1 Zoological Laboratory, State University of Utrecht, Utrecht, The ... In the pineal, the Harderian gland and the retina of the golden hamster ...... Syrian hamster.
Journal&
1Vew'al~
J. Neural Transmission 56, 53-72 (1983)
~
9 by Springer-Verlag 1983
Day/Night Rhythmicity in the Methylating Capacities for Different 5-Hydroxyindoles in the Pineal, the Retina and the Harderian Gland of the Golden Hamster (Mesocricetus Auratus) During the Annual Seasons M . G . M . B a l e m a n s 1, P. P ~ v e t 2, J. v a n B e n t h e m I, C . H a l d a r - M i s r a 2. , I. S m i t h 3, a n d H . H e n d r i k s 1
1 Zoological Laboratory, State University of Utrecht, Utrecht, The Netherlands, 2 Netherlands Institute for Brain Research, Amsterdam, The Netherlands, 3 Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School,: London, England With 9 Figures Received July 26, 1982 Summary In the pineal, the Harderian gland and the retina of the golden hamster the day/night capacity for the synthesis of different methoxyindoles is investigated under natural conditions for one 24-hour period in the months of December, March and June. The amounts of the methoxyindoles and the diurnal rhythms in the synthesis are different in the various months during which the tests were performed. There is a striking increase in the synthesis of melatonin and methoxytryptophol in all three organs in June as compared with December. Equally striking is the high synthesis of methoxytryptamine in the pineal in June, whereas this compound was not formed in the Harderian gland and the retina in this month. Methoxytryptophan synthesis was not observed in June in any of the three organs. Methoxyindoleacetic acid rhythmicity shows a pattern more or less identical to that of melatonin and methoxytryptophol. A high synthesis exists at the end of the June day, but greater fluctuations during the days of the two other months. Acetylmethoxytryptophol is synthesized in the pineal during the * IBRO/Unesco fellow.
0300-9564/83/0056/0053/$ 04.00
54
M.G.M. Balemanset al.
night in March, but in the late afternoon in June. The largest quantity of this compound is found in the Harderian gland in December, in the pineal in March and in the retina in June. The largest amounts of melatonin and methoxytryptophol are synthesized when gonadal weight is largest. The possible correlation between the amounts synthesized, the rhythmicities in synthesis and the reproductive system is discussed. Key words: Pineal, retina, Harderian gland, methoxyindoles. Introduction Mainly due to its seasonal rhythmicity in reproduction the hamster is one of the most interesting and studied animals in pineal research. The testes of male hamsters show atrophy in autumn and winter, a recrudescence in the beginning of spring and activity at the end of spring and in summer (Czyba et al., 1964). These fluctuations in testicular size and function are initiated by short and long photoperiods respectively (Hoffman and Reiter, 1965 a, b). The fact that pinealectomy prevents testicular atrophy in wintertime focussed attention on the pineal as a regulator of this phenomenon (Czyba et al., 1964). The regulating compound is, as yet, unknown. At present, peptides, pteridines and indole derivatives have been considered biologically active components of the pineal. Considering the seasonal rhythmicity of serotonin content (Philo and Rdter, 1980), N-acetyltransferase activity (Illnerova and Vanecek, 1980) and hydroxyindoleO-methyltransferase activity (Wesson et al., 1979; Balemans et al., 1980), indole metabolism seems to be most promising for the study of reproductive seasonal rhythmicity. Chronic melatonin (Reiter et al., 1977, 1978) or methoxytryptamine (P&et and Haldar-Misra, 1982 a) treatment of hamsters in the sexual quiescent period prevents testicular atrophy, whereas melatonin (Tamarkin et al., 1976; Reiter et al., 1976) and methoxytryptamine injections in the late afternoon during long photoperiods inhibit gonadal activity (Pdvet et al., 1981 a, b). Besides this discrepancy between stimulation and inhibition by two different methoxyindoles at different times of the year, yet another is present. When investigating pineal melatonin content in relation to its circadian light/dark rhythmicity, its concentration strikingly shows a peak late in the dark period (Panke et al., 1978, 1979, 1980; Rollag et al., 1980 b) independent of the lengths of the artificial light/dark periods. However, to obtain an inhibitory effect on the gonads it is necessary to inject melatonin (Reiter et al., 1976; Tamarkin et al., 1976; Bridges et al., 1976) or methoxytryptamine (P&et et al., 1981 a, b) either in the late afternoon, or late in the dark period (Tamarkin et al.,
Day/Night Rhythmicityin the MethylatingCapacities
55
1977 a, b). At all other times of the day this effect does not show (Tamarkin et aL, 1977 a). Thus, it is observed either in the late afternoon when little or no melatonin is present in the pineal, or late in the dark phase, when a peak of pineal melatonin content is observed. Furthermore, melatonin injected in the morning during long photoperiods prevents the effects ofmelatonin (Richardson et al., 1981; Chen et aL, 1980) or methoxytryptamine (Pdvet and Haldar-Misra, 1982 b) administered in the late afternoon. However, melatonin injected in the morning during short photoperiods retards testicular regression in hamsters, although this result is not observed if the injection is given in the afternoon (Turek and Pappas, 1980). Identical results have been obtained with methoxytryptamine (P&et and Haldar-Misra, 1982 c). Moreover, changing the light/dark schedule (10 L-14 D to 14 L--10 D), which causes marked changes in testicular weight, has no effect on pineal melatonin rhythmicity, amplitude or pineal content (Tamarkin et al., 1979). The question therefore arises what the significance ofmelatonin, and/or other methoxyindoles synthesized in the pineal is in the regulation of seasonal reproductive rhythmicity. Another question is whether or not the indole metabolism in the Harderian gland and the retina are involved in this rhythmicity. Therefore, we investigated the HIOMT capacity for synthesizing different methoxyindoles in the pineal, retina and Harderian glands of hamsters kept under nearly natural conditions during March, June and December. Material and Methods 3-H-S-adenosyl-methionine was obtained from The Radiochemical Centre Ltd., Amersham, England. Synthetic methoxyindoles were run as markers in the TLC experiments. Methoxytryptophan (MW), methoxytryptamine (MT), melatonin (aMT), methoxyindoleacetic acid (MA) and methoxytryptophol (ML) were obtained from Sigma Chemical Company, U.S.A. while acetylmethoxytryptophol (aML) was synthetized by us. The abbreviations are those introduced by Smith in Balemans (1981). Experiments were performed using young adult male hamsters obtained from TNO, Zeist, The Netherlands, weighing 60-70 g on the day of arrival (see Table 1). For experiment I the animals were placed immediately outdoors under natural conditions of light and temperature; food and tap-water were administered ad libitum. For experiments II and III the animals were first acclimatized for two or four weeks (Table 1) in a room under ambient temperature and light conditions, but protected from wind and snow. After this period of acclimatization the animals were placed outdoors in a cage containing hay for hibernation. Here they remained until the end of the
56
M. G. M. Balemans et al. Table 1. General data
Exp. I Exp. II Exp. III
N
Day of arrival
Date placed outside
End of experiment
27 40 41
11. 6.1980 5.11.1980 7. 1.1981
11. 6.1980 20.11.1980 7. 2.1981
18./19.12.1980
12./13. 3.1981 4./ 5. 6.1981
experiments. The light/dark periods, the degrees of sunheight and the temperature are shown in Figs. 1, 2 and 3, respectively. With respect to the critical timing regarding gonadal atrophy or recrudescence it must be considered that differences of 12 hours between sunrise and sunset occur on September 25th, 1980 and March 18th, 1981. As twilight could not be indicated although this may have been of influence, the present data should be considered a general indication and not exact critical points. In the three experiments performed the sun's altitude is of most importance. The first experiment has been done during decrease of the sun's altitude, the third experiment during sunrise and the second experiment at sunset. The consequences o f the position of the sun on light intensity and spectral changes may be important in studying seasonal reproductivity. The mean of the minimum and maximum temperatures (Fig. 3) is indicated over a 10-day period. Lowest temperatures were measured on November 3rd, 1980 (-4.9 ~ December 7th, 1980 (-10 ~ and February 21st, 1981 (-6.9 ~ Highest temperatures were measured on July 26, 1980 (29 ~ and May 20st, 1981 (27.2 ~
24
Natural light/dark period
22 20 18 16. 14 12 I0 8 6 4 2
Fig. 1. Sunrise and sunset from June 1,1980, until July 1,1981. Middle European summertime is used in the Netherlands (before September 27th and after March 29th, 1981). Note therefore the difference of one hour during these periods. Twilight is not indicated. The duration of the three experiments is indicated by the horizontal lines at the bottom of the figure, the months of the year by capital letters
Day/Night Rhythmicity in the Methylating Capacities
57
At the end of the experiments the hamsters were sacrificed by decapitation. The superficial pineal (here termed pineal), the retinae and the Harderian glands were quickly extirpated, frozen in liquid nitrogen and conserved at - 8 0 ~ until they were processed. During darkness the animals were killed in darkness, but the organs were dissected in light. Degrees 65_ 60_ 55 50_ 45 40 35 30 25 20_ 15 10 5
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Fig. 2. The sun height from June 1,1980,until July 1,1981,in Amsterdam, The Netherlands. The duration of the three experiments is indicated by the horizontal lines, the months of the year by capital letters oC
Fig. 3. Minimum and maximum temperature in a 24-hour period (mean of 10 days) from June 1,1980, until July 1,1981.The duration of the three experiments is indicated by the horizontal lines, the months of the year by capital letters
58
M.G.M. Balemans et aL
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Day/Night Rhythmicity in the Methylating Capacities
59
The capacity of HIOMT to synthesize the different methoxyindoles was investigated by the method ofBalemans et al. (1978). This allows for the determination of the methylation of all hydroxyindoles present in the pineal, the retina and the Harderian gland. HIOMT capacity was investigated in the entire pineal and in both retinae. The Harderian gland was cut into pieces of about 6 mg. Several pieces of different parts of the glands were investigated and calculated for the weight of the two glands. No extra substrates were added to the incubation medium. Thus, only the capacity of HIOMT to synthesize the methoxyindoles of which the precursors are present in the pineal, the retina or the Harderian gland, Can be investigated by this method. The slightly disrupted pineals, retinae or pieces of 6 mg of the Harderian gland were incubated in 20/A 0.1M phosphate buffer (pH 7.9) and 10/al of 3H-S-adenosyl methionine containing 1.0/aCi in HeNO 4 (pH 2.5) at 37 ~ for 60 min. Separation of the methoxyindoles was performed by bidimensional thin layer chromatography. The spots of methoxytryptophan, methoxytryptamine, methoxyindoleacetic acid, melatonin, methoxytryptophol and O-acetylmethoxytryptophol were scraped and the radioactivity of the scrapings determined.
Results The results obtained are shown in Tables 2 and 3 and Figures 4 - 9 . Specific correlations will be referred to in the discussion. O n l y the main points will be m e n t i o n e d here. Most striking is the fact that the Harderian gland synthesize 10-50 times more o f practically all methoxyindoles w h e n compared with the pineal gland and the retina. This holds for the whole year for aMT, ML, aML and MA. However, in wintertime M W and M T synthesis are very high in the Table 3. Increase (+), no change 0 or decrease (-) in 5-methoxyindole synthesis compared with the mean annual values in the Harderian gland, the retina and the pineal during the months in which the tests were performed
Methoxytryptophan Methoxytryptamine Acetylmethoxytryptophol Methoxyindoleacetic acid Melatonin Methoxytryptophol Testicular weight/g
Dec.-,March
March+June
June-~Dec.
HG
E
PG
HG
E
PG
HG
E
PG
---+ + +
---
+ +
+ + + ----
+ +
+ --
+ +
-+ + + +
+ + -
+ + +
0.601+ 0.035 -+0.996 _ 0.011 ~ 3.206 _+ 0.008 -+0.601 + 0.035 (Dec.) (March) (June) (Dec.)
HG
E
PG
T
20OO0 18000
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