Circadian pattern of plasma melatonin concentrations in the marmoset monkey (Callithrix jacchus).код для вставкиСкачать
American Journal of Primatology 17:73-79 (1989) BRIEF REPORTS Circadian Pattern of Plasma Melatonin Concentrations in the Marmoset Monkey (Callithrixjacchus) G.E. WEBLEY', D.H. ABBOTT', L.M. GEORGE1,J.P. HEAR", AND H. MEHL2 'MRCIAFRC Comparative Physiology Research Group, Institute of Zoology, London; ZInstitute of Hormone and Fertility Research, Hamburg, Federal Republic of Germany This study describes the concentrations of melatonin in plasma samples taken from marmoset monkeys (Cullithrix jucchus) every 4 h over three 24-h periods. A circadian pattern of secretion was apparent, with higher levels recorded a t night (20.00-08.00 h) than during the day (08.00-20.00 h) and a peak concentration at 20.00 h. There was a significant difference in the mean day and night concentrations (32.5 2 4.5 pg/ml versus 49.0 k 6.9 pg/ml, respectively) with individual concentrations ranging between <lo-60 pglml in the day and 15-200 pg/ml a t night. Circadian plasma melatonin concentrations were similar over the three 24-h periods, in male (n = 3) and female (n = 3) monkeys, and in dominant (cyclic, n = 5 ) and subordinate (acyclic, n = 4) females. The results show a less pronounced circadian profile in the marmoset than is seen in the human but a similar profile to that in the seasonally breeding rhesus monkey. Key words: marmoset monkey, melatonin, social status INTRODUCTION In all animals so far investigated the pineal hormone melatonin follows a circadian pattern of secretion [Tamarkin et al., 19851. In seasonally breeding species such as the sheep this circadian pattern of melatonin is believed to mediate the influence of seasonal changes in photoperiod on reproductive cyclicity, acting on the luteinizing hormone-releasing hormone (LHRH) pulse generator [Bittman et al., 19851 through two neuroendocrine mechanisms. One mechanism involves a change in sensitivity to the negative feedback action of estradiol [Bittman et al., 1983; Legan et al., 19771, while the other is independent of sex steroids and involves the endogenous opioid peptides [Yang et al., 19881. Among primates, the role of melatonin is less established, but the human shows a clear circadian pattern in melatonin concentrations [Arendt, 1985; Webley et al., 1985; Webley & Leidenberger, 19861. The rhesus monkey, a strict seasonal breeder, also exhibits a Received for publication June 23, 1988; revision accepted September 21, 1988. Address reprint requests to Dr. G.E. Webley, Institute of Zoology, Regent's Park, London NW14RY, UK. 0 1989 Alan R. Liss, Inc. 74 I WebIey et ai. circadian change in plasma [Jenkin et al., 19801 and cerebrospinal fluid [Reppert et al., 19791 concentrations. The objectives of the present study were to establish whether there is a circadian pattern of melatonin in the marmoset monkey, a primate species that breeds throughout the year both in captivity and in the wild [Hearn, 1983; Stevenson, 19781; to establish the physiological levels of circulating melatonin; and to examine any gross changes in the circadian pattern of melatonin in dominant (cyclic) compared with subordinate (acyclic)female monkeys. MATERIALS AND METHODS Animals Adult male and female marmoset monkeys (Callithrixjacchus) were from the self-sustaining breeding colony maintained a t the Institute of Zoology as described previously [Hearn, 19831. The monkeys were housed either as breeding family groups or groups of unrelated adults (social groups). When females are housed in social groups one will become dominant and continue to have regular ovarian cycles whereas the other females become subordinate and acyclic [Abbott & Hearn, 1978; Abbott et al., 19881. Blood samples were taken from the femoral vein without sedation, as described previously [Hearn, 19773, every 4 h over 24 h starting at 08.00 h. A preliminary study was carried out in May 1987 with three female and three male marmosets. In a subsequent study five dominant and four subordinate female marmosets were sampled over two 24-h periods, 2 weeks apart, on June 30lJuly 1 and July 14/15 1987. In the first of these two 24-h periods, two of the dominant females were in the luteal phase and three were in the follicular phase-this was reversed during the second 24-h study. The male and female marmosets studied in May were maintained under conditions of natural light, supplemented with artificial light from 08.00 t o 20.00 h (dark phase from 20.00 h to 06.00 h). In the study of dominant and subordinate females, the animals were solely maintained under conditions of artificial light with a dark phase from 20.00 h to 08.00 h. In all studies the blood samples collected during the dark phase were taken under dim red light during the 1min required to obtain the sample. The start of the ovarian cycle was controlled by administration of a single intramuscular (i.m.) injection of 0.5 pg cloprostenol (Estrumate; ICI Pharmaceuticals Division, Maccclesfield, Cheshire) [Summers et al., 19851to synchronize the cycles and to ensure that the dominant females were studied in the follicular and luteal phases. The ovarian cycles were monitored by measurement of plasma progesterone concentrations, 2 to 3 times per week, as the marmoset monkey does not menstruate and vaginal smear cytology does not accurately reflect the stage of the ovarian cycle [Hearn & Renfree, 19751.The ovarian cycle of the marmoset is 28 days with an 8- to 9-day follicular phase and a 19- to 20-day luteal phase [Harlow et al., 19831. Assays Melatonin was measured using a direct tritiated radioimmunoassay as described previously [Webley et al., 19851. Plasma and serum samples gave similar values in the assay (data not shown) demonstrating that there was no effect of the concentration of heparin used (surface coating only) in taking the blood sample. Plasma volumes of 100 p1, made up to 250-p1 sample volumes with assay buffer, were assayed in duplicate. Plasma samples diluted in parallel to the standard curve, a plasma pool, from male marmosets, assayed at volumes of 200 pl, 100 pl, Melatonin in the Marmoset Monkey I 75 and 50 p1 gave values of 49,47.8, and 52 pglml, respectively. Recovery of melatonin added to a plasma pool a t concentrations of 20 pg/ml and 40 pg/ml gave recoveries of 101%to 111%.The assay had a sensitivity of 2.0 pg/ml and intra- and interassay coefficients of variation of <6% and <16%, respectively. Progesterone concentrations in marmoset plasma were measured using a direct enzyme immunoassay as described previously [Hodges et al., 19881. The sensitivity of the assay was 1.1 ng/ml, and intra- and interassay coefficients of variation were <9% and <16%, respectively. Analysis of Results A log transformation of plasma melatonin concentrations was carried out to increase linearity of the data and to reduce the heterogeneity of variance [Helwig & Council, 1979; Sokal & Rohlf, 19811. Plasma melatonin values from the study in May were subjected to a two-way analysis of variance for repeated measures. No significant difference was found between the sexes so the data for both sexes were combined and subjected to a one-way analysis of variance. Plasma melatonin values in the June/July study were initially put through a nested three-way analysis of variance with replication [Sokal & Rohlf, 19811. As no significant difference was found between the days, the data were combined and subjected to a two-way analysis for repeated measures to test for differences between females over the 24-h period. For this analysis four outlying melatonin values of 140, 150, 170, and 205 pg/ml from three animals were excluded. These values greatly exceeded the overall mean (L SD) of 32.0 k 29.7 pmol/liter and were identified by an outlier test [Sokal & Rohlf, 19811. In all cases comparison of individual transformed means were made post hoc using Duncan’s Multiple Range test [Helwig & Council, 19791. To determine whether there was any change in the total exposure to melatonin over 24 h (melatonin index), as described previously in women [Webley & Leidenberger, 19861, the area under the melatonin curve over 24 h was calculated, using the nadir in melatonin concentrations as the baseline. The melatonin index was compared between days and a t different stages in the cycle using Student’s paired t test. RESULTS The plasma concentrations of melatonin in the marmoset monkey showed a circadian pattern of secretion with significantly higher levels recorded during the hours of darkness (F(6,35) = 2.59, P < .035). A peak concentration of 49.0 6.9 pg/ml (antilog of transformed mean ? SEM) was recorded a t 20.00 h. There was no significant difference in the melatonin concentrations between males and females (F(1,4) = 1.43, P < .3) so the data were combined and are illustrated in Figure 1. The difference in the mean melatonin concentrations over 24 h was not marked, changing by only 50%. There was, however, individual variation, which is also evident from the circadian profiles of melatonin shown in Figure 2 for the dominant and subordinate females. The melatonin concentrations recorded over both May and June/July studies ranged between <10 and 60 pg/ml during the day and 15 to 200 pg/ml during the night. There was no significant difference in the plasma melatonin concentrations between the two 24-h periods in June and July (F(1,2) = 00.31, P > .05), so the data for both days were combined. There was also no difference in melatonin concentrations between any of the three female groups: dominant females (follicular phase), dominant females (luteal phase) and acyclic subordinate females (F(2,ll) = 00.02, P > .97). Combining all data from dominant and subordinate * 76 I Webley et al. 1 0800 1 . . 12.00 ICKX) - m 1 2ooo 2400 W ocoo v 00 Time (h) -40 z B i A 2 Fig. 1. Concentrations of melatonin (mean ? confidence limits) in plasma samples taken every 4 h over 24 h from three female and three male marmoset monkeys. The data are expressed as the antilog of the transformed means with 95%confidence limits. *P < .05 vs. 12.00 and 08.00 h (day 2) (Duncan’s multiple range test). The dark period ended at 06.00h, 2 h before the artificial lights were switched on, because of natural daylight supplementation. Fig. 2. Concentrations of melatonin (mean -C confidence limits) in plasma samples taken every 4 h over 24 h from five dominant (cyclic) females and four subordinate (acyclic) female marmoset monkeys. The data are expressed as the antilog of the transformed mean with 95%confidence limits. a, P < .05 vs b and c; b, P < .05 vs. a and c; c, P < .05 vs. a and b (Duncan’s multiple range test). Melatonin in the Marmoset Monkey I 77 females gave a circadian pattern of plasma melatonin concentrations with significantly higher values recorded at 20.00 h than a t other times of day (F(6,64) = 4.29, P < .0011) (Fig. 2). Calculation of the melatonin index for the individual 24-h profiles showed that there was no significant difference in the values for the follicular (52 f. 14 pgday, mean 2 SEM, n = 5,) and luteal phase (104 2 40 pg.day, n = 5) of the cycle in dominant females and no significant difference between dominant (78 rt 22 pgday, n = 10) and subordinate females (104 2 23, pg.day, n = 8). DISCUSSION This study describes a circadian pattern of melatonin in the marmoset monkey with significantly higher plasma concentrations recorded a t night (20.00-08.00 h) than during the day (08.00-20.00 h). The pattern is similar to that described in the human [Arendt, 1985; Webley et al., 1985; Webley & Leidenberger, 19861 and rhesus monkey [Jenkin et al., 19801 but differs in the timing of the peak concentration: a peak melatonin level recorded a t 20.00 h in the marmoset monkey, 04.00 h in the human [Webley et al., 19851 and 05.30 h in the rhesus monkey [Jenkin et al., 19801. The earlier peak level in the marmoset may be related to activity since the animals habitually enter their nestboxes to sleep a t between 18.00 and 19.00 h. A relationship between the excretion pattern of melatonin and the activity pattern has been described in the hamster [Korenman et al., 19881. A variation in concentrations of melatonin of up to twofold in the marmoset is similar to that described in the rhesus monkey in which mean levels ranged from 115 to 368 pmolfliter [Jenkin et al., 19801. In contrast the difference in mean daylnight levels was considerably greater in the human with an increase from between 20 and 200 pmol/liter in the day to up to 850 pmol/liter a t night [Webley et al., 1985; Webley & Leidenberger, 19861. The marmoset monkeys were exposed to a similar light/dark cycle to that in their equatorial habitat. We demonstrated previously the ability of melatonin to stimulate the secretion of progesterone by the corpus luteum of the marmoset monkey when perfused in vivo [Webley 8z Hearn, 1987; Hearn & Webley, 19871. The present study confirms that the concentrations of melatonin tested were within the physiological range of concentrations in the peripheral circulation. An association between elevated melatonin levels and the luteal phase was suggested from our study of peripheral melatonin concentrations in women [Webley & Leidenberger, 19861 in which there was an increase in the circadian output (melatonin index) during the luteal phase of the menstrual cycle. In the present study, it was not possible, from the number of dominant animals investigated at any particular stage of the cycle, to establish whether any such changes were occuring across the cycle in the marmoset. Experiments are now underway in vivo to test the involvement of melatonin as part of the luteotrophic complex in the marmoset and human. The possibility that changes in the circadian pattern of melatonin might be associated with the socially induced acyclicity in subordinate female marmosets was not supported by our results. Earlier studies in our laboratory showed that the immediate cause of acyclicity was suppressed pituitary LH secretion associated with impaired GnRH secretion [Abbott, 1987, 19881. Two components of the GnRH/LH suppression were identified, and these were similar to the mechanisms controlling LH secretion during seasonal anoestrus in the sheep [Legan et al., 1977; Yang et al., 19881. One is manifest as an increase in sensitivity to estradiol negative feedback [Abbott, 19881, and the other is apparently independent of ovarian negative feedback and involves the endogenous opioid peptides [Abbott, in 78 I Webley et al. press]. In the sheep changes in the duration of the nocturnal elevation of melatonin have been shown to mediate the influence of photoperiod on changes in the LH secretion [Bittman & Karsch, 19841 by their actions on hypothalamic GnRH secretion [Robinson et al., 19861. The results in the present study suggest that normal changes in the circadian pattern of melatonin are not perturbed in the acyclic, subordinate marmoset and thus appear to play no part in the social suppression of GnRH/LH secretion. CONCLUSIONS 1. The marmoset monkey shows a circadian pattern of plasma melatonin concentrations. 2. Night concentrations of melatonin (mean 49.0 6.9 pg/ml) significantly exceeded mean concentrations during the day (32.5 t 4.5 pg/ml). 3. There was no significant difference in plasma melatonin concentrations, over the circadian profile, in dominant (cyclic) and subordinate (acyclic) female marmoset monkeys. * ACKNOWLEDGMENTS We thank Professor F.A. Leidenberger for his interest and support. The work was funded by a joint MRC/AFRC programme grant (to J.P.H.). REFERENCES Abbott, D.H. Behaviourally mediated suppression of reproduction in female primates. JOURNAL OF ZOOLOGY 213:455470,1987. Abbott, D.H. Natural suppression of fertility. Pp. 7-28 in REPRODUCTION AND DISEASE IN CAPTIVE WILD ANIMALS. J.P. Hearn; G.R. Smith, eds. Oxford, Oxford University Press, 1988. Abbott, D.H. Social conflict and reproductive suppression in marmoset and tamarin monkeys. Pp. in PRIMATE SOCIAL CONFLICT. W.A. Mason; S.P. Mendoza, eds. New York, Alan R. Liss, Inc., in press. Abbott, D.H.; Hearn, J.P. Physical, hormonal and behavioural aspects of sexual develoDment in the marmoset monkey (CaZliijtrix jacchus). JOURNAL OF REPRODUCTION AND FERTILITY 53:155166, 1978. Abbott, D.H.; Hodges, J.K.: George, L.M. Social status controls LH secretion and ovulation in female marmoset monkeys (Callithriz jacchus). JOURNAL OF ENDOCRINOLOGY 117~329-339,1988. Arendt, J. Mammalian pineal rhythms. PINEAL RESEARCH REVIEWS 3:161-170, 1985. Bittman, E.L.; Karsch, F.J. Nightly duration of pineal melatonin secretion determines the response to daylength in the ewe. BIOLOGY OF REPRODUCTION 30: 585493,1984. Bittman. E.L.: Karsch. F.J.: Honkins. J.W. Role of the pineal gland in oviAe photoperiodism: Regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. ENDOCRINOLOGY 113:329-336, 1983. Bittman, E.L.; Kaynard, A.H.; Olster, D.H.; Robinson, J.E.; Yellon, S.M.; Karsch, F.J. Pineal melatonin mediates photoperiodic control of pulsatile luteinizing hormone secretion in the ewe. NEUROENDOCRINOLOGY 40:409-418,1985. Harlow, C.R.; Gems, S.; Hodges, J.K.; Hearn, J.P. The relationship between plasma progesterone and the timing of ovulation and early embryonic development in the marmoset monkey (Callithriz jacchw). JOURNAL OF ZOOLOGY 201: 273-282,1983. Hearn, J.P. A device for restraining small monkeys. LABORATORY ANIMALS 11: 261-262,1977. Hearn, J.P. The common marmoset (Callithr& jacchus). Pp. 181-215 in REPRODUCTION IN NEW WORLD PRIMATES. J.P. Hearn. ed. Lancaster. MTP Press, 1983. Hearn, J.P.; Renfree, M.B. Prealbumins in the vaginal flushinas of the marmoset monkey, Callithrizja&us. JOURNAL OF REPRODUCTION AND FERTILITY 43: 159-161, 1975. Hearn, J.P.; Webley, G.E. Regulation of the corpus luteum of early pregnancy in the marmoset monkey: Local interactions of luteotrophic and luteolytic hormones in vivo and their effects on the secretion of progesterone. JOURNAL OF ENDOCRINOLOGY 114:231-239,1987. Melatonin in the Marmoset Monkey I 79 Helwig, J.T.; Council, K.A. SAS USER’S GUIDE, 1979 edition. SAS Institute Inc., Cary, 1979. Hodges, J.K.; Green, D.I.; Cottingham, P.G.; Sauer, M.J.; Edwards, C.; Lightman, S.L. Induction of luteal regression in the marmoset monkey (Callithrix jacchus) by a gonadotrophin-releasing hormone antagonist and the effects on subsequent follicular development. JOURNAL OF REPRODUCTION AND FERTILITY 82:743-752,1988. Jenkin, G.; Mitchell, M.D.; Hopkins, P.; Matthews, C.D.; Thorburn, G.D. Concentrations of melatonin in the plasma of the rhesus monkey (Macaca nuluttu). JOURNAL OF ENDOCRINOLOGY 84:489-494, 1980. Korenman, E.M.D.; Watson, B.W., Silman, R.E. The pattern of motor activity, light and melatonin production in syrian hamsters. JOURNAL OF PINEAL RESEARCH 499-106,1988. Legan, S.J.; Karsch, F.J.; Foster, D.L. The endocrine control of seasonal reproductive function in the ewe: A marked change in response to the negative feedback action of oestradiol on luteinising hormone secretion. ENDOCRINOLOGY 101:818-824, 1977. Reppert, S.M.; Perlow, M.J.; Tamarkin, L.; Klein, D.C. A diurnal melatonin rhythm in primate cerebrospinal fluid. ENDOCRINOLOGY 104:295-301,1979. Robinson, J.E.; Kaynard, A.H.; Karsch, F.J. Does melatonin alter pituitary responsiveness t o gonadotropin-releasing hormone in the ewe? NEUROENDOCRINOLOGY 43: 635-640,1986. Sokal, R.R.; Rohlf, F.J. BIOMETRY. THE PRINCIPLES AND PRACTICE OF STATISTICS IN BIOLOGICAL RESEARCH. New York, W.H. Freeman & Co., 1981. Stevenson, M.F. The behaviour and ecology of the common marmoset (Callithrix jacchus jacchus) in its natural environment. Pp. 278 in BIOLOGY AND BEHAVIOUR OF MARMOSETS. H. Rothe; H.J. Wolters; J.P. Hearn, eds. Gottingen, Eigenverlag Hartmut Rothe, 1978. Summers, P.M.; Wennink, C.J.; Hodges, J.K. Cloprostenol-induced luteolysis in the marix JOURmoset monkey ( C ~ l l i t h ~jacchus). NAL OF REPRODUCTION AND FERTILITY 73:133-138, 1985. Tamarkin, L.; Baird, C.J.; Almeida, O.F.X. Melatonin: A coordinating signal for mammalian reproduction? SCIENCE 227:714720,1985. Webley, G.E.; Leidenberger, F. The circadian pattern of melatonin and its positive relationship with progesterone in women. JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM 63~323328,1986. Webley, G.E.; Hearn, J.P. Local production of progesterone by the corpus luteum of the marmoset monkey in response to perfusion with chorionic gonadotrophin and melatonin in uiuo. JOURNAL OF ENDOCRINOLOGY 112:449-457, 1987. Webley, G.E.; Mehl, H.; Willey, K.P. Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species. JOURNAL OF ENDOCRINOLOGY 106:387-394,1985. Yang, K.; Haynes, N.B.; Lamming, G.E.; Brooks, A.N. Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons. JOURNAL OF REPRODUCTION AND FERTILITY 83:129-139,1988.