Circadian rhythms of melatonin secretion from superfused goldfish (Carassius auratus) pineal glands in vitro

GENERAL

AND

Circadian

COMPARATIVE

83, 1.52-158 (191)

Rhythms of Melatonin Secretion from Superfused (Carassius auratus) Pineal Glands in Vitro

MASAYUKI Department

ENDOCRINOLOGY

11~0,’

HIROAKI

KEZUKA,

of Fisheries, Faculty of Agriculture.

KATSUMI

AIDA,

Goldfish

AND ISAO HANYU

The University of Tokyo, Bunkyo, Tokyo 113, Japan

Accepted August 14, 1990 A flow-through, whole-organ culture (superfusion) system was developed, and goldfish pineal glands were maintained at 25” under light-dark (LD) 12:12 cycles, reversed LD 12:12 cycles, continuous dark (DD), or continuous light (LL) conditions for 48 hr. Under LD 12: 12 and reversed LD 12:12 cycles, superfused pineal glands showed a rhythmic melatonin secretion: Scotophase was associated with high titers and photophase with low titers. The melatonin secretion rhythms persisted for two cycles under DD conditions, whereas nocturnal rises were suppressed under LL conditions. After the transition from LL to DD conditions on the third day, melatonin showed a nocturnal increase. These results indicate that melatonin secretion from the superfused goldfish pineal gland is directly photosensitive and that the goldfish pineal gland harbors a circadian oscillator which generates melatonin secretion rhythms. o 1991 Academic press. hc.

Melatonin levels in the plasma and pineal mation into both hormonal (melatonin) and gland show clear diurnal rhythms that are nervous signals (Hanyu and Niwa, 1970; synchronous with a given photoperiod. Hanyu et al., 1978; Falcon et al., 1981, High levels of melatonin were observed 1984; Falcon and Meissel, 1981). Thus, fish during the scotophase while low levels pineal glands are considered a “photoneuwere observed during the photophase in roendocrine transducer.” vertebrate species (in rat, Ozaki et al., The diurnal plasma melatonin rhythm 1976; in sheep, Rollag and Niswender, found in goldfish was abolished by pine1976; in the japanese quail Coturnix alectomy (Kezuka, 1988). This diurnal coturnixjuponica, Underwood et al., 1984; rhythm persisted for at least a few days afin the green sea turtle Chelonia mydas, ter transfer from light-dark (LD) 12:12 cyOwens et al., 1980; in the frog Rana perezi, cles to continuous dark (DD) conditions. In Delgado and Vivien-Roels, 1989; in the addition, goldfish pineal glands in organ rainbow trout Oncorhynchus mykiss (for- culture secreted melatonin in a similar fashmerly Salmo gairdneri), Gern et al., 1978; ion under DD conditions (Kezuka et al., in the pike Esox Lucius, Falcon et al., 1986; 1989). These in vivo and in vitro data indiin the goldfish Carassius auratus, Kezuka, cate that the pineal gland is the main organ 1988; in the common carp Cyprinus carpio, that generates plasma melatonin rhythms in Kezuka et al., 1988). this species and suggest the existence of The teleostean pineal gland is a photocircadian rhythms in melatonin secretion. sensitive organ and transduces photic inforRecently, Falcon et al. (1989) suggested that the pineal gland of the pike contains a circadian oscillator which generates the ’ To whom correspondence should be addressed at rhythms in serotonin iV-acetyltranferase the present address: Department of Anatomy, St. Mar(NAT) activity and of melatonin release. ianna University School of Medicine, Sugao 2-16-1, Miyamaeku, Kawasaki 216. Japan. Superfused trout pineal glands secrete 152 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

MELATONIN

SECRETION

melatonin reflecting photoperiod under LD 12:12 cycles; however, an endogenous rhythmicity in melatonin secretion was not detected under DD conditions (Gern and Greenhouse, 1988). Therefore, it is not clear whether fish pineal glands have endogenous melatonin secretion rhythms. Chicken pineal glands maintained in organ and cell culture conditions showed rhythmic changes in NAT activity (Binkley et al., 1978; Deguchi, 1979b; Kasal et al., 1979) and in melatonin secretion under DD conditions (Takahashi et al., 1980). In arrhythmic pinealectomized sparrows, Passer domesticus, transplantation of pineal tissue into the eye chamber recovered rhythmicity in locomotor activity which is synchronous with the phase of the donor bird’s rhythm (Zimmerman and Menaker, 1979). These results indicate the existence of a circadian oscillator in the avian pineal gland. In this investigation, a flow-through, whole-organ culture (superfusion) system for the pineal gland was developed and the amount of melatonin secreted into perfusates was determined by radioimmunoassay (RIA) in order to confirm the existence of endogenous circadian rhythms in melatonin secretion in goldfish. MATERIALS

AND METHODS

Male goldfish (Carassius aug were used in these experiments. Fish were reared in 50-liter experimental aquaria under LD 12: 12 cycles (lights on 0600-1800 hr) at 25” for more than 2 weeks prior to the start of the superfusion culture. Illumination was supplied by a white fluorescent bulb (10 W) for the light phase. Light intensity measured at the surface of water was about 1000 lux during photophase. Superficsion pineal culture. Fish were sacrificed by decapitation and pineal glands were dissected out between 1500 and 1700 hr by a procedure previously described (Kezuka et al.. 1989). Pineal glands were washed with culture medium and preincubated under light conditions until the start of superfusion at 1800 hr. One liter of culture medium (pH 7.5) contained 7.013 g NaCI, 0.224 g KCI. 0.308 g MgSO, ’ 7H20, 0.006 g NaH,PO, . 2H,O, 0.168 g NaHCO,, 0.111 g CaCI,. 4.766 g Hepes (Wako Junyaku Kougyo, Japan),

RHYTHMS

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0.5 g glucose, 0.88 g Eagle’s MEM (Nissui Seiyaku, Japan), 0.2 g gentamycin sulfate (Sigma), and 0.0025 g amphotericin B (Sigma). The entire superfusion culture apparatus shown in Fig. 1 was placed in an incubator controlled at 25“. A disposable l-ml syringe (Nipro, Japan) was cut at the height of 0.4 ml to serve as a culture chamber. A pineal gland was placed on sterile glass wool in the chamber. The upper end of the chamber was closed with a rubber stopper and the upper and lower ends were connected with disposable 19-gauge needles (Nipro, Japan). A peristaltic pump (Bio Rad), the chamber, and a fraction collector (Atto, Japan) were connected by sterile silicon tubes (0.02” i.d. x 0.83” o.d. and 1 .Omm i.d. x 2.0 mm 0.d.). The volume of the chamber was 0.2 ml and that of the silicon tube from the chamber to the fraction collector was 0.5 ml. Pineal glands were maintained for 2 days under LD 12: 12 cycles which were used for acclimation (lights on 0600-1800 hr), reversed LD 12:12 cycles (lights on 1800-0600 hr), DD conditions, or LL conditions. Room lights (36 W white fluorescent bulbs X 9) were always on during the experiments and a white fluorescent bulb (10 W) in the photo-proof incubator was turned on during the photophase and off during the scotophase. Light intensity at the surface of the incubation chamber was about 1000 lux during the light phase. The chamber was covered with aluminum foil for the DD group to assure complete darkness. The photoperiod of the LL group was changed to DD conditions on Day 3 and the culture was continued for an additional 24 hr. The culture medium was continuously pumped at a rate of 1 mI/hr and the perfusate was collected hourly in glass tubes. RfA procedrrre. The amount of melatonin secreted was determined by the RIA as previously described (Kezuka et al.. 1988) with modifications. Melatonin Penstahc

Experimental animals. ratus) weighing 43-127

Pump

Silicon

Tube

Incubator

1. Diagram of the flow-through whole-organ (superfusion) culture apparatus. The entire apparatus was placed into an incubator controlled at 25”. Medium was continuously supplied into the chamber at a rate of 1 ml/hr and perfusates were collected hourly. FIG.

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was extracted from each perfusate (1 ml) by the addition of chloroform (3 ml). The mixture was vortexed for 15 min and centrifuged at 1000 rpm for 5 min. After discarding the aqueous phase, the organic phase was evaporated with a vacuum evaporator. The residue was dissolved in 0.5 ml of gel-PBS (20 mM phosphate buffer containing 0.1% gelatin and 140 mM NaCl) and 0.2-ml aliquots were subjected to the RIA. RIA validation. Parallelism of inhibition curves was examined between serial twofold dilutions of melatonin standard and extracted samples. The inhibition curve for the melatonin extract was parallel to the curve for the melatonin standard as shown in Fig. 2. The relationship between the quantity of melatonin added and the quantity recovered was analyzed using linear regression analysis and results are shown in Fig. 3. A significant correlation (r = 0.994, P < 0.001) was obtained between amounts of melatonin added and recovered. Mean recovery rate was 100.4%. Intra- and interassay coefficients of variation at three dose levels (BIB, = 15, 40, and 70%. approximately) were 14.6, 7.4,and 16.l%and 11.4.6.5. and 13.1%. respectively. These results demonstrated the validity of the RIA system employed.

50001 r=0.994 Y=1.056X+0.116

=4000E 5 ,P = 3000: 8 z

1000 MELATONIN

Ijl, 2000 ADDED

I

I

3000

4000

(p+~Il)

FIG. 3. Recoveries of melatonin added. Each value represents the mean and standard error of eight samples.

RESULTS Under LD 12:12 Cycles

Melatonin profiles under LD 12: 12 cycles (lights on 06OG1800 hr) are shown in Fig. 4. Superfused goldfish pineal glands showed rhythmic melatonin secretion according to a given photoperiod in this group. Scotophase was associated with high melatonin MELATONIN 1001

I

62.5 I

1

ADDED 250 I

I

(pg/ml) 1000

1

I

4000

,

titers and photophase with low melatonin titers. Peak values of melatonin secretion during the scotophase ranged from 8.7 to 18.9 ng/hr. Under Reversed LD 12:12 Cycles

Melatonin profiles under reversed LD 12:12 cycles (lights on 18OO-0600 hr) are shown in Fig. 5. Super-fused goldfish pineal glands showed rhythmic melatonin secretion according to a given photoperiod in this group. Scotophase was associated with high melatonin titers and photophase with low melatonin titers. Peak values of melatonin secretion during scotophase ranged from 1.2 to 15.9 ng/hr. Under DD Conditions

o-1 ,

, 64

MELATONIN

,

,

,

256 STANDARD

, 1024

(

,

4096 (pg/ml)

FIG. 2. Inhibition curves of the melatonin standard and melatonin extract. BIB,,; relative binding (radioactivity of sample tube x lOO/total binding).

Melatonin profiles under DD conditions are shown in Fig. 6. Free-running rhythms in melatonin secretion were present. The pattern of changes in melatonin secretion was dependent on the environmental photoperiod used for acclimation. High melatonin levels were observed during the pe-

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c 18

18

6 CLOCK

TIME

6

18

(hr)

FIG. 4. Profiles in melatonin secretion under LD 12:12 cycles. The amount of melatonin secreted into perfusates (ng/hr: Y-axis) was plotted against clock time (hr; X-axis). Dark bars along the X-axis represent scotophase and open bars indicate photophase.

riod corresponding to the scotophase while low levels were observed during the period corresponding to the photophase. Peak values of melatonin secretion ranged from 3.1 to 12.6 ng/hr. Under LLIDD Transitions

Melatonin profiles under LL/DD transitions are shown in Fig. 7. A nocturnal rise in melatonin secretion was suppressed under LL conditions and rhythms in melatonin secretion were not observed. When the photoperiod was changed from LL to DD conditions on the third day, melatonin secretion was rapidly activated and melatonin was maintained at high levels during the period corresponding to the scotophase in the acclimation photoperiod. Peak values of melatonin secretion during DD exposure ranged from 1.7 to 14.0 ng/hr.

18

6

18

CLOCK

TIME

6

18

(hr)

FIG. 5. Profiles in melatonin secretion under reversed LD 12: 12 cycles. The amount of melatonin secreted into perfusates (ng/hr; Y-axis) was plotted against clock time (hr; X-axis). Dark bars along the X-axis represent scotophase and open bars indicate photophase.

DISCUSSION

In this investigation, it has been ascertained that the superfused goldfish pineal gland secretes melatonin into perfusates in response to a given photoperiod. The present results are parallel to that obtained in a previous organ culture study (Kezuka et al., 1989). Connection with the central nervous system (CNS) is not required to maintain rhythmic melatonin secretion. Under LD 12: 12 or reversed LD 12: 12 cycles, melatonin secretion in vitro is clearly scotophase dependent: the dark phase was associated with high melatonin titers and the light phase with low melatonin titers. While peak melatonin titers were characteristic of individual pineal glands, profiles of melatonin secretion in vitro are consistent

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18

6

6

18

CLOCK

TIME

18

(hr)

FIG. 6. Profiles in melatonin secretion under DD conditions. The amount of melatonin secreted into perfusates (ng/hr; Y-axis) was plotted against clock time (hr; X-axis). The dark bar along the X-axis represents scotophase and the open bar indicates photophase.

with circulating melatonin levels (Get-n et al., 1978; Kezuka, 1988; Kezuka et al., 1988), melatonin content, and NAT activity in the pineal gland (Falcon et al., 1986, 1987). Twenty-four hour cycles in melatonin secretion were observed both in LD 12:12 and in reversed LD 12:12 groups. Under DD conditions, the pineal gland continued to secrete melatonin in a rhythmic fashion. The pattern of changes in melatonin secretion from superfused pineal glands depended on the environmental lighting schedule to which goldfish had been acclimated. The rhythm observed under DD conditions was considered to be circadian. Melatonin secretion was suppressed under LL conditions. When the photoperiod was changed from LL to DD conditions on the third day, melatonin showed a nocturnal increase. These results indicate that light directly suppressed melatonin secretion from the superfused goldfish pineal gland, and that a 2-day exposure to light

caused no phase shift in melatonin secretion. In the case of teleost fish, Gern and Greenhouse (1988) reported that endogenous rhythms in melatonin secretion were not detected under DD conditions from the superfused rainbow trout pineal gland and that the lack of endogenous rhythmicity may be inherent in the strain of rainbow trout used. In the pineal gland of the pike, night-time values in NAT activity were higher than day-time values under DD conditions (Falcon et al., 1987), and freerunning rhythms in NAT activity and in melatonin secretion were observed (Falcon et al., 1989). In this investigation, circadian rhythms in melatonin secreted from goldfish pineal glands exist in vitro. These results indicate that goldfish and pike pineal glands contain a circadian oscillator and that the pineal gland itself is a “biological clock” in these species. The existence of a circadian oscillator in the pineal gland has been demonstrated in chickens (Binkley et al., 1978; Deguchi 1979a,b; Kasal et al., 1979; Takahashi et al., 1980) and in reptiles (Menaker and Wisner, 1983). In combination with these results, it is suggested that the pineal gland of nonmammalian vertebrates contains a oscillator which generates circadian rhythms in melatonin secretion. The physiological roles of the pineal gland and rhythmic secretion of melatonin in fish remain yet unknown. The pineal gland has been implicated in the control of circadian organization and rhythmicity in behavioral activities (Kavaliers, 1979a,b. 1980, 1981). Recently, Kezuka et al. (1989) reported that ovulation which ordinarily occurs synchronously at midnight in matured female goldfish (Stacy et al., 1979; Kobayashi et al., 1985) occurred randomly in pinealectomized females, suggesting that the pineal gland is involved in determining the timing of the preovulatory gonadotropin surge. Since the photoreceptor cells in the pineal gland transduce photoperiodic infor-

MELATONIN

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6

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IN

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7. Profiles in melatonin secretion under LL/DD transition. The amount of melatonin secreted into perfusates (ng/hr; I’-axis) was plotted against clock time (hr; X-axis). The dark bar along the X-axis represents scotophase and the open bar indicates photophase. FIG.

mation into both rhythmic secretion of melatonin (Gem er al., 1978; Kezuka et al., 1988) and nervous signals into the CNS (Hanyu and Niwa, 1970; Hanyu et al, 1978), it remains to be clarified whether melatonin rhythm or rhythmical nervous discharge is important in the regulation of the timing of the preovulatory gonodotropin surge in female goldfish. This is the first demonstration that the goldfish pineal gland contains an oscillator which generates circadian rhythms in melatonin secretion. Further investigations will be required to characterize the nature of the circadian clock located in the pineal gland of teleost fish. ACKNOWLEDGMENTS We express our thanks to M. N. Wilder. Department of Agriculture, The University of Tokyo, for reading the manuscript. This study was supported in part by a grant-in-aid (Bio Media Program 90-H-2-4) from the Ministry of Agriculture, Forestry. and Fisheries.

Neuroendocrinology

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