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1983 Circadian Systems in Development and Photoperiodism in Japanese Quail. Albert C. Russo Jr Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Russo, Albert C. Jr, "Circadian Systems in Development and Photoperiodism in Japanese Quail." (1983). LSU Historical Dissertations and Theses. 3907. https://digitalcommons.lsu.edu/gradschool_disstheses/3907
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Russo, Albert C., Jr.
CIRCADIAN SYSTEMS IN DEVELOPMENT AND PHOTOPERIODISM IN JAPANESE QUAIL
The Louisiana State University and Agricultural and Mechanical Col. Ph.D. 1983
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University Microfilms International
CIRCADIAN SYSTEMS IN DEVELOPMENT AND PHOTOPERIODISM IN JAPANESE QUAIL
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Zoology and Physiology
by Albert C. Russo, Jr. B.S., Louisiana State University, 1971 M.S., Louisiana State University, 1974 August, 1983 ACKNOWLEDGEMENTS
I would like to express my sincere thanks and appreciation to Dr.
Albert H. Meier for his guidance, patience, friendship, and enthusiasm
for science which made every day fun and exciting. I thank Drs. J.
Caprio, J.P. Woodring, K. Corkum, A. Aammond, and W. Johnson for
reviewing the manuscript. I further thank Dr. W. Johnson for supplying
the Japanese quail for this study as well as teaching me how to care for
them. I thank all of my fellow graduates students, especially John M.
Wilson for help along the way. Above all, I thank my parents, Mr. and
Mrs. Albert C. Russo, Sr., for giving me life, and L. Blair Roy for
saving it. TABLE OF CONTENTS
Page
Acknowledgements ...... il
List of Tables ...... v
List of Figures ...... vl
Section I. Maturation of the Circadian Rhythm of Adrenal Cortical Hormone and Thyroxine in Japanese Quail: Effect of hypo- and hyperthyroidism ...... 1
Abstract ...... 2
Introduction ...... 3
Materials and Methods ...... 5
Results ...... 9
Discussion...... 36
Literature Cited ...... 43
Section II. Relation of the Circadian Rhythm of Adrenal Cortical Hormone and Photosensitivity in Japanese Quail ...... 48
A b s t r a c t ...... 49
Introduction ...... 50
Materials and M e t h o d s ...... 52
R e s u l t s ...... 54
Discussion...... 63
Literature Cited ...... 68
Section III. The Roles of the Eyes, the Rhythm of Adrenal Cortical Hormone, and Thyroxine in Photo- periodism in Japanese Quail ...... 70
A b s t r a c t ...... 71
Introduction ...... 72
Materials and Methods ...... 74
R e s u l t s ...... 77
iii Page
Discussion...... 92
Literature Cited ...... 100
V i t a ...... 105
iv LIST OF TABLES
Page
Section I:
Table 1 Body weights, paired testes weights, and ovary weights of 7-, 17-, 27- and 37-day old Japanese quail treated with thiouracil or thyroxine...... 15
Section II:
Table 1 Paired testes weights of intact and enucleated Japanese quail injected with TRH or s a l i n e ...... 56
Section III:
Table 1 Body weights, paired testes weights and ovary weights of Japanese quail pretreated with LD 8:16 for three weeks and then transferred to continuous darkness (DD) . . 80
Table 2 Plasma thyroxine concentrations of reproductively developed or regressed enucleated Japanese quail maintained on different daylengths and sampled at six times of d a y ...... 81
v LIST OF FIGURES
Page
Section I:
Figure 1. Daily rhythm of plasma ACH concentration in 7-, 17-, 27-, and 37-day old Japanese quail maintained on LD 1 6 : 8 ...... 16
Figure 2. Effects of 0.2% 2-thlouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma ACH concen tration in 7-day old Japanese quail maintained on LD 1 6 : 8 ...... 18
Figure 3. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma ACH concen tration in 17-day old Japanese quail maintained on LD 1 6 : 8 ...... 20
Figure 4. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma ACH concen tration in 27-day old Japanese quail maintained on LD 1 6 : 8 ...... 22
Figure 5. Effects of 0.2% thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma ACH concen tration in 37-day old Japanese quail maintained on LD 1 6 : 8 ...... 24
Figure 6. Daily rhythm of plasma thyroxine concentration in 7-, 17-, 27-, and 37-day old Japanese quail maintained on LD 16:8 ...... 26
Figure 7. Effects of 0.2% thiouracil in the diet on the daily rhythm of plasma thyroxine concentration in 7-day old Japanese quail maintained on LD 1 6 : 8 ...... 28
Figure 8. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma thyroxine concentration in 17-day old Japanese quail maintained on LD 1 6 : 8 ...... 30
Figure 9. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma levels of thyroxine in 27-day old Japanese quail maintained on LD 1 6 : 8 ...... 32
Figure 10. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine in the diet on the daily rhythm of plasma thyroxine concentration in 37-day old Japanese quail maintained on LD 1 6 : 8 ...... 34
vi Page
Section II:
Figure 1. Plasma corticosteroid concentrations in repro- ductively developed (#) or reproductively regressed CO) Japanese quail maintained on either LD 8:16 (top) or LD 16:8 ( b o t t o m ) ...... 57
Figure 2. Volume of the left testis (expressed as % of initial volume) of Japanese quail after 15 daily injections of corticosterone (C) or saline (S) at 4 or 16 hours after the onset of light (LD 16:8). uninjected controls: U ...... 59
Figure 3. A model depicting the temporal relations of circadian corticosteroid injections with the LD cycle in reproductively regressed and developed Japanese quail. The photoinducible phase (PIP) occurs 12-20 hours following the daily peak of the plasma corticosteroid concentration (Cen) and the time of corticosterone injection (Cex). Coincidence of light with the PIP is stimulatory for reproductive development whereas coincidence with dark is i n h i b i t o r y ...... 61
Section III:
Figure 1. Left testes volume (LTV) of Japanese quail enucleated after 6 weeks on LD 16:8 and then transferred to LD 8:16. LTV was determined 3 weeks and 32 weeks after transfer to LD 8:16 and expressed as a % of the LTV determined at the time of transfer to LD 8 : 1 6 8 2
Figure 2. Left testes volume (LTV) of enucleated Japanese quail treated with 0.2% 2-thiouracil or 0.001% L-thyroxine in the food. The quail were exposed to LD 8:16 for 8 weeks after hatching, at which point they were enucleated and placed on LD 16:8. After 3 weeks, the LTV was determined, the photoperiod was changed to LD 8:16 and treatment began. LTV was redetermined 3 and 6 weeks after treat ment onset and expressed as the % of initial LTV deter mined at treatment o n s e t ...... 84
Figure 3. Left testes volume (LTV) of enucleated Japanese quail treated with 0.2% 2-thiouracil or 0.001% L-thyroxine in the food. The quail were exposed to LD 16:8 for 12 weeks after hatching, at which time they were placed on LD 8:16. After 3 weeks LTV was determined and treat ment began. LTV was redetermined 3 and 6 weeks after treatment onset and expressed as the % of initial LTV determined at treatment onset ...... 86
vii Page
Figure 4. Plasma adrenal cortical hormone concentrations in reproductively developed (©) or regressed (©) enucleated Japanese quail maintained on either LD 8:16 (top) or LD 16:8 ( b o t t o m ) ...... 88
Figure 5. Plasma thyroxine concentrations in intact reproductively developed (©) or regressed (Q) Japanese quail maintained on either LD 8:16 (top) or LD 16:8 (bottom) ...... 90
viii SECTION I
Maturation of the Circadian Rhythm of Adrenal Cortical Hormone and
Thyroxine in Japanese Quail: Effect of hypo- and hyperthyroidism.
1 Abstract
The daily rhythm of plasma levels of adrenal corticol hormones
(ACH) differed in phase and amplitude in Japanese quail of different ages and reproductive condition. Treatment with thiouracil accelerated and thyroxine delayed maturation of the ACH rhythm and development of the reproductive system. In contrast to the ACH rhythm, the rhythm of plasma thyroxine concentrations was similar at all ages tested.
2 Introduction
Circadian neuroendocrine mechanisms are an integral component of the systems controlling seasonal metabolic and reproductive conditions in birds (reviews, Gwinner, 1973, 1974, 1977; Assenmacher and Jallageas, 1980; Meier, Ferrell, and Miller, 1980). Despite the fundamental nature of these processes, very little information is available concerning their ontogeny. In birds, only the ontogeny of the daily rhythm of plasma levels of adrenal cortical hormones (ACH) has been studied (Russo, 1974; Wilson et. a l ., 1982). The daily rhythm of ACH is an expression of a circadian neuroendocrine oscillation in a number of avian species including the Japanese quail
(Boissin and Assenmacher, 1970), pigeons (Joseph and Meier, 1973), chickens (Beuving and Vonder, 1977), turkeys (El Halawani et^. al.,
1973), and white-throated sparrows (Dusseau and Meier, 1971) and may be an important component of the photoperiodic mechanism in the white-throated sparrow (Meier and Fivizzani, 1975) and the Japanese quail (Section 2). Developmental changes in the rhythm of ACH have been studied only in theJapanese quail (Russo, 1974) and Khaki
Cambell duck (Wilson et. a l ., 1982). The phase and the amplitude of the ACH rhythm changed during different developmental stages in both species, perhaps reflecting maturational changes in the neuroendocrine mechanisms driving the rhythm of ACH.
The thyroid plays a very well documented role in many developmental processes. The thyroid is required for the regulation of growth and reproductive development, and in a number of avian species, thyroidectomy severely inhibited normal maturation of these processes (review, Assenmacher, 1973). The thyroid also plays an important role in the expression of circadian rhythms. Thyroxine was required for the expression of the daily rhythm of corticosterone in hypophysectomized rats (Meier, 1976). In pigeons maintained on constant light, thyroxine was necessary for the integrity of the circadian rhythms of fattening and cropsac response to prolactin
(John et^. a l ., 1972). In addition, thyroxine increased and thiourea decreased the period of the free-running rhythm of activity in canaries (Wahlstrom, 1965).
Thus, it seems likely that the thyroid is involved in the development of circadian neuroendocrine mechanisms. The experiments herein were designed to explore the ontogeny of the circadian rhythms of ACH and thyroxine and to determine the effects of induced hypo- and hyperthyroidism on the maturation of the daily rhythm of plasma concentrations of ACH and thyroxine in the Japanese quail (Coturnix coturnix japonica). Materials and Methods
Japanese quail (Coturnix coturnix japonica) were obtained from the LSU Poultry Science Department on the day of hatching. The quail were divided into three groups and placed on a photophase of LD16:8.
All groups of quail were fed Purina Wild Gamebird starter: the first group received starter alone; the second group received starter with
10 mg of L-thyroxine (Sigma, sodium salt) per kg of starter for the first 14 days, and starter alone thereafter; the third group was fed starter with 2 g of 2-thiouracil (Sigma) per kg of starter until day
27 and 3 g per kg of starter thereafter. Thiouracil is a goitrogen that inhibits iodination of the tyrosyl residues (Goodman and Gilman,
1970). All groups received water ad libitum. The chicks were brooded at 37°C for the first week. The temperature was lowered 3°C each week for 4 weeks and maintained at 25°C for the duration of the experiment.
On days 7, 17, 27 and 37 body weights and paired testes or ovary weights of 10 quail were measured. In addition, blood samples were collected in heparinized capillary tubes from the wing vein of 8 quail every four hours throughout a 24-hour period beginning at the onset of light (0900, 1300, 1700, 2100, and 0500). Each bird was bled only once, and less than 30 sec was required for the completion of each sample, or it was discarded. The plasma was separated and stored at -20°C until assay.
Concentrations of adrenal corticoids were determined in samples
5 from individual birds using a modification of the competitive protein binding technique of Murphy (Meier and Fivizzani, 1975). Duplicate
10 ul samples were extracted with 1.0 ml of absolute ethanol. The samples were placed in a 45°C water bath and dried under air.
Standards containing 0.0, 0.1, 0.5, and 1.0 ng of corticosterone in
0.1 ml absolute ethanol were prepared in triplicate and dried in the same manner. One ml of CBG-solution containing 1.0% male human 3 plasma and 0.04 uCi H-corticosterone (47.5 Ci/mmole) in distilled water, was added to each tube. The tubes were vortexed for 15 sec and incubated at 45°C for 5 min. The tubes were vortexed again for
15 sec. and incubated in an ice-water bath for 30 min. While still in the ice-water bath, 40 mg of Florisil was added to each tube. The tubes were then vortexed for 30 sec. A 0.5 ml aliquot was removed from each tube and added to 5.5 ml of liquid scintillation fluid containing 0.4% p-terphenyl and 33% Triton X-100 in spectrograde toluene.
The samples were counted to 2% error on a Beckman LS-8000. The mean cpm for the replicates were expressed as a per cent of the cpm in the standards with 0.0 corticosterone. Corticosterone concentrations were estimated using a standard curve of per cent of
0.0 cpm versus ng corticosterone in the standards. The extraction efficiency was nearly 100%. The limit of detectability was 0.05 ug%.
The intra-assay variability was 5%, and the interassay variability was 8%.
For thyroxine determinations, 3 plasma pools for each sample time were constructed from 10 ul of plasma from each of 3 quail. Total thyoxine concentrations in each pool were determined using a solid-phase radioimmunoassay kit (Gamraa-coat, Clinical assays, Inc.).
Standards were prepared by adding 0, 0.4, 2.0, 6.0, and 18.0 ug%
L-thyroxine (free acid, Sigma) to stripped quail plasma. Duplicate lOul samples or triplicate standards were added to assay tubes that had antibody to thyroxine immobilized on the lower inner wall of the tube. The samples and standards were incubated for 30 min. with 1 ml 125 of buffer containing ANS, salicylate, and I-thyroxine. Contents of the tubes were aspirated, and the tubes counted for 1 minute on a
Beckman Gamma Counter. The concentrations of thyroxine were estimated using a standard curve prepared by plotting the mean cpm of the standards on the arithmetic axis and the total thyroxine concentrations of the standards on the logarithmic axis of semilog graph paper. All samples were analyzed at the same time to avoid interassay variability. Intraassay variability was 3.2% and the sensitivity was 0.47ug%.
The hormone data were analyzed by a 3 Way Factorial Analysis of
Variance (ANOVA). The factors time (0100, 0500, 0900, 1300, 1700,
2100), treatment (thyroxine, thiouracil, untreated) and age (7, 17,
27, 37 days) were fit by the full model in which all main effects and all possible interactions were tested. Treatments were applied in a completely randomized manner. Comparisons among treatment groups were done by the computation of Least Squares means and t-tests of the probability of difference between the LS means. Although all possible comparisons were calculated only those associated with the preplanned program were used (Freund and Littell, 1981). Body weight 8
and gonad weights were compared using the Student's t-test (Snedecor and Cochran, 1967). Results
ACH Concentration
The daily rhythm of plasma levels of ACH differed in phase and amplitude in Japanese quail of different ages and reproductive condition. Treatment with thiouracil accelerated and thyroxine delayed maturation of the ACH rhythm and development of the reproductive system. In contrast to the ACH rhythms, the rhythm of thyroxine concentration was similar in phase in all ages tested.
Plasma levels of ACH did not vary during the day in untreated quail at 7 days of age (Figure 1). By 17 days of age there was a significant rhythm of ACH (P < .01). Levels increased more than
2-fold from the low point at the onset of light to peak levels 12 hours later (4 hours before the onset of dark), and dropped sharply from that point during the next four hours. The phase of the ACH rhythm was delayed by 4 hours in quail 27 days of age compared with
17-day old quail. Peak levels occurred at the onset of darkness in this group instead of 4 hours earlier. The ACH rhythm of 37-day old quail was further delayed 4 hours after the onset of dark, 8 hours later than the peak at day 17. The amplitude of the ACH rhythm at this age was dramatic. Levels rose from a trough of 2.51 + 0.7 ug% at
4 hours after the onset of light to a peak of 10.11 +1.6 ug% 16 hours later. Also, the overall baseline mean of the 37-day old untreated quail (i.e., the mean of all samples at that age and treatment) was nearly twice as high (P < .01) as the overall means of the other
9 untreated groups which were not different from each other.
Treatment with thyroxine or thiouracil induced differences in the phase and/or the amplitude of the rhythm of ACH at nearly every age tested. At 7 days of age, only quail treated with thyroxine had a significant daily rhythm of ACH levels (Fig. 2). In this group, the level at the onset of light was more than twice as high as the mean of the other sample times (6.85 + 1.9 ug% vs. 2.81 + 0.2 ug%). The ACH levels of the untreated or thiouracil treated groups did not vary significantly during the day. However, the overall mean of the thiouracil-treated quail (0.7 + 0.2 ug%) was significantly lower (P <
.001) than either the thyroxine-treated quail (3.49 + 0.7 ug%) or the untreated group (2.87 +0.1 ug%) at this age.
By 17 days of age, the plasma levels of ACH varied significantly in all groups. However, the phase of the rhythm was different for each treatment. In the thyroxine-treated group (Figure 3), the levels were highest 4 hours after the onset of light (4 hours later than the peak of thyroxine-treated quail at day 7, while lowest levels occurred
8 hours later. In the thiouracil-treated group, dramatic peak concentrations (9.5 + 1.3 ug%) occurred at the onset of darkness, 4 hours after the peak in the untreated group. Lowest levels occurred at the onset of light in both the thiouracil and the untreated groups.
The overall mean of the thiouracil-treated group (5.27 + 1.1 ug%) was significantly greater (P < .001) than that of either the thyroxine-treated group (2.8 + 0.6 ug%) or the untreated group (1.23 +
0.5 ug%).
At 27 days of age in the thyroxine-treated group (Figure 4), highest levels of ACH occurred 8 hours after the onset of light (4 hours later than this group at day 17, and 4 hours after the onset of darkness in the thiouracil-treated group (also 4 hours later than this group at day 17. Recall that in the untreated group at this age, peak concentrations of ACH occurred at the onset of darkness (4 hours later than this group at day 17). Of particular interest is the 4-hour phase delay observed in all groups at 27 days of age as compared to the same treatment groups at 17 days. It may also be noted that the phase of the thiouracil-treated group is delayed 4 hours compared with the untreated group, similar to the situation at 17 days of age. The overall mean of the thiouracil-treated group (4.59 + 0.6 ug%) in
27-day old quail was significantly higher (P < .001) than that of the thyroxine-treated group (2.54 + 0.4 ug%) and the untreated group (2.62
+0.4 ug%).
At 37 days of age, the ACH concentrations were highest in all groups 4 hours after the onset of darkness (Figure 5). Compared with the 27-day old quail, this represents another 4-hour phase delay in the untreated group, a 12-hour phase delay in the thyroxine-treated group, and the same phase in the thiouracil-treated group. Lowest concentration occurred at the onset of light in the thiouracil-treated group, and 4 hours later in the untreated group. In the thyroxine-treated group, except for the peak concentration 4 hours after the onset of darkness, the ACH concentrations were very low. In fact, the overall mean of the thyroxine-treated group (1.40 + 0.5 ug%) was less than 25% of the overall mean of either the thiouracil-treated group (6.57 + 0.7 ug%) or the untreated group (5.97 + 1.0 ug%). At this age, the amplitudes of the thiouracil-treated group (4.90 + 0.6 ug% - 9.50 + 1.3 ug%) and the untreated group (2.51 + 0.7 ug% - 10.11
+1.6 ug%) were remarkable. Thus, it appears that treatment with thiouracil accelerated the maturation of the rhythm of ACH so that thiouracil-treated quail 17 days old had a phase similar to that of untreated quail 27 days old, and 27-day old thiouracil-treated quail had a phase characteristic of 37 day old untreated quail. Treatment with thyroxine, on the other hand, appears to delay maturation of the rhythm of ACH.
Thyroxine Concentration
Plasma concentrations of thyroxine varied significantly (P < .05) throughout the day in each of the three treatment groups at each of the ages tested, except the concentrations of the 7-day thyroxine-treated group which were not tabulated.
In the untreated groups, the concentrations of thyroxine were highest four hours after the onset of darkness at all ages (Figure 6).
The overall means of the 7-day (2.1 + 0.3 ug%) and the 17-day (2.3 +
0.3 ug%) quail were similar. By day 27, the overall mean (3.44 + 0.4 ug%) was significantly higher (P < .01) than those of both day 7 and day 17, but lower (P < .01) than that at day 37 (6.36 + 0.3 ug%).
In 7-day old quail treated with thiouracil, highest concentrations of thyroxine occurred at the onset of darkness (Figure
7). Lowest concentrations occurred 8 hours after the onset of light.
Thus, at this age thiouracil treatment induced a 4 hour phase advance compared with untreated quail. The overall means of the 7-day groups 13
were not different despite thiouracil treatment. At all other ages examined the overall means of the thiouracil-treated groups were about
50% lower than those of the thyroxine-treated or untreated groups.
The concentrations of thyroxine for 7-day old thyroxine-treated quail were not tabulated because those quail were receiving thyroxine in their diet at that time, and the concentrations were all extremely high.
At day 17, the highest levels of thyroxine occurred 4 hours after the onset of light in the thiouracil-treated group (Figure 8), whereas higher levels occurred at the onset of darkness at 27 days of age
(Figure 9) and 4 hours after the onset of darkness at day 37. Highest concentrations of thyroxine occurred at the onset of light at 17 and
27 days of age, and 4 hours after the onset of darkness on day 37
(Figure 10) in thyroxine-treated quail. The overall mean concentrations of thyroxine increased significantly (P < .01) from day
17 (2.79 + 0.2 ug%) to day 27 (3.79 + 0.4 ug%) and from day 27 to day
37 (5.59 + 0.7 ug%; P < .001).
Treatment with thiouracil accelerated the growth rate and the rate of development of the reproductive system while treatment with thyroxine delayed maturation of these events (Table 1). Although, at
7 days of age, quail treated with thiouracil weighed less (P < .05) than untreated quail 19.3 + l.lg vs. 31.19 + 1.3g). Male quail treated with thyroxine had significantly (P < .05) smaller paired testes (2.68 + 0.2mg) than the thiouracil treated (5.93 + 0.7mg) or the untreated groups (6.54 + l.lmg), which were similar. There were no significant differences in ovary weights among the groups at this 14
age. By 17 days of age, the group treated with thiouracil had
significantly (P < .05) greater body weights and ovary weights (69.3 +
2.3 g and 52.12 + 1.3 mg) than the untreated group (67.04 + 1.9 g and
37.8 + 3.9 mg) or the thyroxine-treated group (54.2 + 3.5 g and 24.8 +
2.9 mg). The paired testes weights of the thiouracil-treated group
(52.4 +1.3 mg) were greater than those of the thyroxine-treated group
(11.46 + 2.1 g) but were similar to those of the untreated group (44.9
+ 3.9 g). At 27 days of age, the body and paired testes weights of the thiouracil-treated group (119.0 + 3.5 g and 577.4 + 135.5 mg) were greater than (P < .001) the thyroxine7treated group (102.3 + 2.7 g and
47.7 + 11.8 mg), but were not statistically different from the untreated group (119.9 + 3.1 g and 284.14 + 68.9 mg). Ovary weights of the thiouracil-treated quail (157.9 + 14.4 mg) were about 50% larger (P < .05) than those of the thyroxine-treated quail (108.6 +
10.9 mg) and the untreated quail (104.6 + 6.7 mg). By 37 days of age, there were no differences in body weights or testes weights among the groups. However, ovary weights of the thiouracil treated quail were larger (P<.05) than those in either the untreated or thyroxine-treated groups. (183.3 + 30.4 g vs. 135.0 + 10.3 and 136.8 + 33.5 g). Table 1
Body weights, paired testes weights, ^nd ovary weights of 7-, 17-, 27- and 37- day old Japanese quail treated with thiouracil or thyroxine .
Measurement Treatment Age (days) 17 27 37
Body wt (g) untreated 31.2 + 1.3* 67.0 + 1.9a 119.9 + 3.1* 111.5 + 2.4 Thiouracil 19.3 + 69.3 + 119.0 + 104.8 + 2. 6£ lmlb 2*3b 3‘5b Thyroxine 23.2 + 0.8 54.2 + 3.5 102.3 + 2.7 111.5 + 2.4s
Testes wts (mg) untreated 6.5 + 1.1* 44.9 + 3.9* 284.1 + 68.9* 1195.6 + Thiouracil 5.9 + 0.7* 52.4 + 3.2* 577.4 + l35 . f 1299.0 + a Thyroxine 2.7 + 0.2 11.5 + 2.1 47.7 + 10.9 924.0 +
Ovary wts (mg) untreated 15.7 + 0.8* 37.8 + 3.9 104.6 + 6.7b 135.0 + 10.3 Thiouracil 12.9 + 2-9! 52.1 + 1.3* 157.9 + 14.4*: 183.8 + 30.4* Thyroxine 11.4 + 1.4 24.8 + 2.9C 108.6 + 10.9 136.8 + 33.5
1. 5 animals per group 2. Quail received 2 g of 2-thiouracil per kg of starter for the first 27 days, and then 3 g per kg of starter thereafter. 3. Quail received 10 mg L-thyroxine per kg of starter for the first 14 days, and starter alone thereafter. 4. Mean weight + standard error of the mean. Means with different superscripts were significantly different (P<.05). 16
Figure 1. Daily rhythm of plasma ACH concentration in 7-,
17-, 27-, and 37-day old Japanese quail maintained
on LD 16:8. 17 0500
0100
2100
Time 1700
1300
7 7 Day 17 17 Day 37 Day 27 Day 0900 8 10 [%Bn] HOV emseid 18
Figure 2. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma ACH
concentration in 7-day old Japanese quail
maintained on LD 16:8. Plasma ACH [ug%] 0900 hrxn O Thyroxine Thiouracil oto ® Control
1300
1700 Time
2100
0100
0500 19 Figure 3. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma ACH
concentration in 17-day old Japanese quail
maintained on LD 16:8. Plasma ACH [ug% 10 8 0900 horcl ■ Thiouracil hrxn O Thyroxine otrl • rol Cont
1300
1700 Time
2100
0100 -S.E.
0500 21 22
Figure 4. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma ACH
concentrations in 27-day old Japanese quail
maintained on LD 16:8. Plasma ACH fug%] 10 8 0900 hrxn Q Thyroxine horcl ■ Thiouracil Control Control
1300 ®
1700 Time
2100
S.E— 0100
0500 23 24
Figure 5. Effects of 0.2% thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma ACH
concentration in 37-day old Japanese quail
maintained on LD 16:8. Plasma ACH [ug%] 10 2 8 ■ - 0900 horcl ■ Thiouracil hrxn O Thyroxine o rl • trol Con
1300
1700 i Time
2100
0100 S.E: 0500 i 25 26
Figure 6. Daily rhythm of plasma thyroxine concentration in
7-, 17-, 27-, and 37-day old Japanese quail
maintainedon LD 16:8. Thyroxine Cug %3 10 8 0900 7 Day 37 Day 27 7 Day 17 Day 7
1300
1700 Time
2100 V •
0100
/ 0500 / 27 28
Figure 7. Effects of 0.2% thiouracil in the diet on the daily
rhythm of plasma thyroxine concentration in 7-day
old Japanese quail maintained on LD 16:8. Thyroxine [ug%] 10 8 0900 horcl ■ Thiouracil otrl ® rol Cont
1300
1700 Time 2100
0100 —S.E.
0500 29 30
Figure 8. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma thyroxine
concentration in 17-day old Japanese quail
maintained on LD 16:8. Thyroxine tug%] 10 8 - 0900 hrxn Q Thyroxine horcl ■ Thiouracil I oto ® Control
1300
1700
Time 2100 i
0100 T
0500 T 31 32
Figure 9. Effects of 0.2% 2-thiouracil and 0.001% L-thyroxine
in the diet on the daily rhythm of plasma levels of
thyroxine in 27-day old Japanese quail maintained
on LD 16:8. Thyroxine tug%] 0 1 - 0900 hrxn O Thyroxine horcl ■ Thiouracil Control
1300
1700
Time 2100
0100
0500 ? 33 Figure 10. Effects of 0.2% 2-thiouracil and 0.001%
L-thyroxine in the diet on the daily rhythm of
plasma thyroxine concentration in 37-day old
Japanese quail maintained on LD 16:8. Thyroxine [ug%] 10 8 Tyoie O Thyroxine - 0900 horcl ■ Thiouracil —S.E. oto ® Control
1300
1700 Time
2100
0100
0500 35 Discussion
Maturation of the circadian rhythm of plasma corticosterone concentration appears to be correlated with maturation of the reproductive system in normally developing Japanese quail. In addition, both processes are accelerated by experimentally induced hypothyroidism and delayed by induced hyperthyroidism.
The ACH rhythm apparently develops between day 7 when no rhythm was apparent and day 17 when the peak concentration occurred 4 hours before the onset of darkness (LD 16:8). With further development, the phase of the rhythm was gradually delayed so that the peak occurred at the onset of darkness on day 27, and 4 hours after the onset of darkness on day 37.
The phase shift of the ACH rhythm during development agrees with preliminary findings in Japanese quail (Russo, 1974). However, in the previous study the daily peaks occurred earlier in the photoperiod during most of the maturational period. In the adult quail, the ACH peak occurred at 0 to 4 hours after dark onset (Russo, 1974), which is similar to our findings in the present study for 37-day old quail.
Similar maturational changes in phase of the ACH rhythm have been reported in rats (Ramaley, 1973) and Khaki Cambell ducks (Wilson et. al., 1982).
Although it is not yet possible to decide whether maturation of the ACH rhythm is an important factor regulating development, several lines of evidence suggest that it may have such a role. For example, adrenalectomy delayed onset of puberty in rats (Ramaley, 1974), and
36 37
treatment with corticosterone influenced the time of hatching in chickens (Wentworth and Hussin, 1982). In addition, daily injections of corticosterone at specific times relative to prolactin injections delayed growth and development of the reproductive system in Japanese quail (Russo, 1974).
In the present study, the quail were in different stages of reproductive development at each age tested. In quail maintained on a light-dark cycle with 14 hours or more of light, gonadotropin production begins about day 14 (Siopes et. al., 1979), spermatozoa first appear about day 26 (Mather and Wilson, 1964), and full spermatogenesis is not observed until day 32 (Purull and Wilson, 1975).
This raises questions concerning the relationship of the reproductive system and the maturation of the daily rhythm of ACH concentration.
Are the changes in the ACH rhythm necessary for maturation of the reproductive system? Is maturation of the reproductive system responsible for maturation of the ACH rhythm? Are both processes dependent on the maturation of another system and essentially independent of one another?
Reports of gonadal hormone influences on the plasma concentration of ACH are numerous, although not at all consistent. For example, ACH levels were highest during the breeding season in domestic ducks.
During the non-breeding seasons, levels of ACH were low, and injections of testosterone induced higher levels of ACH similar to those observed during the breeding season (Daniel and Assenmacher, 1976). On the other hand, in the same species, castration increases ACH levels (Chan and Phillips, 1973) or had no effect (Deviche et. al., 1980). Testosterone injections decreased ACH levels in several avian species
(Peczely, 1979), while in Japanese quail ovariectomy decreased concentrations of ACH.
The confusion that exists over this issue suggests that the one dimensional approach utilized by these investigators where no attention is given to time of day may have detected only a small facet of the total change that occurred. Perhaps, gonadal hormones induce phase shifts rather than, or in addition to baseline concentration changes.
If so, then depending on the time of sampling, a phase shift could easily be misinterpreted as a baseline change, or a baseline change and a phase shift could give the impression that no change occurred at all.
In addition, phase alterations of circadian rhythms induced by gonadal hormones have been demonstrated in hamsters (Morin et. al., 1976) and mice (Daan et^. al_., 1975). In rats, estrogen induced phase shifts in the rhythm of CRF (Hiroshige et. al., 1973). In birds, testosterone altered the phase of the circadian locomotor activity rhythm (Gwinner,
1974).
In addition to effects of gonadal hormones on adrenal function, several studies suggest that the adrenal can exert important effects on the reproductive system as well. Implants of corticosterone in the basal hypothalamus induced gonadal regression in tree sparrows (Wilson and Follett, 1975). Systemic injections of corticosterone inhibited gonadal growth in ducks maintained on LD 16:8, but increased secretion of LH in ducks maintained on LD 8:16. The adrenal has been further implicated in timing ovulation in the chicken (van Tienhoven, 1961;
Soliman and Huston, 1974; Wilson and Cunningham, 1980). 39
A series of studies by Meier and coworkers have indicated that the
daily rhythm of ACH may be an expression of a neuroendocrine mechanism
controlling seasonal conditions in the white-throated sparrow (review,
Meier et. a l ., 1981). In these studies, injections of corticosterone
at a time of day corresponding with the time of the daily peak
concentrations of endogenous hormone for a specific season induced in
birds out of season metabolic and reproductive conditions
characteristic of that season. Injections of corticosterone apparently
set the phase of a central circadian neuroendocrine oscillation by way
of feedback influences on serotonergic neurons. Finally, the daily
rhythm of ACH is a component of the mechanisms involved in
photoperiodism in the Japanese quail (Section 2). Injections of
corticosterone entrained the photoinducible phase of the
photosensitivity rhythm into or out of the light, depending on the time
of corticosterone injection with respect to the photoperiod. Thus, the time of day when corticosterone is injected is signally important in ascertaining corticosteroid influences on the reproductive system.
Although thyroidal activity may well be necessary for normal development as indicated by previous researchers, mild hypothyroidism
(present study) accelerated maturation of both the daily rhythm of ACH and of the reproductive system. The phase of the ACH rhythm of
thiouracil-treated 17-day old quail was similar to that of 27-day old untreated quail, and the phase of the ACH rhythm of the 27-day old
thiouracil-treated quail was similar to that of 37-day old untreated quail. Although the quail were treated with thyroxine for only 14 days, the delays in maturation of the ACH rhythm and reproductive AO
development were still present at day 27.
Three of the possible explanations of thyroidal involvement in the
maturation of the daily rhythm of ACH are the following: 1) The
thyroid hormones may influence neural circuits which direct
maturational changes of the ACH rhythm. 2) The thyroid hormones may
influence development of one or more systems that regulate reproductive
development, which indirectly influence maturation of the ACH rhythm by
way of reproductive hormones. 3) The thyroid hormones may have a
direct effect on the adrenal, perhaps altering responsiveness to ACTH.
Relatively few studies have examined the thyroid-adrenal
relationship. In rats the thyroid appears to stimulate adrenal
function. Thyroxine was necessary for the expression of the daily
rhythm of ACH in hypophysectomized rats (Meier, 1976). Thyroxine
injections in immature rats accelerated the development of the adrenal
stress response (Schapiro, 1968) and maturation of the daily rhythm of
ACH (Lenguari et. al., 1977). Studies in birds that did not take time
into consideration suggested that the thyroid had little (Peczely and
Daniel, 1979) or no effect (Peczely, 1979) on adrenal function.
However, experimentally induced hypothyroidism delayed the phase of the
ACH rhythm by 8 hours in adult Japanese quail maintained on LD6:18 as
compared to untreated controls (Kovacs and Peczely, 1983). These
results in adult quail are very similar to those of the present study
in immature quail, and serve to reemphasize the importance of time as a
physiological dimension.
There have been an enormous number of often contradictory reports
on the thyroid-gonad relationship. On the one hand, thyroid hormones are required for reproductive development and activity. Thyroidectomy
led to gonadal collapse in adult birds (Aron and Benoit, 1934; Blivaiss
and Domm, 1942) or delayed development of the reproductive system in
immature chickens (Woitkiwitch 1940) and ducks (Benoit and Aron, 1934).
On the other hand, the thyroid exerts inhibitory influences at several
levels. Thyroidectomy induced development of the reproductive system
in reproductively regressed birds (Pandha and Thapliyal, 1964;
Thapliyal and Pandha, 1965; Wieselthier and van Tienhoven, 1972).
Thyroxine suppressed gonadal hormone levels in Japanese quail (Peczely et. al., 1979), perhaps by increasing metabolic clearance of
testosterone (Jallageas, 1975) or inhibiting LH secretion (Chaisson et. al., 1979).
Inhibition of thyroid function with goitrogens has produced variable results on reproductive indices. Treatment of immature
Japanese quail with thiouracil either inhibited reproductive development (Marks and Lepore, 1968), or had no effect (Howarth and
Marks, 1973). Treatment with methimasol either accelerated or delayed development of the reproductive system of immature chickens (Singh and
Parshad, 1978).
In the present study thiouracil reduced thyroxine levels by about
50%. Apparently, normal amounts of thyroxine during maturation have an inhibitory influence on development and this effect is reduced by the lower thyroxine levels in thiouracil-treated quail.
The daily rhythms of plasma levels of thyroxine and ACH appear to be controlled differently. The phase of the rhythm of thyroxine was essentially the same at all ages tested. The pattern, with low levels 42
during the light and higher concentrations during the dark, is similar
to the pattern in immature (Newcomer, 1974) and adult chickens (Sharp and Klandorf, 1981), and in adult Japanese quail (Almeida and Thomas,
1981).
In the present study, the overall mean thyroxine concentration was similar in 7- and 17-day old quail. The levels increased significantly between day 17 and 27, and again between day 27 and day 37. Thiouracil treatment prevented these maturational increases in overall mean thyroid levels in our study, and perhaps in doing so removed some inhibitory influences as discussed earlier. In chickens, after a decline from high levels on the day of hatching, there was no change in the level of thyroxine during development (Davison, 1976).
Unfortunately, the likelihood of detecting changes was reduced due to single sampling times.
The results of this study are consistent with the idea that the ontogeny of the circadian rhythm of plasma levels of ACH is an expression of the neuroendocrine mechanisms which drive the rhythm, and perhaps regulate development of the reproductive system. Maturation of these systems can be accelerated by inducing mild hypothyroidism with thiouracil, or delayed by inducing hyperthyroidism by adding thyroxine to the food.
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Relation of the Circadian Rhythm of Adrenal Cortical Hormone
and Photosensitivity in Japanese Quail.
48 ABSTRACT
The roles of circadian rhythms in photoperiodism as they relate
to adrenal cortical hormone (ACH) were examined in male Japanese quail, Coturnix coturnix japonica. Plasma ACH concentration was found to undergo circadian variations, which varied in phase with respect to the daily photoperiod depending on the length of photoperiod and condition of the reproductive system. The ACH peak occurred at light onset in sexually regressed quail maintained on LD
8:16 and at 12 and 16 hours after light onset in sexually developed quail kept on LD 8:16 or LD 16:8 respectively. In blind, sexually developed quail maintained on LD 16:8, daily injections of corticosterone for 15 days given early during the photoperiod (4 hours after light onset) induced a 40% reduction in the left testis volume (LTV) whereas injections 16 hours after light onset were ineffective.
Thus, it appears that the daily rhythm of plasma concentrations of ACH and the photosensitivity rhythm are expressions of the same neural oscillation, and that injections of corticosterone at different times with respect to the photoperiod can entrain the oscillation so as to shift the photoinducible phase into or out of the light.
49 Introduction
A primary role for circadian rhythms in photoperiodism was first
II enunciated by Bunning (1936) on the basis of plant and animal
ii experiments. Eventually Bunning's ideas were tested and corroborated in an avian species (Hamner, 1964). It is generally believed that a circadian rhythm of photosensitivity is entrained by the daily light-dark (LD) cycle, and that coincidence of light with a photoinducible phase of the photosensitivity rhythm initiates the sequence of events that results in the photoperiodic (e.g. reproductive) responses (Pittendrigh and Minis, 1964).
An internal coincidence model has been constructed to explain circadian neuroendocrine mechanisms in seasonality largely on the basis of experiments with the white-throated sparrow, Zonotrichia albicollis (Meier and Ferrell, 1978; Meier et al., 1981). The rationale proposed is that there are two circadian neuroendocrine oscillations which interact temporally to regulate seasonal responsiveness to daylength. One oscillation, to which a rhythm of photosensitivity is coupled, is usually entrained by the 24-hour LD cycle. When light coincides with the photoinducible phase, gonadal stimulation occurs. The circadian rhythm of plasma adrenal cortical hormone (ACH) concentration is thought to be coupled to the same oscillation as the photosensitivity rhythm and daily injections of
ACH are believed to reentrain, by way of feedback influences on the nervous system, the oscillation to which the endogenous rhythm of ACH is coupled.
The following experiments were designed to examine these
postulates: 1) If the daily rhythm of ACH is an expression of a
neural oscillation whose phase plays a role in determining the
reproductive condition of the animal, then the phase of the rhythm of
ACH should vary in birds in different reproductive conditions. 2) If
injections of corticosterone entrain a circadian oscillation to which
a rhythm of photosensitivity is coupled, then injections of
corticosterone at different times with respect to the photoperiod should have variable effects on reproductive indices of birds held on
a daily light-dark cycle. 3). If the daily rhythm of either
Thyrotropin Releasing Hormone (TRH) or thyroxine is an expression of
the same neural oscillation as the daily rhythm of ACH, then injections of TRH at different times with respect to the photoperiod should have variable effects on reproductive indices of birds held on a daily light-dark cycle. Materials and Methods
Male Japanese quail (Coturnix coturnix japonica) were obtained from the LSU Poultry Science Department on the day of hatching. The quail were fed Purina Wild Gamebird ration and water aJ libitum.
In Experiment I, the quail were divided into three groups 10 to
15 weeks after hatching: reproductively developed quail maintained on a LD 16:8 regimen from hatching; reproductively developed quail held on LD 8:16 for 15 weeks after hatching; and reproductively regressed quail held on LD 16:8 for six weeks after hatching and then transferred to LD 8:16 for six weeks to induce regression of the reproductive system before sampling. Blood samples were collected every 4 hours in heparinized capillary tubes from the wing vein.
Each bird was bled only once. The plasma was separated and stored at
-20°C until assay by a modification of the competitive protein binding technique of Murphy (Meier and Fivizzani, 1975; Section 1).
In Experiment II, adult Japanese quail were enucleated and placed on a 16-hour daily photoperiod (light onset: 0500) for 6 weeks. Corticosterone (150 ug/0.05 ml 0.9 saline) was injected daily for 15 days either 4 or 16 hours after the onset of light. The injection times were chosen to simulate the peak of corticosteroid observed in Experiment I. The length and width of the left testis were measured with Vernier calipers following unilateral laparotomy before and after the injections. The left testis volume (LTV) was 2 calculated using the formula 4/3 pi (width/2) /(length/2). In Experiment III, adult Japanese quail maintained on LD 16:8 (Light
onset: 0500) were divided into 8 groups. Groups 1 and 2 were
enucleated and injected with 0.4 mg/kg of TRH (Sigma) at 0900 or
2100. Groups 3 and 4 were also enucleated, but were injected with
0.05 ml 0.9 saline at 0900 or 2100. Groups 5 and 6 remained intact and were injected with 0.4 mg/kg TRH at 0900 or 2100. Groups 7 and
8 , also intact, were injected with saline at 0900 or 2100. After 15 days of injection, testes weights of all quail were measured.
The data in Experiment I and II were analyzed by one way analysis of variance (ANOVA) or Student's t where indicated. Data in
Experiment III were analyzed by Student's t. Results
Experiment I. Circadian variations (p < 0.05 for each group:
ANOVA) in plasma ACH concentration were observed in the group held on
long daylengths (LD 16:8) as well as in both groups held on short daylengths (LD 8:16) (Fig. 1). A point of particular interest is
that the phase of the hormone rhythm with respect to the daily photoperiod varied among the three groups.
In reproductively stimulated quail (paired testis weights: 3.6
+ 0.6 g) held on long daylengths, plasma ACH concentration increased sharply beginning twelve hours after light onset and peaked four hours later at the onset of darkness. The levels fell precipitously during the ensueing four hours and remained low throughout the rest of the day.
In quail maintained on LD 8:16, both overall levels of plasma
ACH and phase of the rhythm differed between sexually developing
(paired testis weights: 1.7 + 0.4 g) and undeveloped (paired testis weights: 0.2 + 0.1 g) quail (Fig. 1). The mean ACH concentration for all sampling times was almost five times greater in the developing quail (3.4 ug%) compared with that of birds with regressed testes (0.7 ug%) (p < 0.001, Students' t). In sexually regressed quail, the ACH concentration rose above near baseline levels only at the onset of light. In sexually developing quail, plasma concentrations rose from the nadir at four hours after light onset and peaked eight hours later, four hours after the onset of darkness (12 hours after light onset).
Experiment II. Immediately prior to experimental treatment, laparotomies revealed that the testes of the enucleated quail were 3 moderately well developed (mean volume of left testes: 1400 mm +
168). Daily injections of corticosterone for 15 days made 4 hours after onset of the 16-hour daily photoperiod produced a 40% reduction in left testis volume (p < 0.05: Students' t). However, corticosterone injections at 16 hours after light onset had no appreciable effect. Saline control injections at 4 or 16 hours after light onset were similarly ineffective in reducing testis volume
(Fig. 2).
Experiment III. Daily injections of TRH or saline made 4 or 16 hours after the onset of light (LD 16:8) had no effects on testes weights of intact or enucleated Japanese quail. 56
Table 1
Paired testes weights of intact and enucleated Japanese quail * injected with TRH or saline.
Intact or Enucleated Treatment Paired Testes Weights (g)
09003 2100
Intact
TRH 5.2 + 0.2(7)4 5.1 + 0.5(7) Saline 5.2 + 0.4(6) 5.3 + 0.7(5)
Enucleated TRH 5.0 + 0.8(8) 5.0 + 0.7(7) Saline 6.0 + 0.8(5) 4.5 + 0.5(4)
1. Quail were maintained on LD 16:8. (Light onset: 0900). 2. Quail were injected for 15 days with 0.4 mg/kg of TRH' in 0.05 ml saline, or 0.05 ml of 0.9% saline 3. Time of injection 4. Mean testes weight + standard error (n) Figure 1: Plasma corticosteroid concentrations in reproductively
developed (#) or reproductively regressed (O) Japanese
quail maintained on either LD 8:16 (top) or LD 16:8
(bottom). Plasma ACH [ug%] 0900
1300 ie f Day of Time 1700
2100 0100 o • developed • regressed 0500 58
59
Figure 2: Volume of the left testis (expressed as % of initial volume)
of enucleated Japanese quail after 15 daily injections of
corticosterone (C) or saline (S) at 4 or 16 hours after the
onset of light (LD 16:8). uninjected controls: U. ON o of of initial volume) Left Testis Left Testis Volume
4 4 hours 16 hours 61
Figure 3: A model depicting the temporal relations of circadian
corticosteroid injections with the LD cycle in
reproductively regressed and developed Japanese quail. The
photoinducible phase (PIP) occurs 12-20 hours following the
daily peak of the plasma corticosteroid concentration (Cen)
and the time of corticosterone injection (Cex). Coincidence
of light with the PIP is stimulatory for reproductive
development whereas coincidence with dark is inhibitory. Exogenous Entrainment Endogenous Relation 0 0 0 0 0 Inhibitory Stimulatory Stimulatory Stimulatory
Inhibitory
8 8 8 8 8
or atr ih Onset Light after Hours 16 16 16 16 16
0 0 0 0 0
8 8 8 8 8
16 16 16 16 16
62 0 0 0 0 0
Discussion
Our results support the hypothesis that the ACH rhythm and the photosensitivity rhythm are expressions of the same neural oscillation and that the phases of these rhythms with respect to the daily photoperiod vary predictably in quail in different reproductive conditions. If one assumes that the photoinducible phase occurs during light in reproductively stimulated quail held on long daylengths (LD 16:8), then there is a 12- to 20-hour interval between the peak of the ACH rhythm (16 hours after light onset; Figure 1) and the photoinducible phase (Figure 3). Similarly, in reproductively regressed quail held on short daylengths (LD 8:16), an interval of 12 or 20 hours after the corticosteroid peak at light onset (Figure 1) would place the photoinducible phase in the dark (Figure 3).
A further test of the relation between the ACH rhythm and the photoinducible phase was made possible by the findings that the reproductive system of Japanese quail is developed at ten weeks of age when the quail are maintained on short daylengths from hatching
(Slopes and Wilson, 1974; Konishl, et a l ., 1965). In our study as well, the testes were also enlarged in quail held on LD 8:16 for 15 weeks from hatching. The peak of the ACH rhythm in these reproductively stimulated birds occurred 12 hours after light onset
(Figure 1) rather than at light onset as found in reproductively regressed quail maintained on LD 8:16. Furthermore, a 12- to 20-hour interval from the ACH peak of the reproductively stimulated quail
63 would place the photoinducible phase in the light (Figure 3). The location of the photoinducible phase has been determined in Japanese quail using night interruption experiments in which each group of birds was exposed to a nonstimulatory LD 6:18 or LD 8:16 together with a 0.5 to 4 hour light pulse provided at different times during the dark in different groups. When the light pulse coincided with the photoinducible phase gonadal growth occurred (Follett and Sharp,
1969; Slopes and Wilson, 1980; Wada, 1979). Under these conditions the photoinducible phase occurred 12 to 18 hours after the onset of light. These results are consistant with the results of the present study in which the photoinducible phase occurred 12 to 20 hours after the peak of ACH concentration which occurred at the onset of light in nonstimulated quail held on LD 8:16.
If there is a 12- to 20-hour relation between the peak of ACH rhythm and the photoinducible phase in quail exposed to LD 16:8, then the photoinducible phase must occur during the early part of the light. This conclusion is supported by the result of an elegant experiment in which Cain and Wilson (1970) put Japanese quail on a photocycle consisting of 14 hours of dim green light, which did not indure gonadal growth, and 10 hours of dark. In addition, each group received 3 hours of bright white light during different parts of the dim light. Three hours of bright light at the beginning of the dim green light phase stimulated the reproductive system more effectively than bright light at any other time during the dim light. Thus, the photoinducible phase occurred in this experiment as well.
Apparently, the dim green light phase entrained the rhythm of 65
photosensitivity to bright light as it does in house sparrows
(Menaker, and Eskin, 1967).
These data also support the conclusion that daily injections of
corticosterone can be substituted for photoperiodic entrainment of
the photosensitivity rhythm (Meier and Dusseau, 1973). It was
suggested that the circadian rhythms of ACH and of photosensitivity
are expressions of the same oscillation and that corticosterone
injections set the phase of this oscillation by way of negative
feedback influences on neurotransmitter activities, particularly
those of serotonergic neurons. (Meier, Ferrell and Miller, 1980;
Meier and Horseman, 1978). Injections of corticosterone stimulated
synthesis of serotonin, either by stimulating activity of tryptophan
hydroxylase, the rate limiting enzyme in serotonin synthesis
(Azmitia, et al., 1970), or by stimulating uptake of tryptophan into
the neuron (Neckers and Sze, 1975). Accordingly, corticosterone
injections may be expected to set the phase of the photosensitivity
rhythm and thereby influence the reproductive system positively or
negatively as a function of whether the photoinducible phase
coincides with light or darkness.
The dual functions of light in photoperiodism, entrainment of a
photosensitivity rhythm and photoinduction of photoperiodic events when light coincides with a photoinducible phase, are mediated by two
transducing system. Photoperiodic entrainment of the
photosensitivity rhythm appears to be by way of the eye in Japanese
quail, whereas photoinduction of reproductive stimulation is by way
of extraoccular receptors in the brain (Homma al^., 1979, Eomraa et al., 1980). Thus our attempt to set the phase of a photosensitivity rhythm by corticosterone injections was carried out in enucleated quail in order to remove photoperiodic interference.
The injections of corticosterone may well have entrained a circadian rhythm of photosensitivity (Experiment II). Whereas corticosterone injections were inhibitory for testes weights when given four hours after light (LD 16:8), injections at 16 hours were ineffective (Figure 2). Thus one might conclude that corticosterone sets the phase of the photosensitivity rhythm so that the photoinducible phase occurs >12 to 20 hours after the daily injections
(Figure 4). Corticosterone injections at four hours after light onset cause the photoinducible phase to coincide with darkness with a resultant decrease of support for the reproductive system.
Injections at 16 hours after light onset cause the photoinducible phase to coincide with light and thereby maintain reproductive stimulation by the long daylength. Injections of TRH failed to induce any change in the size of the testes of the Japanese quail.
These results suggest that either TRH injections were not capable of entraining the neural oscillation to which the rhythm of photosensitivity is coupled, or that the timing of the injections was not appropriate for re-entrainment.
It should be emphasized that this model does not ascribe direct effects of costicosteroid hormone on reproduction. The major effect of corticosterone injections is probably indirect by way of its influences on a neural oscillation and thereby influences the phases of its many circadian expressions (Meier and Horseman, 1979). Thus hormone injections appear to entrain a neuroendocrine oscillation, and the circadian rhythms of the endogenous hormones might be considered markers for the temporal relations of neuroendocrine oscillations, yet the hormone rhythms themselves may not be essential components of photostimulation. Literature Cited
Azmitia.^E.C., Algeri, S., an^ Costa, E. (1970). In vivo conversion of H-L-tryptophan into H-serotonin in brain areas of adrenalectomized rats. Science 169, 201-203.
II Bunning, E. (1936). Die endogene Tagesrhythmik als Gundlage der photoperiodischen Reaktion. Ber. Deut. Bot. Ges. 54, 590-607.
Cain, J.R., and Wilson, W.O. (1971). Phases of photosensitivity in the circadian rhythm of Coturnix Quail. J. Interdiscipl. Cycle Res. 2, 427-436.
Follett, B.K., and Sharp, P.J. (1969). Circadian rhythmicity in photoperiodically induced gonadotrophin secretion and gonadal growth in the quail. Nature 223, 968-971.
Hamner, W.H. (1963). Diurnal rhythm and photoperiodism in testicular recrudescense of the house finch. Science 142, 1294-1295.
Homraa, K . , Ohta, M . , and Shkakibara, Y. (1979). Photoinducible phase of the Japanese quail detected by direct stimulation to the brain. In: "Biological rhythms and their central mechanisms", pp. 85-94. (Suda, M . , Hayaishi, 0., Nakagawa, H . , eds.) Elsevier, Amsterdam.
Homma, K . , Ohta, M., and Sakakibara, Y. (1980). Surface and deep photoreceptors in photoperiodism in birds. In "Biological Rhythms in Birds" (Y. Tanabe, K. Tanaka, and T. Ookawa, eds.) pp. 149-156. Japan Scientific Societies Press, Tokyo.
Konishi, T., Ishigiero, T . , Kato, M (1965). Photoperiodical studies on the testicular development of Japanese quail. Zool. Mag. 74, 54-62.
Meier, A.H., and Dusseau, J.W. (1973). Daily entrainment of the photoinducible phases for photostimulation of the reproductive system of the sparrows, Zonotrichia albicolis and Passer domesticus. Biol. Reproduction 8, 400-410.
Meier, A.H., Ferrell, B.R. and Miller, L.J. (1981). Circadian components of the circannual mechanism in the white-throated sparrow. In "Proceeding of XVII International Ornithological Congress", pp. 458-462. Deutsche Ornithologen-Gesellschaft.
Meier, A.H., and Ferrell, B.R. (1978). Avian Endocrinology. In "Chemical Zoology: Aves." A.H. Brush, ed. pp. 214-260. Academic Press, New York.
68 Meier, A.H., and Fivizzani, A.J. (1975). Changes in the daily rhythm of plasma corticosterone concentration related to seasonal conditions in the white-throated sparrow, Zonotrichia albicollls. Proc. Soc. Exp. Biol. Med. 150, 356-362.
Meier, A.H., and Horseman, N.D. (1978). Circadian mechanisms in reproduction. In "Animal Models for Research on Contraception and Fertility". (N. Alexander, ed.) pp. 33-45. Harper and Row, 1978.
Menaker, M., and Eskin, A. (1967) Cirqadian clocks in photoperiodic time measurement: A test of the Bunning hypothesis. Science 157, 1182-1185.
Neckers, L . , and Sze, P.Y. (1975). Regulation of 5-hydroxytryptamine metabolism in mouse brain by adrenal glucocorticoids. Brain Res. 93, 123-132.
Pittendrigh, C.S., and Minis, D.H. (1964). The entrainment of circadian oscillations by light and their role as photoperiodic clocks. Amer. Natur. 98, 261-294.
Siopes, T.D., and Wilson, W.O. (1974). Extraoccular modification of photoreception in intact and pinealectomized Coturnix. Poultry Sci. 53, 2035-2041.
Siopes, T.D., and Wilson, W.O. (1980). Participation of the eyes in the photosexual response of Japanese quail. Biol. Reprod. 23, 352-357.
Wada, M. (1979). Photoperiodic control of LH secretion in Japanese Quail with special reference to the photoinducible phase. Gen. Comp. Endocrinol. 39, 141-149. Section III
The Roles of the Eyes, the Rhythm of Adrenal Cortical Hormone,
and Thyroxine in Photoperiodism in Japanese Quail.
70 Abstract
The roles of the eyes and the daily rhythm of adrenal cortical hormone (ACH) and thyroxine in photoperiodism were examined in male
Japanese quail. The reproductive system of quail developed in continuous darkness (DD) after pretreatment with LD 8:16 for 3 weeks.
The reproductive system of Japanese quail raised and enucleated after
6 weeks exposure to LD 16:8 regressed initially in response to LD
8:16, but recrudesced during the next 8 months exposure to LD 8:16.
Reproductively undeveloped quail raised and enucleated on LD 8:16, developed reproductively when exposed to LD 16:8 and did not regress on subsequent exposure to LD 8:16 unless treated with 0.2%
2-thiouracil in the food. Concentrations of thyroxine were lower in intact reproductively developed quail exposed to LD 16:8 compared with intact quail (reproductively regressed or stimulated) exposed to
LD 8:16. Significant daily variations in plasma thyroxine concentration occurred in all intact quail groups, but did not occur in any enucleated quail. In addition, there were no significant daily variations in plasma ACH concentration in enucleated quail.
These results demonstrate that reproductive development can occur in the absence of light or the eyes entrainment of the daily ACH and thyroxine rhythms appear to require the eyes and a hypothyroid condition may be necessary to sensitize enucleated quail to the inhibitory effects of short daylenths.
71 Introduction
The importance of light in controlling reproductive activity in
birds was first demonstrated by Rowan in 1926. Since then, it has
been demonstrated many times that birds use circadian mechanisms to measure photoperiodic time (reviews, Hamner 1966; Farner and Gwinner
1980; Assenmacher and Jallageas, 1980; and Meier et al., 1981). It
is generally believed that a circadian rhythm of photosensitivity is
entrained by the daily light-dark cycle, and coincidence of light with the photoinducible phase of the photosensitivity rhythm results
in development of the reproductive system. This type of model has
been termed an "external coincidence" model (Pittendrigh and Minis,
1964). However, this model does not adequately explain the results
of experiments in which photoperiodic reactions are induced even in
the absence of light. For example, gonadal development was maintained in constant darkness (DD) in slate-colored juncos
(Wolfson, 1966) and Japanese quail (Cain and Wilson, 1971) pretreated
on LD 16:8 or LD 14:10 for 8 days or 2 weeks. Such inadequacies in
the "external coincidence" model stimulated construction of an
"internal coincidence" model to explain circadian neuroendocrine mechanism in photoperiodism (Meier and Ferrell, 1978). The rationale
proposed is that there are two circadian neuroendocrine oscillations
that interact temporally to regulate the reproductive system.
Changes in the phase relations of the oscillations in response to
daylength alter the complex of neural and hormonal messages released
72 and thereby adjust reproductive indices. One oscillation, to which a rhythm of photosensitivity is coupled, is entrained by the 24-hour light dark cycle. The other oscillation is coupled to the first in various phase relations usually by interaction of light with the photoinducible phase of the photosensitivity rhythm. When light coincides with the photoinducible phase, the oscillations are set into a stimulatory relation for the photoperiodic response (eg. gonadal development). In addition, the oscillations may be set into a stimulatory or inhibitory relation by means other than light (eg. hormone injections).
The daily rhythm of plasma concentration of adrenal cortical hormone (ACH) is an expression of the same neural oscillation as the photosensitivity rhythm (Meier et al., 1981; Section 2). Further, entrainment of the photosensitivity rhythm in Japanese quail (Homma et al., 1979) and the ACH rhythm in mice (Haus et al., 1967) appears to involve photoreceptors in the eye. In addition, the phase of the
ACH rhythm can be altered in immature (Section 1) or adult Japanese quail (Kovacs and Peczeley, 1983) by thyroid hormones. The experiments reported herein examine the roles of the eyes, the rhythms of ACH, and thyroxine in photoperiodism in Japanese quail in order to test several aspects of the "internal coincidence" model. Materials and Methods
Japanese quail (Coturnix coturnix japonica) were obtained from
the LSU Poultry Science Department on the day of hatching. The
chicks were brooded at 37° C for the first week, and the temperature
was lowered 3° C each week for 4 weeks and maintained at 25° C for
the duration of the experiment. The quail were supplied with water
and Purina Wild Gamebird starter ad^ libitum.
In Experiment I, male the quail were placed on LD 8:16 for 3
weeks after hatching, and on continuous darkness (DD) thereafter. At
60, 80 and 125 days of age body and gonad weights of 7 male and 7
female quail were measured.
In Experiment II, quail were maintained on LD 16:8 for the first
6 weeks. At that point the quail were enucleated, the left testis
volume (LTV) was determined via laparotomy, and the photoperiod was
altered to LD 8:16. LTV was calculated using the formula 4/3
(width/2)^ (length/2). After three weeks on LD 8:16 the LTV was
determined again, and blood samples were collected from the wing vein
every 4 hours in heparinized capillary tubes. Each bird was bled
only once, and less than 30 sec was required for the collection or
the sample was discarded. The plasma was separated and stored at
-20° C until assay for ACH and thyroxine. After 8 months on LD 8:16
blood samples were retaken and LTV determined again.
In Experiment III, male quail were maintained on LD 8:16 for 2 months at which time they were enucleated and placed on LD 16:8. In
74 75
addition, testes weights of 5 quail were measured to determine the
state of reproductive development. After 3 weeks on LD 16:8, LTV was
determined, blood samples were taken for hormone determinations, and
the quail were divided into 3 groups and placed on LD 8:16. Group
one received 10 mg of L-thyroxine (Sigma, sodium salt) per kg of
starter feed; group 2 received 2g of 2-thiouracil per kg started;
group 3 continued to receive starter alone. The LTV for all groups
was determined 3 and 6 weeks after the beginning of drug treatment.
In Experiment IV, quail were maintained on LD 16:8 for 3 months
at which time they were enucleated and placed on LD 8:16. Testes weights of 5 quail were measured to determine the state of
reproductive development. After 3 weeks on LD 8:16, the LTV's were
determined and the quail were divided into 3 groups, as in Experiment
II.
In addition to blood samples taken from enucleated quail (Exp. 2
and 3), blood samples were also collected, for thyroxine
determinations from sighted quail under several conditions:
reproductively developed quail maintained on LD 16:8 for 10 weeks;
reproductively developed quail maintained on LD 8:16 for 15 weeks;
and reproductively regressed quail held on LD 16:8 for six weeks and
then transferred to LD 8:16 for another six weeks to induce
regression of the reproductive system before blood samples were
collected.
ACH concentrations were determined in samples from individual
birds using a modification of the competitive protein binding
technique of Murphy (Meier and Fivizzani, 1975; Section 1). Thyroxine concentrations were determined using a solid-phase radioimmunoassay kit (Gamma Coat, Clinical Assays, Inc.). Thyroxine standards were prepared in stripped quail plasma (Section 1).
The results of Experiment I-IV were analyzed by Student _t tests.
The hormone data was analyzed by one-way Analyses of Variance
(Snedecor and Cochran, 1967). RESULTS
Experiment I. Reproductive development was slow initially in intact Japanese quail maintained for 2 weeks on LD 8:16 and then transferred to DD (Table 1) as compared to quail raised on LD 16:8
(Mather et al., 1964). Paired testes and ovary weights after 6 weeks were 25.4 + 2.7 mg and 156.0 + 15.5 mg and were 148.1 + 49.3 mg and
155.5 + 22.5 mg after 9 weeks. However, after 12 weeks on DD paired testes were well developed (2278.4 + 303.4 mg) and 5 out of 7 females were laying regularly.
Experiment II. The LTV decreased 83.2% (P < .01) 3 weeks after quail raised on LD 16:8 were transferred to LD 8:16 (from 2565.9 +
3 3 246.2 mm to 288.1 + 60.5 mm ) (Figure 1). After 8 months on LD 8:16 the LTV was 82.5% (1769.1 + 254.8 mm^) of the initial volume.
Experiment III. Quail which were reproductively regressed
(234.3 + 15.6 mg) after 2 months on LD 8:16 were enucleated and
3 placed on LD 16:8. Three weeks later the LTV was 3093 + 196.5 mm
(Figure 2). At this point the quail were placed on LD 8:16 and treatment was begun. After 3 weeks on LD 8:16 the LTV of the thiouracil-treated group had regressed to 48.5% and 59.1% after 6 weeks as compared to the LTV measured after exposure to LD 16:8. The
LTV of quail treated with thyroxine (81.8% and 77.2%) or untreated
(76.1% and 88.0%) approached that of the initial LTV.
Experiment IV. Reproductively developed quail (paired testes weight, 6380.0 + 300 mg) were enucleated after 3 months on LD 16:8,
77 and placed on LD 8:16. Six weeks later, after 3 weeks of treatment, the LTV of the thiouracil-treated group was 32.6% (P < .05) of the initial LTV (Figure 3). The thyroxine treated had only 3 quail remaining at this point, and consequently was not statistically different from the initial LTV.
ACH Concentrations.
No significant daily rhythms of plasma concentrations of ACH occurred in blind quail (Figure 4). The overall mean (the mean of all values of a group across time) of blind, sexually regressed (1.48
+ 0.3 ug%) quail maintained,on LD 16:8 (0.57 + 0.1 ug%) was significantly lower (P < .001) than the overall mean of the sexually stimulated (2.73 + 0.3 ug%) or sexually regressed quail maintained on
LD 8:16.
Thyroxine Concentrations.
Significant (P < 0.5) daily variations of plasma concentrations of thyroxine occurred in intact, reproductively stimulated or regressed Japanese quail maintained on LD 8:16, and in reproductively stimulated quail maintained on LD 16:8 (Figure 5). In both LD 8:16 groups, thyroxine concentrations were highest at the onset of light and declined during the next 4 to 8 hours. Highest concentrations occurred 4 hours after onset of darkness and lowest levels occurred at the onset of light in the LD 16:8 group. The overall mean was lowest in reproductively stimulated quail maintained on LD 16:8 (1.42
+ 0 . 1 ug%) as compared to reproductively stimulated (2.11 + 0.2 ug%) or regressed (2.11 + 9.2 ug%) quail maintained on LD 8:16. There were no significant daily variations or overall mean differences of thyroxine concentrations among the enucleated quail groups (Table 2). 80
Table 1
Body Yeights, paired testes weights and ovary weights of Japanese quail pretreated with LD 8:16 for three weeks and then transferred to continuous darkness (DD).
Measurement Weeks of Exposure to DD
8 12 16
Body Weight (g) 145.0 + 3.32 148.6 + 2.7 157.0 + 4.2
Testes Weight (mg) 25.4 + 2.7 148.1 + 49.3 2278.4 + 303.4
Ovary Weight (mg) 156.0 + 15.5 155.5 + 22.5 5 of 7 laying
1. 7 males and 7 females were measured at each sampling time 2. Mean + standard error 81
Table 2
Plasma thyroxine concentrations of^reproductively developed or regressed enucleated Japanese quail maintained on different daylengths and sampled at six times of day.
Thyroxine Concentrations
Sample Reproductively Reproductively Time Developed Regressed
LD 8:162 LD 16:83 LD
0900 0.8 + 0.15 4.0 + 1.1 2.1 0.2 1300 1.1 + 0.2 1.8 + 0.4 1.5 0.1 1700 1.2 + 0.2 1.3 + 0.3 1.4 0.1 2100 1.4 + 0.2 1.1 + 0.1 1.4 0.1 0100 1.6 + 0.2 2.3 + 0.8 1.6 0.2 0500 1.4 + 0.1 1.5 + 0.4 1.6 0.1
1. ug % 2. 8 animals per group 3. Light onset: 0900; Light offset: 1700 4. Light onset: 0900; Light offset: 0100 5. Mean + standard error Figure 1: Left testes volume (LTV) of Japanese quail enucleated after
6 weeks on LD 16:8 and then transferred to LD 8:16. LTV
was determined 3 weeks and 32 weeks after transfer to LD
8:16 and expressed as a % of the LTV determined at the time
of transfer to LD 8:16. % of Initial LTV 100 0 4 0 6 0 8 20 ek atr Enucleation after Weeks 2 3 3 S.E. — 83 84
Figure 2: Left testes volume (LTV) of enucleated Japanese quail
treated with 0.2% 2-thiouracil or 0.001% L-thyroxine in the
food. The quail were exposed to LD 8:16 for 8 weeks after
hatching, at which point they were enucleated and placed on
LD 16:8. After 3 weeks, the LTV was determined, the
photoperiod was changed to LD 8:16 and treatment began.
LTV was redetermined 3 and 6 weeks after treatment onset
and expressed as the % of initial LTV determined at
treatment onset. % of Initial LTV 100 120 60 80 40 20 Control 3
6 S.E. Thiouracil 3
6 Thyroxine 3
6 85 Figure 3: Left testes volume (LTV) of enucleated Japnese quail
treated with 0.2% 2-thiouracil or 0.001% L-thyroxine in the
food. The quail were exposed to LD 16:8 for 12 weeks after
hatching, at which time they were enucleated and placed on
LD 8:16. After 3 weeks LTV was determined and treatment
began. LTV was redetermined 3 and 6 weeks after treatment
onset and expressed as the % of initial LTV determined at
treatment onset. 3 6 3 6 3 6 Control Thiouracil Thyroxine 88
Figure 4: Plasma adrenal cortical hormone concentrations in
reproductively developed (©) or regressed (©) enucleated
Japanese quail maintained on either LD 8:16 (top) or LD
16:8 (bottom). Plasma ACH Iug %) ■ O developed O O O developed regressed ^ 0 0 5 0 0 0 1 0 0 0 1 2 0 0 7 1 0 0 3 1 0 0 9 0 V
-O' _L_ I ie f Day of Time 0 1 o I 89 90
Figure 5 Plasma thyroxine concentrations in intact reproductively
developed (©) or regressed (©) Japanese quail maintained
on either LD 8:16 (top) or LD 16:8 (bottom). Plasma Thyroxine lug%| 0 0 1 2 0 0 7 1 0 0 3 1 0 0 9 0 O 1-S.E. ie f Day of Time _L o 0100 Odeveloped regressed • developed O 0 0 5 0 91
Discussion
The reproductive system of Japanese quail (Coturnix coturnix
japonica) usually develops in response to daylengths of more than 12
hours of light and regresses to daylengths less than 12 hours (Mather
and Wilson, 1964; Tanada et^ al_., 1965). This so called "photosexual
reflex" was believed to be so predictable that this species is often
used as a model for the study of photoperiodic mechanisms. However,
the results of several studies through the years indicate exceptions
to the "photosexual rule" mentioned above. For example, the
reproductive system of maturing Japanese quail developed despite
exposure to a usually nonstimulatory LD 8:16 (Konishi et^ aT., 1965;
Scopes and Wilson, 1974; Section 2). In addition, adult Japanese
quail exposed to alternating periods (6-14 weeks) of long (LD 16:8)
and short (LD 8:16) daylengths become refractory to the inhibitory
effects of short days (Oishi, 1978; Oishi and Konishi, 1983).
Further, the testes of adult Japanese quail increased in size during
exposure to DD after pretreatment with LD 14:10. Thus, it appears
that hypotheses which envision a strict requirement of coincidence of
light with a photosensitive phase in order to elicit gonadotrophin
release (external coincidence model) would seem untenable.
In the present study, reproductive development occurred in
Japanese quail maintained on DD for 4 months after pretreatment with
LD 8:16 for 3 weeks after hatching. Paired testes weights were 2.3
+ 0.30 g, and 5 of 7 females were laying regularly. This is
92 particularly significant in light of a statement by Gwinner (1981) that "significant (gonadal) growth has apparently never been rigorously documented under such conditions" (i.e. exposure to DD).
Another important point is that the quail were pretreated with a usually nonstimulatory (LD 8:16) photoperiod in the present study, whereas in the earlier study with quail, pretreatment consisted of exposure to a stimulatory (LD 14:10) photoperiod. Continued development in DD after transfer from a long stimulatory daylength has been termed a "carry over" effect of photoperiodic stimulation
(Farner and Lewis, 1971).
Development of the reproductive system under normally nonstimulatory photoperiodic conditions might best be explained using the "internal coincidence" model for photoperiodism. As stated in the introduction, induction of gonadal growth occurs when 2 neuroendocrine oscillations are in a stimulatory relation. Usually, the neuroendocrine oscillations are entrained into, or out of, a stimulatory relation by the daily photoperiod, although entrainment by other means, hormone injections, for example (Meier, 1981, Section
2), is possible.
The location and participation of the photoreceptors involved in the gonadal response to light remains a topic of great debate.
Originally, the testes and the skin of the foot were thought to contain the photoreceptors (Bissonnette, 1930). Since that time numerous studies have demonstrated the presence of 2 types of photoreceptors involved in the reproductive response to light in birds: retinal receptors and extraretinal receptors probably located in the hypothalamus (reviews, Benoit, 1964; Homma et al., 1979;
Oliver and Bayle, 1982.
In house sparrows, the eyes do not appear to have any influence
on the photosexual response (menaker, 1968; Menaker and Keats, 1968;
Menaker et al., 1970, and McMillan et al., 1975). While the eyes are
not necessary for the development of the reproductive system in
response to light in ducks (Benoit, 1964) quail (Oishi et al., 1966),
and white- and golden-crowned sparrows (Gwinner et^ 1975; Yokoyama and Farner, 1976), the eyes do appear to exert a contributory influence in these species. In Japanese quail, photoperiodic entrainment of the rhythm of photosensitivity appears to be mediated by retinal photoreceptors, whereas photoperiodic induction of gonadal growth is a function of the hypothalamic receptors (Homma et al., 1979). The state of development of the reproductive system and/or the photoperiodic history at the time of enucleation appear to affect the response of Japanese quail to subsequent exposure to short daylengths (Homma et^ al_., 1972). Quail maintained on LD 16:8 and enucleated while reproductivity developed, regressed when exposed to LD 8:16. On the other hand, quail maintained on LD 8:16 and enucleated while reproductively regressed, became reproductively developed when exposed to LD 16:8 and did not regress on subsequent exposure to LD 8:16. However, in another experiment, reproductively developed quail enucleated after exposure to LD 16:8 did not regress when exposed to LD 8:16 (Siopes and Wilson, 1979). In Experiments II and IV of the present study, the testes of quail enucleated after exposure to LD 16:8 regressed in response to LD 8:1,6. However, in Experiment II gonadal recrudescense occurred spontaneously and was maintained during an extended exposure to LD 8:16. Wada (1979) has suggested that the photoinducible phase of Japanese quail can increase in length. Perhaps in the absence of the entraining influences of the retinal photoreceptor, the photoinducible phase lengthens and free-runs so as to be partially within the light much of the time. One of the purposes of the present study was to determine whether thyroid hormones are involved in the response of enucleated quail to daylength. Several lines of evidence suggest a photoperiodic role for the thyroid in intact birds. For example, thyroid hormones exert a wide range of stimulatory and inhibitory effects on the reproductive system of birds (reviews, Assenmacher, 1973; Assenmacher and Jallageas, 1980; Pethes et al., 1981). In immature (Section 1) and adult (Kovacs and Peczely, 1983) Japanese quail, thyroid hormones can alter the phase of the ACH rhythm, which we believe is an expression of the same neuroendocrine oscillation as the rhythm of photosensitivity (Meier et al., 1981). In addition, thyroid hormones appear to be involved in the annual rhythm of reproductive events in several species of birds. In the spotted munia, the phase of the annual rhythm of thyroid hormone levels is inversely related to the annual gonadal cycle (Chandola and Thaphyal, 1973). In Peking duck and teal, plasma thyroxine concentrations gradually increase during the breeding season to a sharp peak at the very end of the breeding season (Jallageas et al., 1978). In Experiment III, Japanese quail raised on LD 8:16 were enucleated and placed on LD 16:8 for 3 weeks, and then re-exposed to LD 8:16. As in previous studies (Homma et al., 1972, Scopes and Wilson, 1974) gonadal regression did not occur in response to short daylengths in untreated quail. However, significant gonadal regression did occur in quail treated with thiouracil. Perhaps a period of low concentration of thyroxine is necessary to restore the sensitivity of the reproductive system to short photoperiods. This conclusion is supported by the observation that thyroxine levels are low in intact reproductively developed quail maintained on long daylengths (Figure 5). Quail under these conditions usually respond to the inhibitory effects of short daylengths. On the other hand, enucleated quail exposed to LD 16:8 did not have lower levels of thyroxine as compared to intact quail maintained on LD 16:8 or enucleated quail exposed to LD 8:16, and did not respond to the inhibitory effects of short daylengths. The observation that thyroxine did not prevent the inhibitory effects of short photoperiods in quail enucleated during exposure to LD 16:8 (Figure 3) suggests that the period of low thyroxine levels must precede the exposure to short daylengths if it is to resensitize the reproductive system. In the present study, thyroxine treatment began 3 weeks after the quail were transferred to LD 8:16. Intact quail exposed to LD 16:8 had significantly lower overall mean thyroxine levels than intact reproductively regressed or developed quail exposed to LD 8:16. In previous studies, intact Japanese quail exposed to long days had lower thyroxine levels than quail exposed to short days (Sharp and Klandorf, 1981; Peczely et al., 1979; Almeida, 1982). The lower thyroxine levels were attributed to an inhibitory influence of gonadal hormones. In the present study, however, the length of the photoperiod appeared to exert a greater influence than the reproductive state of the quail; reproductively developed intact quail exposed to LD 8:16 had significantly higher overall thyroxine levels than reproductively developed quail exposed to LD 16:8. In addition, the presence of the eyes appear necessary for long daylengths to exert an inhibitory effect on thyroxine levels. In the present study, enucleated quail maintained on LD 16:8 had higher levels of thyroxine than quail on LD 8:16. Although there were no significant daily variations of plasma thyroxine concentration in any of the enucleated groups (Table 2), significant daily rhythms did occur in all intact groups (Figure 5). Peak levels of thyroxine occurred at the end of the dark in the groups exposed to LD 8:16, or 4 hours before in LD 16:8 group. These patterns are similar to those observed in chickens (Newcomer, 1974), and Japanese quail (Almeida and Thomas, 1981). These results and ours indicate an inverse relation between thyroxine concentrations and locomotor activity in these diurnal animals and support the suggestion that thyroid hormone rhythms are consequences of both rhythms of secretion and utilization (review, Meier, 1975). The overall mean ACH levels in enucleated quail were significantly affected by the length of the photoperiod. Enucleated reproductively developed quail exposed to LD 16:8 had significantly lower ACH concentrations as compared to enucleated quail (developed and regressed) exposed to LD 8:16. The inhibitory effect of long days on overall ACH levels cannot be explained in terms of effects of gonadal hormones; one of the groups maintained on LD 8:16 was reproductively developed. However, the inhibitory effect of long daylengths does seem to involve the absence of the eyes. In intact reproductively regressed quail maintain on LD 8:16, the overall ACH concentrations were much lower than in reproductively developed quail maintained on LD 8:16 and LD 16:8 (Section 2). The daily rhythm of plasma ACH concentration appears to be an expression of the same neuroendocrine oscillation as the rhythm of photosensitivity in the white-throated sparrow (Meier et al., 1981) and the intact Japanese quail (Section 2). The phase of the ACH rhythm varied predictably with respect to the photoperiod and the photoinducible phase in quail in different reproductive conditions (Section 2). In enucleated Japanese quail, there was no significant daily rhythm of plasma concentration of ACH. These results suggest that the ACH rhythm was either damped out in individual enucleated quail or that the rhythm was free-running and the colony had become desynchronized. The high degree of variability supports the latter explanation. Enucleation has been shown to abolish the rhythms of ACTH and ACH in rats and mice (Saba et al., 1963; Haus, 1964; Halberg 1969). If the relationship between the photoinducible phase and the rhythm of ACH is maintained in enucleated quail, then the photosensitivity rhythm is free-running as well. This conclusion is further supported by the results of Section 2 in which corticosterone injections were shown to entrain the rhythm of photosensitivity in enucleated quail. The results of this study suggest that the mechanisms controlling reproductive development can be divorced from the daily light-dark schedule so that gonadal growth can occur even in continuous darkness. It appears that enucleated Japanese quail tend toward reproductive development, and become refractory to the inhibitory effects of short photoperiods. Also, the rhythm of ACH concentration seems to free-run in enucleated quail supporting the idea that the ACH rhythm and the photosensitivity rhythm are entrained by the daily photoperiod via retinal photoreceptors. There is an indication that the photoinducible phase can become lengthened (Wada, 1979). Perhaps in enucleated quail the photoinducible lengthens, and although free-running is partially within the light at most times. However, the demonstration that reproductive development can occur in DD casts serious doubt on the premise that light is necessary for the induction of reproductive development in Japanese quail. Finally, there is a suggested role for thyroid hormones in the mediation of the inhibitory effects of short daylengths. Literature Cited Almeida, O.F.X. (1982). Hypothalamic secretion of thyroprophin releasing hormone is decreased in male Japanese quail exposed to long daily photoperiods. J. Endocr. 92, 261-265. Almeida, O.F.X., and Thomas, D.H. (1981). 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Extaocular modification of photoreception in intact and pinealectomized Coturnix. Poultry Sci. 53, 2035-2041. 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. Snedecor, G.W., and Cochran, W.G. (1967). "Statistical Methods," ed. 6 . pp. 481. Iowa State University Press. Ames, Iowa. Tanaka, K . , Mather, F.B., Wilson, W.O., and McFarland, L.Z. (1965). Effect of photoperiods on early growth of gonads and on potency of gonadotropins of the anterior pituitary in Coturnix. Poultry Sci. 44, 662-665. Thapliyal, J.P. (1969). Thyroid in avian reproduction. Gen. Comp. Endocrinol. Suppl. 2, 111-122. Thapliyal, J.P., and Pandha, S.K. (1965). Thyroid-gonad relationship in spotted munia, Uroloncha punctulata. Gen. Grasp. Endocrinol. 8 , 84-93. Turek, F.W. (1975). Extraretinal photoreceptionuring the gonadal photorefractory period in the golden-crowned sparrwo. J. Comp. Physiol. 96, 27-36. Wada, M. (1979). Photoperiodic control of LH secretion in Japanese quail with special reference to the photoinducible phase. Gen. Comp. Endocrinol. 39, 141-149. Wolf son, A. (1966). Environmental and neuroendocrine regulation of annual gonadal cycles and migratory behavior in birds. Recent Prog, in Horm. Res. 12, 177-244. Yokoyama, K . , and Famer, D.S. (1976). Photoperiodic responses in bilaterally enucleated female white-crowned sparrows, Zonotrlchia leucophrys gambelii. Gen. Comp. Endocr. 30, 528-533 (1976). Yokoyama, K . , Oksche, A., Dardene, T.R., Farner, D.S. (1978). The site of encephalic photoreception in photoperiodic induction of the growth of the testes in the white-crowned sparrow, Zonotrichia leucophrys gambelii. Cell. Tiss, Res. 189, 441-467. VITA Albert C. Russo, Jr. was born November 12, 1949, in Abbeville, La. In the fall of 1967, after graduation from Abbeville High School, he entered Louisiana State University from where he was graduated with Bachelor of Science degree in Zoology in 1971. In Fall of 1971 he entered graduate school at LSU under the direction of Dr. Albert H. Meier and was granted a Master of Science degree in Zoology in 1974. In 1976, Mr. Russo entered the doctoral degree program in the Department of Zoology and Physiology. He has been employed as an Instructor in the Department of Zoology and Physiology since 1981, and is also a candidate for the degree of Doctor of Philosophy at Louisiana State University. 105 EXAMINATION AND THESIS REPORT Candidate: Albert C. Russo, Jr. Major Field: Physi ology Title of Thesis: Circadian Systems in Development and Photoperiodism in Japanese Quail Approved: AMatjl+A Major Professor and^Chairman i\J aIa-*"-. • . I V]AA Dean of the Gradu. ate (school EXAMINING COMMITTEE: Date of Examination: July 19. 1985