GENERAL
AND
Isoelectric
COMPARATIVE
(1979)
Focusing and Gel Filtration Studies on the Heterogeneity of Avian Pituitary Luteinizing Hormone MASAAKI
Hormone
39,215-221
ENDOCRINOLOGY
Assay
Center,
HATTORI
Institute
AND KATSUMI
of Endocrinology,
Gunma
WAKABAYASHI University,
Maebashi,
371, Japan
Accepted June 7, 1979 The heterogeneity of avian pituitary LH was shown by means of isoelectric focusing and gel filtration studies coupled with radioimmunoassay. The immunoreactive LH (IR-LH) in anterior pituitary extracts from male quail and chickens contained four components with isoelectric points of 9.8 (component I), 9.4 (component II), 9.0 (component III), and 8.5 (component IV). All IR-LH components possessed the ability to bind to rat ovarian LH receptors but the estimates of potency obtained by the radioreceptor assay did not agree with those from radioimmunoassay. Sephadex G-100 gel filtration of the four individual components obtained from chicken pituitary LH indicated the presence of an LH with a molecular weight of 23,500-25,000 together with a component having double the molecular weight. The components differed in the relative amounts of the larger molecular weight component. The large component was also found in a gel filtration of the extract of chicken anterior pituitary glands. The amounts of the various components were changed in male quail by photostimulation. After 21 days of photostimulation, the amounts of all the components, especially of component I, had increased markedly.
Some investigators who have purified glycoprotein hormones have noticed a heterogeneity in their purified preparations. Working on luteinizing hormone (LH), Ward et al. (1959) obtained biologically active highly purified ovine LH preparations containing different amounts of glucosamine. Braselton and McShan (1970) purified equine LH and found four biologically active components after isoelectric focusing separation. Reichert (1971) examined the isoelectric points (pls) of a number of LH preparations and found that his human LH preparation had four components with different pZ values. Yoshida and Ishii (1973) also found three biologically active components with different pZs in a partially purified bovine LH preparation. In order to observe the heterogeneity of immunoreactive LH (IR-LH) in a state as natural as possible, one of the present authors (Wakabayashi) analyzed neutral saline extracts of rat anterior pituitaries by isoelectric focusing and measured IR-LH by radioimmunoassay (1977). He found that rat IR-LH in the anterior pituitary con-
tained seven components with different pls. Furthermore, the relative amounts of these components were changed by orchidectomy, estradiol benzoate or progesterone treatment. Individual components of LH have been also isolated from human (Roos et al., 1975), whale (Tamura-Takahashi and Ui, 1977), and bovine (Yora and Ui, 1978) pituitaries. In contrast, there have been no reports concerning the heterogeneity of avian LH. The present report first describes an isoelectric focusing study on avian LH and on the changes in the various components caused by photostimulation, and secondly gel filtration studies of these components for heterogeneity in molecular size. METHODS Male Japanese quail were purchased from a commercial breeder at 3 weeks of age and subjected to a photoperiod of 8L:16D (SD, 8 hr light and 16 hr darkness daily) for 2 weeks. Then, the birds were divided into two groups. One group was kept on a regime of SD, and the other group was transferred to a regime of 16L:8D(LD) on Day 0 (designated as SD 0 or LD 0). Birds.
Sampling for studies. After
isoelectric
focusing
and gel filtration
blood samples were collected from the
215 0016~6480/79/100215-07$01.00/O Copyright
0 1979 by Academic
Press, Inc.
216
HATTORI
AND
jugular vein on Days 0, 7, and 2 1, the birds were killed and the anterior pituitary glands immediately removed, pooled in 1 ml of phosphate-buffered saline (PBS), pH 7.5 (containing 0.01 M sodium phosphate, 0.14 M NaCl, and 0.01% merthiolate) and stored at -70” until examination by isoelectric focusing. The sera were stored at -30” until radioimmunoassay. The anterior pituitary glands of broiler chickens (about 1000 glands) were also collected at a slaughterhouse and stored at -70” until use. Radioimmunoassay. The radioimmunoassay was carried out by a double-antibody method with fraction IRC-2 (Gunma) as standard preparation (Hattori et al., 1975) and an anti-avian LH serum AL-MH#l. A further purified preparation, IEF-1, which was obtained by preparative isoelectric focusing of IRC-2, was used for radioiodination. The preparation IEF-1 had a single pZ of pH 9.8 and was free from IEF-2 which had low immunoreactivity, when assayed with the radioiodinated IEF-1 as labeled hormone. The iodination rate of IEF-1 was very high by chloramine-T method (Greenwood et al., 1963). The assay system consisted of 400 ~1 of 0.1% gelatin in PBS (Gel-PBS). 200 ~1 of standard or sample dissolved in 0.1% Gel-PBS, 100 ~1 of diluted antiserum (l:lOO,OOO) in 1% normal rabbit serum-O.05 M EDTA-PBS, and 100 ~1 of 1311-labeled hormone in 0.1% Gel-PBS. Before the addition of 13’1-LH, the preincubation was carried out at 4” overnight. A goat anti-rabbit y-globulin serum, H-4, prepared in our laboratory, was employed as the second antibody. Results were expressed as nanograms of the preparation IRC-2 (Gunma). Radioreceptor assay fur LH. The radioreceptor assay was performed according to Lee and Ryan (1973) using PMS- and HCG-primed rats (Parlow, 1961). “jI-labeled rat LH was prepared by radioiodination of NIAMDD Rat LH-I-4 (supplied by Rat Pituitary Hormone Distribution Program, NIAMDD, NIH) with lactoperoxidase according to the method of Miyachi et al. (1972), with minor modification. Nonspecific binding was determined by using excess unlabeled avian hormone (IRC-2 (Gunma), 20 pg). Results were expressed as nanograms of the preparation IRC-2 (Gunma). Isoelectric focusing. A preparative isoelectric focusing column, size 110 ml, of Kato Manufacturing Company, Osaka, Japan, was employed. As carrier ampholytes, Ampholines pH 9- 11, pH 7-9, and pH 5-7 (LKB Produkter) were mixed to make up a pH gradient from pH 7 to 11. For the stabilization of the isoelectric focusing solution, a sorbitol density gradient from 5 to 50% was employed. The anterior pituitary extracts were prepared by homogenizing the glands with PBS, followed by freezing and thawing and centrifugation at 15,000 rpm to remove insoluble fragments. The extracts of the anterior pituitaries of quail were added to the isoelectric focusing solution in an
WAKABAYASHI
amount equivalent to 20-25 glands; in the case of the broiler chickens, equivalents of 5 glands, were added. For preparation of the samples for Sephadex gel liltration and radioreceptor assay analyses, the extract of 500 glands of the anterior pituitaries of broiler chickens was applied to isoelectric focusing. Isoelectric focusing with the anode at the top of the column was run for 65-70 hr under cooling at 2-4”, the voltage applied being increased stepwise from 400 to 1450 V. After focusing, the solution was eluted at a flow rate of 30 ml/hr, and 2.5-ml fractions were collected. Each fraction was vortexed well and 100 ~1 of the fraction was removed before pH measurement and was diluted to 1: 10 with Gel-PBS, and stored at -30” until assay. This stored sample solution was further diluted to 1: 10 with Gel-PBS prior to assay. Sephadex gel~ltration. A 15 x 900-mm Sephadex G- 100 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) column was equilibrated with PBS containing 0.1% sodium azide, pH 7.5. The column was coated with 10 ml of normal rat serum. LH components obtained by isoelectric focusing were separately applied to the column with BSA and cytochrome ( (Boehringer-Mannheim) as calibration proteins and eluted with the same buffer. Fractions of 1.5 ml of the eluate were collected and stored at -30” until LH assay. The PBS extract of the anterior pituitaries from broiler chickens was also applied to the same column. The anterior pituitary extract was prepared in the same manner as described in isoelectric focusing followed by ultracentrifugation at 105,OOOgfor 1 hr. The supernatant fluid was applied to the column, and elution and fractionation were carried out similarly.
RESULTS Isoelectric F’ocusing Patterns of IR-LH of the Anterior Pituitary Extracts
Figure 1 illustrates the distribution of IR-LH in the anterior pituitary extracts from male Japanese quail exposed to 16L:8D for 2 weeks and chicken in the pH 7- 11 Ampholine gradient. IR-LH in both species was fractionated into four distinct components with pZ values of 9.8, 9.4, 9.0, and 8.5; these are tentatively termed: component I, II, III, and IV, respectively. Among them, I is the major component in amount; II, III, and IV follow in descending order in both extracts. Examination of the IR-LH Components by Radioreceptor Assay
Chicken LH (preparation (Gunma)) showed a parallel inhibition
IRC-2 curve
HETEROGENEITY
OF
20 Froclion
AVIAN
217
LH
30 Number
40
50
PH z 300 \ .-s ; 7E 200
ChIchen
-\
N k2 5 5 r;-
too
0
I IO
20 Fraction
40
30 Number
50
FIG. 1. Isoelectric focusing patterns of the anterior pituitary extracts of male Japanese quail photostimulated for 2 weeks and of chicken. Isoelectric focusing was performed for 68-70 hr in the pH 7- 11 Ampholine gradient. The PBS extract of quail anterior pituitary glands was applied in an amount equivalent to 23 glands, while that of chicken glands, equivalent to 5 glands, was applied to the column. After isoelectric focusing, the solution was eluted at a flow rate of 30 mkhr, and 2.5-ml fractions were collected.
to that of rat LH in rat LH radioreceptor assay, though the receptor-binding activity of the former was poor as compared with rat LH. All IR-LH components possessed the ability to bind rat LH receptor (Table 1). However, the values obtained by radioreceptor assay (b) did not agree with those obtained by radioimmunoassay (a),
as reflected by b/a ratio. The ratio was the highest with component IV, and I, II, and III followed in descending order. Gel Filtration of IR-LH Components the Anterior Pituitary Extract on Sephadex G-100 Each fraction containing an IR-LH
TABLE
1
ESTIMATIONSOFTHE FOUR IR-LH COMPONENTS DETERMINEDBYRADIOIMMUNOASSAYAND
Component I
II III IV
Radioimmunoassay 355.0 288.6 232.0 106.4
rf. 14.7 (5)* zk 5.1 (5) 2 6.0 (5) ? 2.4 (5)
(a)”
INTHECHICKENANTERIORPITUITARY RADIORECEFTORASSAY
Radioreceptor 977.4 634.8 386.0 494.6
+ k k ?
n All the assay values are expressed as ngxIRC-2/fraction/AP. h Mean 2 SE (No. of tubes). c Ratio + SE.
assay (b)”
21.0 24.1 61.0 25.3
(5)b (5) (5) (5)
bla
2.75 2.20 1.67 4.64
k * k ”
0.13’ 0.09 0.27 0.26
and
com-
218
HATTORI
AND
WAKABAYASHI
10
COMPONENT
II
COMPONENT
IV
”
KiiV COMPOliEFlT
111
;
FIG. 2. Gel filtration of the immunoreactive LH components on Sephadex G-100. A 15 X 900-mm Sephadex G-100 column was equilibrated with PBS containing 0.1% sodium azide, pH 7.5. Each fraction containing the immunoreactive LH components obtained by isoelectric focusing was diluted to 1:lO with 0.1% gelatin-PBS, and 1 ml of the solution was subjected to the column with BSA and cytochrome c as calibration proteins.
ponent was applied to a Sephadex G-100 column to compare relative molecular sizes of IR-LH components. Two discrete peaks were found with all the components as shown in Fig. 2. The slowly eluted peaks of these four IR-LH components, which we tentatively term “little LH,” were of the same molecular size of 23,500-25,000 within the range of error. The rapidly eluted peaks, designated tentatively as “big LH,” had a molecular size of about twice that of “little LH.” The relative amounts of “big LH” in these components were not similar. In component I, the relative amount of “big LH” was 15% of the total; 32% in component II; 40% in component III; and 30% in component IV. The gel filtration of IR-LH in chicken pituitary extract also represented two peaks as shown in Fig. 3. These peaks seemed to correspond to “big LH”
and “little nents.
LH”
found in electrical
compo-
Amounts of IR-LH Components of Male Quail in Some Physiological States When male quail maintained in SD, IR-LH levels in sera remained low (a range of 0.64-0.78 ng x IRC-2/ml). In these birds, amounts of four IR-LH components increased moderately and evenly (Table 2). Amounts of the components in SD 21 were about twice as much as that in SD 0. In contrast to SD quail, amounts of the components changed markedly in LD quail (Table 2). In LD 7, when IR-LH was released vigorously to the circulation (4.34 f 0.35 &ml), amounts of the components changed only slightly, suggesting the release and biosynthesis of these components were almost balanced. In LD 21, when
HETEROGENEITY
FIG. 3. Gel filtration pattern of immunoreactive LH in the chicken anterior pituitary extract on Sephadex G-100. The anterior pituitary extract was prepared by homogenizing the glands with PBS. After freezing and thawing and centrifugation at 15,000 rpm for 20 min to remove insoluble fragments, the supematant fluids were subjected to ultracentrifugation at 105,OOOgfor 1 hr. The supematant fluids were applied to the column (15 x 900 mm), and fractions of 1.5 ml of the eluate were collected.
serum IR-LH was also high (4.23 + 0.47 r&ml), amounts of all the components have increased markedly. Especially, the increment of component I was the largest among the four, suggesting biosynthetic rate of component I was the highest. DISCUSSION
Many investigators have reported pZs of LH preparations with different degrees of purity obtained from several species of animals, including sheep (Sherwood et al., 1970; Reichert, 1971), cattle (Reichert, 1971; Yoshida and Ishii, 1973), horse (Braselton and McShan, 1970; Reichert, TABLE
2
ACCOUNTS OF THE FOUR IR-LH COMWNENTS IN THE ANTERIOR PITUITARY OF MALE JAPANESE QUAIL MAINTAINED IN SD OR LD
Components” Group
I
II
III
IV
SD 0 (LD 0)
50.10
21.04
14.59
7.08
SD 7 LD 7
60.69 46.78
16.26 14.34
12.01 18.88
4.87 10.00
SD 21 1-D 21
95.82 209.10
49.26 64.33
25.83 58.62
17.61 22.77
” The values are expressed as ngxIRC-2iAP.
OF AVIAN
LH
219
1971), pig (Reichert, 1971), human (Bettendorf et al., 1968; Rathnam and Saxena, 1970; Reichert, 1971). However, pZ values of the preparations even from the same species of animals are not always identical. Since pituitary LH is composed of several components, some of them are eliminated in the course of extraction and purification carried out to obtain a homogenous preparation. This fact may account for the varying pZ values reported. The recent observation by Wakabayashi (1977) that IR-LH in rat pituitary gland homogenate was resolvable into seven components with different pZ values supports the concept of electrical heterogeneity of LH in the pituitary gland. On the other hand, there have been no reports as to the heterogeneity in avian LH. Our evidence indicates that IR-LH in quail and chicken anterior pituitary extracts is resolvable into four components with pZs of 9.8, 9.4, 9.0, and 8.5 which we have tentatively termed components I, II, III, and IV, respectively. The presence of IR-LH components in avian pituitary introduces a question whether these components have biological activity or not. Because of the low sensitivity and large variation of bioassays, we used a radioreceptor assay to estimate biological activity in the present study, though radioreceptor assay does not always reflect accurately the biological activity. The radioreceptor assay employing homogenates of rat luteinized ovary as a receptor fraction indicated that all the components had the ability to bind rat LH receptor. However, the assay results of all the components were not parallel with those obtained by radioimmunoassay, indicating that these components had different relative affinities for the antibody and/or the receptor. We could not find any significant differences in molecular sizes of the components in gel filtration study. However, it is of interest that “big LH,” possibly a dimeric form of “little LH,” was found in all the components, and that these components
220
HATTORI
AND
were different in the relative proportion of “big LH.” Size heterogeneity has also been shown in human pituitary and urinary LH (Rabinowitz et al., 1974) and human serum LH (Graesslin et al., 1976). Goodman et al. (1974) observed that isolated “big GH” transformed into “little GH” during storage. Such a phenomenon has not yet been found in glycoprotein hormones. However, it could not be excluded that storage of tissues might cause interconversion between “big and little LH.” However, “big LH” was not formed during isoelectric focusing and the preservation of the fractions, because “big LH” was also found in gel filtration of the PBS extract of chicken pituitary glands. The problem of size heterogeneity has to be further studied. A further important finding is that isoelectric focusing patterns of IR-LH in male quail pituitary changed during some physiological and experimental states, suggesting changes of biosynthesis and release of the components. In male quail sexually suppressed in a regime of 8L: 16D no abrupt increases of amounts the components were observed, while in photostimulated quail in 16L:8D changes became apparent, especially in component I after photostimulation for 21 days. In studies of electrical heterogeneity of rat LH (Wakabayashi, 1977), it was also observed that the relative amounts of the seven components did not remain constant, but that several of the components changed in proportion greatly during some physiological states. The biological significance of these phenomena remains obscure, though some of them might be due to sex steroid hormone action. Bogdanove et al. (1975) reported that FSH was pleomorphic in the rat and that feedback control in pituitary-gonadal system was not only quantitative but qualitative. The changes of the hormone in biological and chemical characteristics, the index of discrimination (bioassay-radioimmunoassay comparison), apparent molecular size, and capacity to survive in the circulation, were
WAKABAYASHI
affected by alterations in the gonadal steroids environment. At present, the heterogeneity or polymorphism of glycoprotein hormones has not been fully explained. TamuraTakahashi and Ui (1977) examined the chemical properties of four components of whale LH and found that molecular size, the contents of acidic, basic, and neutral amino acid residues, and of carbohydrates, especially in sialic acid content, which affects electric charge of protein, were not significantly different among those components. When Reichert partially purified human LH preparations with neuraminidase to deprive them of sialic acid, he found that its four components with different pls were apparently converted into a single one having a pZ of 9.35 (1971). Sialic acid-free HCG prepared from the native HCG complex by treatment with neuraminidase also showed a single band on gel electrofocusing (Graesslin et al., 1973). On the other hand, Webster et al. (1972) carried out the same experiment with human TSH and reported results different from those of Reichert. Therefore, variation in sialic acid content does not explain the heterogeneity of all glycoprotein hormones. ACKNOWLEDGMENTS We are grateful to Dr. A. F. Parlow, Rat Pituitary Hormone Program, NIAMDD, NIH, for the kind supply of radioimmunoassay kit for rat LH. We also acknowledge with thanks the technical assistance of Miss K. Sakamoto. This work was supported by Ford Foundation Research Grant 740-0405, and by the Ministry of Education Research Grant.
REFERENCES Bettendorf, G., Breckwoldt, M., Czygan, P. J., Fock, A., and Kumasaka, T. (1968). Fractionation of human pituitary gonadotropins (extraction, gelfiltration and electrofocusing). In “Gonadotropins” (E. Rosemberg ed.), pp. 13-23. Geron-X, Los Altos. Bogdanove, E. M., Nolin, J. M., and Campbell, G. T. (1975). Qualitative and quantitative gonadpituitary feedback. Recent Progr. Horm. Res. 31, 567-626. Braselton, W. E., Jr., and McShan, W. H. (1970).
HETEROGENEITY Purification and properties of follicle-stimulating and luteinizing hormones from pituitary glands. Arch. Biochem. Biophys. 139, 45-58. Goodman, A. D., Tanenbaum, R., Wreight, D. R., Trimble, K. D., and Rabinowitz, D. (1974). Existence of “big” and “little” forms of immunoreactive growth hormone in human plasma. In “Heterogeneity of Polypeptide Hormones” (D. Rabinowitz and J. Roth, eds.), pp. 48-56. Academic Press, New York. Graesslin, D., Weise, H. C., and Braendle, W. (1973). The microheterogeneity of human chorionic gonadotropin (HCG) reflected in the p-subunits. F‘EBS lett. 31, 214-216. Graesslin, D., Leidenberger, F. A., Lichtenberg, V., Glismann, N., Hess, N., Czygan, P. J., and Bettendorf, G. (1976). Existence of big and little forms of luteinizing hormone in human serum. Acta
Endocrinol.
(Kbh)
83, 466-482.
Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963). The preparation of i3’I labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114- 123. Hartori, M., Koga, O., and Nishiyama, H. (1975). The biological property of chicken pituitary luteinizing hormone. Sci. Bull. Far. Agr. Kyushu Univ. 30, 137- 142. Lee, C. Y., and Ryan, R. J. (1973). Luteinizing hormone receptors in luteinized rat ovaries. In “Receptors for Reproductive Hormones” (B. W. O’Malley and A. R. Means, eds.), pp. 419-430. Plenum, New York/London. Miyachi, Y., Vaitukaitis, J. L., Nieschlag, E., and Lipsett, B. (1972). Enzymatic radioiodination of gonadotropins. J. Clin. Endocrinol. 34, 23-28. Parlow, A. F. (1961). Bio-assay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In “Human Pituitary Gonadotropins” (A. Albert, ed.), pp. 300-310. Thomas, Springfield, III. Rabinowitz, D., Benveniste, R., and Bell, J. (1974). Heterogeneity of human luteinizing hormone: Behavior in radioimmunoassay and radioligandreceptor assay systems and evidence for the presence of a-subunit in serum. In “Heterogeneity of Polypeptide Hormones” (D. Rabinowitz and J. Roth, eds.), pp. 90-97. Academic Press, New York.
OF AVIAN
LH
221
Rathnam, P., and Saxena, B. B. (1970). Isolation and physicochemical characterization of luteinizing hormone from human pituitary glands. J. Biol. Chem.
245, 3725-373
1.
Reichert, L. E., Jr. (1971). Electrophoretic properties of pituitary gonadotropins as studied by electrofocusing. Endocrinology 88, 1029- 1044. Roos, P., Nyberg, L., Wide, L., and Gemzell, C. (1975). Human pituitary luteinizing hormone: Isolation and characterization of four glycoproteins with luteinizing activity. Biochim. Biophys. Acta
405, 363-379.
Sherwood, 0. D., Grimek, H. J., and McShan, W. H. ( 1970). Purification of luteinizing hormone from sheep pituitary glands and evidence for several physicochemically distinguishable active components. Biochim. Biophys. Acta 221, 87- 106. Tamura-Takahashi, H., and Ui, N. (1977). Purification and properties of four biologically active components of whale luteinizing hormone. J. Biochem. 81, 1155-1160. Wakabayashi, K. (1977). Heterogeneity of rat luteinizing hormone revealed by radioimmunoassay and electrofocusing studies. Endocrinol. Japon.
24. 473-485.
Ward, D. N., McGregor, R. F., and Griffin, A. C. (1959). Chromatography of luteinizing hormone from sheep pituitary glands. Biochim. Biophys. Acta 32, 305-314. Webster, B. R., Hummel, B. C. W., McKenzie, J. M., Brown, G. M., and Paice, J. C. (1972). Isoelectric focusing of human thyrotropin: Identification of multiple components with dissociation of biological and immunological activities. In “Structure-Activity Relationships of Protein and Polypeptide Hormones” (M. Dubuisson, A. Niget, and H. van Cauwenberge, eds.), pp. 369-378. Excerpta Medica, Amsterdam/London. Yora, T., and Ui. N. (1978). Purification and subfractionation of bovine thyrotropin and lutropin using radioimmunoassay for evaluation of the puritication processes. J. B&hem. 83, 1173- 1190. Yoshida, T., and Ishii, S. (1973). Electrofocusing of partially purified bovine luteinizing hormone. Endocrinol.
Japon.
20, 625-633.
AND
Isoelectric
COMPARATIVE
(1979)
Focusing and Gel Filtration Studies on the Heterogeneity of Avian Pituitary Luteinizing Hormone MASAAKI
Hormone
39,215-221
ENDOCRINOLOGY
Assay
Center,
HATTORI
Institute
AND KATSUMI
of Endocrinology,
Gunma
WAKABAYASHI University,
Maebashi,
371, Japan
Accepted June 7, 1979 The heterogeneity of avian pituitary LH was shown by means of isoelectric focusing and gel filtration studies coupled with radioimmunoassay. The immunoreactive LH (IR-LH) in anterior pituitary extracts from male quail and chickens contained four components with isoelectric points of 9.8 (component I), 9.4 (component II), 9.0 (component III), and 8.5 (component IV). All IR-LH components possessed the ability to bind to rat ovarian LH receptors but the estimates of potency obtained by the radioreceptor assay did not agree with those from radioimmunoassay. Sephadex G-100 gel filtration of the four individual components obtained from chicken pituitary LH indicated the presence of an LH with a molecular weight of 23,500-25,000 together with a component having double the molecular weight. The components differed in the relative amounts of the larger molecular weight component. The large component was also found in a gel filtration of the extract of chicken anterior pituitary glands. The amounts of the various components were changed in male quail by photostimulation. After 21 days of photostimulation, the amounts of all the components, especially of component I, had increased markedly.
Some investigators who have purified glycoprotein hormones have noticed a heterogeneity in their purified preparations. Working on luteinizing hormone (LH), Ward et al. (1959) obtained biologically active highly purified ovine LH preparations containing different amounts of glucosamine. Braselton and McShan (1970) purified equine LH and found four biologically active components after isoelectric focusing separation. Reichert (1971) examined the isoelectric points (pls) of a number of LH preparations and found that his human LH preparation had four components with different pZ values. Yoshida and Ishii (1973) also found three biologically active components with different pZs in a partially purified bovine LH preparation. In order to observe the heterogeneity of immunoreactive LH (IR-LH) in a state as natural as possible, one of the present authors (Wakabayashi) analyzed neutral saline extracts of rat anterior pituitaries by isoelectric focusing and measured IR-LH by radioimmunoassay (1977). He found that rat IR-LH in the anterior pituitary con-
tained seven components with different pls. Furthermore, the relative amounts of these components were changed by orchidectomy, estradiol benzoate or progesterone treatment. Individual components of LH have been also isolated from human (Roos et al., 1975), whale (Tamura-Takahashi and Ui, 1977), and bovine (Yora and Ui, 1978) pituitaries. In contrast, there have been no reports concerning the heterogeneity of avian LH. The present report first describes an isoelectric focusing study on avian LH and on the changes in the various components caused by photostimulation, and secondly gel filtration studies of these components for heterogeneity in molecular size. METHODS Male Japanese quail were purchased from a commercial breeder at 3 weeks of age and subjected to a photoperiod of 8L:16D (SD, 8 hr light and 16 hr darkness daily) for 2 weeks. Then, the birds were divided into two groups. One group was kept on a regime of SD, and the other group was transferred to a regime of 16L:8D(LD) on Day 0 (designated as SD 0 or LD 0). Birds.
Sampling for studies. After
isoelectric
focusing
and gel filtration
blood samples were collected from the
215 0016~6480/79/100215-07$01.00/O Copyright
0 1979 by Academic
Press, Inc.
216
HATTORI
AND
jugular vein on Days 0, 7, and 2 1, the birds were killed and the anterior pituitary glands immediately removed, pooled in 1 ml of phosphate-buffered saline (PBS), pH 7.5 (containing 0.01 M sodium phosphate, 0.14 M NaCl, and 0.01% merthiolate) and stored at -70” until examination by isoelectric focusing. The sera were stored at -30” until radioimmunoassay. The anterior pituitary glands of broiler chickens (about 1000 glands) were also collected at a slaughterhouse and stored at -70” until use. Radioimmunoassay. The radioimmunoassay was carried out by a double-antibody method with fraction IRC-2 (Gunma) as standard preparation (Hattori et al., 1975) and an anti-avian LH serum AL-MH#l. A further purified preparation, IEF-1, which was obtained by preparative isoelectric focusing of IRC-2, was used for radioiodination. The preparation IEF-1 had a single pZ of pH 9.8 and was free from IEF-2 which had low immunoreactivity, when assayed with the radioiodinated IEF-1 as labeled hormone. The iodination rate of IEF-1 was very high by chloramine-T method (Greenwood et al., 1963). The assay system consisted of 400 ~1 of 0.1% gelatin in PBS (Gel-PBS). 200 ~1 of standard or sample dissolved in 0.1% Gel-PBS, 100 ~1 of diluted antiserum (l:lOO,OOO) in 1% normal rabbit serum-O.05 M EDTA-PBS, and 100 ~1 of 1311-labeled hormone in 0.1% Gel-PBS. Before the addition of 13’1-LH, the preincubation was carried out at 4” overnight. A goat anti-rabbit y-globulin serum, H-4, prepared in our laboratory, was employed as the second antibody. Results were expressed as nanograms of the preparation IRC-2 (Gunma). Radioreceptor assay fur LH. The radioreceptor assay was performed according to Lee and Ryan (1973) using PMS- and HCG-primed rats (Parlow, 1961). “jI-labeled rat LH was prepared by radioiodination of NIAMDD Rat LH-I-4 (supplied by Rat Pituitary Hormone Distribution Program, NIAMDD, NIH) with lactoperoxidase according to the method of Miyachi et al. (1972), with minor modification. Nonspecific binding was determined by using excess unlabeled avian hormone (IRC-2 (Gunma), 20 pg). Results were expressed as nanograms of the preparation IRC-2 (Gunma). Isoelectric focusing. A preparative isoelectric focusing column, size 110 ml, of Kato Manufacturing Company, Osaka, Japan, was employed. As carrier ampholytes, Ampholines pH 9- 11, pH 7-9, and pH 5-7 (LKB Produkter) were mixed to make up a pH gradient from pH 7 to 11. For the stabilization of the isoelectric focusing solution, a sorbitol density gradient from 5 to 50% was employed. The anterior pituitary extracts were prepared by homogenizing the glands with PBS, followed by freezing and thawing and centrifugation at 15,000 rpm to remove insoluble fragments. The extracts of the anterior pituitaries of quail were added to the isoelectric focusing solution in an
WAKABAYASHI
amount equivalent to 20-25 glands; in the case of the broiler chickens, equivalents of 5 glands, were added. For preparation of the samples for Sephadex gel liltration and radioreceptor assay analyses, the extract of 500 glands of the anterior pituitaries of broiler chickens was applied to isoelectric focusing. Isoelectric focusing with the anode at the top of the column was run for 65-70 hr under cooling at 2-4”, the voltage applied being increased stepwise from 400 to 1450 V. After focusing, the solution was eluted at a flow rate of 30 ml/hr, and 2.5-ml fractions were collected. Each fraction was vortexed well and 100 ~1 of the fraction was removed before pH measurement and was diluted to 1: 10 with Gel-PBS, and stored at -30” until assay. This stored sample solution was further diluted to 1: 10 with Gel-PBS prior to assay. Sephadex gel~ltration. A 15 x 900-mm Sephadex G- 100 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) column was equilibrated with PBS containing 0.1% sodium azide, pH 7.5. The column was coated with 10 ml of normal rat serum. LH components obtained by isoelectric focusing were separately applied to the column with BSA and cytochrome ( (Boehringer-Mannheim) as calibration proteins and eluted with the same buffer. Fractions of 1.5 ml of the eluate were collected and stored at -30” until LH assay. The PBS extract of the anterior pituitaries from broiler chickens was also applied to the same column. The anterior pituitary extract was prepared in the same manner as described in isoelectric focusing followed by ultracentrifugation at 105,OOOgfor 1 hr. The supernatant fluid was applied to the column, and elution and fractionation were carried out similarly.
RESULTS Isoelectric F’ocusing Patterns of IR-LH of the Anterior Pituitary Extracts
Figure 1 illustrates the distribution of IR-LH in the anterior pituitary extracts from male Japanese quail exposed to 16L:8D for 2 weeks and chicken in the pH 7- 11 Ampholine gradient. IR-LH in both species was fractionated into four distinct components with pZ values of 9.8, 9.4, 9.0, and 8.5; these are tentatively termed: component I, II, III, and IV, respectively. Among them, I is the major component in amount; II, III, and IV follow in descending order in both extracts. Examination of the IR-LH Components by Radioreceptor Assay
Chicken LH (preparation (Gunma)) showed a parallel inhibition
IRC-2 curve
HETEROGENEITY
OF
20 Froclion
AVIAN
217
LH
30 Number
40
50
PH z 300 \ .-s ; 7E 200
ChIchen
-\
N k2 5 5 r;-
too
0
I IO
20 Fraction
40
30 Number
50
FIG. 1. Isoelectric focusing patterns of the anterior pituitary extracts of male Japanese quail photostimulated for 2 weeks and of chicken. Isoelectric focusing was performed for 68-70 hr in the pH 7- 11 Ampholine gradient. The PBS extract of quail anterior pituitary glands was applied in an amount equivalent to 23 glands, while that of chicken glands, equivalent to 5 glands, was applied to the column. After isoelectric focusing, the solution was eluted at a flow rate of 30 mkhr, and 2.5-ml fractions were collected.
to that of rat LH in rat LH radioreceptor assay, though the receptor-binding activity of the former was poor as compared with rat LH. All IR-LH components possessed the ability to bind rat LH receptor (Table 1). However, the values obtained by radioreceptor assay (b) did not agree with those obtained by radioimmunoassay (a),
as reflected by b/a ratio. The ratio was the highest with component IV, and I, II, and III followed in descending order. Gel Filtration of IR-LH Components the Anterior Pituitary Extract on Sephadex G-100 Each fraction containing an IR-LH
TABLE
1
ESTIMATIONSOFTHE FOUR IR-LH COMPONENTS DETERMINEDBYRADIOIMMUNOASSAYAND
Component I
II III IV
Radioimmunoassay 355.0 288.6 232.0 106.4
rf. 14.7 (5)* zk 5.1 (5) 2 6.0 (5) ? 2.4 (5)
(a)”
INTHECHICKENANTERIORPITUITARY RADIORECEFTORASSAY
Radioreceptor 977.4 634.8 386.0 494.6
+ k k ?
n All the assay values are expressed as ngxIRC-2/fraction/AP. h Mean 2 SE (No. of tubes). c Ratio + SE.
assay (b)”
21.0 24.1 61.0 25.3
(5)b (5) (5) (5)
bla
2.75 2.20 1.67 4.64
k * k ”
0.13’ 0.09 0.27 0.26
and
com-
218
HATTORI
AND
WAKABAYASHI
10
COMPONENT
II
COMPONENT
IV
”
KiiV COMPOliEFlT
111
;
FIG. 2. Gel filtration of the immunoreactive LH components on Sephadex G-100. A 15 X 900-mm Sephadex G-100 column was equilibrated with PBS containing 0.1% sodium azide, pH 7.5. Each fraction containing the immunoreactive LH components obtained by isoelectric focusing was diluted to 1:lO with 0.1% gelatin-PBS, and 1 ml of the solution was subjected to the column with BSA and cytochrome c as calibration proteins.
ponent was applied to a Sephadex G-100 column to compare relative molecular sizes of IR-LH components. Two discrete peaks were found with all the components as shown in Fig. 2. The slowly eluted peaks of these four IR-LH components, which we tentatively term “little LH,” were of the same molecular size of 23,500-25,000 within the range of error. The rapidly eluted peaks, designated tentatively as “big LH,” had a molecular size of about twice that of “little LH.” The relative amounts of “big LH” in these components were not similar. In component I, the relative amount of “big LH” was 15% of the total; 32% in component II; 40% in component III; and 30% in component IV. The gel filtration of IR-LH in chicken pituitary extract also represented two peaks as shown in Fig. 3. These peaks seemed to correspond to “big LH”
and “little nents.
LH”
found in electrical
compo-
Amounts of IR-LH Components of Male Quail in Some Physiological States When male quail maintained in SD, IR-LH levels in sera remained low (a range of 0.64-0.78 ng x IRC-2/ml). In these birds, amounts of four IR-LH components increased moderately and evenly (Table 2). Amounts of the components in SD 21 were about twice as much as that in SD 0. In contrast to SD quail, amounts of the components changed markedly in LD quail (Table 2). In LD 7, when IR-LH was released vigorously to the circulation (4.34 f 0.35 &ml), amounts of the components changed only slightly, suggesting the release and biosynthesis of these components were almost balanced. In LD 21, when
HETEROGENEITY
FIG. 3. Gel filtration pattern of immunoreactive LH in the chicken anterior pituitary extract on Sephadex G-100. The anterior pituitary extract was prepared by homogenizing the glands with PBS. After freezing and thawing and centrifugation at 15,000 rpm for 20 min to remove insoluble fragments, the supematant fluids were subjected to ultracentrifugation at 105,OOOgfor 1 hr. The supematant fluids were applied to the column (15 x 900 mm), and fractions of 1.5 ml of the eluate were collected.
serum IR-LH was also high (4.23 + 0.47 r&ml), amounts of all the components have increased markedly. Especially, the increment of component I was the largest among the four, suggesting biosynthetic rate of component I was the highest. DISCUSSION
Many investigators have reported pZs of LH preparations with different degrees of purity obtained from several species of animals, including sheep (Sherwood et al., 1970; Reichert, 1971), cattle (Reichert, 1971; Yoshida and Ishii, 1973), horse (Braselton and McShan, 1970; Reichert, TABLE
2
ACCOUNTS OF THE FOUR IR-LH COMWNENTS IN THE ANTERIOR PITUITARY OF MALE JAPANESE QUAIL MAINTAINED IN SD OR LD
Components” Group
I
II
III
IV
SD 0 (LD 0)
50.10
21.04
14.59
7.08
SD 7 LD 7
60.69 46.78
16.26 14.34
12.01 18.88
4.87 10.00
SD 21 1-D 21
95.82 209.10
49.26 64.33
25.83 58.62
17.61 22.77
” The values are expressed as ngxIRC-2iAP.
OF AVIAN
LH
219
1971), pig (Reichert, 1971), human (Bettendorf et al., 1968; Rathnam and Saxena, 1970; Reichert, 1971). However, pZ values of the preparations even from the same species of animals are not always identical. Since pituitary LH is composed of several components, some of them are eliminated in the course of extraction and purification carried out to obtain a homogenous preparation. This fact may account for the varying pZ values reported. The recent observation by Wakabayashi (1977) that IR-LH in rat pituitary gland homogenate was resolvable into seven components with different pZ values supports the concept of electrical heterogeneity of LH in the pituitary gland. On the other hand, there have been no reports as to the heterogeneity in avian LH. Our evidence indicates that IR-LH in quail and chicken anterior pituitary extracts is resolvable into four components with pZs of 9.8, 9.4, 9.0, and 8.5 which we have tentatively termed components I, II, III, and IV, respectively. The presence of IR-LH components in avian pituitary introduces a question whether these components have biological activity or not. Because of the low sensitivity and large variation of bioassays, we used a radioreceptor assay to estimate biological activity in the present study, though radioreceptor assay does not always reflect accurately the biological activity. The radioreceptor assay employing homogenates of rat luteinized ovary as a receptor fraction indicated that all the components had the ability to bind rat LH receptor. However, the assay results of all the components were not parallel with those obtained by radioimmunoassay, indicating that these components had different relative affinities for the antibody and/or the receptor. We could not find any significant differences in molecular sizes of the components in gel filtration study. However, it is of interest that “big LH,” possibly a dimeric form of “little LH,” was found in all the components, and that these components
220
HATTORI
AND
were different in the relative proportion of “big LH.” Size heterogeneity has also been shown in human pituitary and urinary LH (Rabinowitz et al., 1974) and human serum LH (Graesslin et al., 1976). Goodman et al. (1974) observed that isolated “big GH” transformed into “little GH” during storage. Such a phenomenon has not yet been found in glycoprotein hormones. However, it could not be excluded that storage of tissues might cause interconversion between “big and little LH.” However, “big LH” was not formed during isoelectric focusing and the preservation of the fractions, because “big LH” was also found in gel filtration of the PBS extract of chicken pituitary glands. The problem of size heterogeneity has to be further studied. A further important finding is that isoelectric focusing patterns of IR-LH in male quail pituitary changed during some physiological and experimental states, suggesting changes of biosynthesis and release of the components. In male quail sexually suppressed in a regime of 8L: 16D no abrupt increases of amounts the components were observed, while in photostimulated quail in 16L:8D changes became apparent, especially in component I after photostimulation for 21 days. In studies of electrical heterogeneity of rat LH (Wakabayashi, 1977), it was also observed that the relative amounts of the seven components did not remain constant, but that several of the components changed in proportion greatly during some physiological states. The biological significance of these phenomena remains obscure, though some of them might be due to sex steroid hormone action. Bogdanove et al. (1975) reported that FSH was pleomorphic in the rat and that feedback control in pituitary-gonadal system was not only quantitative but qualitative. The changes of the hormone in biological and chemical characteristics, the index of discrimination (bioassay-radioimmunoassay comparison), apparent molecular size, and capacity to survive in the circulation, were
WAKABAYASHI
affected by alterations in the gonadal steroids environment. At present, the heterogeneity or polymorphism of glycoprotein hormones has not been fully explained. TamuraTakahashi and Ui (1977) examined the chemical properties of four components of whale LH and found that molecular size, the contents of acidic, basic, and neutral amino acid residues, and of carbohydrates, especially in sialic acid content, which affects electric charge of protein, were not significantly different among those components. When Reichert partially purified human LH preparations with neuraminidase to deprive them of sialic acid, he found that its four components with different pls were apparently converted into a single one having a pZ of 9.35 (1971). Sialic acid-free HCG prepared from the native HCG complex by treatment with neuraminidase also showed a single band on gel electrofocusing (Graesslin et al., 1973). On the other hand, Webster et al. (1972) carried out the same experiment with human TSH and reported results different from those of Reichert. Therefore, variation in sialic acid content does not explain the heterogeneity of all glycoprotein hormones. ACKNOWLEDGMENTS We are grateful to Dr. A. F. Parlow, Rat Pituitary Hormone Program, NIAMDD, NIH, for the kind supply of radioimmunoassay kit for rat LH. We also acknowledge with thanks the technical assistance of Miss K. Sakamoto. This work was supported by Ford Foundation Research Grant 740-0405, and by the Ministry of Education Research Grant.
REFERENCES Bettendorf, G., Breckwoldt, M., Czygan, P. J., Fock, A., and Kumasaka, T. (1968). Fractionation of human pituitary gonadotropins (extraction, gelfiltration and electrofocusing). In “Gonadotropins” (E. Rosemberg ed.), pp. 13-23. Geron-X, Los Altos. Bogdanove, E. M., Nolin, J. M., and Campbell, G. T. (1975). Qualitative and quantitative gonadpituitary feedback. Recent Progr. Horm. Res. 31, 567-626. Braselton, W. E., Jr., and McShan, W. H. (1970).
HETEROGENEITY Purification and properties of follicle-stimulating and luteinizing hormones from pituitary glands. Arch. Biochem. Biophys. 139, 45-58. Goodman, A. D., Tanenbaum, R., Wreight, D. R., Trimble, K. D., and Rabinowitz, D. (1974). Existence of “big” and “little” forms of immunoreactive growth hormone in human plasma. In “Heterogeneity of Polypeptide Hormones” (D. Rabinowitz and J. Roth, eds.), pp. 48-56. Academic Press, New York. Graesslin, D., Weise, H. C., and Braendle, W. (1973). The microheterogeneity of human chorionic gonadotropin (HCG) reflected in the p-subunits. F‘EBS lett. 31, 214-216. Graesslin, D., Leidenberger, F. A., Lichtenberg, V., Glismann, N., Hess, N., Czygan, P. J., and Bettendorf, G. (1976). Existence of big and little forms of luteinizing hormone in human serum. Acta
Endocrinol.
(Kbh)
83, 466-482.
Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963). The preparation of i3’I labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114- 123. Hartori, M., Koga, O., and Nishiyama, H. (1975). The biological property of chicken pituitary luteinizing hormone. Sci. Bull. Far. Agr. Kyushu Univ. 30, 137- 142. Lee, C. Y., and Ryan, R. J. (1973). Luteinizing hormone receptors in luteinized rat ovaries. In “Receptors for Reproductive Hormones” (B. W. O’Malley and A. R. Means, eds.), pp. 419-430. Plenum, New York/London. Miyachi, Y., Vaitukaitis, J. L., Nieschlag, E., and Lipsett, B. (1972). Enzymatic radioiodination of gonadotropins. J. Clin. Endocrinol. 34, 23-28. Parlow, A. F. (1961). Bio-assay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In “Human Pituitary Gonadotropins” (A. Albert, ed.), pp. 300-310. Thomas, Springfield, III. Rabinowitz, D., Benveniste, R., and Bell, J. (1974). Heterogeneity of human luteinizing hormone: Behavior in radioimmunoassay and radioligandreceptor assay systems and evidence for the presence of a-subunit in serum. In “Heterogeneity of Polypeptide Hormones” (D. Rabinowitz and J. Roth, eds.), pp. 90-97. Academic Press, New York.
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Rathnam, P., and Saxena, B. B. (1970). Isolation and physicochemical characterization of luteinizing hormone from human pituitary glands. J. Biol. Chem.
245, 3725-373
1.
Reichert, L. E., Jr. (1971). Electrophoretic properties of pituitary gonadotropins as studied by electrofocusing. Endocrinology 88, 1029- 1044. Roos, P., Nyberg, L., Wide, L., and Gemzell, C. (1975). Human pituitary luteinizing hormone: Isolation and characterization of four glycoproteins with luteinizing activity. Biochim. Biophys. Acta
405, 363-379.
Sherwood, 0. D., Grimek, H. J., and McShan, W. H. ( 1970). Purification of luteinizing hormone from sheep pituitary glands and evidence for several physicochemically distinguishable active components. Biochim. Biophys. Acta 221, 87- 106. Tamura-Takahashi, H., and Ui, N. (1977). Purification and properties of four biologically active components of whale luteinizing hormone. J. Biochem. 81, 1155-1160. Wakabayashi, K. (1977). Heterogeneity of rat luteinizing hormone revealed by radioimmunoassay and electrofocusing studies. Endocrinol. Japon.
24. 473-485.
Ward, D. N., McGregor, R. F., and Griffin, A. C. (1959). Chromatography of luteinizing hormone from sheep pituitary glands. Biochim. Biophys. Acta 32, 305-314. Webster, B. R., Hummel, B. C. W., McKenzie, J. M., Brown, G. M., and Paice, J. C. (1972). Isoelectric focusing of human thyrotropin: Identification of multiple components with dissociation of biological and immunological activities. In “Structure-Activity Relationships of Protein and Polypeptide Hormones” (M. Dubuisson, A. Niget, and H. van Cauwenberge, eds.), pp. 369-378. Excerpta Medica, Amsterdam/London. Yora, T., and Ui. N. (1978). Purification and subfractionation of bovine thyrotropin and lutropin using radioimmunoassay for evaluation of the puritication processes. J. B&hem. 83, 1173- 1190. Yoshida, T., and Ishii, S. (1973). Electrofocusing of partially purified bovine luteinizing hormone. Endocrinol.
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20, 625-633.
