British Poultry Science
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Effect of progesterone on the magnum proteins during primary stimulation of chick oviduct V. K. Goel & B. C. Joshi To cite this article: V. K. Goel & B. C. Joshi (1978) Effect of progesterone on the magnum proteins during primary stimulation of chick oviduct, British Poultry Science, 19:5, 591-594, DOI: 10.1080/00071667808416518 To link to this article: http://dx.doi.org/10.1080/00071667808416518
Published online: 08 Nov 2007.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [University of Florida]
Date: 06 November 2015, At: 10:15
Br. Poult. Sci., 19: 591-594. 1978
Longman: printed in Great Britain
EFFECT OF PROGESTERONE ON THE MAGNUM PROTEINS DURING PRIMARY STIMULATION OF CHICK OVIDUCT V. K. GOEL AND B. C. JOSHI Indian Veterinary Research Institute, Izatnagar, U.P., India
Downloaded by [University of Florida] at 10:15 06 November 2015
Received for publication 25th August 1977
1. The effect of progesterone on the secretion of protein by the magnum of 5-d-old, female chicks was determined. 2. The supernatant prepared by centrifuging an homogenate of the magnum at 105 000g was found, by immunodiffusion, to contain an antigenic component which precipitated the antisera for conalbumin 1, conalbumin 2 and ovalbumin after 5 d treatment with progesterone: there was no reaction to ovomucoid, lysozyme and avidin antisera. 3. Disc-electrophoresis of the homogenate revealed two bands at the site of ovalbumin. 4. Incorporation of 3H-lysine into the magnum proteins of progesterone-treated chicks did not differ from that of controls. 5. The secretion available in the magnum may be only a transudate from the serum and not a true secretory product. Progesterone behaved qualitatively as oestrogen in this study although the action is much less pronounced and was delayed. INTRODUCTION
Progesterone, together with oestrogen, is involved in the control of the secretion of magnum proteins (albumen proteins) in fowl and is essential for the production of avidin (Hertz, 1950). O'Malley and McGuire (1968) and O'Malley et al. (1969) have suggested that the initiation of the synthesis of most albumen proteins is stimulated by oestrogen alone, progesterone being required only for avidin formation. Palmitter (1971), however, observed that progesterone caused an increase in the relative rate of synthesis of ovalbumin and conalbumin. Dorfman and Dorfman (1963) suggested that progesterone acts synergistically with oestrogen at some dose levels and antagonistically at others. Cox and Sauerwein (1970) found that progesterone alone can differentiate several types of cells. These findings have been supported by Palmitter and Wrenn (1971) but they failed to detect secretory granules as seen by the former authors. Further, no synthesis of ovalbumin was detectable after 2 or 5 d of progesterone treatment. In the present study it is shown that while progesterone alone appears to induce the synthesis of protein in the magnum, the presence of protein is probably 591
592
V. K. GOEL AND B. C. JOSHI
due to changes in the permeability of the cells thereby allowing plasma proteins to pass into the magnum.
Downloaded by [University of Florida] at 10:15 06 November 2015
MATERIALS AND
METHODS
Twenty White Leghorn female chicks aged 4 or 5 d were divided into two groups. The control group was injected subcutaneously with 0-2 ml olive oil daily. The experimental group was injected with 5 mg progesterone in 0-2 ml olive oil daily. The dose was chosen for maximal response and was the same as that used by O'Malley et al. (1967£). Five chicks from each group were killed 24 h after the second and the fifth injection respectively. Each magnum was disected out, homogenised in Tris-HGl buffer (pH 7-4) and the 105 000^ supernatant collected, freeze dried and reconstituted in 2 ml distilled water. The protein concentration was determined by the method of Lowry et al. (1951). Disc electrophoresis was done in polyacrylamide gels using 7-5% acrylamide and tris-glycine buffer (pH 8-6) (see Davis, 1964). The samples were tested for the presence of conalbumin 1, conalbumin 2, ovalbumin, ovomucoid, lysozyme and avidin by their respective antiserum on double immunodiffusion on Ouchterlony plates. The antigen was placed in the central well and the antisera in the peripheral wells. A minimum of 48 h at room temperature (20 to 29 °C) was allowed for patterns to develop. Incorporation of 3H lysine in the magnum proteins under the influence of progesterone
White Leghorn female chicks aged 4 to 5 d were divided into two groups of 15. Tht birds of the control group were injected with 0-2 ml olive oil daily for 5 d while the experimental birds received 5 mg progesterone dissolved in the olive oil. The chicks were killed 24 h after the last injection. Two hours before the birds were killed each chick received 10 /iCi 3H-lysine in 0-5 ml saline intraperitoneally. The magnum of each chick was dissected out and five of them were pooled, giving three replicates per group. The tissue was then homogenised in Tris-HCl buffer (pH 7-4) and was processed as detailed in the flow diagram shown in the Fig. Tissue homogenate
1
Add an equal volume of 20% trichloroacetic acid (TCA) Mix and centrifuge at 2000 rpm for 600s Wash precipirate twice with cold 10% TCA and centrifuge Wash residue with 10% TGA at 80 °: centrifuge for 600s
1
Repeat cold 10% TCA wash and centrifuge
i
E x t r a c t residue w i t h e t h a n o l a t 70 ° followed b y cold e t h a n o l : e t h e r ( 3 : 1 ) a n d cold ether
1
Centrifuge and dissolve residue in 1-1-5 ml of N NaOH FIG.—Flow diagram for the preparation of the magnum for analysis.
The total protein in the sample was determined on a sample (0-2 ml) by the method of Lowry et al. (1951). Another sample (0-5 ml) was added to 7 ml scintillation fluid (Bray, 1960) and its radioactivity determined using 0-5 ml NaOH and
PROGESTERONE AND MAGNUM PROTEINS
593
7 ml of scintillation fluid as a control. The correction for quenching was done by the channel ratio method. The specific radioactivity (DPM/mg protein) was then calculated. RESULTS
The 105 000# supernatant
Downloaded by [University of Florida] at 10:15 06 November 2015
Only after five injections were single precipitin lines found in the immunodiffusion plates. The lines were against conalbumin 1, conalbumin 2 and ovalbumin antisera. Disc electrophoresis yielded two bands at the site of ovalbumin bands on the 5th day. Incorporation of zH-lysine
The average specific radioactivity (DPM/mg protein) was 4 243 for the control group and 3 903 for the progesterone-injected group: the difference was not significant {P> 0-05). DISCUSSION
Five days stimulation with progesterone, 5 mg/d, caused the appearance of the first antigenic components for conalbumin 1, conalbumin 2 and ovalbumin as shown by immunodiffusion. Ovalbumin was also detected by disc electrophoresis after 5 d. These results conflict with those of Palmitter and Wrenn (1971) who failed to detect the presence of ovalbumin after 2 or 5 d of treatment with progesterone alone. Small amounts of avidin are known to occur in the unstimulated, immature chick given progesterone alone (Korenman and O'Malley, 1968; O'Malley et al., 1967a). O'Malley et al. (1971) showed the specific progesterone binding component to be present in the oviduct nuclei of oestrogen-treated chicks. Later, Spelsberg et al. (1971, 1972) showed that the presence of progesterone-binding sites in the oviduct was not dependent on oestrogen stimulation. This indicates that progesterone is able to stimulate synthesis by itself. Cox and Sauerwein (1970) found that cytodifferentiation of the oviduct of 8-week-old females begins after 2-d treatment with progesterone even in the absence of oestrogen. Palmitter and Wrenn (1971) confirmed these findings and have propounded that cytodifferentiation proceeds to an intermediate stage and in directions other than the formation of mature tubular gland cells. Our results suggest that the action of progesterone in the first few days of primary stimulation may simply be to modify the permeability of the cells of the magnum thereby causing the albumen fractions to infiltrate into the magnum. This would account for the presence of one antigenic component of ovalbumin and conalbumin out of a possible three (Goel, 1975). The action of progesterone on cell permeability is delayed, compared with that of the oestrogens since it was not until the 5th day that the presence of egg white fraction was noted. Further support to our view comes from the experiment conducted on the incorporation of 3H-lysine into the magnum proteins of chicks treated with progesterone. No increase in
594
V. K. GOEL AND B. C. JOSHI
specific activity was noted indicating that the substance identified by immune-diffusion and gel electrophoresis was not the result of active secretion but was merely a product which had migrated from the plasma. ACKNOWLEDGEMENTS
The authors thank Dr N. K. Bhattacharyya and Dr B. Panda for their interest in the work. They also thank the Director of the Indian Veterinary Research Institutes, Izatnagar, for providing the laboratory facilities.
Downloaded by [University of Florida] at 10:15 06 November 2015
REFERENCES BRAY, G. A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Annals of Biochemistry and Experimental Medicine, 1: 279-285. Cox, R. F. AND SAUERWEIN, H. (1970). Studies on the mode of action of progesterone on chicken oviduct epithelium: I. Morphological changes associated with early differentiation of the tissue. Experimental Cell Research, 6 1 : 79-90. DAVIS,
B. J. (1964). Disc electrophoresis. II. Method and application to human serum proteins.
Annals of the New York Academy of Sciences, 121: 404-427. DORFMAN, R. I. AND DORFMAN, A. S. (1963). Steroids, 1: 528-531. GOEL, V. K. (1975). Hormonal control on the secretion of magnum proteins in fowl. Ph.D. Thesis, Agra University, India. HERTZ, R. (1950). Endocrine and vitamin factors in hormone induced tissue growth. Texas Reports on Biology and Medicine, 8: 154-158. KORENMAN, S. G. AND O'MALLEY, B. W. (1968). Progesterone action: Regulation of avidin biosynthesis by hen oviduct in vivo and in vitro. Endocrinology, 83: 11-17. LOWRY, O. H., ROSEBROUGH, J., FARR, A. L. AND RANDALL, R. J . (1951).
Protein measurement
with the folin phenol reagent. Journal of Biological Chemistry, 193: 265-268. O'MALLEY, B. W. AND MCGUIRE, W. L. (1968). Studies on the mechanism of action of progesterone in regulation of the synthesis of specific protein. Journal of Clinical Investigation, 47: 654-664. O'MALLEY, B. W., MCGUIRE, W. L. AND KORENMAN, S. G. (1967a). Estrogen stimulation of syn-
thesis of specific protein and RNA polymerase activity in the immature chick oviduct. Biochemica et Biophysica Acta, 145: 204-207. O'MALLEY, B. W., MCGUIRE, W. L. AND MIDDLETON, P. A. (19674). Structure function relationship
of various steroids relative to induction of specific oviduct protein (avidin). Endocrinology, 8 1 : 677-678. O'MALLEY, B. W., MCGUIRE, W. L., KOHLER, P. O. AND KORENMAN, S. G. (1969).
Studies on the
mechanism of steroid hormone regulation of synthesis of specific protein. Recent Progress in Hormone Research, 25: 105-160. O'MALLEY, B. W., TOFT, D. O. AND SHERMAN, M. R. (1971). Progesterone binding components of
chick oviduct. I I . Nuclear components. Journal of Biological Chemistry, 246: 1117-1122. PALMITTER, R. D. (1971). Interaction of estrogen, progesterone and testosterone in the regulation of protein synthesis in chick oviduct. Biochemistry 10: 4399-5404. PALMITTER, R. D. AND WRENN, J . T. (1971). Interaction of estrogen and progesterone in chick oviduct development. I I I . Tubular gland cell cytodifferentiation. Journal of Cell Biology, 50: 598-615. SPELSBERG, T. C , STEGGLES, A. W. AND O'MALLEY, B. W. (1971).
Progesterone binding com-
ponents of chick oviduct. I I I . Chromatin acceptor sites. Journal of Biological Chemistry, 246: 4188-4197. SPELSBERG, T. C , STEGGLES, A. W., CHYTIL, F. AND O'MALLEY, B. W. (1972). Progesterone binding
components of chick oviduct. V. Exchange of progesterone binding capacity from target to non-target tissue chromatins. Journal of Biological Chemistry, 247: 1368-1374.
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Effect of progesterone on the magnum proteins during primary stimulation of chick oviduct V. K. Goel & B. C. Joshi To cite this article: V. K. Goel & B. C. Joshi (1978) Effect of progesterone on the magnum proteins during primary stimulation of chick oviduct, British Poultry Science, 19:5, 591-594, DOI: 10.1080/00071667808416518 To link to this article: http://dx.doi.org/10.1080/00071667808416518
Published online: 08 Nov 2007.
Submit your article to this journal
Article views: 8
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [University of Florida]
Date: 06 November 2015, At: 10:15
Br. Poult. Sci., 19: 591-594. 1978
Longman: printed in Great Britain
EFFECT OF PROGESTERONE ON THE MAGNUM PROTEINS DURING PRIMARY STIMULATION OF CHICK OVIDUCT V. K. GOEL AND B. C. JOSHI Indian Veterinary Research Institute, Izatnagar, U.P., India
Downloaded by [University of Florida] at 10:15 06 November 2015
Received for publication 25th August 1977
1. The effect of progesterone on the secretion of protein by the magnum of 5-d-old, female chicks was determined. 2. The supernatant prepared by centrifuging an homogenate of the magnum at 105 000g was found, by immunodiffusion, to contain an antigenic component which precipitated the antisera for conalbumin 1, conalbumin 2 and ovalbumin after 5 d treatment with progesterone: there was no reaction to ovomucoid, lysozyme and avidin antisera. 3. Disc-electrophoresis of the homogenate revealed two bands at the site of ovalbumin. 4. Incorporation of 3H-lysine into the magnum proteins of progesterone-treated chicks did not differ from that of controls. 5. The secretion available in the magnum may be only a transudate from the serum and not a true secretory product. Progesterone behaved qualitatively as oestrogen in this study although the action is much less pronounced and was delayed. INTRODUCTION
Progesterone, together with oestrogen, is involved in the control of the secretion of magnum proteins (albumen proteins) in fowl and is essential for the production of avidin (Hertz, 1950). O'Malley and McGuire (1968) and O'Malley et al. (1969) have suggested that the initiation of the synthesis of most albumen proteins is stimulated by oestrogen alone, progesterone being required only for avidin formation. Palmitter (1971), however, observed that progesterone caused an increase in the relative rate of synthesis of ovalbumin and conalbumin. Dorfman and Dorfman (1963) suggested that progesterone acts synergistically with oestrogen at some dose levels and antagonistically at others. Cox and Sauerwein (1970) found that progesterone alone can differentiate several types of cells. These findings have been supported by Palmitter and Wrenn (1971) but they failed to detect secretory granules as seen by the former authors. Further, no synthesis of ovalbumin was detectable after 2 or 5 d of progesterone treatment. In the present study it is shown that while progesterone alone appears to induce the synthesis of protein in the magnum, the presence of protein is probably 591
592
V. K. GOEL AND B. C. JOSHI
due to changes in the permeability of the cells thereby allowing plasma proteins to pass into the magnum.
Downloaded by [University of Florida] at 10:15 06 November 2015
MATERIALS AND
METHODS
Twenty White Leghorn female chicks aged 4 or 5 d were divided into two groups. The control group was injected subcutaneously with 0-2 ml olive oil daily. The experimental group was injected with 5 mg progesterone in 0-2 ml olive oil daily. The dose was chosen for maximal response and was the same as that used by O'Malley et al. (1967£). Five chicks from each group were killed 24 h after the second and the fifth injection respectively. Each magnum was disected out, homogenised in Tris-HGl buffer (pH 7-4) and the 105 000^ supernatant collected, freeze dried and reconstituted in 2 ml distilled water. The protein concentration was determined by the method of Lowry et al. (1951). Disc electrophoresis was done in polyacrylamide gels using 7-5% acrylamide and tris-glycine buffer (pH 8-6) (see Davis, 1964). The samples were tested for the presence of conalbumin 1, conalbumin 2, ovalbumin, ovomucoid, lysozyme and avidin by their respective antiserum on double immunodiffusion on Ouchterlony plates. The antigen was placed in the central well and the antisera in the peripheral wells. A minimum of 48 h at room temperature (20 to 29 °C) was allowed for patterns to develop. Incorporation of 3H lysine in the magnum proteins under the influence of progesterone
White Leghorn female chicks aged 4 to 5 d were divided into two groups of 15. Tht birds of the control group were injected with 0-2 ml olive oil daily for 5 d while the experimental birds received 5 mg progesterone dissolved in the olive oil. The chicks were killed 24 h after the last injection. Two hours before the birds were killed each chick received 10 /iCi 3H-lysine in 0-5 ml saline intraperitoneally. The magnum of each chick was dissected out and five of them were pooled, giving three replicates per group. The tissue was then homogenised in Tris-HCl buffer (pH 7-4) and was processed as detailed in the flow diagram shown in the Fig. Tissue homogenate
1
Add an equal volume of 20% trichloroacetic acid (TCA) Mix and centrifuge at 2000 rpm for 600s Wash precipirate twice with cold 10% TCA and centrifuge Wash residue with 10% TGA at 80 °: centrifuge for 600s
1
Repeat cold 10% TCA wash and centrifuge
i
E x t r a c t residue w i t h e t h a n o l a t 70 ° followed b y cold e t h a n o l : e t h e r ( 3 : 1 ) a n d cold ether
1
Centrifuge and dissolve residue in 1-1-5 ml of N NaOH FIG.—Flow diagram for the preparation of the magnum for analysis.
The total protein in the sample was determined on a sample (0-2 ml) by the method of Lowry et al. (1951). Another sample (0-5 ml) was added to 7 ml scintillation fluid (Bray, 1960) and its radioactivity determined using 0-5 ml NaOH and
PROGESTERONE AND MAGNUM PROTEINS
593
7 ml of scintillation fluid as a control. The correction for quenching was done by the channel ratio method. The specific radioactivity (DPM/mg protein) was then calculated. RESULTS
The 105 000# supernatant
Downloaded by [University of Florida] at 10:15 06 November 2015
Only after five injections were single precipitin lines found in the immunodiffusion plates. The lines were against conalbumin 1, conalbumin 2 and ovalbumin antisera. Disc electrophoresis yielded two bands at the site of ovalbumin bands on the 5th day. Incorporation of zH-lysine
The average specific radioactivity (DPM/mg protein) was 4 243 for the control group and 3 903 for the progesterone-injected group: the difference was not significant {P> 0-05). DISCUSSION
Five days stimulation with progesterone, 5 mg/d, caused the appearance of the first antigenic components for conalbumin 1, conalbumin 2 and ovalbumin as shown by immunodiffusion. Ovalbumin was also detected by disc electrophoresis after 5 d. These results conflict with those of Palmitter and Wrenn (1971) who failed to detect the presence of ovalbumin after 2 or 5 d of treatment with progesterone alone. Small amounts of avidin are known to occur in the unstimulated, immature chick given progesterone alone (Korenman and O'Malley, 1968; O'Malley et al., 1967a). O'Malley et al. (1971) showed the specific progesterone binding component to be present in the oviduct nuclei of oestrogen-treated chicks. Later, Spelsberg et al. (1971, 1972) showed that the presence of progesterone-binding sites in the oviduct was not dependent on oestrogen stimulation. This indicates that progesterone is able to stimulate synthesis by itself. Cox and Sauerwein (1970) found that cytodifferentiation of the oviduct of 8-week-old females begins after 2-d treatment with progesterone even in the absence of oestrogen. Palmitter and Wrenn (1971) confirmed these findings and have propounded that cytodifferentiation proceeds to an intermediate stage and in directions other than the formation of mature tubular gland cells. Our results suggest that the action of progesterone in the first few days of primary stimulation may simply be to modify the permeability of the cells of the magnum thereby causing the albumen fractions to infiltrate into the magnum. This would account for the presence of one antigenic component of ovalbumin and conalbumin out of a possible three (Goel, 1975). The action of progesterone on cell permeability is delayed, compared with that of the oestrogens since it was not until the 5th day that the presence of egg white fraction was noted. Further support to our view comes from the experiment conducted on the incorporation of 3H-lysine into the magnum proteins of chicks treated with progesterone. No increase in
594
V. K. GOEL AND B. C. JOSHI
specific activity was noted indicating that the substance identified by immune-diffusion and gel electrophoresis was not the result of active secretion but was merely a product which had migrated from the plasma. ACKNOWLEDGEMENTS
The authors thank Dr N. K. Bhattacharyya and Dr B. Panda for their interest in the work. They also thank the Director of the Indian Veterinary Research Institutes, Izatnagar, for providing the laboratory facilities.
Downloaded by [University of Florida] at 10:15 06 November 2015
REFERENCES BRAY, G. A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Annals of Biochemistry and Experimental Medicine, 1: 279-285. Cox, R. F. AND SAUERWEIN, H. (1970). Studies on the mode of action of progesterone on chicken oviduct epithelium: I. Morphological changes associated with early differentiation of the tissue. Experimental Cell Research, 6 1 : 79-90. DAVIS,
B. J. (1964). Disc electrophoresis. II. Method and application to human serum proteins.
Annals of the New York Academy of Sciences, 121: 404-427. DORFMAN, R. I. AND DORFMAN, A. S. (1963). Steroids, 1: 528-531. GOEL, V. K. (1975). Hormonal control on the secretion of magnum proteins in fowl. Ph.D. Thesis, Agra University, India. HERTZ, R. (1950). Endocrine and vitamin factors in hormone induced tissue growth. Texas Reports on Biology and Medicine, 8: 154-158. KORENMAN, S. G. AND O'MALLEY, B. W. (1968). Progesterone action: Regulation of avidin biosynthesis by hen oviduct in vivo and in vitro. Endocrinology, 83: 11-17. LOWRY, O. H., ROSEBROUGH, J., FARR, A. L. AND RANDALL, R. J . (1951).
Protein measurement
with the folin phenol reagent. Journal of Biological Chemistry, 193: 265-268. O'MALLEY, B. W. AND MCGUIRE, W. L. (1968). Studies on the mechanism of action of progesterone in regulation of the synthesis of specific protein. Journal of Clinical Investigation, 47: 654-664. O'MALLEY, B. W., MCGUIRE, W. L. AND KORENMAN, S. G. (1967a). Estrogen stimulation of syn-
thesis of specific protein and RNA polymerase activity in the immature chick oviduct. Biochemica et Biophysica Acta, 145: 204-207. O'MALLEY, B. W., MCGUIRE, W. L. AND MIDDLETON, P. A. (19674). Structure function relationship
of various steroids relative to induction of specific oviduct protein (avidin). Endocrinology, 8 1 : 677-678. O'MALLEY, B. W., MCGUIRE, W. L., KOHLER, P. O. AND KORENMAN, S. G. (1969).
Studies on the
mechanism of steroid hormone regulation of synthesis of specific protein. Recent Progress in Hormone Research, 25: 105-160. O'MALLEY, B. W., TOFT, D. O. AND SHERMAN, M. R. (1971). Progesterone binding components of
chick oviduct. I I . Nuclear components. Journal of Biological Chemistry, 246: 1117-1122. PALMITTER, R. D. (1971). Interaction of estrogen, progesterone and testosterone in the regulation of protein synthesis in chick oviduct. Biochemistry 10: 4399-5404. PALMITTER, R. D. AND WRENN, J . T. (1971). Interaction of estrogen and progesterone in chick oviduct development. I I I . Tubular gland cell cytodifferentiation. Journal of Cell Biology, 50: 598-615. SPELSBERG, T. C , STEGGLES, A. W. AND O'MALLEY, B. W. (1971).
Progesterone binding com-
ponents of chick oviduct. I I I . Chromatin acceptor sites. Journal of Biological Chemistry, 246: 4188-4197. SPELSBERG, T. C , STEGGLES, A. W., CHYTIL, F. AND O'MALLEY, B. W. (1972). Progesterone binding
components of chick oviduct. V. Exchange of progesterone binding capacity from target to non-target tissue chromatins. Journal of Biological Chemistry, 247: 1368-1374.
