Jean-Claude Thiéry

LHRH-immunoreactive structures in the sheep brain

SummaryNeural structures containing luteinizing hormone-releasing hormone (LHRH) are characterized in adult ewe and female lamb brains. Three anti-LHRH antisera are used in an immunofluorescent or immunoperoxidase method. On our preparations, all three gave the same results, expressed as number of labelled cells (about 2500 in a whole brain). It was found that 95% of the LHRH-immunoreactive cells are located in the preoptico-hypothalamic area, where cell bodies are localized mainly (50%) in the area surrounding the organum vasculosum of the lamina terminalis (OVLT); they are also found in a more anterior section of the medial part of the olfactory tubercle and the medial septum (14%), in a more posterior situation in the anterior and lateral hypothalamus (16%), and in the mediobasal hypothalamus (15%). Fibres originating in various part of the whole preoptico-hypothalamic group reach the OVLT and the median eminence. The remaining cells (5%) and fibres are found in various tel-, di-, and mesencephalic areas.

Role of hypothalamic catecholamines in the regulation of luteinizing hormone and prolactin secretion in the ewe during seasonal anestrus.

Separate studies with ewes have shown that catecholamines play an inhibitory role in the control of LH secretion during anestrus, and that there are structures in the lateral retrochiasmatic area (L-RCh), which could be involved in the regulation of gonadotrophin secretion. These observations have led to the hypothesis that the catecholaminergic structures in the L-RCh mediate the inhibition of pulsatile LH secretion by estradiol in the anestrous ewe. We tested this hypothesis by injecting 6-hydroxydopamine (6OH-DA) into the L-RCh of ovariectomized ewes during the anestrous season, and comparing the secretion of LH and prolactin in these animals with that in sham (injected with vehicle) and control (no injection) animals, in the presence and absence of exogenous estradiol. Finally, the effectiveness of the toxin was assessed by immunocytochemical techniques. When the ewes were treated with estradiol, LH pulse frequency was significantly lower in the controls (mean 1.1 pulses/4 h) and shams (0 pulses/4 h) than in the ewes treated with 6OH-DA (3.1 pulses/4 h). When the estradiol implants were removed, the frequencies increased to 5.1 pulses/4 h for the controls and 5.7 pulses/4 h in the ewes treated with 6OH-DA. These were not significantly different. Plasma prolactin levels were significantly reduced by 6OH-DA treatment. The 6OH-DA ewes recovered their response to estradiol by 14 weeks after the injection. The anatomical study at the end of the experiment revealed a difference between treated and control ewes of only 15% in the numbers of dopaminergic cells in the L-RCh.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Multiunit Activity in the Anterior Median Eminence and Adjacent Areas of the Hypothalamus of the Ewe in Relation to LH Secretion

Changes in multiunit activity (MUA) in the anterior median eminence and adjacent tissues just posterior to the optic chiasma were recorded in lightly anaesthetized, ovariectomized ewes after an i.m. injection either of oil (group I) or of 50 microgram oestradiol benzoate (ODB) in oil (group II). Changes in plasma LH concentration that occurred during the recording were determined by radioimmunoassay. In both groups, when the electrodes reached the correct site, the MUA showed a bursting pattern of firing with a frequency of 0.5-1.5 bursts/s. In group I (10 ewes), the MUA showed circhoral variations at intervals of 55.1 +/- 2.8 min similar to those for LH pulses. Plasma LH levels, measured every 10 min, for 2 h in 8 ewes, showed a total of 14 pulses which were always preceded by an increase in MUA. Hormonal values and bioelectrical activity were positively correlated (p < 0.01). When cumulated hourly, the mean MUA in group I remained stable, from 10.5 to 22.5 h after oil treatment. Then, intravenous injection of 100 microgram of ODB induced a rapid increase in MUA. During the 2 h after this injection, we observed only two pulses of LH in the 8 studied ewes. In animals injected i.m. with ODB before the recording (group II, n = 16), the mean MUA (cumulated every hour) from 10.5 to 22.5 postinjection was significantly higher (p < 0.001) than in animals from group I (control). Within group II, 5 ewes discharged an obvious peak of LH, 18-24 h after ODB injection and showed a shortening of the MUA circhoral period (which varied from 10 to 20 min, at the time of initiation of the surge) followed by an increase in the mean amplitude. These 5 ewes showed a significantly higher increase (p < 0.03) in the MUA per hour (from 10.5 to 22.5 h after ODB) than the 9 which did not show an LH peak. Thus, the changes in MUA were clearly related in time to changes in LH secretion whether occurring as spontaneous pulses or as peaks induced by ODB.

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.

Monoamine Content of the Stalk‐Median Eminence and Hypothalamus in Adult Female Sheep as Affected by Daylength

In the ewe, plasma luteinizing hormone and prolactin concentrations exhibit seasonal variations. During long days, inhibition of pulsatile luteinizing hormone secretion is mediated by monoamines. In a model of ovariectomized ewes bearing a subcutaneous oestradiol implant, we previously showed that the steroid‐dependent inhibition of luteinizing hormone involves the A15 dopaminergic nucleus of the retrochiasmatic area. In the present work, we compared the aminergic activities of tele‐diencephalic structures in groups of ovariectomized ewes under artificial illumination for short versus long days (8 versus 16 h/day of light, respectively). Half the animals in each group were bearing a subcutaneous oestradiol implant. Using high‐performance liquid chromatography and electrochemical detection, we measured the levels of amines and amine metabolites in ‘punches’ of tissues from regions containing luteinizing hormone‐releasing hormone axon terminals or cell bodies and catecholaminergic structures. Concurrently, we checked the pulsatile luteinizing hormone release and plasma prolactin concentration to assess the ability of our model to mimic seasonal changes in the hormonal status.

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

Several neurotransmitters are implicated in the photoperiodic regulation of prolactin and luteinising hormone (LH) secretion in the ewe. This work investigated whether catecholamines, γ‐amino butyric acid (GABA), excitatory amino acids and serotonin diencephalic contents are affected by photoperiod and how such changes relate to the seasonal effects of photoperiod on LH and prolactin secretions. Moreover, to determine whether photoperiod can influence catecholamine biosynthesis, the activity of its rate limiting enzyme, tyrosine hydroxylase (TH) was also investigated. TH activity and the tissue content of the monoamines and their metabolites were measured in stalk‐median eminence (SME), preoptic area (POA) and the mediobasal, mediodorsal and laterobasal aspects of the hypothalamus. Investigation of excitatory amino acids and GABA was limited to the POA and the SME. Ovariectomized ewes were initially maintained in long days (LD) for 70 days. Thereafter half the ewes remained exposed to long days and the other half were transferred onto short days (SD) for 63 to 66 days to induce a stimulation of LH secretion and an inhibition of prolactin secretion. In each photoperiodic regime, half the ewes were treated with a subcutaneous oestradiol implant (+E) and half were not (−E). As expected, short days induced a decrease in prolactin and an increase in pulsatile LH secretion. These neuroendocrine changes were associated with a decrease in the TH activity of the SME in both oestradiol treated and non treated animals (146.5±24.1, 167.6±26.5 U TH/g of tissue in LD‐E and LD+E vs 83.5±12.4 and 95.0±30.2 U TH/g of tissue in SD‐E and SD+E animals; P≤0.01). A similar and parallel short day‐induced decrease was observed in the tissue content of dopamine and its metabolite, 3,4‐dihydroxy‐phenylacetic acid (SD level were 55% of LD levels, P<0.05). In POA, a short day‐induced decrease in dopamine (18%; P≤0.05) and GABA (16.4%; P≤0.05) content and an oestradiol‐induced decrease in aspartate (15.6%; P≤0.05) content were found. This study provides the first report of a photoperiodic control of the synthesis activity of catecholaminergic neurones of the SME in the ewe. The photoperiod‐induced changes in dopaminergic activity at the level of the SME were associated with changes in LH and prolactin secretion indicating that TH activity of dopaminergic neurones of the SME could be a critical component of the photoperiodic regulation of LH and/or prolactin secretion. In particular, this finding is in agreement with the hypothesis that photoperiod can control a dopaminergic pathway inhibitory of LH secretion and which ends in the median eminence.

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...

Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep.

Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohisto-chemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibers were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase-immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep.

Estradiol Increases Multiunit Electrical Activity in the A15 Area of Ewes Exposed to Inhibitory Photoperiods

Abstract Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E2). This increase in the inhibitory effects of E2 is controlled by photoperiod and appears to be mediated, in part, by dopaminergic neurons in the retrochiasmatic area of the hypothalamus (A15 group). This study was designed to test the hypothesis that E2 increases multiunit electrical activity (MUA) in the A15 during inhibitory long days. MUA was monitored in the retrochiasmatic area of 14 ovariectomized ewes from 4 h before to 24 h after insertion of an E2-containing implant subcutaneously. In six of these ewes, MUA activity was also monitored before and after insertion of blank implants. Three of the 14 ewes were excluded from analysis because E2 failed to inhibit LH. When MUA was recorded within the A15, E2 produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E2 did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E2 increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E2 negative feedback during long photoperiods.