Comparative plasma lipid response of pullets and laying hens to estradiol and progesterone

TOXICOLOGY

AND APPLIED PHARMACOLOGY

76,483-489

(1984)

Comparative Plasma Lipid Response of Pullets and Laying Hens to Estradiol and Progesterone R. C. HAGAN,* D. E. LESZCZYNSKJ-~AND F. A. KuMMERow*,t *Burnsides Research Laboratory, Department of Food Science, University of Illinois, Urbana, Illinois 61801, and tHarlan E. Moore Heart Research Foundation, Champaign, Illinois 61820

Received May 10, 1984: accepted July 26, 1984 Comparative Plasma Lipid Response of Pullets and Laying Hens to Ektradiol and Progesterone. R. C., LESZCZYNSKI, D. E., AND KUMMEROW, F. A. (1984). Toxicol. Appl. Pharmacol. 76, 483489. The effects of estradiol and progesterone treatment on plasma hormone and lipid concentrations were measured in laying hens and sexually immature pullets. Pullets and hens were divided into three groups and injected with estradiol (1 mg/kg bw), progesterone (4 mg/ kg bw), or vehicle (propylene glycol) once each day for 14 days. Blood samples were collected before treatment and 24 hr after the 7th and 14th treatments. Plasma progesterone (P), estradiol (E2), triglycerides (TG), cholesterol (C), and phospholipids (PL) were measured. Er treatments elevated hen plasma TG 7.2X, PL 5.1X, and C 7.2X; and pullet plasma TG 6.8X, PL 3.7X, and C 2.5X. However, because hen plasma was initially mildly hyperlipidemic, the E,-treated hens developed severe hyperlipidemia, but egg production was unaffected. Progesterone treatments of pullets had little or no effect on plasma lipids, but progesterone treatment of hens significantly reduced initial plasma TG and PL and also reduced egg producton. No substantial differences were found in circulating Ez or P in hormone-treated hens and pullets, which indicated no extreme differences in plasma hormone clearance rates. These results indicate that long-term rather than short-term hepatic priming may account for observed differences in layer and pullet response to estradiol t~atnWIt. 0 1984 Academic PBS, h. HAGAN,

Estrogen is well known as a hyperlipidemic agent in chickens. The estrogen-stimulated hyperlipidemic response is mediated by highaffinity estrogen receptors in liver plasma membranes which translocate estrogen to cell nuclei, where specific gene loci related to lipoprotein synthesis are activated (Snow et al., 1978; Kassis and Gorski, 1981). This system is believed to be responsible for the hyperlipidemia of the laying hen (Jensen, 1979). A single estrogen injection of 25 mg/kg to cockerel or rooster has become an acceptable method for studying hepatic lipoprotein response in chickens. In this system, apolipoprotein genes in the liver were found to be induced by estrogen to produce multiple transcripts of messenger RNA, the products

of which generate plasma hyperlipidemia similar to that of the laying hen (Chan et al., 1980; Capony and Williams, 1980; Wiskocil et al., 1980). Studies with hepatectomized cockerels indicated that the elevated plasma phosphoprotein and phospholipid induced by estrogen treatment are exclusively of hepatic origin (Vanstone et al., 1957). These findings are compatible with the belief that lipogenesis in the chicken is almost exclusively hepatic in origin (Pearce, 1977, Bannister, 1979). Thus it appears that estrogen treatment alone can elicit a strong hepatic lipogenic response in the chicken which is quantitatively the same independent of sex or age. Few studies have compared the magnitude of lipogenic response to steroid sex hormones 483

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484

HAGAN,

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between chickens in different states of sexual physiology. Pearson and Butler (1978) found no difference between 7-week-old cockerels and pullets in plasma lipid response to multiple estrogen injections. Clegg et al. (1976), however, reported considerable differences in plasma lipid response to multiple estrogen injections between 1l-week-old cockerels and pullets. Laying hens have higher plasma lipids than roosters, cockerels, or pullets, but the response of hens to estrogen treatment relative to chickens in other states of sexual physiology is unknown. To obtain some information regarding lipogenic response to sex hormones as a function of sexual physiology, we measured plasma lipids of both laying hens and sexually immature pullets treated with equal concentrations of estradiol (E2) and progesterone (P). METHODS Animals and blood samples. White Leghorn 15-weekold puIIets and ZO-month-old layers were caged individually and provided with feed and water ad fibitum and a daily lighting schedule with seven hours of darkness, lights on between 0500 and 2200 hr. All chickens were fed a high-protein corn-soybean-based commercial ration. Blood samples were collected at 10 AM from wing veins into heparinized tubes from which plasma was prepared and stored at -20°C until analysis. Chemicals. [2, 4, 6, 7, 16, 17,-‘H(N)] Estradiol, 140 Ci/mmol, and [I, 2, 6, 7-‘H(N)] progesterone, 101 Ci/ mmol, were obtained from New England Nuclear (Boston, Mass.). Progesterone-( 1I-hemisuccinate-BSA) and I7-@estradiol-(6-0-carboxymethyl)-oxime-BSA conjugate antibodies produced in rabbits were obtained from Calbiochem Behring Corporation (La Jolla, Cahf.) for use in antiserum solutions. Progesterone (P, 0 130) and estradiol (E, 8875) were obtained from Sigma Chemical Company (St. Louis, MO.). Other chemicals were yglobulin, Cohn fraction II (Sigma), Sephadex LH-20, and dextran T-70 (Pharmacia, Piscataway, N.J.), and Norit A charcoal (Fisher Scientific, Chicago, Ill.). Nanograde ether, benzene and isooctane were obtained from J. T. Baker Company (Chicago, Ill.) and pretested for residue interference with the radioimmunoassay (RIA). Hormone treatmenl. Steroid hormones dissolved in propylene glycol were delivered by sc injection in the legs of chickens, with potencies adjusted to initial body weight (bw). Estradiol treatment was 1.0 mg/kg bw, progesterone treatment was 4.0 mg/kg bw, and controls

AND KUMMEROW received only propylene glycof. Treatments were given once per day at IO AM for a total of 14 treatments and adjusted to individual body weights so that each treatment was contained in 0.75 ml propylene glycol vehicle. Plasma lipid analysis. Methods used for determining plasma triglycerides, total cholesterol, and phosphohpid have been reported previously (Leszczynski et a/., 1982). In cases of hyperlipidemic plasma, the bulk of the lipid was removed by cold methanol precipitation (MonvaiGerowdeau ef al., 1970), and fractional steroid losses were determined before the RIA analysis. Hormone RIA assays. Plasma (I ml) was combined with recovery tracer (0.1 ml; 600 to 800 cpm) and extracted two times with 4 vol:I vol diethyl ether for estradiol, and 4 vol: 1 vol petroleum ether for progesterone. Combined ether extracts were evaporated under N2 after which the extract was solubilized in isooctane:benzene:methanol (6220: 18) for estradiol RIA and 90:5:5 for progesterone RIA and then chromatographed over Sephadex LH-20 minicolumns. Appropriate steroid fractions were evaporated to dryness and resuspended in 2 ml absolute ethanol from which duplicate 0.6-m] ahquots were transferred and dried for RIA, and 0.8 ml was dried and counted for calculation of recovery. RIA assayswere performed according to the procedures given with the progesterone and estradiol Radioimmunoassay Paks supplied by New England Nuclear, and based on the methods of Abraham et al. (1971) and Youssefrejadian et al. (1972). Briefly, 0.1 -ml portions of assay buffer (saline phosphate buffer, pH 7.0, containing 1 g/liter each bovine y-globulin and gelatin) were added to the sample and blank tubes followed by O.l-ml portions of assay tracer (3000 to 4000 cpm). Next 0.1 ml antiserum solution was added to each tube, followed by incubation at room temperature for I hr, and 0°C for an additional hour. After incubation, 1.0 ml cold dextran-coated charcoal solution (0.4%) was added, and the tubes were centrifuged 15 min at 12OOgat 4°C after which the clear supematant fractions were removed and counted. Standard curves were prepared by plotting the average percentage bound for standard tubes vs picogram or nanogram standard. Hormone concentrations in plasma were determined by the formula: steroid/ml = (pg or ng from graph)/(fractional recovery) (assay volume) (volume plasma extracted). RIA validation. Cross reactivities of 17-&estradioi antiserum were ~0.6% for estrone, 10.2% for estriol, and undetectable for progesterone; progesterone RIA antiserum had ~17% reactivity with 1I-cr-hydroxyprogesterone, < 16% with 1 1-&hydroxyprogesterone, <2.4% with pregnenolone, ~1.5% with deoxycorticosterone, and ~0.05% with estradiol. Average water blank values for the progesterone RIA were 12 pg, and 25 pg for the estradiol RIA. Assay sensitivity, defined as the concentration of hormone needed to give a response 2 SD units higher than the zero standard response, averaged 20 pg for progesterone and 3 pg for estradiol. Assay precision

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LIPIDS IN HORMONE

was measured by calculating the within- and betweenassay coefficients of variation (CV) from pooled plasma samples; these CV were 3.7% (N = 6) and 11.6% (N = 12) for the progesterone RIA, and 5.6% (N = 6) and 8.2% (N = 3) for the estradiol RIA. Accuracy was tested by comparing the recovery of various amounts of cold hormone against the amount added to pooled plasma; coefficients of correlation (r) were 0.997 for progesterone and 0.998 for estradiol recoveries. These validations ensured the reliability of our assay results. Statislics. Mean plasma hormone, plasma lipid, and organ values obtained from various groups were tested for significant differences within groups by paired t test. The values of hormone-treated groups were compared to the appropriate control values of equal treatment duration by two-tailed t tests (Steel and Torrie, 1960).

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485

HENS

RESULTS Plasma lipid and hormone values of sexually immature 4-month-old White Leghorn pullets and 20-month-old sexually mature White Leghorn layers which received either estradiol, progesterone, or propylene glycol (control) injections once per day for a period of 14 days are presented in Table 1. EZ treatments of 1 mg/kg bw per day resulted in sharp increases of plasma lipids, including triglycerides, phospholipids, and cholesterol, in both pullets and layers; the layer plasma

TABLE 1 MEANPLASMA HORMONEANDLIPIDVALUESFORHORMONE-TREATED Treatment group"

Control

Estradiol (1 a&

N

5

0 7 14

6

0 7 14

5

0 7 14

W

Progesterone (4 mg/kg bw)

Days of treatment

P bg/mU

E2

b-x/ml)

107 + 176 + 346 f

75 27 161

220 + 90 + 227 f 163 + 228 f 254 I?

PL hddl)

(m$l)

20-month-old layers 2.72 f 1.69 1248 + 481 2.03 f 1.76 1166 k 601 1.07 + 0.76 1228 -t 758

190 f 60’ 720 + 70d 1055 + 457d

FEMALECHICKENS C bg/dl)

939 f 218 835 f 283 727 + 414

111 f 29 119 + 36 188 f 72

1.76 f 1.17 1.79 + 0.69 3.27 f 2.67

1303 r 891’ 4550 + 2587d 9381 3- 2007d

847 2 345’ 2369 f 1153d 4282 + 830d

109 f41C 292 + 95d 786 + 263d

106 1.51 f 1.50 44d 4.41 f 3.59 102 3.76 + 2.74

1626 + 694’ 1273 + 565 205 rt 51d

1OOOf 271’ 589 ?I 155 369 f 42

136+31 251 + 99 172 + 25

4-month-old pullets 80 0.185 k 0.081 81 0.922 f 0.770 39 1.25 + 0.53

Control

6

0 7 14

Estradiol (1 me/kg bw)

7

0 7 14

361 f 247’ 0.185 + 0.103 3365 f 4081 0.777 + 0.702 1292 f 615d 2.62 + 1.94

Progesterone (4 Wke bw)

7

0 7 14

241 It 108 1279 + 1654 305 + 102

0.344 + 0.333’ 1.69 + 0.98 3.42 f 1.57d

176 + 170r 248 r

45 41 51

188 + 62’ 761 iz 261d 1282 f 562 163 ~tr 43 189 + 27 230+ 51

214+ 197 f 250 f

26 17 26

105 f 11 109* 7 130 + 10

235 f 43’ 106 + IO’ 515 31 147d 203 f4gd 869 -c 309 266 + 81d 229 + 2592 302 k

31 109 f 14 12d 135+ 13d 35d 142 k 14

Note. E2, estradiol; P, progesterone; TG, triglycerides; PL, phospholipids; C, total cholesterol; N, sample size; bw, body weight. ‘Treatments were given once each day; controls were injected with propylene glycol. Data are presented as X rl: SD. All possible paired t tests within groups and two-tailed t tests between hormone treatment and control groups with the same days of treatment were calculated. b Within this group, two of three possible paired t tests were significantly different (p < 0.05). ’ Within this group, al1 three possible combinations of paired t tests were significantly different (p < 0.05). dHormone-treated group value is significantly different (p c 0.05, two-tailed f test) from comparable control group value.

486

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LESZCZYNSKI,

lipid surged to much higher absolute values compared to pullets, even though circulating E2 concentrations in these groups were not substantially different 24 hr after the 7th and 14th treatments. P treatments of 4 mg/kg bw caused a sharp reduction in both plasma triglycerides and phospholipids of layers. In pullets, where plasma lipid concentrations were already low, P treatments had little effect on plasma lipid except for a possible slight increase in phospholipids. Before treatments were begun, circulating Ez concentrations were no different in pullets compared to layers, but P values were eight times lower in the pullets (X = 0.24, N = 20, vs X = 1.98, N = 16). Circulating EZ, after E2 treatment, was significantly increased in both pullets and layers; but after 2 weeks of treatment, the absolute circulating EZ concentrations of pullets and layers were not different. In layers, circulating P remained relatively constant regardless of treatment, whereas P treatment of pullets resulted in significantly increased circulating P. Absolute circulating P concentrations, however, were

AND KUMMEROW

not significantly different between layers and pullets after either 7 or 14 treatments with either P or Ez. Organ weights presented in Table 2 show that P treatment of pullets had no effect on ovary weight, whereas the same treatment in layers resulted in sharply reduced ovary weights. P treatments also caused noticeable ascites in both pullets and layers. Ez treatment of pullets produced a large increase in oviduct size, but had little or no effect on layer oviducts. Ez had no effect on egg producton of layers; but P treatment severely diminished egg production as indicated in Table 3. The combination of sharply reduced egg production plus sharply reduced plasma lipids would indicate a sharp reduction of hepatic lipogenesis in layers treated with P (Leszczynski et al., 1982). DISCUSSION Average baseline plasma concentrations between 1 and 2 rig/ml have been reported

TABLE 2 NECROP~Y VALUESFROM HORMONE-TREATED FEMALE CHICKENS Treatment*

N

Heart

Control Estradiol (1 w/kg bw.) Progesterone (4 m&kg bw.)

5

3.89 f 0.3gh

6

4.32 f 0.59

5

3.89 + 0.50

6

3.50 f 0.26

7

3.27 + 0.20

7

3.58 k 0.32

Control Estradiol (1 w.k bw.1 Progesterone (4 mg/kg bw.)

Liver

Oviduct

Ovary

22.76 k 13.33

13.89 zk 9.19

29.9 f 6.1

27.62 f

6.70

17.79 + 13.54

33.4 k 8.5

18.51 k

3.18’

2.36 + 0.91~~

0.36 k

0.16

0.54 f

0.11

24.6 + 1.7

8.52 f

3.63*

0.53 f

0.11

22.3 + 1.9

2.02 + 0.93Gd

0.51 f

0.12

20-month-old layers 24.1 f 5.3

4-month-old pullets 23.0 + 1.3

* Treatments were once a day for a 1Cday duration. b Data are X k SD expressed as g/kg bw. c Within either Iayers or pullets, progesterone treatment value was significantly different (p < 0.05, two-tailed ttest) from estradiol treatment. *Within either layers or pullets, hormone treatment value was significantly different (p < 0.05, two-tailed t test) from control.

PLASMA

LIPIDS IN HORMONE

TABLE 3 EGG PRODUCTION

BY LAYERSBEFOREANDDURING EXPERIMENTAL TREATMENT PERIOD

Treatment

N

Before treatment

During treatment

Control Progesterone Estradiol

5 5 6

6.8 + 3.8” 6.6 f 3.1 7.0 + 4.4

7.2 + 4.6 1.8 + l.lb 7.0 * 4. I

a Data are expressed as 2 * SD eggs laid per chicken in the period 2 weeks before the start of treatments, and also during the 2-week treatment period. bSignificant decrease in egg production (p < 0.05, two-tailed I test).

for actively laying hens (Furr et al., 1973; Tanabe and Nakamura, 1980), but information regarding P concentrations in immature pullets is lacking. P values obtained from 20month-old layers in our study were similar to values for layers determined by others; before treatment, plasma P in 15-week-old pullets was five- to tenfold lower than in layers. It also appears that pullet P concentrations were increasing during the experiment, which may be normal for pullets 4 to 5 weeks before the initiation of egg laying. Senior (1974) found that plasma E2 concentrations in sexually immature and mature female chickens were different. At 7 or 10 weeks of age, El values were almost always < 100 pg/ml, then gradually increased and peaked just before the onset of laying, and then dropped to values usually between 100 and 200 pg/ml. In our study, pretreatment Ez in 15-week-old pullets was slightly higher than those of layers, but both pullet and layer EZ values were consistent with the report of Senior (1974). In our experiment, 24 hr after the last injection of l- and 2-week treatment regimes, the plasma concentrations of treatment hormones were generally 2 to 10 times higher compared to controls. The most outstanding difference between pullets and hens was the magnitude of plasma lipid concentrations, especially triglycerides

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487

and phospholipids, in response to equal dosage of Ez. In laying hens, egg production accounts for the major portion of lipid removal from serum (Jensen, 1979). However, since egg production was not affected by EZ treatment, it appears that the treatments produced a greater lipogenic response in the hens compared to the pullets. Chicken liver can be classified as an estrogen target tissue since a high-affinity estrogenbinding protein has been found in the liver cytosol of cockerels (Lazier and Haggarty, 1979). Recent reports indicate that there are differences in sensitivity of the various target tissues which respond to estrogen, and the tissue sensitivity may reflect the relative accessibility of cells to the blood supply. Differences between hen and pullet liver sensitivity cannot be explained by this model, however, as the liver is thoroughly perfused by blood in both cases. Another possibility is that the hen liver is primed for higher activity relative to the pullet liver due to prior induction of hormone sensitive hepatic lipogenesis. In short-term (72 hr) priming experiments with cockerels, Chan et al. ( 1977) found no priming effects of either estradiol or progesterone on the lipogenic response of liver slices to estrogen treatment. In our experiment, pullet plasma TG increased only 521 mg/dl between treatments 8 through 14, compared to 3247 mg/dl between treatments 1 through 7 and a further increase of 4831 mg/dl between treatments 8 through 14 for hens. This experiment demonstrates that short-term priming of pullets, even for a period of 7 days, is not sufficient for stimulating lipogenesis to the capacity exhibited by the hens. Differences observed between hens and pullets in estrogen-stimulated lipogenesis could be due to long-term primed effects which likely involve mechanisms for increasing organ sensitivity that are supplemental to the mechanism of simple hepatic gene induction as described by Chan et al. (1978) for short-term Ez treatment of cockerels. Increased response to E2 stimulation can be

488

HAGAN.

LESZCZYNSKI,

achieved by the long-term increase in absolute amount or mass of tissues involved in lipogenesis. This possibility is indicated by the study of Aprahamian et al. (1980), which found that six treatments of Ez to l-monthold pullets over a l-week period resulted in a 180-fold increase in oviduct fatty acid synthetase and a 3-fold increase in liver fatty acid synthetase, with much of the enzyme increase being due to increased organ mass. Therefore, it is likely that long-term primed changes in the hen, which include increased organ or specialized tissue mass (especially liver and oviduct), were responsible for the accentuated plasma lipid response compared to pullets. It is not known to what extent the mechanisms for plasma lipid removal are affected by estrogen treatment in chickens, but it is apparent from the immediate and accelerated production of hepatic messenger RNA for apolipoproteins that hepatic lipid fatty acid synthesis is a major factor in estrogen-stimulated hyperlipidemia (Snow et al., 1978; Chan et al., 1980). Prolonged treatment with estrogen produces fatty liver (Leszczynski et al., 1982); raised serum estrogen concentrations are believed responsible for fatty liver hemorrhagic syndrome in commercial chickens (Jensen, 1979). In most mammals, fatty acid synthesis can take place in both liver and adipose tissue, with the latter being the major site of lipogenesis (Pearce, 1977). It is noteworthy that, in contrast to the situation in rodents and domestic mammals, the liver is the major site of lipogenesis in chickens and man (Pearce, 1977). This similarity in lipid metabolism has contributed to establishing the chicken as a valuable animal model in atherosclerosis research (Leszczynski et al., 1982). Hyperestrogenemia may be a separate and potent risk factor in human coronary heart disease (Kolata, 1983; Phillips et al.., 1983). It would be interesting to test whether the apparent progesterone antagonism of estrogen-stimulated lipogenesis found in chickens also occurs in man.

AND KUMMEROW

ACKNOWLEDGMENTS This work was funded by grants from the American Heart Association, Illinois Affiliate (D.L.) and the Wallace Genetic Foundation.

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CHAN, L., JACKSON, R. L., AND MEANS, A. R. (1978). Regulation of lipoprotein synthesis: Studies on the molecular mechanisms of lipoprotein synthesis and their regulation by estrogen in the cockerel. Circ. Res. 43,209-2

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YOUSSEFRWADIAN,E., FLORENSA, E., COLLINS, W. P., AND SOMMERVILLE, I. F. (1972). Radioimmunoassay of plasma progesterone. J. Steroid Biochem. 3, 893901.