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Effect of changing photoperiod on plasma thyroxine-binding prealbumin in Japanese quail (Coturnix coturnix japonica)
GENERAL
AND
COMPARATIVE
41, 539-545 (1980)
ENDOCRINOLOGY
Effect of Changing Photoperiod on Plasma Thyroxine-Binding Prealbumin in Japanese Quail (Coturnix coturnix japonica) M. Departtneni
of Biochemistry,
D. J.
EL-SAYED, University
HEAF,
of Liverpool.
Accepted
AND P.O.
February
Box
J.
GLOVER
147, Liverpool.
L69 3BX,
England
15, 1980
The concentration of thyroxine-binding prealbumin (TBPA) in blood plasma of Japanese quail is shown to be inversely related to changing photoperiod over the annual cycle and particularly from September to April. It is suggested that this protein has a key role among the various plasma factors undergoing change in conditioning animals for seasonal breeding. The changes in TBPA are opposite to, but not synchronous with, those of retinol-binding protein (RBP) and there is a wide variation in the TBPAiRBP molar ratio (0.6- 1.6) during the year, which may indirectly influence the turnover rate of vitamin A.
Thyroxine-binding prealbumin (TBPA) occupies a position of intermediate importance among the three main plasma proteins capable of transporting thyroxine in higher animals. It has a greater capacity but lower affinity for the ligand than the highly specific thyroxine-binding globulin (TBG) which is present only in certain species at 5-- 10% of the concentration of TBPA (R.obbins et al., 1978). It has a much greater atlfinity for the hormone but is present in lower concentration than the other less specific carrier, albumin. TBPA has a wider distribution in the animal kingdom than TiBG, which is reported to be absent from avian species (Tata and Shellabarger, 1959; Parer et al., 1962; Tanabe et af., 1969). Presumably its role in the thyroxine transport system in birds must be of greater significance. Another function was assigned to TBPA in connection with the transport of vitamin 4, when it was observed that it formed a 1: 1 complex with retinol-binding protein (RBP) in human plasma (Kanai et al., 1968). The interaction was considered to be of physiological importance in reducing the rate of loss of the vitamin by ultrafiltration of RBP (21,000 daltons) through the kidney. The same two proteins isolated from avian plasma also interact in this way (Abe et af., 1975; Heller, 1976).
Recent studies in our laboratory on the retinol transport system in blood plasma have shown that there is a specific twofold increase in plasma RBP concentration in sheep (Glover et al., 1976), Japanese quail (Heaf and Glover, 1979; Glover et al., 1980), and rat (Kershaw, 1978) as they reach maturity and enter their reproductive period. Retinol is known to be essential for the proper differentiation and growth of the gonads (Thompson et al., 1964) and the increased supply of RBP in plasma is considered necessary for meeting the additional demand of these tissues over the basic requirements of the immature animal. In seasonal breeders, cyclic gonadal development and regression are associated with changing photoperiod and so too are the cyclic changes in RBP. The biochemical links, however, relating the hypothalamic-pituitary-gonadal axis with the liver where the protein is synthesised have not yet been established. TBPA which is also secreted by the liver could perhaps be involved through its interaction with RBP in plasma so affecting the overall vitamin A economy of the animal. This possibility was examined by determining if changing photoperiod could influence the plasma concentration of TBPA in Japanese quail. In addition, albumin concentration was monitored to provide a control over the possibility that
539 00 16-6480/80/080539-07$0 1.0010 Copyright 0 1980 by Academic Press. Inc. All rights of reproduction m any form reserved.
540
EL-SAYED,
HEAF, AND GLOVER
changes in TBPA may be due to more general variations in liver protein synthesis.
phosphate-buffered saline [PBS, 0.15 M, NaCl, 0.05 M PO,, pH 7.5) at 4”. The PBS was decanted and the extraction of the gel pieces was repeated. Pooled PBS extracts were filtered, concentrated by ultrafiltration METHODS to 5 ml, and dialysed overnight against PBS. Small Preparation of Animals portions of the puritied TBPA and albumin showed single bromophenol blue-stained bands when subQuail, hatched in the laboratory, were reared under jected to polyacrylamide disc gel electrophoresis. For natural lighting until they were 3 months old (September-December), and were sexually immature at the standardisation purposes the protein concentration in the purified albumin was determined according to start of the experiment. They were then maintained Lowry et al. (1951) using bovine serum albumin (BSA) under artificial light (4 x 60-W daylight fluorescent tubes) for 18 months at 21” with the photoperiod con- as a standard. Using the Aiwcm value for human TBPA of 14.1 at trolled by a solar dial time switch (Sangamo Weston Ltd., Enfield, Middlesex) which simulated seasonal 280 nm (Raz and Goodman, 1%9) the yield of TBPA was found by UV spectrophotometry to be about 20 daylight changes between sunrise and sunset equivalent to those at S6”N latitude. Blood samples (1 ml) mg. TBPA, 2.5 mg, diluted to 1 ml with PBS was homogenised with 1 ml Freunds complete adjuvant were taken at intervals varying from 7 to 30 days, from (Gibco, Grand Island, N.Y.) and injected at several three male and three female birds in all, from the wing subcutaneous sites into a 3-kg male New Zealand vein, where it crosses the ulna-radial-humeral joint, directly into heparinized capillary tubes. The tubes white rabbit. After 1 month the immunization was rewere sealed at one end, centrifuged to separate the peated and 2 weeks later blood was sampled from an ear vein. Crossed immunoelectrophoresis (Weeke, plasma which was removed, frozen rapidly in liquid 1973) with the serum gave a single precipitin peak with N,, and stored at -20” until required for assay. All purified TBPA, but with quail serum, two peaks were samples were first assayed for TBPA. Thereafter, obtained, one corresponding to TBPA, and the other samples from five dates, corresponding to TBPA to albumin. Rocket immunoelectrophoresis (Laurell, maxima and minima, were assayed for albumin. 1966) of this bispecific antiserum gave two coaxial rockets with quail serum and showed the titre to be Protein Purljkation and Production of low for TBPA but at a usable level for albumin. It was Antisera therefore stored at -20” for use in the quail albumin Chicken serum was separated from blood donated immunoassay. by J. P. Wood Ltd., Llangefni, Anglesey, and stored at TBPA was further purified by a repeat slab gel elec-20” until required. Serum (300 ml) was subjected to trophoresis taking a narrower (5-mm) cut when reDEAE-Sephadex followed by CM-Sephadex (Pharmoving the protein band. After concentration of the macia, Uppsala, Sweden) ion-exchange chromatogragel extracts as before, the purity was checked by phy according to the method of Abe et al. (1975). The SDS-polyacrylamide disc gel electrophoresis (Weber fractions containing TBPA were concentrated to a et al., 1972) and isoelectric focussing (Wrigley, 1968). volume of 20 ml in an Amicon UMlO (Lexington, For standardisation purposes the TBPA concentration Mass.) ultrafiltration cell and applied to the top of a 5% was determined by using an analytical ultracentrifuge polyacrylamide slab gel (width 7 mm, length 200 mm, as a differential interferometer (Babul and Stellwagen, depth 60 mm) with gel and reservoir buffers as de- 1969). The A iv’,, was determined in the same solution scribed previously (Glover et al., 1974). Electrophoreby uv spectrophotometry at 280 nm. sis was carried out at 4” with a current of 100 mA until The further purified TBPA (0. I mg) was used to imthe position of most mobile ions, made visible by a gel munise the same rabbit 31/i months after the first imimpurity as a blue fluorescent band under ultraviolet munisation and blood was sampled 2 weeks after this light, had reached 5 mm from the bottom of the gel. booster dose. The antiserum had a usable titre for Thin slices (3 mm), three in all, were cut from both TBPA (50 pg TBPA precipitated/ml serum) on rocket edges and the centre of the gel, stained with immunoelectrophoresis and the reaction with albumin bromophenol blue, and after destaining were returned was still present, although at a considerably lower to their original positions in the gel. The TBPA stained level. The antiserum contained two separate populaas a thin band close to the blue ultraviolet fluorescent tions of antibodies against the two quail serum proteins band and was well separated from the broad band of as evidenced by the two coaxial rockets which were albumin. Using the stained gel as a guide a 7-mm sec- clearly distinguishable from one another. This comtion containing the TBPA band was cut from the slab. bined with the approximately 100-fold difference in Quail albumin was obtained in a similar manner from concentration between TBPA and albumin avoided mutual interference between the precipitin zones of 50 ml serum collected from 20 quail. The two proteins were extracted separately by cutting the sections into the two proteins when this antiserum was used for small pieces and soaking them overnight in 20 ml of immunoassay of TBPA.
THYROXINE-BINDING
lmmunoassays TBPA and albumin were assayed by radial immunodiffusion (Mancini et al., 1965) on 8 X 20-cm plates coated with 26 ml agarose (lS%, British Drug Houses, Poole, Dorset) containing 0.25 ml of the appropriate antiserum. The gel was punched with 144 wells, 12 of which were reserved for duplicate applications of standards at positions randomly allocated over the plate. The purified TBPA was used to standardise a pooled quail serum which was divided into 50-~1 aliquots and stored at -20”. Purified albumin was used without preparation of a secondary standard. Standards and unknowns were diluted for assays using PBS containing 30 g/liter BSA. Both TBPA and albumin standards were diluted to give six concentrations over the range 5-50 pg/ml. Quail serum samples for assay were diluted 20 times for the TBPA assay and 1000 times for the albumin assay. Three-microliter standards and unknowns were apphed to the plate. Unknowns were applied in duplicate to wells spaced a minimum of 10 cm: apart on the plate. The plates were developed at 37” in a humid atmosphere for 24 hr, and soaked in 0.15 M NaCl for a further 24 hr. After washing with water the plates were dried and stained with Coomassie blue (British Drug Houses) according to Weeke (1973). Each application of quail serum gave single precipitin rings in both assays. The diameter of the rings was measured with a hand lens and graticule calibrated in 0. l-mm divisions and a calibration curve was drawn fram the rings of standards by plotting the square of the diameter against the concentration. The calibration was a straight line up to 50 &ml antigen, and under these assay conditions 15 ng TBPA or albumin per well was readily detectable. The standard error of replicaies in both assays was routinely less than 3%.
RESULTS
The chicken plasma TBPA used to standardise the assay and to complete the production of the anti-TBPA serum was judged ta be pure by SDS-polyacrylamide gel electrophoresis and by isoelectric focusTABLE BLOOD -
PLASMA
ALBUMIN
ANNUAL
CONCENTRATIONS CYCLE
OF DAILY
541
PREALBUMIN
sing. The molecular weight of the TBPA monomer was 13,000 and the intact protein had a pZ of 6.4. The Ajacm value at 280 nm obtained for the purpose of assay standardisation was found to be 10.4. Mean albumin concentrations (Table 1) on five occasions distributed at approximately equal intervals during the 18 months showed only minor fluctuations. There are no statistically significant (paired t test) differences between any pair of means shown in Table 1. The mean concentration of TBPA in plasma was found to change with the photoperiod to which the birds were exposed during the 18 months (Fig. 1). The data for both sexes have been pooled because there was no significant difference between their means at any time during the experiment. The results show that during January and December 1977, and January 1978, mean plasma TBPA was at a high level of approximately 500 Z.&ml. As the photoperiod increased from 7 to 15 hr from the end of January to the end of April in both years, the concentration steadily declined to almost half the winter level, reaching 280 Z.&ml in April 1977 and 240 Z&ml in April 1978. This lower level was maintained throughout the summer months when the photoperiod was in excess of 14 hr from the beginning of May until early September. A small elevation of approximately 5% of the minimum value occurred in June, which was not statistically significant (paired t test), before the concentration passed through a second minimum in September. From mid-September until De1
OF SIX
JAPANESE
PHOTOPERIODS
OVER
QUAIL
AT VARIOUS
18 CONSECUTIVE
TIMES
DURING
AN
MONTHS”
Date
Ailbumin (g/l)
31/l/77
2714177
1619177
1811178
2515178
34 k 2
29 f 6
33 ” 9
35 + 7
32 k 3
-(I Values are means ~fr SD.
542
EL-SAYED,
I
1
FMAMJ
I977
1
I
I
I
HEAF,
I
1
AND
1
GLOVER
1
I
I
I
I
I
I
1
I
JASONDJF;MAMJ MONTH
AND
YEAR
1. Variation in blood plasma TBPA concentration (a) during an annual cycle of daily photoperiods (b) over 18 consecutive months, in Japanese quail. Each point (panel a) shows the mean 2 SEM for the same six quail (three male, three female) housed throughout 18 months at 21”. The vertical arrows indicate the regular intervals of the TBPA cyclic changes in concentrations at which the mean values pass through the 290 &ml level. FIG.
cember as the photoperiod shortened from 13 to 7 hr, the TBPA concentration rose steadily to the winter level again. Thus the concentration of TBPA seems to be inversely related to the photoperiod (or directly to the scotoperiod) over the greater portion of the annual cycle. The strength of the correlation between the two is demonstrated by a linear correlation coefficient Y = -0.7 (n = 26 pairs, P < 0.01) for all the data. Selecting the data which lie outside the summer threshold region, r = -0.9 (n = 17 pairs, P < O.OOl), therefore the plasma TBPA concentration, particularly from autumn to spring, followed inversely but very closely the changing photoperiod. There was, however, a time lag or phase shift of approximately 3 weeks between the appearance of the maximal concentration of TBPA in January following the minimum photoperiod on December 22nd. This was expressed throughout the
cycle where it can be seen that the mean plasma concentration of the protein was the same (290 pg/ml) on the dates corresponding to intervals of 91 and 271 days (0.25 and 0.75 portions of the cycle) before the peak and 91 days after it (vertical arrows, Fig. 1). DISCUSSION
The similar antigenic properties of TBPA from both domestic fowl and quail which were revealed by the cross-reaction of the quail protein with the antiserum raised against domestic fowl TBPA proved helpful in the development of the immunoassay. This is because, in order to provide sufticient material for standardisation and antiserum production, a relatively large serum volume was required, TBPA being only a minor protein constituent, and this was more easily obtained from domestic fowl. The molecular weight was the same as that found by Abe et al. (1975) and the isoelec-
THYROXINE-BINDING
tric focussing technique provided further evidence that the preparation contained a single protein. The A :“c,, value at 280 nm is somewhat less than the values shown for human (Raz and Goodman, 1969) or domestic fowl TBPA (Abe et al., 1975) but this may be due to our estimation of the TBPA concentration by differential inteferometry, a method which is reliable for several proteins differing in the relative proportions of amino acids (Babul and Stellwagen, 1969). The results show that TBPA is another plasma protein in addition to RBP, whose synthesis in the liver of both sexes of Japanese quail is subject to some control by photoperiod above a minimal level. The changes in plasma TBPA, however, run quite differently and opposite to those for RBP, which were reported previously (Heaf and Glover, 1979; Glover et al., 1980). An additional indication as to the specificity of the TBPA cycle is shown by the albumin results. At times when mean TBPA concentrations were at maxima and minima those of albumin showed no significant change. Whereas the concentration of TBPA declines in spring with increasing photoperiod, that of RBP rises and remains elevated from March to July, by whieh time TBPA attains its minimal level. In August-September RBP concentration falls to minimum, while TBPA remains virtually unchanged. Then from October to December TBPA returns dramatically again to its peak winter level, but RBP increases only slightly. Since the changing photoperiod initiates the stimulus resulting in the varied synthesis and secretion of both liver proteins over the annual cycle, it is clear that the factors controlling the final stages of their biosynthesis must be quite different. Those controlling TBPA with its relatively smooth periodicity, particularly when the photoperiod is less than 14 hr (r = -0.9) would appear to have more closely tied links with the photoreceptor system
PREALBUMIN
543
than those involved with RBP where the correlation coefficient r = +0.3 was not significant (Heaf and Glover, 1979). The regular fluctuations in plasma TBPA concentration together with the fact that the protein specifically accommodates the thyroxine molecule in its structure (Blake and Oatley, 1977) implies that the protein has a much more important role in relation to thyroxine transport and metabolism than merely assisting in vitamin A transport by forming a 1:l complex with RBP. Although in man the molar ratio of TBPAl RBP remains fairly constant about 2, in the Japanese quail, it varied from 1.6 in December when TBPA is maximal to 0.6 in April when RBP reaches its peak value. At this time the high-affinity binding site for RBP would be saturated leaving only the low-affinity sites (Kopelman et al., 1976) to interact with the excess RBP, much of which must remain free as has been observed in chronic renal disease in man (Smith and Goodman, 1971). In the quail, however, this unbound RBP would tend to be metabolised faster in the normal kidney than the bound form as in the monkey (Vahlquist, 1972). Our own observations show that quail, like other birds (Tata and Shellabarger, 1959; Farer et al., 1962; Tanabe et al., 1969), lack the plasma thyroxine-binding globulin present in higher animals. TBPA and albumin are the main carriers of the hormone and if the affinity constant of the former is about 100 times that of the latter, as in man (Robbins et al., 1978) and domestic fowl (Bhat and Cama, 1978), then variations in TBPA concentration alone are very likely to influence the free thyroid hormone content of blood. A pronounced seasonal variation in the plasma thyroid hormones in the woodchuck has been attributed in part to seasonal variation in plasma thyroid hormone-binding capacity (Young et al., 1979). It seems probable that the protein has a key role in modulating the free thyroxine
544
EL-SAYED,
HEAF,
level and so creates the right environmental conditions for the metabolic changes occurring in birds for the onset of development of the gonads in the spring and for their regression in the autumn. Thyroid-gonadal interactions are relatively strong in birds and it is well known that thyroid hormones are essential for normal gonadal maturation. Thyroidectomy in the quail impairs photostimulated gonadal growth and exposure of quail to long days decreases plasma thyroxine (Peczely et al., 1979). Although the reported rise in testosterone may account for the latter, another explanation might be the fall in TBPA concentration which follows exposure to long days. It is also unlikely that the rapid changes in TBPA arise secondary to those of testosterone since they occur equally strongly in both sexes. Furthermore, under similar conditions of photoperiod TBPA shows major fluctuations during September to April when testosterone concentration is steady and the latter changes most rapidly during May to August (Follett and Maung, 1978) when TBPA remains constant. However, clarification of this issue depends on identifying the factors, presumably hormonal, which are responsible for mediating the effect of photoperiod on prealbumin synthesis. The time lag of approximately 3 weeks for the change in TBPA synthesis to become evident after changes in photoperiod in the above experiment suggests that several intermediate stages must be involved in transforming the daily fluctuations in photoperiod to the remarkably smooth annual rhythm in TBPA and these are under investigation. In further experiments to be reported later, it has been confirmed that the concentration of plasma TBPA in Japanese quail can in fact alter more quickly in response to artificially accelerated changes in photoperiod emphasising the above close correlation between the two and indicating also that a strong link
AND
GLOVER
exists between TBPA concentration and the capacity of gonads to develop and regress. ACKNOWLEDGMENTS We thank the Agricultural Research Council for their support, Grant AG 26/l 17. M. El-Sayed received a traveling research fellowship from the British Council. We are also grateful to J. Carroll for his technical assistance and to J. P. Wood Ltd. for the donation of chicken blood.
REFERENCES Abe, T., Muto, Y., and Hosoya, N. (1975). Vitamin A transport in chicken plasma: isolation and characterisation of the retinol-binding protein (RBP), prealbumin (PA). and RBP-PA complex. J. Lipid. Res. 16, 200-210. Babul, J., and Stellwagen, E. (1969). Measurement of protein concentration by interference optics. Anal. Biochem. 28, 216-221. Bhat, M. K., and Cama, H. R. (1978). Vitamin A and thyroxine carrier proteins in chicken plasma. Steady-state control of the plasma level of free retinol-binding protein and free thyroxine. Biochim. Biophys. Acta 541, 199-210. Blake, C. C. F. and Oatley, S. J. (1977). Protein-DNA and protein-hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor? Nature (London) 268, 115 - 120. Farer, L. S., Robbins, J., Blumberg, R. S., and Rail, J. E. (1962). Thyroxine-serum protein complexes in various animals. Endocrinology 70, 686-696. Follett, B. K. and Maung, S. L. (1978). Rate of testicular maturation in relation to gonadotrophin and testosterone levels, in quail exposed to various artificial photoperiods and to natural daylengths. J. Endocrinol. 78, 267-280. Glover, J., Moxley, L., Muhilal, H., and Weston, S. (1974). Micromethod for fluorimetric assay of retinal-binding protein in blood plasma. C/in. Chim. Acta 50, 371-380. Glover, J., Jay, C., Kershaw, R. C., and Reilly, P. E. B. (1976). Seasonal changes in the plasma retinol-binding holoprotein concentration of sheep. Brit. J. Nurr. 36, 137-141. Glover, J., Heaf, D. J., and Large, S. (1980). Seasonal changes in plasma retinol-binding holoprotein in Japanese quail. Brit. J. Nutr. 43, 357-366. Heaf, D. J., and Glover, J. (1979). Circannual changes in plasma concentrations of immunoreactive retinol-binding protein and luteinizing hormone in male and female Japanese quail. J. Endocrinol. 83, 323-330. Heller, J. (1976). Purification and evidence for the
THYROXINE-BINDING
identity of chicken plasma and egg yolk retinolbinding protein-prealbumin complex. Develop. Biol.
51, 1-8.
Kanai, M., Raz, A., and Goodman, D. S. (1966). Retinal-binding protein: the transport protein for vitamin A in human plasma. J. Chin. Inresr. 47, 2025-2044.
Kershaw, R. C. (1978). “Factors Controlling Plasma Retinol-Binding Protein.” Ph.D. Thesis, University of Liverpool. Kopelman, M.. Cogan, U., Mokady, S., and Shinitzky, M. (1976). The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization. Biochim. Biophys. Acta
439,
449-460.
265-275.
Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Zmmunochemistry
2, 235-254.
Pecztly, P., Astier, H., and Jallageas, M. (1979). Reciprocal interactions between testis and thyroid in male Japanese quail. Gen. Comp. Endocrinol. 37, 400-402.
Raz and Goodman (1969). interaction of thyroxin with human plasma PA-RBP complex. Biol. Chem. 244,
3230-3237.
Robbins, J., Cheng, S.-Y., Gershengorn, M. C., Glinoer, D., Cahnmann, H. J., and Edelnoch, H. (1978). Plasma thyroxine transport proteins. Rec. Prog.
Horm.
Res.
Smith, F. R., and Goodman, D. S. (1971). The effects of diseases of the liver thyroid and kidneys on the transport of vitamin A in human plasma. J. C/in. Invest.
34,
477-520.
50,
2426-2436.
Tanabe, Y., lshii, T., and Tamaki, Y. (1969). Comparison of thyroxine-binding plasma proteins of various vertebrates and their evolutionary aspects. Gen. Comp. Endocrinol. 13, 14-21. Tata, J. R., and Shellabarger, C. J. (1959). An explanation of the differences between the responses of mammals and birds to thyroxine and triiodothyronine. Biochem. J. 72, 608-613. Thompson, J. N., Howell, J. McC., and Pitt, G. A. J. (1964). Vitamin A and reproduction in rats. Proc. Roy.
Laurell, C.-B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,
545
PREALBUMIN
Sot.
Ser.
B 159, 510-535.
Vahlquist, A. (1972). Metabolism of the vitamin A transporting protein complex: Turnover of retinol-binding protein, prealbumin and vitamin A in a primate (Macaca bus). Stand. J. C/in. Lab. lnr,est.
30,
349-360.
Weber, K., Pringle, J. R., and Osborn, M. (1972). Measurement of molecular weight by electrophoresis on SDS-acrylamide gel. In “Methods in Enzymology” (C. H. W. Hirs and S. M. Timasheff, eds.), Vol. 26, pp. 3-27. Academic Press, New York. Weeke, B. (1973). Crossed Immunoelectrophoresis. Stand. J. Immunol. (Suppl. I), 2, 76-59. Wrigley, C. (1968). Gel electrofocussing: A technique for analysing protein samples by isoelectric focussing. Sci. Tools, 15, 17-23. Young, R. A., Darnforth, E., Jr., Vagenakis, A. G., Krupp, P. P., Frink, R., and Sims, E. A. H. (1979). Seasonal variation and influence of body temperature of plasma concentrations and binding of thyroxine and triiodothyronine in the woodchuck. Endocrinology 104, 996-999.
AND
COMPARATIVE
41, 539-545 (1980)
ENDOCRINOLOGY
Effect of Changing Photoperiod on Plasma Thyroxine-Binding Prealbumin in Japanese Quail (Coturnix coturnix japonica) M. Departtneni
of Biochemistry,
D. J.
EL-SAYED, University
HEAF,
of Liverpool.
Accepted
AND P.O.
February
Box
J.
GLOVER
147, Liverpool.
L69 3BX,
England
15, 1980
The concentration of thyroxine-binding prealbumin (TBPA) in blood plasma of Japanese quail is shown to be inversely related to changing photoperiod over the annual cycle and particularly from September to April. It is suggested that this protein has a key role among the various plasma factors undergoing change in conditioning animals for seasonal breeding. The changes in TBPA are opposite to, but not synchronous with, those of retinol-binding protein (RBP) and there is a wide variation in the TBPAiRBP molar ratio (0.6- 1.6) during the year, which may indirectly influence the turnover rate of vitamin A.
Thyroxine-binding prealbumin (TBPA) occupies a position of intermediate importance among the three main plasma proteins capable of transporting thyroxine in higher animals. It has a greater capacity but lower affinity for the ligand than the highly specific thyroxine-binding globulin (TBG) which is present only in certain species at 5-- 10% of the concentration of TBPA (R.obbins et al., 1978). It has a much greater atlfinity for the hormone but is present in lower concentration than the other less specific carrier, albumin. TBPA has a wider distribution in the animal kingdom than TiBG, which is reported to be absent from avian species (Tata and Shellabarger, 1959; Parer et al., 1962; Tanabe et af., 1969). Presumably its role in the thyroxine transport system in birds must be of greater significance. Another function was assigned to TBPA in connection with the transport of vitamin 4, when it was observed that it formed a 1: 1 complex with retinol-binding protein (RBP) in human plasma (Kanai et al., 1968). The interaction was considered to be of physiological importance in reducing the rate of loss of the vitamin by ultrafiltration of RBP (21,000 daltons) through the kidney. The same two proteins isolated from avian plasma also interact in this way (Abe et af., 1975; Heller, 1976).
Recent studies in our laboratory on the retinol transport system in blood plasma have shown that there is a specific twofold increase in plasma RBP concentration in sheep (Glover et al., 1976), Japanese quail (Heaf and Glover, 1979; Glover et al., 1980), and rat (Kershaw, 1978) as they reach maturity and enter their reproductive period. Retinol is known to be essential for the proper differentiation and growth of the gonads (Thompson et al., 1964) and the increased supply of RBP in plasma is considered necessary for meeting the additional demand of these tissues over the basic requirements of the immature animal. In seasonal breeders, cyclic gonadal development and regression are associated with changing photoperiod and so too are the cyclic changes in RBP. The biochemical links, however, relating the hypothalamic-pituitary-gonadal axis with the liver where the protein is synthesised have not yet been established. TBPA which is also secreted by the liver could perhaps be involved through its interaction with RBP in plasma so affecting the overall vitamin A economy of the animal. This possibility was examined by determining if changing photoperiod could influence the plasma concentration of TBPA in Japanese quail. In addition, albumin concentration was monitored to provide a control over the possibility that
539 00 16-6480/80/080539-07$0 1.0010 Copyright 0 1980 by Academic Press. Inc. All rights of reproduction m any form reserved.
540
EL-SAYED,
HEAF, AND GLOVER
changes in TBPA may be due to more general variations in liver protein synthesis.
phosphate-buffered saline [PBS, 0.15 M, NaCl, 0.05 M PO,, pH 7.5) at 4”. The PBS was decanted and the extraction of the gel pieces was repeated. Pooled PBS extracts were filtered, concentrated by ultrafiltration METHODS to 5 ml, and dialysed overnight against PBS. Small Preparation of Animals portions of the puritied TBPA and albumin showed single bromophenol blue-stained bands when subQuail, hatched in the laboratory, were reared under jected to polyacrylamide disc gel electrophoresis. For natural lighting until they were 3 months old (September-December), and were sexually immature at the standardisation purposes the protein concentration in the purified albumin was determined according to start of the experiment. They were then maintained Lowry et al. (1951) using bovine serum albumin (BSA) under artificial light (4 x 60-W daylight fluorescent tubes) for 18 months at 21” with the photoperiod con- as a standard. Using the Aiwcm value for human TBPA of 14.1 at trolled by a solar dial time switch (Sangamo Weston Ltd., Enfield, Middlesex) which simulated seasonal 280 nm (Raz and Goodman, 1%9) the yield of TBPA was found by UV spectrophotometry to be about 20 daylight changes between sunrise and sunset equivalent to those at S6”N latitude. Blood samples (1 ml) mg. TBPA, 2.5 mg, diluted to 1 ml with PBS was homogenised with 1 ml Freunds complete adjuvant were taken at intervals varying from 7 to 30 days, from (Gibco, Grand Island, N.Y.) and injected at several three male and three female birds in all, from the wing subcutaneous sites into a 3-kg male New Zealand vein, where it crosses the ulna-radial-humeral joint, directly into heparinized capillary tubes. The tubes white rabbit. After 1 month the immunization was rewere sealed at one end, centrifuged to separate the peated and 2 weeks later blood was sampled from an ear vein. Crossed immunoelectrophoresis (Weeke, plasma which was removed, frozen rapidly in liquid 1973) with the serum gave a single precipitin peak with N,, and stored at -20” until required for assay. All purified TBPA, but with quail serum, two peaks were samples were first assayed for TBPA. Thereafter, obtained, one corresponding to TBPA, and the other samples from five dates, corresponding to TBPA to albumin. Rocket immunoelectrophoresis (Laurell, maxima and minima, were assayed for albumin. 1966) of this bispecific antiserum gave two coaxial rockets with quail serum and showed the titre to be Protein Purljkation and Production of low for TBPA but at a usable level for albumin. It was Antisera therefore stored at -20” for use in the quail albumin Chicken serum was separated from blood donated immunoassay. by J. P. Wood Ltd., Llangefni, Anglesey, and stored at TBPA was further purified by a repeat slab gel elec-20” until required. Serum (300 ml) was subjected to trophoresis taking a narrower (5-mm) cut when reDEAE-Sephadex followed by CM-Sephadex (Pharmoving the protein band. After concentration of the macia, Uppsala, Sweden) ion-exchange chromatogragel extracts as before, the purity was checked by phy according to the method of Abe et al. (1975). The SDS-polyacrylamide disc gel electrophoresis (Weber fractions containing TBPA were concentrated to a et al., 1972) and isoelectric focussing (Wrigley, 1968). volume of 20 ml in an Amicon UMlO (Lexington, For standardisation purposes the TBPA concentration Mass.) ultrafiltration cell and applied to the top of a 5% was determined by using an analytical ultracentrifuge polyacrylamide slab gel (width 7 mm, length 200 mm, as a differential interferometer (Babul and Stellwagen, depth 60 mm) with gel and reservoir buffers as de- 1969). The A iv’,, was determined in the same solution scribed previously (Glover et al., 1974). Electrophoreby uv spectrophotometry at 280 nm. sis was carried out at 4” with a current of 100 mA until The further purified TBPA (0. I mg) was used to imthe position of most mobile ions, made visible by a gel munise the same rabbit 31/i months after the first imimpurity as a blue fluorescent band under ultraviolet munisation and blood was sampled 2 weeks after this light, had reached 5 mm from the bottom of the gel. booster dose. The antiserum had a usable titre for Thin slices (3 mm), three in all, were cut from both TBPA (50 pg TBPA precipitated/ml serum) on rocket edges and the centre of the gel, stained with immunoelectrophoresis and the reaction with albumin bromophenol blue, and after destaining were returned was still present, although at a considerably lower to their original positions in the gel. The TBPA stained level. The antiserum contained two separate populaas a thin band close to the blue ultraviolet fluorescent tions of antibodies against the two quail serum proteins band and was well separated from the broad band of as evidenced by the two coaxial rockets which were albumin. Using the stained gel as a guide a 7-mm sec- clearly distinguishable from one another. This comtion containing the TBPA band was cut from the slab. bined with the approximately 100-fold difference in Quail albumin was obtained in a similar manner from concentration between TBPA and albumin avoided mutual interference between the precipitin zones of 50 ml serum collected from 20 quail. The two proteins were extracted separately by cutting the sections into the two proteins when this antiserum was used for small pieces and soaking them overnight in 20 ml of immunoassay of TBPA.
THYROXINE-BINDING
lmmunoassays TBPA and albumin were assayed by radial immunodiffusion (Mancini et al., 1965) on 8 X 20-cm plates coated with 26 ml agarose (lS%, British Drug Houses, Poole, Dorset) containing 0.25 ml of the appropriate antiserum. The gel was punched with 144 wells, 12 of which were reserved for duplicate applications of standards at positions randomly allocated over the plate. The purified TBPA was used to standardise a pooled quail serum which was divided into 50-~1 aliquots and stored at -20”. Purified albumin was used without preparation of a secondary standard. Standards and unknowns were diluted for assays using PBS containing 30 g/liter BSA. Both TBPA and albumin standards were diluted to give six concentrations over the range 5-50 pg/ml. Quail serum samples for assay were diluted 20 times for the TBPA assay and 1000 times for the albumin assay. Three-microliter standards and unknowns were apphed to the plate. Unknowns were applied in duplicate to wells spaced a minimum of 10 cm: apart on the plate. The plates were developed at 37” in a humid atmosphere for 24 hr, and soaked in 0.15 M NaCl for a further 24 hr. After washing with water the plates were dried and stained with Coomassie blue (British Drug Houses) according to Weeke (1973). Each application of quail serum gave single precipitin rings in both assays. The diameter of the rings was measured with a hand lens and graticule calibrated in 0. l-mm divisions and a calibration curve was drawn fram the rings of standards by plotting the square of the diameter against the concentration. The calibration was a straight line up to 50 &ml antigen, and under these assay conditions 15 ng TBPA or albumin per well was readily detectable. The standard error of replicaies in both assays was routinely less than 3%.
RESULTS
The chicken plasma TBPA used to standardise the assay and to complete the production of the anti-TBPA serum was judged ta be pure by SDS-polyacrylamide gel electrophoresis and by isoelectric focusTABLE BLOOD -
PLASMA
ALBUMIN
ANNUAL
CONCENTRATIONS CYCLE
OF DAILY
541
PREALBUMIN
sing. The molecular weight of the TBPA monomer was 13,000 and the intact protein had a pZ of 6.4. The Ajacm value at 280 nm obtained for the purpose of assay standardisation was found to be 10.4. Mean albumin concentrations (Table 1) on five occasions distributed at approximately equal intervals during the 18 months showed only minor fluctuations. There are no statistically significant (paired t test) differences between any pair of means shown in Table 1. The mean concentration of TBPA in plasma was found to change with the photoperiod to which the birds were exposed during the 18 months (Fig. 1). The data for both sexes have been pooled because there was no significant difference between their means at any time during the experiment. The results show that during January and December 1977, and January 1978, mean plasma TBPA was at a high level of approximately 500 Z.&ml. As the photoperiod increased from 7 to 15 hr from the end of January to the end of April in both years, the concentration steadily declined to almost half the winter level, reaching 280 Z.&ml in April 1977 and 240 Z&ml in April 1978. This lower level was maintained throughout the summer months when the photoperiod was in excess of 14 hr from the beginning of May until early September. A small elevation of approximately 5% of the minimum value occurred in June, which was not statistically significant (paired t test), before the concentration passed through a second minimum in September. From mid-September until De1
OF SIX
JAPANESE
PHOTOPERIODS
OVER
QUAIL
AT VARIOUS
18 CONSECUTIVE
TIMES
DURING
AN
MONTHS”
Date
Ailbumin (g/l)
31/l/77
2714177
1619177
1811178
2515178
34 k 2
29 f 6
33 ” 9
35 + 7
32 k 3
-(I Values are means ~fr SD.
542
EL-SAYED,
I
1
FMAMJ
I977
1
I
I
I
HEAF,
I
1
AND
1
GLOVER
1
I
I
I
I
I
I
1
I
JASONDJF;MAMJ MONTH
AND
YEAR
1. Variation in blood plasma TBPA concentration (a) during an annual cycle of daily photoperiods (b) over 18 consecutive months, in Japanese quail. Each point (panel a) shows the mean 2 SEM for the same six quail (three male, three female) housed throughout 18 months at 21”. The vertical arrows indicate the regular intervals of the TBPA cyclic changes in concentrations at which the mean values pass through the 290 &ml level. FIG.
cember as the photoperiod shortened from 13 to 7 hr, the TBPA concentration rose steadily to the winter level again. Thus the concentration of TBPA seems to be inversely related to the photoperiod (or directly to the scotoperiod) over the greater portion of the annual cycle. The strength of the correlation between the two is demonstrated by a linear correlation coefficient Y = -0.7 (n = 26 pairs, P < 0.01) for all the data. Selecting the data which lie outside the summer threshold region, r = -0.9 (n = 17 pairs, P < O.OOl), therefore the plasma TBPA concentration, particularly from autumn to spring, followed inversely but very closely the changing photoperiod. There was, however, a time lag or phase shift of approximately 3 weeks between the appearance of the maximal concentration of TBPA in January following the minimum photoperiod on December 22nd. This was expressed throughout the
cycle where it can be seen that the mean plasma concentration of the protein was the same (290 pg/ml) on the dates corresponding to intervals of 91 and 271 days (0.25 and 0.75 portions of the cycle) before the peak and 91 days after it (vertical arrows, Fig. 1). DISCUSSION
The similar antigenic properties of TBPA from both domestic fowl and quail which were revealed by the cross-reaction of the quail protein with the antiserum raised against domestic fowl TBPA proved helpful in the development of the immunoassay. This is because, in order to provide sufticient material for standardisation and antiserum production, a relatively large serum volume was required, TBPA being only a minor protein constituent, and this was more easily obtained from domestic fowl. The molecular weight was the same as that found by Abe et al. (1975) and the isoelec-
THYROXINE-BINDING
tric focussing technique provided further evidence that the preparation contained a single protein. The A :“c,, value at 280 nm is somewhat less than the values shown for human (Raz and Goodman, 1969) or domestic fowl TBPA (Abe et al., 1975) but this may be due to our estimation of the TBPA concentration by differential inteferometry, a method which is reliable for several proteins differing in the relative proportions of amino acids (Babul and Stellwagen, 1969). The results show that TBPA is another plasma protein in addition to RBP, whose synthesis in the liver of both sexes of Japanese quail is subject to some control by photoperiod above a minimal level. The changes in plasma TBPA, however, run quite differently and opposite to those for RBP, which were reported previously (Heaf and Glover, 1979; Glover et al., 1980). An additional indication as to the specificity of the TBPA cycle is shown by the albumin results. At times when mean TBPA concentrations were at maxima and minima those of albumin showed no significant change. Whereas the concentration of TBPA declines in spring with increasing photoperiod, that of RBP rises and remains elevated from March to July, by whieh time TBPA attains its minimal level. In August-September RBP concentration falls to minimum, while TBPA remains virtually unchanged. Then from October to December TBPA returns dramatically again to its peak winter level, but RBP increases only slightly. Since the changing photoperiod initiates the stimulus resulting in the varied synthesis and secretion of both liver proteins over the annual cycle, it is clear that the factors controlling the final stages of their biosynthesis must be quite different. Those controlling TBPA with its relatively smooth periodicity, particularly when the photoperiod is less than 14 hr (r = -0.9) would appear to have more closely tied links with the photoreceptor system
PREALBUMIN
543
than those involved with RBP where the correlation coefficient r = +0.3 was not significant (Heaf and Glover, 1979). The regular fluctuations in plasma TBPA concentration together with the fact that the protein specifically accommodates the thyroxine molecule in its structure (Blake and Oatley, 1977) implies that the protein has a much more important role in relation to thyroxine transport and metabolism than merely assisting in vitamin A transport by forming a 1:l complex with RBP. Although in man the molar ratio of TBPAl RBP remains fairly constant about 2, in the Japanese quail, it varied from 1.6 in December when TBPA is maximal to 0.6 in April when RBP reaches its peak value. At this time the high-affinity binding site for RBP would be saturated leaving only the low-affinity sites (Kopelman et al., 1976) to interact with the excess RBP, much of which must remain free as has been observed in chronic renal disease in man (Smith and Goodman, 1971). In the quail, however, this unbound RBP would tend to be metabolised faster in the normal kidney than the bound form as in the monkey (Vahlquist, 1972). Our own observations show that quail, like other birds (Tata and Shellabarger, 1959; Farer et al., 1962; Tanabe et al., 1969), lack the plasma thyroxine-binding globulin present in higher animals. TBPA and albumin are the main carriers of the hormone and if the affinity constant of the former is about 100 times that of the latter, as in man (Robbins et al., 1978) and domestic fowl (Bhat and Cama, 1978), then variations in TBPA concentration alone are very likely to influence the free thyroid hormone content of blood. A pronounced seasonal variation in the plasma thyroid hormones in the woodchuck has been attributed in part to seasonal variation in plasma thyroid hormone-binding capacity (Young et al., 1979). It seems probable that the protein has a key role in modulating the free thyroxine
544
EL-SAYED,
HEAF,
level and so creates the right environmental conditions for the metabolic changes occurring in birds for the onset of development of the gonads in the spring and for their regression in the autumn. Thyroid-gonadal interactions are relatively strong in birds and it is well known that thyroid hormones are essential for normal gonadal maturation. Thyroidectomy in the quail impairs photostimulated gonadal growth and exposure of quail to long days decreases plasma thyroxine (Peczely et al., 1979). Although the reported rise in testosterone may account for the latter, another explanation might be the fall in TBPA concentration which follows exposure to long days. It is also unlikely that the rapid changes in TBPA arise secondary to those of testosterone since they occur equally strongly in both sexes. Furthermore, under similar conditions of photoperiod TBPA shows major fluctuations during September to April when testosterone concentration is steady and the latter changes most rapidly during May to August (Follett and Maung, 1978) when TBPA remains constant. However, clarification of this issue depends on identifying the factors, presumably hormonal, which are responsible for mediating the effect of photoperiod on prealbumin synthesis. The time lag of approximately 3 weeks for the change in TBPA synthesis to become evident after changes in photoperiod in the above experiment suggests that several intermediate stages must be involved in transforming the daily fluctuations in photoperiod to the remarkably smooth annual rhythm in TBPA and these are under investigation. In further experiments to be reported later, it has been confirmed that the concentration of plasma TBPA in Japanese quail can in fact alter more quickly in response to artificially accelerated changes in photoperiod emphasising the above close correlation between the two and indicating also that a strong link
AND
GLOVER
exists between TBPA concentration and the capacity of gonads to develop and regress. ACKNOWLEDGMENTS We thank the Agricultural Research Council for their support, Grant AG 26/l 17. M. El-Sayed received a traveling research fellowship from the British Council. We are also grateful to J. Carroll for his technical assistance and to J. P. Wood Ltd. for the donation of chicken blood.
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THYROXINE-BINDING
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Robbins, J., Cheng, S.-Y., Gershengorn, M. C., Glinoer, D., Cahnmann, H. J., and Edelnoch, H. (1978). Plasma thyroxine transport proteins. Rec. Prog.
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Tanabe, Y., lshii, T., and Tamaki, Y. (1969). Comparison of thyroxine-binding plasma proteins of various vertebrates and their evolutionary aspects. Gen. Comp. Endocrinol. 13, 14-21. Tata, J. R., and Shellabarger, C. J. (1959). An explanation of the differences between the responses of mammals and birds to thyroxine and triiodothyronine. Biochem. J. 72, 608-613. Thompson, J. N., Howell, J. McC., and Pitt, G. A. J. (1964). Vitamin A and reproduction in rats. Proc. Roy.
Laurell, C.-B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,
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Vahlquist, A. (1972). Metabolism of the vitamin A transporting protein complex: Turnover of retinol-binding protein, prealbumin and vitamin A in a primate (Macaca bus). Stand. J. C/in. Lab. lnr,est.
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Weber, K., Pringle, J. R., and Osborn, M. (1972). Measurement of molecular weight by electrophoresis on SDS-acrylamide gel. In “Methods in Enzymology” (C. H. W. Hirs and S. M. Timasheff, eds.), Vol. 26, pp. 3-27. Academic Press, New York. Weeke, B. (1973). Crossed Immunoelectrophoresis. Stand. J. Immunol. (Suppl. I), 2, 76-59. Wrigley, C. (1968). Gel electrofocussing: A technique for analysing protein samples by isoelectric focussing. Sci. Tools, 15, 17-23. Young, R. A., Darnforth, E., Jr., Vagenakis, A. G., Krupp, P. P., Frink, R., and Sims, E. A. H. (1979). Seasonal variation and influence of body temperature of plasma concentrations and binding of thyroxine and triiodothyronine in the woodchuck. Endocrinology 104, 996-999.