B. Malpaux

EVIDENCE THAT MELATONIN ACTS IN THE PREMAMMILLARY HYPOTHALAMIC AREA TO CONTROL REPRODUCTION IN THE EWE : PRESENCE OF BINDING SITES AND STIMULATION OF LUTEINIZING HORMONE SECRETION BY IN SITU MICROIMPLANT DELIVERY

Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.

Melatonin Enters the Cerebrospinal Fluid through the Pineal Recess

The pineal recess (PR), a third ventricle (IIIV) evagination penetrating into the pineal gland, could constitute a site of melatonin passage to the cerebrospinal fluid (CSF) and explain the high concentrations of melatonin in this fluid. To test this hypothesis, we characterized melatonin distribution in the IIIV of sheep by CSF collection in the ventral part of IIIV (vIIIV) and in PR. At 30 l/min collection rate, melatonin concentrations were much higher in PR than in vIIIV (19,934 6,388 vs. 178 70 pg/ml, mean SEM, respectively, P < 0.005), and they increased in vIIIV when CSF collection stopped in the PR (P < 0.05). At 6 l/min, levels increased to 1,682 585 pg/ml in vIIIV and were not influenced by CSF collection in the PR. This concentration difference between sites and the influence of PR collection on vIIIV levels suggest that melatonin reaches the PR and then diffuses to the IIIV. To confirm the role of PR, we demonstrated that its surgical sealing off decreased IIIV melatonin levels (1,020 305 pg/ml, compared with 5,984 1,706 and 6,917 1,601 pg/ml in shams or animals with a failed sealing off, respectively, P < 0.01) without changes in blood levels. Therefore, this study identified the localization of the main site of penetration of melatonin into the CSF, the pineal recess. (Endocrinology 143: 84 –90, 2002)

Control of sheep and goat reproduction: Use of light and melatonin

Abstract Breeds of sheep and goats from temperate latitudes exhibit seasonal variations of breeding activity which are controlled by annual photoperiodic changes. Short days (SD) stimulate sexual activity, but prolonged exposure results in refractoriness to short days and subsequent cessation of reproductive activity. Refractoriness can be broken by exposing the animals to long days (LD), thus alternations between LD and SD are essential for the photoperiodic control of seasonal reproduction. Light pulses during the dark period can mimic a long day (‘LD’) and melatonin treatments can mimic a short day. For out-of-season control of sexual activity, treatments using the succession ‘LD’-decreasing days or ‘LD’-melatonin were very effective in advancing puberty in young rams in which sperm production was increased, permitting these animals to be submitted earlier for progeny testing, using artificial insemination (AI). In adult rams, such treatments also caused an important increase in testicular weight and sperm production in the spring. In the female goat, the succession ‘LD’-melatonin treatment efficiently induces and maintains oestrous and ovulatory activities in spring, leading to high fecundity after natural mating. This treatment so far appears to be less effective in the seasonal sheep breeds of Northern Europe than in goats. However, melatonin alone can be used after the end of May to advance the breeding season and to increase fecundity. Induction of permanent reproductive activity in rams and bucks was made possible by the observation that monthly alternations between LD and SD (short light cycles) abolished seasonality of behavioural and spermatogenic activities. These males could be used all the year round to produce a high number of AI doses without variations in sperm quality and with no variation in fertility. Short light cycles can be used in open barns by alternating ‘LD’ and melatonin. In contrast, in the ewe, such short light cycles were unable to abolish seasonality of ovulatory activity. Knowledge of the different effects of photoperiod on neuroendocrine pathways and the reproductive activity in sheep and goats has therefore allowed us to successfully apply light treatments to control seasonal reproductive activity in field conditions and in males raised in AI centres.

Male Reproductive Condition Is the Limiting Factor of Efficiency in the Male Effect During Seasonal Anestrus in Female Goats

Abstract Two experiments were conducted to determine whether the failure of males to induce sexual activity in goats during seasonal anestrus is due to unresponsiveness of females to male stimulus or insufficient stimulation from males. In the first study, one group of males (sexually inactive, SI; n = 4) was kept under natural photoperiod while the other (sexually active, SA; n = 4) was subjected to 2.5 mo of long days (16L:8D) and received 2 s.c. implants of melatonin. Two mo later, 2 different flocks of anovulatory goats previously separated from bucks were exposed to either SI (n = 34) or SA (n = 40) bucks. Progesterone assays and estrous behavior were used to determine ovarian and behavioral responses of the females to teasing. Of the goats exposed to SI males, only 2 ovulated, and none showed estrous behavior during the 35 days of the study. In contrast, all females (40 of 40) in contact with SA males ovulated and showed at least one estrous behavior during the first 11 days following male introduction (P < 0.001). Overall, 38 of 40 females stimulated with SA bucks were diagnosed pregnant at Day 35, according to progesterone assay (versus 0 in SI-treated group: P < 0.001). To control for a possible difference of responsiveness between flocks, the experiment was repeated 1 yr later using a single flock of goats divided into 2 groups. Again, over the first 14 days, 1 of 33 goats showed estrous behavior in the SI-treated group versus 27 of 33 in the SA-treated group (P < 0.001). Therefore, treating bucks with long days and melatonin increased their teasing capacity to induce sexual activity in females during anestrus. These results indicate that the absence of response to teasing at this time of the year is not due to female unresponsiveness, but to insufficient stimulation from the male.

Evidence for an annual reproductive rhythm independent of food availability in male creole goats in subtropical northern mexico

The aim of this study was to determine if there is a seasonal pattern of sexual activity dependent on food availability in male Creole goats in subtropical Mexico. The study was conducted in the Laguna Region in the State of Coahuila, Mexico (26 degrees N). Male Creole goats (n = 8) were kept in a shed, fed alfalfa ad libitum and given 200 g of concentrate daily throughout the study. Live weight and testicular weight were determined every 2 wk. Sexual behavior and sperm production were determined monthly. Blood samples were obtained weekly to determine testosterone plasma concentrations. All variables were subjected to sinusoidal modeling procedures and showed important seasonal variations (P < 0.0001) with different phase angles for body weight, testicular weight and testosterone plasma concentrations. The nadir of live weight occurred in November and the peak in May. The lowest testicular weight (90 g) and testosterone plasma concentrations (0.1 ng/mL) were observed in January and February, respectively, while the peaks were observed in July and August (145 g and 10 ng/mL, respectively). Ejaculation latency also varied during the study, being low between May and November (96 sec) and reaching a peak in April (183 sec). Minimum number of spermatozoa per ejaculate occurred between February and April (1.4 x 10(9) cells/ejaculate) while the maximum number was observed between May and September (2.8 x 10(9) spermatozoa/ejaculate). Progressive sperm motility was low between January and April (3.04 on average) and high between May and November (about 3.55 on average). The percentage of live spermatozoa diminished between January and April (68% in April) and then increased to values around 80% between May and November. These results lead us to conclude that male Creole goats in Northern Mexico, fed constantly throughout the year, exhibit seasonality in their reproductive activity. Intense sexual activity occurred between May and December.

Control of the circannual rhythm of reproduction by melatonin in the ewe

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.

Seasonal breeding in sheep: Mechanism of action of melatonin

Abstract Melatonin, through its duration of nocturnal secretion, transduces the effects of daylength on the reproductive axis by modifying the pulsatile secretion of luteinising hormone-releasing hormone (LHRH). It is not yet known where or how this pineal indoleamine acts to exert this effect. Although melatonin binding sites are preferentially localised in the pars tuberalis (PT) of the adenohypophysis, which suggests that this may be the site of action, the direct delivery of melatonin into the PT does not influence LH secretion. In contrast, melatonin delivered in the mediobasal hypothalamus mimics the effects on LH secretion of peripherally administered melatonin. These results suggest that, despite the presence of high melatonin binding in the PT, the mediobasal hypothalamus contains the physiological target site of melatonin. The action of melatonin on LHRH secretion does not seem to be directly on LHRH neurones; rather, it appears to involve a complex circuit of interneurones. Both the A15 dopaminergic cell group and dopaminergic neuronal terminals of the median eminence are parts of that network but their anatomical and functional relationships are still unknown. Serotonin and excitatory amino acids also appear to be involved in the seasonal regulation of LHRH secretion. Characterisation of this neuronal circuit is a key to understanding photoperiodic regulation of reproduction.

Characterization of the Short Day-Induced Decrease in Median Eminence Tyrosine Hydroxylase Activity in the Ewe: Temporal Relationship to the Changes in Luteinizing Hormone and Prolactin Secretion and Short Day-Like Effect of Melatonin1

In the ewe, photoperiod modulates LH and PRL secretion as well as median eminence (ME) dopaminergic activity. The studies reported here were designed to characterize the functional significance of this photoperiodic modulation of ME dopaminergic neuron activity in relation to the regulation of LH and PRL secretion. The aim of the first experiment was to assess whether photoperiodic changes in hypothalamic dopaminergic activity were temporally linked to changes in either PRL or LH secretion. The purpose of the second experiment was to determine whether melatonin mimicked the effects of photoperiod on ME dopaminergic activity. In the first experiment, LH and PRL secretion, hypothalamic tyrosine hydroxylase (TH) activity, and catecholamine contents were determined in ovariectomized estradiol-treated ewes either during long days (LD; control group) or after 5, 25, and 76 short days (SD). SD were associated with a stimulation of LH secretion and a decrease in ME TH activity, which were both expressed only in t...

Does a short loop feedback mechanism for the control of luteinizing hormone secretion exist in the ewe

It is not known whether a short loop feedback mechanism for the regulation of LH exists in sheep. This study on ovariectomized ewes investigated whether a bolus injection (10, 1, and 0.1 μg LH or 1μ g BSA; n = 4) or a 3-h continuous infusion of exogenous LH (100 or 1 ng/min; n = 7) into the third ventricle through a permanent indwelling cannula could influence the activity of the GnRH pulse generator, as determined by measurement of endogenous LH secretion. To assess the potential for involvement in a LH short loop feedback system and to estimate the level of LH in the hypothalamic milieu, the concentrations of LH in the peripheral circulation, portal circulation, and third ventricle were measured during an estradiol-induced preovulatory LH surge (n = 4). Neither the bolus nor continuous administration of LH into the third ventricle had any effect on the mean interpulse interval, nadir, pulse amplitude, or circulating level of systemic LH. Furthermore, despite portal LH concentrations being more than 20-f...