Estrogen Induction of Plasma Vitellogenin in the Cockerel: Studies with a Phosvitin Antibody RICHARD L. JACKSON, HU-YU LIN, J. T. SIMON MAO, LAWRENCE CHAN, AND ANTHONY R. MEANS Departments of Cell Biology and Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030 phosvitin produced no detectable immunoprecipitate in the plasma. However, after a single sc injection of diethylstilbestrol (2.5 mg), plasma vitellogenin levels began to increase at 4 h and reached a maximum 20-30 h after hormone administration. The increase in plasma levels of triglyceride paralleled those of vitellogenin. These studies suggest that there is no significant time lag in the estrogenic induction of plasma vitellogenesis in the cockerel; the longer lag periods observed by other investigators may be a function of the sensitivity of the assays used for detecting vitellogenin. (Endocrinology 101: 849, 1977)

ABSTRACT. The effects of estrogen on plasma vitellogenin have been studied in the cockerel by immunoprecipitation techniques using an antiserum prepared against the egg yolk phosphoprotein, phosvitin. The antiserum gave precipitin lines of complete identity to phosvitin and to vitellogenin which was isolated from hen plasma by DEAEcellulose chromatography and by affinity chromatography using anti-phosvitin coupled to Sepharose 4B. The cross-reactivity of vitellogenin and phosvitin adds support to the concept that plasma vitellogenin is the precursor phosphoprotein of egg yolk phosvitin. In the three-week old cockerel, anti-

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T IS NOW well known that the adminis- and two molecules of phosvitin (molecular tration of estrogen to immature pullets weight 30,000 each); vitellogenin is cleaved or to cockerels is associated with dramatic in the oocyte to lipovitellin and phosvitin increases in the levels of plasma lipids, (10). Phosvitin is unique in that it contains lipoproteins and phosphoproteins (1-8). 50% serine of which almost all are phosThe increase in plasma triglycerides is phorylated. Since estrogen so dramatically accompanied by an increased rate of syn- increases the synthesis of triglycerides and thesis of very low density lipoproteins phosphoprotein in the cockerel, this system (VLDL) (5,8). Chan et al. (8) found that has been used as a model for studies the estrogen effect was mediated at the level related to the mechanism of hormone acof transcription by enhancing the accumu- tion. In previous studies from this laboralation of a specific mRNA for one of the tory, we have described the effects of estroVLDL proteins. The major estrogen-in- gen on VLDL synthesis. In order to deterduced phosphoprotein is vitellogenin, the mine if estrogen induces vitellogenin and precursor of the egg yolk proteins, phos- VLDL synthesis by a common mechanism, vitin and lipovitellin (9,10). Vitellogenin we have measured the effect of hormone is synthesized in the liver of laying hens on plasma vitellogenin. This report deor estrogen-treated chickens and is then scribes the preparation and characterization secreted and transported via the circula- of an antiserum prepared against egg yolk tory system to the ovary where it is deposited phosvitin. Using this specific antiserum, in the developing oocyte. Plasma vitello- the accumulation of plasma vitellogenin genin contains, within its single polypep- has been measured in the estrogen-treated tide chain, one molecule of lipovitellin cockerel. (molecular weight approximately 180,000) Materials and Methods Received January 3, 1977. Supported by Health, Education and Welfare Research grant HL 16512-02 and Grant-in-Aid 75-914 of the American Heart Association.

Animals and collection of plasma White Leghorn hens or 21-day old cockerels were used throughout the study. In those studies

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were removed by low-speed centrifugation and the plasma stored at 4 C.

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Preparation of phosvitin Phosvitin was isolated from fresh White Leghorn egg yolk by modifications of the procedures of Bernardi and Cook (16) and Beuving and Gruber(17). To yolks (1100 ml) from 60 eggs were added at 4 C three volumes of 0.4M MgSO4, and then this solution was diluted with an equal volume of water. The precipitate which developed was collected by centrifugation at 5000 rpm for 30 min; it was washed with 1500 ml of 0.4M MgSO4, diluted with an equal volume of water and recentrifuged. After lyophilization, the precipitate (30 g) was delipidated with diethylether: ethanol (3:1). In a typical experiment, 10 g of precipitate was delipidated with 500 ml of ether:ethanol by continuous stirring at 4 C overnight. The mixture was then centrifuged and the pellet washed two times with ether. After centrifugation, the pellet was taken to dryness with nitrogen. The partially purified phosvitin (2.6 g) was then dissolved in water and the insoluble material removed by centrifugation. The soluble phosvitin was lyophilized and subjected to DEAE-cellulose chromatography using the conditions described in the legend to Fig. 1A. The major phosphoruscontaining fraction was resubjected to chromatography on a column (1.6 x 200 cm) of Sephadex G-100 (Fig. IB).

FIG. 1. (A) DEAE-cellulose chromatography of partially purified phosvitin. A crude preparation of phosvitin was isolated from egg yolk by MgSO4 precipitation, delipidation and extraction as described in the text. 25 mg of protein in 5 ml of 0.15M sodium citrate-O.lUM NaCl, pH 5.0 was applied to a column (1.6 x 30 cm) of DEAE-cellulose in the same buffer, which was run at room temperature. After application of the sample, the flow rate was adjusted to 25 ml/h and 5 ml fractions were collected. The column was eluted stepwise first with the same citrate buffer, which was used to equilibrate the DEAE. At tube 48, the elution solution Preparation of antisera against phosvitin and was then changed to 0.01M Tris-HCl, 0.5M NaCl, pH determination of equivalence point 8.0. Phosphorus was determined by the method of A single adult male goat was used for the Bartlett (19). (B) Sephadex G-100 chromatography of purified phosvitin from DEAE-cellulose (Fig. 1A). preparation of antiphosvitin. The antigen (5 mg) The column, 1.6 x 200 cm, was equilibrated with in 1 ml of 0.15M NaCl was mixed with an 0.1M ammonium bicarbonate. The sample (6 mg) was equal volume of complete Freund's adjuvant applied and eluted with the equilibrating buffer at (Difco) and the emulsion injected into 50 differa flow rate of 20 ml/h. ent sites on the back of the goat. After six weeks, a second injection of 2 mg phosvitin in in which vitellogenin synthesis was studied, the complete Freund's adjuvant was given im. A animals were given a single sc injection of third injection of 2 mg was given two weeks diethylstilbestrol (DES), 25 mg in sesame oil. later. Finally, after a further 2 weeks, 10 mg All animals were fed a standard chicken diet of antigen were given and the goat was sacrificed and were housed in a room that was lighted 10 days later. Immunoglobulins were isolated daily from 0700 to 1900 h. All animals were from the serum by ammonium sulfate precipitakilled by decapitation and the blood was col- tion (50%) (8) and then dialyzed against 0.01M lected in a polyethylene beaker containing 15 mg Tris-HCl, pH 8.0, containing 0.15M NaCl. trisodium citrate and 50 /xg of phenylmethylTo define the conditions for optimal precipitasulfonyl fluoride per ml of blood. The red cells tion of phosvitin or vitellogenin by the anti-

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DES INDUCTION OF COCKEREL VITELLOGENIN serum, increasing amounts of the phosphoproteins were added to a constant amount of antiserum (50 /ul) in 0.20 ml of 0.1M Tris-HCl, pH 8.0, containing 0.15M NaCl. The mixtures were allowed to incubate for 1 h at 37 C and then overnight at 4 C. The immunoprecipitates were collected by centrifugation at 2,000 x g for 10 min. The supernatant fluid was decanted and the precipitates washed three times with 5 ml of the incubation buffer containing, in addition to the Tris-HCl and NaCl, 1% Triton X-100. The pellet was dissolved in 1 ml of 1% acetic acid and protein determined by absorbance at 280 nm or by the method of Lowry et al. (18); phosphorus was determined by the method of Bartlett (19). Isolation of vitellogenin The procedure used for the purification of vitellogenin was identical to that of Deeley et al. (10). Hen plasma was collected and the VLDL were removed by ultracentrifugation (8). The infranatant fraction was then subjected to chromatography on DEAE-cellulose using the conditions of Deeley et al. (10). The fractions which contained phosphorus were pooled and concentrated by an Amicon concentrator using a UM100 membrane. Vitellogenin was also purified by affinity chromatography on an anti-phosvitin column. The adsorbent was prepared by mixing 300 mg of antiphosvitin gamma immunoglobulin with 10 ml (3 g) of cyanogen bromide-activated Sepharose 4B. After 30 min at 4 C, 90% of the protein was coupled to the Sepharose. The gamma immunoglobulin-Sepharose 4B resin was poured into a column (0.9 x 15 cm) and washed extensively with 0.01M Tris-HCl, 0.15M NaCl, pH 8.0. To the column was applied 1 ml of a d > 1.006 fraction from hen plasma which was diluted to 5 ml with the equilibration buffer. After the sample entered the column, it was washed with the same equilibration buffer. The vitellogenin was eluted from the column by 0.15M NaCl, pH 11.0, adjusted with NaOH. Other methods Very low density, low density and high density lipoproteins were isolated from hen plasma by ultracentrifugation as described previously (8,20). Plasma triglycerides were determined by autoanalyzer techniques (21).

TABLE 1. Amino acid compositions of phosvitin and vitellogenin1

Amino acid Aspartic acid Threonine Serine5 Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine % Phosphorus (wt/wt)

Phosvitin2 (This study)

Phosvitin (Clark and Joubert)3

Vitellogenin4 (This study)

14.3 1.8 123.0 14.7 3.0 5.5 7.2 — 3.5 0.9 1.6 3.0 0.9 0.9 15.0 12.8 10.0

13 2 125 12 3 5 8 — 3 1 2 3 1 1 15 12 10

96 51 138 109 18 63 85 — 61 32 56 90 21 26 66 25 63

10.3

9.4

3.2

1

The values represent duplicate analyses on samples hydrolyzed for 24 h. 2 Residues; based on a molecular weight of 35,000. 3 From Clark and Joubert (14). 4 Based on 103 residues. 5 Corrected for 10% destruction.

Results Purification of phosvitin Phosvitin was partially purified by an aqueous extraction of delipidated egg yolk granules (16). Further purification was achieved by chromatography on DEAEcellulose (Fig. 1A). Phosvitin was eluted from the resin with 0.01M Tris-HCl, 0.5M NaCl, pH 8.0. To show that the phosvitin was homogeneous, it was further purified on Sephadex G-100 (Fig. IB); only one protein fraction was eluted from the column. Furthermore, the phosphorus content coincided with the absorbance at 226 nm. The amino acid composition of the Sephadex fraction (Table 1) was nearly identical to that reported by Clark and Joubert (14). The purified phosvitin contained 123 residues of serine per molecular weight of 35,000. Phosphorus analysis indicated that 95% of the serine was phosphorylated.

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molecular weight species in the plasma. Egg yolk lipovitellin did not react with antiphosvitin. To determine if the plasma phosphoprotein was associated with any of the plasma lipoproteins, we next fractionated hen plasma by ultracentrifugation in KBr at d 1.210 (8,20). At this density, all of the plasma lipoproteins float and can be separated by tube slicing. As shown in Fig. 3, right (well C), no precipitin line was observed against the lipoprotein fraction. However, the d > 1.210 fraction gave a precipitin line of complete identity to plasma (wells E and D). These results show that the protein in plasma which reacts with

antiphosvitin is not associated with the major plasma lipoproteins. Isolation of plasma vitellogenin We next isolated the major phosphoprotein from hen plasma by chromatography on DEAE-cellulose. As described by Deeley FIG. 2. Immunochemical purity of phosvitin and et al. (10), great care was taken in this anti-phosvitin. The antigen (10 /xg) was first subjected study for the collection of plasma and in to electrophoresis in a 1.0% agarose support. The plate running the DEAE column as quickly as was then cleaned and the agarose replaced with 2% goat antibody against phosvitin in 1.0% agarose and possible. Figure 4 shows the elution profile when 5 ml of the d > 1.006 fraction was the electrophoresis repeated. The plates were stained with Coomassie blue. chromatographed on DEAE. More than 90% of the absorbance at 280 nm was eluted 0.05M sodium citrate, pH 5.5 (Fracwith Preparation of anti-phosvitin tion A). Two major fractions were eluted Antibodies against phosvitin were raised with a NaCl gradient; only Fraction C conin the goat only after repeated injections tained phosphorus. Fraction C was pooled, of the antigen. The immunochemical purity concentrated and subjected to further purifiof the antigen and antibody was determined cation on Sephadex G-150 (not shown); the by double diffusion analyses according to phosphoprotein eluted at the void volume the method of Ouchterlony (22) and by of the column indicating that the molecular crossed immunoelectrophoresis (23). As weight was in excess of 100,000. Under the shown in Fig. 2, a single immunoprecipitin same chromatographic conditions, the puriarc was obtained with the antiserum and fied phosvitin was included in the Sephadex the purified phosvitin. Single precipitin and was clearly separated from the vitellolines of complete identity were obtained genin. Polyacrylamide gel electrophoresis by double diffusion analyses (Fig. 3, left) of Fraction C (Fig. 4) in sodium dodecyl against the purified phosvitin and against sulfate as described by Deeley et al. (10) three different hen plasmas. Although the gave a protein band with a high molecular precipitin lines which formed with plasma weight consistent with these investigators' were identical to phosvitin, the plasma findings. Since our results are identical to antigen did not migrate to the center well those of Deeley et al. (10), we have termed as rapidly as phosvitin, suggesting a higher the phosphoprotein, vitellogenin. The isoThe Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 14:54 For personal use only. No other uses without permission. . All rights reserved.

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FIG. 3. (Left) Immunochemical reactivity of phosvitin and hen plasma against anti-phosvitin. The center well contained 7.5 fi\ of purified anti-phosvitin. The outer wells contained 7.5 fxl of the following: A, phosvitin (1 mg/ml). B, C, D, three different hen plasmas. (Right) Immunochemical reactivity of various plasma samples against anti-phosvitin. The center well contained 7.5 /xl of purified anti-phosvitin. The outer wells contained 7.5 /xl of the following: A, vitellogenin isolated by affinity chromatography. B, vitellogenin isolated by DEAEcellulose. C, d < 1.210 fraction. D, d > 1.210 infranatant fraction. E, plasma.

lated vitellogenin gave a single precipitin line with anti-phosvitin (Fig. 3, right, well B). The amino acid composition of the purified vitellogenin is given in Table 1. FiG. 4. Chromatography of 5 ml of the d > 1.006 infranatant fraction on DEAE52-cellulose. Five ml of hen plasma was subjected to ultracentrifugation at plasma density at 45,000 rpm in a Beckman SW 50.1 rotor operated at 8 C for 18 h. The VLDL was removed and the infranatant fraction was diluted with 45 ml of 0.05M sodium citrate buffer, pH 5.5. The sample was applied to a column (1.6 x 50 cm) at 4 C of DEAE-52-cellulose which had been equilibrated with the same buffer. After the sample had entered the DEAE, it was washed with 100 ml of a 0.1M sodium citrate buffer, pH 5.5. At tube 10, a NaCl gradient (0 to 0.25M NaCl) was initiated and consisted of a two-chambered apparatus with 250 ml of the eluting buffer on one side of the gradient and 250 ml of the same buffer, but containing 0.25M NaCl, on the other side. The flow rate was 100 ml per hour and 10 ml fractions were collected. Of the three fractions pooled, only Fraction C contained phosphoprotein. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (10) of Fraction C (insert) indicated one major high molecular weight protein band.

The phosphoprotein contained 3.2% phosphorus (wt/wt). Plasma vitellogenin was also purified by affinity chromatography on a column of anti-

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1.006 fraction from hen plasma (Fig. 4) was diluted to 5 ml with 0.01M Tfis-HCl, 0.15M NaCl, pH 8.0, and applied to a column (0.9 x 15 cm) containing 10 ml of anti-phosvitin-Sepharose 4B. After the sample entered the column, it was eluted with O.OlM Tris-HCl, 0.15M NaCl, pH 8.0. At tube 50, the phosphoprotein was eluted with 0.15M NaCl, pH 11.0.

phosvitin Sepharose 4B (Fig. 5). One ml of the d > 1.006 infranatant was applied to the column and the column was eluted with 0.15M NaCl; greater than 95% of the protein applied to the column was not retained by the antibody. Vitellogenin was eluted at pH 11.0. The isolated vitellogenin gave a faint precipitin line against anti-phosvitin (Fig. 3, right, well A). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid analysis indicated that the vitellogenin isolated by affinity chromatography was identical to that isolated by DEAE-cellulose. The immunochemical purity of the vitellogenin isolated by affinity chromatography was determined by crossimmunoelectrophoresis and is shown in Fig. 6. A single arc was found indicating one major antigenic determinant.

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FlG. 6. Immunochemical purity of vitellogenin. Vitellogenin (10 /ng) which was isolated by DEAE-cellulose (Fig. 4) was first subjected to electrophoresis in a 1.0% agarose support. The plate was then cleaned and replaced with 2% goat antibody against phosvitin in 1.0% agarose and the electrophoresis repeated. The plate was stained as described in Fig. 2.

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Induction of vitellogenin In Fig. 8 is depicted the plasma levels of vitellogenin and triglycerides in the cockerel following the administration of estrogen. As shown previously (8), plasma triglycerides increased some seven-fold over control values; the maximum amount of plasma triglycerides occurred 24 h after DES injection and was followed by a drop to near baseline values by 48 h. The accumulation of vitellogenin, as determined

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FIG. 8. Effects of DES on plasma triglyceride and vitellogenin. Groups of three cockerels were treated with a single injection of DES (2.5 mg) sc. Animals were decapitated at the indicated times and plasma collected as described in the text. To 0.1 ml of plasma was added 50 fil of anti-phosvitin and the immunoprecipitates collected, washed and the absorbance and phosphorus measured as described in Materials and Methods.

50

100

150

pg VITELLOGENIN

FIG. 7. Determination of equivalence for phosvitin (A) and vitellogenin (B). Increasing amounts of phosphoprotein were added to 50 fi\ of goat anti-phosvitin. Incubation, washing, and protein and phosphorus determinations on the precipitates were carried out as described in Materials and Methods.

by immunoprecipitation, also peaked at 24 h but remained at a high level at 48 h. As determined by measuring the absorbance by 280 nm of the vitellogeninanti-phosvitin complex, the accumulation of vitellogenin appeared to parallel that of plasma triglycerides. By measuring the phosphorus content of the antigen-antibody complex, there was no apparent accumulation of phosphoprotein between 0 and 8 h after DES injection. However, when the experiment was repeated with more plasma (0.5 ml), there was a clear increase in vitellogenin accumulation even after four hours of DES (Fig. 9). This increase was accompanied by a parallel increase in the phosphorus content of the immunoprecipitates. Discussion In the present study, a goat antibody was prepared against hen egg yolk phos-

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TIME AFTER INJECTION (HOURS)

FIG. 9. Effects of DES on plasma vitellogenin. Groups of five cockerels were treated with a single injection of DES (2.5 mg) and the animals were sacrificed, plasma collected and immunoprecipitates collected from 0.5 ml plasma as described in Fig. 8.

vitin. Only two other reports (24,25) in the literature have described the preparation of anti-phosvitin. However, in these studies, no attempt was made to characterize the antibody or to use it to measure phosphoprotein induction in the estrogeninduced cockerel. Deeley et al. (10) have attempted to prepare anti-phosvitin in rabbits and sheep but were unsuccessful even though they rendered phosvitin more immunogenic by a variety of different methods. The finding that anti-phosvitin precipitates vitellogenin from hen plasma adds further support to the notion (9,10) that phosvitin is an integral part of a higher molecular weight polypeptide, i.e., vitellogenin, in plasma. Most studies in the literature (3,10,2432) which are concerned with the induction of vitellogenin in the estrogen-induced cockerel have used antibodies raised against egg yolk lipovitellin. Using this antibody, Jost et al. (32) found a lag phase of about 12 h before there was any accumulation of vitellogenin in the plasma. A similar lag period was found by Bergink et al. (31). No information was available in the latter

study as to whether the anti-lipovitellin used by these investigators cross-reacted with vitellogenin. Bergink et al. (9) found only a 3 to 4 h lag period in vitellogenin synthesis. However, phosphoprotein was measured indirectly by labeling with 32P phosphate in vivo. An intriguing aspect of the present study was the finding that there was only a short lag in the accumulation of plasma vitellogenin as determined directly with the anti-phosvitin; prior to injection of DES, there was no detectable immunoprecipitable material using antiphosvitin, but by 4 h, a definite immunoprecipitate was obtained, accompanied by a parallel increase in the phosphorus content of the precipitate. Another interesting finding in this study was that the accumulation of plasma triglycerides coincided with that of vitellogenin. This observation suggests that estrogen induces the synthesis of VLDL and vitellogenin by a common mechanism. Acknowledgments L. Chan and R. L. Jackson are Established Investigators of the American Heart Association. A. R. Means is a Faculty Research Awardee of the American Cancer Society. We would like to thank Ms. Debbie Mason for typing the manuscript.

References 1. Bergink, E. W., H. J. Kloosterboer, M. Gruber, and G. Ab, Biochim Biophys Ada 294: 497, 1973. 2. Jost, J.-P., G. Pehling, and O. G. Baca, Biochem Biophys Res Commun 62: 957, 1975. 3. Bos, E. S., R. J. Vonk, M. Gruber, and G. Ab, FEBS Lett 24: 197, 1972. 4. Hillyard, L. A., C. Entenman, and I. L. Chaikoff, J Biol Chem 223: 359, 1956. 5. Luskey, K. L., M. S. Brown, and J. L. Goldstein, J Biol Chem 249: 5939, 1974. 6. Kudzma, D. J., P. M. Hegstad, and R. E. Stoll, Metab Clin Exp 22: 423, 1973. 7. Schjeide, O. A., and M. R. Urist, Science 124: 1242, 1956. 8. Chan, L., R. L. Jackson, B. W. O'Malley, and A. R. MeansJ Clin Invest 58: 368, 1976. 9. Bergink, E. W., R. A. Wallace, J. A. van de Berg, E. S. Bos, M. Gruber, and G. Ab, Am Zool 14: 1177, 1974.

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DES INDUCTION OF COCKEREL VITELLOGENIN 10. Deeley, R. G., K. P. iMullinix, W. Wetekam, H. M. Kronenberg, M. Meyers, J. D. Eldridge, and R. F. Goldberger,/ Biol Chem 250: 9060, 1975. 11. Shainkin, R., and G. E. Perlman, J Biol Chem 246: 2278, 1971. 12. Shainkin, R., and G. E. Perlman, Arch Biochem Biophys 145: 693, 1971. 13. Clark, R. C., Biochim Biophys Acta 310: 174, 1973. 14. Clark, R. C , and F. J. Joubert, FEBS Lett 13: 225, 1971. 15. Clark, R. C , Biochem J 118: 537, 1970. 16. Bernardi, G., and W. H. Cook, Biochim Biophys Acta 44: 96, 1960. 17. Beuving, G., and M. Gruber, Biochim Biophys Acta 232: 524, 1971. 18. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. RandallJ Biol Chem 193: 265, 1951. 19. Bartlett, G. R . J Biol Chem 234: 466, 1959. 20. Jackson, R. L., H.-Y. Lin, L. Chan, and A. Means, Biochim Biophys Acta 420: 342, 1976. 21. Manual of Laboratory Methods, Lipid Research Clinics Program, University of North Carolina, Chapel Hill, N.C., 1972.

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22. Ouchterlony, O., In Ackroyd, J. F. (ed.), Immunological Methods, Backwell, Oxford, 1964, p. 55. 23. Laurell, C. B., Anal Biochem 15: 45, 1966. 24. Carinci, P., C. E. Grossi, and P. Locci,/ Embryol Morph 29: 713, 1973. 25. Carinci, P., P. Locci, M. A. Bodo, and A. Caruso, Experientia 88: 300, 1974. 26. Mullinix, K. P., W. Wetekam, R. G. Deeley, J. I. Gordon, M. Meyers, K. A. Kent, and R. F. Goldberger, Proc Natl Acad Sci USA 73: 1442, 1976. 27. Jost, J.-P., and G. Pehling, Eur J Biochem 62: 299, 1976. 28. Jost, J.-P., and G. Pehling, Eur] Biochem 66: 399, 1976. 29. Wetekam, W., K. P. Mullinix, R. G. Deeley, H. M. Kronenberg, j . D. Eldridge, M. Meyers, and R. F. Goldberger, Proc Natl Acad Sci USA 72: 3364, 1975. 30. Ab, G., W. G. Roskam, J. Dijkstra, J. Mulder, M. Willems, A. van der Ende, and M. Gruber, Biochim Biophys Acta 454: 67, 1976. 31. Bergink, E. W., H. J. Kloosterboer, M. Gruber, and G. Ab, Biochim Biophys Acta 294: 497, 1973. 32. Jost, J.-P., R. Keller, and C. Dierks-Ventling, J Biol Chem 248: 5262, 1973.

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Estrogen induction of plasma vitellogenin in the cockerel: studies with a phosvitin antibody.

Estrogen Induction of Plasma Vitellogenin in the Cockerel: Studies with a Phosvitin Antibody RICHARD L. JACKSON, HU-YU LIN, J. T. SIMON MAO, LAWRENCE...
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