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Control of the Circannual Rhythm of Reproduction by Melatonin in the Ewe
Brain Research Bulletin, Vol. 44, No. 4, pp. 431– 438, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00
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Control of the Circannual Rhythm of Reproduction by Melatonin in the Ewe B. MALPAUX,*1 C. VIGUIE´,*2 D. C. SKINNER,† J. C. THIE´RY* AND P. CHEMINEAU* *INRA, Neuroendocrinologie Sexuelle, Station de Physiologie de la Reproduction des Mammife`res Domestiques, 37380 Nouzilly, France †Department of Neurobiology, The Babraham Institute, Cambridge CB2 4AT, UK ABSTRACT: Annual variations in day length are responsible for seasonal changes in reproductive activity in sheep. However, in constant photoperiodic conditions, ewes express an endogenous rhythm characterized by alternations of reproductive activity and quiescence that are not synchronized among animals. Thus, the main role of photoperiod in the natural environment appears to be the synchronization of this endogenous rhythm. Photoperiodic information is processed through a complex nervous and endocrine pathway to modulate reproductive activity. Light information perceived at the level of the retina is transformed through neural processing into an endocrine signal by the pineal gland: the nocturnal increase in melatonin release. Recent studies strongly suggest that melatonin has a hypothalamic target to modulate the reproductive neuroendocrine axis. Most LHRH perikarya are located in the preoptic area, but this region is devoid of melatonin receptors, and microimplants of melatonin placed in the preoptic area do not effect LHRH release. Thus, melatonin influences LHRH neurones indirectly and must involve interneurons. Good evidence now exists to demonstrate that a population of dopaminergic neurons with axons projecting to the median eminence is one of these interneurons. © 1997 Elsevier Science Inc.
PHOTOPERIODIC CONTROL OF REPRODUCTION Seasonal Variations in Gonadotropin Secretion and in Estradiol Negative Feedback Seasonal variations in reproductive activity result from changes in gonadotropin secretion [12]. Season influences the frequency of LH pulsatile release, the most important characteristic of that secretion, by two complementary mechanisms: one that is steroid dependent, and the other steroid independent. In ovariectomized (OVX) ewes, LH pulse frequency is lower during anestrus than during the breeding seasons (one vs. two pulses per hour) [32,38]. This change in LH pulsatility between the anestrous and breeding seasons is increased dramatically when OVX ewes are treated with estradiol (E). Indeed, in OVX ewes treated with an E implant that elevates blood E levels to those of follicular phase intact ewes, one pulse is observed every 12 to 24 h during anestrus and every 30 min during the breeding season [12]. The shift in E negative feedback, therefore, is the main mechanism responsible for reproductive seasonality. This dramatic seasonal change in the ability of E to inhibit LH secretion has led to the development of a widely used experimental model: the OVX ewe treated with a subcutaneous constant-release implant of E. LH concentrations in this model are correlated with ovulatory activity in intact ewes [12]. Sheep also display large seasonal variations in prolactin secretion, with highest levels produced in early summer and lowest produced in early winter [34]. However, in this species, these changes in prolactin secretion do not seem to play any causal role in the expression of seasonal variations of reproductive activity [4,49,61].
KEY WORDS: Sheep, Reproduction, Circannual, Melatonin, Dopamine, LHRH.
INTRODUCTION In temperate climates, most primitive breeds of sheep exhibit a strong seasonal pattern of reproduction with a short breeding season (November–December) [8]. Although domestication has attenuated the strict confines of this seasonal pattern, most breeds have maintained some distinction between the breeding and anestrous seasons with ewes sexually active from late summer to late winter and births occurring in Spring after a 5-month pregnancy (Fig. 1; [12]). For example, in the Ile-de-France breed, ovulation starts in August and ends in January [48]. In this article, we will describe the evidence for the implication of photoperiod and an endogenous rhythm of reproduction in the expression of seasonality in the ewe. We shall then analyze the neuroendocrine mechanisms by which melatonin modulates reproductive activity. 1 2
Photoperiod as the Main External Factor Controlling Reproduction Photoperiod is the main environmental factor controlling the seasonality of reproduction in sheep. Indeed, reproductive activity can be driven by modifying only this component of the environment; the reversal of the annual photoperiodic cycle causes the breeding cycle to phase shift by 6 months, and the reduction of its period to 6 months leads to the appearance of two breeding seasons per year [29,50]. Reproductive activity can also be driven by simply exposing the animals to an alternation of constant short days and long days; when animals are exposed sequentially to 90 long days (16 h light: 8 h darkness; (16L:8D)) and to 90 short days
To whom requests for reprints should be addressed. Present address: Reproductive Science Program, The University of Michigan, 300 N. Ingalls Building, Ann Arbor, MI 48109-0404.
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432 (8L:16D), breeding activity or increased LH secretion in OVX1E ewes commences after 40 to 60 short days and terminates after 20 to 30 days (Fig. 1) [12,47]. Photoperiod also influences prolactin secretion; short days inhibit prolactin release whereas long days stimulate it [49]. Does Photoperiod Drive Reproductive Activity or Synchronize an Endogenous Rhythm? Based on the timing of the breeding season (shortening days of late summer and autumn) and on the demonstration of a stimulatory effect of short days in artificial photoperiodic conditions, the concept of a sheep being a short-day breeder has been postulated. This concept implies that in natural conditions reproductive activity is driven by daylength and that the breeding season is the result of the stimulatory effect of shortening days. Although this concept remains useful from a descriptive perspective, it now appears that it may be misleading from a mechanistic viewpoint. Indeed, if ewes are kept under constant long days from the summer solstice onwards, the breeding season or the seasonal activation of LH secretion starts at the same time as it does in controls ewes exposed to natural daylength [39,61]. The decrease in daylength occurring after the summer solstice, therefore, is not cueing the onset of the breeding season. Furthermore, if ewes are exposed to constantly increasing daylengths from the spring equinox, they also initiate their period of reproductive activity at the normal time [26]. The same conclusion applies for the cessation of the breeding season that occurs in February, whether animals are kept in constant short
MALPAUX ET AL. days after the winter solstice or in constantly decreasing daylengths from the autumn equinox [28,36,60]. These studies suggest that both the initiation and termination of the breeding season are obligatory processes that cannot be prevented by modifying the ambient photoperiod. This apparent ‘‘refractoriness’’ could be the expression of an underlying endogenous rhythm of reproduction. The existence of such a rhythm has been substantiated by the observation that animals kept in constant photoperiodic conditions continue to display variations in reproductive activity. This has been demonstrated with animals exposed to constant photoperiods (long or short) or have been enucleated or pinealectomized [6,17,59]. For instance, when ewes were maintained on constant short days (8L:16D) for 4 years, they displayed cyclical changes in neuroendocrine reproductive activity [13]. These cycles were not synchronized among ewes and had a period of less than 1 year (9 months on average). It is hypothesized, therefore, that the annual breeding cycle in natural conditions is the expression of an internal rhythm and that the role of photoperiod is to synchronize this rhythm and impose a period equal to a year [37]. The most powerful support for this role of photoperiod was obtained by reinstating a ‘‘photoperiodic’’ signal (infusion of melatonin) in animals deprived of photoperiodic information by pinealectomy [59]. In particular, it was shown that infusing melatonin in a long-day profile (8 h of infusion every day) for 30 days once a year is sufficient to entrain the endogenous rhythm of reproduction with a period of a year [59]. Subsequent experiments have suggested that all segments of the photoperiodic cycle are not capable of entraining the rhythm [57]. Long days of spring appear to be critical to synchronize the rhythm, especially to time the onset of the breeding season in late summer, and short days act mainly to sustain reproductive activity once it is initiated [26]. NEUROENDOCRINE BASIS FOR PHOTOPERIODIC REGULATION OF LH SECRETION Regulation of LHRH Secretion by Melatonin
FIG. 1. Variations in ovulatory activity in Ile-de-France ewes in natural photoperiod (top, n 5 8) or submitted to alternations between 3 months of long days and 3 months of short days (bottom, n 5 7). Lengths of day and night are depicted at the top of each graph by white and dark areas, respectively (adapted from [47]).
Most studies directed at elucidating the neuroendocrine mechanisms of reproductive seasonality have not accounted directly for the existence of an endogenous rhythm. Animals are usually exposed to alternations between long and short days; a regime where it is not clear whether activity is driven or synchronized by photoperiod. However, in the ewe, the inhibitory effects of long days and the stimulatory effects of short days are well characterized and provide valuable experimental models. It has been clearly shown that in mammals photoperiodic information is received at the level of the retina and is transmitted via a multistep neural pathway to the pineal gland where the message modulates the rhythm of melatonin secretion [12]. Melatonin is released both in the Galen vein and in the cerebrospinal fluid and concentrations in the CSF are 20-fold greater than in the jugular vein [40]. However, the relative importance of these two pathways to modulating reproductive activity remains to be resolved. Melatonin is released only at night and, therefore, the duration of secretion differs between long and short days. This duration of melatonin secretion is then processed neurally to regulate the secretion of LHRH. Indeed, in OVX1E ewes exposed to inhibitory long days (16L: 8D) for 70 to 90 days, LHRH and LH pulse frequency are low (about 1 pulse/6 h). If these ewes are then treated with a melatonin implant, which produces high concentrations of melatonin for 24 h every day and thereby causes a short day-like response by lengthening the duration of melatonin, LHRH and LH pulse frequency increase dramatically to about 10 pulses/6 h (Fig. 2) [53]. This change in LHRH and LH pulse frequency is observed after 40 to 60 days of melatonin treatment and results in an increase in the mean circulating LH levels observed at the same time in these
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FIG. 2. Effect of melatonin on LHRH and LH pulsatile secretion. Examples of individual profiles of LHRH (top profiles) and simultaneous LH (bottom profiles) secretions before (D-1, left panels) or 74 days after (D 1 74, right panels) the insertion of a melatonin implant. Ewes were ovariectomized and treated with a subcutaneous estradiol implant and were exposed to long days (16L:8D). Blood samples were obtained every 10 min for 6 h on each occasion. The closed symbols depict detected pulses (adapted from [53]).
ewes. The action of melatonin on the reproductive axis is mediated, therefore, by a hypothalamic step which is a modulation of LHRH secretion, reflecting a change in the E negative feedback on LHRH secretion. Sites of Action of Melatonin Because the action of melatonin on reproduction involves a hypothalamic neuronal system(s) to regulate LHRH pulse frequency, we initially hypothesized that melatonin acted within the brain and most likely within the hypothalamus. To test this hypothesis, microimplants of melatonin were inserted into regions of the hypothalamo– hypophysial complex, which restricted the delivery of this hormone to discrete sites and to a confined area within that site. Effects of these microimplants on the reproductive axis were compared to those of control animals that had been given melatonin peripherally or against animals that had been exposed to short days. Melatonin microimplants were placed initially in specific hypothalamic and perihypothalamic areas of OVX1E ewes. This study demonstrated that only microimplants positioned in the mediobasal hypothalamus (MBH: 7 out of 12 implanted ewes responded) could stimulate LH secretion [24]. Furthermore, both the timing and the amplitude of the LH increase were similar to those of short day-treated animals [24]. Microimplants placed in the preoptic area or the anterior or dorsolateral hypothalamus had no effect on LH secretion. These results concur with studies on Soay rams exposed to long days in which the placement of melatonin microimplants in the MBH, but not in the preoptic area, induced an increase in FSH secretion and testicular size [22]. However, these results present an apparent dichotomy because in sheep, as in all species (photoperiodic or nonphotoperiodic) investigated to date, high melatonin binding sites are expressed consistently in only one site: the pars tuberalis (PT) of the adeno-
hypophysis [1,5,33,43]. No melatonin binding is detectable in other subdivisions of the adenohypophysis [41]. Although binding has been found in the hypothalamus, the density in the PT is an order of magnitude higher. Furthermore, the recent cloning of the gene for a membrane melatonin receptor has reinforced this preferential distribution by showing the presence of melatonin receptor mRNA in the PT [35]. Because the MBH and PT are positioned closely together, the action of microimplants located in the MBH could be interpreted in two ways: either the sites of action are truly located in the MBH, or else melatonin released from the MBH microimplants diffused to the PT to exert its effects. However, the absence of radioactivity in the PT after placement of microimplants containing radioactive (125I or 3H) melatonin in the MBH does not support the latter conjecture [24,27]. More importantly, melatonin delivered directly to the PT does not appear to modify the secretion of LH: neither the placement of a melatonin microimplant directly against the anterior face of the PT [25] nor the discrete insertion of a microimplant into the PT [27] modified LH secretion in ewes (Fig. 3). In contrast, microimplants placed in the MBH or third ventricle stimulated LH release [25,27]. These studies provide definitive evidence that the MBH, not the PT, is the important melatonin target for transducing the effects of this indoleamine on the reproductive neuroendocrine axis. Moreover, the placement of microimplants in the PT or pars distalis of Soay rams led to similar conclusions [20]. In that study, PT microimplants stimulated FSH secretion but the effect was much weaker than that obtained with MBH implants in a previous study, suggesting that the small effect observed with the PT implants resulted from a passive diffusion of melatonin to the MBH [20,22]. The presence of a site(s) of action in the hypothalamus rekindles interest in the low density of melatonin binding sites observed in this area ([1,3]; Daveau, Malpaux, Chemineau, unpublished).
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FIG. 3. Effect of placing melatonin microimplants into the pars tuberalis (Experiment 1, left panels) or directly against the anterior face of the pars tuberalis (Experiment 2, right panels) on LH secretion in ewes. Mean (6SEM) values of LH concentrations in control ewes (open symbols, repeated in the two panels of each experiment) and in animals that had received a microimplant of melatonin into or against the pars tuberalis (closed symbols of both top panels), in the mediobasal hypothalamus (closed symbols, bottom left panel) or in the third ventricle (closed symbols, bottom right panel). Ewes were ovariectomized and treated with a subcutaneous estradiol implant and were exposed to long days (16L:8D). Blood samples were obtained twice weekly and melatonin treatment started on day 0 (adapted from [25,27]).
The presence of two types of melatonin receptors (Mel1a and Mel1b) has already been demonstrated in mammals, and it is likely that additional subtypes will be found [58]. It is possible, therefore, that different subtypes of melatonin receptors may account for the difference in the intensity of binding. Although the above studies suggest that the hypothalamus, not the PT, is the important melatonin target for the observed effects on LH secretion, it is noteworthy that the PT may be responsible, at least in part, for transducing the effects of melatonin on the seasonal change in prolactin secretion. Indeed, the placement of microimplants in the PT caused a suppression of prolactin levels similar to that obtained with MBH implants [27]. Furthermore, after hypothalamo– hypophysial disconnection, seasonal changes in prolactin secretion were still observed and melatonin still inhibits prolactin secretion, which suggests that, in contrast to its action on the reproductive axis, the effect of melatonin on prolactin secretion could be mediated directly at the level of the pituitary, possibly in the PT [21]. Interestingly, a similar distinction between hypothalamic melatonin target sites involved in the control of
gonadotropin secretion and other sites involved in that of prolactin has also been proposed for the hamster [30]. Dopaminergic Neurons as Possible Relays between Melatonin Targets and LHRH Neurons Several pieces of evidence suggest that melatonin does not act directly on LHRH neurons. First, the distribution of most LHRH neurons does not match that of the putative sites of action of melatonin. Most LHRH neuronal perikarya are located in the preoptic area (60%) with a few located in the MBH (15%); some of these project to the median eminence and abut portal vessels [2,18]. Secondly, several neurotransmitters have been implicated in the regulation of LHRH secretion by melatonin; of these, the potential role of dopamine is particularly well documented. Many studies have suggested that dopamine is involved in transducing the negative feedback of E on LHRH secretion. For instance, systemic injection of a dopamine antagonist (pimozide) during anoestrus (strong negative feedback of E) induced a temporary in-
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FIG. 4. Effects of photoperiodic and estrogen treatments on mean (6SEM) LH pulse frequency and prolactin level (left panels) and tyrosine hydroxylase (TH) activity, dopamine, and noradrenaline contents in the pituitary stalk median eminence (right panels) of ovariectomized ewes. TH activity and monoamine contents were measured individually in the same tissue extract of stalk-median eminence of ovariectomized ewes treated by either 133 to 136 long days (LD) or 63 to 66 short days (SD). For each photoperiodic regime half of the ewes were bearing a 2-cm subcutaneous implant of estradiol (1E) and half did not receive any steroid replacement (2E). LH and prolactin concentrations were determined in serial samples of blood obtained 3 to 6 days before the end of the experiment, i.e., after 60 short days or 130 long days for the SD and LD groups, respectively. *p , 0.05; **p , 0.01 (adapted from [55]).
crease in LH secretion [31]. Similarly, pimozide caused a temporary increase in LH secretion in long-day exposed (photo-inhibited) OVX1E ewes [14]. The A15 hypothalamic cell group appears to be a key dopaminergic structure involved in mediating the inhibitory effects of E because a neurotoxic lesion of this structure during anestrus caused an increase in LH secretion [46]. Furthermore, it has been shown that E increases tyrosine hydroxylase (TH, rate limiting step enzyme of catecholamine synthesis) activity in long day-treated ewes [7] and induces c-fos gene expression in TH-immunoreactive cells of this structure in a season-dependent manner [19]. A similar role has been proposed for the A14 dopaminergic cell group [10]. Because a major effect of melatonin is a modulation of E negative feedback on LHRH secretion, this implication of dopamine makes these neurons likely candidates to act as relays between melatonin target sites and LHRH neurons [45]. To test this hypothesis, we first determined whether photoperiod and melatonin could modulate the activity of some dopaminergic neurons in the hypothalamus. Exposure to stimulatory short days resulted in decreased dopaminergic activity in the median eminence as assessed both by a reduction in dopamine content and in TH activity (Fig. 4) [44,55]. No effect of short-day exposure on noradrenaline content in this structure or on TH activity in the other hypothalamic areas investigated was found [55]. The stim-
ulation of LH secretion by a melatonin implant caused a parallel reduction in TH activity, which suggests strongly that the effect of photoperiod on TH activity is mediated by melatonin [56]. The inhibition of median eminence TH activity by short days or by treatment with a melatonin implant is expressed at a time when the inhibition of prolactin secretion is already maximal (Fig. 5), suggesting that these photoperiod-induced changes in TH activity are independent of the regulation of prolactin secretion [56]. Rather, they appear to be related to the photoperiodic regulation of LH secretion. Indeed, the pharmacological blockade of TH locally in the median eminence of long-day photo-inhibited ewes led to an increase in LH secretion [52]. This finding is consistent with the effect of pimozide implants in the median eminence of anestrous ewes [9]. These data suggest, therefore, that a reduction in TH activity in the median eminence is an important component of the stimulatory effect of melatonin on LHRH output. However, such modulation of TH activity appears to be E independent because the photoperiod-induced changes in TH activity are similar in OVX and OVX1E ewes [55]. Thus, in contrast to the A15 and possibly A14 dopaminergic nucleus, which are involved in the modulation of the E negative feedback, the dopaminergic neurons of the median eminence appear to be involved upstream relative to the integration of the E signal. The median eminence in sheep is a
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MALPAUX ET AL. structure rich in dopaminergic terminals but contains no THimmunoreactive perikarya [51]. The localization of the cell bodies projecting their axons to the median eminence, and more generally the anatomical and functional relationship between the A14 and A15 nuclei and the median eminence, have yet to be determined. In addition to dopamine, other neurotransmitters have been implicated in the seasonal neuroendocrine changes. These include serotonin, particularly via 5HT2 receptors [15,16], excitatory aminoacids [23,54], and NPY [42]. Other neuronal systems that are possible transducers of the E negative feedback effects [10,11] may also be involved. CONCLUSION The regulation of LHRH secretion by melatonin is central to the photoperiodic regulation of reproductive activity. The target sites of melatonin appear to be located in the hypothalamus. Melatonin does not act directly on LHRH neurons; rather, its action is probably mediated by a complex network of interneurons. Within that network, the dopaminergic terminals of the median eminence play an important role. The unravelling of this ‘‘black box’’ linking melatonin target sites and the LHRH neurones is a challenge to understand the interaction between melatonin and the endogenous rhythm of reproduction. Of particular interest will be the distinction between the mechanisms involved in generating this circannual rhythm and those involved in mediating the synchronizing effect of melatonin. ACKNOWLEDGEMENTS
Original research performed in this laboratory was supported in part by a grant from Re´gion Centre. C.V. was in receipt of a Ph.D. grant from INRA and Re´gion Centre. Collaboration with DCS was made possible by INRA/BBSRC and ALLIANCE exchange schemes.
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FIG. 5. Mean (6SEM) difference in the number of LH pulses/6 h between the control LD period and the end of the experiment (top), mean (6SEM) plasma prolactin concentrations at the end of the experiment (middle), and mean (6SEM) TH activity in the median eminence of ewes. All ewes were ovariectomized and treated with a subcutaneous implant of oetradiol. They were pretreated with long days (16L:8D) and then allocated to one of four groups treated with 0, 5, 25, or 76 short days. LH secretory profiles were determined from LH concentration measured in blood samples obtained every 10 min for 6 h at two different periods of the photoperiodic treatment: (1) 7 days before the end of the last LD period, and (2) 3 to 4 days before the end of the experiment (i.e., in LD or on average after 3, 23, or 74 SD). Prolactin concentrations were determined in every third sample during the last 3 h of the second sampling period. TH activity was measured individually in the stalk-median eminence of the same ovariectomized ewes (n 5 7 per group) *p , 0.05; **p , 0.01 (adapted from [56]).
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438 53. Viguie´, C.; Caraty, A.; Locatelli, A.; Malpaux, B. Regulation of LHRH secretion by melatonin in the ewe. I. Simultaneous delayed increase in LHRH and LH pulsatile secretion. Biol. Reprod. 52: 1114 –1120; 1995. 54. Viguie´, C.; Caraty, A.; Locatelli, A.; Malpaux, B. Regulation of LHRH secretion by melatonin in the ewe. II. Changes in N-Methyl-D,Laspartic acid-induced LHRH release during the stimulation of LH secretion by melatonin. Biol. Reprod. 52:1156 –1161; 1995. 55. Viguie´, C.; Thibault, J.; Thie´ry, J. C.; Tillet, Y.; Malpaux, B. Photoperiodic modulation of monoamines and amino-acids involved in the control of prolactin and LH secretion in the ewe: Evidence for a regulation of tyrosine hydroxylase activity. J. Neuroendocrinol. 8:465– 474; 1996. 56. Viguie´, C.; Thibault, J.; Thie´ry, J. C.; Tillet, Y.; Malpaux, B. Characterization of the short day-induced decrease in median eminence tyrosine hydroxylase activity in the ewe: Temporal relationship with the changes in LH and prolactin secretion and short-day like effect of melatonin. Endocrinology 138:499 –506; 1997.
MALPAUX ET AL. 57. Wayne, N. L.; Malpaux, B.; Karsch, F. J. Photoperiodic requirements for timing the onset and duration of the breeding season of the ewe: Synchronization of an endogenous rhythm of reproduction. J. Comp. Physiol. A 166:835– 842; 1990. 58. Weaver, D. R.; Liu, C.; Reppert, S. M. Nature’s knockout: the Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters. Mol. Endocrinol. 10:1478 –1487; 1996. 59. Woodfill, C. J. I.; Robinson, J. E.; Malpaux, B.; Karsch, F. J. Synchronization of the circannual reproductive rhythm of the ewe by discrete photoperiodic signals. Biol. Reprod. 45:110 –121; 1991. 60. Worthy, K.; Haresign, W. Evidence that the onset of seasonal anoestrus in the ewe may be independent of increasing prolactin concentrations and daylength. J. Reprod. Fertil. 69:41– 48; 1983. 61. Worthy, K.; Haresign, W.; Dodson, S.; McLeod, B. J.; Foxcroft, G. R.; Haynes, N. B. Evidence that the onset of the breeding season in the ewe may be independent of decreasing plasma prolactin concentrations. J. Reprod. Fertil. 75:237–246; 1985.
PII S0361-9230(97)00223-2
Control of the Circannual Rhythm of Reproduction by Melatonin in the Ewe B. MALPAUX,*1 C. VIGUIE´,*2 D. C. SKINNER,† J. C. THIE´RY* AND P. CHEMINEAU* *INRA, Neuroendocrinologie Sexuelle, Station de Physiologie de la Reproduction des Mammife`res Domestiques, 37380 Nouzilly, France †Department of Neurobiology, The Babraham Institute, Cambridge CB2 4AT, UK ABSTRACT: Annual variations in day length are responsible for seasonal changes in reproductive activity in sheep. However, in constant photoperiodic conditions, ewes express an endogenous rhythm characterized by alternations of reproductive activity and quiescence that are not synchronized among animals. Thus, the main role of photoperiod in the natural environment appears to be the synchronization of this endogenous rhythm. Photoperiodic information is processed through a complex nervous and endocrine pathway to modulate reproductive activity. Light information perceived at the level of the retina is transformed through neural processing into an endocrine signal by the pineal gland: the nocturnal increase in melatonin release. Recent studies strongly suggest that melatonin has a hypothalamic target to modulate the reproductive neuroendocrine axis. Most LHRH perikarya are located in the preoptic area, but this region is devoid of melatonin receptors, and microimplants of melatonin placed in the preoptic area do not effect LHRH release. Thus, melatonin influences LHRH neurones indirectly and must involve interneurons. Good evidence now exists to demonstrate that a population of dopaminergic neurons with axons projecting to the median eminence is one of these interneurons. © 1997 Elsevier Science Inc.
PHOTOPERIODIC CONTROL OF REPRODUCTION Seasonal Variations in Gonadotropin Secretion and in Estradiol Negative Feedback Seasonal variations in reproductive activity result from changes in gonadotropin secretion [12]. Season influences the frequency of LH pulsatile release, the most important characteristic of that secretion, by two complementary mechanisms: one that is steroid dependent, and the other steroid independent. In ovariectomized (OVX) ewes, LH pulse frequency is lower during anestrus than during the breeding seasons (one vs. two pulses per hour) [32,38]. This change in LH pulsatility between the anestrous and breeding seasons is increased dramatically when OVX ewes are treated with estradiol (E). Indeed, in OVX ewes treated with an E implant that elevates blood E levels to those of follicular phase intact ewes, one pulse is observed every 12 to 24 h during anestrus and every 30 min during the breeding season [12]. The shift in E negative feedback, therefore, is the main mechanism responsible for reproductive seasonality. This dramatic seasonal change in the ability of E to inhibit LH secretion has led to the development of a widely used experimental model: the OVX ewe treated with a subcutaneous constant-release implant of E. LH concentrations in this model are correlated with ovulatory activity in intact ewes [12]. Sheep also display large seasonal variations in prolactin secretion, with highest levels produced in early summer and lowest produced in early winter [34]. However, in this species, these changes in prolactin secretion do not seem to play any causal role in the expression of seasonal variations of reproductive activity [4,49,61].
KEY WORDS: Sheep, Reproduction, Circannual, Melatonin, Dopamine, LHRH.
INTRODUCTION In temperate climates, most primitive breeds of sheep exhibit a strong seasonal pattern of reproduction with a short breeding season (November–December) [8]. Although domestication has attenuated the strict confines of this seasonal pattern, most breeds have maintained some distinction between the breeding and anestrous seasons with ewes sexually active from late summer to late winter and births occurring in Spring after a 5-month pregnancy (Fig. 1; [12]). For example, in the Ile-de-France breed, ovulation starts in August and ends in January [48]. In this article, we will describe the evidence for the implication of photoperiod and an endogenous rhythm of reproduction in the expression of seasonality in the ewe. We shall then analyze the neuroendocrine mechanisms by which melatonin modulates reproductive activity. 1 2
Photoperiod as the Main External Factor Controlling Reproduction Photoperiod is the main environmental factor controlling the seasonality of reproduction in sheep. Indeed, reproductive activity can be driven by modifying only this component of the environment; the reversal of the annual photoperiodic cycle causes the breeding cycle to phase shift by 6 months, and the reduction of its period to 6 months leads to the appearance of two breeding seasons per year [29,50]. Reproductive activity can also be driven by simply exposing the animals to an alternation of constant short days and long days; when animals are exposed sequentially to 90 long days (16 h light: 8 h darkness; (16L:8D)) and to 90 short days
To whom requests for reprints should be addressed. Present address: Reproductive Science Program, The University of Michigan, 300 N. Ingalls Building, Ann Arbor, MI 48109-0404.
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432 (8L:16D), breeding activity or increased LH secretion in OVX1E ewes commences after 40 to 60 short days and terminates after 20 to 30 days (Fig. 1) [12,47]. Photoperiod also influences prolactin secretion; short days inhibit prolactin release whereas long days stimulate it [49]. Does Photoperiod Drive Reproductive Activity or Synchronize an Endogenous Rhythm? Based on the timing of the breeding season (shortening days of late summer and autumn) and on the demonstration of a stimulatory effect of short days in artificial photoperiodic conditions, the concept of a sheep being a short-day breeder has been postulated. This concept implies that in natural conditions reproductive activity is driven by daylength and that the breeding season is the result of the stimulatory effect of shortening days. Although this concept remains useful from a descriptive perspective, it now appears that it may be misleading from a mechanistic viewpoint. Indeed, if ewes are kept under constant long days from the summer solstice onwards, the breeding season or the seasonal activation of LH secretion starts at the same time as it does in controls ewes exposed to natural daylength [39,61]. The decrease in daylength occurring after the summer solstice, therefore, is not cueing the onset of the breeding season. Furthermore, if ewes are exposed to constantly increasing daylengths from the spring equinox, they also initiate their period of reproductive activity at the normal time [26]. The same conclusion applies for the cessation of the breeding season that occurs in February, whether animals are kept in constant short
MALPAUX ET AL. days after the winter solstice or in constantly decreasing daylengths from the autumn equinox [28,36,60]. These studies suggest that both the initiation and termination of the breeding season are obligatory processes that cannot be prevented by modifying the ambient photoperiod. This apparent ‘‘refractoriness’’ could be the expression of an underlying endogenous rhythm of reproduction. The existence of such a rhythm has been substantiated by the observation that animals kept in constant photoperiodic conditions continue to display variations in reproductive activity. This has been demonstrated with animals exposed to constant photoperiods (long or short) or have been enucleated or pinealectomized [6,17,59]. For instance, when ewes were maintained on constant short days (8L:16D) for 4 years, they displayed cyclical changes in neuroendocrine reproductive activity [13]. These cycles were not synchronized among ewes and had a period of less than 1 year (9 months on average). It is hypothesized, therefore, that the annual breeding cycle in natural conditions is the expression of an internal rhythm and that the role of photoperiod is to synchronize this rhythm and impose a period equal to a year [37]. The most powerful support for this role of photoperiod was obtained by reinstating a ‘‘photoperiodic’’ signal (infusion of melatonin) in animals deprived of photoperiodic information by pinealectomy [59]. In particular, it was shown that infusing melatonin in a long-day profile (8 h of infusion every day) for 30 days once a year is sufficient to entrain the endogenous rhythm of reproduction with a period of a year [59]. Subsequent experiments have suggested that all segments of the photoperiodic cycle are not capable of entraining the rhythm [57]. Long days of spring appear to be critical to synchronize the rhythm, especially to time the onset of the breeding season in late summer, and short days act mainly to sustain reproductive activity once it is initiated [26]. NEUROENDOCRINE BASIS FOR PHOTOPERIODIC REGULATION OF LH SECRETION Regulation of LHRH Secretion by Melatonin
FIG. 1. Variations in ovulatory activity in Ile-de-France ewes in natural photoperiod (top, n 5 8) or submitted to alternations between 3 months of long days and 3 months of short days (bottom, n 5 7). Lengths of day and night are depicted at the top of each graph by white and dark areas, respectively (adapted from [47]).
Most studies directed at elucidating the neuroendocrine mechanisms of reproductive seasonality have not accounted directly for the existence of an endogenous rhythm. Animals are usually exposed to alternations between long and short days; a regime where it is not clear whether activity is driven or synchronized by photoperiod. However, in the ewe, the inhibitory effects of long days and the stimulatory effects of short days are well characterized and provide valuable experimental models. It has been clearly shown that in mammals photoperiodic information is received at the level of the retina and is transmitted via a multistep neural pathway to the pineal gland where the message modulates the rhythm of melatonin secretion [12]. Melatonin is released both in the Galen vein and in the cerebrospinal fluid and concentrations in the CSF are 20-fold greater than in the jugular vein [40]. However, the relative importance of these two pathways to modulating reproductive activity remains to be resolved. Melatonin is released only at night and, therefore, the duration of secretion differs between long and short days. This duration of melatonin secretion is then processed neurally to regulate the secretion of LHRH. Indeed, in OVX1E ewes exposed to inhibitory long days (16L: 8D) for 70 to 90 days, LHRH and LH pulse frequency are low (about 1 pulse/6 h). If these ewes are then treated with a melatonin implant, which produces high concentrations of melatonin for 24 h every day and thereby causes a short day-like response by lengthening the duration of melatonin, LHRH and LH pulse frequency increase dramatically to about 10 pulses/6 h (Fig. 2) [53]. This change in LHRH and LH pulse frequency is observed after 40 to 60 days of melatonin treatment and results in an increase in the mean circulating LH levels observed at the same time in these
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FIG. 2. Effect of melatonin on LHRH and LH pulsatile secretion. Examples of individual profiles of LHRH (top profiles) and simultaneous LH (bottom profiles) secretions before (D-1, left panels) or 74 days after (D 1 74, right panels) the insertion of a melatonin implant. Ewes were ovariectomized and treated with a subcutaneous estradiol implant and were exposed to long days (16L:8D). Blood samples were obtained every 10 min for 6 h on each occasion. The closed symbols depict detected pulses (adapted from [53]).
ewes. The action of melatonin on the reproductive axis is mediated, therefore, by a hypothalamic step which is a modulation of LHRH secretion, reflecting a change in the E negative feedback on LHRH secretion. Sites of Action of Melatonin Because the action of melatonin on reproduction involves a hypothalamic neuronal system(s) to regulate LHRH pulse frequency, we initially hypothesized that melatonin acted within the brain and most likely within the hypothalamus. To test this hypothesis, microimplants of melatonin were inserted into regions of the hypothalamo– hypophysial complex, which restricted the delivery of this hormone to discrete sites and to a confined area within that site. Effects of these microimplants on the reproductive axis were compared to those of control animals that had been given melatonin peripherally or against animals that had been exposed to short days. Melatonin microimplants were placed initially in specific hypothalamic and perihypothalamic areas of OVX1E ewes. This study demonstrated that only microimplants positioned in the mediobasal hypothalamus (MBH: 7 out of 12 implanted ewes responded) could stimulate LH secretion [24]. Furthermore, both the timing and the amplitude of the LH increase were similar to those of short day-treated animals [24]. Microimplants placed in the preoptic area or the anterior or dorsolateral hypothalamus had no effect on LH secretion. These results concur with studies on Soay rams exposed to long days in which the placement of melatonin microimplants in the MBH, but not in the preoptic area, induced an increase in FSH secretion and testicular size [22]. However, these results present an apparent dichotomy because in sheep, as in all species (photoperiodic or nonphotoperiodic) investigated to date, high melatonin binding sites are expressed consistently in only one site: the pars tuberalis (PT) of the adeno-
hypophysis [1,5,33,43]. No melatonin binding is detectable in other subdivisions of the adenohypophysis [41]. Although binding has been found in the hypothalamus, the density in the PT is an order of magnitude higher. Furthermore, the recent cloning of the gene for a membrane melatonin receptor has reinforced this preferential distribution by showing the presence of melatonin receptor mRNA in the PT [35]. Because the MBH and PT are positioned closely together, the action of microimplants located in the MBH could be interpreted in two ways: either the sites of action are truly located in the MBH, or else melatonin released from the MBH microimplants diffused to the PT to exert its effects. However, the absence of radioactivity in the PT after placement of microimplants containing radioactive (125I or 3H) melatonin in the MBH does not support the latter conjecture [24,27]. More importantly, melatonin delivered directly to the PT does not appear to modify the secretion of LH: neither the placement of a melatonin microimplant directly against the anterior face of the PT [25] nor the discrete insertion of a microimplant into the PT [27] modified LH secretion in ewes (Fig. 3). In contrast, microimplants placed in the MBH or third ventricle stimulated LH release [25,27]. These studies provide definitive evidence that the MBH, not the PT, is the important melatonin target for transducing the effects of this indoleamine on the reproductive neuroendocrine axis. Moreover, the placement of microimplants in the PT or pars distalis of Soay rams led to similar conclusions [20]. In that study, PT microimplants stimulated FSH secretion but the effect was much weaker than that obtained with MBH implants in a previous study, suggesting that the small effect observed with the PT implants resulted from a passive diffusion of melatonin to the MBH [20,22]. The presence of a site(s) of action in the hypothalamus rekindles interest in the low density of melatonin binding sites observed in this area ([1,3]; Daveau, Malpaux, Chemineau, unpublished).
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FIG. 3. Effect of placing melatonin microimplants into the pars tuberalis (Experiment 1, left panels) or directly against the anterior face of the pars tuberalis (Experiment 2, right panels) on LH secretion in ewes. Mean (6SEM) values of LH concentrations in control ewes (open symbols, repeated in the two panels of each experiment) and in animals that had received a microimplant of melatonin into or against the pars tuberalis (closed symbols of both top panels), in the mediobasal hypothalamus (closed symbols, bottom left panel) or in the third ventricle (closed symbols, bottom right panel). Ewes were ovariectomized and treated with a subcutaneous estradiol implant and were exposed to long days (16L:8D). Blood samples were obtained twice weekly and melatonin treatment started on day 0 (adapted from [25,27]).
The presence of two types of melatonin receptors (Mel1a and Mel1b) has already been demonstrated in mammals, and it is likely that additional subtypes will be found [58]. It is possible, therefore, that different subtypes of melatonin receptors may account for the difference in the intensity of binding. Although the above studies suggest that the hypothalamus, not the PT, is the important melatonin target for the observed effects on LH secretion, it is noteworthy that the PT may be responsible, at least in part, for transducing the effects of melatonin on the seasonal change in prolactin secretion. Indeed, the placement of microimplants in the PT caused a suppression of prolactin levels similar to that obtained with MBH implants [27]. Furthermore, after hypothalamo– hypophysial disconnection, seasonal changes in prolactin secretion were still observed and melatonin still inhibits prolactin secretion, which suggests that, in contrast to its action on the reproductive axis, the effect of melatonin on prolactin secretion could be mediated directly at the level of the pituitary, possibly in the PT [21]. Interestingly, a similar distinction between hypothalamic melatonin target sites involved in the control of
gonadotropin secretion and other sites involved in that of prolactin has also been proposed for the hamster [30]. Dopaminergic Neurons as Possible Relays between Melatonin Targets and LHRH Neurons Several pieces of evidence suggest that melatonin does not act directly on LHRH neurons. First, the distribution of most LHRH neurons does not match that of the putative sites of action of melatonin. Most LHRH neuronal perikarya are located in the preoptic area (60%) with a few located in the MBH (15%); some of these project to the median eminence and abut portal vessels [2,18]. Secondly, several neurotransmitters have been implicated in the regulation of LHRH secretion by melatonin; of these, the potential role of dopamine is particularly well documented. Many studies have suggested that dopamine is involved in transducing the negative feedback of E on LHRH secretion. For instance, systemic injection of a dopamine antagonist (pimozide) during anoestrus (strong negative feedback of E) induced a temporary in-
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FIG. 4. Effects of photoperiodic and estrogen treatments on mean (6SEM) LH pulse frequency and prolactin level (left panels) and tyrosine hydroxylase (TH) activity, dopamine, and noradrenaline contents in the pituitary stalk median eminence (right panels) of ovariectomized ewes. TH activity and monoamine contents were measured individually in the same tissue extract of stalk-median eminence of ovariectomized ewes treated by either 133 to 136 long days (LD) or 63 to 66 short days (SD). For each photoperiodic regime half of the ewes were bearing a 2-cm subcutaneous implant of estradiol (1E) and half did not receive any steroid replacement (2E). LH and prolactin concentrations were determined in serial samples of blood obtained 3 to 6 days before the end of the experiment, i.e., after 60 short days or 130 long days for the SD and LD groups, respectively. *p , 0.05; **p , 0.01 (adapted from [55]).
crease in LH secretion [31]. Similarly, pimozide caused a temporary increase in LH secretion in long-day exposed (photo-inhibited) OVX1E ewes [14]. The A15 hypothalamic cell group appears to be a key dopaminergic structure involved in mediating the inhibitory effects of E because a neurotoxic lesion of this structure during anestrus caused an increase in LH secretion [46]. Furthermore, it has been shown that E increases tyrosine hydroxylase (TH, rate limiting step enzyme of catecholamine synthesis) activity in long day-treated ewes [7] and induces c-fos gene expression in TH-immunoreactive cells of this structure in a season-dependent manner [19]. A similar role has been proposed for the A14 dopaminergic cell group [10]. Because a major effect of melatonin is a modulation of E negative feedback on LHRH secretion, this implication of dopamine makes these neurons likely candidates to act as relays between melatonin target sites and LHRH neurons [45]. To test this hypothesis, we first determined whether photoperiod and melatonin could modulate the activity of some dopaminergic neurons in the hypothalamus. Exposure to stimulatory short days resulted in decreased dopaminergic activity in the median eminence as assessed both by a reduction in dopamine content and in TH activity (Fig. 4) [44,55]. No effect of short-day exposure on noradrenaline content in this structure or on TH activity in the other hypothalamic areas investigated was found [55]. The stim-
ulation of LH secretion by a melatonin implant caused a parallel reduction in TH activity, which suggests strongly that the effect of photoperiod on TH activity is mediated by melatonin [56]. The inhibition of median eminence TH activity by short days or by treatment with a melatonin implant is expressed at a time when the inhibition of prolactin secretion is already maximal (Fig. 5), suggesting that these photoperiod-induced changes in TH activity are independent of the regulation of prolactin secretion [56]. Rather, they appear to be related to the photoperiodic regulation of LH secretion. Indeed, the pharmacological blockade of TH locally in the median eminence of long-day photo-inhibited ewes led to an increase in LH secretion [52]. This finding is consistent with the effect of pimozide implants in the median eminence of anestrous ewes [9]. These data suggest, therefore, that a reduction in TH activity in the median eminence is an important component of the stimulatory effect of melatonin on LHRH output. However, such modulation of TH activity appears to be E independent because the photoperiod-induced changes in TH activity are similar in OVX and OVX1E ewes [55]. Thus, in contrast to the A15 and possibly A14 dopaminergic nucleus, which are involved in the modulation of the E negative feedback, the dopaminergic neurons of the median eminence appear to be involved upstream relative to the integration of the E signal. The median eminence in sheep is a
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MALPAUX ET AL. structure rich in dopaminergic terminals but contains no THimmunoreactive perikarya [51]. The localization of the cell bodies projecting their axons to the median eminence, and more generally the anatomical and functional relationship between the A14 and A15 nuclei and the median eminence, have yet to be determined. In addition to dopamine, other neurotransmitters have been implicated in the seasonal neuroendocrine changes. These include serotonin, particularly via 5HT2 receptors [15,16], excitatory aminoacids [23,54], and NPY [42]. Other neuronal systems that are possible transducers of the E negative feedback effects [10,11] may also be involved. CONCLUSION The regulation of LHRH secretion by melatonin is central to the photoperiodic regulation of reproductive activity. The target sites of melatonin appear to be located in the hypothalamus. Melatonin does not act directly on LHRH neurons; rather, its action is probably mediated by a complex network of interneurons. Within that network, the dopaminergic terminals of the median eminence play an important role. The unravelling of this ‘‘black box’’ linking melatonin target sites and the LHRH neurones is a challenge to understand the interaction between melatonin and the endogenous rhythm of reproduction. Of particular interest will be the distinction between the mechanisms involved in generating this circannual rhythm and those involved in mediating the synchronizing effect of melatonin. ACKNOWLEDGEMENTS
Original research performed in this laboratory was supported in part by a grant from Re´gion Centre. C.V. was in receipt of a Ph.D. grant from INRA and Re´gion Centre. Collaboration with DCS was made possible by INRA/BBSRC and ALLIANCE exchange schemes.
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FIG. 5. Mean (6SEM) difference in the number of LH pulses/6 h between the control LD period and the end of the experiment (top), mean (6SEM) plasma prolactin concentrations at the end of the experiment (middle), and mean (6SEM) TH activity in the median eminence of ewes. All ewes were ovariectomized and treated with a subcutaneous implant of oetradiol. They were pretreated with long days (16L:8D) and then allocated to one of four groups treated with 0, 5, 25, or 76 short days. LH secretory profiles were determined from LH concentration measured in blood samples obtained every 10 min for 6 h at two different periods of the photoperiodic treatment: (1) 7 days before the end of the last LD period, and (2) 3 to 4 days before the end of the experiment (i.e., in LD or on average after 3, 23, or 74 SD). Prolactin concentrations were determined in every third sample during the last 3 h of the second sampling period. TH activity was measured individually in the stalk-median eminence of the same ovariectomized ewes (n 5 7 per group) *p , 0.05; **p , 0.01 (adapted from [56]).
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