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Changes in plasma levels of luteinizing hormone, steroid, and thyroid hormones during the postfledging development of white-crowned sparrows, Zonotrichia leucophrys
GENERAL
AND
COMPARATIVE
41, 372-377 (1980)
ENDOCRINOLOGY
Changes in Plasma Levels of Luteinizing Hormone, Steroid, and Thyroid Hormones during the Postfledging Development of White-Crowned Sparrows, Zonotrichia leucophrys JOHNC.WINGFIELD,JAN Department
of Zoology,
University
P. SMITH,
AND DONALDS.
Washington,
of
Seattle,
FARNER
Washington
98195
Accepted August 10, 1979 Soon after becoming independent from their parents, young white-crowned sparrows molt their cryptic juvenile plumage and replace it with the first-year plumage, which is similar to that of the adult except that crown is striped with brown and tan instead of the black and white. During this molt there is in both sexes a transitory increase in the plasma level of immunoreactive luteinizing hormone, which is followed closely by an equally transient increase in circulating levels of 17/3-hydroxy-Sa-androstan-3-one (DHT). In contrast, testosterone remains at a level below the sensitivity of our assay. Corticosterone levels remain low throughout the molt. There is a significant elevation in thyroxine levels during midmolt in females only, whereas plasma levels of triiodothyronine remain unchanged in both sexes. Our observations and data suggest that the increases in plasma levels of LH and DHT in both sexes may play a role in the development of song and social behavior that permits the integration of the young into winter flocks.
Following the nestling phase of lo-12 days, young white-crowned sparrows leave the nest with the body feathers fully grown but with remiges and rectrices still developing. By the time the latter are fully formed the bird is 25-30 days old and becoming independent of its parents (Banks, 1959; Blanchard, 1941; DeWolfe, 1967; Lewis, 1971; Morton et al., 1972). It then begins the postjuvenile molt of the body feathers, or first basic molt of Humphrey and Parkes (1959). Except for stripes of brown and tan instead of black and white on the crown, the resulting plumage is essentially adult. In 1974 during a study of the Puget Sound white-crowned sparrow, Zonotrichia leucophrys pugetensis (Wingfield and Farner, 1976, 1977, 1978a) we collected blood samples from juvenile birds and found that plasma levels of immunoreactive luteinizing hormone (irLH) were as much as 12-fold as great as in adults in postnuptial or prebasic molt at the same time (J. C. Wingfield and D. S. Farner, unpublished observations). Therefore, in 1975 and again in 1976 during 0016-6480/80/070372-06$01.00/O Copyright All rights
Q 1980 by Academic Press. Inc. of reproduction in any form reserved.
an investigation of Z. 1. gambelii in central Alaska, we collected extensive series of blood samples from juvenile birds with particular attention to molt and stage of development. The results, which we communicate herein, are of particular interest in the light of recent demonstrations that the hypophysial-gonad axis plays a role in the development of voice or song and possibly of sexual imprinting in several species of birds (Abs, 1975; Lieberburg and Nottebohm, 1979; Nottebohm, 1969; Prove and Sossinka, 1978). MATERIALS
AND METHODS
.Study areas. During the summer of 197.5, blood samples were collected from a feral population of Zonotrichia leucophrys pugetensis, a short-distance migrant, on Camano Island, Island County, Washington (48”N) and in 1976 from naturally breeding populations of Z. 1. gambelii. a long-distance migrant, near Fairbanks, Alaska (64”N). Collection of samples. Birds were captured in Japanese mist nets or in live traps. A blood sample was removed from the wing vein of each into microhematocrit capillary tubes as soon as possible after capture (usually 5-10 min). Each bird was then weighed, laparotomized (gambelii only) to determine
372
PLASMA
HORMONE
LEVELS
sex and state of development of the reproductive system, and individually marked with a numbered aluminum band (U.S. Fish and Wildlife Service) and a unique combination of color bands. State in molt was determined as follows: appearance of many feather buds (pin feathers) in the ventral tracts, relatively few on the dorsal tracts, and none on the crown was designated early molt. Erupted feather buds (sheath) on both ventral and dorsal tracts and some pin feathers on the crown were designated as midmolt, and late molt was characterized by virtually fully grown feathers on the ventral and dorsal tracts and both pin and sheath feathers on the crown. The first-year plumage (aspect) is apparent in late molt. The extent of depot fat in the furculum and abdomen was designated on an arbitrary scale in which 0 is no visible fat and 5 represents bulging fat bodies. After capture and processing, birds were released for subsequent observation and in some cases recapture at a later date. Blood was centrifuged in the field and, if necessary, plasma was stored frozen on dry ice until return to the laboratory where the samples were stored at -30” until assayed. These techniques have been described in detail earlier (Wingfield and Farner, 1976). Hormone crssays. Plasma levels of LH were measured by a double antibody radioimmunoassay technique using chicken LH standards (Follett et al., 1972) and modified for use on white-crowned sparrow blood by Follett et al. (1975). Testosterone and l7/3-hydroxy-5o-androstan-3-one (DHT) were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique after chromatographic separation on Celite:propylene glycol:ethylene glycol microcolumns. For details of validation of the assays, see Wingfield and Farner (1975). Because the high levels of irLH in fledglingpugerensir might possibly be a result of a cross-reaction of thyroid-stimulating hormone (TSH) with the LH antiserum (see Follett et a/., l972), we thought it necessary to measure plasma levels of thyroxine (T4) and triiodothyronine (T3) in the blood samples from gambe/ii. If high LH levels were due to cross-reaction with increased levels of TSH, then high concentrations of T4 and also possibly T3 should also be present. T4 and T3 were measured by the radioimmunoassay procedure of Dickoff et al. (1978). Statisfics. The data were subjected to an analysis of variance, and the levels of significance determined by Newman-Keuls multiple range test for unequal sampies. Field conditions do not permit a programming of the time of day at which blood samples are taken from previously marked birds. Consequently our samples are distributed essentially randomly through the daylight hours. Obviously it is not posible to obtain samples during twilight or night. Plots of plasma levels for each of the hormones as functions of time of day at
IN JUVENILE
Zonofrichia
373
which the samples were taken provide no evidence for regular changes in levels during the daylight hours. This, of course, does not preclude the possibility that significantly different levels occur during the night. This can be approached only indirectly i.e., under controlled conditions in the laboratory. Under these conditions we have unpublished data on the plasma levels of LH and corticosterone. There are trends, mostly not statistically significant, toward higher levels at night and lower, but more stable, levels during the day. These differences, if they prove to be real, are of substantially lesser magnitude than those that we report here, and are similarly lesser than previously communicated seasonal changes in plasma levels of these hormones in this species (Wingfield and Farner, 1977, 1978a. b). We are, therefore, confident that the changes in plasma levels of hormones that we report herein cannot be attributed, in any appreciable extent, to changes in phase of entrained circadian cycles.
RESULTS
Because of the incomplete synchrony among breeding pairs and the emergence of fledglings over a period of 2-3 weeks, we have analyzed our data as a function of stage of development. There is a significant, but transient, increase in plasma irLH (P < 0.02, Fig. 1) during the first basic molt of pugetensis with maximum levels in mid molt. The ensuing increase in DHT, which cannot be evaluated statistically, represents an increase to detectable level (100 pg/ml) in our assay system. Plasma levels of testosterone remain below the detectable limit (100 pg/ml) of the assay throughout this period. The plasma levels of corticosterone, which are high following fledging, decline thereafter (P < O.OOl), whereas body weight increases (P < 0.001) to adult levels by the onset of first basic molt. Among juvenile male gambelii (Fig. 2), circulating levels of thyroid hormones, which we did not measure for pugetensis, do not change significantly although there appears to be a trend toward higher plasma levels of T4 during midmolt. There is a transitory increase in plasma level of irLH (P < 0.05). In females (Fig. 3) the increasing trend of plasma levels of T4 during the molt is significant (P < O.OS), whereas T3, as in males, does not change. Again there is
374
WINGFIELD,
SMITH,
AND
FARNER
t
FIG. 1. Body weight and plasma levels of LH, DHT, testosterone, and corticosterone in Zonorrichiu leucophrys pugefensis on Camano Island (1975) in relation to age and molt. Means + SE. Stub tails and half-tails are fledglings just out of the nest (lo- 15 days old). Juveniles are approximately 30 days old and postjuvenile molt requires 30-40 days.
a transitory increase in the circulating level of irLH (P < 0.05) followed closely by a similar change in the level of DHT (P < 0.05). In both males and females, plasma concentrations of testosterone remain below the sensitivity of the assay (30-40 pg/ml in assays for gambelii). Plasma levels of corticosterone remain low throughout the molt (Figs. 2 and 3) but increase significantly during migration (P < 0.05). Just prior to the onset of migration, the amount of depot fat and, correspondingly, body weight, increase significantly (P < 0.05). DISCUSSION
The transitory increase in the plasma level of T4 in female Zonorrichia feucophrys gambelii, and the similar trend in males (Figs. 2 and 3), raises the possibility that the increased level of LH might be, at least in part, an artifact from cross-reaction of TSH with the LH antiserum. Although this cannot be precluded with absolute certainty, it should be emphasized that the
FIG. 2. Body weight, fat depots, and plasma levels of LH, DHT, testosterone, corticosterone, T3, and T4 in fledgling male Zonotrichia Ieucophrys gambefii in central Alaska (1976). Means 2 SE representing stage in development or molt and spaced according to the mean time taken for first basic molt and other functions in this race. (See Results for details.) The dark bar represents a period of complete first-year plumage followed in the next period by premigratory hyperphagia and fattening.
cross-reaction of this antiserum to TSH from the domestic fowl is very slight (Follett et al., 1972). Moreover the ensuing elevation in plasma level of DHT is most probably induced by the prior increase of LH, since avian LH is steroidogenic (e.g. Jenkins et al., 1978; Maung and Follett, 1977). Furthermore, LH levels are basal during prebasic molt in adults (Wingfield and Farner, 1978b) at a time when T4 levels and presumably TSH are high (Smith, 1978). We therefore regard the increases in LH (Figs. 1- 3) as real rather than the result of interference by TSH in the assay system. It is puzzling that we found no parallel changes in plasma levels of testosterone, although such could be occurring at levels below the sensitivity of the assay with rapid conversion to DHT.
PLASMA
HORMONE
LEVELS
FIG. 3. Body weight, fat depots, and plasma levels of LH, DHT, testosterone, corticosterone, T3, and T4 in fledgling female 2. 1. gambelii in central Alaska (1976). Means + SE representing stage in development or molt and spaced according to the mean time taken for first basic molt and other functions in this species. (See Results for details.) The dark bar represents a period of complete first-year plumage followed in the next period by premigratory hyperphagia and fat-
We have three hypotheses, not necessarily mutually exclusive, to explain the transitory elevations in plasma levels of LH and DHT. The first supposes a maturation of the hypothalamo-hypophysial-gonad axis, resulting in an increase in plasma levels of LH which, in turn, stimulate an increased rate of secretion of DHT and hence an elevated plasma level, or an increased rate of secretion of testosterone that is rapidly converted to DHT. A negative feedback system is established, resulting in a stabilization of plasma levels after the initially high levels. Very little information is available, but Woods et al. (1977) have shown that the adenohypophysialgonad axis is established as early as Day 13.5 of incubation in the chick embryo. Furthermore, plasma testosterone and LH
IN
JUVENILE
Zonotrichia
375
levels are elevated in ducklings (Storey and Nicholls, 1977) and juvenile domestic fowl (Sharp, 1975), also suggesting that this axis is established at a very early age. A second hypothesis proposes that the hypothalamo - hypophysial -gonadal axis is established and that the LH and DHT levels in the blood become elevated as a result of development of photosensitivity. Days are long at this time of year (July and August) and stimulatory in terms of gonadotropin secretion (Follett et al., 1975). However, this hypothesis must also provide a very rapid development of photorefractoriness to avoid an aseasonal gonadal maturation. It is known that first-year gambelii are photorefractory, at least after completion of the first basic molt, and become photosensitive in the late fall at the same time as adults (Farner and Mewaldt, 1955). The third hypothesis, mutually compatible with either the first or second, ascribes a behavioral function to the increases in plasma levels of LH and DHT. Field observations (J. C. Wingfield, unpublished) indicate that young in the first basic molt begin to form flocks and to use communal roosts, often involving many hundred individuals. Numerous interactions, such as chases, fighting, and supplanting, occur at this time. Also both male and female young begin to sing. At first the song is characterized by hoarse whistles and burry notes, but by completion of molt the song is entirely recognizable as that of a whitecrowned sparrow. It lacks the pure whistles and well-defined trills of the sexual and territorial song of the breeding males, and thus is better designated as subsong or winter song. This song occurs throughout the winter and is especially noticeable in evenings as birds begin to accumulate at roosts. Perhaps it affords some sort of individual recognition, since males and females, adults and young, are integrated together in these flocks. It is now established that androgens are necessary for normal development of sex-
376
WINGFIELD,
SMITH,
ual song or at least the frequency of singing (e.g., Arnold, 1975; Nottebohm, 1969; Prove, 1974), and also for the “solitary” song of the zebra finch, Poephila guttata (Prove, 1974). Furthermore, androgen uptake has been demonstrated in areas of the brain related to vocal control (Zigmond et al., 1973; Arnold et al., 1976) and also in the muscles of the syrinx (Lieberburg and Nottebohm, 1979). Elevated plasma levels of LH and androgens during postfledging development have also been noted in the domestic pigeon (Abs et al., 1977), in which the level of plasma LH increases just prior to the breaking of voice at about 50 days of age, possibly accompanied by a rise in plasma androgen (Abs and Dahlmann, 1974). Similarly, in the zebra finch, Prove and Sossinka (1978) found a transitory elevation of plasma testosterone levels at 31-40 days of age, a time that is important for sexual imprinting in this species. Therefore, we suggest that the transitory increase in the plasma level of DHT in fledglings of Zonotrichia leucophrys, both males and females, is involved in the development of at least the winter song and possibly other social behavior. Young male white-crowned sparrows learn the sexual song at a very early age but do not reproduce it until the following spring (Marler, 1967), presumably under the influence of the very high vernal levels of circulating testosterone (Wingfield and Farner, 1977, 1978a, b). Females do not normally express sexual song and do not develop very high levels of testosterone (Wingfield and Farner, 1977, 1978a, b), but can be induced to sing in spring by injections of testosterone propionate (Kern and King, 1972). Thus, another question can be raised: Is there a qualitative difference in that DHT, which occurs at approximately the same levels in both sexes, may stimulate winter song, whereas testosterone is required in high concentrations, normally characteristic of males only, for the development of
AND
FARNER
sexual song? However, it is also possible that winter song has a much lower threshold to testosterone than sexual song (Prove, 1974). ACKNOWLEDGMENTS We are indebted to Professor George West and the Institute of Arctic Biology, University of Alaska, College, Alaska, for provision of facilities and advice while conducting field work in central Alaska. Thanks are also due to Mr. P. W. Mattocks, Jr., who assisted with the assay of luteinizing hormone, and to Professor B. K. Follett who provided the LH antiserum and LH standards. The investigations reported herein were supported by Grant PCM 77-17690 from the National Science Foundation, and by a grant from the Graduate School Research Fund, University of Washington.
REFERENCES Abs, M. (1975). Zur Entwicklung der Lautausserungen bei der Haustaube (Columba livia domestica). Verb.
Deut.
Zool.
Ges.
1974,
347-350.
Abs, M., Ashton, S. A., and Follett, B. K. (1977). Plasma luteinizing hormone in juvenile pigeons around the time of breaking of the voice. Horm. Behac. 9, 1899192. Abs, M., and Dahlmann, B. (1974). Der Zusammenhang zwischen dem sogenannten Stimmbruch und der taglichen Ketosteroidauscheidung bei juvenilen Haustauben (Columba /ivia domestica). Experentia
30, %9-970.
Arnold, A. P. (1975). The effects of castration on song development in zebra finches (Poephila guttata). J. Exp. Zool. 191, 261-278. Arnold, A. P., Nottebohm, F., and Pfaff, D. W. (1976). Hormone concentrating cells in vocal control and other areas of the brain of the zebra finch (Poephila guttata). J. Comp. Neurol. 165, 487-512. Banks, R. C. (1959). Development of nestbng whitecrowned sparrows in central coastal California. Condor
61,
96-
109.
Blanchard, B. D. (1941). The white-crowned sparrows (Zonotrichiu letrcophrys) of the Pacific seaboard: Environment and annual cycle. Univ. Caiif. Pub/. Zool. 46, 1-178. DeWolfe, B. B. (1967). Biology of white-crowned sparrows in late summer at College, Alaska. Condor 69, 1 lo- 132. Dickhoff, W. W., Folmar, L. C., and Gorbman, A. (1978). Changes in plasma thyroxine during smoltitication of coho salmon, Onchorhynchus kisutch. Gen.
Comp.
Endocrinol.
36, 229-232.
Farner, D. S., and Mewaldt, L. R. (1955). The natural termination of the refractory period in the whitecrowned sparrow. Condor 57, 112- 116.
PLASMA
HORMONE
LEVELS
Follett, B. K., Farner, D. S., and Mattocks, P. W., Jr. (1975). Luteinizing hormone in the plasma of white-crowned sparrows, Zonotrichia leucophrys gambelii, during artificial photostimulation. Gen. Comp. Endocrinol. 26, 126- 134. Follett, B. K., Scanes, C. G., and Cunningham, F. J. (1972). A radioimmunoassay for avian luteinizing hormone. J. Endocrinol. 52, 359-378. Humphrey, P. S., and Parkes, K. C. (1959). An approach to the study of molts and plumages. Auk 76, l-31. Jenkins, N., Sumpter, J. P., and Follett, B. K. (1978). The effects of vertebrate gonadotropins on androgen release in vitro from testicular cells of Japanese quail and a comparison with their radioimmunoassay activities. Gen. Camp. Endocrinol. 35, 309-321. Kern, M. D., and King, J. R. (1972). Testosteroneinduced singing in female white-crowned sparrows. Condor 74, 204-209. Lewis, R. A. (1971). “The Temporal Organization of Reproductive and Associated Cycles of the Puget Sound Sparrow, Zonotrichia leucophrys pugetensis.” Ph.D. Dissertation. University of Washington, Seattle. Lieberburg, I., and Nottebohm, F. (1979). Highaffhtity androgen binding proteins in syringeal tissues of songbirds. Gen. Comp. Endocrinol; 37, 286-293. Marler, P. (1967). Comparative study of song development in sparrows. In “Proceedings of the XIV International Ornithological Congress at Oxford” (D. W. Snow, ed.), pp. 231-244. Blackwell, Oxford/Edinburgh. Maung, 2. W., and Follett, B. K. (1977). Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testis. Gen. Comp. Endocrinol.
33, 242-253.
Morton, M. L., Orejuela, J., and Budd, S. (1972). The biology of immature mountain white-crowned sparrows (Zonotrichia leucophrys oriantha), on the breeding ground. Condor 74, 423-430. Nottebohm, F. (1969). The “critical period” for song learning. Ibis 111, 386-387. Prove, E. (1974). Der Einfluss von Kastration und Testosteronsubstitution auf das Sexualverhalten
IN JUVENILE
377
Zonotrichia
mannlicher
Zebrafinken (Taeniopygia guttata Gould). J. Ornithol. 115, 338-347. Prove, E., and Sossinka, R. (1978). Testosterontiter in der Entwicklung junger Zebrafinken-6 . J. Ornithol. 119, 235. Sharp, P. J. (1975). A comparison of variations in plasma luteinizing hormone concentrations in male and female domestic chickens (Callus domesticus) from hatch to sexual maturity. J. Endocrinol. 67, 211-223. Smith, J. P. (1978). Annual cycles of thyroid hormones in the plasma of white-crowned sparrows (Zonotrichia Ieucophrys gambelii) and house sparrows (Passer domesticus). Amer. Zool. 18, 591. Storey, C. R., and Nicholls, T. J. (1977). Plasma luteinizing hormone levels following ovariectomy of juvenile female mallard (Anas platyrhynchos). Gen. Comp. Endocrinol. 33, 8- 12. Wingfield, J. C., and Farner, D. S. (1975). The determination of five steroids in avian plasma by radioimmunoassay and competitive proteinbinding. Steroids 26, 3 11-327. Wingfield, J. C., and Farner, D. S. (1976). Avian endocrinology-Field investigations and methods. Condor 78, 570-573. Wingfield, J. C., and Farner, D. S. (1977). Zur Endokrinologie des Fortpflanzungszyklus von Zonocastanotis
trichia derheff)
leucophrys 29, 25-32.
pugetensis.
Vogelwarte
(Son-
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, 189-205. Wingfield, J. C., and Farner, D. S. (1978;;. The annual cycle in plasma irLH and steroid hormones in feral populations of the white-crowned sparrow, Zonotrichia
leucophrys
gamhelii.
Biol.
Reprod.
19, 1046- 1056. Woods, J. E., Podczaski, E. S., Erton, L. H., Rutherford, J. E., and McCarter, C. F. (1977). Establishment of the adenohypophyseal-testitular axis in the chick embryo. I. Testicular androgen levels. Gen. Comp. Endocrinol. 32, 390-394. Zigmond, R. E., Nottebohm, F., and Pfaff, D. W. (1973). Androgen concentrating cells in the midbrain of a song bird. Science 179, 1005- 1007.
AND
COMPARATIVE
41, 372-377 (1980)
ENDOCRINOLOGY
Changes in Plasma Levels of Luteinizing Hormone, Steroid, and Thyroid Hormones during the Postfledging Development of White-Crowned Sparrows, Zonotrichia leucophrys JOHNC.WINGFIELD,JAN Department
of Zoology,
University
P. SMITH,
AND DONALDS.
Washington,
of
Seattle,
FARNER
Washington
98195
Accepted August 10, 1979 Soon after becoming independent from their parents, young white-crowned sparrows molt their cryptic juvenile plumage and replace it with the first-year plumage, which is similar to that of the adult except that crown is striped with brown and tan instead of the black and white. During this molt there is in both sexes a transitory increase in the plasma level of immunoreactive luteinizing hormone, which is followed closely by an equally transient increase in circulating levels of 17/3-hydroxy-Sa-androstan-3-one (DHT). In contrast, testosterone remains at a level below the sensitivity of our assay. Corticosterone levels remain low throughout the molt. There is a significant elevation in thyroxine levels during midmolt in females only, whereas plasma levels of triiodothyronine remain unchanged in both sexes. Our observations and data suggest that the increases in plasma levels of LH and DHT in both sexes may play a role in the development of song and social behavior that permits the integration of the young into winter flocks.
Following the nestling phase of lo-12 days, young white-crowned sparrows leave the nest with the body feathers fully grown but with remiges and rectrices still developing. By the time the latter are fully formed the bird is 25-30 days old and becoming independent of its parents (Banks, 1959; Blanchard, 1941; DeWolfe, 1967; Lewis, 1971; Morton et al., 1972). It then begins the postjuvenile molt of the body feathers, or first basic molt of Humphrey and Parkes (1959). Except for stripes of brown and tan instead of black and white on the crown, the resulting plumage is essentially adult. In 1974 during a study of the Puget Sound white-crowned sparrow, Zonotrichia leucophrys pugetensis (Wingfield and Farner, 1976, 1977, 1978a) we collected blood samples from juvenile birds and found that plasma levels of immunoreactive luteinizing hormone (irLH) were as much as 12-fold as great as in adults in postnuptial or prebasic molt at the same time (J. C. Wingfield and D. S. Farner, unpublished observations). Therefore, in 1975 and again in 1976 during 0016-6480/80/070372-06$01.00/O Copyright All rights
Q 1980 by Academic Press. Inc. of reproduction in any form reserved.
an investigation of Z. 1. gambelii in central Alaska, we collected extensive series of blood samples from juvenile birds with particular attention to molt and stage of development. The results, which we communicate herein, are of particular interest in the light of recent demonstrations that the hypophysial-gonad axis plays a role in the development of voice or song and possibly of sexual imprinting in several species of birds (Abs, 1975; Lieberburg and Nottebohm, 1979; Nottebohm, 1969; Prove and Sossinka, 1978). MATERIALS
AND METHODS
.Study areas. During the summer of 197.5, blood samples were collected from a feral population of Zonotrichia leucophrys pugetensis, a short-distance migrant, on Camano Island, Island County, Washington (48”N) and in 1976 from naturally breeding populations of Z. 1. gambelii. a long-distance migrant, near Fairbanks, Alaska (64”N). Collection of samples. Birds were captured in Japanese mist nets or in live traps. A blood sample was removed from the wing vein of each into microhematocrit capillary tubes as soon as possible after capture (usually 5-10 min). Each bird was then weighed, laparotomized (gambelii only) to determine
372
PLASMA
HORMONE
LEVELS
sex and state of development of the reproductive system, and individually marked with a numbered aluminum band (U.S. Fish and Wildlife Service) and a unique combination of color bands. State in molt was determined as follows: appearance of many feather buds (pin feathers) in the ventral tracts, relatively few on the dorsal tracts, and none on the crown was designated early molt. Erupted feather buds (sheath) on both ventral and dorsal tracts and some pin feathers on the crown were designated as midmolt, and late molt was characterized by virtually fully grown feathers on the ventral and dorsal tracts and both pin and sheath feathers on the crown. The first-year plumage (aspect) is apparent in late molt. The extent of depot fat in the furculum and abdomen was designated on an arbitrary scale in which 0 is no visible fat and 5 represents bulging fat bodies. After capture and processing, birds were released for subsequent observation and in some cases recapture at a later date. Blood was centrifuged in the field and, if necessary, plasma was stored frozen on dry ice until return to the laboratory where the samples were stored at -30” until assayed. These techniques have been described in detail earlier (Wingfield and Farner, 1976). Hormone crssays. Plasma levels of LH were measured by a double antibody radioimmunoassay technique using chicken LH standards (Follett et al., 1972) and modified for use on white-crowned sparrow blood by Follett et al. (1975). Testosterone and l7/3-hydroxy-5o-androstan-3-one (DHT) were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique after chromatographic separation on Celite:propylene glycol:ethylene glycol microcolumns. For details of validation of the assays, see Wingfield and Farner (1975). Because the high levels of irLH in fledglingpugerensir might possibly be a result of a cross-reaction of thyroid-stimulating hormone (TSH) with the LH antiserum (see Follett et a/., l972), we thought it necessary to measure plasma levels of thyroxine (T4) and triiodothyronine (T3) in the blood samples from gambe/ii. If high LH levels were due to cross-reaction with increased levels of TSH, then high concentrations of T4 and also possibly T3 should also be present. T4 and T3 were measured by the radioimmunoassay procedure of Dickoff et al. (1978). Statisfics. The data were subjected to an analysis of variance, and the levels of significance determined by Newman-Keuls multiple range test for unequal sampies. Field conditions do not permit a programming of the time of day at which blood samples are taken from previously marked birds. Consequently our samples are distributed essentially randomly through the daylight hours. Obviously it is not posible to obtain samples during twilight or night. Plots of plasma levels for each of the hormones as functions of time of day at
IN JUVENILE
Zonofrichia
373
which the samples were taken provide no evidence for regular changes in levels during the daylight hours. This, of course, does not preclude the possibility that significantly different levels occur during the night. This can be approached only indirectly i.e., under controlled conditions in the laboratory. Under these conditions we have unpublished data on the plasma levels of LH and corticosterone. There are trends, mostly not statistically significant, toward higher levels at night and lower, but more stable, levels during the day. These differences, if they prove to be real, are of substantially lesser magnitude than those that we report here, and are similarly lesser than previously communicated seasonal changes in plasma levels of these hormones in this species (Wingfield and Farner, 1977, 1978a. b). We are, therefore, confident that the changes in plasma levels of hormones that we report herein cannot be attributed, in any appreciable extent, to changes in phase of entrained circadian cycles.
RESULTS
Because of the incomplete synchrony among breeding pairs and the emergence of fledglings over a period of 2-3 weeks, we have analyzed our data as a function of stage of development. There is a significant, but transient, increase in plasma irLH (P < 0.02, Fig. 1) during the first basic molt of pugetensis with maximum levels in mid molt. The ensuing increase in DHT, which cannot be evaluated statistically, represents an increase to detectable level (100 pg/ml) in our assay system. Plasma levels of testosterone remain below the detectable limit (100 pg/ml) of the assay throughout this period. The plasma levels of corticosterone, which are high following fledging, decline thereafter (P < O.OOl), whereas body weight increases (P < 0.001) to adult levels by the onset of first basic molt. Among juvenile male gambelii (Fig. 2), circulating levels of thyroid hormones, which we did not measure for pugetensis, do not change significantly although there appears to be a trend toward higher plasma levels of T4 during midmolt. There is a transitory increase in plasma level of irLH (P < 0.05). In females (Fig. 3) the increasing trend of plasma levels of T4 during the molt is significant (P < O.OS), whereas T3, as in males, does not change. Again there is
374
WINGFIELD,
SMITH,
AND
FARNER
t
FIG. 1. Body weight and plasma levels of LH, DHT, testosterone, and corticosterone in Zonorrichiu leucophrys pugefensis on Camano Island (1975) in relation to age and molt. Means + SE. Stub tails and half-tails are fledglings just out of the nest (lo- 15 days old). Juveniles are approximately 30 days old and postjuvenile molt requires 30-40 days.
a transitory increase in the circulating level of irLH (P < 0.05) followed closely by a similar change in the level of DHT (P < 0.05). In both males and females, plasma concentrations of testosterone remain below the sensitivity of the assay (30-40 pg/ml in assays for gambelii). Plasma levels of corticosterone remain low throughout the molt (Figs. 2 and 3) but increase significantly during migration (P < 0.05). Just prior to the onset of migration, the amount of depot fat and, correspondingly, body weight, increase significantly (P < 0.05). DISCUSSION
The transitory increase in the plasma level of T4 in female Zonorrichia feucophrys gambelii, and the similar trend in males (Figs. 2 and 3), raises the possibility that the increased level of LH might be, at least in part, an artifact from cross-reaction of TSH with the LH antiserum. Although this cannot be precluded with absolute certainty, it should be emphasized that the
FIG. 2. Body weight, fat depots, and plasma levels of LH, DHT, testosterone, corticosterone, T3, and T4 in fledgling male Zonotrichia Ieucophrys gambefii in central Alaska (1976). Means 2 SE representing stage in development or molt and spaced according to the mean time taken for first basic molt and other functions in this race. (See Results for details.) The dark bar represents a period of complete first-year plumage followed in the next period by premigratory hyperphagia and fattening.
cross-reaction of this antiserum to TSH from the domestic fowl is very slight (Follett et al., 1972). Moreover the ensuing elevation in plasma level of DHT is most probably induced by the prior increase of LH, since avian LH is steroidogenic (e.g. Jenkins et al., 1978; Maung and Follett, 1977). Furthermore, LH levels are basal during prebasic molt in adults (Wingfield and Farner, 1978b) at a time when T4 levels and presumably TSH are high (Smith, 1978). We therefore regard the increases in LH (Figs. 1- 3) as real rather than the result of interference by TSH in the assay system. It is puzzling that we found no parallel changes in plasma levels of testosterone, although such could be occurring at levels below the sensitivity of the assay with rapid conversion to DHT.
PLASMA
HORMONE
LEVELS
FIG. 3. Body weight, fat depots, and plasma levels of LH, DHT, testosterone, corticosterone, T3, and T4 in fledgling female 2. 1. gambelii in central Alaska (1976). Means + SE representing stage in development or molt and spaced according to the mean time taken for first basic molt and other functions in this species. (See Results for details.) The dark bar represents a period of complete first-year plumage followed in the next period by premigratory hyperphagia and fat-
We have three hypotheses, not necessarily mutually exclusive, to explain the transitory elevations in plasma levels of LH and DHT. The first supposes a maturation of the hypothalamo-hypophysial-gonad axis, resulting in an increase in plasma levels of LH which, in turn, stimulate an increased rate of secretion of DHT and hence an elevated plasma level, or an increased rate of secretion of testosterone that is rapidly converted to DHT. A negative feedback system is established, resulting in a stabilization of plasma levels after the initially high levels. Very little information is available, but Woods et al. (1977) have shown that the adenohypophysialgonad axis is established as early as Day 13.5 of incubation in the chick embryo. Furthermore, plasma testosterone and LH
IN
JUVENILE
Zonotrichia
375
levels are elevated in ducklings (Storey and Nicholls, 1977) and juvenile domestic fowl (Sharp, 1975), also suggesting that this axis is established at a very early age. A second hypothesis proposes that the hypothalamo - hypophysial -gonadal axis is established and that the LH and DHT levels in the blood become elevated as a result of development of photosensitivity. Days are long at this time of year (July and August) and stimulatory in terms of gonadotropin secretion (Follett et al., 1975). However, this hypothesis must also provide a very rapid development of photorefractoriness to avoid an aseasonal gonadal maturation. It is known that first-year gambelii are photorefractory, at least after completion of the first basic molt, and become photosensitive in the late fall at the same time as adults (Farner and Mewaldt, 1955). The third hypothesis, mutually compatible with either the first or second, ascribes a behavioral function to the increases in plasma levels of LH and DHT. Field observations (J. C. Wingfield, unpublished) indicate that young in the first basic molt begin to form flocks and to use communal roosts, often involving many hundred individuals. Numerous interactions, such as chases, fighting, and supplanting, occur at this time. Also both male and female young begin to sing. At first the song is characterized by hoarse whistles and burry notes, but by completion of molt the song is entirely recognizable as that of a whitecrowned sparrow. It lacks the pure whistles and well-defined trills of the sexual and territorial song of the breeding males, and thus is better designated as subsong or winter song. This song occurs throughout the winter and is especially noticeable in evenings as birds begin to accumulate at roosts. Perhaps it affords some sort of individual recognition, since males and females, adults and young, are integrated together in these flocks. It is now established that androgens are necessary for normal development of sex-
376
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SMITH,
ual song or at least the frequency of singing (e.g., Arnold, 1975; Nottebohm, 1969; Prove, 1974), and also for the “solitary” song of the zebra finch, Poephila guttata (Prove, 1974). Furthermore, androgen uptake has been demonstrated in areas of the brain related to vocal control (Zigmond et al., 1973; Arnold et al., 1976) and also in the muscles of the syrinx (Lieberburg and Nottebohm, 1979). Elevated plasma levels of LH and androgens during postfledging development have also been noted in the domestic pigeon (Abs et al., 1977), in which the level of plasma LH increases just prior to the breaking of voice at about 50 days of age, possibly accompanied by a rise in plasma androgen (Abs and Dahlmann, 1974). Similarly, in the zebra finch, Prove and Sossinka (1978) found a transitory elevation of plasma testosterone levels at 31-40 days of age, a time that is important for sexual imprinting in this species. Therefore, we suggest that the transitory increase in the plasma level of DHT in fledglings of Zonotrichia leucophrys, both males and females, is involved in the development of at least the winter song and possibly other social behavior. Young male white-crowned sparrows learn the sexual song at a very early age but do not reproduce it until the following spring (Marler, 1967), presumably under the influence of the very high vernal levels of circulating testosterone (Wingfield and Farner, 1977, 1978a, b). Females do not normally express sexual song and do not develop very high levels of testosterone (Wingfield and Farner, 1977, 1978a, b), but can be induced to sing in spring by injections of testosterone propionate (Kern and King, 1972). Thus, another question can be raised: Is there a qualitative difference in that DHT, which occurs at approximately the same levels in both sexes, may stimulate winter song, whereas testosterone is required in high concentrations, normally characteristic of males only, for the development of
AND
FARNER
sexual song? However, it is also possible that winter song has a much lower threshold to testosterone than sexual song (Prove, 1974). ACKNOWLEDGMENTS We are indebted to Professor George West and the Institute of Arctic Biology, University of Alaska, College, Alaska, for provision of facilities and advice while conducting field work in central Alaska. Thanks are also due to Mr. P. W. Mattocks, Jr., who assisted with the assay of luteinizing hormone, and to Professor B. K. Follett who provided the LH antiserum and LH standards. The investigations reported herein were supported by Grant PCM 77-17690 from the National Science Foundation, and by a grant from the Graduate School Research Fund, University of Washington.
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