Photoperiod, Pineal, Melatonin and Reproduction in Hamsters

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Photoperiod, Pineal, Melatonin and Reproduction in Hamsters KLAUS HOFFMANN

Max-Planck-Institutefur Verhaltensphysiologie,8131 Andechs (F.R.G.)

In many mammalian species, especially in those living in medium or higher latitudes, a marked annual cycle in reproductive activity has been observed, often accompanied by distinct structural changes (for reviews see Aschoff, 1955; Sadleir, 1969; Chapman, 1970; Lodge and Salisbury, 1970). Figure 1 shows the seasonal changes occurring in the Djungarian hamster. There is a clear annual cycle in testicular weight and in tubular diameter. The weight changes in accessory glands and in the ventral marking gland can be considered an indirect indicator of changes in androgen levels. The histological appearance of the testes closely follows testis weight and tubular diameter, full spermatogenesis is only observed in summer (Fig. 2). Corresponding seasonal variations are found in the females of this species. Such an annual cycle can be considered an adaptation to the seasonal changes in the environment, assuring that birth of young and juvenile development are restricted to the favourable seasons.

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Fig. 1. Annual cycle of (left) testis weight (both testes combined), tubular diameter, pelage colour (% of animals in full summer pelage) and (right) weight of accessory glands, ventral marking gland and body weight in male Djungarian hamsters. Mean and standard error of 15 animals for each point. The hamsters were maintained at constant temperature, but exposed to the natural illumination and its changes.

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Fig. 2. Microscopic appearance of testes of Djungarian hamsters. u, In summer (full spermatogenesis); b , in autumn (regression); c in winter (maximal regression), d in early spring (recrudescence). Mayer’s haemalum, S-rtm sections.

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Not only the reproductive system, but also other functions may show such seasonal changes. Figure 1 also demonstrates a marked annual cycle in body weight with a decrease of about 30% in winter. This weight cycle is only partially controlled by gonadal steroids (Hoffmann, 1978~).In addition, a seasonal change in pelage colour from a brownish summer fur to a whitish winter pelage is indicated (for details see Figala et al., 1973). The change of pelage colour in the stoat is another well-known example. PHOTOPERIODIC PHENOMENA In an increasing number of mammalian species it has been shown that the photoperiod, i.e., the length of the daily light period and its seasonal changes, regulates the annual cycle (for reviews see Aschoff, 1955; Thibault et al., 1966; Farner et al. 1973; Reiter, 1974). The data given in Fig. 1 were obtained in hamsters kept indoors in constant temperature and maintained on a standard diet; however, the animals were exposed to the natural illumination and its changes. This suggests that photoperiod is the regulating factor, as was demonstrated in experiments with artificial photoperiods (Fig. 3). When in summer male Djungarian hamsters are exposed to short photoperiods, testes and accessory glands regress while long photoperiods maintain gonadal size and activity. In winter, at a time when gonads and glands are regressed, exposure to long photoperiods leads to rapid recrudescence (Hoffmann, 1972, 1973, 1974). Corresponding results were obtained for body weight and pelage colour (Hoffmann, 1973, 1977). Similar reactions have been described in other mammalian species whose phase of sexual activity lies in spring or summer. The first pioneering studies were performed by Baker and Ranson (1932) in voles and by Bissonette (1932) in ferrets nearly half a century ago. Winter Nov 29 - J a n 16

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Fig. 3. Weight of testes and accessory glands after exposure to long (LD 16 : 8 = 16 h of light and 8 h of dark per day) and short (LD 8 : 16) photoperiods. In winter long photoperiods cause rapid recrudescence, in summer short photoperiods lead to regression. (After Hoffmann, 1977,1978b.)

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However, in a number of larger species like sheep or deer, sexual activity is restricted to fall or winter, a time when the photoperiod decreases. Here it has been shown that long or increasing photoperiods instigate cessation of sexual activity and anoestrus, while short or decreasing photoperiods hasten full gonadal development (French et al., 1960; Pelletier and Ortavant, 1975; Lincoln and Davidson, 1977; Lincoln et al., 1978). Such species-specific differences in reaction to the photoperiod are understandable if one visualizes that the photoperiod is not the ultimate factor providing the selective pressure that brought about the timing. The photoperiod is only a signal giving the most noisefree information on season and its changes, and it has been exploited for timing of vital functions in organisms as diverse as plants, insects and several vertebrate groups. Since its function is to signal in advance changes to be expected later, it is not surprising that, depending on gestation time and other factors, in some species short photoperiods bring about gonadal regression and cessation of sexual activity, while in others they induce recrudescence of gonads and onset of sexual activity. It should also be mentioned that the photoperiod is not the only factor that can influence the annual cycle. In several cases, strong influences of temperature have been shown (Sadleir, 1969; Frehn and Liu, 1970; Blackshaw, 1977), though sometimes temperature only accentuates the photoperiodic effects (Lynch, 1973). Diet may also influence the annual cycle. Thus, in the vole Microtus montanus chemical signals from the foodplants may initiate as well as terminate the reproductive effort in natural populations (Negus and Berger, 1977; Berger et al., 1977). However, photoperiod is certainly the most widespread and most important regulator of the annual cycle in many mammals. Since a large portion of the experimental work on the photoperiodic mechanism and on pineal involvement in this mechanism has been performed in species with breeding seasons in spring or summer, and

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here especially in hamsters, most of the further discussion w ill be restricted to investigations in two hamster species, the golden hamster (Mesocricetus uurutus) and the Djungarian hamster (Phodopus sungorus). Photoperiodic effects are not only observed in adult mammals. In some species the age at which puberty is reached depends upon the time of birth (Sadleir, 1969; Lincoln and MacKinnon, 1976). In typical cases, animals born early in the breeding season mature rapidly and may reproduce within the same season, while animals born late in the breeding season mature slowly and do not reach puberty until the following spring when the next breeding season starts. This delay of puberty may also be controlled by photoperiod. Figure 4 shows this for the Djungarian hamster. In long photoperiods testicular development is rapid, and smears from the epididymal caudae contain mature spermatozoa from about 35 days of age onward. In short photoperiods, on the other hand, testicular development is arrested for some time, and final testicular size and activity is reached only at an age of about 160-200 days (Hoffmann, 1978a). A similar though less drastic delay of puberty after exposure to short photoperiods has also been described in several vole species (see Hoffmann, 1978a for references). Marked species differences may occur even in related forms. Thus, while in adult golden and Djungarian hamsters photoperiodic manipulations render nearly identical results, drastic differences exist in juveniles. In the golden hamster, sexual development towards puberty is independent of photoperiodic conditions (Gaston and Menaker, 1967; Reiter et al., 1970), while in the Qungarian hamster it is controlled by the photoperiod (see Fig. 4). This difference is not due to differences in development of the pineal gland and its innervation in the two species (Van Veen et al., 1978). THE PHOTOPERIODIC MECHANISM In both hamster species there is a rather distinct critical photoperiod dividing light periods that maintain gonadal activity or cause recrudescence from those that lead to involution (Gaston and Menaker, 1967; Elliott, 1974, 1976; Hoffmann, unpublished). Figure 5 shows the results of experiments in the two species. In the golden hamster the critical photoperiod lies between 12 and 121 h per day, in the Djungarian hamster it is at about 13 h. Such distinct turning points in the reaction to length of the daily photoperiod imply that the animals are able to measure this period rather precisely. The underlying mechanism was analyzed only recently in two mammalian species. For a long time it had been assumed that photoperiodic time measurement was based on an “hourglass” or “interval timer” which determines the length of the light or the dark portion in each daily cycle or the ratio of light to dark (Follet, 1973). Recent work in the golden hamster and in the field vole, however, has shown that photoperiodic time measurement is based on a circadian rhythm of photosensitivity (Elliott et al., 1972; Stetson et al., 1976; Grocock and Clarke, 1974), a mechanism that was suggested for plants by Bunning (1936) more than 40 years ago. The most elegant demonstration of the circadian basis of photoperiodic time measurement in mammals is the work of Elliott (1974, 1976) in the golden hamster. Elliott was able to show that one hour of light daily could either maintain testicular activity, or lead to regression depending on the phase of the circadian cycle into which the light pulse fell. The circadian cycle of locomotor activity was recorded simultaneously. A daily repeated light pulse hitting the time between shortly before onset of activity and 12 h thereafter maintained testicular size and activity or stimulated recrudescence. If the daily light pulse fell within the phase of the running cycle normally corresponding to daytime, the

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Fig. 5. Mean paired testis weight in golden (4) and in Djungarian (b)hamsters after exposure to the photoperiods indicated. Values after 89-96 days (4) or 45 days ( b ) in the corresponding photoperiod. Note the abrupt change from maintenance of testicular size to regression (4, from Elliott, 1976); b , from Hoffmann, unpubl.)

testes regressed. These findings have to be taken into consideration when the underlying physiological mechanism is analysed. Two other points need be mentioned. In both hamster species short photoperiods, leading t o regression, do not maintain this state indefinitely. After several months spontaneous recrudescence occurs in spite of the short photoperiods (Reiter, 1972a; Hoffmann, 1973; Turek et d., 1975b; Ellis and Turek, 1979). Similarly, in juvenile Phodopus short photoperiods supress testicular development for a long time, but finally spontaneous development sets in (Hoffmann, 1978a). Similarly, winter pelage induced by short photoperiods is not maintained forever, but is molted spontaneously at about the time when the gonads have started to recrudesce (Figala et al., 1973; Hoffmann, 1978a). No spontaneous regression has been observed in the two hamster species and a true endogenous circannual cycle seems to be lacking. In longer living mammalian species evidence for the existence of such a cycle accumulates (Gwinner, 1979). In ferrets that were permanently maintained in short photoperiods or were blinded, onset of oestrus as well as termination of oestrus could be observed, albeit rather irregularly and not showing a connection to the change in seasons. These findings suggest that in this species photoperiodic stimuli are not essential for bringing about onset and end of oestrus while they are indispensable for the synchronization with the seasons. Observations in the golden hamster show that there is photorefractoriness to the effect of short photoperiods (Reiter, 1972a). After the gonads recrudesce, a prolonged exposure to long photoperiods is necessary to again render the animals sensitive to the effect of short days. The mechanism underlying the termination of photorefractoriness is also based on a circadian cycle of photosensitivity (Stetson et al., 1977). On the other hand, no photorefractoriness to long photoperiods has been observed (Hoffmann, 1978b). Regression can be interrupted at any stage by exposure to long photoperiods.

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PHYSIOLOGICAL ASPECTS All available evidence suggests that in mammals the light stimuli bringing about the photoperiodic reaction are perceived by the retina. This statement is not trivial, since in birds extraretinal photoreceptors are mainly or exclusively responsible for the photoperiodic effects (Benoit, 1964; Gwinner et al., 1971; Oishi and Lauber, 1973; Menaker and Underwood, 1976). In mammals, however, bilateral enucleation or sectioning of the optic nerves always led to the same result as keeping the animals in continuous dark (Hoffman and Reiter, 1965; Reiter, 1974a; Rusak and Morin, 1976; Herbert et al., 1978). In the golden hamster there are 3 projections from the retina, the primary optic tract, the superior accessory optic tract (no inferior tract has been found in this species) and a retinohypothalamic projection terminating in the suprachiasmatic nuclei (Eichler and Moore, 1974). Recent experiments suggest that the pathway essential for the photoperiodic effect is the retinohypothalamic tract, and that the suprachiasmatic nuclei are an essential component of the photoperiodic mechanism (Rusak and Morin, 1976; Stetson and WatsonWhitmyre, 1976). Figure 6 shows experiments in the golden hamster illustrating these points. As can be seen, blinding leads to regression regardless of the photoperiodic condition, whereas destruction of the primary and accessory optic tracts does not interfere with the photoperiodic response. From these findings it can be concluded that the retinohypothalamic tract is the pathway involved. So far it has not been possible to selectively interrupt this tract without damaging the suprachiasmatic nuclei. And these nuclei are also an integral part of the reaction. As Fig. 6 demonstrates, their destruction prohibits the effect of short photoperiods leading to regression. This cannot be explained by the simultaneous interruption of the retinohypothalamic projection, since destruction of the nuclei has not only the opposite effect of blinding, but even prevents gonadal regression normally brought about by enucleation. A participation of the suprachiasmatic nuclei in the photoperiodic mechanism seems plausible since the photoperiodic effects depend upon a circadian cycle of photosensitivity (see above), and these nuclei are involved in the regulation of rhythmic functions. After bilateral destruction, a large number of circadian cycles are abolished. The present evidence suggests that the suprachiasmatic nuclei are circadian pacemakers in the mammalian brain driving a number of rhythmic functions (Zucker et al., 1976; Moore, 1977, 1978). This

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Fig. 6. Testis sue in golden hamsters after 9 weeks in long (LD 16 : 8) or shoit (LD 2 : 22) photoperiods after different surgical procedures. C, untreated control; BL, bilaterally enucleated;POT/SAOT, lesion of primary and superior accessory optic tract; SCN,destruction of suprachiasmatic nuclei. (Modified after Rusak and Morin, 1975.)

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includes the circadian rhythm of serotonin N-acetyltransferase (NAT) activity in the pineal gland, NAT being the rate-limiting enzyme in melatonin production (Klein, 1973; Axelrod, 1974). Destruction of both suprachiasmatic nuclei prevents the nocturnal rise of NAT activity and thus of melatonin production. The latter effect may be important in the photoperiodic mechanism since the pineal gland and possibly melatonin are involved in the transduction of photoperiodic stimuli. THE PINEAL GLAND It has been demonstrated many times in golden hamsters, first by Hoffman and Reiter (1965), that pinealectomy prevents the gonadal regression normally brought about by short photoperiods or by blinding. Figure 7 shows the same effect in the Djungarian hamster. Here not only the involution of testes but also the molt into winter pelage is prevented by.pinealectomy. No effect is discernible if the operation is performed in hamsters maintained in long photoperiods. The same results ensue if the pineal is left intact but deprived of its sympathetic input by removal of the superior cervical ganglia (Reiter and Hester, 1966). In general, in hamsters gonadal regression by short photoperiods is only obtained if the nervous pathways from the suprachiasmatic nuclei to the pineal are intact and functional (Reiter, 1972b, Reiter and Sorrentino, 1972; Reiter, 1974a, 1978). In the vole Microtus ugrestis, pinealectomy and superior cervical ganglionectomy also prevent gonadal involution in short photoperiods (Charlton et al., 1976). From these and other findings it has been concluded that the pineal inhibits gonadal function, and that this inhibition is intensified by short photoperiods or continuous darkness, while it is overcome by long photoperiods or continuous illumination or by interrrupting the nervous pathways to the pineal. Especially Reiter (1973, 1978) has always maintained that by long photoperiods the animals are “physiologically pinealectomized”. This would imply that the pineal transmits exclusively antigonadotrophic effects. However, it has been shown in several experiments with Djungarian hamsters that the pineal may also be involved in conveying stimulatory effects of long photoperiods (Hoffmann and Kuderling, 1975,1977; Brackmann and Hoffmann, 1977; Hoffmann, 1978b). Figure 8 shows the results of 3 replicate experiments in which male hamsters whose testes had regressed due to exposure to Pelage Colour

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Fig. 7. Effect of pinealectomy in Djungarian hamsters. Testicular regression (left) and molt into winter pelage (right) in short photoperiods are prevented by pinealectomy. (After Hoffmann, 1974, 1977.)

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Fig. 8. Weight of testes and accessory glands in Djungarian hamsters after exposure to long or short photoperiods. Experiments are started with animals whose testes and accessory glands had regressed due to exposure to short natural (above and below) or artificial (middle) photoperiods. Hamsters were pinealectomized (Px), sham-operated (Sh) or left untreated (C) before the beginning of the experiment. Note that the development of testes and accessory glands was significantly delayed in pinealectomized animals in LD 16 : 8. Development in LD 8 : 16 is due to start of spontaneous recrudescence. (From Hoffmann, 1978b.)

short natural or artificial photoperiods were pinealectomized, and then gonadal development was stimulated by long photoperiods. If the effect of long photoperiods was solely to suppress the antigonadotrophic influences of the pineal, pinealectomized hamsters regardless of photoperiod, and control hamsters exposed to long photoperiods, should yield identical results. This was not the case. In all experiments development of testes and accessory glands was slightly but significantly retarded in the pinealectomized animals exposed to long photoperiods. While in golden hamsters no such results have been reported, in female ferrets Herbert (1971) has shown that premature oestrus which,can be brought about by long photoperiods is greatly retarded in animals that were pinealectomized prior to photoperiodic stimulation. From these results I conclude that at least in some photoperiodic mammals the pineal conveys not only the inhibitory effect of short photoperiods, but is also involved, at least partially, in the transduction of the stimulatory effects of long photoperiods. The latter effect is not simply a suppression of antigonadotrophic pineal influences, but a positively stimulating process.

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MELATONIN Two groups of pineal compounds have been discussed in conjunction with the role of the pineal gland in the photoperiodic mechanism of mammals, indolamines and polypeptides. Especially the effect of melatonin has been studied intensively in hamsters. While in early experiments no effect of melatonin was found (Reiter, 1969, 1974b), in more recent investigations drastic effects were reported. Since the halflife of melatonin in the organism is very short, in most of these experiments melatonin was implanted, either in beeswax or in silastic tubing. Figure 9 gives an example. In juvenile Djungarian hamsters in which sexual development is controlled by the photoperiod, implantation of melatonin retarded testicular development as do short photoperiods. The effect was dose-dependent. Similar effects have Testis weight L =8hr

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Fig. 9. Testis weight in young Djungarian hamsters maintained in long (LD 16 : 8) photoperiods from birth on. At 6 days of age the animals received implants of silastic tubing of 1, 2, or 3 cm length filled with melatonin (Mel); empty tubes (Contr), or no implants (U).Right: values for untreated animals raised in short photoperiods (LD 8 : 16). (From Brackmann, 1977.) Fig. 10.Testicular wqight in Djungarian hamsters after implantation of melatonin in beeswax (M) or 6eeswax only (C). Prior to the experiment all animals were kept in natural illumination (see Fig. 1).Above: testes regressed at the beginning of the experiment (Jan. 2), end of experiment after 37 days. Middle: testes large and active at beginning of experiment (July lo), end of experiment after 2 months. Below: testes still large at beginning of the experiment, but first signs of regression noticable (Aug. 22), end of experiment after 1 month. Note that the same treatment has different effects in different seasons. (From Hoffmann, 1977;for method see Hoffmann, 1973.)

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been observed in several experiments with adult Qungarian hamsters. In these cases, implantation of melatonin led to involution of testes in spite of long photoperiods, or prevented or at least retarded recrudescence in such photoperiods (Hoffmann, 1972, 1973; Hoffmann and Kuderling, 1977). Such findings might suggest that melatonin has antigonadotrophic effects. However, results that could be considered demonstrating a progonadotrophic effect of melatonin have also been reported. Figure 10 shows the results of three experiments with adult Djungarian hamsters at 3 different seasons. Prior to each experiment the hamsters had been held under natural illumination. In winter when the gonads were regressed, melatonin inhibited or retarded testicular recrudescence in animals stimulated by long photoperiods. In summer, however, when the gonads were large and functional, melatonin failed not only to induce regression in long photoperiods, but even prevented involution normally brought about by short photoperiods. In late summer, at a time when the testes were still large but first indications of involution could be discerned, melatonin instigated rapid regression in spite of long photoperiods, while in controls testicular size and activity was maintained. In the golden hamster, similarly, prevention of regression or even stimulation of recrudescence in short photoperiods or instigation @fregression in long photoperiods have been described (Reiter et al., 1974,1975; Turek et al., 1975a, 1976a, b; Turek and Losee, 1978). In general the effects of melatonin implantation depended on dose, physiological state of the animal or the phase of its annual cycle, and upon the photoperiodic conditions. Similar results could often be obtained in different laboratories and in different species under otherwise comparable conditions (Hoffmann, 1977). Strong effects of melatonin on gonadal function have also been foundin two mustelid species, the short-tail weasel and the ferret (Rust and Meyer, 1969; Herbert, 1971; Herbert and Klinowska, 1978). At present it is difficult to interpret the conflicting results obtained after implantation of melatonin, and only the following generalizations seem possible, Melatonin may have drastic effects upon gonadal conditions and activity in photoperiodic mammals, while in nonphotoperiodic mammals only slight or no effects are usually found. This is supported by experiments of Turek et al. (1976a) who reported that melatonin implanted in silastic tubing induced testicular regression in spite of long photoperiods in two photoperiodic rodent species, the golden hamster and the grasshopper mouse, but was totally ineffective in two non-photoperiodic species, the laboratory rat and the house mouse. Even in photoperiodic species melatonin can only influence the gonads if they can be influenced by the photoperiod. Thus, melatonin was unable to prevent spontaneous recrudescence in both hamster species when gonadal regression had been obtained by short photoperiods, though it prevented the effect of long photoperiods accelerating this process (Hoffmann, 1973; Turek and Losee, 1978). In juvenile Djungarian hamsters in which short photoperiods can drastically delay gonadal development, implantation of melatonin can also significantly retard this development (see Fig.9), while in golden hamsters in which sexual development in juveniles is independent of the photoperiod, melatonin has no effect (Turek, 1979). In general, thus,' melatonin does not seem to have anti- or progonadotrophic action per se, but its implantation may interfere with, and in some cases mimic, photoperiod effects. Synthesis and release of melatonin in the pineal show a' marked daily cycle, with high values at night time and low values during the day in all species studied so far. Implantation of melatonin and thus sustained release can therefore be considered unphysiological. It is possible that melatonin is the factor responsible for the transduction of the photoperiodic effects, but that not only the amount but also the temporal pattern of melatonin release are important, and that the conflicting picture resulting from experiments with melatonin implantation is due to the unnatural pattern of melatonin availability. Such an assumption is

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Fig. 11. Effect of daily injections of melatonin on testis weight in golden hamsters. a, Time of day of injection; b , effect on testes. Injection in the morning (A) had no effect, the same dosage in the afternoon (B) led to testicular regression; injection of only 2.5 g melatonin in the afternoon (B) led to regression in only 2 of 5 animals. Injections were repeated daily for 7 weeks. (After data in Bridges et al., 1976.)

supported by recent findings of Tamarkin et al. (1976, 1977a, b) and Bridges et al. (1976) in the golden hamster that the effect of daily injections of melatonin depends on the time of day of injection (Fig. 11). When melatonin was injected in the morning no effect ensued, while the same injection in the afternoon caused rapid regression. This and similar findings in general support the hypothesis that not only the amount of available melatonin but also its temporal pattern of release are important. Such a hypothesis is further supported by the fact that all surgical procedures that suppress the effect of short photoperiods upon gonads in the hamster, i.e., destruction of the suprachiasmatic nuclei, superior cervical ganglionectomy, or any interruption of the nervous pathway between the suprachiasmatic nuclei and the pineal, have also been found to prevent the nocturnal rise of serotonin N-acetyl-transferase (NAT) activity and thus of melatonin synthesis, albeit in the rat (Klein, 1973; Axelrod, 1974). Another strong argument for an involvement of the pattern of melatonin formation in the photoperiodic process is the strong light dependence of melatonin production. Light at night-time leads to a rapid decrease of NAT activity and of melatonin secretion, and just one minute of light suffices to produce drastic changes in the pattern of NAT activity (Klein and Weller, 1972; Illnerovi et al., 1979; IlInerovti and VaiiEcek, 1979; VaiilScek and Illnerovti, 1979). All these findings were made in the rat, but they are in good agreement with Elliott’s (1974, 1976) report in the hamster that one hour of light could mimic the effects of long or short photoperiods, depending on the phase of the circadian cycle into which light fell. In the hamster the circadian rhythm of NAT activity is qualitatively similar to that of the rat, though with a much smaller amplitude (Rudeen et al., 1975). Cycles with very short photophases may double the nocturnal maximum of NAT activity as compared to LD 12 : 12 (Rudeen and Reiter, 1977). Further progress will undoubtedly be made once a reliable RIA has been developed for melatonin in hamsters. In sheep, strong differences in the pattern of serum melatonin have been reported in different Seasons and, thus, different photoperiods (Arendt, 1979). The physiological site of action of melatonin is unknown. Most investigators assume that

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Fig. 12. Weight of both testes (above) and accessory glands (below) in hamsters kept in long photoperiods and implanted with melatonin in beeswax (M)or beeswax only (C). The hamsters were pinealectomized (Px), sham-operated (Sh) or left unoperated (U) prior to the experiment. Note that melatonin-implanted animals show lower values in all 3 groups. Before the experiment, hamsters were kept in natural illumination. Time of experiment corresponds to “late summer” in Fig. 10. Significantly lower values in pinealectomized C-animals is due to the fact that the pineal is also involved in transducing stimulatory effects of long photoperiods, see Fig. 8 and p. 404. (After Hoffmann and Kiiderling, 1977.)

it acts somewhere in the hypothalamus. In view of the reported antigonadotrophic effects of pineal compounds other than melatonin, it has been suggested that the main effect of melatonin might be in the pineal itself where it regulates the synthesis and/or release of such compounds (Quay, 1974; Reiter et al., 1976). This view was not supported by experiments with pinealectomized hamsters (Fig. 12). When melatonin implantation led to regression or prevented recrudescence in long photoperiods, the same effects were observed in shamoperated and pinealectomized animals (Hoffmann and Kuderling, 1977: Turek, 1977). Similarly, repeated daily injections of melatonin could bring about testicular regression in pinealectomized golden hamsters (Tamarkin et al., 1977a). CONCLUDING REMARKS This report has nearly exclusively dealt with experiments in male hamsters. It should be noted, however, that corresponding results have been obtained in females in nearly all cases, regardless of whether photoperiodic effects, the results after pineal manipulations or after application of melatonin are considered (Reiter, 1973, 1974a, b, 1978; Reiter et al., 1975; Bridges et al., 1976; Tamarkin et al., 1976; Turek and Campbell, 1979). Only gonadal reactions have been discussed here. However, corresponding changes have been observed in the hormonal state in many cases (Reiter, l973,1974b, 1978; Turek and Campbell, 1979). The recent review by Turek and Campbell (1979) should be consulted on the effects of photoperiodic manipulations on the neuroendocrine-gonadal axis. SUMMARY Many mammalian species show a marked annual cycle of gonadal and other functions. In a number of cases it has been shown that the photoperiod, i.e., the length of the daily

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light cycle and its changes, are involved in the regulation of this cycle. In spring and summer breeders short photoperiods lead to gonadal regression, and long photoperiods stimulate recrudescence, while the opposite may hold for autumn and winter breeders. The photoperiod may also regulate sexual development towards puberty. In two rodent species it has been shown that the mechanism underlying photoperiodic time measurement is a circadian rhythm of photosensitivity. Only one hour of light per day may bring about effects corresponding to those of long or short photoperiods, depending on the phase of the photosensitive cycle with which light coincides. In mammals the photoperiodic signals are perceived by the retina and conveyed to the brain by the retinohypothalamic projection to the suprachiasmatic nuclei. Intact suprachiasmatic nuclei are essential for the effects of short photoperiods. This may be due to the participation of these nuclei in the regulation of circadian rhythms. The pineal has been shown to participate in the transduction of photoperiodic effects of short photoperiods leading to regression and also of long photoperiods stimulating recrudescence. The latter effect is not only a suppression of antigonadotrophic effects from the pineal, but a positive stimulation. The exact role of melatonin in the photoperiodic mechanism and its site of action are still unclear. Strong effects of melatonin application have been found in photoperiodic mammals. Recent experiments suggest that not only the amount of melatonin, but its pattern of synthesis and release may be important in the conveyance of photoperiodic effects. No support for the assumption that the site of action of melatonin is the pineal itself has been found in experiments with pinealectomized animals. REFERENCES Arendt, J. (1979) Radioimmunoassayable melatonin: circulating patterns in man and sheep. In: J. Ariens Kappers and P. PBvet (Us.), The Pineal Gland of Vertebrates including Man (Prog. in Brain, Research, Vol. 52). Elsevier, Amsterdam, pp. 249-258. Aschoff, J. (1955) Jahresgang der Fortpflanzung beim Warmbluter. Studium generale, 8: 742-775. Axelrod, J. (1974) The pineal gland: A neurochemical transducer. Science, 184: 1341-1348. Baker, J. and Ranson, R. (1932) Factors affecting the breeding of the field mouse (Microtus agresris), I. Light.hoc. roy. SOC.Ser. B., 110: 113-322. Benoit, J. (1964) The role of the eye and the hypothalamus in the photostimulation of gonads in the duck.Ann. N.Y.Acad. Sci., 117: 204-215. Berger, P.J., Sanders, E.H., Gardner, P.D. and Negus, N.C. (1977) Phenolic plant compounds functioning as reproductive inhibitors in Microtus montanus. Science, 195: 575-577. Bissonette, T.H. (1932) Modification of mammalian sexual cycles. I. Reactions of ferrets (Atrorius vulgaris) of both sexes to electric light added after dark in November and December. Proc. roy. SOC.Ser. B , 110: 322-336. Blackshaw, A.W. (1977) Temperature and seasonal influences. In: A.D. Johnson, W.R. Gomes and N.L. Van Demark (Us.), The Testis, Vol. 4. Academic Press, New York, pp. 517-545. Brackmann, M. and Hoffmann, K. (1977) Pinealectomy and photoperiod influence testicular development in the Djungarian hamster. Naturwissenschaften, 64: 341-342. Bridges, R., Tamarkin, L.and Goldman, B. (1976) Effects of photoperiod and melatonin on reproduction in the Syrian hamster. Ann. Bwl. anim. Biochem. Bwphys., 16: 399-408. Bunning, E. (1 936) Die endogene Tagesrhythmik als Grundlage der photoperiodischen Reaktion. Ber. dtsch. bot. Ges., 54: 590-607. Chapman, D.I. (1970) Seasonal changes in the gonads and accessory glands of male mammals. Mammal. Rev., 1: 231-248. Charlton, H.M., Grocock, C.A. and Ostberg, A. (1976) The effects of pinealectomy and superior cervical ganglionectorny on the testis of the vole. Microtus agrestis. J. Reprod. Fert., 48: 377-379.

411 Eichler, V.B. and Moore, R.Y. (1974) The primary and accessory optic systems in the golden hamster, Mesocricetus auratus, Acta Anat. (Basel), 89: 359-371. Elliott, J.A. (1974) Photoperiodic Regulation o f Testis Function in the Golden Hamster: Relation to the Circadian System. Ph.D. Thesis, University of Texas at Austin. Elliott, J.A. (1976) Circadian rhythms and photoperiodic time measurement in mammals. Fed. Proc., 35: 2339-2346. Elliott, J.A., Stetson, M.J. and Menaker, M. (1972) Regulation of testis function in golden hamster: A circadian clock measures photoperiodic time. Science, 178: 771-773. Ellis, G.B. and Turek, F.W. (1979) Time course of the photoperiod-induced change in sensitivity of the hypothalamic-pituitary axis to testosterone feedback in castrated male hamsters. Endocrinology, 104: 625-630. Farner, D.S., Lewis, R.A. and Darden, T.R. (1973) Photoperiodic control mechanisms: homoiothermic animals. In: P.L. Altman and D.S. Dittmer (Eds.), Biology Data Book, Vol. I1 2nd edition. Fed. Amer. SOC.Exp. Biol., Bethesda, MA, pp. 1047-1052. Figala, J., Hoffmann, K. and Goldau, G. (1973) Zur Jahresperiodik beim Dsungarischen Zwerghamster Phodopus sungorus Pallas. Oecologia, 12: 89-1 18. Follet, B.K. (1973) Circadian rhythms and photoperiodic time measurement in birds. J. Reprod. Fertil., SUPPI.1 9 , ~5-18. ~ . Frehn, J.L. and Liu, C.C. (1970) Effects of temperature, photoperiod, and hibernation on the testes of hamsters. J. exp. Zool., 174: 317-324. French, C.E., McEwen, L.C., Magruder, N.D., Radar, T., Long, T.A. and Swift, R.W. (1960) Responses of white-tailed bucks to added artificial light. J. Mammal., 41: 23-29. Gaston, S. and Menaker, M. (1967) Photoperiodic control of hamster testis. Science, 158: 925-928. Grocock, C.A. and Clarke, J.R. (1974) Photoperiodic control of testis activity in the vole, Microtus agrestis. J. Reprod. Fert., 39: 337-347. Gwinner, E. (1979) Cicannual rhythms. In: J. Aschoff (Ed.) Handbook of Behavioral Neurobiology, Vol. 5 (Biological Rhythms). Plenum Publ. Corp., New York, In press. Gwinner, E., Turek, F.W. and Smith, S.D. (1971) Extraocular light perception in photoperiodic responses of the white-crowned (Zonotrichia leucophrys) and of the golden-crowned sparrow (Z. atricapilla). Z. Vergl. Physiol., 75: 323-331. Herbert, J. (1971) The role of the pineal gland in the control by light of the reproductive cycle of the ferret. In: G.E.W. Wolstenholme and J. Knight (Eds.): The Pineal Gland (A Ciba Foundation Symposium). Churchill Livingstone, Edinburgh, pp. 303-327. Herbert, J. and Klinowska, M. (1978) Daylength and the annual cycle in the ferret (Mustela furo): the role of the pineal body. In: I. Assenmacher and D.S. Farner (Eds.), Environmental Endocrinology. Springer-Verlag, Berlin, pp. 87-93. Herbert, J., Stacey, P.M. and Thorpe, D.H. (1978) Recurrent breeding seasons in pinealectomized or optic-nerve-sectioned ferrets. J. Endocr., 78: 389-397. Hoffman, R.A. and Reiter, R.J. (1965) Pineal gland: Influence on the gonads of male hamsters. Science, 148: 1609-1611. Hoffmann, K. (1972) Melatonin inhibits photoperiodically induced testis development in a dwarf hamster. Naturwissenschaften, 59: 21 8-21 9. Hoffmann, K. (1973) The influence of photoperiod and melatonin on testis sue, body weight, and pelage colour in the Djungarian hamster (Phodopus sungorus). J. comp. Physiol., 85: 267-282. Hoffmann, K. (1974) Testicular involution in short photoperiods inhibited by melatonin. Naturwissenschaften, 61: 364-365. Hoffmann, K. (1977) Die Funktion des Pineals bei der Jahresperiodik der Sauger. Nova Acta Leopold., NF 4 6 : 217-229. Hoffmann, K. (1978a) Effects of short photoperiods on puberty, growth and moult in the Djungarian hamster (Phodopus sungorus), J. Reprod. Fert., 54: 29-35. Hoffmann, K. (1978b) Photoperiodic mechanisms in hamsters: the participation of the pineal gland. In: I. Assenmacher and D.S. Farner (Eds.), Environmental Endocrinology. Springer-Verlag, Berlin, pp. 94-102. Hoffmann, K. ( 1 9 7 8 ~ )Effect of castration on photoperiodically induced weight gain in the Djungarian hamster. Naturwissenschaften, 65: 494. Hoffmann, K. and Kuderling, I. (1975) Pinealectomy inhibits stimulation of testicular development by long photoperiods in a hamster (Phodopus sungorus). Experientia (Basel). 31 : 122-123.

412 Hoffmann, K. and Kuderling, I. (1977) Antigonadal effects of melatonin in pinealectomized Djungarian hamsters. Natunvissenschaften. 64: 339-340. Illnerovi, H. and VangEek, J. (1979) Effect of one-minute exposure to light at night on rat pineal serotonin N-acetyltransferase. In J. Ariens Kappers and P. Pkvet (Eds.), The Pineal Gland o f Vertebrates including Man (Progr. in Brain Research, Vol. 52). Elsevier, Amsterdam, pp. 241-243. Illnerovi, H., Vanzfek, J., Kre'cek, J., Wetterberg, L. and Saaf, J. (1979) Effect of one minute exposure to light at night on rat pineal serotonin N-acetyltransferase and melatonin. J. Neurochem., In press. Klein, D.C. (1973) The role of serotonin N-acetyltransferase in the adrenergic regulation of indole metabolism in the pineal gland. In: J. Barchas and E. Usdin (Eds.), Serotonin and Behavior. Academic Press, New York, pp. 109-1 19. Klein, D.C. and Weller, J.L. (1972) Rapid light-induced decrease in pineal serotonin N-acetyltransferase activity. Science, 177: 532-533. Lincoln, G.A. and Davidson, W. (1977) The relationship between sexual and aggressive behaviour and pituitary and testicular activity during the seasonal sexual cycle of rams, and the influence of photoperiod. J. Reprod. Fert., 49: 267-276. Lincoln, G.A. and MacKinnon, P.C.B. (1976) A study of seasonally delayed puberty in the male hare, Lepus europaeus. J. Reprod. Fert., 46: 123-128. Lincoln, G.A., McNeilly, A.S. and Cameron, C.L. (1978) The effects of a sudden decrease in daylength on prolactin secretion in the ram. J. Reprod. Fert., 52: 305-31 1. Lodge, J. and Salisbury, G. (1970) Seasonal variation and male reproductive efficiency. In: A.D. Johnson, W.R. Comes and N.L. Van Demark (Eds.), The Testis, Vol. 3. Academic Press, New York, pp. 139-167. Lynch, G.R. (1973) Effect of simultaneous exposure to differences in photoperiod and temperature on the seasonal molt and reproductive system of the white-footed mouse, Peromyscus leucopus. Comp. Biochem. Physiol., 44a: 1373-1376. Menaker, M. and Underwood, H. (1976) Extraretinal photoreception in birds. Photochem. Photobiol., 23: 299-306. Moore, R.Y. (1977) Central neural control of circadian rhythms. In: W.F. Ganong and L. Martini (Eds.) Frontiers in Neuroendocrinology. Vol. 5. Raven Press, New York, pp. 185-206. Moore, R.Y. (1978) The Innervation of the Pineal Gland (Progr. in Reproduction Biology, Vol. 4). Karger, Basel, pp. 1-29. Negus, N.C. and Berger, P.J. (1977) Experimental triggering of reproduction in a natural population of Microtus montanus. Science, 196: 1236-1231. Oishi, T. and Lauber, J.K. (1973) Photoreception in the photosexual response of quail. I. Site of photoreceptor.Amer. J. Physiol., 225: 155-158. Pelletier, J. and Ortavant, R. (1975) Photoperiodic control of LH release in the ram. I. Influence of increasing and decreasing light photoperiods. Acta endocr. (Kbh.), 78: 435-441. Quay, W.B. (1974) Pineal Chemistry in Celhlar and Physiological Mechanisms. Charles C. Thomas, Springfield, IL. Reiter, R.J. (1969) Pineal-gonadal relationship in male rodents. In: C. Gual (Ed.), Progress in Endocrinology. Excerpta Medica Foundation, Amsterdam, pp. 631 -635. Reiter, R.J. (1972a) Evidence for refractoriness of the pituitary-gonadal axis to the pineal gland in golden hamsters and its possible implications in annual reproductive rhythms. Anat. Rec., 173: 365-371. Reiter, R.J. (1972b) Surgical procedures involving the pineal gland which prevent gonadal degeneration in adult male hamsters.Ann. Endocr. (Paris), 33: 571-581. Reiter, R. (1973) Comparative physiology: Pineal gland. Ann. Rev. Physiol., 35: 305-328. Reiter, R.J. (1974a) Circannual reproductive rhythms in mammals related to photoperiod and pineal function: a review. Chronobiologia, 1 : 365-395. Reiter, R.J. (1974b) Pineal regulation of hypothalamicopituitary axis: gonadotrophins. In: E. Knobil and W.H. Sawyer (Us.). Handbook of Physiology, Endocrinology ZV,Part 2. American Physiological Society, Washington, DC, pp. 519-550. Reiter, R.J. (1978) Interaction of photoperiod, pineal and seasonal reproduction as exemplified by findings in the hamster. In: Bogress in Reproduction Biology, Vol. 4. Karger, Basel, pp. 169-190. Reiter, R.J. and Hester, R.J. (1966) Interrelationships of the pineal gland, the superior cervical ganglia and the photoperiod in the regulation of the endocrine systems of hamsters. Endocrinalogy, 79: 1168-1170. Reiter, R.J. and Sorrentino, Jr., S. (1972) Prevention of pineal-mediated reproductive responses in light-

41 3 deprived hamsters by partial or total isolation of the medial basal hypothalamus. J. neuro-vise. Rel., 32: 355-367. Reiter, R.J., Sorrentino, Jr., S. and Hoffman, R.A. (1970) Early photoperiodic conditions and pineal antigonadal function in male hamsters.Int. J. Ferr., 15: 163-170. Reiter, R.J., Vaughan, M.K., Blask, D.E. and Johnson, I.Y. (1974) Melatonin: its inhibition of pineal antigonadotrophic activity in male hamsters. Science, 185: 1169-1 171. Reiter, R.J., Vaughan, M., Blask, D. and Johnson, L. (1975) Pineal methoxyindoles: New evidence concerning their function in the control of pineal-mediated changes in the reproductive physiology of male golden hamsters. Endocrinology, 96: 206-21 3. Reiter, R.J., Blask, D.E., Johnson, L.Y., Rudeen, P.K., Vaughan, M.K. and Waring, P.J. (1976) Melatonin inhibition of reproduction in the male hamster: its dependency on time of day of administration and on an intact and sympathetically innervated pineal gland. Neuroendocrinology, 22: 107- 116. Rudeen, P.K. and Reiter, R.J. (1977) Effect of shortened photoperiods on pineal serotonin N-acetyltransferase activity and rhythmicity. J. interdisc. Cycle Res., 8: 47-54. Rudeen, P.K., Reiter, R.J. and Vaughan, M.K. (1975) Pineal serotonin N-acetyltransferase activity in four mammalian species. Neurosci. Lett., 1 : 225-230. Rusak, B. and Morin, L.P. (1976) Testicular responses to photoperiod are blocked by lesions of the suprachiasmatic nuclei in golden hamsters. Biol. Reprod., 15: 366-374. Rust. C.C. and Meyer, R. (1969) Hair colour, molt and testis size in male, short tailed weasels treated with melatonin. Science, 165: 9 2 1 9 2 2 . Sadleir, R.M.F.S. (1969) The Ecology of Reproduction in Wild and Domestic Mammals. Methuen, London. Stetson, M.H. and Watson-Whitmyre, M. (1976) Nucleus suprachiasmaticus: the biological clock in the hamster? Science, 191: 197-199. Stetson, M.H., Watson-Whitmyre, M. and Matt, K.S. (1977) Termination of photorefractoriness in golden hamsters - photoperiodic requirements. J. exp. Zool., 202: 81 -88. Tamarkin, L.,Westrom, W., Hamill, A. and Goldman, B. (1976) Effect of melatonin on the reproductive systems of male and female Syrian hamsters: a diurnal rhythm in sensitivity to melatonin. Endocrinology, 99: 15 34- 154 1. Tamarkin, L., Hollister, C.W., Lefebvre, N.G. and Goldman, B.D. (1977a) Melatonin induction of gonadal quiescence in pinealectomized Syrian Hamsters. Science, 198: 953-955. Tamarkin, L., Lefebvre, N.G., Hollister, C.W. and Goldman, B.D. (1977b) Effect of melatonin administered during the night on reproductive function in the Syrian Hamster. Endocrinology, 101: 631634. Thibault, C., Courot, M., Martinet, L., Mauleon, P., Mesnil du Boisson, F. du, Ortavant, R., Pelletier, J. and Signoret, J.P. (1966) Regulation of breeding season and estrous cycles by light and external stimuli in some mammals. J. anim. Sci., Suppl. 25, pp. 119-142. Turek, F.W. (1977) Antigonadal effect of melatonin in pinealectomized and intact male hamsters. B o C . SOC.exp. Biol. Med., 155: 31-34. Turek, F.W. (1979) Effect of melatonin on photic-independent and photic dependent testicular growth in juvenile and adult male golden hamsters. Biol. Reprod., In press. Turek, F.W. and Campbell, C.S. (1979) Photoperiodic regulation of neuroendocrinegonadal activity. Biol. Reprod., 20: 32-50. Turek, F.W. and Losee, S.H. (1978) Melatonin-induced testicular growth in golden hamsters maintained on short days. Biol. Reprod., 18: 229-305. Turek, F.W., Desjardins, C. and Menaker, M. (1975a) Melatonin: antigonadal and progonadal effects in male golden hamsters. Science, 190: 280-282. Turek, F.W., Elliot, J.A., Alvis, J.D. and Menaker, M. (1975b) Effects of prolonged exposure to nonstimulatory photoperiods on the activity of the neuroendocrine-testicular axis o f golden hamsters. Biol. Reprod., 13: 475-481. Turek, F.W., Desjardins, C. and Menaker, M. (1976a) Differential dffects of melatonin on the testes of photoperiodic and nonphotoperiodic rodents. Biol. Reprod., 15: 94-97. Turek, F.W., Desjardins, C. and Menaker, M. (1976b) Melatonin-induced inhibition of testicular function in adult golden hamsters. Boc. Soc. exp. Biol. Med., 151: 502-506. VanZEek, J. and Illnerova', H. (1979) Changes of a rhythm in rat pineal serotonin N-acetyltransferase following a one-minute light pulse at night. In: J. Ariens Kappers and P. Pivet (Eds.), The Pineal Gland of Vertebrates including Man (Progr. in Brain Research, Vol. 52). Elsevier, Amsterdam, pp. 245-248.

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Van Veen, T., Brackmann, M. and Moghimzadeh, E. (1978) Postnatal development of the pineal organ in the hamsters Phodopus sungorus and Mesocricetusauratus. Cell Tiss. Res., 189: 241-250. Zucker, I., Rusak, B. and King, R.G. (1976) Neural basis for circadian rhythms in rodent behavior. In: A. Riesen and R.F. Thompson (Eds.) Advances in Psychobiology. Vol. 3. Wiley-Interscience, New York, NY, pp. 35-74.

DISCUSSION G. CREMER-BARTELS: Do you think that different HIOMT enzymes which differ in their sensitivity to light or pteridineae pigments may be involved in photoperiodic regulation?

K. HOFFMANN: Since after bilateral enucleation, regardless of photoperiodic conditions, the effects of exposure to constant dark are observed, I do not think that direct light effects on the pineal are involved in the photoperiodic reaction, at least in mammals. B. MESS: How do you explain that the same compound, melatonin, has under certain circumstances an inhibitory, under other circumstances a stimulatory effect on the gonads, at least in photoperiodic animals like the hamster.

K. HOFFMANN: Implantation and thus sustained release of melatonin is certainly not the normal pattern. Thus experiments of this sort might cause artefacts and interfere with the normal reaction. I also want to stress that in such cases the application of melatonin apparently mimics, or interferes with, the photoperiodic effects, but that this does not support the assumption of a pro- or antigonadotropic action of melatonin per se.

D.S. CARTER: Administration of melatonin in beeswax implants is clearly aphysiological. Therefore the “progonadotrophic” effects of melatonin demonstrated in two species of hamsters must be interpreted with extreme caution. It is probable that the constant release of melatonin from the implant in some instances interferes with usual interaction between the brain and the pineal gland. K. HOFFMANN: I would agree. Nevertheless the fact that melatonin has drastic effects in photoperiodic but not in non-photoperiodic mammals; that all operations interfering with synthesis of melatonin in the pineal also interfere with the photoperiodic reaction; that the light schedule may drastically change the pattern of NAT activity and thus of melatonin synthesis; that daily injections of melatonin may have different effects depending on the phase of the circadian cycle in which they are performed, and that melatonin may affect the gonads in pinealectomized photoperiodic mammals, suggests to me that its amount and its pattern of release may be a major component of the photoperiodic process conveying stimuli from the light schedule to the neuroendocrine-gonadal axis. CH. BARTSCH: We arrive at the basic question when speaking about the synchronization between the external environmental clock and the internal clock via the pineal gland. What is the nature of the regulation of this internal clock; is it just part of a feedback mechanism or are we confronted with important pulsations typical of a living organism which come from “within”?

K. HOFFMANN: The question is very broad and not easy to answer. We know that there is an endogenous circadian clock in most eukaryotic organisms. This circadian clock can be entrained, within a range of entrainment, by external cycles like the light-dark cycle. The cellular mechanism is unknown. In mammals apparently the suprachiasmatic nuclei are, or are involved in, an endogenous master-clock driving other circadian rhythms, including those in the pineal (Moore, 1977, 1978; Zucker et al., 1976). In some longer lived mammals endogenous zircannual rhythms have been described @winner, 1979). The mechanism of these circannual rhythms is unknown, however they have to be taken into consideration in all experimentations. There is evidence that these circannual rhythms can be entrained by external factors, especially by photoperiod and its changes. At least in the hamster and in a vole it has been shown that the mechanism of photoperiodic time measurement, which regulates the annual cycle, is based on a circadian cycle of photosensitivity. It must be mentioned, however, that in these species no true cir-

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cannual cycle has been demonstrated. The problem that the daily light-dark cycle entrains the circadian cycle, and that at the same time the light-dark relationship in this cycle has photoperiodic effects that normally regulate the annual cycle, has been more fully discussed by Follet (1973),and by Elliot (1974).

J.P. RAVAULT: Have you measured the pattern of prolactin secretion and did you measure the number of LH receptors in testes (as reported by Bartke)? K. HOFFMANN: No, we did not. We are just starting with hormone determinations in Phodopus.