a 14L:10D photoperiod. A commercial game bird ration (18 to 24% protein; Big Spring Mills, Shawsville, VA) and water were available ad libitum. Eggs were incubated at 39 6 1° and .90% relative humidity in a forced-air incubator (Humidaire Hatchette Incubator; New Madison, OH). Hens were dosed with hormone solutions using a tuberculin syringe fitted with a 2-in. piece of tygon tubing to deliver the dose into the hen’s crop.
Experimental Design Maternal thyroid hormone deposition in yolk. To determine the number of days thyroid hormones could be deposited in the yolk of an individual egg, adult Japanese quail hens (n 5 8) were dosed orally at 0800 hr for 10 days, alternating two lipid soluble dyes (technique modified from Bacon and Cherms, 1968). Either Sudan black B or Sudan IV red was administered (200 µl of 0.01 g dye/ml of vegetable oil; dyes from Sigma Chemical Co., St. Louis, MO). Eggs were collected daily and boiled for 20 min. Boiled eggs were sliced longitudinally to count the colored rings of yolk. Manipulation of hens’ thyroid status and egg hormone content. To determine if hens deposit hormones in eggs in proportion to their own thyroid status, laying hens (n 5 12 per treatment) were made hyperthyroid by oral dosing with L-T4 (Sigma Chemical Co.) in 200 µl of saline (0.9% NaCl) at 0800 and 1800 hr. Dosages were a multiple (either 1 or 33) of the normal daily thyroid secretion rate of T4 (TSR T4) for adult Japanese quail (2.78 µg/100 g body weight/day; Singh et al., 1967). Control hens were dosed with 200 µl of saline at the same times. Twice-daily dosing was chosen based on a preliminary experiment in which blood from hens was collected before dosing and then every 3 hr for 12 hr after the 0800-hr dose of T4. This experiment indicated that T4 needed to be administered twice daily to maintain elevated plasma T4 concentrations. Blood was collected weekly from control (saline)-, 13 TSR T4-, and 33 TSR T4-dosed hens (3 to 4 hr after the morning dose). For yolk hormone analysis, eggs were collected (labeled by hen and date) from salineand T4-dosed hens on the day blood was sampled. One set of eggs collected from saline- and T4-dosed hens was incubated until hatch. Hatchlings were inspected for gross morphology, weighed, and measured
Thyroid Hormones in Egg Yolk
(length in mm, crown to rump), and the approximate time of hatch was recorded. Another set of eggs from the hens on each treatment was incubated and sampled for hormone measurements on Days 7 and 15 for embryos, on Days 0, 7 and 15 for yolk, on Days 0 and 7 for albumen, on Day 7 for allantois, and on Days 9 and 14 for embryonic blood. Two sets of eggs (from hens receiving each treatment) were incubated for the studies of pelvic cartilage. For wet- and dry-weight measurements, pelvic cartilages were sampled from Day-7 to -13 embryos (Day 7 was the first day when the fragile cartilages could be effectively trimmed for accurate weight determinations). For alkaline phosphatase activity, sampling started on Day 6 but extended only to Day 12 (the last day when it was practical to homogenize the cartilages with a glass tissue grinder). In a single experiment using T3, laying hens were dosed at 0800 and 1800 hr with 1 µg T3 in 200 µl 0.9% NaCl. Blood was collected prior to dosing and then at 1200 and 1800 hr on the first day of dosing. Eggs were collected from these hens prior to dosing and on Days 5 and 6 of the dosing; only Day 12 embryos were sampled. To determine if hypothyroid hens produce thyroid hormone-deficient eggs, and to attempt to obtain eggs with low thyroid hormone content, adult laying hens (n 5 8) were given an oral dose of 4 mg of methimazole (a goitrogen that inhibits thyroid hormone synthesis; Sigma Chemical Co.) in 200 µl saline at 0800 hr daily for 30 days. Because this treatment did not lower plasma thyroid hormone concentrations, the dose was increased to 8 mg/day for an additional 40 days. Soon after this increase in methimazole dosage, the hens ceased laying. At this point the experiment was ended, and the hens were sacrificed and weighed and their thyroid glands removed and weighed.
Sampling and Analyses Blood was collected from the brachial vein of laying hens and from the chorioallantoic artery of embryos. Samples were collected in heparinized capillary tubes and centrifuged, and the plasma was stored at 220°. For the studies of embryonic pelvic cartilage, the cartilage was dissected out of embryos, connective tissue and muscle were removed, and the cartilage was blotted and weighed. Some pelvic cartilages were dried to constant weight at 100° for 18–24 hr. For
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alkaline phosphatase analyses, other pelvic cartilages were homogenized in 0.9% NaCl (73 v/w) using a 0.5-ml glass tissue grinder, and the homogenate centrifuged (12,500g, 4°, 2 min). The postmitochondrial supernatant fraction (PMF) was removed and stored at 220° until analysis for alkaline phosphatase activity. Yolk thyroid hormone extraction. Thyroid hormones were extracted from egg yolk using a modification of a methanol/chloroform extraction procedure previously used for amphibian larvae by Denver (1993). Egg yolk was weighed, diced with scissors, and homogenized using a 50-cc glass syringe with an 18-gauge needle; each yolk was drawn into and expelled from the syringe three times. A 0.5-g sample of homogenized yolk was transferred to a 13-ml (17 3 100 mm) test tube (all tubes used were polypropylene), and 2 ml of methanol containing 1 mM propylthiouracil (PTU; an inhibitor of deiodination; Sigma Chemical Co.) was added. Labeled thyroid hormone (,2000 cpm of either 125I-T4 or 125I-T3; high specific activity: T4, 1250 µCi/µg; T3, 1200 µCi/µg; New England Nuclear; Boston, MA) was added, and the sample was vortexed and counted for 10 min. After a 10-min extraction on a multitube shaker (150 oscillations/min), the sample was centrifuged (1700g, 4°, 10 min) and the supernatant was decanted into a 50-ml graduated conical polypropylene tube. The precipitate was resuspended in 1 ml of methanol (with 1 mM PTU), shaken for 10 min, and centrifuged, and the supernatant decanted into a second 50-ml tube. The two separate supernatants each received 5.0 ml of chloroform (CHCl3) and 0.5 ml ammonium hydroxide (2 N NH4OH) and were shaken and centrifuged as above; the upper phase (methanol/aqueous phase) from both tubes was removed and combined in a 13-ml tube. The CHCl3 phase remaining in each tube was extracted again with NH4OH as above and the upper phase from each was added to the methanol/aqueous pool for each sample and then dried under a filtered air stream (for 6–7 hr) in a fume hood. When dry, the sample was resuspended in 1 ml 2 N NH4OH, briefly vortexed, shaken, and centrifuged (as above), and the supernatant decanted into a 13-ml tube. The general extraction procedure was then repeated with 1 ml CHCl3, and the upper phase was removed, dried, resuspended in 150 µl 75% EtOH, and counted (10 min) to determine extraction efficiency (recovery of the labeled thyroid
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hormone added to the original homogenate). The extraction efficiency was consistent; 63 6 1% (mean 6 SE) for 125I-T4 and 61 6 3% for 125I-T3 (n 5 6 for each hormone). These recoveries are comparable to others from similar extraction techniques for yolk or tissue thyroid hormones [45 to 70% recovery: Tagawa and Hirano (1990), fish; Niinuma et al. (1991) and Denver (1993), amphibians]. Individual recoveries were determined for all samples and used to calculate thyroid hormone concentrations. The reconstituted extracts were stored at 220° until analysis. To measure embryonic hormones, embryos were decapitated to remove the area containing the thyroid gland, and the carcass was homogenized in a volume of methanol (with 1 mM PTU) twice the embryo weight using a Brinkmann tissue homogenizer (Model PT 10/35 with PTA 10 generator; Brinkmann Instruments, Westbury, NY). After homogenization, the blades were rinsed with an equivalent volume of methanol (with 1 mM PTU) which was added to the homogenate. Thyroid hormones in the embryo, albumen, and allantois homogenates were extracted and assayed as described above for yolk. Thyroid hormone radioimmunoassays (RIAs). Plasma T4 and T3 concentrations were measured with a double antibody RIA by the method of McNabb and Hughes (1983) using hormone standards prepared in hormone-free chicken plasma or in 75% ethanol for
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plasma and extract samples, respectively. Primary antibodies were purchased from Endocrine Sciences (Calabasas, CA), 125I-T4 and 125I-T3 were from New England Nuclear (Boston, MA; high specific activity: T4, 1250 µCi/µg; T3, 1200 µCi/µg), and carrier immunoglobulin was from Antibodies Incorporated (Davis, CA). Secondary antibody was kindly provided by Dr. John McMurtry (USDA; Beltsville, MD). Assay volumes were 12.5 µl for T4 and 25 µl for T3. The plasma assay was validated previously (McNabb and Hughes, 1983). To validate the RIA for use on yolk extract, a pooled extract sample was diluted with 75% ethanol or spiked with hormone standards prepared in 75% ethanol. Precision tests performed on yolk extracts show 6 2 SE was 13.7% of the mean for T4 (n 5 10) and 14.8% of the mean for T3 (n 5 8). A Thiel–Sen test showed no significant difference between the slopes of the measured and predicted (slope of 1.0) lines for both RIAs (T4: k 5 3, P 5 0.431, Fig. 1a; T3: k 5 9.0, P 5 0.376, Fig. 1b), indicating consistent measurements over a range of hormone concentrations. Alkaline phosphatase (ALP) analysis. An ALP assay, adapted from the method of Suvarna et al.(1993), was validated by demonstrating linearity of enzyme activity with time and proportionality between enzymatic activity and PMF concentration for pelvic cartilages from 6-, 9-, and 12-day embryos. The validation studies indicated that pelvic cartilage PMF required
FIG. 1. Validation of radioimmunoassays for measuring thyroid hormones in extracts of egg yolk. (a) T4; (b) T3. Circles, measured values of yolk extract spiked with known amounts of hormone; squares, measured values of dilutions of yolk extract. The dashed lines are Theil–Sen regression lines of the measured points; the solid (predicted) line has a slope of 1.0. The measured and predicted lines do not differ significantly for either hormone.
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Thyroid Hormones in Egg Yolk
dilution (with 0.9% NaCl) by 1:1 for 6- and 7-day embryos and by 1:19 for Days 8 to 12. For the assay, a 20-µl volume of diluted PMF was added to 300 µl of ALP reagent (Sigma Diagnostic Alkaline Phosphatase Components Kit; Sigma Chemical Co.) in a narrow light path cuvette, the solution was mixed by rapid shaking, and absorbance readings were taken at 0 and 30 min. The hydrolysis of phosphate by ALP converts the substrate p-nitrophenyl phosphate into p-nitrophenol and inorganic phosphate, resulting in a color change which was measured at l 5 405 nm (25°) with a Beckman DU-640 spectrophotometer. The precision of this technique on 10 aliquots of a pooled sample of pelvic cartilage PMF showed that 62 SE was 8.3% of the mean. A unit of alkaline phosphatase activity is defined as ‘‘that amount of alkaline phosphatase which will produce one µmol of p-nitrophenol per min at 25°9 (Sigma Diagnostics, 1990).
Statistical Analyses
Thyroid Status of Hens and Their Egg Thyroid Hormone Content The administration of T4 to hens produced a hyperthyroid condition (increased plasma T4) and elevated the yolk T4 content in their eggs. Hens given a single dose of 13 TSR T4 or 33 TSR T4 had plasma T4 concentrations 8- to 15-fold those of controls by 3 hr after dosing, but by 9–12 hr plasma T4 had decreased 2to 3-fold. Thus, twice-daily dosing was needed to sustain high plasma T4 over a 24-hr period, and all subsequent experiments used twice-daily dosing. The twice-daily 13 TSR T4 dose significantly increased T4 but not T3 in the hens’ plasma compared to controls; the 33 TSR T4 dose resulted in significant increases in both hormones in the hens’ plasma. Yolk from eggs of hens on each of the T4 doses had significantly higher concentrations of both T4 and T3 than yolk from eggs of control hens (Fig. 2). However, examination of the relationship between yolk T4 in individual eggs and the hen’s plasma T4 at the time of laying each egg indicated that yolk T4 concentrations
Student’s t tests were used to evaluate the effects of treatment of hens on egg yolk and plasma thyroid hormones. A Theil–Sen simple linear regression procedure was used to compare the slope of regression lines to a slope of 1 (the Theil–Sen test statistic is reported as k). Pelvic cartilage wet and dry weights expressed as a percentage of individual embryonic body weight were normally distributed (Shapiro–Wilks test for normality); the effects of treatments on embryonic pelvic cartilages were compared by ANOVA (general linear models procedure). A value of P # 0.05 was considered indicative of statistically significant differences.
RESULTS Time Course of Egg Yolk Deposition Eggs from hens given alternating doses of two lipid-soluble dyes showed alternating rings of dye in egg yolk. For hens that laid one egg each day, five to six alternating, colored rings were present in each egg yolk after 5 to 10 days of dosing with the dyes. This number of days of yolk deposition is consistent with that previously reported by Bacon and Koontz (1971).
FIG. 2. The relationship between thyroid hormone concentrations in hen plasma (a) and egg yolk (b). Top panels, T4; bottom panels, T3. C, controls dosed with saline; 13 T4, hens dosed twice daily with 13 the daily thyroid secretion rate (TSR) of T4; 33 T4, hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 38–68 for each mean); different lowercase letters above the bars indicate significant differences between means.
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did not increase in simple proportion to increases in hen plasma T4 [i.e., Theil–Sen regression line slopes were significantly ,1.0 for each treatment group (P 5 0.00001); Figs. 3b–3d]. Instead, yolk T4 concentrations showed a stepwise pattern of increase with increasing T4 treatment of the hens (Fig. 3a). Variation between yolk T4 concentrations in eggs of hens was similar in the three treatment groups when standard deviations were expressed as a percentage of the mean (controls, 24%; 13 TSR, 19%; 33 TSR, 30%; based on 5–6 hens per group). Some individual hens in each treatment group had much higher variation among their eggs than did others. When standard deviations were expressed as a percentage of the mean, values ranged from 8 to 43% for different individual hens (based on analysis of 17 hens; four eggs per hen). Hens dosed with 1 µg T3 showed small, but significant, increases (1.7-fold) in plasma T3 concentrations
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by 4 hr after dosing; plasma T4 concentrations were significantly less (3.3-fold) than that of controls by 4 hr after dosing. Plasma hormone concentrations decreased in the period from 4 to 8 hr after dosing, so this experiment utilized twice-daily dosing with 1 µg T3/dose. The T3 and T4 content of the yolk of eggs from the T3 treated hens did not differ significantly from that of controls. The administration of methimazole at 4 mg/day for 1 month and 8 mg/day for a second month did not significantly decrease hen plasma thyroid hormone concentrations compared to controls. However, the thyroid glands of the methimazole-treated hens were significantly heavier (,9-fold) than those from control hens. Yolk of eggs laid by hens receiving either 4 or 8 mg/day methimazole did not differ significantly in either T4 or T3 from the yolk of eggs laid by control hens. This experiment was stopped soon after the start of the 8-mg dose because the hens stopped laying.
FIG. 3. The relationship between T4 concentrations in individual eggs and plasma T4 concentration of the Japanese quail hen that laid each egg. (a) All data; open circles, control hens dosed twice daily with saline; closed squares, hens dosed twice daily with 13 the daily thyroid secretion rate of T4; closed circles, hens dosed twice daily with 33 TSRT4. For the bottom panels the lines are Theil–Sen regression lines. (b) Controls (slope 5 0.08); (c) 13 TSR T4 dose (slope 5 0.05); (d) 33 TSR T4 dose (slope 5 0.25).
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Thyroid Hormones in Egg Yolk
Egg Compartments during Development The total weight of the embryo, albumen, and allantois on Days 0, 7, and 15 did not differ in control and high T4 eggs from hens which received either T4 dose. The weight of the embryo and egg compartments changed in the directions expected during development: embryonic body weight increased 9-fold from Days 7 to 15, albumen weight decreased 2.8-fold from Days 7 to 15, and yolk weight increased 1.4-fold from Days 0 to 7 and then decreased 3-fold by Day 15. This pattern of yolk weight increase followed by a decrease occurs as a result of water influx into the yolk from the albumen, followed by the uptake of yolk by the embryo during the latter part of embryonic development (Romanoff, 1967). Allantoic weight did not differ in control and high T4 eggs on Day 7; the allantois could not be dissected out for measurement at later stages of development. Yolk T4 content of high T4 eggs decreased dramatically during incubation. Before incubation the T4 content in eggs from the 13 TSR T4-dosed hens was 7.5-fold, and that of the 33 TSR T4-dosed hens was 26.5-fold greater than that in controls. By Day 15 of incubation, the yolk T4 content in eggs from 13 TSR T4-treated hens did not differ from that in controls. Yolk T4 content in eggs from 33 TSR T4-dosed hens, although markedly decreased, remained significantly higher (P 5 0.002; Fig. 4a) than that in control eggs. Yolk T3 content did not change between Days 0, 7, and 15 in control embryos or in embryos from eggs of 13 TSR T4-dosed hens. However, in the experiment shown in Fig. 4a, the T3 content of the yolk on Day 7 in the eggs of hens on the 33 TSR dose was significantly greater than that of controls. We repeated this experiment and found that the yolk content of T3 at Day 7 did not differ between groups in this second experiment. Before incubation, the albumen T4 content was 4.2 ng per egg for controls, 5.9 ng for 13 TSR T4, and 18.3 ng for 33 TSR T4 eggs; albumen T3 content was 1.1–1.2 ng per egg for all treatment groups. Thus the albumen in eggs from the highest T4 dosage group contained about 4-fold more T4 than that in control eggs. However, this is a small increase compared to that of yolk T4. In yolk, the T4 content of eggs from the highest dosage group was about 26-fold that in control eggs. Allantoic content of T4 was 0.56 6 0.07 ng/egg in
FIG. 4. Thyroid hormone content of egg yolk and embryos (carcass without thyroid gland) of Japanese quail. (a) Thyroid hormone content of egg yolk. (b) Thyroid hormone content of embryos (without thyroid gland). Top panels, T4; bottom panels, T3. Open circles, control hens dosed twice daily with saline; closed squares, hens dosed twice daily with 13 the daily thyroid secretion rate of T4; open triangles, hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 4–7); asterisks indicate a significant difference from control values on the same day of incubation.
controls, 1.12 6 0.44 ng/egg in 13 TSR, and 0.84 6 0.17 ng/egg in 33 TSR on Day 7 of incubation. In the experiment where T3 was administered to hens, there were no significant differences in the embryo weight or in the plasma concentrations of T4 and T3 in Day-12 embryonic plasma from their eggs compared to controls (Table 1).
Embryonic Growth and Thyroid Development Chicks hatched from control and high T4 eggs (from hens on either T4 dosage) showed no significant differences in gross morphology, crown to rump length, or body weight. Hatchability of chicks from control and high T4 eggs was similar (control vs 13 TSR T4, 75% vs 68%, respectively; control vs 33 TSR T4, 65% vs 66%, respectively; n 5 50 eggs per treatment). Chicks from eggs of control hens began to hatch several hours earlier than those from eggs of 13 TSR T4- and 33 TSR T4-dosed hens. The T4 and T3 contents of embryos (with the thyroid
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TABLE 1 Body Growth, Thyroid Hormones, and Pelvic Cartilage Development in 12-Day Embryos from Eggs of Control and T3-Dosed Japanese Quail Hens Control Egg weight (g) Body weight (mg) Plasma T4 (ng/ml) Plasma T3 (ng/ml) Pelvic cartilage wet wt. (mg) Pelvic cartilage wet wt. as % of embryo body wt. Pelvic cartilage ALP activity (units/µg cartilage)
8.0 6 0.17 (16) 3.1 6 0.07 (16) 4.1 6 0.70 (8) 0.65 6 0.08 (9)
T3 Dose 7.9 6 0.17 (20) 3.3 6 0.07 (20) 4.1 6 0.35 (10) 1.7 6 0.65 (12)
P value 0.06 0.06 0.97 0.19
80.7 6 2.4 (16)
88.3 6 2.6 (20)
0.05*
2.61% 6 0.07 (16)
2.69% 6 0.08 (20)
0.46
181.9 6 16.5 (16)
243.4 6 17.3 (20)
0.02*
Note. Values are the mean 6 SE (n). Asterisks represent significant differences (P , 0.05) within rows. A unit of alkaline phosphatase activity is defined as that amount of alkaline phosphatase which will produce 1 µmol of p-nitrophenol/min at 25°.
glands removed) from control and high T4 eggs were very low, but detectable, on Day 7 of incubation and were not significantly different in control and high T4 eggs. By Day 15, the embryonic T4 and T3 contents had increased significantly from Day 7 (T4, <60-fold; T3 < 25-fold), but did not differ significantly in embryos from control and high T4 eggs (Fig. 4b). The concentration of T4 and T3 in the plasma of Day 9 embryos was below detection (,1.25 and 0.125 ng/ml, respectively) in control and high T4 eggs. Both thyroid hormones were detectable in the plasma of Day 14 embryos, but the plasma hormone concentrations of embryos from control and high T4 eggs did not differ significantly for either T4 or T3 (Fig. 5).
FIG. 5. Embryonic plasma thyroid hormone concentrations on Day 14 of incubation in Japanese quail. Top panel, T4; bottom panel, T3. C, embryos from control hens dosed twice daily with saline; 13 T4, embryos from hens dosed twice daily with 13 the daily thyroid secretion rate of T4; 33 T4, embryos from hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 4–6). Bars with the same lowercase letters do not differ significantly.
Embryonic Pelvic Cartilage Development In embryos of eggs from hens dosed with 33 TSR T4, embryonic pelvic cartilage wet weights (relative to body weight; Fig. 6) and dry weights (data not shown) were significantly greater than those of controls (ANOVA, P 5 0.04 and 0.03, for wet and dry weights, respectively; P 5 0.01 and 0.002 for weight ratios, respectively). There were no significant differences in these variables in embryos from eggs of the control hens vs those on the 13 TSR T4 dose despite signifi-
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FIG. 6. Pelvic cartilage wet weights as a percentage of embryonic body weights in Japanese quail embryos. Light bars, embryos from eggs of control hens dosed twice daily with saline; dark bars, embryos from hens dosed twice daily with 33 the daily thyroid secretion rate of T4. Values are means 6 SE (n 5 4–6). ANOVA showed pelvic cartilage wet weights in embryos from 33 TSR T4-dosed hens were significantly greater than those in controls (P 5 0.013).
Thyroid Hormones in Egg Yolk
cantly higher yolk T4 content in the eggs from 13 TSR T4-dosed hens. The ALP activity in the pelvic cartilages of 6- to 12-day embryos from eggs of 33 TSR T4-dosed hens was significantly higher than that in controls (ANOVA, P 5 0.005; Fig. 7); in pelvic cartilage of embryos from eggs of 13 TSR T4-dosed hens, ALP did not differ from controls. In the single T3 experiment, the wet weights of pelvic cartilages were significantly greater (P 5 0.05) in 12-day embryos from eggs of hens dosed with T3 than those for controls; however, the weights of these pelvic cartilages as a percentage of embryonic body weight did not differ from those of controls (Table 1). In this experiment, ALP activity was significantly higher in pelvic cartilages from Day-12 embryos from eggs of the dosed hens than from controls (P 5 0.02; Table 1).
DISCUSSION Our study of Japanese quail eggs supports and extends previous studies of chicken eggs that have
FIG. 7. Alkaline phosphatase activity in pelvic cartilages of Day 12 Japanese quail embryos. Units are the amount of enzyme which will produce 1 µmol of p-nitrophenol/min at 25° (Sigma Diagnostics, 1990). Values are means 6 SE (n 5 6). Circles, embryos from eggs of control hens dosed twice daily with saline; triangles, embryos from eggs of hens dosed twice daily with 33 TSR T4. ANOVA showed that pelvic cartilage alkaline phosphatase activity was significantly greater in embryos from 33 TSR T4-dosed hens than that in control embryos (P 5 0.005).
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suggested that thyroid hormones of maternal origin are transferred into the egg prior to laying [Hilfer and Searls (1980), Sechman and Bobeck (1988), and Prati et al. (1992)]. The T4 and T3 concentrations we found in the yolk of Japanese quail eggs were similar to those reported by Prati et al. (1992; T4, 3.8 6 0.3 ng/g; T3, 1.5 6 0.2 ng/g) and Sechman and Bobeck (1988; T4, 6.0–10.0 ng/g; T3, 1.5–2.3 ng/g) for chicken eggs. Our study and the latter two studies listed above all chemically extracted thyroid hormones from yolk. In contrast, Hilfer and Searls (1980), who obtained very different results (T4, 15.0–20.0 ng/g), used yolk samples directly in the RIAs. Thus, the high yolk hormone concentrations they reported may have been inflated as a result of lipid interference with primary antibody binding in the RIA. To our knowledge, our study is the first that addresses the relationship between the hen’s thyroid status and the deposition of thyroid hormones in her eggs. Overall, the data show that hyperthyroid hens, with high concentrations of T4 in their plasma, deposit increased amounts of maternal thyroid hormones in the yolk of their eggs. However, when individual eggs are considered relative to the hens’ plasma thyroid hormones at the time of laying, there is evidence of some regulation of thyroid hormone deposition. Within each treatment, hens regulate yolk T4 concentrations independent of variations in their plasma T4 concentrations. However, more work is needed to further document the capability of yolk hormone regulation and to determine how such regulation of yolk hormones is achieved. Our attempts to study the effects of hen hypothyroidism on yolk T4 content suggest that thyroid hormonedeficient eggs may not be produced. Laying hens were very resistant to the effects of the goitrogen, methimazole, and there was no significant reduction in the thyroid hormone concentrations in either their plasma or the yolk of their eggs during 30 days of treatment at the first dosage used. It is not clear whether these hens continued to synthesize some hormone or whether hormone reserves in the thyroid gland maintained blood hormone concentrations and supplied hormones for deposition in the eggs. When the methimazole dose was doubled (second month), egg laying became sporadic and quickly ceased but plasma thyroid hormones remained in the normal range. When
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the experiment was terminated and hens were sacrificed, thyroid gland hypertrophy was observed, suggesting that the goitrogen had been effective and the pituitary was detecting hormone decreases too subtle to be measured. Overall, these results suggest that hypothyroid hens cease egg laying, so the production of eggs with low thyroid hormone content seems unlikely. Some studies have addressed the potential mechanism(s) for the transfer of thyroid hormones from the hen to the egg yolk. Lipoproteins in the plasma of chicken hens bind small but significant amounts of plasma thyroid hormones and are responsible for the transport of these hormones into the yolk of developing follicles (Mitchell, 1984; Mitchell et al., 1985; Mitchell and Stiles, 1985). This proposed mechanism of transfer is plausible, because low-density lipoproteins are synthesized by the liver and deposited in oocytes during follicle development, (Griffin et al., 1984) and the lipoproteins found in hen plasma and egg yolk are very similar (Griffin et al., 1984). Vitellogenin also binds thyroid hormones in hen plasma and binds to receptors on ovarian membranes in chickens (Barber et al., 1991). Transthyretin (thyroxine-binding prealbumin) found in egg yolk is derived from the hen’s plasma and there is a putative transthyretin receptor on the oocyte membrane (Vieira et al., 1995). Thus, it appears that several plasma proteins and lipoproteins may be involved in the transfer of maternal thyroid hormones into the egg and could be important in regulating the amount of thyroid hormone deposited in the yolk. If yolk thyroid hormones are taken up by embryos prior to hormone release by the thyroid gland, they could be playing essential roles in early tissue development. In a study of chicken eggs, Prati et al. (1992) found detectable amounts of T4 in 4-day embryos (2.48 pg/embryo) when one sample consisting of 30 embryos was extracted and analyzed. They also analyzed three pools of 6-day embryos and found 7.4 pg T4/ embryo (in embryos with the neck, and thus presumably the thyroid gland, removed). Based on the assumption that the embryonic thyroid gland does not secrete hormones until after Day 9, Prati et al. assert that their data demonstrate embryonic uptake of maternal hormones from the yolk. However, Thommes and Hylka (1978) found T4 (0.23 ng/ml) in the blood of 6.5-day
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Wilson and McNabb
chicken embryos and thyrotropin stimulation increased the concentration of T4 by about sevenfold. Likewise, the finding of Thommes and Tonetta (1979) that thiourea administration on Day 5.5 of incubation markedly reduced embryonic plasma T4 concentrations on Day 7.5 argues for much of the plasma T4 coming from the embryonic thyroid gland. These studies provide evidence that the thyroid is producing and releasing hormones at this time. However, no one has yet devised a way of determining the proportions of embryonic hormones contributed by the embryonic thyroid gland vs the maternal hormone stores of the yolk. We attempted to determine if yolk hormones are taken up early in development, by examining our data from Day 7 quail embryos (16.5-day incubation). At this stage the embryonic thyroid gland contains very small amounts of thyroid hormones (Day 8 of the 16.5-day incubation: 1.7 ng T4/gland pair and 1.5 ng T3/gland pair; McNichols and McNabb, 1988) and is relatively inactive (based on radioiodine uptake studies; McNabb et al., 1981). Although a very large amount of T4 was available in the yolk of eggs from 33 TSR T4-dosed hens (<400 ng/yolk) we found no evidence of differences in hormone content in Day 7 embryos from control vs high T4 eggs. This lack of differential thyroid hormone content in embryos from eggs of very different hormone content seems to argue against hormone uptake from the yolk at this stage. What is not clear is whether the embryo is taking up T4 and degrading it (yolk T4 decreases significantly from preincubation values to Day 7 of incubation) or whether T4 is degraded within the yolk itself. Whatever the site of degradation, our data do not support the idea that this disappearance of the extra T4 in high T4 eggs results in T3 production, as neither the yolk nor the embryo has higher T3 in the high T4 eggs than in the controls. One of our experiments did show significantly higher T3 in the yolk of high T4 eggs at Day 7 of incubation, but this result was not confirmed in a repeat study. To our knowledge, the only study in which deiodinase activities in eggs have been investigated is that of Reddy et al. (1992) on tilapia eggs. In an in vitro assay system with unlabeled T4 as substrate and measurement of product (T3) by RIA, these workers were unable to detect any 58-deiodinase (58D) activity in fertilized eggs. However, they found that T4
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Thyroid Hormones in Egg Yolk
decreased rapidly in eggs even before fertilization. If the T4 was 5-deiodinated to rT3, their assay would not have detected this degradation. The lack of T4 in the allantois in our study suggested the T4 was not being excreted from the embryo without degradation. In the latter half of embryonic development, maternal hormones taken up from the yolk could supplement endogenous embryonic thyroid hormones. In high hormone eggs one might expect accelerated development of thyroid hormone-responsive tissues. Pelvic cartilages from chicken embryos have previously been shown to be thyroid hormone-responsive with respect to both their growth and differentiation (Burch and Lebovitz, 1982). We found accelerated growth (significantly increased cartilage wet and dry weights) and evidence of accelerated cartilage differentiation (increased ALP activity) in embryos from eggs of hens on the highest T4 dose. In contrast to these effects on pelvic cartilage, high T4 content in eggs did not affect hatchability, body length, body weight, or the gross morphological appearance of quail chicks at hatch. These results may not be surprising because thyroid hormones appear to be permissive with respect to the stimulation of general body growth, and there is no simple relationship between growth and thyroid hormone concentrations (McNabb and King, 1993). It is generally thought that most thyroid hormone action in birds is triggered by T3, as is the case in mammals (McNabb and King, 1993). Thyroxine in the yolk of high T4 eggs might be deiodinated in embryonic tissues to T3, to inactive reverse-T3, or to one or both of these products, followed by further deiodinations. Thus, if there were appreciable 58-deiodination in embryos, we might expect to see higher embryonic T3 content in the embryos from high T4 eggs than in controls. This was not the case in the present study. This finding is consistent with in vitro studies that show little 58D activity in tissues such as liver until the beginning of the perihatch period (review, McNabb and King, 1993). Thus it appears that high egg T4 does not lead to overall high embryo T3 in quail embryos. The embryos may be deiodinating T4 to r T3 because that enzymatic capability is known to exist in liver during the latter half of embryonic life (chicken embryos, Borges et al., 1981; Galton and Hiebert, 1987; Darras et al., 1992). The deiodination capabilities of
individual hormone-responsive tissues, such as the pelvic cartilage used in this study, have not been investigated. In summary, our study indicates that thyroid hormone deposition in egg yolk varies with the thyroid status of the hen, but that there is some regulation of yolk hormone concentrations. In addition, high concentrations of thyroid hormone in egg yolk may affect the growth and differentiation of some thyroid hormoneresponsive tissues (such as the pelvic cartilage used in this study), but do not influence general body growth. In the high T4 eggs, yolk T4 disappears progressively during embryonic development, but does not lead to increased T4 or T3 in the embryo. These results suggest the need for further investigations to understand hormone deposition in eggs and the effects of maternal hormones on embryonic development: (1) how the mechanisms of transfer of maternal thyroid hormones from the hen to the yolk are influenced by differences in the hen’s thyroid status, (2) the extent to which maternal thyroid hormones in the yolk enter the embryo at different stages of development, and (3) the possibility and/or extent of deiodination of T4 (and the pathways involved) in the yolk and the embryo.
ACKNOWLEDGMENTS The following people volunteered their time to assist with several aspects of the project and deserve special thanks: Allison Wolf, Melissa Goode, Melissa Catena, Heather Solari, Osama Abed, Steve Sklarew, Eric Belling, Megan Harris, Emilio Martinez-Lezama, Cynthia Tate, Matt Stinchcomb, Judy McCord, Amy Wood, David Mullins, Memuna Khan, Kevin Simon, Matt Lovern, and Steve Nunez. Drs. P. B. Siegel, D. M. Denbow, and C. L. Rutherford provided helpful perspective on many parts of the study. This research was supported by funding from Sigma Xi Grants-In-Aid, the Graduate Research Development Project of Virginia Tech., and the Biology Department of Virginia Tech.
REFERENCES Bacon, W. L., and Cherms, F. L. (1968). Ovarian follicular growth and maturation in the domestic turkey. Poultry. Sci. 47, 1303–1314. Bacon, W. L., and Koontz, M. (1971). Ovarian follicular growth and maturation in Coturnix quail. Poultry Sci. 50, 233–236.
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164 Barber, D. L., Sanders, E. J., Aebersold, R., and Schneider, W. J. (1991). The receptor for yolk lipoprotein deposition in the chicken oocyte. J. Biol. Chem. 266, 18761–18770. Borges, M., LaBourene, J., and Ingbar, S. H. (1980). Changes in hepatic iodothyronine metabolism during ontogeny of the chick embryo. Endocrinology 107, 1751–1761. Brown, C. L., Doroshov, S. I., Nunez, J. M., Hadley, C., Vaneenennaam, J., Nishioka, R. S., and Bern, H. A. (1988). Maternal triiodothyronine injections cause increases in swimbladder inflation and survival rates in larval striped bass, Morone saxatilis. J. Exp. Zool. 248, 168–176. Brown, C. L., Sullivan, C. V., Bern, H. A., and Dickhoff, W. W. (1987). Occurrence of thyroid hormones in early developmental stages of teleost fish. Am. Fish. Soc. Symp. 2, 144–150. Burch, W. M., and Lebovitz, H. E. (1982). Triiodothyronine stimulation of in vitro growth and maturation of embryonic chick cartilage. Endocrinology 111, 462–468. Darras, V. M., Visser, T. J., Berghmans, L. R., and Ku¨hn, E. R. (1992). Ontogeny of type I and type II deiodinase activities in embryonic and posthatch chicks: Relationship with changes in plasma triiodothyronine and growth hormone levels. Comp. Biochem. Physiol. 103, 131–136. Denver, R. J. (1993). Acceleration of anuran amphibian metamorphosis by corticotrophin-releasing hormone-like peptides. Gen. Comp. Endocrinol. 91, 38–51. Fisher, D. A., Dussault, J. H., Stack, J., and Chopra, I. J. (1977). Ontogenesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheep, and rat. Rec. Prog. Horm. Res. 33, 59–116. Galton, V. A., and Hiebert, A. (1987). The ontogeny of the enzyme systems for the 58- and 5-deiodination of thyroid hormones in chick embryo liver. Endocrinology 120, 2604–10. Gorbman, A., Dickhoff, W. W., Vigna, S. R., Clark, N. B., and Ralph, C. L. (1983). The thyroid gland. In ‘‘Comparative Endocrinology,’’ pp. 185–276. Wiley, New York. Griffin, H. D., Perry, M. M., and Gilbert, A. B. (1984). ‘‘Physiology and Biochemistry of the Domestic Fowl,’’ Vol. 5, pp. 345–380. Academic Press, New York. Hilfer, S. R., and Searls, R. L. (1980). Differentiation of the thyroid in the hypophysectomized chick embryo. Dev. Biol. 79, 107–118. Kobuke, L., Specker, J. L., and Bern, H. A. (1987). Thyroxine content of eggs and larvae of coho salmon, Oncorhynchus kisutch. J. Exp. Zool. 242, 89–94. McNabb, F. M. A. (1992). ‘‘Thyroid Hormones.’’ Prentice Hall, New Jersey. McNabb, F. M. A., and Hughes, T. E. (1983). The role of serum binding proteins in determining free thyroid hormone concentrations during development in quail. Endocrinology 113, 957–963. McNabb, F. M. A., and King, D. B. (1993). Thyroid hormone effects on growth, development, and metabolism. In ‘‘The Endocrinology of Growth, Development, and Metabolism in Vertebrates,’’ pp. 393–417. Academic Press, New York. McNabb, F. M. A., Weirich, R. T., and McNabb, R. A. (1981). Thyroid function in embryonic and perinatal Japanese quail. Gen. Comp. Endocrinol. 43, 218–226. McNichols, M. J., and McNabb, F. M. A. (1988). Development of thyroid function and its pituitary control in embryonic and
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Wilson and McNabb hatchling precocial Japanese quail and altricial Ring doves. Gen. Comp. Endocrinol. 69, 109–118. Mitchell, M. A. (1984). Binding of tri-iodothyronine and thyroxine to b-lipoprotein in chicken plasma. J. Physiol. 343, 86P. Mitchell, M. A., and Stiles, M. S. (1985). Some new observations on the binding of thyroxine and triiodothyronine to plasma proteins and lipoproteins in the domestic fowl. Gen. Comp. Endocrinol. 57, 309–319. Mitchell, M. A., Raza, A., and Uglow, P. (1985). Transfer of maternal thyroid hormones to the embryo in the domestic fowl. J. Physiol. 360, 74P. Morreale de Escobar, G., Obregon, M. J., Ruiz de Ona, C., and Escobar del Rey, F. (1988). Transfer of thyroxine from the mother to the rat fetus near term: Effects on brain 3,5,38-triiodothyronine deficiency. Endocrinology 122, 1521–1531. Morreale de Escobar, G., Pastor, R., Obregon, M. J., and Escobar del Rey, F. (1985). Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, before and after onset of fetal thyroid function. Endocrinology 117, 1890–1900. Niinuma, K., Tagawa, M., Hirano, T., and Kikuyama, S. (1991). Changes in tissue concentrations of thyroid hormones in metamorphosing toad larvae. Zool. Sci. 8, 345–350. Prati, M., Calvo, R., and Morreale de Escobar, G. (1992). L-Thyroxine and 3,5,38-triiodothyronine concentrations in the chicken egg and in the embryo before and after the onset of thyroid function. Endocrinology 130, 2651–2659. Reddy, P. K., Brown, C. L., Leatherland, J. F., and Lam, T. J. (1992). Role of thyroid hormones in tilapia larvae (Oreochromis mossambicus): II. Changes in the hormones and 58-monodeiodinase activity during development. Fish Physiol. Biochem. 9, 487–496. Romanoff, A. L. (1967). ‘‘Biochemistry of the Avian Embryo—A Quantitative Analysis of Prenatal Development.’’ Wiley, New York. Schwartz, H. L. (1983). Effects of thyroid hormone on growth and development. In ‘‘Molecular Basis of Thyroid Hormone Action’’ (J. H. Oppenheimer and H. H. Samuels, Eds.), pp. 413–444. Academic Press, New York. Sechman, A., and Bobek, S. (1988). Presence of iodothyronines in the yolk of the hen’s egg. Gen. Comp. Endocrinol. 69, 99–105. Sigma Diagnostics. (1990). Alkaline phosphatase (procedure No. 245). Sigma Chemical Co., St. Louis. Singh, A., Reineke, E. P., and Ringer, R. K. (1967). Thyroxine and triiodothyronine turnover in the chicken and the bobwhite and Japanese quail. Gen. Comp. Endocrinol. 9, 353–361. Suvarna, S., McNabb, F. M. A., Dunnington, E. A., and Siegel, P. B. (1993). Intestinal 58deiodinase activity of developing and adult chickens selected for high and low body weight. Gen. Comp. Endocrinol. 91, 2590–266. Tagawa, M., and Hirano, T. (1987). Presence of thyroxine in eggs and changes in its content during early development of Chum salmon, Onorhychus keta. Gen. Comp. Endocrinol. 68, 129–135. Tagawa, M., and Hirano, T. (1990). Changes in tissue and blood concentrations of thyroid hormones in developing chum salmon. Gen. Comp. Endocrinol. 76, 437–443. Thommes, R. C. (1987). Ontogenesis of thyroid function and regulation in the developing chick embryo. J. Exp. Zool. Suppl. 1, 273–79.
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Thommes, R. C., and Hylka, V. W. (1978). Hypothalamo-adenohypophyseal-thyroid interrelationship in the chick embryo. I. TRH and TSH sensitivity. Gen. Comp. Endocrinol. 34, 193–200. Thommes, R. C., and Tonetta, S. A. (1979). Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. II. Effects of thiourea treatment on plasma total thyroxine levels and thyroidal 125I uptake. Gen. Comp. Endocrinol. 37, 167–176.
165 Thommes, R. C., Vieth, R. L., and Levasseur, S. (1977). The effects of hypophysectomy by means of surgical decapitation on thyroid function in the developing chick embryo. Gen. Comp. Endocrinol. 31, 29–36. Vieira, A. V., Sanders, E. J., and Schneider, W. J. (1995). Transport of serum transthyretin into chicken oocytes. J. Biol. Chem. 270, 2952–2956.
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Maternal Thyroid Hormones in Japanese Quail Eggs and Their Influence on Embryonic Development C. Morgan Wilson and F. M. Anne McNabb1 Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406 Accepted February 26, 1997
We addressed the relationship between the thyroid status of hens and the thyroid hormone content of their eggs, as well as the influences of egg hormones on embryonic development. Methods for measuring thyroid hormones in egg yolk were verified by demonstrating consistency in the recovery of yolk thyroid hormones following a methanol/chloroform extraction and in the measurement of thyroid hormones by RIA for a range of hormone concentrations in yolk extracts. Untreated hens produced eggs with yolk thyroxine (T4) concentrations that were low relative to plasma T4, but yolk triiodothyronine (T3) concentrations comparable to those of plasma. Hens dosed twice daily with T4 (1 or 33 the daily thyroid secretion rate, TSR, of T4 per dose) had significantly higher plasma and egg yolk T4 concentrations than did control hens dosed with saline. In general, the T4 concentration of egg yolk varied with the thyroid status of the hen. When the relationship between each hen’s plasma T4 and the yolk T4 concentration of her eggs was examined, hens appeared to regulate T4 deposition into yolk at ‘‘levels’’ characteristic of the ‘‘levels’’ of thyroid status produced by the different doses of T4. Embryonic pelvic cartilage, a thyroid hormone-responsive tissue, showed enhanced growth and differentiation in embryos from eggs of hens given the highest dose of T4. Specifically, alkaline phosphatase activity (a marker of differentiation) and pelvic cartilage wet and dry weights were significantly greater in embryos from high T4 eggs (hens on the 33 TSR dose) than those in controls. However, 1
To whom correspondence should be addressed. Fax: (540) 2319307. 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
embryos from high T4 eggs did not differ in general body growth (body weight, length, and general morphology) or hatchability compared to controls. In a single T3 experiment, hens were dosed twice daily with 1 mg T3. The embryos from eggs of these hens had accelerated differentiation/maturation of pelvic cartilages (sampled at Day 12) compared to those from control eggs; body growth did not differ from that of controls. r 1997 Academic Press
Thyroid hormones are essential for the control of development in all vertebrate classes (Gorbman et al., 1983; McNabb, 1992; McNabb and King, 1993). However, some early mammalian studies suggested that maternal thyroid hormones did not cross the placenta, and therefore they were presumed not to play a role in embryonic development (Fisher et al., 1977; Schwartz, 1983). It is now known that maternal thyroid hormones are available to embryos of many vertebrates. Maternal thyroid hormones are transferred into fish eggs (Brown et al., 1987; Kobuke et al., 1987; Tagawa and Hirano, 1987) and cross the placenta in rats (Morreale de Escobar et al., 1985). In striped bass, treatment of prespawning females with T3 is associated with accelerated larval development, swimbladder inflation, and enhanced larval survival (Brown et al., 1988). Maternal thyroid hormones influence fetal development in rats prior to the time of appreciable fetal thyroid gland function (Morreale de Escobar et al., 1985, 1988). Although thyroid hormones are known to affect the
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development of avian embryos and chicks (review, McNabb and King, 1993), few studies have investigated maternal thyroid hormones in avian eggs. The first reports of thyroid hormones in avian eggs (chickens; Hilfer and Searls, 1980; Sechman and Bobeck, 1987) are difficult to evaluate because they provided no validation showing that the methods used effectively extracted or accurately measured yolk thyroid hormones. However, the data from these studies, as well as those from a more recent study of chicken eggs using validated techniques (Prati et al., 1992), indicated that maternal thyroid hormones are present in avian egg yolk. If utilized by the embryo, maternal thyroid hormones in egg yolk could influence development, especially before appreciable synthesis and release of hormones by the thyroid gland. During the first one-third to one-half of incubation, the thyroid gland appears to possess a very limited capability to synthesize hormones (chickens: Thommes, 1987; Japanese quail: McNichols and McNabb, 1988). Thommes and Hylka (1978) found detectable T4 in embryonic chicken plasma by Day 6.5 of the 21-day incubation period, and Prati et al. (1992) found extremely small amounts of T4 and T3 in pooled chicken embryos on Days 4 and 6. It is not clear whether these small amounts of thyroid hormones in early embryos are of maternal or embryonic origin or both. It is clear, however, that after linking of the hypothalamic–pituitary–thyroid (HPT) axis (Days 10.5–11.5 in chicken embryos) thyroid hormones from the embryonic thyroid gland predominate (Thommes et al., 1977). The objectives of this study were: (1) to validate methods for the extraction of thyroid hormones from avian egg yolk, (2) to determine the relationship between the thyroid status of the hen and thyroid hormones in the yolk of her eggs, and (3) to determine if thyroid hormones in the egg yolk affect thyroid hormone-responsive development of embryos.
MATERIALS AND METHODS Animals Adult Japanese quail (Coturnix japonica), ages 8 to 12 months, from a random-bred colony, were housed in
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Wilson and McNabb
pairs and maintained under a 14L:10D photoperiod. A commercial game bird ration (18 to 24% protein; Big Spring Mills, Shawsville, VA) and water were available ad libitum. Eggs were incubated at 39 6 1° and .90% relative humidity in a forced-air incubator (Humidaire Hatchette Incubator; New Madison, OH). Hens were dosed with hormone solutions using a tuberculin syringe fitted with a 2-in. piece of tygon tubing to deliver the dose into the hen’s crop.
Experimental Design Maternal thyroid hormone deposition in yolk. To determine the number of days thyroid hormones could be deposited in the yolk of an individual egg, adult Japanese quail hens (n 5 8) were dosed orally at 0800 hr for 10 days, alternating two lipid soluble dyes (technique modified from Bacon and Cherms, 1968). Either Sudan black B or Sudan IV red was administered (200 µl of 0.01 g dye/ml of vegetable oil; dyes from Sigma Chemical Co., St. Louis, MO). Eggs were collected daily and boiled for 20 min. Boiled eggs were sliced longitudinally to count the colored rings of yolk. Manipulation of hens’ thyroid status and egg hormone content. To determine if hens deposit hormones in eggs in proportion to their own thyroid status, laying hens (n 5 12 per treatment) were made hyperthyroid by oral dosing with L-T4 (Sigma Chemical Co.) in 200 µl of saline (0.9% NaCl) at 0800 and 1800 hr. Dosages were a multiple (either 1 or 33) of the normal daily thyroid secretion rate of T4 (TSR T4) for adult Japanese quail (2.78 µg/100 g body weight/day; Singh et al., 1967). Control hens were dosed with 200 µl of saline at the same times. Twice-daily dosing was chosen based on a preliminary experiment in which blood from hens was collected before dosing and then every 3 hr for 12 hr after the 0800-hr dose of T4. This experiment indicated that T4 needed to be administered twice daily to maintain elevated plasma T4 concentrations. Blood was collected weekly from control (saline)-, 13 TSR T4-, and 33 TSR T4-dosed hens (3 to 4 hr after the morning dose). For yolk hormone analysis, eggs were collected (labeled by hen and date) from salineand T4-dosed hens on the day blood was sampled. One set of eggs collected from saline- and T4-dosed hens was incubated until hatch. Hatchlings were inspected for gross morphology, weighed, and measured
Thyroid Hormones in Egg Yolk
(length in mm, crown to rump), and the approximate time of hatch was recorded. Another set of eggs from the hens on each treatment was incubated and sampled for hormone measurements on Days 7 and 15 for embryos, on Days 0, 7 and 15 for yolk, on Days 0 and 7 for albumen, on Day 7 for allantois, and on Days 9 and 14 for embryonic blood. Two sets of eggs (from hens receiving each treatment) were incubated for the studies of pelvic cartilage. For wet- and dry-weight measurements, pelvic cartilages were sampled from Day-7 to -13 embryos (Day 7 was the first day when the fragile cartilages could be effectively trimmed for accurate weight determinations). For alkaline phosphatase activity, sampling started on Day 6 but extended only to Day 12 (the last day when it was practical to homogenize the cartilages with a glass tissue grinder). In a single experiment using T3, laying hens were dosed at 0800 and 1800 hr with 1 µg T3 in 200 µl 0.9% NaCl. Blood was collected prior to dosing and then at 1200 and 1800 hr on the first day of dosing. Eggs were collected from these hens prior to dosing and on Days 5 and 6 of the dosing; only Day 12 embryos were sampled. To determine if hypothyroid hens produce thyroid hormone-deficient eggs, and to attempt to obtain eggs with low thyroid hormone content, adult laying hens (n 5 8) were given an oral dose of 4 mg of methimazole (a goitrogen that inhibits thyroid hormone synthesis; Sigma Chemical Co.) in 200 µl saline at 0800 hr daily for 30 days. Because this treatment did not lower plasma thyroid hormone concentrations, the dose was increased to 8 mg/day for an additional 40 days. Soon after this increase in methimazole dosage, the hens ceased laying. At this point the experiment was ended, and the hens were sacrificed and weighed and their thyroid glands removed and weighed.
Sampling and Analyses Blood was collected from the brachial vein of laying hens and from the chorioallantoic artery of embryos. Samples were collected in heparinized capillary tubes and centrifuged, and the plasma was stored at 220°. For the studies of embryonic pelvic cartilage, the cartilage was dissected out of embryos, connective tissue and muscle were removed, and the cartilage was blotted and weighed. Some pelvic cartilages were dried to constant weight at 100° for 18–24 hr. For
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alkaline phosphatase analyses, other pelvic cartilages were homogenized in 0.9% NaCl (73 v/w) using a 0.5-ml glass tissue grinder, and the homogenate centrifuged (12,500g, 4°, 2 min). The postmitochondrial supernatant fraction (PMF) was removed and stored at 220° until analysis for alkaline phosphatase activity. Yolk thyroid hormone extraction. Thyroid hormones were extracted from egg yolk using a modification of a methanol/chloroform extraction procedure previously used for amphibian larvae by Denver (1993). Egg yolk was weighed, diced with scissors, and homogenized using a 50-cc glass syringe with an 18-gauge needle; each yolk was drawn into and expelled from the syringe three times. A 0.5-g sample of homogenized yolk was transferred to a 13-ml (17 3 100 mm) test tube (all tubes used were polypropylene), and 2 ml of methanol containing 1 mM propylthiouracil (PTU; an inhibitor of deiodination; Sigma Chemical Co.) was added. Labeled thyroid hormone (,2000 cpm of either 125I-T4 or 125I-T3; high specific activity: T4, 1250 µCi/µg; T3, 1200 µCi/µg; New England Nuclear; Boston, MA) was added, and the sample was vortexed and counted for 10 min. After a 10-min extraction on a multitube shaker (150 oscillations/min), the sample was centrifuged (1700g, 4°, 10 min) and the supernatant was decanted into a 50-ml graduated conical polypropylene tube. The precipitate was resuspended in 1 ml of methanol (with 1 mM PTU), shaken for 10 min, and centrifuged, and the supernatant decanted into a second 50-ml tube. The two separate supernatants each received 5.0 ml of chloroform (CHCl3) and 0.5 ml ammonium hydroxide (2 N NH4OH) and were shaken and centrifuged as above; the upper phase (methanol/aqueous phase) from both tubes was removed and combined in a 13-ml tube. The CHCl3 phase remaining in each tube was extracted again with NH4OH as above and the upper phase from each was added to the methanol/aqueous pool for each sample and then dried under a filtered air stream (for 6–7 hr) in a fume hood. When dry, the sample was resuspended in 1 ml 2 N NH4OH, briefly vortexed, shaken, and centrifuged (as above), and the supernatant decanted into a 13-ml tube. The general extraction procedure was then repeated with 1 ml CHCl3, and the upper phase was removed, dried, resuspended in 150 µl 75% EtOH, and counted (10 min) to determine extraction efficiency (recovery of the labeled thyroid
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hormone added to the original homogenate). The extraction efficiency was consistent; 63 6 1% (mean 6 SE) for 125I-T4 and 61 6 3% for 125I-T3 (n 5 6 for each hormone). These recoveries are comparable to others from similar extraction techniques for yolk or tissue thyroid hormones [45 to 70% recovery: Tagawa and Hirano (1990), fish; Niinuma et al. (1991) and Denver (1993), amphibians]. Individual recoveries were determined for all samples and used to calculate thyroid hormone concentrations. The reconstituted extracts were stored at 220° until analysis. To measure embryonic hormones, embryos were decapitated to remove the area containing the thyroid gland, and the carcass was homogenized in a volume of methanol (with 1 mM PTU) twice the embryo weight using a Brinkmann tissue homogenizer (Model PT 10/35 with PTA 10 generator; Brinkmann Instruments, Westbury, NY). After homogenization, the blades were rinsed with an equivalent volume of methanol (with 1 mM PTU) which was added to the homogenate. Thyroid hormones in the embryo, albumen, and allantois homogenates were extracted and assayed as described above for yolk. Thyroid hormone radioimmunoassays (RIAs). Plasma T4 and T3 concentrations were measured with a double antibody RIA by the method of McNabb and Hughes (1983) using hormone standards prepared in hormone-free chicken plasma or in 75% ethanol for
Wilson and McNabb
plasma and extract samples, respectively. Primary antibodies were purchased from Endocrine Sciences (Calabasas, CA), 125I-T4 and 125I-T3 were from New England Nuclear (Boston, MA; high specific activity: T4, 1250 µCi/µg; T3, 1200 µCi/µg), and carrier immunoglobulin was from Antibodies Incorporated (Davis, CA). Secondary antibody was kindly provided by Dr. John McMurtry (USDA; Beltsville, MD). Assay volumes were 12.5 µl for T4 and 25 µl for T3. The plasma assay was validated previously (McNabb and Hughes, 1983). To validate the RIA for use on yolk extract, a pooled extract sample was diluted with 75% ethanol or spiked with hormone standards prepared in 75% ethanol. Precision tests performed on yolk extracts show 6 2 SE was 13.7% of the mean for T4 (n 5 10) and 14.8% of the mean for T3 (n 5 8). A Thiel–Sen test showed no significant difference between the slopes of the measured and predicted (slope of 1.0) lines for both RIAs (T4: k 5 3, P 5 0.431, Fig. 1a; T3: k 5 9.0, P 5 0.376, Fig. 1b), indicating consistent measurements over a range of hormone concentrations. Alkaline phosphatase (ALP) analysis. An ALP assay, adapted from the method of Suvarna et al.(1993), was validated by demonstrating linearity of enzyme activity with time and proportionality between enzymatic activity and PMF concentration for pelvic cartilages from 6-, 9-, and 12-day embryos. The validation studies indicated that pelvic cartilage PMF required
FIG. 1. Validation of radioimmunoassays for measuring thyroid hormones in extracts of egg yolk. (a) T4; (b) T3. Circles, measured values of yolk extract spiked with known amounts of hormone; squares, measured values of dilutions of yolk extract. The dashed lines are Theil–Sen regression lines of the measured points; the solid (predicted) line has a slope of 1.0. The measured and predicted lines do not differ significantly for either hormone.
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Thyroid Hormones in Egg Yolk
dilution (with 0.9% NaCl) by 1:1 for 6- and 7-day embryos and by 1:19 for Days 8 to 12. For the assay, a 20-µl volume of diluted PMF was added to 300 µl of ALP reagent (Sigma Diagnostic Alkaline Phosphatase Components Kit; Sigma Chemical Co.) in a narrow light path cuvette, the solution was mixed by rapid shaking, and absorbance readings were taken at 0 and 30 min. The hydrolysis of phosphate by ALP converts the substrate p-nitrophenyl phosphate into p-nitrophenol and inorganic phosphate, resulting in a color change which was measured at l 5 405 nm (25°) with a Beckman DU-640 spectrophotometer. The precision of this technique on 10 aliquots of a pooled sample of pelvic cartilage PMF showed that 62 SE was 8.3% of the mean. A unit of alkaline phosphatase activity is defined as ‘‘that amount of alkaline phosphatase which will produce one µmol of p-nitrophenol per min at 25°9 (Sigma Diagnostics, 1990).
Statistical Analyses
Thyroid Status of Hens and Their Egg Thyroid Hormone Content The administration of T4 to hens produced a hyperthyroid condition (increased plasma T4) and elevated the yolk T4 content in their eggs. Hens given a single dose of 13 TSR T4 or 33 TSR T4 had plasma T4 concentrations 8- to 15-fold those of controls by 3 hr after dosing, but by 9–12 hr plasma T4 had decreased 2to 3-fold. Thus, twice-daily dosing was needed to sustain high plasma T4 over a 24-hr period, and all subsequent experiments used twice-daily dosing. The twice-daily 13 TSR T4 dose significantly increased T4 but not T3 in the hens’ plasma compared to controls; the 33 TSR T4 dose resulted in significant increases in both hormones in the hens’ plasma. Yolk from eggs of hens on each of the T4 doses had significantly higher concentrations of both T4 and T3 than yolk from eggs of control hens (Fig. 2). However, examination of the relationship between yolk T4 in individual eggs and the hen’s plasma T4 at the time of laying each egg indicated that yolk T4 concentrations
Student’s t tests were used to evaluate the effects of treatment of hens on egg yolk and plasma thyroid hormones. A Theil–Sen simple linear regression procedure was used to compare the slope of regression lines to a slope of 1 (the Theil–Sen test statistic is reported as k). Pelvic cartilage wet and dry weights expressed as a percentage of individual embryonic body weight were normally distributed (Shapiro–Wilks test for normality); the effects of treatments on embryonic pelvic cartilages were compared by ANOVA (general linear models procedure). A value of P # 0.05 was considered indicative of statistically significant differences.
RESULTS Time Course of Egg Yolk Deposition Eggs from hens given alternating doses of two lipid-soluble dyes showed alternating rings of dye in egg yolk. For hens that laid one egg each day, five to six alternating, colored rings were present in each egg yolk after 5 to 10 days of dosing with the dyes. This number of days of yolk deposition is consistent with that previously reported by Bacon and Koontz (1971).
FIG. 2. The relationship between thyroid hormone concentrations in hen plasma (a) and egg yolk (b). Top panels, T4; bottom panels, T3. C, controls dosed with saline; 13 T4, hens dosed twice daily with 13 the daily thyroid secretion rate (TSR) of T4; 33 T4, hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 38–68 for each mean); different lowercase letters above the bars indicate significant differences between means.
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did not increase in simple proportion to increases in hen plasma T4 [i.e., Theil–Sen regression line slopes were significantly ,1.0 for each treatment group (P 5 0.00001); Figs. 3b–3d]. Instead, yolk T4 concentrations showed a stepwise pattern of increase with increasing T4 treatment of the hens (Fig. 3a). Variation between yolk T4 concentrations in eggs of hens was similar in the three treatment groups when standard deviations were expressed as a percentage of the mean (controls, 24%; 13 TSR, 19%; 33 TSR, 30%; based on 5–6 hens per group). Some individual hens in each treatment group had much higher variation among their eggs than did others. When standard deviations were expressed as a percentage of the mean, values ranged from 8 to 43% for different individual hens (based on analysis of 17 hens; four eggs per hen). Hens dosed with 1 µg T3 showed small, but significant, increases (1.7-fold) in plasma T3 concentrations
Wilson and McNabb
by 4 hr after dosing; plasma T4 concentrations were significantly less (3.3-fold) than that of controls by 4 hr after dosing. Plasma hormone concentrations decreased in the period from 4 to 8 hr after dosing, so this experiment utilized twice-daily dosing with 1 µg T3/dose. The T3 and T4 content of the yolk of eggs from the T3 treated hens did not differ significantly from that of controls. The administration of methimazole at 4 mg/day for 1 month and 8 mg/day for a second month did not significantly decrease hen plasma thyroid hormone concentrations compared to controls. However, the thyroid glands of the methimazole-treated hens were significantly heavier (,9-fold) than those from control hens. Yolk of eggs laid by hens receiving either 4 or 8 mg/day methimazole did not differ significantly in either T4 or T3 from the yolk of eggs laid by control hens. This experiment was stopped soon after the start of the 8-mg dose because the hens stopped laying.
FIG. 3. The relationship between T4 concentrations in individual eggs and plasma T4 concentration of the Japanese quail hen that laid each egg. (a) All data; open circles, control hens dosed twice daily with saline; closed squares, hens dosed twice daily with 13 the daily thyroid secretion rate of T4; closed circles, hens dosed twice daily with 33 TSRT4. For the bottom panels the lines are Theil–Sen regression lines. (b) Controls (slope 5 0.08); (c) 13 TSR T4 dose (slope 5 0.05); (d) 33 TSR T4 dose (slope 5 0.25).
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Thyroid Hormones in Egg Yolk
Egg Compartments during Development The total weight of the embryo, albumen, and allantois on Days 0, 7, and 15 did not differ in control and high T4 eggs from hens which received either T4 dose. The weight of the embryo and egg compartments changed in the directions expected during development: embryonic body weight increased 9-fold from Days 7 to 15, albumen weight decreased 2.8-fold from Days 7 to 15, and yolk weight increased 1.4-fold from Days 0 to 7 and then decreased 3-fold by Day 15. This pattern of yolk weight increase followed by a decrease occurs as a result of water influx into the yolk from the albumen, followed by the uptake of yolk by the embryo during the latter part of embryonic development (Romanoff, 1967). Allantoic weight did not differ in control and high T4 eggs on Day 7; the allantois could not be dissected out for measurement at later stages of development. Yolk T4 content of high T4 eggs decreased dramatically during incubation. Before incubation the T4 content in eggs from the 13 TSR T4-dosed hens was 7.5-fold, and that of the 33 TSR T4-dosed hens was 26.5-fold greater than that in controls. By Day 15 of incubation, the yolk T4 content in eggs from 13 TSR T4-treated hens did not differ from that in controls. Yolk T4 content in eggs from 33 TSR T4-dosed hens, although markedly decreased, remained significantly higher (P 5 0.002; Fig. 4a) than that in control eggs. Yolk T3 content did not change between Days 0, 7, and 15 in control embryos or in embryos from eggs of 13 TSR T4-dosed hens. However, in the experiment shown in Fig. 4a, the T3 content of the yolk on Day 7 in the eggs of hens on the 33 TSR dose was significantly greater than that of controls. We repeated this experiment and found that the yolk content of T3 at Day 7 did not differ between groups in this second experiment. Before incubation, the albumen T4 content was 4.2 ng per egg for controls, 5.9 ng for 13 TSR T4, and 18.3 ng for 33 TSR T4 eggs; albumen T3 content was 1.1–1.2 ng per egg for all treatment groups. Thus the albumen in eggs from the highest T4 dosage group contained about 4-fold more T4 than that in control eggs. However, this is a small increase compared to that of yolk T4. In yolk, the T4 content of eggs from the highest dosage group was about 26-fold that in control eggs. Allantoic content of T4 was 0.56 6 0.07 ng/egg in
FIG. 4. Thyroid hormone content of egg yolk and embryos (carcass without thyroid gland) of Japanese quail. (a) Thyroid hormone content of egg yolk. (b) Thyroid hormone content of embryos (without thyroid gland). Top panels, T4; bottom panels, T3. Open circles, control hens dosed twice daily with saline; closed squares, hens dosed twice daily with 13 the daily thyroid secretion rate of T4; open triangles, hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 4–7); asterisks indicate a significant difference from control values on the same day of incubation.
controls, 1.12 6 0.44 ng/egg in 13 TSR, and 0.84 6 0.17 ng/egg in 33 TSR on Day 7 of incubation. In the experiment where T3 was administered to hens, there were no significant differences in the embryo weight or in the plasma concentrations of T4 and T3 in Day-12 embryonic plasma from their eggs compared to controls (Table 1).
Embryonic Growth and Thyroid Development Chicks hatched from control and high T4 eggs (from hens on either T4 dosage) showed no significant differences in gross morphology, crown to rump length, or body weight. Hatchability of chicks from control and high T4 eggs was similar (control vs 13 TSR T4, 75% vs 68%, respectively; control vs 33 TSR T4, 65% vs 66%, respectively; n 5 50 eggs per treatment). Chicks from eggs of control hens began to hatch several hours earlier than those from eggs of 13 TSR T4- and 33 TSR T4-dosed hens. The T4 and T3 contents of embryos (with the thyroid
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TABLE 1 Body Growth, Thyroid Hormones, and Pelvic Cartilage Development in 12-Day Embryos from Eggs of Control and T3-Dosed Japanese Quail Hens Control Egg weight (g) Body weight (mg) Plasma T4 (ng/ml) Plasma T3 (ng/ml) Pelvic cartilage wet wt. (mg) Pelvic cartilage wet wt. as % of embryo body wt. Pelvic cartilage ALP activity (units/µg cartilage)
8.0 6 0.17 (16) 3.1 6 0.07 (16) 4.1 6 0.70 (8) 0.65 6 0.08 (9)
T3 Dose 7.9 6 0.17 (20) 3.3 6 0.07 (20) 4.1 6 0.35 (10) 1.7 6 0.65 (12)
P value 0.06 0.06 0.97 0.19
80.7 6 2.4 (16)
88.3 6 2.6 (20)
0.05*
2.61% 6 0.07 (16)
2.69% 6 0.08 (20)
0.46
181.9 6 16.5 (16)
243.4 6 17.3 (20)
0.02*
Note. Values are the mean 6 SE (n). Asterisks represent significant differences (P , 0.05) within rows. A unit of alkaline phosphatase activity is defined as that amount of alkaline phosphatase which will produce 1 µmol of p-nitrophenol/min at 25°.
glands removed) from control and high T4 eggs were very low, but detectable, on Day 7 of incubation and were not significantly different in control and high T4 eggs. By Day 15, the embryonic T4 and T3 contents had increased significantly from Day 7 (T4, <60-fold; T3 < 25-fold), but did not differ significantly in embryos from control and high T4 eggs (Fig. 4b). The concentration of T4 and T3 in the plasma of Day 9 embryos was below detection (,1.25 and 0.125 ng/ml, respectively) in control and high T4 eggs. Both thyroid hormones were detectable in the plasma of Day 14 embryos, but the plasma hormone concentrations of embryos from control and high T4 eggs did not differ significantly for either T4 or T3 (Fig. 5).
FIG. 5. Embryonic plasma thyroid hormone concentrations on Day 14 of incubation in Japanese quail. Top panel, T4; bottom panel, T3. C, embryos from control hens dosed twice daily with saline; 13 T4, embryos from hens dosed twice daily with 13 the daily thyroid secretion rate of T4; 33 T4, embryos from hens dosed twice daily with 33 TSR T4. Values are means 6 SE (n 5 4–6). Bars with the same lowercase letters do not differ significantly.
Embryonic Pelvic Cartilage Development In embryos of eggs from hens dosed with 33 TSR T4, embryonic pelvic cartilage wet weights (relative to body weight; Fig. 6) and dry weights (data not shown) were significantly greater than those of controls (ANOVA, P 5 0.04 and 0.03, for wet and dry weights, respectively; P 5 0.01 and 0.002 for weight ratios, respectively). There were no significant differences in these variables in embryos from eggs of the control hens vs those on the 13 TSR T4 dose despite signifi-
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FIG. 6. Pelvic cartilage wet weights as a percentage of embryonic body weights in Japanese quail embryos. Light bars, embryos from eggs of control hens dosed twice daily with saline; dark bars, embryos from hens dosed twice daily with 33 the daily thyroid secretion rate of T4. Values are means 6 SE (n 5 4–6). ANOVA showed pelvic cartilage wet weights in embryos from 33 TSR T4-dosed hens were significantly greater than those in controls (P 5 0.013).
Thyroid Hormones in Egg Yolk
cantly higher yolk T4 content in the eggs from 13 TSR T4-dosed hens. The ALP activity in the pelvic cartilages of 6- to 12-day embryos from eggs of 33 TSR T4-dosed hens was significantly higher than that in controls (ANOVA, P 5 0.005; Fig. 7); in pelvic cartilage of embryos from eggs of 13 TSR T4-dosed hens, ALP did not differ from controls. In the single T3 experiment, the wet weights of pelvic cartilages were significantly greater (P 5 0.05) in 12-day embryos from eggs of hens dosed with T3 than those for controls; however, the weights of these pelvic cartilages as a percentage of embryonic body weight did not differ from those of controls (Table 1). In this experiment, ALP activity was significantly higher in pelvic cartilages from Day-12 embryos from eggs of the dosed hens than from controls (P 5 0.02; Table 1).
DISCUSSION Our study of Japanese quail eggs supports and extends previous studies of chicken eggs that have
FIG. 7. Alkaline phosphatase activity in pelvic cartilages of Day 12 Japanese quail embryos. Units are the amount of enzyme which will produce 1 µmol of p-nitrophenol/min at 25° (Sigma Diagnostics, 1990). Values are means 6 SE (n 5 6). Circles, embryos from eggs of control hens dosed twice daily with saline; triangles, embryos from eggs of hens dosed twice daily with 33 TSR T4. ANOVA showed that pelvic cartilage alkaline phosphatase activity was significantly greater in embryos from 33 TSR T4-dosed hens than that in control embryos (P 5 0.005).
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suggested that thyroid hormones of maternal origin are transferred into the egg prior to laying [Hilfer and Searls (1980), Sechman and Bobeck (1988), and Prati et al. (1992)]. The T4 and T3 concentrations we found in the yolk of Japanese quail eggs were similar to those reported by Prati et al. (1992; T4, 3.8 6 0.3 ng/g; T3, 1.5 6 0.2 ng/g) and Sechman and Bobeck (1988; T4, 6.0–10.0 ng/g; T3, 1.5–2.3 ng/g) for chicken eggs. Our study and the latter two studies listed above all chemically extracted thyroid hormones from yolk. In contrast, Hilfer and Searls (1980), who obtained very different results (T4, 15.0–20.0 ng/g), used yolk samples directly in the RIAs. Thus, the high yolk hormone concentrations they reported may have been inflated as a result of lipid interference with primary antibody binding in the RIA. To our knowledge, our study is the first that addresses the relationship between the hen’s thyroid status and the deposition of thyroid hormones in her eggs. Overall, the data show that hyperthyroid hens, with high concentrations of T4 in their plasma, deposit increased amounts of maternal thyroid hormones in the yolk of their eggs. However, when individual eggs are considered relative to the hens’ plasma thyroid hormones at the time of laying, there is evidence of some regulation of thyroid hormone deposition. Within each treatment, hens regulate yolk T4 concentrations independent of variations in their plasma T4 concentrations. However, more work is needed to further document the capability of yolk hormone regulation and to determine how such regulation of yolk hormones is achieved. Our attempts to study the effects of hen hypothyroidism on yolk T4 content suggest that thyroid hormonedeficient eggs may not be produced. Laying hens were very resistant to the effects of the goitrogen, methimazole, and there was no significant reduction in the thyroid hormone concentrations in either their plasma or the yolk of their eggs during 30 days of treatment at the first dosage used. It is not clear whether these hens continued to synthesize some hormone or whether hormone reserves in the thyroid gland maintained blood hormone concentrations and supplied hormones for deposition in the eggs. When the methimazole dose was doubled (second month), egg laying became sporadic and quickly ceased but plasma thyroid hormones remained in the normal range. When
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the experiment was terminated and hens were sacrificed, thyroid gland hypertrophy was observed, suggesting that the goitrogen had been effective and the pituitary was detecting hormone decreases too subtle to be measured. Overall, these results suggest that hypothyroid hens cease egg laying, so the production of eggs with low thyroid hormone content seems unlikely. Some studies have addressed the potential mechanism(s) for the transfer of thyroid hormones from the hen to the egg yolk. Lipoproteins in the plasma of chicken hens bind small but significant amounts of plasma thyroid hormones and are responsible for the transport of these hormones into the yolk of developing follicles (Mitchell, 1984; Mitchell et al., 1985; Mitchell and Stiles, 1985). This proposed mechanism of transfer is plausible, because low-density lipoproteins are synthesized by the liver and deposited in oocytes during follicle development, (Griffin et al., 1984) and the lipoproteins found in hen plasma and egg yolk are very similar (Griffin et al., 1984). Vitellogenin also binds thyroid hormones in hen plasma and binds to receptors on ovarian membranes in chickens (Barber et al., 1991). Transthyretin (thyroxine-binding prealbumin) found in egg yolk is derived from the hen’s plasma and there is a putative transthyretin receptor on the oocyte membrane (Vieira et al., 1995). Thus, it appears that several plasma proteins and lipoproteins may be involved in the transfer of maternal thyroid hormones into the egg and could be important in regulating the amount of thyroid hormone deposited in the yolk. If yolk thyroid hormones are taken up by embryos prior to hormone release by the thyroid gland, they could be playing essential roles in early tissue development. In a study of chicken eggs, Prati et al. (1992) found detectable amounts of T4 in 4-day embryos (2.48 pg/embryo) when one sample consisting of 30 embryos was extracted and analyzed. They also analyzed three pools of 6-day embryos and found 7.4 pg T4/ embryo (in embryos with the neck, and thus presumably the thyroid gland, removed). Based on the assumption that the embryonic thyroid gland does not secrete hormones until after Day 9, Prati et al. assert that their data demonstrate embryonic uptake of maternal hormones from the yolk. However, Thommes and Hylka (1978) found T4 (0.23 ng/ml) in the blood of 6.5-day
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Wilson and McNabb
chicken embryos and thyrotropin stimulation increased the concentration of T4 by about sevenfold. Likewise, the finding of Thommes and Tonetta (1979) that thiourea administration on Day 5.5 of incubation markedly reduced embryonic plasma T4 concentrations on Day 7.5 argues for much of the plasma T4 coming from the embryonic thyroid gland. These studies provide evidence that the thyroid is producing and releasing hormones at this time. However, no one has yet devised a way of determining the proportions of embryonic hormones contributed by the embryonic thyroid gland vs the maternal hormone stores of the yolk. We attempted to determine if yolk hormones are taken up early in development, by examining our data from Day 7 quail embryos (16.5-day incubation). At this stage the embryonic thyroid gland contains very small amounts of thyroid hormones (Day 8 of the 16.5-day incubation: 1.7 ng T4/gland pair and 1.5 ng T3/gland pair; McNichols and McNabb, 1988) and is relatively inactive (based on radioiodine uptake studies; McNabb et al., 1981). Although a very large amount of T4 was available in the yolk of eggs from 33 TSR T4-dosed hens (<400 ng/yolk) we found no evidence of differences in hormone content in Day 7 embryos from control vs high T4 eggs. This lack of differential thyroid hormone content in embryos from eggs of very different hormone content seems to argue against hormone uptake from the yolk at this stage. What is not clear is whether the embryo is taking up T4 and degrading it (yolk T4 decreases significantly from preincubation values to Day 7 of incubation) or whether T4 is degraded within the yolk itself. Whatever the site of degradation, our data do not support the idea that this disappearance of the extra T4 in high T4 eggs results in T3 production, as neither the yolk nor the embryo has higher T3 in the high T4 eggs than in the controls. One of our experiments did show significantly higher T3 in the yolk of high T4 eggs at Day 7 of incubation, but this result was not confirmed in a repeat study. To our knowledge, the only study in which deiodinase activities in eggs have been investigated is that of Reddy et al. (1992) on tilapia eggs. In an in vitro assay system with unlabeled T4 as substrate and measurement of product (T3) by RIA, these workers were unable to detect any 58-deiodinase (58D) activity in fertilized eggs. However, they found that T4
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Thyroid Hormones in Egg Yolk
decreased rapidly in eggs even before fertilization. If the T4 was 5-deiodinated to rT3, their assay would not have detected this degradation. The lack of T4 in the allantois in our study suggested the T4 was not being excreted from the embryo without degradation. In the latter half of embryonic development, maternal hormones taken up from the yolk could supplement endogenous embryonic thyroid hormones. In high hormone eggs one might expect accelerated development of thyroid hormone-responsive tissues. Pelvic cartilages from chicken embryos have previously been shown to be thyroid hormone-responsive with respect to both their growth and differentiation (Burch and Lebovitz, 1982). We found accelerated growth (significantly increased cartilage wet and dry weights) and evidence of accelerated cartilage differentiation (increased ALP activity) in embryos from eggs of hens on the highest T4 dose. In contrast to these effects on pelvic cartilage, high T4 content in eggs did not affect hatchability, body length, body weight, or the gross morphological appearance of quail chicks at hatch. These results may not be surprising because thyroid hormones appear to be permissive with respect to the stimulation of general body growth, and there is no simple relationship between growth and thyroid hormone concentrations (McNabb and King, 1993). It is generally thought that most thyroid hormone action in birds is triggered by T3, as is the case in mammals (McNabb and King, 1993). Thyroxine in the yolk of high T4 eggs might be deiodinated in embryonic tissues to T3, to inactive reverse-T3, or to one or both of these products, followed by further deiodinations. Thus, if there were appreciable 58-deiodination in embryos, we might expect to see higher embryonic T3 content in the embryos from high T4 eggs than in controls. This was not the case in the present study. This finding is consistent with in vitro studies that show little 58D activity in tissues such as liver until the beginning of the perihatch period (review, McNabb and King, 1993). Thus it appears that high egg T4 does not lead to overall high embryo T3 in quail embryos. The embryos may be deiodinating T4 to r T3 because that enzymatic capability is known to exist in liver during the latter half of embryonic life (chicken embryos, Borges et al., 1981; Galton and Hiebert, 1987; Darras et al., 1992). The deiodination capabilities of
individual hormone-responsive tissues, such as the pelvic cartilage used in this study, have not been investigated. In summary, our study indicates that thyroid hormone deposition in egg yolk varies with the thyroid status of the hen, but that there is some regulation of yolk hormone concentrations. In addition, high concentrations of thyroid hormone in egg yolk may affect the growth and differentiation of some thyroid hormoneresponsive tissues (such as the pelvic cartilage used in this study), but do not influence general body growth. In the high T4 eggs, yolk T4 disappears progressively during embryonic development, but does not lead to increased T4 or T3 in the embryo. These results suggest the need for further investigations to understand hormone deposition in eggs and the effects of maternal hormones on embryonic development: (1) how the mechanisms of transfer of maternal thyroid hormones from the hen to the yolk are influenced by differences in the hen’s thyroid status, (2) the extent to which maternal thyroid hormones in the yolk enter the embryo at different stages of development, and (3) the possibility and/or extent of deiodination of T4 (and the pathways involved) in the yolk and the embryo.
ACKNOWLEDGMENTS The following people volunteered their time to assist with several aspects of the project and deserve special thanks: Allison Wolf, Melissa Goode, Melissa Catena, Heather Solari, Osama Abed, Steve Sklarew, Eric Belling, Megan Harris, Emilio Martinez-Lezama, Cynthia Tate, Matt Stinchcomb, Judy McCord, Amy Wood, David Mullins, Memuna Khan, Kevin Simon, Matt Lovern, and Steve Nunez. Drs. P. B. Siegel, D. M. Denbow, and C. L. Rutherford provided helpful perspective on many parts of the study. This research was supported by funding from Sigma Xi Grants-In-Aid, the Graduate Research Development Project of Virginia Tech., and the Biology Department of Virginia Tech.
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