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Physiologic properties of steroid hormone-binding proteins in avian blood
GENERAL
AND
COMPARATIVE
Physiologic
Properties
JOHN C. WINGFIELD," The
53, 28 l-292 (1984)
ENDOCRINOLOGY
Rockefeller University Field fDepartment of Zoology,
of Steroid Hormone-Binding in Avian Blood
KATHLEEN Research University
S. MATT,~
AND DONALD
Proteins S. FARNER~
Center, Tyrrel Road, Millbrook, New of Washington, Seattle, Washington
York 12545, 98195
and
Accepted March 17, 1983 The physiologic properties of steroid hormone-binding proteins have been partially characterized in plasmas of 23 avian species (8 orders and 12 families). A specific sex hormonebinding protein (SBP) with high affinity and low capacity, as found in amphibians and some reptiles and mammals, could not be ‘identified in any of the avian species investigated. In addition SBP appeared to be totally absent in the blood of posthatching and juvenile stages of twa species, and in the embryonic blood of three species. For birds it is concluded that sex steroid hormones circulate bound to plasma albumins with low affinity (& = 10d5 mol/ liter) and very high capacity. There are two protein-binding systems for corticosterone in the blood of all species investigated, including samples collected from embryos. One is saturable and specific with low capacity (1O-*-1O-g mohliter) and high affinity (& = 10d7 - 10eg mohliter), whereas the other is a nonspecific component with very. high capacity and low affinity (Kd = 10m5moliliter). Specificity studies show that the high-affinity binding system for corticosterone also binds progesterone with virtually identical affinity, whereas testosterone and estradiol are bound with lower affinity. These data suggest that the highaffinity binding protein in avian blood has physiologic properties similar to mammalian corticosteroid-binding globulin (CBP).
The circulating concentrations of steroid hormones are functions of the rates of secretion, metabolic clearance, and uptake by target tissues. The latter two processes can be modified considerably, however, if the steroid hormones are bound to blood proteins. Steroid hormone-binding proteins fall into three major classes: albumins that bind most steroid hormones with low, affinity and very high capacity (Burton and Westphal, 1972; .Plager, 1965); corticosteroidbinding globulin (CBP), which binds primarily glucocorticosteroid hormones and progesterone (Burton and Westphal, 1972; Funder et al., 1973); and sex hormonebinding globulin (SBP), which binds sex steroid hormones with affinity and specificity that vary among species (Anderson, 1974; Petra and Schiller, 1977; Sernia et al., 1979). When bound to the high-affinity binding systems, steroid hormones are generally
thought to be biologically inactive since only free hormone can enter cells and encounter cytoplasmic receptors. A&bough it has been demonstrated recently that some protein- steroid complexes may enter the cytoplasm of target cells (Bordin and Petra, 1980), the significance of this uptake remains to be assessed. In addition, steroid hormones bound to plasma proteins are cleared less rapidly, a process that may he@ to stabilize the unbound concentration of the steroid hormone, thus providing for more precise assessment by t organ (Burton and Westphal, 1972; Anderson, 1974; Hoffman et al., 1969, Raynaud, 1973; Keller et al., 1969; A.cs and Stark, 1973). Thus the ratio of bound to unbound hormone in plasma may,have important implica&ons for endocrine investigations especially since assay techniques for plasma levels of steroid harm&es tend to measure total concentrations. Changes
281
0016-6480/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All tights of reproduction in any form resewed.
282
WINGFIELD,
MATT,
in total plasma levels may not necessarily reflect changes in the unbound levels. Variations in plasma levels of binding proteins may provide at least partial explanations of certain anomalies in endocrine investigations of avian reproduction. For example, plasma levels of testosterone do not always vary synchronously with those of luteinizing hormone (Jallageas et al., 1974; Wingfield and Farner, 1977, 1978a, b; Lam and Farner, 1976). In the whitecrowned sparrow, Zonotrichia leucophrys, plasma levels of corticosterone increase during the breeding season whereas the interrenal tissue shows histological signs of regression (Lorenzen and Farner, 1964; Wingfield and Farner, 1977, 1978a, b). However, before the physiological significance of changes in plasma levels of binding proteins can be understood, it is crucial that the physiologic properties of those proteins be understood. Although the presence of corticosteroidbinding proteins in avian blood is well established (e.g., Seal and Doe, 1966; Gould and Siegel, 1978; Daniel and Assenmacher, 1974; Wingfield et al., 1980) very little is known of the physiologic properties of binding in comparison with mammalian CBP. The presence of sex hormone-binding proteins in avian blood is somewhat controversial. Corvol and Bardin (1973) failed to detect specific high-affinity binding of testosterone in the blood of the domestic fowl, Gallus domesticus, and pigeon, Columba Zivia. On the other hand, Wenn et al. (1977) did find trace amounts of a testosterone-binding protein in the blood of pigeons and turkeys, Meleagris gallopavo, but not in the domestic fowl and mallard, Anas platyrhynchos. Information on the presence of SBP is apparently lacking for wild avian species. In this communication we report the results of an investigation of the presence and physiological properties of steroid hormone-binding proteins in the blood of 23
AND FARNER
avian species of diverse orders and families. MATERIALS
AND METHODS
Colleciion of plasma samples. Blood samples were taken from a wing vein into heparinized capillary tubes or into heparinized syringes. In some cases blood was collected via cardiac puncture. After centrifugation at 2000 rpm for 10 min, plasma was removed and stored at - 20” until analyzed. Standard and tritiated steroids. The standard steroid hormones were purchased from Sigma Chemical Company (St. Louis, MO.) and Research Plus Steroids Laboratories, Inc. (Denville, N.J.). Tritiated steroid hormones were obtained from New England Nuclear Corporation (Boston, Mass.) and purified on Celiteglycol columns before use (see Wingheld and Famer, 1975). Both standard and 3H-labeled steroid solutions were made up in phosphate-buffered saline (PBS) for dialysis and in phosphate-buffered saline plus 0.1% gelatin (PBSG) for other procedures. So-
TABLE
1
SUMMARY OF THE DISTRIBUTION OF STEROID HORMONE-BINDING PROTEINS IN AVIAN BLOOD Binding protein Corticosterone
Species Procellariiiormes
+
ND
+
+
ND
+
+
ND
+ +
f +
ND ND
+ + + + + + + +
+ + + + + + + +
ND PT ND ND PT PT ND ND
livia macroura
+ +
+ +
PT PT
auratus
+
+
ND
+ + + + + + +
+ + + + + + +
ND ND ND ND ND ND ND
homochroa
platyrhynchos
Ciconiiormes Bubulcus
ibis
Galliformes Gallus domesticus Coturnix coturnix
Charadriiformes Larus occident& Rissa tridactyla Rissa brevirostris Thalasseus maximus Uris aalge Uris lomvia Endomvchura hvooleuca Aethia &&la I’
Cohunbiformes Columba Zenaidura
Piciformes Colaptes
Testosterone
+
Oceanodroma
Anseriformes Anas
Progesterone
Passeriformes Turdus merula Molothrus ater Agelaius phoeniceus Sturnella neglecta Passer domesticus Pooecetes gramineus Zonotrichia leucophrys
Note. ND = not detectable; PT = possible trace. Estradiol17p was bound by the plasma of none of these species.
STEROID-BINDING
PROTEINS
IN AVIAN
BLOOD
.25
LD Specific
Mole
Pool
component
capacity 7.588 x IdeM/ Kd= 2.4331 x 10-gM/I Non-specific Kd= 2.785
ON
component x 10-5M/I
0 --a---
0
moles of corticosterone bound x l$?lOO ml FIG. 1. A Scatchard plot for the binding of corticosterone to protein in a pool of plasma cullected from male white-crowned sparrows, Z. leucophrys gambelii. The total binding curve, represented by the dotted line (0), reflects two binding components. Specific binding, represented by the solid line (0) was determined by the methods of Chamness and McGuire (1975). dium azide (0.1%) was added to all solutions to prevent bacterial degradation. Protein-binding studies. Initially, up to 0.5 ml of plasma samples was treated with 1.0 ml dextrancoated charcoal suspended in PBS (6.25 g charcoalNo&a, and 0.625 g Dextran T-70 in 500 ml PBS) and the mixture incubated at 40” for 30 min. Endogenous steroids are adsorbed to the charcoal that forms a pellet during centrifugation leaving in the supernatant an essentially steroid-free plasma. The uptake of steroid hormones by proteins in the steroid-free plasma was assessed as follows: unless
stated otherwise plasma was diluted to 2% in PBSG and incubated with approximately 10,000 ‘cpm of 3Hlabeled steroid at 4” for 3 hr. After incubation, bound and unbound steroids were separated by addition of 0.5 ml dextran-coated charcoal at 4” and centrifugation after an incubation time of about 10 min~(King, 1980). The supematant, containing bound hormone, was decanted into a scintillation vial, 5 ml of scintillation liquid added, and activity counted to a 2% accuracy. To determine optimal conditions for incubation, the concentration of plasma, incubation temperature, time, and concentration of 3H-labeled steroid hormone
284
WINGFIELD,
MATT,
AND FARNER
dition of charcoal as described above. The percentage [3H]corticosterone bound was then plotted versus the logarithmic dose of added steroid thus generating a series of inhibition curves. Such curves provide a more precise assessment of the specificity of binding and a quantitative measure of the competition of the various steroids. Affinity and capacity. The affinity of binding and the number of binding sites available (capacity) in avian plasma were assessed as follows: Duplicate 0.5 ml aliquots of plasma diluted to 2% in PBS were incubated with increasing quantities of unlabeled corticosterone (0.1-100 ng) and a constant amount (ca. 10,000 cpm) of [3H]corticosterone for 2 hr at 4”. Bound and free radioactivity were separated by the charcoaladsorption method. For each duplicate the bound/tinbound ratio can then be calculated and, since the total mass of steroid hormone added to each duplicate is known, the bound/unbound ratio can then be used to calculate the total mass of hormone bound (Scatchard, 1949). The slope of a line fitted to the curve by the method of least squares is the association constant (K,) and its reciprocal is the dissociation constant (ZQ. All binding affinities are expressed as & in moli liter. The intersection of the line with the abscissa is the binding capacity in moliliter. All lines fitted to these curves were corrected for nonspecific binding as suggested by Chamness and McGuire (1975). Ontogeny of steroid hormone-binding proteins. Percentage bound = 100 x 1 - (D . V, IR * V,), Thirty-six eggs from domestic fowl were incubated at 36” and 60% relative humidity. Blood samples were where R and D are the total amounts of radioactivity collected from vitellogenic veins into heparinized capinside and outside the bag, respectively, and V, and Vd illary tubes on Days 12, 17, and 19 of incubation, eight are the corresponding volumes (Plager, 1965). The eggs being sampled on each day. equilibrium dialysis technique is also useful for estiSamples were also collected from posthatching Japmating low-affinity binding. The use of dextran-coated anese quail, Coturnix coturnix, and from juvenile charcoal to separate bound and unbound moeities by white-crowned sparrows, Zonotrichia leucophrys. The adsorption is useful only if the affinity of binding is presence of steroid hormone-binding proteins was ashigh (10e7 molfliter or higher). sessed as described above. Specificity studies. An important property of highaffinity binding proteins is specificity. In general, high specificity indicates that few steroid compounds comTABLE 2 pete for binding sites on the protein. For CBP the perAFFINITY (Kd) AND CAPACITY OF CORTICOSTERONEcentage cross-reactions of a variety of steroid comBINDING PROTEIN IN PLASMAS FROM SELECTED pounds were determined as follows: Known quantities AVIAN SPECIES (lo- 100 mg) of each compound tested were incubated with diluted plasma and [3H]corticosterone at 4” for 2 Capacity Kd hr. Bound and free hormones were then separated by Species (mol/liter) (mol/liter) addition of dextran-coated charcoal. Inhibition of [3H]corticosterone uptake by the various compounds Lams occidentalis 4.47 x 10-s 3.55 x 10-s tested was expressed as a percentage cross-reaction. Columbia livia 4.78 x lO-7 7.71 x lO-s Specificity was investigated further in plasmas from Gallus domesticus 5.59 x 10-s 1.44 x 10-s four species as follows: Diluted plasma was incubated Coturnix coturnix with respective [3H]corticosterone and a series of con(male) 8.44 x lO-8 7.35 x lo-* centrations (0.001-1.00 nM) of potentially cross-reCoturnix coturnix acting steroid hormones (as determined in the first (female) 4.02 x 1O-8 6.46 x 1O-v series of specificity studies). After incubation at 4” for 7.52 x 1O-v 2.91 x lo+ Anus platyrhynchos 2 hr, bound and free moeities were separated by ad-
were varied. Nonspecific binding (e.g., albumin binding of low affinity and very high capacity) was determined by addition of a large excess on nonradioactive hormone (100 ng) to a parallel series of incubation vessels. All determinations were in duplicate or triplicate. To obtain the amount of 3H-labeled steroid hormone bound specifically by high-affinity binding proteins, the nonspecific component was subtracted from the total amount bound. Preliminary studies indicated that for the binding of corticosterone and progesterone, samples diluted to 2% in PBS and incubated with 10,000 cpm of the respective 3H-labeled steroid hormone for 2 hr at 4-5” gave reproducible results; It is possible that the dissociation of the steroid hormones from binding proteins is rapid especially when free steroid is adsorbed by charcoal, thus altering the equilibrium of bound and unbound hormone. To verify this, samples of diluted plasma (2 and 20%) from four species were placed inside dialysis bags (0.5 ml of plasma in size No. 8 dialysis tubing prewashed in distilled water) and dialyzed against a solution of 3H-labeled steroid hormone in PBS for 24-48 hr at 40”. Nonspecific binding was assessed by dialyzing a parallel series of samples against a solution of 3H-labeled steroid hormone and nonradioactive hormone (concentration 100 &ml). The percentage of steroid hormone bound can then be calculated as
Pregnenolone Deoxycorticosterone (DOC) Progesterone 17e.,21-Dihydroxy-4-pregnen3,20,dione 20o-Hydroxy-4-pregnen-3-one 5o-Pregnan-3,20-dione 17a-Hydroxyprogesterone Cortisone Cortisol Adrenosterone Androsterone Dehydroisoandrosterone A5-Androsten-38-17$-diool %Androstan-3B-17B-diol Sn-Androstan-3ol-17$-diol Etiocholanone Cholesterol Testosterone 5olDHT Estradiol Estrone _I__ I___-
Steroid
3 Sruurns
1.1 13.8 53.8 14.0 10.8 1.37 13.7 14.1 10.3 0.1 1.3 1.0 0.4 0.5 0.9 1.4 0.6 38.9 24.0 Cl.0 Cl.0
11.6 3.7 1.21 11.8 11.1 2.4 co.1 0.4 0.3 0.2 0.4 0.8 1.1 CO.1 Cl.0 Cl.0 1.6 1.9
Gallus domesticus
11.2 10.7 1.2 11.1 9.7 7.4 co.1 0.6 0.4
1.0 10.5 102.0
Colwnba livia d
18.7 3.0 1.2 11.1 11.2 3.6 0.3 0.5 0.6 co.1 0.4 0.6 1.2 co. 1 5.76
9.1 10.6 94.8
Aethia pusilla d
41.3 41.8 3.8 29.7 38.0 46.7 4.1 3.8 3.9 2.4 3.9 0.1 1.5 4.0 3.8 co. 1 -
3.4 34.3 85.0
Anus platyrhynchos 0
____-
15.4 7.8 1.4 14.4 14.8 7.3 0.4 7.7 0.2
0.8 13.8 200 +
6
Coturnix coturnix
0.7 69.3 107.0 66.7 37.8 14.4 154.9 61.5 132.2 0.2 0.4 0.3 0.1 0.3 0.3 0.4 0.2 23.0 3.5 13.9 12.0
10.3 5.9 0.2 co.1 CO.1 5.21 0.3 0.5 0.9 0.2 0.5 1.0 1.0 0.6 28.9 15.6 -
0
1.8 1.8 2.1 1.8 45.9 28.7 41.2 23.2
1.9 1.4
2.4
26.6 12.0 67.8 90.5 122.4 1.5
161.1
6.3 153.0 140.3
Zonotrichia Ieucophrys 6
protein
0.4 9.3 -
0
Percentage cross-reaction of selected steroid compounds with corticosterone-binding
1.04 11.4 102.6
8, ?
Lams occidentalis
TABLE SPECIFICITY
-
2
is 8 u
E
s
l ce Q
z (3 8
57
g
2
286
WINGFIELD,
+---
too
& ----*-------L-----o-
MATT,
AND FARNER
I-----c----~L_,~~*~
____ --DC ---* --__ A---*-----7% \ \ -ST _ _ _ - ____ 0-e-m o- --__ -. E-s N \ \ a\ \E2
-4----
-,
\
*\
\
‘1
$
‘El
\:
75
\
W
5 .$ b Y lo=
0’
\ I\ \ \
50
\ Y \ *‘El
25
.OOi
.OI steroid (nM)
.I
t
FIG. 2. Inhibition of [3H]corticosterone binding by unlabeled dihydrotesterone (DHT), testosterone (T), estradiol-17@ (Ez), estrone (El), and corticosterone (B) in plasma from western gulls (L. occident&).
RESULTS General aspects. Presence of a binding protein is indicated if the specific binding component is greater than 50% of the total amount of hormone bound. Although components binding corticosterone and progesterone were found in all 23 avian species investigated, sex hormone binding appeared to be absent in all except five (Table 1). Plasma from Rissa tridactyla, Uris aalge, U. lomvia, C. livia, and Zenaidura macroura all showed some specific uptake of testosterone indicating a possible trace of SHBP. However, none of the species investigated showed significant binding of estradiol. Physiologic properties of corticosterone-
binding protein. A Scatchard
plot for the binding of corticosterone to protein in a plasma pool collected from male whitecrowned sparrows revealed two obvious binding systems (Fig. 1). The steep part of the curve indicates high-affinity, low-capacity binding (Kd = 2.43 x 10e9 mol/liter, and 7.59 x lo-* mol/liter, respectively) and the shallow portion of the curve indicates low-affinity, nonspecific binding (Kd = 2.79 x 10e5 mol/liter) with very high capacity. Among the species examined affinity ranged from 4.78 x 1O-7 molfliter in the pigeon to 7.52 x lop9 mol/liter in the mallard, and the capacity from 1.44 x lo-* mol/liter in the domestic fowl to 6.46 x 10M9 mol/liter in the Japanese quail (Table 2). Volumes of plasma samples from the
STEROID-BINDING
PROTEINS
IN AVIAN
01
BLOOD
.l I
t.006)
(.032)
287
I
(431
steroid InM) FIG. 3. Inhibition of [3H]corticosterone binding by unlabeled androstenedione (A), dihydrotestosterone (DHT), testosterone (T), corticosterone (B), and progesterone (P) in plasma from male Japanese quail (C. coturnix).
other avian species investigated were generally insufficient for further analysis. With the exception of 2. leucophtys and A. platyrhynchos CBP appeared to be highly specific for corticosterone and progesterone in the seven species examined (Table 3). All other progestins, C21 steroid hormones, androgens, and estrogens bind much less strongly. Curiously, plasma from Z. Zeucophrys and A. platyrhynchos had a much wider cross-reaction with a variety of progestins, as well as with DOC, cortisol, and cortisone. In 2. Eeucophrys these steroid compounds were bound equally as well as corticosterone. Both, testosterone and estradiol were bound, but somewhat less strongly than corticosterone.
The specificity of CBP was investigated more rigorously in three species. The western gull, Lams occidentalis, TV+ found to have a CBP that was highly specific for corticosterone and progesterone (Table 31, a conclusion confirmed by the inhibition curves (Fig. 2). On the other hand plasma from some species reacted also with testosterone which was consistent with the inhibition curves for CBP from C. coturnix (Fig. 3), and 2. leucophrys (Fig. 4). Metahelites of testosterone (dihydrotestosterone and androstenedione) bound much less well. The plasma of all avian species investi-, gated showed binding of’ [3Hjprogesterone, a reaction that was inhibited by addition of
288
WINGFIELD,
MATT,
AND FARNER
I--
\
I--
,--
(02)
(.0046]
(.;6)
steroid (nM) FIG. 4. Inhibition of [3H]corticosterone binding by unlabeled dihydrotesterone (DHT), testosterone (T), and cortisterone (B) in plasma from male white-crowned sparrows (2. Zeuchophrys gambelii).
TABLE COMPETITIVE-BINDING ~ITII UNLABELED WHITE-CROWNED
SPARROWS,Zonotrichia
Mass of corticosterone added (ng) Male Female
4
STUDY OF [3H]~~~~~~~~~~ CORTICOSTERONE IN PLASMA
0 1 5 0 1 5
OF
leucophrys
Percentage binding of progesteronea 100 86.3 63.6 100 69.4 37.8
Note. Approximately 10,000 cpm of [3H]progesterone was added in each incubation. (2Each value is the mean of these determinations.
an excess of unlabeled progesterone. Although the specificity data (Table 3) suggest that progesterone binds to CBP, it is also possible that a specific-binding component for progesterone exists in avian plasma. If progesterone does bind mostly to CBP and not to a separate specific protein, it should be possible to inhibit uptake of [3H]progesterone by addition of unlabeled corticosterone. In Table 4 it can be seen that relatively small concentrations of corticosterone depressed uptake of [3H]progesterone in the plasma of both male and female Z. leucophrys, supporting the hypothesis that CBP binds corticosterone and progesterone
STEROID-BINDING
TABLE
PROTEINS
5
EQUILIBRIUM DIALYSIS OF AVIAN PLASMA FROM FOUR
SPECIES
Percentage bound Species Gallus domesticus
Total NS Total
Testosterone
Estradiol
67.1
89.1
62.3 67.2 64.2 63.8 57.2 53.3 48.8
88.3 83.4 82.0 75.0 76.6 64.7 64.2
IN
AVIAN
BLOOD
289
ticosterone. Addition of 10 and 25 ng unlabeled corticosterone did decrease the uptake of [3H]testosterone in plasma from Zenaidura, U. aalge, and U. lomvia (Table 6) suggesting that testosterone is binding to CBP rather than to a separate moiety. Steroid hormone-binding pi-oteins during development. Binding of testosterone and
estradiol by protein could not be demonstrated in plasma from either embryos or NS Columba &via Total chicks, with the possible exception of a NS trace of testosterone uptake in fledglings of Zonotrichia Total Zonotrichia (Table 7). However, uptake of leucophrys NS corticosterone was apparent in the blood of Note. Total = total % binding; NS = nonspecific embryos, chicks, and fledglings with an apbinding after addition of a large excess of unlabeled proximate capacity, based on percentage hormone (JO0 &ml). binding versus binding in adult blood, that was well within the range given for adults and arguing against the presence of a sep- (Table 2). There was insufficient plasma for arate progesterone-binding protein. determination of affinity and specificity. Sex hormone-binding protein. No spe- Progesterone uptake was also demoncific uptake of estradiol was found in strated in embryonic blood. plasma from any of the avian species inDISCUSSION vestigated (Table 1). However, in five species, very slight uptake of testosterone was The failure of these investigations to detected suggesting that SBP may be demonstrate the presence of SBP in avian present but not detectable by the charcoalTABLE 6 separation method. Accordingly, plasma COMPETITIVE-BINDING STUDY OF [3HI~s~~~~~~o~~ from four specieswere tested using equilibWITH UNLABELED GORTICCISTERONEINJ?LA~MAOF rium dialysis (Table 5). Again, addition of a SELECTED AV~AN SPECIES large excess of testosterone only inhibited Mass of Percentage the uptake of [3H]testosterone very slightly corticosterone binding of suggesting that most testosterone and esSpecies added (ng) [3H]testosteronea tradiol circulate bound to low-affinity and 0 100 very high-capacity proteins, e.g., albumin. Zenaidura macroura 1 84.4 It is also possible that the low-level up10 59.4 take af testosterone in some species (Table 25 40.6 1) was a result of binding to CBP (see Table Uris aalge 0 100 1 112.2 3, Figs. 3 and 4), especially since the 10 63.4 plasma tested had been treated with dex25 53.7 tran-coated charcoal to remove endoge- Vria lomvia 100 0 nous steroid hormones thus eliminating 1 107.5 competition for binding sites. If the uptake 10 41.5 ~ 34.0 2.5 of testosterone were a result of cross-reaction with CBP, then binding of 13H]testos- Note. Approximately 10,000 cpm of 13H]testosterone should be inhibited by addition of terone was added to each incubation. relatively small quantities of unlabeled cor0 Each value is the mean of three determinations. Laws
occidentalis
290
WINGFIELD,
MATT,
AND FARNER
TABLE 7 STEROID HORMONE-BINDING PROTEINS IN AVIAN BLOOD AT VARIOUS STAGES OF DEVELOPMENT
Species and stage Gallus domesticus embryo Day 12 Day 17 Day 19 Thalasseus maximus embryo Days 19-26 Bubulcus ibis embryo Days 15-18 Coturnix coturnti chicks Male 2 weeks Female 2 weeks Male 3.5 weeks Female 3.5 weeks Zonotrichia leucophrys fledglings Premolt, 30 days Midmolt, 45 days Late molt, 60 days
Corticosterone capacity (moliliter)
Progesterone
Testosterone
1.13 x 10-s 1.50 x 10-s 1.32 x lo-*
+ + +
ND ND ND
6.23 x 1O-s
+
ND
5.55 x 10-S
+
ND
lo+ 10-s 1O-8 10-s
NM NM NM NM
ND ND ND ND
1.13 x 10-S 6.30 x 1O-8 6.06 x 10-s
NM NM NM
ND FT ND
1.58 1.30 1.29 1.28
x x x x
Note. Capacity is only approximate and calculated from percentage binding of corticosterone in adult plasma. ND = not detectable; NM = not measured; PT = possible trace.
plasma from 23 species is consistent with the findings of Corvol and Bardin (1973) and Renoir et al. (1980) in domestic chickens and pigeons. Wenn et al. (1977) also failed to find SBP in the blood of domestic fowl and domestic ducks, although they did identify a trace of testosterone uptake in the plasma of turkeys and pigeons following electrophoresis on agarose gel. There is also evidence for a trace of testosterone uptake in plasma from pigeons and a related columbid species, 2. macroura, as well as from three additional species in this investigation. However, competitive binding studies showed that testosterone probably binds to CBP in these species, although the possibility that very small quantities of SBP are present cannot be precluded at this time. Nevertheless, it is unlikely that such small quantities of SBP, if present, are capable of binding significant quantities of testosterone. Thus, it appears that in avian blood, sex steroid hormones circulate bound mostly to blood proteins with low affinity and very high capacity. It
is unlikely, therefore, that the low-affinity uptake of sex steroid hormones has a significant role in the reproductive physiology of avian species. It is also apparent that SBP is absent from avian blood during development, although testosterone is present in the blood of the embryos of the domestic fowl and Japanese quail; in 2- to 3-week-old chicks of the latter and in fledgling white-crowned sparrows (Woods et al., 1975; Ottinger and Bakst, 1981; Wingfield et al., 1980). Thus, the class Aves appears to be unique among tetrapod vertebrates in that SBP iS absent at all stages of the life cycle, at least in the wide range of species studies. In contrast, SBP with varying specificity has been identified in all amphibian and reptilian species investigated to date (Martin and Ozon, 1975; Martin et al., 1971, 1972; Ozon et al., 1971; Corvol and Bardin, 1973; Sernia, 1978). However, the distribution of SBP in mammalian blood is by no means uniform and, in addition, varies greatly in specificity (see Sernia, 1978; Sernia et al., 1979;
STEROID-BINDING
PROTEINS
Burton and Westphal, 1973; Anderson, 1974; Petra and Schiller, 1977; Danzo and Eller, 1975; Raynaud, 1973; Westphal et al., 1977; see also Wingfield, 1980). The reason for such variability in the presence and properties of SBP in homeothermic vertebrates is obscure, although it has been suggested tentatively that SBP,as found in amphibians and reptiles, has been lost in higher vertebrates, and that the diverse properties of SBP in some mammals may represent secondary evolutionary development of SBP with rather specific functions (Wingfield, 1980). The physiologic properties of avian CBP appear to be identical to those of mammalian CBP and to those of other vertebrate species (e.g., Seal and Doe, 1966; Corvol and Bardin, 1973) and consonant with the findings of Daniel and Assenmacher (1974), Gould and Siegel (1974, 1978), Siegel and Gould (1976), Pdczely (1979), and Peczely and Daniel (1979) for mostly domesticated avian species. The possible functions of avian CBP in relation to the physiologic actions of corticosterone and progesterone are currently under investigation. ACKNOWLEDGMENTS These investigations were supported by Grant R805409 from the United States Environmental Protection Agency and a Cell and Molecular Biology Training Grant (GM 07270-06) to Kathleen S . Matt . Dr. Marilyn Ramenofsky kindly provided plasma samples from posthatching Japanese quail. Plasma samples from Rissa tridactyla, R. brevirostris, U. aalge, U. lomvia, and Aethia pusilla were provided by Dr. George L. Hunt, Jr.; those from Turdus merula, by Dr. Hubert Schwabl; and those from Bubulcus ibis and Thalasseus maximus, by Dr. David Vleck and Dr. Carol Vleck.
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Bordin, S., and Petra, P. H. (1980). Immunological localization of sex steroid-binding protein of plasma
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291
BLOOD
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Sci.
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Chamness, G. C., and McGuire, W. L. (1975). Scatchard plots: Common errors in correction and interpretation. Steroids 26, 538-542. Corvol, P, and Bardin, C. W. (1973). Species distribution of testosterone-binding globulin. Biol. Reprod.
8, 277-282.
Daniel, J. Y., and Assenmacher, I. (1974). Hotmortal effects on plasma binding capacity in ducks. J. Ster.
Biochem.
5, 399.
Danzo, B. J., and Eller, B. C. (1975). Steroid-binding proteins in rabbit plasma: Separation of testosterone-binding gfobulin (TeBG) from corticosteroid-binding globulin (CBG), preliminary characterization of TeBG, and changes in TeBG coneentration during sexual maturation. Mol. Cell. Endocrinol. 2, 351-368. Funder, J. W., Feldman, D., and Edelman, I. S. (1973). The roles of plasma binding and receptor specificity in the mineralocorticoid action of aldosterone. Endocrinology 92, 994-1004. Gould, N. R., and Siegel, H. S. (1974). Age variation in corticosteroid binding by serum proteins from growing chickens. Gen. Comp. Endocrinol. 24, 177-182. Gould, N. R.,.and Siegel, H. S. (1978). EfTect of age and sex on the association constant and binding capacity of chicken serum for corticosteroid. Paul.
Sci. 57, 778-784.
Hoffman, W., Forbes, T. R., and Westphal, U. (1969). Biological inactivation of progesterone by interaction with corticosteroid-binding ~globulin and with albumin. Endocrinology L&778-781. Jallageas, M., Assenmacher, J., and FoIlett, B. K. (1974). Testosterone secretion and plasma luteinizing hormone concentration during a sexual cycle in the Pekin duck, and after thyroxine .treatment. Gen.
Camp.
Endocrinol.
23, 472-475.
King, R. J. B. (1980). Quality control of estradiol receptor analysis. Cancer 46, 2822-2824. Keller, N., Richardson, U. I., and Yates, F. E. (1969). Protein binding and the biological activity ‘of corticosteroids: Zn vivo induction of l&pa& and pancreatic alanine amino transferaaes by ~corticosteroids in normal and estrogen-treated rats. Endocrinology
84, 49-62.
Lam, F., and Famer D. S., (1976). The ultratimcture of the cells of Leydig in the white-crowned sparrow (Zonotrichiu leucophrys gambeiii) in relation to plasma levels of hrteinizing hormone and testosterone. Cell Tiss. Res. 169, 93-109.
292
WINGFIELD,
MATT,
Lorenzen, L. C., and Famer, D. S. (1964). An annual cycle in the interrenal tissue of the adrenal gland of the white-crowned sparrow, Zonotrichia leucophrys gambelii. Gen. Comp. Endocrinol. 4, 253-263. Martin, B., and Ozon, R. (1975). Steroid-protein interactions in nonmammalian vertebrates II. Steroid-binding proteins in the serum of amphibians, a physiological approach. Biol. Reprod. 13, 371-380. Martin, B., Gasc, J. M., and Thibier, M. (1977). Czisteroid binding proteins and progesterone levels in chicken plasma during ontogenesis. J. Steroid Biochem. 8, 161-166. Martin, B., Lacombe, G., van Thiem, N., and Ozon, R. (1972). Mise en evidence dans le serum d’un amphibien d’une proteine liant la testosterone (PLT). Separation par chromatographie d’affinite de la PLT et de la proteine liant les steroides sexuels (ASBP). C.R. Acad. Sci. (Paris) 275, 10591062. Martin, B., Ozon, R., and Boffa, Cl. A. (1971). Liason des steroides sexuels au proteines seriques chez 1’Amphibien Urodele Pleurodeles waltlii Michah. Influence du sexe. C.R. Acad. Sci. Ser. D. 272, 1413-1416. Ottinger, M. A., and Bakst, M. R. (1981). Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail. Gen. Comp. Endocrinol. 43, 170-177. Ozon, R., Martin, B., and Boffa, G. A. (1971). Protein binding of estradiol and testosterone in newt serum (Pleurodeles waltlii Michah). Gen. Comp. Endocrinol. 17, 566-570. Peczely, P (1979). Effect of testosterone and thyroxine on corticosterone and transcortin plasma levels in different bird species. Acta Physiol. Acad. Sci. Hung. 53, 9-15. Peczely, P., and Daniel, J. Y. (1979). Interactions reciproques testothyroido-surrenaliennes chez la Caille male. Gen. Comp. Endocrinol. 39, 164-173. Petra, P H., and Schiller, H. S. (1977). Sex steroid binding protein in the plasma of Macaca nemestrina. J. Steroid Biochem. 8, 655-661. Plager, J. E. (1965). The binding of androsterone sulphate, etiocholanone sulphate and dehydroisoandrosterone sulphate by human plasma protein. J. Clin. Invest. 44, 1234-1239. Raynaud, J. P. (1973). Influence of rat estradiol binding plasma protein (EBP) on uterotrophic activity. Steroids 21, 249-258. Renoir, J.-M., Mercier-Bodard, C., and Baulieu, E. E. (1980). Hormonal and immunological aspects of the phylogeny of sex steroid-binding plasma protein. Proc. Natl. Acad. Sci. USA 77, 4578-4582. Scatchard, G. (1949). The attractions of proteins for
AND FARNER small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-672. Seal, U. S., and Doe, R. P. (1966). Corticosteroid binding globulin: Biochemistry, physiology and phylogeny. In “Steroid Dynamics” (G. Pincus, N. Nakeo, and J. E Tait, eds.), pp. 63-90. Academic Press, New York/London. Siegel, H. S., and Gould, N. R. (1976). Chick embryonic plasma proteins and binding capacity for corticosterone. Dev. Biol. 50, 510-516. Sernia, C. (1978). Steroid-binding proteins in the plasma of the echidna, Tachyglossus aculeatus, with comparative data for some marsupials and reptiles. Aust. Zool. 20, 87-98. Sernia, C., Bradley, A. J., and MacDonald, I. R. (1979). High affinity binding of adrenocortical and gonadal steroids by plasma proteins of Australian marsupials. Gen. Comp. Endocrinol. 38,496-503. Wenn: R. V., Kamberi, I. A., Keyvanja, M., and Joharmes, A. (1977). Distribution of testosterone-estradiol binding globulin (TeBG) in higher vertebrates. Endokrinologie 69, 151- 156. Westphal, U., Stroupe, S. D., Kute, T., and Cheng, S.-L. (1977). Steroid interactions with progesterone-binding globulin. J. Steroid Biochem. 8, 367-374. Wingfield, J. C. (1980). Sex steroid-binding proteins in vertebrate blood. In “Hormones, Adaptation and Evolution” (S. Ishii, T. Hirano, and M. Wada, eds.), pp. 135-144. Japan Scientific Societies Press, Tokyo/Springer-Verlag, Berlin/New York. Wingtield, J. C., and Famer, D. S. (1975). The determination of five steroids in avian plasma by radioimmunoassay and competitive protein binding. Steroids 26, 311-327. Wingtield, J. C., and Farner, D. S. (1977). Zur Endokrinologie des Fortpflanzungszyklus von Zonotrichiu leucophtys pugetensis. Vogelwarte (Sonderheft) 29, 25-32. Wingfield, J. C., and Farner, D. S. (1978a). The endocrinology of a natural breeding population of the white-crowned sparrow (Zonotrichia leucophrys pugetensis). Physiol. Zool. 51, 188-205. Wingtield, J. C., and Famer, D. S. (1978b). The annual cycle of plasma irLH and steroid hormones in feral populations of white-crowned sparrow, Zonotrichia leucophrys gambelii. Biol. Reprod. 19, 1046-1056. Wingfield, J. C., Smith, J. P., and Farner, D. S. (1980). Changes in plasma levels of luteinizing hormone, steroid, and thyroid hormones during postfledging development of white-crowned sparrows, Zonotrichia leucophrys Gen. Comp. Endocrinol. 41, 372-377. Woods, J. E., Simpson, R. M., and Moore, P. L. (1975). Plasma testosterone levels in the chick embryo. Gen. Comp. Endocrinol. 27, 543-547.
AND
COMPARATIVE
Physiologic
Properties
JOHN C. WINGFIELD," The
53, 28 l-292 (1984)
ENDOCRINOLOGY
Rockefeller University Field fDepartment of Zoology,
of Steroid Hormone-Binding in Avian Blood
KATHLEEN Research University
S. MATT,~
AND DONALD
Proteins S. FARNER~
Center, Tyrrel Road, Millbrook, New of Washington, Seattle, Washington
York 12545, 98195
and
Accepted March 17, 1983 The physiologic properties of steroid hormone-binding proteins have been partially characterized in plasmas of 23 avian species (8 orders and 12 families). A specific sex hormonebinding protein (SBP) with high affinity and low capacity, as found in amphibians and some reptiles and mammals, could not be ‘identified in any of the avian species investigated. In addition SBP appeared to be totally absent in the blood of posthatching and juvenile stages of twa species, and in the embryonic blood of three species. For birds it is concluded that sex steroid hormones circulate bound to plasma albumins with low affinity (& = 10d5 mol/ liter) and very high capacity. There are two protein-binding systems for corticosterone in the blood of all species investigated, including samples collected from embryos. One is saturable and specific with low capacity (1O-*-1O-g mohliter) and high affinity (& = 10d7 - 10eg mohliter), whereas the other is a nonspecific component with very. high capacity and low affinity (Kd = 10m5moliliter). Specificity studies show that the high-affinity binding system for corticosterone also binds progesterone with virtually identical affinity, whereas testosterone and estradiol are bound with lower affinity. These data suggest that the highaffinity binding protein in avian blood has physiologic properties similar to mammalian corticosteroid-binding globulin (CBP).
The circulating concentrations of steroid hormones are functions of the rates of secretion, metabolic clearance, and uptake by target tissues. The latter two processes can be modified considerably, however, if the steroid hormones are bound to blood proteins. Steroid hormone-binding proteins fall into three major classes: albumins that bind most steroid hormones with low, affinity and very high capacity (Burton and Westphal, 1972; .Plager, 1965); corticosteroidbinding globulin (CBP), which binds primarily glucocorticosteroid hormones and progesterone (Burton and Westphal, 1972; Funder et al., 1973); and sex hormonebinding globulin (SBP), which binds sex steroid hormones with affinity and specificity that vary among species (Anderson, 1974; Petra and Schiller, 1977; Sernia et al., 1979). When bound to the high-affinity binding systems, steroid hormones are generally
thought to be biologically inactive since only free hormone can enter cells and encounter cytoplasmic receptors. A&bough it has been demonstrated recently that some protein- steroid complexes may enter the cytoplasm of target cells (Bordin and Petra, 1980), the significance of this uptake remains to be assessed. In addition, steroid hormones bound to plasma proteins are cleared less rapidly, a process that may he@ to stabilize the unbound concentration of the steroid hormone, thus providing for more precise assessment by t organ (Burton and Westphal, 1972; Anderson, 1974; Hoffman et al., 1969, Raynaud, 1973; Keller et al., 1969; A.cs and Stark, 1973). Thus the ratio of bound to unbound hormone in plasma may,have important implica&ons for endocrine investigations especially since assay techniques for plasma levels of steroid harm&es tend to measure total concentrations. Changes
281
0016-6480/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All tights of reproduction in any form resewed.
282
WINGFIELD,
MATT,
in total plasma levels may not necessarily reflect changes in the unbound levels. Variations in plasma levels of binding proteins may provide at least partial explanations of certain anomalies in endocrine investigations of avian reproduction. For example, plasma levels of testosterone do not always vary synchronously with those of luteinizing hormone (Jallageas et al., 1974; Wingfield and Farner, 1977, 1978a, b; Lam and Farner, 1976). In the whitecrowned sparrow, Zonotrichia leucophrys, plasma levels of corticosterone increase during the breeding season whereas the interrenal tissue shows histological signs of regression (Lorenzen and Farner, 1964; Wingfield and Farner, 1977, 1978a, b). However, before the physiological significance of changes in plasma levels of binding proteins can be understood, it is crucial that the physiologic properties of those proteins be understood. Although the presence of corticosteroidbinding proteins in avian blood is well established (e.g., Seal and Doe, 1966; Gould and Siegel, 1978; Daniel and Assenmacher, 1974; Wingfield et al., 1980) very little is known of the physiologic properties of binding in comparison with mammalian CBP. The presence of sex hormone-binding proteins in avian blood is somewhat controversial. Corvol and Bardin (1973) failed to detect specific high-affinity binding of testosterone in the blood of the domestic fowl, Gallus domesticus, and pigeon, Columba Zivia. On the other hand, Wenn et al. (1977) did find trace amounts of a testosterone-binding protein in the blood of pigeons and turkeys, Meleagris gallopavo, but not in the domestic fowl and mallard, Anas platyrhynchos. Information on the presence of SBP is apparently lacking for wild avian species. In this communication we report the results of an investigation of the presence and physiological properties of steroid hormone-binding proteins in the blood of 23
AND FARNER
avian species of diverse orders and families. MATERIALS
AND METHODS
Colleciion of plasma samples. Blood samples were taken from a wing vein into heparinized capillary tubes or into heparinized syringes. In some cases blood was collected via cardiac puncture. After centrifugation at 2000 rpm for 10 min, plasma was removed and stored at - 20” until analyzed. Standard and tritiated steroids. The standard steroid hormones were purchased from Sigma Chemical Company (St. Louis, MO.) and Research Plus Steroids Laboratories, Inc. (Denville, N.J.). Tritiated steroid hormones were obtained from New England Nuclear Corporation (Boston, Mass.) and purified on Celiteglycol columns before use (see Wingheld and Famer, 1975). Both standard and 3H-labeled steroid solutions were made up in phosphate-buffered saline (PBS) for dialysis and in phosphate-buffered saline plus 0.1% gelatin (PBSG) for other procedures. So-
TABLE
1
SUMMARY OF THE DISTRIBUTION OF STEROID HORMONE-BINDING PROTEINS IN AVIAN BLOOD Binding protein Corticosterone
Species Procellariiiormes
+
ND
+
+
ND
+
+
ND
+ +
f +
ND ND
+ + + + + + + +
+ + + + + + + +
ND PT ND ND PT PT ND ND
livia macroura
+ +
+ +
PT PT
auratus
+
+
ND
+ + + + + + +
+ + + + + + +
ND ND ND ND ND ND ND
homochroa
platyrhynchos
Ciconiiormes Bubulcus
ibis
Galliformes Gallus domesticus Coturnix coturnix
Charadriiformes Larus occident& Rissa tridactyla Rissa brevirostris Thalasseus maximus Uris aalge Uris lomvia Endomvchura hvooleuca Aethia &&la I’
Cohunbiformes Columba Zenaidura
Piciformes Colaptes
Testosterone
+
Oceanodroma
Anseriformes Anas
Progesterone
Passeriformes Turdus merula Molothrus ater Agelaius phoeniceus Sturnella neglecta Passer domesticus Pooecetes gramineus Zonotrichia leucophrys
Note. ND = not detectable; PT = possible trace. Estradiol17p was bound by the plasma of none of these species.
STEROID-BINDING
PROTEINS
IN AVIAN
BLOOD
.25
LD Specific
Mole
Pool
component
capacity 7.588 x IdeM/ Kd= 2.4331 x 10-gM/I Non-specific Kd= 2.785
ON
component x 10-5M/I
0 --a---
0
moles of corticosterone bound x l$?lOO ml FIG. 1. A Scatchard plot for the binding of corticosterone to protein in a pool of plasma cullected from male white-crowned sparrows, Z. leucophrys gambelii. The total binding curve, represented by the dotted line (0), reflects two binding components. Specific binding, represented by the solid line (0) was determined by the methods of Chamness and McGuire (1975). dium azide (0.1%) was added to all solutions to prevent bacterial degradation. Protein-binding studies. Initially, up to 0.5 ml of plasma samples was treated with 1.0 ml dextrancoated charcoal suspended in PBS (6.25 g charcoalNo&a, and 0.625 g Dextran T-70 in 500 ml PBS) and the mixture incubated at 40” for 30 min. Endogenous steroids are adsorbed to the charcoal that forms a pellet during centrifugation leaving in the supernatant an essentially steroid-free plasma. The uptake of steroid hormones by proteins in the steroid-free plasma was assessed as follows: unless
stated otherwise plasma was diluted to 2% in PBSG and incubated with approximately 10,000 ‘cpm of 3Hlabeled steroid at 4” for 3 hr. After incubation, bound and unbound steroids were separated by addition of 0.5 ml dextran-coated charcoal at 4” and centrifugation after an incubation time of about 10 min~(King, 1980). The supematant, containing bound hormone, was decanted into a scintillation vial, 5 ml of scintillation liquid added, and activity counted to a 2% accuracy. To determine optimal conditions for incubation, the concentration of plasma, incubation temperature, time, and concentration of 3H-labeled steroid hormone
284
WINGFIELD,
MATT,
AND FARNER
dition of charcoal as described above. The percentage [3H]corticosterone bound was then plotted versus the logarithmic dose of added steroid thus generating a series of inhibition curves. Such curves provide a more precise assessment of the specificity of binding and a quantitative measure of the competition of the various steroids. Affinity and capacity. The affinity of binding and the number of binding sites available (capacity) in avian plasma were assessed as follows: Duplicate 0.5 ml aliquots of plasma diluted to 2% in PBS were incubated with increasing quantities of unlabeled corticosterone (0.1-100 ng) and a constant amount (ca. 10,000 cpm) of [3H]corticosterone for 2 hr at 4”. Bound and free radioactivity were separated by the charcoaladsorption method. For each duplicate the bound/tinbound ratio can then be calculated and, since the total mass of steroid hormone added to each duplicate is known, the bound/unbound ratio can then be used to calculate the total mass of hormone bound (Scatchard, 1949). The slope of a line fitted to the curve by the method of least squares is the association constant (K,) and its reciprocal is the dissociation constant (ZQ. All binding affinities are expressed as & in moli liter. The intersection of the line with the abscissa is the binding capacity in moliliter. All lines fitted to these curves were corrected for nonspecific binding as suggested by Chamness and McGuire (1975). Ontogeny of steroid hormone-binding proteins. Percentage bound = 100 x 1 - (D . V, IR * V,), Thirty-six eggs from domestic fowl were incubated at 36” and 60% relative humidity. Blood samples were where R and D are the total amounts of radioactivity collected from vitellogenic veins into heparinized capinside and outside the bag, respectively, and V, and Vd illary tubes on Days 12, 17, and 19 of incubation, eight are the corresponding volumes (Plager, 1965). The eggs being sampled on each day. equilibrium dialysis technique is also useful for estiSamples were also collected from posthatching Japmating low-affinity binding. The use of dextran-coated anese quail, Coturnix coturnix, and from juvenile charcoal to separate bound and unbound moeities by white-crowned sparrows, Zonotrichia leucophrys. The adsorption is useful only if the affinity of binding is presence of steroid hormone-binding proteins was ashigh (10e7 molfliter or higher). sessed as described above. Specificity studies. An important property of highaffinity binding proteins is specificity. In general, high specificity indicates that few steroid compounds comTABLE 2 pete for binding sites on the protein. For CBP the perAFFINITY (Kd) AND CAPACITY OF CORTICOSTERONEcentage cross-reactions of a variety of steroid comBINDING PROTEIN IN PLASMAS FROM SELECTED pounds were determined as follows: Known quantities AVIAN SPECIES (lo- 100 mg) of each compound tested were incubated with diluted plasma and [3H]corticosterone at 4” for 2 Capacity Kd hr. Bound and free hormones were then separated by Species (mol/liter) (mol/liter) addition of dextran-coated charcoal. Inhibition of [3H]corticosterone uptake by the various compounds Lams occidentalis 4.47 x 10-s 3.55 x 10-s tested was expressed as a percentage cross-reaction. Columbia livia 4.78 x lO-7 7.71 x lO-s Specificity was investigated further in plasmas from Gallus domesticus 5.59 x 10-s 1.44 x 10-s four species as follows: Diluted plasma was incubated Coturnix coturnix with respective [3H]corticosterone and a series of con(male) 8.44 x lO-8 7.35 x lo-* centrations (0.001-1.00 nM) of potentially cross-reCoturnix coturnix acting steroid hormones (as determined in the first (female) 4.02 x 1O-8 6.46 x 1O-v series of specificity studies). After incubation at 4” for 7.52 x 1O-v 2.91 x lo+ Anus platyrhynchos 2 hr, bound and free moeities were separated by ad-
were varied. Nonspecific binding (e.g., albumin binding of low affinity and very high capacity) was determined by addition of a large excess on nonradioactive hormone (100 ng) to a parallel series of incubation vessels. All determinations were in duplicate or triplicate. To obtain the amount of 3H-labeled steroid hormone bound specifically by high-affinity binding proteins, the nonspecific component was subtracted from the total amount bound. Preliminary studies indicated that for the binding of corticosterone and progesterone, samples diluted to 2% in PBS and incubated with 10,000 cpm of the respective 3H-labeled steroid hormone for 2 hr at 4-5” gave reproducible results; It is possible that the dissociation of the steroid hormones from binding proteins is rapid especially when free steroid is adsorbed by charcoal, thus altering the equilibrium of bound and unbound hormone. To verify this, samples of diluted plasma (2 and 20%) from four species were placed inside dialysis bags (0.5 ml of plasma in size No. 8 dialysis tubing prewashed in distilled water) and dialyzed against a solution of 3H-labeled steroid hormone in PBS for 24-48 hr at 40”. Nonspecific binding was assessed by dialyzing a parallel series of samples against a solution of 3H-labeled steroid hormone and nonradioactive hormone (concentration 100 &ml). The percentage of steroid hormone bound can then be calculated as
Pregnenolone Deoxycorticosterone (DOC) Progesterone 17e.,21-Dihydroxy-4-pregnen3,20,dione 20o-Hydroxy-4-pregnen-3-one 5o-Pregnan-3,20-dione 17a-Hydroxyprogesterone Cortisone Cortisol Adrenosterone Androsterone Dehydroisoandrosterone A5-Androsten-38-17$-diool %Androstan-3B-17B-diol Sn-Androstan-3ol-17$-diol Etiocholanone Cholesterol Testosterone 5olDHT Estradiol Estrone _I__ I___-
Steroid
3 Sruurns
1.1 13.8 53.8 14.0 10.8 1.37 13.7 14.1 10.3 0.1 1.3 1.0 0.4 0.5 0.9 1.4 0.6 38.9 24.0 Cl.0 Cl.0
11.6 3.7 1.21 11.8 11.1 2.4 co.1 0.4 0.3 0.2 0.4 0.8 1.1 CO.1 Cl.0 Cl.0 1.6 1.9
Gallus domesticus
11.2 10.7 1.2 11.1 9.7 7.4 co.1 0.6 0.4
1.0 10.5 102.0
Colwnba livia d
18.7 3.0 1.2 11.1 11.2 3.6 0.3 0.5 0.6 co.1 0.4 0.6 1.2 co. 1 5.76
9.1 10.6 94.8
Aethia pusilla d
41.3 41.8 3.8 29.7 38.0 46.7 4.1 3.8 3.9 2.4 3.9 0.1 1.5 4.0 3.8 co. 1 -
3.4 34.3 85.0
Anus platyrhynchos 0
____-
15.4 7.8 1.4 14.4 14.8 7.3 0.4 7.7 0.2
0.8 13.8 200 +
6
Coturnix coturnix
0.7 69.3 107.0 66.7 37.8 14.4 154.9 61.5 132.2 0.2 0.4 0.3 0.1 0.3 0.3 0.4 0.2 23.0 3.5 13.9 12.0
10.3 5.9 0.2 co.1 CO.1 5.21 0.3 0.5 0.9 0.2 0.5 1.0 1.0 0.6 28.9 15.6 -
0
1.8 1.8 2.1 1.8 45.9 28.7 41.2 23.2
1.9 1.4
2.4
26.6 12.0 67.8 90.5 122.4 1.5
161.1
6.3 153.0 140.3
Zonotrichia Ieucophrys 6
protein
0.4 9.3 -
0
Percentage cross-reaction of selected steroid compounds with corticosterone-binding
1.04 11.4 102.6
8, ?
Lams occidentalis
TABLE SPECIFICITY
-
2
is 8 u
E
s
l ce Q
z (3 8
57
g
2
286
WINGFIELD,
+---
too
& ----*-------L-----o-
MATT,
AND FARNER
I-----c----~L_,~~*~
____ --DC ---* --__ A---*-----7% \ \ -ST _ _ _ - ____ 0-e-m o- --__ -. E-s N \ \ a\ \E2
-4----
-,
\
*\
\
‘1
$
‘El
\:
75
\
W
5 .$ b Y lo=
0’
\ I\ \ \
50
\ Y \ *‘El
25
.OOi
.OI steroid (nM)
.I
t
FIG. 2. Inhibition of [3H]corticosterone binding by unlabeled dihydrotesterone (DHT), testosterone (T), estradiol-17@ (Ez), estrone (El), and corticosterone (B) in plasma from western gulls (L. occident&).
RESULTS General aspects. Presence of a binding protein is indicated if the specific binding component is greater than 50% of the total amount of hormone bound. Although components binding corticosterone and progesterone were found in all 23 avian species investigated, sex hormone binding appeared to be absent in all except five (Table 1). Plasma from Rissa tridactyla, Uris aalge, U. lomvia, C. livia, and Zenaidura macroura all showed some specific uptake of testosterone indicating a possible trace of SHBP. However, none of the species investigated showed significant binding of estradiol. Physiologic properties of corticosterone-
binding protein. A Scatchard
plot for the binding of corticosterone to protein in a plasma pool collected from male whitecrowned sparrows revealed two obvious binding systems (Fig. 1). The steep part of the curve indicates high-affinity, low-capacity binding (Kd = 2.43 x 10e9 mol/liter, and 7.59 x lo-* mol/liter, respectively) and the shallow portion of the curve indicates low-affinity, nonspecific binding (Kd = 2.79 x 10e5 mol/liter) with very high capacity. Among the species examined affinity ranged from 4.78 x 1O-7 molfliter in the pigeon to 7.52 x lop9 mol/liter in the mallard, and the capacity from 1.44 x lo-* mol/liter in the domestic fowl to 6.46 x 10M9 mol/liter in the Japanese quail (Table 2). Volumes of plasma samples from the
STEROID-BINDING
PROTEINS
IN AVIAN
01
BLOOD
.l I
t.006)
(.032)
287
I
(431
steroid InM) FIG. 3. Inhibition of [3H]corticosterone binding by unlabeled androstenedione (A), dihydrotestosterone (DHT), testosterone (T), corticosterone (B), and progesterone (P) in plasma from male Japanese quail (C. coturnix).
other avian species investigated were generally insufficient for further analysis. With the exception of 2. leucophtys and A. platyrhynchos CBP appeared to be highly specific for corticosterone and progesterone in the seven species examined (Table 3). All other progestins, C21 steroid hormones, androgens, and estrogens bind much less strongly. Curiously, plasma from Z. Zeucophrys and A. platyrhynchos had a much wider cross-reaction with a variety of progestins, as well as with DOC, cortisol, and cortisone. In 2. Eeucophrys these steroid compounds were bound equally as well as corticosterone. Both, testosterone and estradiol were bound, but somewhat less strongly than corticosterone.
The specificity of CBP was investigated more rigorously in three species. The western gull, Lams occidentalis, TV+ found to have a CBP that was highly specific for corticosterone and progesterone (Table 31, a conclusion confirmed by the inhibition curves (Fig. 2). On the other hand plasma from some species reacted also with testosterone which was consistent with the inhibition curves for CBP from C. coturnix (Fig. 3), and 2. leucophrys (Fig. 4). Metahelites of testosterone (dihydrotestosterone and androstenedione) bound much less well. The plasma of all avian species investi-, gated showed binding of’ [3Hjprogesterone, a reaction that was inhibited by addition of
288
WINGFIELD,
MATT,
AND FARNER
I--
\
I--
,--
(02)
(.0046]
(.;6)
steroid (nM) FIG. 4. Inhibition of [3H]corticosterone binding by unlabeled dihydrotesterone (DHT), testosterone (T), and cortisterone (B) in plasma from male white-crowned sparrows (2. Zeuchophrys gambelii).
TABLE COMPETITIVE-BINDING ~ITII UNLABELED WHITE-CROWNED
SPARROWS,Zonotrichia
Mass of corticosterone added (ng) Male Female
4
STUDY OF [3H]~~~~~~~~~~ CORTICOSTERONE IN PLASMA
0 1 5 0 1 5
OF
leucophrys
Percentage binding of progesteronea 100 86.3 63.6 100 69.4 37.8
Note. Approximately 10,000 cpm of [3H]progesterone was added in each incubation. (2Each value is the mean of these determinations.
an excess of unlabeled progesterone. Although the specificity data (Table 3) suggest that progesterone binds to CBP, it is also possible that a specific-binding component for progesterone exists in avian plasma. If progesterone does bind mostly to CBP and not to a separate specific protein, it should be possible to inhibit uptake of [3H]progesterone by addition of unlabeled corticosterone. In Table 4 it can be seen that relatively small concentrations of corticosterone depressed uptake of [3H]progesterone in the plasma of both male and female Z. leucophrys, supporting the hypothesis that CBP binds corticosterone and progesterone
STEROID-BINDING
TABLE
PROTEINS
5
EQUILIBRIUM DIALYSIS OF AVIAN PLASMA FROM FOUR
SPECIES
Percentage bound Species Gallus domesticus
Total NS Total
Testosterone
Estradiol
67.1
89.1
62.3 67.2 64.2 63.8 57.2 53.3 48.8
88.3 83.4 82.0 75.0 76.6 64.7 64.2
IN
AVIAN
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289
ticosterone. Addition of 10 and 25 ng unlabeled corticosterone did decrease the uptake of [3H]testosterone in plasma from Zenaidura, U. aalge, and U. lomvia (Table 6) suggesting that testosterone is binding to CBP rather than to a separate moiety. Steroid hormone-binding pi-oteins during development. Binding of testosterone and
estradiol by protein could not be demonstrated in plasma from either embryos or NS Columba &via Total chicks, with the possible exception of a NS trace of testosterone uptake in fledglings of Zonotrichia Total Zonotrichia (Table 7). However, uptake of leucophrys NS corticosterone was apparent in the blood of Note. Total = total % binding; NS = nonspecific embryos, chicks, and fledglings with an apbinding after addition of a large excess of unlabeled proximate capacity, based on percentage hormone (JO0 &ml). binding versus binding in adult blood, that was well within the range given for adults and arguing against the presence of a sep- (Table 2). There was insufficient plasma for arate progesterone-binding protein. determination of affinity and specificity. Sex hormone-binding protein. No spe- Progesterone uptake was also demoncific uptake of estradiol was found in strated in embryonic blood. plasma from any of the avian species inDISCUSSION vestigated (Table 1). However, in five species, very slight uptake of testosterone was The failure of these investigations to detected suggesting that SBP may be demonstrate the presence of SBP in avian present but not detectable by the charcoalTABLE 6 separation method. Accordingly, plasma COMPETITIVE-BINDING STUDY OF [3HI~s~~~~~~o~~ from four specieswere tested using equilibWITH UNLABELED GORTICCISTERONEINJ?LA~MAOF rium dialysis (Table 5). Again, addition of a SELECTED AV~AN SPECIES large excess of testosterone only inhibited Mass of Percentage the uptake of [3H]testosterone very slightly corticosterone binding of suggesting that most testosterone and esSpecies added (ng) [3H]testosteronea tradiol circulate bound to low-affinity and 0 100 very high-capacity proteins, e.g., albumin. Zenaidura macroura 1 84.4 It is also possible that the low-level up10 59.4 take af testosterone in some species (Table 25 40.6 1) was a result of binding to CBP (see Table Uris aalge 0 100 1 112.2 3, Figs. 3 and 4), especially since the 10 63.4 plasma tested had been treated with dex25 53.7 tran-coated charcoal to remove endoge- Vria lomvia 100 0 nous steroid hormones thus eliminating 1 107.5 competition for binding sites. If the uptake 10 41.5 ~ 34.0 2.5 of testosterone were a result of cross-reaction with CBP, then binding of 13H]testos- Note. Approximately 10,000 cpm of 13H]testosterone should be inhibited by addition of terone was added to each incubation. relatively small quantities of unlabeled cor0 Each value is the mean of three determinations. Laws
occidentalis
290
WINGFIELD,
MATT,
AND FARNER
TABLE 7 STEROID HORMONE-BINDING PROTEINS IN AVIAN BLOOD AT VARIOUS STAGES OF DEVELOPMENT
Species and stage Gallus domesticus embryo Day 12 Day 17 Day 19 Thalasseus maximus embryo Days 19-26 Bubulcus ibis embryo Days 15-18 Coturnix coturnti chicks Male 2 weeks Female 2 weeks Male 3.5 weeks Female 3.5 weeks Zonotrichia leucophrys fledglings Premolt, 30 days Midmolt, 45 days Late molt, 60 days
Corticosterone capacity (moliliter)
Progesterone
Testosterone
1.13 x 10-s 1.50 x 10-s 1.32 x lo-*
+ + +
ND ND ND
6.23 x 1O-s
+
ND
5.55 x 10-S
+
ND
lo+ 10-s 1O-8 10-s
NM NM NM NM
ND ND ND ND
1.13 x 10-S 6.30 x 1O-8 6.06 x 10-s
NM NM NM
ND FT ND
1.58 1.30 1.29 1.28
x x x x
Note. Capacity is only approximate and calculated from percentage binding of corticosterone in adult plasma. ND = not detectable; NM = not measured; PT = possible trace.
plasma from 23 species is consistent with the findings of Corvol and Bardin (1973) and Renoir et al. (1980) in domestic chickens and pigeons. Wenn et al. (1977) also failed to find SBP in the blood of domestic fowl and domestic ducks, although they did identify a trace of testosterone uptake in the plasma of turkeys and pigeons following electrophoresis on agarose gel. There is also evidence for a trace of testosterone uptake in plasma from pigeons and a related columbid species, 2. macroura, as well as from three additional species in this investigation. However, competitive binding studies showed that testosterone probably binds to CBP in these species, although the possibility that very small quantities of SBP are present cannot be precluded at this time. Nevertheless, it is unlikely that such small quantities of SBP, if present, are capable of binding significant quantities of testosterone. Thus, it appears that in avian blood, sex steroid hormones circulate bound mostly to blood proteins with low affinity and very high capacity. It
is unlikely, therefore, that the low-affinity uptake of sex steroid hormones has a significant role in the reproductive physiology of avian species. It is also apparent that SBP is absent from avian blood during development, although testosterone is present in the blood of the embryos of the domestic fowl and Japanese quail; in 2- to 3-week-old chicks of the latter and in fledgling white-crowned sparrows (Woods et al., 1975; Ottinger and Bakst, 1981; Wingfield et al., 1980). Thus, the class Aves appears to be unique among tetrapod vertebrates in that SBP iS absent at all stages of the life cycle, at least in the wide range of species studies. In contrast, SBP with varying specificity has been identified in all amphibian and reptilian species investigated to date (Martin and Ozon, 1975; Martin et al., 1971, 1972; Ozon et al., 1971; Corvol and Bardin, 1973; Sernia, 1978). However, the distribution of SBP in mammalian blood is by no means uniform and, in addition, varies greatly in specificity (see Sernia, 1978; Sernia et al., 1979;
STEROID-BINDING
PROTEINS
Burton and Westphal, 1973; Anderson, 1974; Petra and Schiller, 1977; Danzo and Eller, 1975; Raynaud, 1973; Westphal et al., 1977; see also Wingfield, 1980). The reason for such variability in the presence and properties of SBP in homeothermic vertebrates is obscure, although it has been suggested tentatively that SBP,as found in amphibians and reptiles, has been lost in higher vertebrates, and that the diverse properties of SBP in some mammals may represent secondary evolutionary development of SBP with rather specific functions (Wingfield, 1980). The physiologic properties of avian CBP appear to be identical to those of mammalian CBP and to those of other vertebrate species (e.g., Seal and Doe, 1966; Corvol and Bardin, 1973) and consonant with the findings of Daniel and Assenmacher (1974), Gould and Siegel (1974, 1978), Siegel and Gould (1976), Pdczely (1979), and Peczely and Daniel (1979) for mostly domesticated avian species. The possible functions of avian CBP in relation to the physiologic actions of corticosterone and progesterone are currently under investigation. ACKNOWLEDGMENTS These investigations were supported by Grant R805409 from the United States Environmental Protection Agency and a Cell and Molecular Biology Training Grant (GM 07270-06) to Kathleen S . Matt . Dr. Marilyn Ramenofsky kindly provided plasma samples from posthatching Japanese quail. Plasma samples from Rissa tridactyla, R. brevirostris, U. aalge, U. lomvia, and Aethia pusilla were provided by Dr. George L. Hunt, Jr.; those from Turdus merula, by Dr. Hubert Schwabl; and those from Bubulcus ibis and Thalasseus maximus, by Dr. David Vleck and Dr. Carol Vleck.
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