GENERAL
AND
Plasma
COMPARATIVE
ENDOCRINOLOGY
37, 493-500 (1979)
Melatonin in the Scincid Lizard, Trachydosaurus rugosus: Die1 Rhythm, Seasonality, and the Effect of Constant Light and Constant Darkness B. T. FIRTH,' D. J. KENNAWAY,~ AND M. A. M. ROZENBILDS,~
Departments
of Anatomy
and Histology and Obstetrics Adelaide, South Australia
and Gynaecology. 5001, Australia
University
of Adelaide.
Accepted January 9, 1979 Melatonin was assayed in the plasma of the scincid lizard, Trachydosaurus rugosus, using a specific radioimmunoassay. The levels of this indole exhibited a daily fluctuation similar to that observed in many other vertebrates. Plasma melatonin titers were low during the light phase and elevated during the dark phase when exposed to a lighting regimen of 13 hr light and 11 hr dark. In lizards captured in spring and transferred to this regimen, the amplitude of the plasma melatonin oscillation appeared damped in comparison to those sampled in the autumn. This damping was attributed to higher light phase and lower dark phase concentrations in spring. In one experiment, a correlation between size and plasma melatonin concentration was evident, higher levels being present in smaller animals. In contrast to birds and mammals, where a rhythm in blood melatonin content persists in constant dark, the plasma melatonin rhythm of T. rugosus was abolished by both constant light and constant darkness under constant temperature conditions.
It is generally held that the pineal organ elf many nonmammalian vertebrates (fishes, amphibians, and some reptiles) is directly plhotosensory while in other vertebrates (some reptiles, birds, and mammals) it is supplied with photoreceptor information viia sympathetic pathways from the lateral e/yes (Kappers, 1971; Collin, 1971). A direct photosensory capacity of pineal cells does nlot preclude a second, secretory, function. Por example there is now evidence to indicate that the pineal organs of all vertebrate cilasses are capable of synthesizing and se1 Present address: Department of Zoology and Entamology, Colorado State University, Fort Collins, CiA. 80523. * Present address: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, R3E OW3, Clmada. 3 Present address: Department of Pathology, The Flinders University of South Australia, Bedford Park, SA. 5042, Australia.
creting indoleamines, the most widely studied of which is melatonin (N-acetyl-5methoxytryptamine), a putative hormone secreted principally although not exclusively by the pineal organ (Ozaki and Lynch, 1976; Kennaway et al.. 1977; Gern et al., 1978a). Pineal and plasma content of melatonin of birds and mammals have been shown to vary rhythmically, with highest levels occurring during darkness and corresponding with increases in pineal enzyme activity (Arendt et al., 1975; Ralph, 1976; Rollag and Niswender, 1976; Ozaki and Lynch, 1976; Kennaway et al., 1977). Melatonin has also been found in the cerebrospinal fluid of calves (Hedlund et al., 1977), sheep (Rollag et al., 1978a), humans (Smith et al., 1976; Arendt et al., 1977), and turtles (Owens, Gern and Ralph, unpublished). The first positive identification of melatonin in the pineal organ of an ec-
493 0016~6480/79/040493-08$0 I .00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
494
FIRTH,
KENNAWAY,
tothermic vertebrate was in the Pacific salmon (Oncorhynchus tshawytscha) by the method of thin-layer chromatography and fluorometry (Fenwick, 1970). Other studies have demonstrated the presence of the enzyme (hydroxyindole-0-methyltransferase or HIOMT) responsible for the synthesis of melatonin in a number of vertebrate species (Quay, 1965) including cyclostomes (Joss, 1977), teleost fishes (Smith and Weber, 1974; Hafeez and Quay, 1970), amphibians (Eichler and Moore, 1975), and reptiles (Quay et al., 1971). Some of these reports detail a photoperiod-dependent rhythm in pineal HIOMT activity, with the highest levels being recorded in the dark period and the lowest levels in the light period of a daily light cycle (Joss, 1977, in cyclostomes; Smith and Weber, 1974, in teleost fishes). Other investigators found no such photoperiod-dependent fluctuation in HIOMT in frogs (Eichler and Moore, 1975), lizards (Quay et al., 1971), and chickens (Binkley et al., 1973). Studies in birds and mammals indicate that the light-dependent changes in pineal HIOMT activity do not parallel those of melatonin. Indeed, the enzyme serotonin, N-acetyl-transferase, appears to more accurately reflect changes in circulating melatonin (Binkley et al., 1975; Binkley, 1976; Klein, 1978). Apart from the studies in birds and mammals previously mentioned, a die1 rhythmic variation in plasma or serum melatonin content has recently been shown in a wide variety of vertebrate species including trout (Gern et al., 1978a), turtles (Owens et al., unpublished), and lizards (Kennaway et al., 1977). The major current concept of the action of melatonin is that it is an hormonal transducer of environmental photic input signals to the pineal and enables the organism to adapt its reproductive system to the environment via interactions with the hypothalamic-hypophyseal-gonadal axis (Reiter, 1974). The present report characterizes plasma melatonin concentrations
AND
ROZENBILDS
measured by radioimmunoassay (RIA) in the scinid lizard, Trachydosaurus rugosus (also known as Tiliqua rugosa), under an imposed. photoperiod as well as constant light and constant darkness. MATERIALS General Procedures
AND METHODS
Truchydosaurus rugosus (Gray) were collected in the spring and early summer of 1975 and 1976 from the Murray river and Yorke Peninsula area within a 150-km radius of Adelaide, South Australia. The sex was recorded and animals weighed and snout-vent length (S-V) determined as an indicator of age. They were housed in cages which were partially exposed to natural light and temperature, and a diet of fruit and vegetables was supplied biweekly. Prior to blood sampling, the lizards were acclimated in an environmentally controlled light-proof box 1.8 m long, 0.9 m wide, and 0.6 m high, provided with a thermostatically controlled heater-fan and incandescent lights connected to a time switch (lights on 0530 hr, lights off 1830 hr, i.e., 13 hr light, 11 hr dark: 13L: 1ID). The light intensity for all experiments was 240 lux. The animals were fed at the commencement of acclimation and provided with water ad libitum
.
Three to six milliliters of blood was collected by cardiac puncture following tribromoethanol anaesthesia (Sigma Chemical Co.). Three grams of tribromoethanol was dissolved in 3 ml of tertiary amyl alcohol made up to 100 ml in hot distilled water and injected intraperitoneally at the dose of 1 ml/250 g body wt. The anaesthetic procedure employed precluded sampling of all animals at the same time. Thus, blood was taken over a I- to 1.5-hr period around the designated sampling time. This sometimes necessitated removing animals from darkened quarters for blood sampling. The blood was placed in 10 ml EDTA tubes and held in a refrigerator at 4” for up to 2 hr before centrifugation. The plasma was frozen prior to extraction for RIA.
Radioimmunoassay Procedures The melatonin RIA employed in this study has been described by Kennaway et al. (1977) and was used without further modification. Briefly, the assay involved extraction of 2 and 1 ml of plasma with borate buffer pH 10 and chloroform. The dried chloroform extract was reconstituted and chromatographed on columns of Lipidex 5000 (Packard Instrument Co., Downers Grove, Ill.) and the melatonin-rich eluate assayed. Owing to insufficient plasma, the assay has not been rigorously validated in T. rugosus. Kennaway et ul. (1977) have indicated that the only possible interfering indole is N-acetyltryptamine. An indica-
PLASMA
MELATONIN
tion of parallelism with the melatonin standard was evident with the 2 vol of plasma extracted. Pineal glands from this species synthesize chromatogiaphically pure melatonin when incubated with appropriate precursors (Kennaway and Firth, unpublished).
Experimental
Procedures
Experiment I. (a) Twenty-six T. rugosus (S-V, 20.4-31.4 cm) were acclimated to a photoperiod of l!lL: 1lD and constant temperature for 11 days in late snmmer. Blood samples from 13 of these lizards (six males and seven females) were obtained around 1800 hr on February 24 and again around 2400 hr on March 26 following the intervening period in acclimation. Food was withheld 3 days prior to sampling. The remaining 13 lizards (five males and eight females with a similar size distribution to the previous group) were bled around 1200 hr on February 24 and 0600 hr on March 26 after being subjected to the above conditions. Four females were added in the second phase of’ the experiment to replace individuals that did not recover satisfactorily from the initial sampling procedime. (b) Twenty-one lizards (S-V, 2X4-29.0 cm) were acclimated to 13L: 11D in mid-spring 1976. Four males and five females were sampled around 1200 hr and six males and six females around 2400 hr. (c) Twenty-five T. rugosus (S-V, 25.0-31.3 cm) were acclimated to 13L: 11D in late spring 1976. Six 1i;zards (three males and three females) were sampled around each of 0600, 1200, and 1800 hr and seven individuals (three males and four females) around 21100
hr.
Exprriment 2. (a) Thirty-six lizards (S-V, 24.7-30.7 cm) were acclimated to constant light (LL) and a constant temperature of 24.3 ? 0.8” for 9 days following a week in 13L: 1lD in mid-spring 1976. Groups of four males and five females were bled around 0600, 1200, 1800, and 2400 hr. One individual from the 2400 hr sample died soon after and was excluded from the analysis. (b) Thirty-nine lizards (S-V, 26.6-30.7 cm) were acclimated to constant dark (DD) and constant temperature of 22.7 + 0.8’ for 9 days in mid-spring 1976 following a week in 13L: 1lD. Groups of 10 individuals were sampled around 0600 and 1200 hr (four males and six females) at 1800 hr (five males and five females) and nine individuals around 2400 hr (four males and five females).
Wiometrical
Considerations
A two-way analysis of variance (factorial design, with time and sex as the factors) was performed on all data, following a logarithmic transformation (log 10) toI make the variance independent of the mean (see
495
IN LIZARD
Sokal and Rholf, 1969, p. 382). If the analysis of variance showed an acceptable level of significance ( 0.05). Many of the animals used in Expt 2a were later sampled in Expt 2b. A two-way analysis of variance with repeated measures reban Sample Timr vealed no significant interaction of time and FIG. 1. Plasma melatonin of T. rugosus in sex with melatonin. Similarly a two-way 13L:llD at various sampling points in (a) February factorial analysis of variance on all of the (b) October, and (c) November. Solid circles repremelatonin values in Expts 2a and b did not sent means, the vertical lines ? SEM, and the numwith time bers above them the sample size, N. Shaded areas at show any significant interaction the top of the figure represent the dark period of the and sex. 9
200
l-7
lighting cycle. Two mean plasma levels are given at 1800 hr in (a), representing values immediately prior to and after “lights off.” TABLE
loo
1
RESULTS OF STUDENT-NEWMAN-KEULS MEAN SEPARATION TEST ON PLASMA MELATONIN OF T. W~OSUS IN 13L:llD (EXPT. la)
Sampling time (hr) Lights on 1200 0600 1800 Lights off 2400 1800
Plasma melatonin
(mean log,,pg/ml) 0.2258 1.5263 1.7159 2.0088 2.1438
Significance A
80= 60 F -40 ,o .g 20
(a) sopt.
9
1
9
g ,; g 60
B BC C C
a Significance is indicated by letters not in common. Means are adjusted for the covariate of size.
h
40 20
Mean
Sample
Time
FIG. 2. Plasma melatonin of T. rugosus in (a) LL and (b) DD. Conventions as in Fig. 1.
PLASMA
MELATONIN
DISCUSSION
These results are in agreement with those for other vertebrates where a photoperioddependent oscillation of plasma or serum melatonin levels has been documented (Ralph, 1976; Hedlund et al., 1977; Kennamay et al., 1977; Gern et al., 1978b) with low titers occurring in the day and elevated til:ers occurring at night. An interesting feature of the rhythm in plasma melatonin content in T. rugosus is the “anticipated” rise al 1800 hr, before “lights off.” Such a phenomenon has been reported in chickens (Ralph, 1976) and humans (Arendt et al., 1077). There appears to be a difference in the amplitude of the daily rhythm of melatonin levels in the spring compared to that of l$ards sampled in the late summer. This is the result of low midlight levels and high middark levels in late-summer animals, as opposed to higher midlight and lower middark titers in spring animals. Kennaway et ud. (1977) found mean summer (midFbbruary) nighttime melatonin concentnations of 240 2 43 pg/ml in T. rugosus exposed to the same photoperiod and temperature conditions as described here. The highest mean melatonin concentration recorded in the present study was 130 pgml. These results suggest that in T. rugosus there is a seasonal flux in melatonin secretion or degradative capacity, with a rhythm of low amplitude in the spring changing to a rhythm of greater amplitude in the summer and autumn. There is considerable evidence accnmulating in a number of species indicating that there may be seasonal changes in pilneal activity or circulating melatonin concentrations. Owens et al. (unpublished data) recorded much lower serum melatoniln levels in nesting green sea turtles compared to individuals immediately prior to nesting. Studies in male sparrows (Bat-fuss and Ellis, 1971) and male ground squirrels (Ellis and Balph, 1976) have shown that piheal HIOMT was inversely related to the
IN
LIZARD
497
testicular cycle, enzyme activity being lowest during the breeding season. Further evidence suggests a causal relationship between circulating melatonin and gonadal hormones in rats. Oophorectomy elevated serum melatonin concentrations and this rise was suppressed by administration of oestrogen and progesterone (Ozaki et al., 1978). Rollag et al. (1978b) found no seasonal variation in the amplitude of the rhythm of serum melatonin levels measured in ewes during different reproductive states. There were differences, however, in the duration of the nighttime melatonin pulse (due to alterations in photoperiod) between animals exhibiting oestrous cycles and those exhibiting anoestrus. The apparent seasonal variation in melatonin levels in T. rugosus may be related to one or a number of seasonally fluctuating physiological events. For example, the males of this species undergo a marked seasonal cycle of reproduction, spermatogenesis commencing in the autumn and peak sexual activity occurring in the spring. Bourne et al. (1971) and Bourne and Seamark (1975) found plasma testosterone titers of this lizard to be three times higher in spring than at other times of the year. Plasma testosterone was totally absent during the summer. Although little information is available on female T. rugosus, it is likely that they follow a similar cycle with respect to ovarian activity (Bourne, 1972). The reproductive function attributed to melatonin in mammals is well known (see review by Reiter, 1977). There is also some evidence that this substance may participate in reproductive activity of lizards. In female anoles (An& carolinensis), pinealectomy was effective in accelerating ovarian follicular development in the fall and winter but not at other seasons (Levey, 1973). Daily injection of 10 pg of melatonin were shown to block this response. Similarly, injections of melatonin at the dose of 0.3 pglg body wtlday, produced antigonadatrophic effects in male Callisaurus
498
FIRTH,
KENNAWAY,
draconoides (Packard and Packard, 1978). While it may be contended that such doses are pharmacological, Rollag et al. (1978a) have indicated that sheep secrete up to 5 mg of melatonimday. The observation that smaller lizards had more plasma melatonin than larger ones may further suggest a link with gonadal function. Fenwick (1970) found that immature salmon had approximately six times as much pineal melatonin as mature salmon. Similarly, Get-n et al. (1978b) reported that young trout have more than twice the amount of plasma melatonin when compared to those six times their body weight. The positive correlation between size and melatonin in the present study may be tenuous since it was evident in only one experiment. Alternatively, it could be a result of the smaller size range and the smaller sample size of lizards used in the other experiments. The normal die1 rhythm in plasma melatonin concentration in T. rugosus was abolished after 9 days in constant light or constant dark. To our knowledge no one has demonstrated a persistent cycle of melatonin content in ectothermic vertebrates under constant lighting and temperature conditions. Hafeez and Quay (1970) found no difference in the activity of the pineal enzyme HIOMT in two species of fish sampled in a light-dark cycle, constant light, and constant dark. Similarly, pineal HIOMT activity in lizards (Scefoporus occidentalis) sacrificed in a lighting cycle (at midday, and 8 hr after “lights off ‘) in constant light and constant dark were not significantly different (Quay et al., 1971). In view of the demonstration of a photoperiod-dependent rhythm in plasma melatonin content in the present study, it appears that HIOMT activity in lizards bears the same nonparallel relationship to melatonin as is true for some birds and mammals (Binkley, 1976). We did not detect a free running rhythm of plasma melatonin content of T. rugosus
AND ROZENBILDS
in constant dark, although such oscillations have been found in the pineal of chickens (Ralph et al., 1974; Ralph et al., 1975), rats (Ralph et al., 1971), and in the serum of chickens (Ralph et al., 1974). In T. rugosus, the observation that there was an “anticipated” rise in plasma melatonin (viz., Expt la) under a light-dark cycle suggests the presence of an endogenous rhythm (Klein, 1978) but it is possible that the four sampling periods employed in constant dark were insufficient to detect it, or that the rhythm faded out rapidly. In addition to the reproductive functions discussed above, there is an increasing body of evidence which suggests that pineal systems of reptiles and other vertebrates are involved in daily and seasonal thermoregulatory adjustments (Ralph, 1975a,b). Furthermore, melatonin has been implicated in these responses in lizards (Firth and Heatwole, 1976), birds (Binkley, 1974; John et al., 1978), and mammals (Arutyunyan et al., 1964; Fioretti and Martini, 1968; Fioretti et al., 1974; Palmer and Riedesel, 1976). Since both light and temperature are important in the synchronization of reproductive cycles of reptiles (Licht, 1972) a thermoregulatory function for the pineal gland and melatonin may not be incompatible with that of reproduction. ACKNOWLEDGMENTS We are grateful to Marilyn Campion for her time in performing the statistical analyses and to Dr. R. F. Seamark for his interest in this study. We also thank Mr. J. S. Turner, Dr. W. Gem, Dr. D. Owens, Dr. M. Packard, and Dr. C. Ralph for critically reading the manuscript, and to Brian Miller for help in collecting lizards. This work was supported by a University of Adelaide Postdoctoral Fellowship (to B.T.F.) and the Sir John Gellibrand Memorial Scholarship (to D.J.K.).
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40, 347-350.
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Alrendt, J., Wetterberg, L., Heyden, T., Sizonenko, P. C., and Paunier, L. (1977). Radioimmunoassay of melatonin: Human serum and cerebrospinal fluid. Horm. Res. 8, 65-75. Barfuss, D. W., and Ellis, L. C. (1971). Seasonal cycles in melatonin synthesis by the pineal gland as related to testicular function in the house sparrow (Passer domesticus). Gen. Comp. Endocrinol. 17, 183-193. Blnkley, S. A. (1974). Pineal and melatonin: Circadian rhythms and body temperature of sparrows. In “Chronobiology” (L. E. Scheving, F. Halberg, J. E. Pauley, ed.), pp. 382-385. Igaku Shoin Ltd, Tokyo. Blnkley, S. (1976). Comparative biochemistry of the pineal glands of birds and mammals. Amer. Zool. 16, 57-66.
Blnkley, S., MacBride, S. E., Klein, D. C., and Ralph, C. L. (1973). Pineal enzymes: Regulation of avian melatonin synthesis. Science 181, 273275. Binkley, S., MacBride, S. E., Klein, D. C., and Ralph, C. L. (1975). Regulation of pineal rhythms in chickens: Refractory period and nonvisual light perception. Endocrinology %, 848-853. Bourne, A. R. (1972). “Reproductive Endocrinology of the Viviparous Lizard, Tiliqua rugosa.” Ph.D. thesis, University of Adelaide, Australia. Boume, A. R., and Seamark, R. F. (1975). Seasonal changes in 17 /3-hydroxysteroids in the plasma of a male lizard (Tiliqun rtrgosa). Comp. Biochem.
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Bourne. A. R., Kirkland, J. A., and Seamark, R. F. (1971). Seasonal changes in testicular function of the viviparous lizard Tiliqua rugosa (Gray) J. Reprod. Fertil. 24, 138-139. Cbllin, J. P. (1971). Differentiation and regression of the cells of the sensory line in the epiphysis cerebri. In “The Pineal Gland.” (Cl. E. W. Wolstenholme and J. Knight, eds.) pp. 79-125. Churchill-Livingstone, LondoniEdinburgh. Eichler, V. B., and Moore, R. Y. (1975). Studies on hydroxyindole-0-methyltransferase in frog brain and retina: Enzymology, regional distribution and environmental control of enzyme levels. Coma.
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Elilis, L. C., and Balph, D. F. (1976). Age and seasonal differences in the synthesis and metabolism of testosterone by testicular tissue and pineal HIOMT activity of Uinta ground squirrels (Spermophilus
urmutus).
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42-5 1. Fenwick, J. C. (1970). Demonstration and effect of melatonin in fish. Gen. Comp. Endocrinol. 14, 86-97. Fioretti, M. C., and Martini, L. (1968). Effetti della melatonina sulla temperatura corporea. Ann. Fat.
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Fioretti, M. C., Barzi, F., Borgonovo, G., Menconi, E., and Martini, L. (1974). Effetti indotti dalla melatonina sulla temperatura corporea in ratti ipotermici o mantenuti in condizioni diverse di illuminazione. Foliu Endocrinol. (Rome) 27, 390-400. Firth, B. T., and Heatwole, H. (1976). Panting thresholds of lizards: The role of the pineal complex in panting responses in an agamid, Amphibolurus muricatus. Gen. Comp. Endocrinol. 29, 388-401. Gern, W. A., Owens, D. W., and Ralph, C. L. (1978a). Persistence of the nychthemeral rhythm of melatonin secretion in pinealectomized or optic tract-sectioned trout (Salmo gairdneri). J. Exp. Zool. 205, 371-376. Gem, W. A., Owens, D. W., and Ralph, C. L. (1978b). Plasma melatonin in the trout: Day-night change demonstrated by radioimmunoassay. Gen. Camp. Endocrinol.
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Hafeez. M. A.. and Quay, W. B. (1970). Pineal acetylserotonin methyltransferase activity in the Teleost fishes, Hesperoleucus symmetricus and Salmo gairdneri, with evidence for lack of effect of constant light and darkness. Comp. Gen. Pharmacol.
1, 257-262,
Hedlund, L.. Lischko, M. M., Rollag, M. D., and Niswender, G. D. (1977). Melatonin: Daily cycle in plasma and cerebrospinal fluid of calves. Science
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John, T. M., Itoh, S.. and George, J. C. (1978). On the role of the pineal in thermoregulation in the pigeon. Harm. Res. 9, 41-56. Joss, J. M. P. (1977). Hydroxyindole-O-methyltransferase (HIOMT) activity and the uptake of 3H-melatonin in the lamprey, Geotria austrulis Gray. Gen. Comp. Endocrinol. 31, 270-275. Kappers, J. A. (1971). The pineal organ: An introduction. 112 “The Pineal Gland.” (G. E. W. Wolstenbolme and J. Knight, eds), pp. 3-34. Churchill-Livingstone, London/Edinburgh. Kennaway. D. J., Frith, R. G., Phillipou, G., Mathews, C. D., and Seamark, R. F. (1977). A specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography-mass spectrometry. Endocrinology 101, 119-127. Klein, D. C. (1978). The pineal gland: A model of neuroendocrine regulation In “The Hypothalamus” (S. Reichlin, R. J. Baldassaiini and J. B. Martin, eds.), pp. 303-327. Raven Press, New York. Levey, I. L. (1973). Effects of pinealectomy and melatonin injections at different seasons on ovarian activity in the lizard Anolis crrrolinensis. J. hp. Zoo/. 185, 169-174.
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Licht, P. (1972). Problems in experimentation on timing mechanisms for annual physiological cycles in reptiles. In “Hibernation and Hypothermia, Perspectives and Challenges” (F. South, ed.), pp. 681-711. American’Elsevier. New York. Minneman, K. P., and Wurtman. R. J. (1975). Effects of pineal compounds on mammals. Life Sci. 17, 1189-1200. Ozaki. Y., and Lynch, H. J. (1976). Presence of melatonin in plasma and urine of pinealectomized rats. Endocrinology 99, $41-644. Ozaki, Y., Wurtman, R. J., Alonso, R., and Lynch, H. J. (1978). Melatonin secretion decreases during the proestrous stage of the rat estrous cycle. Proc. Nat.
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Packard, M. J., and Packard, G. C. (1978). Antigonadatrophic effect of melatonin in male lizards (Callisaurus draconoides). Experientiu 33, 1665. Palmer, D. L., and Riedesel. M. L. (1976). Responses of whole animal and isolated hearts of ground squirrels, Cifellus lateralis to melatonin. Comp. Biochem.
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Quay, W. B. (1965). Retinal and pineal hydroxyindole-O-methyltransferase activity in vertebrates. Life Sci. 4, 983-991. Quay, W. B., Stebbins, R. C., Kelley. T. D., and Cohen, N. W. (1971). Effects of environmental and physiological factors on pineal acetylserotonin methyltransferase activity in the lizard Sceloporus occidentulis. Physiol. 2001. 44, 241248. Ralph, C. L. (l975a). The pineal complex: A retrospective view. Amer. Zoo/. lS(Suppl. 1). 105116. Ralph, C. L. (1975b). The pineal gland and geographical distribution of animals. Inf. J. Biometeor. 19, 289-303. Ralph, C. L. (1976). Correlations of melatonin content in pineal gland, blood and brain of some birds and mammals. Amer. Zool. 16, 35-43. Ralph, C. L.. Binkley, S.. MacBride, S. E., and Klein,
AND ROZENBILDS D. C. (1975). Regulation of pineal rhythms in chickens: Effect of blinding, constant light, constant dark, and superior cervical ganglionectomy. Endocrinology 96, 1373-1378. Ralph, C. L., Mull, D.. Lynch, H. J., and Hedlund, L. (1971). A melatonin rhythm persists in rat pineals in darkness. Endocrinology 89, 1361-1366. Ralph, C. L., Pelham, R. W., MacBride, S. E.. and Reilly, D. P. (1974). Persistent rhythms of pineal and serum melatonin in cockerals in continuous darkness. J. Endocrinol. 63, 319-324. Reiter, R. J. (1974). Pineal regulation of hypothathalamo-pituitary axis: Gonadotropins. In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.). Vol. IV, Section 7, pp. 519-550. Amer. Physiol. Sot., Washington. Reiter, R. J. (1977). Melatonin-reproductive effects. Irt “The Pineal 1977,” pp. 96-106. Eden Press. Montreal. Rollag, M. D.. and Niswender, G. D. (1976). Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology 98, 482-489. Rollag, M. D., Morgan, R. J., and Niswender. G. D. (1978a). Route of melatonin secretion in sheep. Endocrinology 102, l-8. Rollag, M. D., O’Callaghan, P. L.. and Niswender, Cl. D. (1978b). Serum melatonin concentration during different stages of the annual reproductive cycle in ewes. Biol. Reprod. 18, 279-285. Smith, I., Mullen, P. E., Silman, R. E., Snedden, W.. and Wilson, B. W. (1976). Absolute identification of melatonin in human plasma and cerebrospinal fluid. Nature (London) 260, 718-719. Smith. J. R., and Weber, L. J. (1974). Diurnal fluctuations in acetylserotonin methyltransferase (ASMT) activity in the pineal gland of the steelhead trout (Salmo gnirdneri). Proc. Sot. Exp. Biol. Med. 147, 441-443. Sokal, R. R., and Rohlf. F. J. (1969). “Biometry.” Freeman, San Francisco.
AND
Plasma
COMPARATIVE
ENDOCRINOLOGY
37, 493-500 (1979)
Melatonin in the Scincid Lizard, Trachydosaurus rugosus: Die1 Rhythm, Seasonality, and the Effect of Constant Light and Constant Darkness B. T. FIRTH,' D. J. KENNAWAY,~ AND M. A. M. ROZENBILDS,~
Departments
of Anatomy
and Histology and Obstetrics Adelaide, South Australia
and Gynaecology. 5001, Australia
University
of Adelaide.
Accepted January 9, 1979 Melatonin was assayed in the plasma of the scincid lizard, Trachydosaurus rugosus, using a specific radioimmunoassay. The levels of this indole exhibited a daily fluctuation similar to that observed in many other vertebrates. Plasma melatonin titers were low during the light phase and elevated during the dark phase when exposed to a lighting regimen of 13 hr light and 11 hr dark. In lizards captured in spring and transferred to this regimen, the amplitude of the plasma melatonin oscillation appeared damped in comparison to those sampled in the autumn. This damping was attributed to higher light phase and lower dark phase concentrations in spring. In one experiment, a correlation between size and plasma melatonin concentration was evident, higher levels being present in smaller animals. In contrast to birds and mammals, where a rhythm in blood melatonin content persists in constant dark, the plasma melatonin rhythm of T. rugosus was abolished by both constant light and constant darkness under constant temperature conditions.
It is generally held that the pineal organ elf many nonmammalian vertebrates (fishes, amphibians, and some reptiles) is directly plhotosensory while in other vertebrates (some reptiles, birds, and mammals) it is supplied with photoreceptor information viia sympathetic pathways from the lateral e/yes (Kappers, 1971; Collin, 1971). A direct photosensory capacity of pineal cells does nlot preclude a second, secretory, function. Por example there is now evidence to indicate that the pineal organs of all vertebrate cilasses are capable of synthesizing and se1 Present address: Department of Zoology and Entamology, Colorado State University, Fort Collins, CiA. 80523. * Present address: Department of Physiology, University of Manitoba, Winnipeg, Manitoba, R3E OW3, Clmada. 3 Present address: Department of Pathology, The Flinders University of South Australia, Bedford Park, SA. 5042, Australia.
creting indoleamines, the most widely studied of which is melatonin (N-acetyl-5methoxytryptamine), a putative hormone secreted principally although not exclusively by the pineal organ (Ozaki and Lynch, 1976; Kennaway et al.. 1977; Gern et al., 1978a). Pineal and plasma content of melatonin of birds and mammals have been shown to vary rhythmically, with highest levels occurring during darkness and corresponding with increases in pineal enzyme activity (Arendt et al., 1975; Ralph, 1976; Rollag and Niswender, 1976; Ozaki and Lynch, 1976; Kennaway et al., 1977). Melatonin has also been found in the cerebrospinal fluid of calves (Hedlund et al., 1977), sheep (Rollag et al., 1978a), humans (Smith et al., 1976; Arendt et al., 1977), and turtles (Owens, Gern and Ralph, unpublished). The first positive identification of melatonin in the pineal organ of an ec-
493 0016~6480/79/040493-08$0 I .00/O Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
494
FIRTH,
KENNAWAY,
tothermic vertebrate was in the Pacific salmon (Oncorhynchus tshawytscha) by the method of thin-layer chromatography and fluorometry (Fenwick, 1970). Other studies have demonstrated the presence of the enzyme (hydroxyindole-0-methyltransferase or HIOMT) responsible for the synthesis of melatonin in a number of vertebrate species (Quay, 1965) including cyclostomes (Joss, 1977), teleost fishes (Smith and Weber, 1974; Hafeez and Quay, 1970), amphibians (Eichler and Moore, 1975), and reptiles (Quay et al., 1971). Some of these reports detail a photoperiod-dependent rhythm in pineal HIOMT activity, with the highest levels being recorded in the dark period and the lowest levels in the light period of a daily light cycle (Joss, 1977, in cyclostomes; Smith and Weber, 1974, in teleost fishes). Other investigators found no such photoperiod-dependent fluctuation in HIOMT in frogs (Eichler and Moore, 1975), lizards (Quay et al., 1971), and chickens (Binkley et al., 1973). Studies in birds and mammals indicate that the light-dependent changes in pineal HIOMT activity do not parallel those of melatonin. Indeed, the enzyme serotonin, N-acetyl-transferase, appears to more accurately reflect changes in circulating melatonin (Binkley et al., 1975; Binkley, 1976; Klein, 1978). Apart from the studies in birds and mammals previously mentioned, a die1 rhythmic variation in plasma or serum melatonin content has recently been shown in a wide variety of vertebrate species including trout (Gern et al., 1978a), turtles (Owens et al., unpublished), and lizards (Kennaway et al., 1977). The major current concept of the action of melatonin is that it is an hormonal transducer of environmental photic input signals to the pineal and enables the organism to adapt its reproductive system to the environment via interactions with the hypothalamic-hypophyseal-gonadal axis (Reiter, 1974). The present report characterizes plasma melatonin concentrations
AND
ROZENBILDS
measured by radioimmunoassay (RIA) in the scinid lizard, Trachydosaurus rugosus (also known as Tiliqua rugosa), under an imposed. photoperiod as well as constant light and constant darkness. MATERIALS General Procedures
AND METHODS
Truchydosaurus rugosus (Gray) were collected in the spring and early summer of 1975 and 1976 from the Murray river and Yorke Peninsula area within a 150-km radius of Adelaide, South Australia. The sex was recorded and animals weighed and snout-vent length (S-V) determined as an indicator of age. They were housed in cages which were partially exposed to natural light and temperature, and a diet of fruit and vegetables was supplied biweekly. Prior to blood sampling, the lizards were acclimated in an environmentally controlled light-proof box 1.8 m long, 0.9 m wide, and 0.6 m high, provided with a thermostatically controlled heater-fan and incandescent lights connected to a time switch (lights on 0530 hr, lights off 1830 hr, i.e., 13 hr light, 11 hr dark: 13L: 1ID). The light intensity for all experiments was 240 lux. The animals were fed at the commencement of acclimation and provided with water ad libitum
.
Three to six milliliters of blood was collected by cardiac puncture following tribromoethanol anaesthesia (Sigma Chemical Co.). Three grams of tribromoethanol was dissolved in 3 ml of tertiary amyl alcohol made up to 100 ml in hot distilled water and injected intraperitoneally at the dose of 1 ml/250 g body wt. The anaesthetic procedure employed precluded sampling of all animals at the same time. Thus, blood was taken over a I- to 1.5-hr period around the designated sampling time. This sometimes necessitated removing animals from darkened quarters for blood sampling. The blood was placed in 10 ml EDTA tubes and held in a refrigerator at 4” for up to 2 hr before centrifugation. The plasma was frozen prior to extraction for RIA.
Radioimmunoassay Procedures The melatonin RIA employed in this study has been described by Kennaway et al. (1977) and was used without further modification. Briefly, the assay involved extraction of 2 and 1 ml of plasma with borate buffer pH 10 and chloroform. The dried chloroform extract was reconstituted and chromatographed on columns of Lipidex 5000 (Packard Instrument Co., Downers Grove, Ill.) and the melatonin-rich eluate assayed. Owing to insufficient plasma, the assay has not been rigorously validated in T. rugosus. Kennaway et ul. (1977) have indicated that the only possible interfering indole is N-acetyltryptamine. An indica-
PLASMA
MELATONIN
tion of parallelism with the melatonin standard was evident with the 2 vol of plasma extracted. Pineal glands from this species synthesize chromatogiaphically pure melatonin when incubated with appropriate precursors (Kennaway and Firth, unpublished).
Experimental
Procedures
Experiment I. (a) Twenty-six T. rugosus (S-V, 20.4-31.4 cm) were acclimated to a photoperiod of l!lL: 1lD and constant temperature for 11 days in late snmmer. Blood samples from 13 of these lizards (six males and seven females) were obtained around 1800 hr on February 24 and again around 2400 hr on March 26 following the intervening period in acclimation. Food was withheld 3 days prior to sampling. The remaining 13 lizards (five males and eight females with a similar size distribution to the previous group) were bled around 1200 hr on February 24 and 0600 hr on March 26 after being subjected to the above conditions. Four females were added in the second phase of’ the experiment to replace individuals that did not recover satisfactorily from the initial sampling procedime. (b) Twenty-one lizards (S-V, 2X4-29.0 cm) were acclimated to 13L: 11D in mid-spring 1976. Four males and five females were sampled around 1200 hr and six males and six females around 2400 hr. (c) Twenty-five T. rugosus (S-V, 25.0-31.3 cm) were acclimated to 13L: 11D in late spring 1976. Six 1i;zards (three males and three females) were sampled around each of 0600, 1200, and 1800 hr and seven individuals (three males and four females) around 21100
hr.
Exprriment 2. (a) Thirty-six lizards (S-V, 24.7-30.7 cm) were acclimated to constant light (LL) and a constant temperature of 24.3 ? 0.8” for 9 days following a week in 13L: 1lD in mid-spring 1976. Groups of four males and five females were bled around 0600, 1200, 1800, and 2400 hr. One individual from the 2400 hr sample died soon after and was excluded from the analysis. (b) Thirty-nine lizards (S-V, 26.6-30.7 cm) were acclimated to constant dark (DD) and constant temperature of 22.7 + 0.8’ for 9 days in mid-spring 1976 following a week in 13L: 1lD. Groups of 10 individuals were sampled around 0600 and 1200 hr (four males and six females) at 1800 hr (five males and five females) and nine individuals around 2400 hr (four males and five females).
Wiometrical
Considerations
A two-way analysis of variance (factorial design, with time and sex as the factors) was performed on all data, following a logarithmic transformation (log 10) toI make the variance independent of the mean (see
495
IN LIZARD
Sokal and Rholf, 1969, p. 382). If the analysis of variance showed an acceptable level of significance ( 0.05). Many of the animals used in Expt 2a were later sampled in Expt 2b. A two-way analysis of variance with repeated measures reban Sample Timr vealed no significant interaction of time and FIG. 1. Plasma melatonin of T. rugosus in sex with melatonin. Similarly a two-way 13L:llD at various sampling points in (a) February factorial analysis of variance on all of the (b) October, and (c) November. Solid circles repremelatonin values in Expts 2a and b did not sent means, the vertical lines ? SEM, and the numwith time bers above them the sample size, N. Shaded areas at show any significant interaction the top of the figure represent the dark period of the and sex. 9
200
l-7
lighting cycle. Two mean plasma levels are given at 1800 hr in (a), representing values immediately prior to and after “lights off.” TABLE
loo
1
RESULTS OF STUDENT-NEWMAN-KEULS MEAN SEPARATION TEST ON PLASMA MELATONIN OF T. W~OSUS IN 13L:llD (EXPT. la)
Sampling time (hr) Lights on 1200 0600 1800 Lights off 2400 1800
Plasma melatonin
(mean log,,pg/ml) 0.2258 1.5263 1.7159 2.0088 2.1438
Significance A
80= 60 F -40 ,o .g 20
(a) sopt.
9
1
9
g ,; g 60
B BC C C
a Significance is indicated by letters not in common. Means are adjusted for the covariate of size.
h
40 20
Mean
Sample
Time
FIG. 2. Plasma melatonin of T. rugosus in (a) LL and (b) DD. Conventions as in Fig. 1.
PLASMA
MELATONIN
DISCUSSION
These results are in agreement with those for other vertebrates where a photoperioddependent oscillation of plasma or serum melatonin levels has been documented (Ralph, 1976; Hedlund et al., 1977; Kennamay et al., 1977; Gern et al., 1978b) with low titers occurring in the day and elevated til:ers occurring at night. An interesting feature of the rhythm in plasma melatonin content in T. rugosus is the “anticipated” rise al 1800 hr, before “lights off.” Such a phenomenon has been reported in chickens (Ralph, 1976) and humans (Arendt et al., 1077). There appears to be a difference in the amplitude of the daily rhythm of melatonin levels in the spring compared to that of l$ards sampled in the late summer. This is the result of low midlight levels and high middark levels in late-summer animals, as opposed to higher midlight and lower middark titers in spring animals. Kennaway et ud. (1977) found mean summer (midFbbruary) nighttime melatonin concentnations of 240 2 43 pg/ml in T. rugosus exposed to the same photoperiod and temperature conditions as described here. The highest mean melatonin concentration recorded in the present study was 130 pgml. These results suggest that in T. rugosus there is a seasonal flux in melatonin secretion or degradative capacity, with a rhythm of low amplitude in the spring changing to a rhythm of greater amplitude in the summer and autumn. There is considerable evidence accnmulating in a number of species indicating that there may be seasonal changes in pilneal activity or circulating melatonin concentrations. Owens et al. (unpublished data) recorded much lower serum melatoniln levels in nesting green sea turtles compared to individuals immediately prior to nesting. Studies in male sparrows (Bat-fuss and Ellis, 1971) and male ground squirrels (Ellis and Balph, 1976) have shown that piheal HIOMT was inversely related to the
IN
LIZARD
497
testicular cycle, enzyme activity being lowest during the breeding season. Further evidence suggests a causal relationship between circulating melatonin and gonadal hormones in rats. Oophorectomy elevated serum melatonin concentrations and this rise was suppressed by administration of oestrogen and progesterone (Ozaki et al., 1978). Rollag et al. (1978b) found no seasonal variation in the amplitude of the rhythm of serum melatonin levels measured in ewes during different reproductive states. There were differences, however, in the duration of the nighttime melatonin pulse (due to alterations in photoperiod) between animals exhibiting oestrous cycles and those exhibiting anoestrus. The apparent seasonal variation in melatonin levels in T. rugosus may be related to one or a number of seasonally fluctuating physiological events. For example, the males of this species undergo a marked seasonal cycle of reproduction, spermatogenesis commencing in the autumn and peak sexual activity occurring in the spring. Bourne et al. (1971) and Bourne and Seamark (1975) found plasma testosterone titers of this lizard to be three times higher in spring than at other times of the year. Plasma testosterone was totally absent during the summer. Although little information is available on female T. rugosus, it is likely that they follow a similar cycle with respect to ovarian activity (Bourne, 1972). The reproductive function attributed to melatonin in mammals is well known (see review by Reiter, 1977). There is also some evidence that this substance may participate in reproductive activity of lizards. In female anoles (An& carolinensis), pinealectomy was effective in accelerating ovarian follicular development in the fall and winter but not at other seasons (Levey, 1973). Daily injection of 10 pg of melatonin were shown to block this response. Similarly, injections of melatonin at the dose of 0.3 pglg body wtlday, produced antigonadatrophic effects in male Callisaurus
498
FIRTH,
KENNAWAY,
draconoides (Packard and Packard, 1978). While it may be contended that such doses are pharmacological, Rollag et al. (1978a) have indicated that sheep secrete up to 5 mg of melatonimday. The observation that smaller lizards had more plasma melatonin than larger ones may further suggest a link with gonadal function. Fenwick (1970) found that immature salmon had approximately six times as much pineal melatonin as mature salmon. Similarly, Get-n et al. (1978b) reported that young trout have more than twice the amount of plasma melatonin when compared to those six times their body weight. The positive correlation between size and melatonin in the present study may be tenuous since it was evident in only one experiment. Alternatively, it could be a result of the smaller size range and the smaller sample size of lizards used in the other experiments. The normal die1 rhythm in plasma melatonin concentration in T. rugosus was abolished after 9 days in constant light or constant dark. To our knowledge no one has demonstrated a persistent cycle of melatonin content in ectothermic vertebrates under constant lighting and temperature conditions. Hafeez and Quay (1970) found no difference in the activity of the pineal enzyme HIOMT in two species of fish sampled in a light-dark cycle, constant light, and constant dark. Similarly, pineal HIOMT activity in lizards (Scefoporus occidentalis) sacrificed in a lighting cycle (at midday, and 8 hr after “lights off ‘) in constant light and constant dark were not significantly different (Quay et al., 1971). In view of the demonstration of a photoperiod-dependent rhythm in plasma melatonin content in the present study, it appears that HIOMT activity in lizards bears the same nonparallel relationship to melatonin as is true for some birds and mammals (Binkley, 1976). We did not detect a free running rhythm of plasma melatonin content of T. rugosus
AND ROZENBILDS
in constant dark, although such oscillations have been found in the pineal of chickens (Ralph et al., 1974; Ralph et al., 1975), rats (Ralph et al., 1971), and in the serum of chickens (Ralph et al., 1974). In T. rugosus, the observation that there was an “anticipated” rise in plasma melatonin (viz., Expt la) under a light-dark cycle suggests the presence of an endogenous rhythm (Klein, 1978) but it is possible that the four sampling periods employed in constant dark were insufficient to detect it, or that the rhythm faded out rapidly. In addition to the reproductive functions discussed above, there is an increasing body of evidence which suggests that pineal systems of reptiles and other vertebrates are involved in daily and seasonal thermoregulatory adjustments (Ralph, 1975a,b). Furthermore, melatonin has been implicated in these responses in lizards (Firth and Heatwole, 1976), birds (Binkley, 1974; John et al., 1978), and mammals (Arutyunyan et al., 1964; Fioretti and Martini, 1968; Fioretti et al., 1974; Palmer and Riedesel, 1976). Since both light and temperature are important in the synchronization of reproductive cycles of reptiles (Licht, 1972) a thermoregulatory function for the pineal gland and melatonin may not be incompatible with that of reproduction. ACKNOWLEDGMENTS We are grateful to Marilyn Campion for her time in performing the statistical analyses and to Dr. R. F. Seamark for his interest in this study. We also thank Mr. J. S. Turner, Dr. W. Gem, Dr. D. Owens, Dr. M. Packard, and Dr. C. Ralph for critically reading the manuscript, and to Brian Miller for help in collecting lizards. This work was supported by a University of Adelaide Postdoctoral Fellowship (to B.T.F.) and the Sir John Gellibrand Memorial Scholarship (to D.J.K.).
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Blnkley, S., MacBride, S. E., Klein, D. C., and Ralph, C. L. (1973). Pineal enzymes: Regulation of avian melatonin synthesis. Science 181, 273275. Binkley, S., MacBride, S. E., Klein, D. C., and Ralph, C. L. (1975). Regulation of pineal rhythms in chickens: Refractory period and nonvisual light perception. Endocrinology %, 848-853. Bourne, A. R. (1972). “Reproductive Endocrinology of the Viviparous Lizard, Tiliqua rugosa.” Ph.D. thesis, University of Adelaide, Australia. Boume, A. R., and Seamark, R. F. (1975). Seasonal changes in 17 /3-hydroxysteroids in the plasma of a male lizard (Tiliqun rtrgosa). Comp. Biochem.
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Licht, P. (1972). Problems in experimentation on timing mechanisms for annual physiological cycles in reptiles. In “Hibernation and Hypothermia, Perspectives and Challenges” (F. South, ed.), pp. 681-711. American’Elsevier. New York. Minneman, K. P., and Wurtman. R. J. (1975). Effects of pineal compounds on mammals. Life Sci. 17, 1189-1200. Ozaki. Y., and Lynch, H. J. (1976). Presence of melatonin in plasma and urine of pinealectomized rats. Endocrinology 99, $41-644. Ozaki, Y., Wurtman, R. J., Alonso, R., and Lynch, H. J. (1978). Melatonin secretion decreases during the proestrous stage of the rat estrous cycle. Proc. Nat.
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Packard, M. J., and Packard, G. C. (1978). Antigonadatrophic effect of melatonin in male lizards (Callisaurus draconoides). Experientiu 33, 1665. Palmer, D. L., and Riedesel. M. L. (1976). Responses of whole animal and isolated hearts of ground squirrels, Cifellus lateralis to melatonin. Comp. Biochem.
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