Influence of pinealectomy on plasma and extrapineal melatonin rhythms in young chickens (Gallus domesticus)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

68, 343-356 (1987)

Influence of Pinealectomy on Plasma and Extrapineal Metatonin Rhythms in Young Chickens (Gallus domesZicus)l ‘) LARRY A. COGBURN,SARAWILSON-PLACENTRA,AND

L.ROXANNE

LETCHER

Delaware Agricultural Experiment Station, Department of Animal Science and Agricultural Biochemistry, College of Agricultural Sciences, University of Delaware, Newark, Delaware 19717-1303 Accepted July 10, 1987 A specific radioimmunoassay was validated for the quantitative measurement of melatonin (MT) levels in plasma and homogenates of the pineal gland, Harderian gland, or retinae of young chickens. Single-comb White Leghorn (SCWL) cockerels were raised under a 12L:12D light/dark cycle for two experiments. In Experiment 1, 12 birds were bled and immediately killed for their pineal glands at 4-hr intervals during a single light/dark cycle at 25 days of age (25 da) for simultaneous determination of changes in MT levels in the plasma and pineal gland. Plasma MT levels were low during photophase (100 pg/ml) and reached a distinct peak (390 pg/ml) at mid-scotophase. A parallel MT rhythm was found in pin&l homogenates where the average MT content during scotophase (7.4 rig/gland) was 10 times higher than the average MT content of pineal glands obtained during photophase. In Experiment 2, SCWL cockerels were either pinealectomized or sham-operated (PN) at 8 to 10 da. At 25 da, six birds from each surgical treatment group, including unoperated controls (C), were bled at 4-hr intervals, corresponding to those in Experiment 1, during a single light/ dark cycle. Immediately after being bled, each bird was killed and the eyes and Harderian glands were removed for measurement of their MT contents. Pinealectomy completely abolished the plasma MT rhythm which in intact chicks (PN and C) reached a sharp peak (298 pg/ml) at mid-scotophase. Although not affected by surgical treatment, retinal MT levels showed a higher amplitude rhythm with a prominent peak (4 r&retina) at mid-scotophase that was 15 times higher than the average retinal MT content during photophase. A modest nocturnal MT rhythm was found in the Harderian gland where the average MT level for all surgical treatment groups during scotophase (89 pg/lOO mg wet wt) was only 51% higher than that observed for photophase. These data indicate that the plasma MT rhythm in chickens is derived solely from MT secreted into blood by the pineal gland and that the extrapineal MT produced rhythmically in both the retina and Harderian gland does not contribute to the plasma MT rhythm. Q 1987 Academic press, IX.

Distinct daily rhythms of melatonin (MT) have been found in plasma and the pineal gland of several species of birds. A quantitative bioassay (Ralph and Lynch, 1970) was first used to establish the daily pattern of MT in the pineal gland of Japanese quail (Ralph et al., 1967; Lynch, 1971) and chickens (Lynch, 1971). The rhythmic synthesis of MT in the pineal gland of mammals (Klein and Weller, 1970) and birds (Binkley et al., 1973) is regulated by the activity of N-acetyltransferase (NAT). 1 Published as Miscellaneous Paper No. 1167 from the Delaware Agricultural Experiment Station.

Furthermore, the daily rhythms of MT and NAT activity found in the pineal gland and serum of chickens persists when birds are transferred from an alternating Light/dark cycle (12L: 12D) to continuous darkness (Ralph et al., 1974, 1975; Binkliey and Geller, 1975). Perhaps the most remarkable feature of the chicken pineal gland is its ability to generate circadian rhythms of NAT activity and MT in tissue culture (Binkley et al., 1978; Deguchi, 1979; Wainwright and Wainwright, 1979; %kahashi et al., 1980). Subsequently, prominent circadian rhythms of NAT activity and MT were 343 00166480187 $1.50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any fomt reserved.

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WILSON-PLACENTRA,

identified in the retina of birds (Binkley et al., 1979; Binkley et al., 1980; Hamm and Menaker, 1980). Although independent of the pineal gland, the retinal rhythms of NAT activity and MT are completely in phase with, and have the same circadian characteristics as, those found in the chicken’s pineal gland (Binkley et al., 1980; Hamm and Menaker, 1980). The Harderian glands of birds and mammals also contain high levels of MT during darkness-presumably from synthesis in situ (Pang et al., 1977; Reiter et al., 1981; Vakkuri et al., 1985a, b). Some reports have indicated a compensatory increase in the MT content of these tissues several weeks after pinealectomy (Yu et al., 1981a; Reiter et al., 1983; Vakkuri et al., 1985b; 0~01 et al., 1985). Persistence of the nocturnal rise of MT in plasma or serum of pinealectomized animals has led to the speculation that these extrapineal sources of MT make a significant contribution to the concentration of MT found in circulation (Gem et a1.,1978; Gern and Norris, 1979; Gern and Ralph, 1979; Reppert and Sagar, 1983; Underwood et al., 1984; 0~01 et al,, 1985; Vakkuri et al., 1985b). Earlier studies, in which MT was measured with the tadpole bioassay (Ralph and Lynch, 1970), indicated that pinealectomy completely abolished the MT rhythm in blood (Pelham, 1975; Pelham et al., 1972). Lewy et al. (1980) have also indicated that pineal ablation completely abolishes plasma MT levels in the rat. Thus, there appears to be some disagreement about the source of MT that varies rhythmically in the blood of chickens. Although specific radioimmunoassays (RIA) have been used to measure MT levels in plasma or tissue homogenates of intact or pinealectomized birds, most of the recent studies have provided information and conclusions based on only two time points during the light/dark cycle-mid-light and mid-dark (Pang et al., 1977; Reppert and Sagas-, 1983; Vakkuri et al., 1985a, b; 0~01

AND LETCHER

et al., 1985). Obviously, a more detailed account, which includes at least six time points, is required to fully understand the contribution of pineal and extrapineal tissues to the MT rhythm found in the bloodstream of birds held under alternating light/dark cycles. One purpose of the present study was to validate a specific radioimmunoassay (Rollag and Niswender, 1976) for the quantitative determination of MT in the plasma and homogenates of the pineal gland, Harderian gland, or retinae of chickens maintained under a 12L:12D light/dark cycle. The other objective was to determine the effect of pinealectomy on plasma and extrapineal MT rhythms in young chickens. This work shows that MT rhythmically synthesized by the pineal gland is responsible for the MT rhythm generated in plasma and that pinealectomy does not affect the MT rhythm in either the retina or Harderian gland of chickens. MATERIALS

AND METHODS

Bird management. Day-old Single-comb White Leghorn (SCWL) cockerels were obtained from a commercial hatchery and housed initially in a heated battery brooder under a 12L:12D light/dark cycle with lights on at 0600 hr. Birds were raised under a standard ambient temperature reduction schedule of 33” for the first week with reductions of 3” at weekly intervals. Feed and water were available ad libitum. Experiment I. Seventy-two SCWL cockerels were randomly distributed among 12 pens (six birds/pen) in a battery-brooder unit. Birds were held under the 12L:12D light/dark cycle with an average light intensity of 258 lux at bird level from broad-spectrum fluorescent lights (Vita-Lite, Durotest Corp., North Bergen, NJ). At 25 days of age (25 da), 12 birds (one bird from each pen) were bled by cardiac puncture at 4-hr intervals during a single light/dark cycle (0800, 1200, 1600, 2000, 2400, and 0400 hr). Immediately after the blood sample was obtained, each bird was killed for its pineal gland. Pineal glands were transferred to 1 ml of 0.1 M sodium phosphate buffer (pH 7.1) and then rapidly frozen on dry ice. A 2.5-W fluorescent black light with an emission peak at 365 nm was used for collection of blood and pineal gland samples during scotophase (Cogburn and Harrison, 1980). Plasma and pineal glands were stored frozen (- 20”) until time of assay.

MELATONIN

RHYTHMS

On the day of assay, each pineal gland was thawed and homogenized by sonication for 15 set (Branson Sonifer, Model 200). Pineal homogenates were then centrifuged at 2000g (4”) for 15 min and the supematants (ca. 0.8 ml) were transferred to clean 12 x 76-mm glass test tubes. Test tubes containing pineal homogenates were held in an ice bath during this entire procedure. Homogenates of pineal gland samples taken during scotophase were diluted 1:20 in phosphate buffer prior to assay. Duplicate 200+1 samples of undiluted (photophase) or diluted (scotophase) pineal homogenates were used in the MT RIA. Experiment 2. At 8 to 10 da, 36 SCWL cockerels were either pinealectomized (PX) or sham-operated (PN) by the procedure developed by Cogburn et al. (1976). Upon removal, each pineal gland was closely inspected to ensure that the entire pineal and its stalk (choroid plexus) were removed together; only birds that met this criterion were used. Another group of 36 birds served as unoperated controls (C). At 17 da, six birds from each surgical treatment (PX, PN, and C) were randomly assigned to 1 of 18 wire cages held in two controlled-environment rooms with a common 12L: 12D light/dark cycle. The rooms were illuminated with wide-spectrum fluorescent lights that provided an average light ,intensity of 590 lux at bird level. At 25 da, six birds from each surgical treatment group were weighed, bled by cardiac puncture, and then killed by cervical dislocation at 4-hr intervals during the light/dark cycle. Both eyes and Harderian glands were removed from each bird, wrapped individually in aluminum foil, and immediately frozen on dry ice. During scotophase, blood and tissue samples were obtained under a 2.5-W fluorescent black light. Duplicate microhematocrit tubes were filled from each heparinized blood sample for determination of hematocrit (percentage packed cell volume, %PCV). Plasma and tissue samples were stored frozen (- 20”) until time of assay. Completeness of pinealectomy was confirmed by inspection of the pineal region under a dissection microscope (Pelham, 1975). No residual pineal tissue was found in any of the PX birds. The retina-pigment epithelium was isolated from the eye by the procedure described by Hamm and Menaker (1980). Each retina was placed in 1 ml of phosphate buffer (0.01 M, pH 7.1) in an ice bath. After sonication for 15 set, both retinae from each bird were pooled into a single 12 x 76-mm glass test tube and centrifuged at 2OQOg (4”) for 15 min. Homogenates of retinae collected during photophase were not diluted prior to assay, whereas homogenates of retinae obtained during scotophase were diluted I:10 in phosphate buffer for RIA of the MT content. The two Harderian gland#s taken from each bird were weighed, sonicated in ‘2 ml of phosphate buffer for 1.5 set and centrifuged at 2OOOg(4”) for 1.5min. The supernatants were transferred to clean glass test tubes and assayed for their MT content without further dilution. The MT

IN CHICKENS

34.5

content of plasma or homogenates of the retina and Harderian gland was determined by RIA. The concentration of MT in these homogenates was used to estimate the MT content of each bird’s retina (pgiretina) and Harderian gland (pg/lOO mg wet wt). Statistical analyses. Data from both experiments were analyzed by least-squares regression analysis following transformation of MT values to the natural logarithm. Experiment 1 had a completely randomized design with planned comparisons for the six times of day. The following single degree-of-freedom (SDF) contrasts were made to separate significant differences due to time of day: photophase vs scotophase, 0800 vs 1200 hr, 1200 vs 1600 hr, 2000 vs 2400 hr, and 2400 vs 0400 hr. In Experiment 2, there was a factorial arrangement of three surgical treatments (PX, PN, and C) and six times of day. Significant differences due to surgical treatment were separated by two SDF contrasts [PX vs (PN + C)/2 and PN vs C]. Body weight data in this experiment were analyzed by analysis of variance. The area under each MT profile was calculated from treatment means by Simpson’s rule for numeric integration and expressed as nanograms per milliliter times hour for plasma data or nanograms per tissue times hour for tissue homogenates. Melatonin radioimmunoassay. The equilibrium, double-antibody assay developed by Rollag and Niswender (1976) was modified for quantitative measurement of MT in the plasma and homogenates of the pineal gland, Harderian gland, or retinae from young chickens. Pooled chicken plasma, obtained from -birds maintained under continuous light (LL), was stripped twice with activated charcoal and filtered through a 0.45pm filter. The stripped plasma was spiked with crystalline MT (Regis Chemical Co., Mortoa Grove, IL) to provide nine plasma standards (0, 7.8, 15.6, 31.2, 62.5, 125, 2.50, 500, and 1000 pg/ml). For measurement of MT in tissue homogenates. a set of MT standards (7.8 to 1000 pgiml) was prepared in phosphate-buffered saline (0.01 M, pH 7.0) with 0.1% gelatin (PBS-G) made with chromatography-grade water (Burdick & Jackson Laboratories Inc., Muskegon, MI). Two-hundred-microliter samples of plasma (or tissue homogenate) and plasma (or buffer) MT standards were extracted in duplicate 12 x 76-mm glass test tubes. Ten volumes (2 ml) of glass-distilled chloroform (Burdick & Jackson Laboratories Inc.) were used to extract MT from samples of plasma or tissue homogenate. The chloroform extracts were then sequentially washed with 500 pJ of 0.1 M sodium car” bonate buffer (pH 10.25) and 500 ~1 of chromatography-grade water. One milliliter of the chloroform layer was then transferred to a clean test tube and evaporated to dryness at 40” under a stream of#pure nitrogen gas. The residue was dissolved in 500 ~1 of PBS-G, vortex mixed, and then washed with 2 ml of

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COGBURN,

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glass-distilled petroleum ether (Burdick & Jackson Laboratories Inc.) to remove possible contaminants (Rollag, 1981). After aspiration of petroleum ether, samples were left overnight at room temperature (about 21”) to allow evaporation of any residual ether. An automatic pipet (Model 2000, Micromedic Systems, Horsham, PA) was then used to dispense 100 ~1 of 1251-MT analog (Product IR-1019, Meloy Laboratories Inc., Alexandria, VA), 60 pg of rabbit y-globulin (Product 3530, Antibodies Inc., Davis, CA), and 200 ~1 of a 1:64,000 dilution of primary MT antibody (R-1055; Rollag and Niswender, 1976) simultaneously into each assay tube. Samples were then incubated for 48 hr in an ice bath (W). Diluted sheep anti-rabbit yglobulin (produced in our laboratory) was added to precipitate the 1251-MT bound to primary antibody. Following an additional 24-hr incubation period in an ice bath, 2 ml of cold PBS-G containing 12% polyethylene glycol was added to the reaction mixture and then the samples were centrifuged at 2000g (4”) for 45 min. The supematant was decanted and the radioactivity in the precipitate was quantified with an automatic y-spectrophotometer (Model 1185-Z, Tracer Analytic, Chicago, IL). After correction for nonspecific binding, the amount of radioactivity bound by primary antibody in each sample (B) was expressed as a ratio (BIB,,) with that of plasma (0 pg/ml) or buffer control (B,) tubes. Logit transformation of BIB, and linear regression analysis (logit vs log dose of MT) were used on all data. The quantity of MT in each sample was calculated with the use of a computer program which provides logit-log transformation of the data (Rodbard, 1974). The following procedures were used to validate the Rollag and Niswender (1976) RIA for quantitatively determining MT concentrations in plasma and homogenates of pineal glands, Harderian glands, or retinae from young chickens. Parallelism. An aliquot of pooled chicken plasma, obtained at mid-scotophase (2400 hr) from birds maintained on a 12L:12D light/dark cycle, was twofold serially diluted in PBS-G. One milliliter of charcoalstripped chicken plasma was spiked with 1000 pg of MT and eight twofold serial dilutions were made in PBS-G. Homogenates of a pineal gland, Harderian gland, or retinae obtained from a bird at mid-photophase (1200 hr) were spiked with 1000 pg of MT, centrifuged at 2000g for 15 min to remove particulate matter, and eight twofold serial dilutions of each tissue homogenate were made in PBS-G. All samples were assayed in triplicate test tubes in the equilibrium, double-antibody assay described above. Parallel inhibition curves were obtained for twofold serial dilutions of a mid-scotophase plasma sample vs the 1000 pg/ml plasma standard (Fig. 1) and a mid-scotophase pineal homogenate vs the 1000 pg/ml MT standard in PBS-G (Fig. 2). Although not shown, similar parallel inhibition curves were obtained when ho-

AND

LETCHER

-AI

LOG ~‘1 PLASMA

ASSAYED

3

5

2

4

6

I

. y=-078x+3 . y =-074x

-4j

0

15, r=-0.994 +3.36.

r =-0.993

I LOG :OSE

C-“.- PLA:MA

5 STANDAR;)

7

FIG. 1. Parallelism between a dilution series of MT in a chicken plasma sample obtained at mid-scotophase and a plasma MT standard (1000 pg/ml) diluted in assay buffer (PBS-G). The solid triangles represent the log of the microliter quantity of mid-scotophase plasma assayed (12.5 to 200 ul). The solid circles represent the log dose of the plasma MT standard (1000 pgiml) diluted in assay buffer. Both inhibition curves have similar regression equations and slopes.

mogenates of either the Harderian gland or retinae taken from a chicken at midscotophase were serially diluted in buffer and compared with the MT buffer standard curve. These data indicate that the modified MT assay is specific for MT found in either plasma or tissue homogenates (pineal, Harderian gland, or retinae) of chickens. Quantitative recovery. Serial quantities of MT were added to charcoal-stripped chicken plasma or homogenates of pineal gland, Harderian gland, or retinae obtained from a bird maintained under LL. Triplicate 200+1 samples of plasma or tissue homogenate spiked with MT were subjected to chloroform extraction and RIA. Estimates of MT in each sample were based on appropriate MT standards prepared in either plasma or PBS-G. Quantitative recovery shows that the amount of MT measured by RIA was closely correlated (r = 0.999) to graded amounts of MT added to either charcoal-stripped plasma (slope, 0.966) or pineal homogenate (slope, 0.958) (Figs. 3 and 4). The basal level of endogenous MT in stripped plasma corresponds to the y-intercept (2.6 pgiml). This value is below the sensitivity of the assay (3 pgitube), which indicates that the combination of charcoal stripping and maintenance of birds under LL essentially eliminates endogenous MT from chicken plasma that is used for preparation of plasma MT standards.

MELATONIN -.-

LOG f/I PINEAL HOMOGENATE

2

I

3

RHYTHMS ASSAYED

4

5 I

I

-2 .y= .y=

I I

-4 1 0

\

-0.921x + 3 18, r = -0999 -0863x+4,89,r=-0.999

/

/

I

I

I

I

2

3

4

5

6

7

LOG DOSE C-e- BUFFER

STANDARD)

FIG. 2. Parallelism between a dilution series of a pineal homogenate in assay buffer and the MT standard prepared in assay buffer. The solid triangles represent the log of the microliter quantity of pineal homogenate assayed (6.8 to 100 t.~l). The solid circles represent the log dose of twofold serial dilutions of the 1000 pgiml MT standard in assay buffer. The regression equation for each inhibition curve is shown; the slopes are not significantly different.

Extrnction e&iency. Tritiated MT ([3H]MT, New England Nuclear, Boston, MA) was used to prepare standards (15.6 to 1000 pgiml) of [3H]MT in either charcoal-stripped chicken plasma or PBS-G for tissue

347

IN CHICKENS

homogenates. The PH]MT standards were then taken through the sample extraction procedure. Triplicate 200+1 samples of each washed chloroform extract were counted in 10 ml of scintillation cocktail (Aquasol 2, New England Nuclear) in a S-spectrophotometer (Tracer Analytic, Chicago, IL). The radioactivity extracted at each concentration of [3H]MT was determined for both the plasma and buffer 13H]MT standards. The average extraction efficiency over the entire range of [3H]MT standards (7.8 to IO00 pg/ml) was 96% for plasma and 93% for PBS-G. Correction for extraction efficiency was not made since the plasma MT standards or MT standards in PBS-G (for tissue homogenates) were always extracted aiong with the unknown samples in each assay. Assay precision. As suggested by Rollag (19gl), quality control tubes containing a low (62.5 pgiml), medium (250 pgiml), and high (500 pglml) concentration of MT were included in each assay to monitor accuracy of the RIA. These three dose levels of MT in plasma, or PBS-G (for tissue homogenates), were assayed in triplicate at the beginning, middle, and end of the sample tubes in each RIA. The nine sample tubes, representing each dose level, were used to provide estimates of intraassay (14% CV) and interassay (12% CV) variability. The limit of sensitivity for measurement of MT extracted from plasma or tissue homogenates was 3 pgitube.

RESULTS

I. Parallel rhythms of MT

Experiment IO00

IOOOC

Y= 0.958~17132 i' 0999

250 2500 125 62.5 15.6

1250 625 i,b

/

I

/

15.6 1250

I

2500

/

500.0 Pg MELATONiN

FIG. 3. Quantitative recovery of melatonin (MT) added to charcoal-stripped chicken plasma. The concentration of MT determined by RIA was closely correlated (Y = 0.999) with the amount of MT added to chicken plasma.

I / / 156 125

250

I 1000

500 Pg MELATONIN

1000 0 ADDED

t-

ADDED

4. Quantitative recovery of melatonin (MT) added to the homogenate of a pineal gland obtained from a chicken maintained under continuous light. The concentration of MT assayed was closely correlated (r = 0.999) with the amount of MT added to the pineal homogenate. FIG.

348

COGBURN,

WILSON-PLACENTRA,

were found in the pineal gland and plasma of 25 da SCWL cockerels raised under a 12L:12D light/dark cycle (Fig. 5). The average pineal MT content during scotophase (6.9 rig/gland) was 10 times higher (P < 0.01) than that during photophase (0.7 rig/gland). The pineal MT content at 2000 hr (6 rig/gland) was significantly lower (P < 0.05) than the broad peak of pineal MT (7.4 rig/gland) maintained during middle (2400 hr) and late scotophase (0400 hr). No timewithin-phase differences were found in MT content of pineal glands collected during photophase. The area under the curve for the pineal MT rhythm was 62.0 ng/ gland * hr. The average plasma MT concentration during scotophase (320 pg/ml) was 3.2-fold higher (P < 0.01) than the average MT level during photophase. During scotophase, plasma MT levels rose to 281 pg/ml at 2000 hr, reached a peak at 2400 hr (390 pg/ml), and fell to 288 pg/ml at 0400 hr. Single degree-of-freedom contrast showed that plasma MT levels were higher (P < 0.05) at 2400 hr than those at 2000 hr although no significant difference in plasma MT level

AND LETCHER

was detected between samples taken at 2400 and 0400 hr. The area under the curve of the plasma MT rhythm was 3.76 rig/ml . hr. Experiment 2. Plasma melatonin rhythm in pinealectomized chickens. Pinealectomy

of newly hatched chickens (8 to 10 da) completely abolished (P < 0.01) the daily MT rhythm at 25 da (Fig. 6). The PX birds had similar levels of plasma MT (73 pg/ml) throughout the light/dark cycle. In contrast, the average plasma MT level of PN and C birds during scotophase (222 pg/ml) was 3 times higher (P < 0.01) than their average for photophase. Plasma MT levels reached a peak in both PN (325 pg/ml) and C (270 pg/ml) treatment groups at 2400 hr. The plasma MT peak in PN and C birds at 2400 hr was about 20% higher than the average plasma MT concentration of these groups at 2000 and 0400 hr (185 pg/ml). The failure of PX birds to exhibit the typical nocturnal surge of plasma MT produced an (P < 0.01) between surgical interaction treatment and time of day. Consequently, the main effects of surgical treatment and time of day are negated by the significant

r

O-

0800

1200

1600 CLOCK-TIME

2000

2400

0400

080(

(hr)

FIG. 5. Parallel rhythms of melatonin in the plasma and pineal gland of 25-day-old chickens maintained under a 12L:12D cycle. Each value represents the mean and SEM of 12 birds. The stippled area denotes scotophase.

MELATONIN

”

0800

RHYTHMS

IN CHICKENS

349

1200

1600 2000 2400 0400 0800 CLOCK-TIME (hr) FIG. 6. Absence of a daily melatonin rhythm in plasma of 25day-old pinealectomized chickens. Surgical treatments were pinealectomy (PX), sham operation (PN), and intact controls (C). Each value represents the mean and SEM of six birds. The stippled area denotes scotophase.

two-way interaction. The area under the curve of the plasma MT rhythm in PX birds (1.44 rig/ml . hr) was greatly reduced in comparison to that of the birds receiving PN (2.58 rig/ml * hr) and C (2.56 rig/ml * hr) treatments. Retinal melatonin rhythm in pinealectomized chickens. Pineal ablation did not alter the daily pattern of MT found in the chicken’s retina (Fig. 7). There was a 13-

0800

1200

fold increase (P < 0.01) in the retinal MT content of PN and C birds during darkness (3.39 rig/retina) over that of their photophase average (0.26 ngiretina). Although not significantly different from that in other treatments, only an eightfold increase in retinal MT content over their photophase average (0.34 rig/retina) was shown by PX birds during scotophase. Across surgical treatments, there was a main effect (i” <

1600 2000 2400 0400 0800 CLOCK-TIME (hr) FIG. 7. Daily rhythm of melatonin in the retinae of 2%day-old pinealectomized chickens. Surgical treatments were pinealectomy (PX), sham operation (PN), and intact controls (C). Each value represents the mean and SEM of six birds. The stippled area denotes scotophase.

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0.01) of time of day due to the 11-fold increase in the average night-time MT content of the retina (3.18 rig/retina) above the average MT content for photophase. The retinal MT content reached a zenith at 2400 hr in PX (3.52 rig/retina), PN (4.53 ngl retina), and C (4.10 rig/retina) birds. For all treatment groups, the mid-scotophase peak of retinal MT was 48% higher than the combined average (2.74 rig/retina) of the ascending (2000 hr) and descending (0400 hr) shoulders of the rhythm. Thus, the daily retinal MT rhythm in young chickens has a much higher amplitude (i.e., a 15fold increase) than the plasma MT rhythm (Fig. 6). The area under the curve of the retinal MT rhythm was slightly lower in PX birds (25.9 rig/retina * hr) than in the PN (28.7 ng/ retina * hr) or C (30.2 &retina * hr) treatment groups. Harderian melatonin rhythm in pinealectomized chickens. Pinealectomy did not affect the daily MT rhythm found in the Harderian gland of chickens (Fig. 8). During scotophase, the Harderian gland exhibited only a 51% increase (P -=L0.01) in MT content (89 pg/lOO mg wet wt) above the average MT content for photophase. The characteristic mid-scotophase peak of MT found in the retina was not observed in Harderian glands of PX and PN birds. Al-

AND LETCHER

though there were no significant differences due to surgical treatment, the peak in Harderian MT content of C birds at 2400 hr (117 pg/lOO mg wet wt) was 26% higher than the average MT level at 2000 and 0400 hr. The greatest amount of MT in the Harderian gland of PX birds was found at 2000 hr (93 pg/lOO mg wet wt), whereas the concentration of Harderian MT in PN birds was rather stable during darkness (83 pg/lOO mg wet wt). The area under the curve for Harderian MT profiles was quite different among PX (1.42 ng/lOO mg wet wt. hr), PN (1.34 ng/lOO mg wet wt. hr), and C (1.52 ng/lOO mg wet wt * hr) treatments. Body weight and hematocrit of pinealectomized chickens. The average body weight of PX birds at 25 da (267 g) was lower (P < 0.05) than the average body weight of the PN and C groups (282 g). No differences due to surgical treatment or time of day were found in hematocrit of blood samples taken at 25 da. The average hematocrit across all surgical treatments and times of day was 27% PCV. DISCUSSION The highly sensitive and specific MT RIA developed by Rollag and Niswender (1976) has been used extensively for the

95 4~ 80 OE 0 ze E‘,

40

WCL cl:

0

0800

1200

1600 2000 CLOCK-TIME

2400 (hr)

0400

0800

FIG. 8. Daily melatonin rhythm in the Harderian gland of 25day-old pinealectomized chickens. Surgical treatments were pinealectomy (PX), sham operation (PN), and intact controls (C). Each value represents the mean and SEM of six birds. The stippled area represents scotophase.

MELATONIN

RHYTHMS

quantitative measurement of MT in tissue homogenates and blood plasma or sera of numerous endothermic and ectothermic animals (Rollag, 1981). Recently, this MT assay was validated for the measurement of MT levels in the retinae of chickens (Hamm and Menaker, 1980), the pineal gland (Cockrem and Follett, 1985), plasma and retinae of Japanese quail -(Underwood et al., 1984), and plasma of laying hens (Liou et al., 1987). Our study shows that a modification of this MT RIA (Rollag and Niswender, 1976) is also specific for the quantitative determination of MT in the plasma and homogenates of the pineal gland, Harderian gland, or retinae of young chickens. Parallel inhibition curves and quantitative recovery were successfully achieved for MT contained in either plasma or tissue homogenates (Figs. l-4). Simultaneous measurement of MT in pineal homogenates and plasma of 12 birds killed at 4-hr intervals throughout the 12L: 12D light/dark cycle reveals parallel rhythms of MT in the plasma and pineal gland of young chickens (Fig. 5). The pineal MT content of chickens at mid-scotophase (7.4 rig/gland) was 13.4 times greater than that at mid-photophase (0.55 rig/gland). Our data agrees with that of Osol et al. (1985) who reported an average pineal MT content of 7.1 r&gland at mid-dark that was 5.5 times higher than the mid-light value (1.3 rig/gland) for chickens maintained under a 14L:lOD light/dark cycle. With their MT RIA, Pang et al. (1983) reported pineal MT contents of 0.78 rig/gland at mid-light and 4.48 rig/gland at mid-dark or a 5.7-fold difference. Using the quantitative bioassay, Lynch (1971) found that the MT content of the pineal gland in 2-weekold chickens was 0.6 r&gland at mid-photophase and 11 rig/gland at midscotophase. Binkley et al. (1973) indicated that the peak in pineal MT content of 8-week-old chickens (measured by bioassay) at midscotophase (21 rig/gland) was IO-fold greater than the pineal MT content at mid-

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photophase although there was a 27-fold difference in NAT activity. Apparently, the tadpole bioassay overestimates the pineal MT content since Ralph et al. (1974) also found 21.7 rig/gland in 8-week-old cockerels at mid-scotophase, which was 1l-fold higher than that at 1200 hr. The daily amplitude of pineal MT content in the pigeon represents a 16-fold (Pang et al., 1983) or a ‘I-fold difference between midday and midnight levels according to Vakkuri et al. (1985a). In Japanese quail, the peak in pineal MT content at mid-scotophase varies among different reports from S- to 11-fold higher than the midphotophase level (Pang et al., 1983; Underwood et al., 1984; Cockrem and Follet, 1985; Vakkmi et al., 1985a). Takahashi et al. (1980) estimated that the chicken pineal gland in a superfnsed culture system, under an imposed 12L:12D light/dark cycle, produced MT at a rate of about 1000 r&24 hr (or 42 ng/hr). When the area under the curve of the pineal MT rhythm is integrated (Fig. 5>, we estimate that the pineal gland in situ produces 62 ng MT/hr under the same lighting schedule. In general, the MT content of the avian pineal gland reaches a peak in midscotophase and falls to its nadir in mid-photophase with MT levels changing from 3- to 16-fold, regardless of the method used to measure MT levels. These observations are consistent with the idea that NAT activity regulates MT synthesis in the pineal gland (Klein and Weller, 1970; Binkley el’ al., 1973) which secretes MT directly into the bloodstream (Pang and Ralph, 1975; Rollag et al., 1978; Reppert et al., 1979; Owen? et al., 1980), since all three rhythms are exactly in phase with each other. In the hamster, the pineal MT peak is reached just before the onset of photophase (Tan&kin tt al., 1979; Rollag and Stetson, 1981; Reiter et al., 1980). Another diurnally active mammal-Richardson’s ground squirrelY has a peak in pineal MT content in early scotophase (Reiter et al., 1981). ‘These re-

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ports suggest that the time-measuring mechanism may differ among species of birds or mammals. The nocturnal pattern of plasma MT determined by RIA in cockerels in our study (Fig. 5) is similar to that observed by Pelham (1975), who used a bioassay to measure MT levels in chicken serum. Our study provides the first full account of the plasma MT rhythm in young chickens based on RIA of blood samples taken at six time points during a light/dark cycle. The most recent reports of plasma MT levels in chickens, determined by specific RIA, are based on blood samples taken at two times of day (mid-light and mid-dark) (Kennaway et al., 1977; Reppert and Sagar, 1983; Pang et al., 1983; Osol et al., 1985). Plasma MT concentrations in chickens of various ages and breeds represented by these reports range from 50 to 80 pg/ml for midphotophase and 200 to 500 pg/ml for mid-scotophase. The use of the bioassay (Ralph and Lynch, 1970) to quantify serum MT concentrations in chickens usually gives a lower daily range (10 to 220 pg/ml) (Ralph et al., 1974; Pelham, 1975) than the lightdark range of plasma MT provided by specific RIA (50 to 550 pg/ml) (Kennaway et al., 1977; Reppert and Sagar, 1983; Pang et al., 1983; Osol et al., 1985; Figs. 5 and 6). Nonetheless, the most salient feature of the plasma MT rhythm in young chickens is a sharp peak in mid-scotophase that is four to five times higher than the nadir at 1200 hr (Figs. 5 and 6). It is interesting that the daily pattern of plasma MT in young chickens is similar to the serum MT rhythm described in ectothermic turtles (Owens et al., 1980). Plasma MT concentrations in the pigeon appear to be lower than those in chickens during both phases of the light/ dark cycle (Pang et al., 1983; Vakkuri et al., 1985a). The light-dark variation of MT levels in plasma or serum of the Japanese quail (Pang et al., 1983; Underwood et al., 1984) resembles that of the chicken. The major issue raised by the present

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study is the source of circulating MT in chickens; our study clearly shows that pineal ablation in young chickens completely abolishes the plasma MT rhythm (Fig. 6). Furthermore, pinealectomy does not alter MT rhythms in either the retina or the Harderian gland of chickens (Figs. 7 and 8). Our finding confirms earlier reports of absence of the MT rhythm in plasma or serum of pinealectomized chickens (Pelham et al., 1972; Pelham, 1975) or rats (Lewy et al., 1980). In contrast, there is considerable evidence that the MT rhythm persists in circulation of several species of pinealectomized birds (Reppert and Sagar, 1983; Underwood et al., 1984; Osol et al., 1985; Vakkuri et al., 1985b) and mammals (Ozaki and Lynch, 1976; Kennaway et al., 1977; Yu et al., 1981b). However, the increase in plasma MT levels in pinealectomized animals is not usually observed until several weeks or months after pinealectomy (Kennaway et al., 1977; Osol et al., 1985). In view of the chicken pineal gland’s ability to generate MT rhythms in vitro (Binkley et al., 1978; Deguchi, 1979; Wainwright and Wainwright, 1979; Takahashi et al., 1980) or when autotransplanted into the eye (Pang and Ralph, 1975), it is not surprising that some workers find a residual MT rhythm after pinealectomy. Any pineal tissue not removed or dislodged during pinealectomy in birds would have the potential to synthesize MT rhythmically. The physiological significance of a compensatory increase in the MT content of the retina or Harderian gland occasionally observed after pinealectomy is not known. Pinealectomy did not affect the MT rhythm found in either the retina or Harderian gland of chickens in the present study (Figs. 7 and S), and our samples were taken more than 2 weeks after pinealectomy. A compensatory increase of nocturnal MT levels has been observed in extrapineal tissues of the rat (Yu et al., 1981a; Reiter et al., 1983), chicken (0~01 et al., 1985), and

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pigeon (Vakkuri et al., 1985b). Although a nocturnal rise in plasma MT was observed after pinealectomy in chickens (Reppert and Sagas-,1983) and Japanese quail (Underwood et al., 1984), neither report indicated that pinealectomy caused a compensatory increase in retinal MT content during darkness. Similarly, the retinal rhythm of NAT activity in the chicken was not affected by pinealectomy (Hamm and Menaker, 1980). Vakkuri et al. (1985b) suggested that the Harderian gland of chickens was the major source of MT found in plasma of pigeons after pinealectomy; however, this conclusion was based on tissue samples taken only at midnight, when the Harderian MT content of the sham-operated group was extremely low when compared with Harderian MT levels of normal birds observed in another study (Vakkuri et al., 1985a). Our data do not support this conclusion; we found a modest low-amplitude rhythm of MT in the Harderian gland of chickens. At present, the reason for these discrepancies is not known. If extrapineal tissues normally make a significant contribution to circulating levels of MT during darkness, then one would expect to see persistence of a MT rhythm in plasma of our pinealectomized chickens. The daily rhythm of MT found in the retina and Harderian gland of birds and mammals is apparently derived from in situ synthesis since a parallel rhythm of NAT a,ctivity has also been demonstrated in these tissues in chickens (Binkley et al., 1979; Binkley et al., 1980; Hamm and Menaker, 1980) and rats (Brammer et al., 1978; Binkley et al., 1979). The fact that the retinal MT #rhythm persists after pineal ablation clearly indicates that MT is rhythmically produced within the eye and not accumulated there from blood (Yu et al., 1981a; Reiter et al., 1983; Underwood et al,, 1984). The peak in retinal MT content (4 ng/ retina) of chickens in our study was about 15 times greater than the daytime level

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(Fig. 7). The magnitude of the nocturnal rise in retinal MT content reported for chickens, Japanese quail, and pigeons ranges from 5- to 15-fold higher than photophase levels (Pang et al., 1983; Reppert and Sagar, 1983; Underwood et al., 1984; 0~01 et al., 1985; Vakkuri et al., 1985a). In rats, retinal MT levels have a lower dark/light ratio (2 to 4) and continue to remain high during the early hours of photophase (Pang et al., 1980; Yu et al., 1981a; Reiter et al., 1983). Assuming the same rate of synthesis, we estimate that the amount of MT produced by both eyes in the chicken (60 nglhr) approximates that produced by the pineal gland (62 ng/hr) under a 12L:12 light/dark cycle. However, there is no direct evidence that the retinae have the same synthetic or secretory capacity as the pineal gland. Furthermore, the contribution of retinal MT to the quantity of MT secreted by the pineal gland into the bloodstream will remain doubtful until definitive experiments show the appearance of [3H]MT in blood after intraocular injection and provide estimates of the half-life of intraocular MT. The information currently available on MT synthesis by t,he retinae does not warrant the speculation that the chicken’s retina serves an endocrine function. Our study provides the first full account of the Harderian MT rhythm in chickens (Fig. 8). A daily variation in the MT eontent of the Harderian gland was first identified in rats (Pang et al., 1977; 3ubenik et al., 1978). Although considerable hghtdark variation was found in the MT content of the Harderian gland of Richardson”s ground squirrel, a distinct daily MT rhythm could not be demonstrated (Reiter et al., 1981). In contrast, the Harderian gland of the rat exhibits a distinct MT rhythm with a peak at 2 hr after the onset of photophase which is synchronized to the retinal MT rhythm (Reiter et al., ,1983). At best, the chicken’s Harderian gland shows a modest low-amplitude MT rhythm. Vakkuri et al.

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(1985b) have suggested that the Harderian gland of the pigeon serves as a source of extrapineal MT. Our data on chickens do not support their conclusion since the amount of MT found in the chicken’s Harderian gland could not make a significant contribution to circulating MT levels. Interestingly, the pineal and Harderian glands of the chicken also share another common feature-both tissues contain a remarkable population of immunologically competent B-lymphocytes (Bang and Bang, 1968; Cogburn and Glick, 1981, 1983). The influence of rhythmic MT synthesis in the pineal and Harderian glands on their resident lymphocytes or other lymphoid tissue in chickens is not presently known. We conclude that the retina and Harderian gland of chickens do not contribute to the plasma MT rhythm under normal circumstances. Parallel rhythms of MT were found in the pineal gland and plasma of chickens maintained under a 12L: 12D light/ dark cycle. Pinealectomy of chickens at 8 to 10 days of age does not affect daily MT rhythms found in the retinae or Harderian glands. Our study supports the notion that the pineal gland is the only source of the MT surge found in blood of chickens during scotophase. ACKNOWLEDGMENTS We express our appreciation to Dr. Mark Rollag for his generous gift of anti-melatonin antisera and his assistance in establishing and validating the melatonin radioimmunoassay. This work was supported in part by a NIH Biomedical Research Support grant, administered by the University of Delaware, to L.A.C.

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