GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
82,
78-85 (1991)
Thyroxine Treatment induces Changes in Hypothalamic Gonadotrophin-Releasing Hormone Characteristic of Photorefractoriness in Starlings (Sturnus vulgaris) M.S. AFRC
Research
Group
A.R.
BOULAKOUDAND
on Photoperiodism Bristol
and Reproduction, BS8 1 UG, United
GOLDSMITH'
Department Kingdom
of Zoology,
University
of Bristol.
Accepted May 29, 1990 Photosensitive intact male starlings were transferred from short days (8L: 16D) to 1lL: 13D for 16 weeks, and were therefore sexually mature. Experimental groups were (i) held under 1lL: 13D and given exogenous thyroxine dissolved in the drinking water for 6 weeks or (ii) given thyroxine for 6 weeks and then transferred from 1IL: 13D to long days (18L:6D) for a further 6 weeks, while control groups were transferred to long days (18L:6D) either (iii) for 6 or (iv) for 12 weeks, or were (v) maintained under llL:l3D throughout. Changes in testicular size and plumage molt were monitored at regular intervals during the 12-week period. At the end of the experiment, the birds were killed and hypothalamic gonadotropinreleasing hormone (Gn-RH) content and testicular mass were measured. Treatment with exogenous thyroxine caused rapid testicular regression followed by plumage molt, and after 6 weeks hypothalamic Gn-RH content was much reduced, to an even greater extent than that in control birds exposed to long days for 6 weeks. After 6 weeks of thyroxine treatment, withdrawal of exogenous thyroxine and exposure to long days for a further 6 weeks caused no increase in testicular size, and caused a further reduction in hypothalamic Gn-RH content to a level similar to that in controls after 12 weeks of exposure to long days. The results confirm previous findings that thyroxine induces a state of photorefractoriness in sexually mature starlings and show for the first time that the treatment mimics the effect of long days in reducing Gn-RH content in the hypothalamus. o 19% Academic PESS, IIK.
For many birds that breed at mid and high latitudes, the increasing day length of spring initiates gonadal maturation. However, the breeding season of many species is quite short and terminates before the summer solstice, while day length is still increasing and is, of course, longer than that which initiated gonadal growth. At that stage, such birds are said to have become photorefractory, and exposure to a certain period of short daily photoperiods is required, in most species, before full photosensitivity is restored, as occurs naturally during the decreasing day length of autumn (for reviews see, Farner et al., 1983; Nicholls et al., 1988a). The physiological mechanism(s) underly-
ing photorefractoriness is still far from understood, although such a process is known to be brought about by long days. In the European starling, only two circumstances are known under which reproductive function occurs but the development of photorefractoriness is completely prevented and the birds remain in breeding condition perhaps indefinitely. The first case is where photosensitive starlings are exposed to constant daily photoperiods of 11L: 13D, indicating that the “critical day length” required for inducing refractoriness must be longer than 11 hr (Hamner, 1971; Dawson and Goldsmith, 1983). The second situation occurs when starlings are thyroidectomized before transfer to long days (Wieselthier and van Tienhoven, 1972; Goldsmith and Nicholls, 1984a). The latter observation,
’ To whom all correspondence should be addressed. 78 0016-6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproductmn in any form reserved.
THYROXINE
AND
REFRACTORINESS
first pointed out by Voitkevich (1940) has prompted much recent research on the importance of the thyroid hormones and their involvement in seasonality in both birds (reviews see Nicholls et al., 1985, 1988a) and mammals (Nicholls et al., 1988b). In starlings, attempts have been made to explore the effects of exogenous thyroxine on the process leading to refractoriness, and these have shown that injection of the hormone into sexually mature starlings held under 11L: 13D induces overt manifestations of the refractory condition such as spontaneous gonadal collapse, decrease in plasma gonadotrophin concentration, plumage molt, and increased levels of plasma prolactin (Goldsmith and Nicholls, 1984b; Dawson, 1989a). Because the development of long-day refractoriness in starlings has been repeatedly shown to be characterized by a considerable reduction in hypothalamic gonadotrophin-releasing hormone (Gn-RH) content (Dawson et al., 1985a, 1986; Goldsmith et al., 1989), the current paper reports the effects of exogenous thyroxine in the hypothalamus when given to sexually mature birds maintained on a day length not long enough to induce refractoriness on its own. MATERIALS
Animals and experimental design. Male starlings (Sturnus vulgaris) were captured locally during summer, 1987, and maintained initially in outdoor aviaries. After a few weeks, they were transferred to aluminum
79
STARLINGS
cages (0.6 x 0.5 x 0.4 m) with 6 to 8 birds per cage. The cages were in lightproof rooms illuminated by 80 W fluorescent tubes. Food (chick crumbs) and water were provided at all times. Throughout the first 5 months, the birds were held under short days (8L: 16D), and were therefore in a photosensitive condition. Thirty-two males were then transferred to a longer day length of 11L: 13D for a further 4 months, to induce full sexual maturation. This was confirmed by visual examination of the gonads; all individuals were laparotomized (see below) and all had fully grown testes with an average width of 8 mm. The experiment was begun 3 days later, when the birds were divided at random into five groups of 6 birds each and exposed to the photoperiodic and thyroxine treatments summarized in Table 1. Thyroxine was administered in the drinking water. L-Thyroxine (30 mg of the sodium salt, code T 2501. Sigma, Poole, Dorset, U.K.) was dissolved in 100 ml 0.01 M sodium hydroxide (NaOH), and then made up to I liter with distilled water. A freshly prepared solution was provided thrice weekly. Control birds received a weak solution (0.001 M) of NaOH. Laparotomy was performed through a small incision on the left-hand side of the body between the last pair of ribs after anesthetizing the bird by intramuscular injection of 4.2 mg of sodium pentobarbitone (70 pi Nembutal. CEVA Ltd, Southampton, Hampshire, U.K.). Testicular width was recorded to the nearest 0.5 mm using calipers. Observations were also made of the occurrence of molting of primary flight feathers. Each wing consists of nine primaries which are normally shed in regular sequence, at the time of reproductive regression, beginning with the innermost ones and continuing symmetrically on both wings. Tissue say. At
AND METHODS
IN
preparation
and
Hypothalamic
Gn-RH
As-
the end of the experimental period all birds were killed by decapitation. The skull was immediately skinned and a block of brain tissue (approximately 7 x 7 x 4 mm) containing the hypothalamus and the median eminence was dissected, homogenized in 1.5 ml ice-cold 0.1 N HCI in a glass-glass homoge-
TABLE I PHOTOPERIODANDTHYROXINETREATMENTS IN STARLINGS INITIALLY SEXUALLY MATUREAFTEREXPOSURE TOI~L:~~DFOR~MONTHSBEFORETREATMENTBEGAN Treatment; first 6 weeks Group
n
Photoperiod
Thyroxine”
Treatment; second 6 weeks Photoperiod
C T C C C .---_____~ .-~ a T, thyroxine administered in drinking water (30 mg/hter in 0.001 M NaOH) C, control drinking water (0.001 M NaOH). A B C D E
6 6 6 6 8
18L:6D llL:13D llL:13D llL:13D llL:13D
18L:6D 18L:6D I8L:6D llL:13D llL:13D
Thyroxine C C C T C
for the 6-week period indicated.
80
BOULAKOUD
AND GOLDSMITH
nizer, and centrifuged at 4000g for 1 hr at 4” and the supematant was stored frozen at -20”. The testes were removed and weighed to the nearest milligram. Hypothalamic concentrations of Gn-RH were measured, after thawing and neutralizing the extracts with 0.1 N NaOH, using a homologous radioimmunoassay for chicken LH-RH-1, as recently described by Stansfield and Cunningham (1988) and Goldsmith et al. (1989). The assay was run at a bound:free ratio of 0.8, using an anti-chicken LH-RH-1 antiserum (code 3/3, provided by P. J. Sharp, AFRC Institute of Animal Physiology and Genetics Research, Roslin, Midlothian, U.K.) and labelled chicken LH-RH-I. The peptide for iodination was supplied by Sigma, Ltd. (code L 0637), and the iodination procedure was performed as described by Sharp et al. (1987) and Goldsmith et al. (1989). The assay is specific for chicken LH-RH-1, showing no cross-reaction with either chicken LH-RH-11 or with ovine LH-RH (Goldsmith et al., 1989). Neutralized starling hypothalamic extracts, which dilute parallel with the chicken LH-RH-I standard (Goldsmith et al., 1989), were measured in a single assay at two dilutions (20 and 10 ~1). The lower detection limit of the method is 10 pmohliter (0.2 fmol/ tube), and the intraassay coefficient of variation was 5.6% (n = 7). Statistical analysis.
Data were analyzed by a oneway analysis of variance (ANOVA) using independent or repeated measures designs as appropriate (Winer, 1971). Comparisons between means were made using Fisher’s PLSD test.
RESULTS
Changes in Testicular Size Changes in gonadal size measured during laparotomy are shown in Fig. 1. At the beginning of the experiment all birds had very large testes, with group mean testicular widths ranging from 7.3 to 8.3 mm. Control birds that were kept on 11L: 13D daily photoperiods throughout (group E) all maintained fully grown gonads during the 12week experimental period, and they are not shown in the figure. Birds transferred to long day lengths of 18L:6D on Day 0 (group A) showed a slight but not significant (Z’ > 0.05) increase in the width of their testes during the first 28 days of photostimulation. Within another 14 days all individuals underwent spontaneous gonadal regression, with testes reaching a minimal size of 2.2mm width (P < 0.005) by Day 56 of the experiment. In group B (thyroxine treat-
'01
1 -D
OJ..,..,..,..,..,.....,..,..,.., -10
0
IO
20
30
40
50
60
70
80
90
TREATMENT FIG. 1. Changes in testicular width (mean ? SEM) in sexually mature intact male starlings transferred from llL:l3D to long days (18L:6D) for 12 weeks (A), maintained on 1lL:l3D and given 30 mg/liter of thyroxine in the drinking water for the first 6 weeks and then transferred to long days (18L:6D) without thyroxine for a further 6 weeks (B), maintained on 1 lL:l3D for the first 6 weeks before being moved to long days (18L:6D) for a further 6 weeks (C), or maintained under llL:l3D for 12 weeks and given 30 mgiliter of thyroxine for the last 6 weeks (D). DAYS
OF
ment for 42 days under 11L: 13D and then transfer to 18L:6D for 6 weeks), the testes did not undergo any further enlargement in size, but after 28 days of thyroxine administration these birds showed a sudden gonadal collapse (P < 0.05), which was almost complete after 42 days of treatment. At that stage, thyroxine administration was stopped and the birds were transferred to long days (18L:6D) to test their responsiveness. Two weeks later, the gonads were even smaller (P < O.OOS), and they remained fully regressed thereafter. Thus, the reduction in testicular size under thyroxine treatment on llL:13D (group B) occurred even more rapidly than in untreated birds exposed to long days (group A). There was no significant change (P > 0.05) in the testicular diameter of birds from group C during the first 6 weeks (on llL:13D). However, 28 days following subsequent photostimulation with long days (18L:6D), the gonads were partially regressed (testicular width 3.4 mm) (P < 0.05) and had reached minimum size of 2.4 mm (P < 0.005) 14
THYROXINE
AND REFRACTORINESS
days later. In group D, birds were maintained under llL:13D for 12 weeks but received thyroxine for the second half of this period. The testes remained large throughout the first 6 weeks, but began to regress just 14 days after thyroxine treatment had commenced and were significantly (P < 0.005) smaller after 28 days of treatment. Testicular
Mass
After the birds had been killed, their testes were immediately removed and weighed. The mean combined testicular mass is shown in Fig. 2. All individuals had fully regressed gonads, except for control birds maintained on 1IL: 13D daily photoperiods, which still had fully mature gonads with a combined testicular mass of 700 mg. There were no significant (P > 0.05) differences between the combined testicular masses of birds from groups A, B, C, and D, but they were all, of course, considerably smaller (P < 0.005) than the combined testicular mass of control birds (group E).
600
IN STARLINGS
Hypothalamic
81
Gn-RH Content
Hypothalamic Gn-RH content showed significant variation between groups (P < 0.001; Fig. 3). Control birds maintained throughout on 1 lL:13D had the highest concentrations of hypothalamic Gn-RH (>1400 fmol/hypothalamus), while individuals from the other groups (A, B, C, and D) had much lower hypothalamic Gn-RH contents. Groups A and B, in which birds were either transferred to long day lengths of 18L:6D for 12 weeks or pretreated with thyroxine under 11L: 13D for 6 weeks and then photostimulated by long days for a further 6 weeks, respectively, had virtually identical stores of low hypothalamic Gn-RH concentration (~300 fmol/hypothalmus). Birds in the other two groups, exposed to long days for 6 weeks (group C) or to thyroxine under 11L: 13D for 6 weeks (group D), had intermediate hypothalamic stores of Gn-RH, in both cases significantly (P < 0.001) lower than those in sexually mature birds (group E). Hypothalamic concentrations were significantly (P < 0.05) lower after 6 weeks of
1
1500
GROUPS GROUP
FIG. 2. Testicular mass (mean + SEM) of sexually mature intact male starlings transferred from 1lL:13D to long days (18L:6D) for 12 weeks (A), maintained on 11L: 13D and receiving 30 mg/liter of thyroxine in the drinking water for 6 weeks and then transferred to long days (18L:6D) without thyroxine for another 6 weeks (B) photostimulated with long days (18L:6D) for 6 weeks (C), or kept on 11L: 13D and given 30 mg/liter of thyroxine for 6 weeks (D) and of control birds maintained on llL:13D (E).
3. Concentrations of hypothalamic Gn-RH (fmohhypothalamus) (mean 2 SEM) in sexually mature intact male starlings transferred from 11L: 13D to long days (18L:6D) for 12 weeks (A), maintained on 1lL: 13D and receiving 30 mgiliter of thyroxine in the drinking water for 6 weeks and then transferred to long days 18L:6D) without thyroxine for another 6 weeks (B), photostimulated with long days (18L:6D) for 6 weeks (C), or kept on 1 IL: 13D and given 30 mg/liter of thyroxine for 6 weeks (D) and in control birds maintained on 11L: 13D (E). FIG.
BOULAKOUD
82
AND GOLDSMITH
thyroxine (group D) than after 6 weeks of long days (group C).
onset of treatment (Week 9; that is 3 weeks after thyroxine treatment had started) than the 18L:6D controls.
Primary Molt At the beginning of the experiment none of the birds was molting, and consequently all individuals had complete numbers of primary flight feathers. Control birds maintained throughout on 11L: 13D did not undergo any molting at all. Birds transferred to long daily photoperiods of 18L:6D (group A) had begun to molt after 6 weeks and molt expression was almost complete at the end of the experiment (Fig. 4). Birds transferred from llL:13D to long days at week 6 (group C) had also just begun molting after 6 weeks under 18L:6D (molt score I .2, not shown on figure). Thyroxine treatment facilitated a more rapid onset of molt (group B) than did long days of 18L:6D (group A), and molting continued after withdrawal of thyroxine and transfer to 18L:6D. Similarly, birds of group D (treated with thyroxine from Week 6 to Week 12) began molting sooner after the 9 8-
WEEKS
OF TREATMENT
FIG. 4. Molt score (mean t SEM) in sexually mature intact male starlings transferred from 1 IL: 13D to long days (18L:6D) for 12 weeks (A), maintained on I IL: 13D and receiving 30 mg/liter of thyroxine in the drinking water for 6 weeks and then transferred to long days (18L:6D) without thyroxine for another 6 weeks (B), and in sexually mature birds held under 1lL:13D for 12 weeks and given 30 mgiliter of thyroxine for the last 6 weeks (D). Molt score is the number of primary flight feathers shed, averaged over the two wings; maximum score = 9.
DISCUSSION
The results provide further information on how administration of exogenous thyroxine affects the photoperiodic response of starlings. They have confirmed previous findings that prolonged treatment with thyroxine induces gonadal regression in sexually mature birds maintained under daily photoperiods of 11L: 13D (Goldsmith and Nicholls, 1984b; Dawson, 1989a). More importantly, it is now demonstrated for the first time that thyroxine administration causes a marked reduction in hypothalamic Gn-RH content, thus mimicking the reproductive condition (photorefractoriness) normally induced at the hypothalamic level by long daily photoperiods. Hypothalamic Gn-RH content was significantly reduced after 6 weeks of long days (18L:6D), and was suppressed further still after 12 weeks of long days (18L:6D); a physiological change that has already been reported to be associated with the development of longday refractoriness in intact starlings (Dawson et al., 1985a, 1986; Goldsmith et al., 1989). In thyroxine-treated birds, hypothalamic Gn-RH content was suppressed after 6 weeks of treatment to an even greater extent than in control birds exposed to 6 weeks of long days 18L:6D), suggesting that this high dose of thyroxine (30 mg/liter) had induced particularly rapid refractory response. This was confirmed by monitoring changes in testicular diameter and the onset of molt. In both thyroxine-treated groups (B and D), testicular regression occurred more rapidly than in control birds exposed to long days, and molting also began earlier in thyroxine-treated groups. While treatment with exogenous thyroxine clearly induces all the characteristics of photorefractoriness, there is no particular reason to expect that the time course of
THYROXINE
AND
REFRACTORINESS
events would be the same as under any particular ‘long’ photoperiodic duration. It is quite possible that a lower dose of thyroxine would lead to a slower onset of regression. Transfer of birds after 6 weeks of thyroxine administration under 1 IL: 13D to long days (18L:6D) without thyroxine (group B) confirmed that the treatment had not merely caused suppression of the reproductive axis but had induced photorefractoriness, since the testes remained regressed and hypothalamic Gn-RH content was reduced after 6 weeks on long days to the low level characteristic of controls killed after 12 weeks of long days (18L:6D) (group A). What is the role of the thyroid glands in the development of photorefractoriness under natural long days? One possibility is that exposure to long days induces an increase in the thyroidal activity, and subsequently leads to increased levels of thyroid hormones in the circulation. This, in turn, could trigger an as yet unknown process in the central nervous system (CNS) leading to photorefractoriness and hence reduced Gn-RH output and gonadal regression. Such an explanation requires there to be an increase in plasma thyroxine (T4) in starlings under photostimulatory day lengths, and while this has been demonstrated in some studies (Dawson, 1984; C. Bishop and A. R. Goldsmith, unpublished data), other experiments have failed to demonstrate a clear increase in T4 under long days (Dawson et al., 1985b; Dawson, 1989a). In some other species increases in circulating plasma thyroid hormone levels have been reported following photostimulation (Assenmacher and Jallageas, 1977; Sharp and Klandorf, 1981; Klandorfet al., 1982; Stokkan et al., 1985), but again other reports are negative (Smith, 1982; Sharp et al., 1986). It is probably necessary to measure other components of thyroid function, e.g., triiodothyronine (Ts) and thyroid binding proteins (particularly transthyretin or TBPA), neither of which has yet been described in
IN
STARLINGS
83
starlings, since these are reported to reach maximal levels during the onset of molting in some species (Cookson et al., 1988). However, there is some evidence in starlings to suggest that the development of photorefractoriness does not depend upon increased thyroid hormone levels alone. Lower doses of exogenous thyroxine than that employed in the present study but sufficient to cause high circulating levels are not always effective in inducing photorefractoriness on photoperiods shorter than 12L: 12D (Dawson, 1989a). Nonetheless, there is no question that active thyroid function is essential for the processes leading to the occurrence of photorefractoriness under long days. Thyroidectomized birds stay fully mature under long days (Goldsmith and Nicholls, 1984a), while thyroxine replacement (even in very small amounts) restores the refractory condition, incorporating all the expected physiological events including reduced hypothalamic GnRH content, a decrease in plasma gonadotrophin levels, gonadal regression, increased plasma prolactin, and molting of the plumage: all these changes occurring in the sequence seen in intact birds exposed to long days (Goldsmith et al., 1985; Dawson. 1989b). The exact site of thyroid involvement is unknown, but there is some evidence to suggest that it acts in the higher neural circuitry and possibly in the photoperiodic time perception (clock), rather than having a direct effect on Gn-RH neurones (Follett et al., 1988; Dawson, 1989a. b). Thus, an alternative hypothesis speculates that under natural conditions, long days cause an event in the CNS which requires the presence of T, (or TX) to proceed and which then leads to the inhibition of Gn-RH output. The long day-induced process might also include an increase in sensitivity to endogenous thyroid hormones (Follett et al., 1988). The effect of exogenous thyroxine in intact birds on photoperiods of less than 12 hr per day can be explained if a particularly
84
BOULAKOUD
AND GOLDSMITH
high dose of thyroxine, as in the present experiment, is able to “force” the development of the process which, although thyroid-dependent, normally also requires long day stimulation to occur (Follett et al., 1988; Dawson, 1989a, b). Further investigation of the site and mode of action of thyroid hormones in the CNS may well shed light upon the natural process of photoperiodic-induced reproductive inhibition (photorefractoriness). ACKNOWLEDGMENTS We are very grateful to Dr. P. J. Sharp for providing us with the antiserum, to W. Ivings for her technical assistance, and to the Algerian government for tinancial support to M. S. Boulakoud.
REFERENCES Assenmacher, I., and Jallageas, M. (1977). Annual endocrine cycles and environment in birds with special reference to male ducks. In “Environmental Endocrinology, Proceeding of an International Symposium, Montpelier, France” (I. Assenmacher and D. S. Farner, Eds), pp 52-60. SpringerVerlag, Berlin/Heidelberg/New York. Cookson, E. J., Hall, M. R., and Glover, J. (1988). The transport of plasma thyroxine in white storks (Ciconia ciconia) and the association of high levels of plasma transthyretin (thyroxine-binding prealbumin) with molt. J. Endocrinol. 117, 75-84. Dawson, A. (1984). Changes in plasma thyroxine concentration in male and female starlings (Srurnus vulgaris) during a photo-induced gonadal cycle. Gen. Comp. Endocrinol. 56, 193-197. Dawson, A. (198%). Pharmacological doses of thyroxine simulate the effects of increased day length and thyroidectomy decreased day length, on the reproductive system of European starlings. J. Exp.
Zool.
249, 62-67.
Dawson, A. (1989b). The involvement of thyroxine and day length in the development of photorefractoriness in European starlings. J. Exp. Zool. 249, 68-75. Dawson, A., and Goldsmith, A. R. (1983). Plasma prolactin and gonadotrophins during gonadal development and the onset of photorefractoriness in male and female starlings (Srurnus vulgaris) on artificial photoperiods. J. Endocrinol. 97, 253260. Dawson, A., Follett, B. K., Goldsmith, A. R., and Nicholls, T. J. (1985a). Hypothalamic gonadotrophin-releasing hormone and pituitary and plasma
FSH and prolactin during photostimulation and photorefractoriness in intact and thyroidectomized starlings. J. Endocrinol. 105, 71-77. Dawson, A., Goldsmith, A. R., and Nicholls, T. J. (1985b). Development of photorefractoriness in intact and castrated male starlings (Sfurnus vulgaris) exposed to different periods of long day lengths. Physiol. Zool. 58, 253-261. Dawson, A., Goldsmith, A. R., Nicholls, T. J., and Follett, B. K. (1986). Endocrine changes associated with the termination of photoreftactoriness by short day lengths and thyroidectomy in starlings (Sturnus vulgaris). J. Endocrinol. 110, 7379. Famer, D. S., Donham, R. S., Matt, K. S., Mattocks, P. W., Moore, M. C., and Wingtield, J. C. (1983). The nature of photorefractoriness. In “Avian Endocrinology. Environmental Perspectives” (S. Mikami, K. Homma, and M. Wada, Eds), pp. 149-166. Japan Scientific Societies Press, Tokyo. Follett, B. K., Nicholls, T. J., and Mayes, C. R. (1988). Thyroxine can mimic photoperiodically induced gonadal growth in Japanese quail. J. Comp. Physiol. B 157, 829-835. Goldsmith, A. R., and Nicholls, T. J. (1984a). Thyroidectomy prevents the development of photorefractoriness and the associated rise in plasma prolactin in starlings. Gen. Comp. Endocrinol. 54, 256-263. Goldsmith, A. R., and Nicholls, T. J. (1984b). Thyroxine induces photorefractoriness and stimulates prolactin secretion in European starlings (Sturnus vulgaris). J. Endocrinol. 101, R143. Goldsmith, A. R., Nicholls, T. J., and Plowman, G. (1985). Thyroxine treatment facilitates prolactin secretion and induces a state of photorefractoriness in thyroidectomized starlings. J. Endocrinol. 104,99-103. Goldsmith, A. R., Ivings, W. E., Pearce-Kelly, A. S., Parry, D. M., Plowman, G., Nicholls, T. J., and Follett, B. K. (1989). Photoperiodic control of the development of the LH-RH neurosecretory system of European starlings (Sturnus vulgaris) during puberty and the onset of photorefractoriness. J. Endocrinol. 122, 255-268. Hamner, W. M. (1971). On seeking an alternative to the endogenous reproductive rhythm hypothesis in birds. In “Biochronometry” (M. Menaker, Ed), pp. 44w61. Natl. Acad. Sci., Washington, D.C. Klandorf, H., Stokkan, K-A., and Sharp, P. J. (1982). Plasma thyroxine and triiodothyronine levels during the development of photorefractoriness in Willow ptarmigan (Lagopus lagopus lagopus) exposed to different photoperiods. Gen. Comp. Endocrinol.
Nicholls,
47, 64-69.
T. J., Goldsmith,
A. R., Dawson,
A.,
THYROXINE
AND REFRACTORINESS
Chakraborty, S., and Follett, B. K. (1985). lnvolvement of the thyroid gland in photorefractoriness in starlings. In “The Endocrine System and The Environment” (B. K. Follett, S. Ishi, and A. Chandola, Eds), pp. 127-135. Japan Scientific Societies Press, Tokyo. Nicholls, T. J., Goldsmith, A. R., and Dawson, A. (1988a). Photorefractoriness in birds and comparison with mammals. Physiol. Rev. 68, 133-176. Nicholls, T. J., Follett, B. K., Goldsmith, A. R., and Pearson, H. (1988b). Possible homologies between photorefractoriness in sheep and birds: The effect of thyroidectomy on the length of the ewe’s breeding season. Reprod. Nutr. Dev. 28, (2B), 375-385. Sharp, P. J., and Klandorf, H. (1981). The interaction between day length and the gonads in the regulation of levels of plasma thyroxine and triiodothyronine in the Japanese quail. Gen. Comp. Endocrinol. 45, 504-512. Sharp, P. J., Lea, R. W., Dunn, 1. C., and Trocchi, V. (1986). Photoperiodic and endocrine control of seasonal breeding in grey partridge (Perdix perdix). J. Zool. 209(A), 187-200. Sharp, P. J., Dunn, I. C., and Talbot, R. T. (1987). Sex differences in the LH responses to chicken
LH-RH
IN STARLINGS
85
I and II in the domestic fowl. J. Endo115, 323-331. Smith, J. D. (1982). Changes in blood levels of thyroid hormones in two species of passerine birds. Condor 84, 160-167. Stansfield, S. C., and Cunningham, F. J. (1988). Attenuation of endogenous opoid peptide inhibition of (Gln8) luteinzing hormone releasing hormone secretion during sexual maturation in the cockerel. Endocrinology 123, 787-794. Stokkan, K-A., Harvey, S., Klandorf, H., Unander, S., and Blix, A. S. (1985). Endocrine changes associated with fat deposition and mobilization in svalbard ptarmigan. Gen. Comp. Endocrinoi. 58, 76-80. Voitkevich, A. A. (1940). Dependence of seasonal periodicity in gonadal changes on the thyroid gland in Sturnus vulgaris. L. C. R. Dok. Acad. Sci. URSS 27, 741-745. Wieselthier, A. S., and van Tienhoven, A. (1972). The effect of thyroidectomy on the photorefractory period in the starling (Sturnus vulgaris). .I. Exp. Zoo/. 179, 331-338. Winer, B. J. (1971). “Statistical Principles in Experimental Design,” 2nd ed. McGraw-Hill, New York. crinol.
AND
COMPARATIVE
ENDOCRINOLOGY
82,
78-85 (1991)
Thyroxine Treatment induces Changes in Hypothalamic Gonadotrophin-Releasing Hormone Characteristic of Photorefractoriness in Starlings (Sturnus vulgaris) M.S. AFRC
Research
Group
A.R.
BOULAKOUDAND
on Photoperiodism Bristol
and Reproduction, BS8 1 UG, United
GOLDSMITH'
Department Kingdom
of Zoology,
University
of Bristol.
Accepted May 29, 1990 Photosensitive intact male starlings were transferred from short days (8L: 16D) to 1lL: 13D for 16 weeks, and were therefore sexually mature. Experimental groups were (i) held under 1lL: 13D and given exogenous thyroxine dissolved in the drinking water for 6 weeks or (ii) given thyroxine for 6 weeks and then transferred from 1IL: 13D to long days (18L:6D) for a further 6 weeks, while control groups were transferred to long days (18L:6D) either (iii) for 6 or (iv) for 12 weeks, or were (v) maintained under llL:l3D throughout. Changes in testicular size and plumage molt were monitored at regular intervals during the 12-week period. At the end of the experiment, the birds were killed and hypothalamic gonadotropinreleasing hormone (Gn-RH) content and testicular mass were measured. Treatment with exogenous thyroxine caused rapid testicular regression followed by plumage molt, and after 6 weeks hypothalamic Gn-RH content was much reduced, to an even greater extent than that in control birds exposed to long days for 6 weeks. After 6 weeks of thyroxine treatment, withdrawal of exogenous thyroxine and exposure to long days for a further 6 weeks caused no increase in testicular size, and caused a further reduction in hypothalamic Gn-RH content to a level similar to that in controls after 12 weeks of exposure to long days. The results confirm previous findings that thyroxine induces a state of photorefractoriness in sexually mature starlings and show for the first time that the treatment mimics the effect of long days in reducing Gn-RH content in the hypothalamus. o 19% Academic PESS, IIK.
For many birds that breed at mid and high latitudes, the increasing day length of spring initiates gonadal maturation. However, the breeding season of many species is quite short and terminates before the summer solstice, while day length is still increasing and is, of course, longer than that which initiated gonadal growth. At that stage, such birds are said to have become photorefractory, and exposure to a certain period of short daily photoperiods is required, in most species, before full photosensitivity is restored, as occurs naturally during the decreasing day length of autumn (for reviews see, Farner et al., 1983; Nicholls et al., 1988a). The physiological mechanism(s) underly-
ing photorefractoriness is still far from understood, although such a process is known to be brought about by long days. In the European starling, only two circumstances are known under which reproductive function occurs but the development of photorefractoriness is completely prevented and the birds remain in breeding condition perhaps indefinitely. The first case is where photosensitive starlings are exposed to constant daily photoperiods of 11L: 13D, indicating that the “critical day length” required for inducing refractoriness must be longer than 11 hr (Hamner, 1971; Dawson and Goldsmith, 1983). The second situation occurs when starlings are thyroidectomized before transfer to long days (Wieselthier and van Tienhoven, 1972; Goldsmith and Nicholls, 1984a). The latter observation,
’ To whom all correspondence should be addressed. 78 0016-6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproductmn in any form reserved.
THYROXINE
AND
REFRACTORINESS
first pointed out by Voitkevich (1940) has prompted much recent research on the importance of the thyroid hormones and their involvement in seasonality in both birds (reviews see Nicholls et al., 1985, 1988a) and mammals (Nicholls et al., 1988b). In starlings, attempts have been made to explore the effects of exogenous thyroxine on the process leading to refractoriness, and these have shown that injection of the hormone into sexually mature starlings held under 11L: 13D induces overt manifestations of the refractory condition such as spontaneous gonadal collapse, decrease in plasma gonadotrophin concentration, plumage molt, and increased levels of plasma prolactin (Goldsmith and Nicholls, 1984b; Dawson, 1989a). Because the development of long-day refractoriness in starlings has been repeatedly shown to be characterized by a considerable reduction in hypothalamic gonadotrophin-releasing hormone (Gn-RH) content (Dawson et al., 1985a, 1986; Goldsmith et al., 1989), the current paper reports the effects of exogenous thyroxine in the hypothalamus when given to sexually mature birds maintained on a day length not long enough to induce refractoriness on its own. MATERIALS
Animals and experimental design. Male starlings (Sturnus vulgaris) were captured locally during summer, 1987, and maintained initially in outdoor aviaries. After a few weeks, they were transferred to aluminum
79
STARLINGS
cages (0.6 x 0.5 x 0.4 m) with 6 to 8 birds per cage. The cages were in lightproof rooms illuminated by 80 W fluorescent tubes. Food (chick crumbs) and water were provided at all times. Throughout the first 5 months, the birds were held under short days (8L: 16D), and were therefore in a photosensitive condition. Thirty-two males were then transferred to a longer day length of 11L: 13D for a further 4 months, to induce full sexual maturation. This was confirmed by visual examination of the gonads; all individuals were laparotomized (see below) and all had fully grown testes with an average width of 8 mm. The experiment was begun 3 days later, when the birds were divided at random into five groups of 6 birds each and exposed to the photoperiodic and thyroxine treatments summarized in Table 1. Thyroxine was administered in the drinking water. L-Thyroxine (30 mg of the sodium salt, code T 2501. Sigma, Poole, Dorset, U.K.) was dissolved in 100 ml 0.01 M sodium hydroxide (NaOH), and then made up to I liter with distilled water. A freshly prepared solution was provided thrice weekly. Control birds received a weak solution (0.001 M) of NaOH. Laparotomy was performed through a small incision on the left-hand side of the body between the last pair of ribs after anesthetizing the bird by intramuscular injection of 4.2 mg of sodium pentobarbitone (70 pi Nembutal. CEVA Ltd, Southampton, Hampshire, U.K.). Testicular width was recorded to the nearest 0.5 mm using calipers. Observations were also made of the occurrence of molting of primary flight feathers. Each wing consists of nine primaries which are normally shed in regular sequence, at the time of reproductive regression, beginning with the innermost ones and continuing symmetrically on both wings. Tissue say. At
AND METHODS
IN
preparation
and
Hypothalamic
Gn-RH
As-
the end of the experimental period all birds were killed by decapitation. The skull was immediately skinned and a block of brain tissue (approximately 7 x 7 x 4 mm) containing the hypothalamus and the median eminence was dissected, homogenized in 1.5 ml ice-cold 0.1 N HCI in a glass-glass homoge-
TABLE I PHOTOPERIODANDTHYROXINETREATMENTS IN STARLINGS INITIALLY SEXUALLY MATUREAFTEREXPOSURE TOI~L:~~DFOR~MONTHSBEFORETREATMENTBEGAN Treatment; first 6 weeks Group
n
Photoperiod
Thyroxine”
Treatment; second 6 weeks Photoperiod
C T C C C .---_____~ .-~ a T, thyroxine administered in drinking water (30 mg/hter in 0.001 M NaOH) C, control drinking water (0.001 M NaOH). A B C D E
6 6 6 6 8
18L:6D llL:13D llL:13D llL:13D llL:13D
18L:6D 18L:6D I8L:6D llL:13D llL:13D
Thyroxine C C C T C
for the 6-week period indicated.
80
BOULAKOUD
AND GOLDSMITH
nizer, and centrifuged at 4000g for 1 hr at 4” and the supematant was stored frozen at -20”. The testes were removed and weighed to the nearest milligram. Hypothalamic concentrations of Gn-RH were measured, after thawing and neutralizing the extracts with 0.1 N NaOH, using a homologous radioimmunoassay for chicken LH-RH-1, as recently described by Stansfield and Cunningham (1988) and Goldsmith et al. (1989). The assay was run at a bound:free ratio of 0.8, using an anti-chicken LH-RH-1 antiserum (code 3/3, provided by P. J. Sharp, AFRC Institute of Animal Physiology and Genetics Research, Roslin, Midlothian, U.K.) and labelled chicken LH-RH-I. The peptide for iodination was supplied by Sigma, Ltd. (code L 0637), and the iodination procedure was performed as described by Sharp et al. (1987) and Goldsmith et al. (1989). The assay is specific for chicken LH-RH-1, showing no cross-reaction with either chicken LH-RH-11 or with ovine LH-RH (Goldsmith et al., 1989). Neutralized starling hypothalamic extracts, which dilute parallel with the chicken LH-RH-I standard (Goldsmith et al., 1989), were measured in a single assay at two dilutions (20 and 10 ~1). The lower detection limit of the method is 10 pmohliter (0.2 fmol/ tube), and the intraassay coefficient of variation was 5.6% (n = 7). Statistical analysis.
Data were analyzed by a oneway analysis of variance (ANOVA) using independent or repeated measures designs as appropriate (Winer, 1971). Comparisons between means were made using Fisher’s PLSD test.
RESULTS
Changes in Testicular Size Changes in gonadal size measured during laparotomy are shown in Fig. 1. At the beginning of the experiment all birds had very large testes, with group mean testicular widths ranging from 7.3 to 8.3 mm. Control birds that were kept on 11L: 13D daily photoperiods throughout (group E) all maintained fully grown gonads during the 12week experimental period, and they are not shown in the figure. Birds transferred to long day lengths of 18L:6D on Day 0 (group A) showed a slight but not significant (Z’ > 0.05) increase in the width of their testes during the first 28 days of photostimulation. Within another 14 days all individuals underwent spontaneous gonadal regression, with testes reaching a minimal size of 2.2mm width (P < 0.005) by Day 56 of the experiment. In group B (thyroxine treat-
'01
1 -D
OJ..,..,..,..,..,.....,..,..,.., -10
0
IO
20
30
40
50
60
70
80
90
TREATMENT FIG. 1. Changes in testicular width (mean ? SEM) in sexually mature intact male starlings transferred from llL:l3D to long days (18L:6D) for 12 weeks (A), maintained on 1lL:l3D and given 30 mg/liter of thyroxine in the drinking water for the first 6 weeks and then transferred to long days (18L:6D) without thyroxine for a further 6 weeks (B), maintained on 1 lL:l3D for the first 6 weeks before being moved to long days (18L:6D) for a further 6 weeks (C), or maintained under llL:l3D for 12 weeks and given 30 mgiliter of thyroxine for the last 6 weeks (D). DAYS
OF
ment for 42 days under 11L: 13D and then transfer to 18L:6D for 6 weeks), the testes did not undergo any further enlargement in size, but after 28 days of thyroxine administration these birds showed a sudden gonadal collapse (P < 0.05), which was almost complete after 42 days of treatment. At that stage, thyroxine administration was stopped and the birds were transferred to long days (18L:6D) to test their responsiveness. Two weeks later, the gonads were even smaller (P < O.OOS), and they remained fully regressed thereafter. Thus, the reduction in testicular size under thyroxine treatment on llL:13D (group B) occurred even more rapidly than in untreated birds exposed to long days (group A). There was no significant change (P > 0.05) in the testicular diameter of birds from group C during the first 6 weeks (on llL:13D). However, 28 days following subsequent photostimulation with long days (18L:6D), the gonads were partially regressed (testicular width 3.4 mm) (P < 0.05) and had reached minimum size of 2.4 mm (P < 0.005) 14
THYROXINE
AND REFRACTORINESS
days later. In group D, birds were maintained under llL:13D for 12 weeks but received thyroxine for the second half of this period. The testes remained large throughout the first 6 weeks, but began to regress just 14 days after thyroxine treatment had commenced and were significantly (P < 0.005) smaller after 28 days of treatment. Testicular
Mass
After the birds had been killed, their testes were immediately removed and weighed. The mean combined testicular mass is shown in Fig. 2. All individuals had fully regressed gonads, except for control birds maintained on 1IL: 13D daily photoperiods, which still had fully mature gonads with a combined testicular mass of 700 mg. There were no significant (P > 0.05) differences between the combined testicular masses of birds from groups A, B, C, and D, but they were all, of course, considerably smaller (P < 0.005) than the combined testicular mass of control birds (group E).
600
IN STARLINGS
Hypothalamic
81
Gn-RH Content
Hypothalamic Gn-RH content showed significant variation between groups (P < 0.001; Fig. 3). Control birds maintained throughout on 1 lL:13D had the highest concentrations of hypothalamic Gn-RH (>1400 fmol/hypothalamus), while individuals from the other groups (A, B, C, and D) had much lower hypothalamic Gn-RH contents. Groups A and B, in which birds were either transferred to long day lengths of 18L:6D for 12 weeks or pretreated with thyroxine under 11L: 13D for 6 weeks and then photostimulated by long days for a further 6 weeks, respectively, had virtually identical stores of low hypothalamic Gn-RH concentration (~300 fmol/hypothalmus). Birds in the other two groups, exposed to long days for 6 weeks (group C) or to thyroxine under 11L: 13D for 6 weeks (group D), had intermediate hypothalamic stores of Gn-RH, in both cases significantly (P < 0.001) lower than those in sexually mature birds (group E). Hypothalamic concentrations were significantly (P < 0.05) lower after 6 weeks of
1
1500
GROUPS GROUP
FIG. 2. Testicular mass (mean + SEM) of sexually mature intact male starlings transferred from 1lL:13D to long days (18L:6D) for 12 weeks (A), maintained on 11L: 13D and receiving 30 mg/liter of thyroxine in the drinking water for 6 weeks and then transferred to long days (18L:6D) without thyroxine for another 6 weeks (B) photostimulated with long days (18L:6D) for 6 weeks (C), or kept on 11L: 13D and given 30 mg/liter of thyroxine for 6 weeks (D) and of control birds maintained on llL:13D (E).
3. Concentrations of hypothalamic Gn-RH (fmohhypothalamus) (mean 2 SEM) in sexually mature intact male starlings transferred from 11L: 13D to long days (18L:6D) for 12 weeks (A), maintained on 1lL: 13D and receiving 30 mgiliter of thyroxine in the drinking water for 6 weeks and then transferred to long days 18L:6D) without thyroxine for another 6 weeks (B), photostimulated with long days (18L:6D) for 6 weeks (C), or kept on 1 IL: 13D and given 30 mg/liter of thyroxine for 6 weeks (D) and in control birds maintained on 11L: 13D (E). FIG.
BOULAKOUD
82
AND GOLDSMITH
thyroxine (group D) than after 6 weeks of long days (group C).
onset of treatment (Week 9; that is 3 weeks after thyroxine treatment had started) than the 18L:6D controls.
Primary Molt At the beginning of the experiment none of the birds was molting, and consequently all individuals had complete numbers of primary flight feathers. Control birds maintained throughout on 11L: 13D did not undergo any molting at all. Birds transferred to long daily photoperiods of 18L:6D (group A) had begun to molt after 6 weeks and molt expression was almost complete at the end of the experiment (Fig. 4). Birds transferred from llL:13D to long days at week 6 (group C) had also just begun molting after 6 weeks under 18L:6D (molt score I .2, not shown on figure). Thyroxine treatment facilitated a more rapid onset of molt (group B) than did long days of 18L:6D (group A), and molting continued after withdrawal of thyroxine and transfer to 18L:6D. Similarly, birds of group D (treated with thyroxine from Week 6 to Week 12) began molting sooner after the 9 8-
WEEKS
OF TREATMENT
FIG. 4. Molt score (mean t SEM) in sexually mature intact male starlings transferred from 1 IL: 13D to long days (18L:6D) for 12 weeks (A), maintained on I IL: 13D and receiving 30 mg/liter of thyroxine in the drinking water for 6 weeks and then transferred to long days (18L:6D) without thyroxine for another 6 weeks (B), and in sexually mature birds held under 1lL:13D for 12 weeks and given 30 mgiliter of thyroxine for the last 6 weeks (D). Molt score is the number of primary flight feathers shed, averaged over the two wings; maximum score = 9.
DISCUSSION
The results provide further information on how administration of exogenous thyroxine affects the photoperiodic response of starlings. They have confirmed previous findings that prolonged treatment with thyroxine induces gonadal regression in sexually mature birds maintained under daily photoperiods of 11L: 13D (Goldsmith and Nicholls, 1984b; Dawson, 1989a). More importantly, it is now demonstrated for the first time that thyroxine administration causes a marked reduction in hypothalamic Gn-RH content, thus mimicking the reproductive condition (photorefractoriness) normally induced at the hypothalamic level by long daily photoperiods. Hypothalamic Gn-RH content was significantly reduced after 6 weeks of long days (18L:6D), and was suppressed further still after 12 weeks of long days (18L:6D); a physiological change that has already been reported to be associated with the development of longday refractoriness in intact starlings (Dawson et al., 1985a, 1986; Goldsmith et al., 1989). In thyroxine-treated birds, hypothalamic Gn-RH content was suppressed after 6 weeks of treatment to an even greater extent than in control birds exposed to 6 weeks of long days 18L:6D), suggesting that this high dose of thyroxine (30 mg/liter) had induced particularly rapid refractory response. This was confirmed by monitoring changes in testicular diameter and the onset of molt. In both thyroxine-treated groups (B and D), testicular regression occurred more rapidly than in control birds exposed to long days, and molting also began earlier in thyroxine-treated groups. While treatment with exogenous thyroxine clearly induces all the characteristics of photorefractoriness, there is no particular reason to expect that the time course of
THYROXINE
AND
REFRACTORINESS
events would be the same as under any particular ‘long’ photoperiodic duration. It is quite possible that a lower dose of thyroxine would lead to a slower onset of regression. Transfer of birds after 6 weeks of thyroxine administration under 1 IL: 13D to long days (18L:6D) without thyroxine (group B) confirmed that the treatment had not merely caused suppression of the reproductive axis but had induced photorefractoriness, since the testes remained regressed and hypothalamic Gn-RH content was reduced after 6 weeks on long days to the low level characteristic of controls killed after 12 weeks of long days (18L:6D) (group A). What is the role of the thyroid glands in the development of photorefractoriness under natural long days? One possibility is that exposure to long days induces an increase in the thyroidal activity, and subsequently leads to increased levels of thyroid hormones in the circulation. This, in turn, could trigger an as yet unknown process in the central nervous system (CNS) leading to photorefractoriness and hence reduced Gn-RH output and gonadal regression. Such an explanation requires there to be an increase in plasma thyroxine (T4) in starlings under photostimulatory day lengths, and while this has been demonstrated in some studies (Dawson, 1984; C. Bishop and A. R. Goldsmith, unpublished data), other experiments have failed to demonstrate a clear increase in T4 under long days (Dawson et al., 1985b; Dawson, 1989a). In some other species increases in circulating plasma thyroid hormone levels have been reported following photostimulation (Assenmacher and Jallageas, 1977; Sharp and Klandorf, 1981; Klandorfet al., 1982; Stokkan et al., 1985), but again other reports are negative (Smith, 1982; Sharp et al., 1986). It is probably necessary to measure other components of thyroid function, e.g., triiodothyronine (Ts) and thyroid binding proteins (particularly transthyretin or TBPA), neither of which has yet been described in
IN
STARLINGS
83
starlings, since these are reported to reach maximal levels during the onset of molting in some species (Cookson et al., 1988). However, there is some evidence in starlings to suggest that the development of photorefractoriness does not depend upon increased thyroid hormone levels alone. Lower doses of exogenous thyroxine than that employed in the present study but sufficient to cause high circulating levels are not always effective in inducing photorefractoriness on photoperiods shorter than 12L: 12D (Dawson, 1989a). Nonetheless, there is no question that active thyroid function is essential for the processes leading to the occurrence of photorefractoriness under long days. Thyroidectomized birds stay fully mature under long days (Goldsmith and Nicholls, 1984a), while thyroxine replacement (even in very small amounts) restores the refractory condition, incorporating all the expected physiological events including reduced hypothalamic GnRH content, a decrease in plasma gonadotrophin levels, gonadal regression, increased plasma prolactin, and molting of the plumage: all these changes occurring in the sequence seen in intact birds exposed to long days (Goldsmith et al., 1985; Dawson. 1989b). The exact site of thyroid involvement is unknown, but there is some evidence to suggest that it acts in the higher neural circuitry and possibly in the photoperiodic time perception (clock), rather than having a direct effect on Gn-RH neurones (Follett et al., 1988; Dawson, 1989a. b). Thus, an alternative hypothesis speculates that under natural conditions, long days cause an event in the CNS which requires the presence of T, (or TX) to proceed and which then leads to the inhibition of Gn-RH output. The long day-induced process might also include an increase in sensitivity to endogenous thyroid hormones (Follett et al., 1988). The effect of exogenous thyroxine in intact birds on photoperiods of less than 12 hr per day can be explained if a particularly
84
BOULAKOUD
AND GOLDSMITH
high dose of thyroxine, as in the present experiment, is able to “force” the development of the process which, although thyroid-dependent, normally also requires long day stimulation to occur (Follett et al., 1988; Dawson, 1989a, b). Further investigation of the site and mode of action of thyroid hormones in the CNS may well shed light upon the natural process of photoperiodic-induced reproductive inhibition (photorefractoriness). ACKNOWLEDGMENTS We are very grateful to Dr. P. J. Sharp for providing us with the antiserum, to W. Ivings for her technical assistance, and to the Algerian government for tinancial support to M. S. Boulakoud.
REFERENCES Assenmacher, I., and Jallageas, M. (1977). Annual endocrine cycles and environment in birds with special reference to male ducks. In “Environmental Endocrinology, Proceeding of an International Symposium, Montpelier, France” (I. Assenmacher and D. S. Farner, Eds), pp 52-60. SpringerVerlag, Berlin/Heidelberg/New York. Cookson, E. J., Hall, M. R., and Glover, J. (1988). The transport of plasma thyroxine in white storks (Ciconia ciconia) and the association of high levels of plasma transthyretin (thyroxine-binding prealbumin) with molt. J. Endocrinol. 117, 75-84. Dawson, A. (1984). Changes in plasma thyroxine concentration in male and female starlings (Srurnus vulgaris) during a photo-induced gonadal cycle. Gen. Comp. Endocrinol. 56, 193-197. Dawson, A. (198%). Pharmacological doses of thyroxine simulate the effects of increased day length and thyroidectomy decreased day length, on the reproductive system of European starlings. J. Exp.
Zool.
249, 62-67.
Dawson, A. (1989b). The involvement of thyroxine and day length in the development of photorefractoriness in European starlings. J. Exp. Zool. 249, 68-75. Dawson, A., and Goldsmith, A. R. (1983). Plasma prolactin and gonadotrophins during gonadal development and the onset of photorefractoriness in male and female starlings (Srurnus vulgaris) on artificial photoperiods. J. Endocrinol. 97, 253260. Dawson, A., Follett, B. K., Goldsmith, A. R., and Nicholls, T. J. (1985a). Hypothalamic gonadotrophin-releasing hormone and pituitary and plasma
FSH and prolactin during photostimulation and photorefractoriness in intact and thyroidectomized starlings. J. Endocrinol. 105, 71-77. Dawson, A., Goldsmith, A. R., and Nicholls, T. J. (1985b). Development of photorefractoriness in intact and castrated male starlings (Sfurnus vulgaris) exposed to different periods of long day lengths. Physiol. Zool. 58, 253-261. Dawson, A., Goldsmith, A. R., Nicholls, T. J., and Follett, B. K. (1986). Endocrine changes associated with the termination of photoreftactoriness by short day lengths and thyroidectomy in starlings (Sturnus vulgaris). J. Endocrinol. 110, 7379. Famer, D. S., Donham, R. S., Matt, K. S., Mattocks, P. W., Moore, M. C., and Wingtield, J. C. (1983). The nature of photorefractoriness. In “Avian Endocrinology. Environmental Perspectives” (S. Mikami, K. Homma, and M. Wada, Eds), pp. 149-166. Japan Scientific Societies Press, Tokyo. Follett, B. K., Nicholls, T. J., and Mayes, C. R. (1988). Thyroxine can mimic photoperiodically induced gonadal growth in Japanese quail. J. Comp. Physiol. B 157, 829-835. Goldsmith, A. R., and Nicholls, T. J. (1984a). Thyroidectomy prevents the development of photorefractoriness and the associated rise in plasma prolactin in starlings. Gen. Comp. Endocrinol. 54, 256-263. Goldsmith, A. R., and Nicholls, T. J. (1984b). Thyroxine induces photorefractoriness and stimulates prolactin secretion in European starlings (Sturnus vulgaris). J. Endocrinol. 101, R143. Goldsmith, A. R., Nicholls, T. J., and Plowman, G. (1985). Thyroxine treatment facilitates prolactin secretion and induces a state of photorefractoriness in thyroidectomized starlings. J. Endocrinol. 104,99-103. Goldsmith, A. R., Ivings, W. E., Pearce-Kelly, A. S., Parry, D. M., Plowman, G., Nicholls, T. J., and Follett, B. K. (1989). Photoperiodic control of the development of the LH-RH neurosecretory system of European starlings (Sturnus vulgaris) during puberty and the onset of photorefractoriness. J. Endocrinol. 122, 255-268. Hamner, W. M. (1971). On seeking an alternative to the endogenous reproductive rhythm hypothesis in birds. In “Biochronometry” (M. Menaker, Ed), pp. 44w61. Natl. Acad. Sci., Washington, D.C. Klandorf, H., Stokkan, K-A., and Sharp, P. J. (1982). Plasma thyroxine and triiodothyronine levels during the development of photorefractoriness in Willow ptarmigan (Lagopus lagopus lagopus) exposed to different photoperiods. Gen. Comp. Endocrinol.
Nicholls,
47, 64-69.
T. J., Goldsmith,
A. R., Dawson,
A.,
THYROXINE
AND REFRACTORINESS
Chakraborty, S., and Follett, B. K. (1985). lnvolvement of the thyroid gland in photorefractoriness in starlings. In “The Endocrine System and The Environment” (B. K. Follett, S. Ishi, and A. Chandola, Eds), pp. 127-135. Japan Scientific Societies Press, Tokyo. Nicholls, T. J., Goldsmith, A. R., and Dawson, A. (1988a). Photorefractoriness in birds and comparison with mammals. Physiol. Rev. 68, 133-176. Nicholls, T. J., Follett, B. K., Goldsmith, A. R., and Pearson, H. (1988b). Possible homologies between photorefractoriness in sheep and birds: The effect of thyroidectomy on the length of the ewe’s breeding season. Reprod. Nutr. Dev. 28, (2B), 375-385. Sharp, P. J., and Klandorf, H. (1981). The interaction between day length and the gonads in the regulation of levels of plasma thyroxine and triiodothyronine in the Japanese quail. Gen. Comp. Endocrinol. 45, 504-512. Sharp, P. J., Lea, R. W., Dunn, 1. C., and Trocchi, V. (1986). Photoperiodic and endocrine control of seasonal breeding in grey partridge (Perdix perdix). J. Zool. 209(A), 187-200. Sharp, P. J., Dunn, I. C., and Talbot, R. T. (1987). Sex differences in the LH responses to chicken
LH-RH
IN STARLINGS
85
I and II in the domestic fowl. J. Endo115, 323-331. Smith, J. D. (1982). Changes in blood levels of thyroid hormones in two species of passerine birds. Condor 84, 160-167. Stansfield, S. C., and Cunningham, F. J. (1988). Attenuation of endogenous opoid peptide inhibition of (Gln8) luteinzing hormone releasing hormone secretion during sexual maturation in the cockerel. Endocrinology 123, 787-794. Stokkan, K-A., Harvey, S., Klandorf, H., Unander, S., and Blix, A. S. (1985). Endocrine changes associated with fat deposition and mobilization in svalbard ptarmigan. Gen. Comp. Endocrinoi. 58, 76-80. Voitkevich, A. A. (1940). Dependence of seasonal periodicity in gonadal changes on the thyroid gland in Sturnus vulgaris. L. C. R. Dok. Acad. Sci. URSS 27, 741-745. Wieselthier, A. S., and van Tienhoven, A. (1972). The effect of thyroidectomy on the photorefractory period in the starling (Sturnus vulgaris). .I. Exp. Zoo/. 179, 331-338. Winer, B. J. (1971). “Statistical Principles in Experimental Design,” 2nd ed. McGraw-Hill, New York. crinol.
