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Biological tests on eel “prolactin” separated by gel electrophoresis
GENERAL
AND
COMPARATIVE
Biological
Tests on Eel “Prolactin” by Gel Electrophoresis
P. J. KNIGHT,* *Department TDepartment
36, 30-32 (1978)
ENDOCRINOLOGY
A. CHADWICK,*J
of Pure and Applied of Zoology, University
Separated
AND J. N. BALL?
Zoology, University of Leeds, Leeds LS2 9JT, and of Shef$eld, Sheffield SIO 2TN, United Kingdom
Accepted May 10, 1978 Aqueous extracts of pituitary glands from freshwater eels were subjected to disc electrophoresis. The gels were cut into sections, and the proteins in each section were eluted and tested for prolactin-like activity using the crop-sac test in the pigeon and a xanthophore dispersion test in the goby. In both tests prolactin activity was found associated with the fastest moving of the -major protein bands. in 7% acetic acid which caused it to shrink back to its original size. The frozen gels were placed one by one alongside the reference gel. The origin and the ion front were clearly visible in the frozen gels and served to position them while they were quickly cut into five sections. Homologous sections were pooled and homogenized in 2 ml of distilled water. The homogenates were filtered, the gel particles were rinsed, and refiltered, and the filtrates were combined for each fraction. The solutions were dialyzed overnight against distilled water and lyophilized. Two tests were used to detect prolactin-like activity, the pigeon crop-sac response to local, intradermal injections (Lyons and Page, 1935) and the xanthophore response in the fish Gobius minutus. The lyophilized fractions were dissolved in a drop of 0.1 N NaOH, neutralized with HCl, and made up to 300 ~1 with 0.9% saline. Three injections of 50 ~1 were given to pigeons at 4-hr intervals on two successive days. Each fraction was injected into two different birds. On the third day the birds were killed, and the results were assessed visually and then by using frozen sections stained for lipid and wax sections, The xanthophore test was a modification of that described by Sage and Bern (1972) for the goby Gillichthys mirabilis. Twenty-microliter aliquots were injected into 3. to 5-cm-long fish. Two different fish were used to test each fraction, the color change over the whole body being observed and recorded at lo-min intervals for a period of 1 hr.
It has previously been reported that disc electrophoresis of eel (Angdlu mzgzda) pituitary homogenates yields three major protein bands (Knight et al., 1970). When the rostra1 and proximal regions of the pars distalis were treated separately the fastest moving protein was associated primarily with the rostra1 region. This region contains mainly one type of acidophil cell, the eta cell, which probably secretes prolactin (Ball and Baker, 1969). Preliminary experiments suggested that the fast moving band was prolactin, and the present report describes further experiments to investigate this suggestion. MATERIALS
AND METHODS
Freshwater eels were obtained from the River Severn and maintained in running tap-water. Whole pituitary glands were removed from freshly killed eels, and 12 mg were homogenized in 300 ~1 of 0.6% saline. Fifty-microliter aliquots were subjected to disc electrophoresis in polyacrylamide gel columns essentially in the manner described by Davies (1964). On completion of the run the gels were removed from the glass tubes under distilled water, and all except one were frozen immediately with liquid nitrogen and stored at -2V. The remaining gel was stained with amido black. After destaining in 7% acetic the reference gel was placed in a solution of 30% ehtyl alcohol 1 Author dressed.
RESULTS
Figure la shows the appearance of a typical gel after electrophoretic separation of the proteins in 50 ~1 of eel pituitary
to whom reprint requests should be ad30
0016-6480/78/0361-0030$01.00/0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROLACTIN
1 FIG.
fractions
23
IN
4
THE
3i
EEL
5
1. (a] Proteins of the eel pituitary gland after separation on acrylamide. The gels were cut into l-5 as indicated. (bj The prolactin band is marked . Material from fraction 4 after electrophoresis.
“F’.
homogenate. The five fractions tested in the pigeon and the goby are also indicated. Fraction 4 was effective in producing cropsac proliferation in the pigeon although lipid droplets were absent from the cells. The other fractions had no stimulating effect on the crop-sac epithelium. Figure lb shows that only one protein, with an Rf of 0.46, was visible when an aliquot of fraction 4 was subjected to gel electrophoresis under the same conditions. In the goby test an ip injection of an homogenate of 60 pg wet weight of fresh goby pituitary induced the diffuse orange skin coloration, which is the end point of the test, within IO min. Each goby received the equivalent of 2 mg wet weight of eel pituitary gland. Table 1 shows that fraction 4 was the most potent although some activity was seen in fractions 3 and 5. DISCUSSION
Observations reported earlier concerning the electrophoretic mobilities of eel pituitary proteins have been confirmed (Knight et al., 1970). These workers suggested that circumstantial evidence pointed strongly to one of the bands being prolactin. The results of the pigeon and the goby tests reported here suggest that the protein band with an electrophoretic mobility of Rf 0.46 represents eel prolactin. Nagahama et a/. (1975) observed that the mobilities reported
TABLE XANTHOPHORE DISPERSION FOLLOWING INJECTION EEL PITUITARY
1 IN Gobius r6u~tu.x OE EXTWCT~ 0~ GLANDS
Fraction
Scorea
1 2 3 4 5
0.0 0.0 0.5 2.5 0.5
~-
-n Fractions were tested on two fish, and responses were assessed as follows: 0, no effect; I, very siight increase in orange color; 2, marked increase in orange color; 3, very striking increase in orange color.
for supposed teleost prolactins varied between Rf 0.12 and 0.80, but that they were usually lower than those of mammaiian prolactins. Ingleton et al. (1973) used the band Rf 0.46 after electrophoresis of eel i?z V~PQ pituitary incubations to demonstrate the reciprocal relationship between prolactin secretion and osmotic pressure of the medium in this species. The pigeon crop test &es appear to be specific for prolactin even though the response obtained with lower vertebrate preparations is atypical @Go11 and Bern, 1968; Chadwick, 1970). As the areas of gel adjacent to Rf 0.46 faiIed to show positive in the crop-sac test, it is unlikely that a false positive, such as those
32
KNIGHT,
CHADWICK,
discussed by Bahn and Bates (1956) and Nicoll and Bern (1964) is the explanation for the prolactin-like activity apparent in fraction 4. Doubts have been raised recently as to the specificity of the goby assay for prolactin (Farmer et ul., 1975). However, under the conditions of electrophoresis employed in these experiments only relatively simple protein hormones such as prolactin and growth hormone are likely to be present in the gel and so able to account for the responses observed. The results of the two tests taken together strongly support the suggestion that the electrophoretic band with a mean of Rf0.46 represents eel prolactin. REFERENCES Bahn, R. C., and Bates, R. W. (1956). Histologic criteria for detection of prolactin: Lack of prolactin in blood and mine of human subjects. .Z. C&r. Endocrinol. 16, 1137-1345. Ball, J. N., and Baker, B. I. (1969). Zn “Fish PhysioIogy” (W. S. Hoar and D. J. Randall, eds.), Vol. 2, pp. l-110. Academic Press, New York. Chadwick, A. (1970). Pigeon crop sac-stimulating activity in the pituitary of the flounder (Pleuronectes flesus). J. Endocrinol. 47, 463-469. Davies, B. J. (1964). Disc electrophoresis. II. Method
AND
BALL
and application to human seturn samples. Ann. N.Y. Acad. Sri. 121, 404-427. Farmer, S. W., Clark, W. C., Papkoff, H., Nishioka, R. S., Bern, H. A., and Li, C. H. (1975). Studies on the purificatio,n and properties of teleost prolactin. L$e Sci. 16, 149-158. Ingleton, P. M., Baker, B. I., and Ball, J. N. (1973). Secretion of prolactin and growth hormone by teleost pituitaries m vitro. 1. Effects of sodium concentration and osmotic pressure during shortterm incubations. J. Comp. Physiol. 87, 317-328 Knight, P. J., Ingleton, P. M., Ball, J. N., and Hancock, M. P. (1970). Separation and identification of eel prolactin by disc electrophoresis. J. Endocrinol. 48, Proceedings XXIX-XXXI. Lyons, W. R., and Page, E. (1935). Detection of mammotrophin in the urine of lactating women. Proc. Sot. Exp. Biol. Med. 32, 1049-1050. Nagahama, G., Nishioki, R. S., Bern, H. A., and Gunther, R. L. (1975). Control of prolactin secretion in teleosts, with special reference to G% lichthys mirabilis ion Tilapia mossambica. Gen. Comp. Endocrinol. 25, 166-168. Nicoll, C. S., and Bern, H. A, (1964). “Prolactin” and the pituitary glands of fishes. Gen. Comp. Endocrinol. 4, 457-471. Nicoll, C. S., and Bern, H. A. (1968). The comparative endocrinology of prolactin. Recent Progr. Horm. Res. 24, 681-720. Sage, M., and Bern, H. A. (1972). Assay of prolactin in vertebrate pituitaries by its dispersion of xanthophore pigment in the teleost Gillichthys mirabilis. .I. Exp. 2001. 180, 169-174.
AND
COMPARATIVE
Biological
Tests on Eel “Prolactin” by Gel Electrophoresis
P. J. KNIGHT,* *Department TDepartment
36, 30-32 (1978)
ENDOCRINOLOGY
A. CHADWICK,*J
of Pure and Applied of Zoology, University
Separated
AND J. N. BALL?
Zoology, University of Leeds, Leeds LS2 9JT, and of Shef$eld, Sheffield SIO 2TN, United Kingdom
Accepted May 10, 1978 Aqueous extracts of pituitary glands from freshwater eels were subjected to disc electrophoresis. The gels were cut into sections, and the proteins in each section were eluted and tested for prolactin-like activity using the crop-sac test in the pigeon and a xanthophore dispersion test in the goby. In both tests prolactin activity was found associated with the fastest moving of the -major protein bands. in 7% acetic acid which caused it to shrink back to its original size. The frozen gels were placed one by one alongside the reference gel. The origin and the ion front were clearly visible in the frozen gels and served to position them while they were quickly cut into five sections. Homologous sections were pooled and homogenized in 2 ml of distilled water. The homogenates were filtered, the gel particles were rinsed, and refiltered, and the filtrates were combined for each fraction. The solutions were dialyzed overnight against distilled water and lyophilized. Two tests were used to detect prolactin-like activity, the pigeon crop-sac response to local, intradermal injections (Lyons and Page, 1935) and the xanthophore response in the fish Gobius minutus. The lyophilized fractions were dissolved in a drop of 0.1 N NaOH, neutralized with HCl, and made up to 300 ~1 with 0.9% saline. Three injections of 50 ~1 were given to pigeons at 4-hr intervals on two successive days. Each fraction was injected into two different birds. On the third day the birds were killed, and the results were assessed visually and then by using frozen sections stained for lipid and wax sections, The xanthophore test was a modification of that described by Sage and Bern (1972) for the goby Gillichthys mirabilis. Twenty-microliter aliquots were injected into 3. to 5-cm-long fish. Two different fish were used to test each fraction, the color change over the whole body being observed and recorded at lo-min intervals for a period of 1 hr.
It has previously been reported that disc electrophoresis of eel (Angdlu mzgzda) pituitary homogenates yields three major protein bands (Knight et al., 1970). When the rostra1 and proximal regions of the pars distalis were treated separately the fastest moving protein was associated primarily with the rostra1 region. This region contains mainly one type of acidophil cell, the eta cell, which probably secretes prolactin (Ball and Baker, 1969). Preliminary experiments suggested that the fast moving band was prolactin, and the present report describes further experiments to investigate this suggestion. MATERIALS
AND METHODS
Freshwater eels were obtained from the River Severn and maintained in running tap-water. Whole pituitary glands were removed from freshly killed eels, and 12 mg were homogenized in 300 ~1 of 0.6% saline. Fifty-microliter aliquots were subjected to disc electrophoresis in polyacrylamide gel columns essentially in the manner described by Davies (1964). On completion of the run the gels were removed from the glass tubes under distilled water, and all except one were frozen immediately with liquid nitrogen and stored at -2V. The remaining gel was stained with amido black. After destaining in 7% acetic the reference gel was placed in a solution of 30% ehtyl alcohol 1 Author dressed.
RESULTS
Figure la shows the appearance of a typical gel after electrophoretic separation of the proteins in 50 ~1 of eel pituitary
to whom reprint requests should be ad30
0016-6480/78/0361-0030$01.00/0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROLACTIN
1 FIG.
fractions
23
IN
4
THE
3i
EEL
5
1. (a] Proteins of the eel pituitary gland after separation on acrylamide. The gels were cut into l-5 as indicated. (bj The prolactin band is marked . Material from fraction 4 after electrophoresis.
“F’.
homogenate. The five fractions tested in the pigeon and the goby are also indicated. Fraction 4 was effective in producing cropsac proliferation in the pigeon although lipid droplets were absent from the cells. The other fractions had no stimulating effect on the crop-sac epithelium. Figure lb shows that only one protein, with an Rf of 0.46, was visible when an aliquot of fraction 4 was subjected to gel electrophoresis under the same conditions. In the goby test an ip injection of an homogenate of 60 pg wet weight of fresh goby pituitary induced the diffuse orange skin coloration, which is the end point of the test, within IO min. Each goby received the equivalent of 2 mg wet weight of eel pituitary gland. Table 1 shows that fraction 4 was the most potent although some activity was seen in fractions 3 and 5. DISCUSSION
Observations reported earlier concerning the electrophoretic mobilities of eel pituitary proteins have been confirmed (Knight et al., 1970). These workers suggested that circumstantial evidence pointed strongly to one of the bands being prolactin. The results of the pigeon and the goby tests reported here suggest that the protein band with an electrophoretic mobility of Rf 0.46 represents eel prolactin. Nagahama et a/. (1975) observed that the mobilities reported
TABLE XANTHOPHORE DISPERSION FOLLOWING INJECTION EEL PITUITARY
1 IN Gobius r6u~tu.x OE EXTWCT~ 0~ GLANDS
Fraction
Scorea
1 2 3 4 5
0.0 0.0 0.5 2.5 0.5
~-
-n Fractions were tested on two fish, and responses were assessed as follows: 0, no effect; I, very siight increase in orange color; 2, marked increase in orange color; 3, very striking increase in orange color.
for supposed teleost prolactins varied between Rf 0.12 and 0.80, but that they were usually lower than those of mammaiian prolactins. Ingleton et al. (1973) used the band Rf 0.46 after electrophoresis of eel i?z V~PQ pituitary incubations to demonstrate the reciprocal relationship between prolactin secretion and osmotic pressure of the medium in this species. The pigeon crop test &es appear to be specific for prolactin even though the response obtained with lower vertebrate preparations is atypical @Go11 and Bern, 1968; Chadwick, 1970). As the areas of gel adjacent to Rf 0.46 faiIed to show positive in the crop-sac test, it is unlikely that a false positive, such as those
32
KNIGHT,
CHADWICK,
discussed by Bahn and Bates (1956) and Nicoll and Bern (1964) is the explanation for the prolactin-like activity apparent in fraction 4. Doubts have been raised recently as to the specificity of the goby assay for prolactin (Farmer et ul., 1975). However, under the conditions of electrophoresis employed in these experiments only relatively simple protein hormones such as prolactin and growth hormone are likely to be present in the gel and so able to account for the responses observed. The results of the two tests taken together strongly support the suggestion that the electrophoretic band with a mean of Rf0.46 represents eel prolactin. REFERENCES Bahn, R. C., and Bates, R. W. (1956). Histologic criteria for detection of prolactin: Lack of prolactin in blood and mine of human subjects. .Z. C&r. Endocrinol. 16, 1137-1345. Ball, J. N., and Baker, B. I. (1969). Zn “Fish PhysioIogy” (W. S. Hoar and D. J. Randall, eds.), Vol. 2, pp. l-110. Academic Press, New York. Chadwick, A. (1970). Pigeon crop sac-stimulating activity in the pituitary of the flounder (Pleuronectes flesus). J. Endocrinol. 47, 463-469. Davies, B. J. (1964). Disc electrophoresis. II. Method
AND
BALL
and application to human seturn samples. Ann. N.Y. Acad. Sri. 121, 404-427. Farmer, S. W., Clark, W. C., Papkoff, H., Nishioka, R. S., Bern, H. A., and Li, C. H. (1975). Studies on the purificatio,n and properties of teleost prolactin. L$e Sci. 16, 149-158. Ingleton, P. M., Baker, B. I., and Ball, J. N. (1973). Secretion of prolactin and growth hormone by teleost pituitaries m vitro. 1. Effects of sodium concentration and osmotic pressure during shortterm incubations. J. Comp. Physiol. 87, 317-328 Knight, P. J., Ingleton, P. M., Ball, J. N., and Hancock, M. P. (1970). Separation and identification of eel prolactin by disc electrophoresis. J. Endocrinol. 48, Proceedings XXIX-XXXI. Lyons, W. R., and Page, E. (1935). Detection of mammotrophin in the urine of lactating women. Proc. Sot. Exp. Biol. Med. 32, 1049-1050. Nagahama, G., Nishioki, R. S., Bern, H. A., and Gunther, R. L. (1975). Control of prolactin secretion in teleosts, with special reference to G% lichthys mirabilis ion Tilapia mossambica. Gen. Comp. Endocrinol. 25, 166-168. Nicoll, C. S., and Bern, H. A, (1964). “Prolactin” and the pituitary glands of fishes. Gen. Comp. Endocrinol. 4, 457-471. Nicoll, C. S., and Bern, H. A. (1968). The comparative endocrinology of prolactin. Recent Progr. Horm. Res. 24, 681-720. Sage, M., and Bern, H. A. (1972). Assay of prolactin in vertebrate pituitaries by its dispersion of xanthophore pigment in the teleost Gillichthys mirabilis. .I. Exp. 2001. 180, 169-174.