Photoperiodic variations of the polysialylated form of neural cell adhesion molecule within the hypothalamus and related reproductive output in the ewe.

Cellular mechanisms induced by melatonin to synchronise seasonal reproduction in several species, including sheep, remain unclear. We sought to evaluate the scale and physiological significance of neural plasticity in order to explain the delay between the change of duration of melatonin secretion and the change of reproductive status following a transition from long days (LD, 16 h light/24 h) to short days (SD, 8 h light/24 h) and from SD to LD. Using Western blots in ovariectomised oestradiol-replaced ewes, we evaluated the content of the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a plasticity marker, in the hypothalamus. From day 15 following a transition to SD, most hypothalamic areas showed a decrease of PSA-NCAM level that was particularly significant in the preoptic area (POA). Following a transition to LD, PSA-NCAM content increased at day 15 in most regions except in the premammillary hypothalamic area (PMH) in which a significant decrease was noted. The functional importance of PSA-NCAM variations for seasonal reproduction was assessed for the PMH and POA. PSA-NCAM was degraded by stereotaxic injections of endoneuraminidase N and luteinising hormone (LH) secretion was recorded in treated and control ewes. Degradation of PSA-NCAM in the PMH in SD-treated ewes failed to produce a significant effect on LH secretion, whereas a similar treatment in the POA before a transition to SD delayed activation of the gonadotroph axis in two-thirds of the ewes. Our results suggest that the photoperiod controls variations of the hypothalamic content of a plasticity marker and that these might be important for the regulation of seasonal reproduction, particularly in the POA.

Estradiol increases multiunit electrical activity in the A15 area of ewes exposed to inhibitory photoperiods.

Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E(2)). This increase in the inhibitory effects of E(2) 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 E(2) 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 E(2)-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 E(2) failed to inhibit LH. When MUA was recorded within the A15, E(2) produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E(2) did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E(2) increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E(2) negative feedback during long photoperiods.

Estradiol acts locally within the retrochiasmatic area to inhibit pulsatile luteinizing-hormone release in the female sheep during anestrus.

In the present study we have identified a site of action of estradiol in the inhibition of LH secretion during anestrus in the ewe. In the first experiment, we studied six sites: the medial preoptic area, the lateral preoptic area, the ventromedial hypothalamus, the ventrolateral hypothalamus, the retrochiasmatic area (RCh), and the periventricular posterior hypothalamus. We compared the changes in parameters of pulsatile LH secretion (interpulse interval, mean nadir, mean amplitude, and mean area under curve) during three 6-h sampling periods: before and 30-36 h and 9 days after intracerebral implantation of crystalline estradiol. Animals that received estradiol in the RCh (n = 5) showed a significantly greater increase in both the intervals between pulses of LH (up 116%, p < 0.03) and the area under the curve (up 180%, p < 0.01) than any of the other groups of 7 animals. In the second experiment, implantation of estradiol in the RCh (n = 6) induced an increase in the intervals between pulses of LH (p < 0.03), whereas receiving an empty implant (n = 6) had no effect, showing that estradiol specifically induced increases in the intervals between pulses. Thus, estradiol appears to act in the RCh where the dopaminergic A15 nucleus, known to inhibit pulsatile LH release, is located.

Initiation of the oestradiol-induced inhibition of pulsatile LH secretion in ewes under long days: comparison of peripheral versus central treatment and neurochemical correlates.

In the ewe, the inhibition of pulsatile LH secretion by oestradiol during long days depends on dopaminergic activity and could involve amino acid transmitters. In the first experiment of the present study we observed the changes in LH secretion in ovariectomised ewes under long days immediately after subcutaneous implantation of oestradiol (peripheral treatment). In the second experiment, in order to identify the site of action of oestradiol, we observed the LH changes following intracerebral infusion of oestradiol through a microdialysis membrane (central treatment) within the preoptic area, the mediobasal hypothalamus (MBH) or the retrochiasmatic area (RCh) and measured amino acids and catecholaminergic transmitters and metabolites within the dialysates. With peripheral treatment, the amplitude, the nadir and the area under the LH pulse curve decreased within 4 to 8 h of the insertion of a subcutaneous oestradiol implant. After 18 h, the amplitude and the area under the pulses increased, as well as the intervals between pulses (from 49.9 + 1.4 min to 75.6 +/- 5.9 min). With central oestradiol treatment. LH changes were similar whatever the site of oestradiol infusion, suggesting either multiple sites of action or diffusion between structures. Twenty hours after the beginning of intracerebral oestradiol treatment, the amplitude and the area under the pulses increased, as did the interval between LH pulses (from 49.5 +/- 4.1 min to 73.2 +/- 14.2 min). Comparison of peripheral with central oestradiol treatment suggested that the long-lasting decrease in the nadir, as well as the transitory decrease in the amplitude and area, before 18 h in experiment 1 are reflections of hypophysial effects. In contrast, the increases in amplitude and area under the LH pulse curve seen 18-20 h after oestradiol in the two experiments could be due to the higher amplitude of LHRH pulses, as a result of an early stimulatory effect of oestradiol. After central oestradiol infusion, there was a decline in the concentration in the dialysate of two metabolites of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the RCh, suggesting an early inhibition of monoamine oxidase by the steroid. During the inhibition of LH pulsatility the concentration of gamma-aminobutyric acid in the dialysate from the RCh and the MBH increased, suggesting the participation of this transmitter in the changes induced by oestradiol under long days.

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, gamma-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 < or = 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 < or = 0.05) and GABA (16.4%; P < or = 0.05) content and an oestradiol-induced decrease in aspartate (15.6%; P < or = 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.

Effect of hypothalamic infusion of a dopamine D1 receptor antagonist on prolactin secretion in the ewe.

In this study we investigated whether dopamine D1 receptors in the hypothalamus are involved in the control of prolactin secretion in ovariectomised, oestradiol implanted ewes. The D1 antagonist SCH23390 or vehicle was infused into either the preoptic area (POA) or the ventromedial hypothalamus (VMH). During infusion into the VMH, prolactin concentrations declined significantly and did not return to control values until more than 60 min after the infusions had stopped. In contrast, infusion into the POA had no significant effect. These results are in accord with the hypothesis that dopaminergic pathways within the hypothalamus stimulate prolactin secretion via dopamine D1 receptors in the VMH.

Dopaminergic control of LH secretion by the A15 nucleus in anoestrous ewes.

Annual variations in the secretion of LH are responsible for seasonal changes in ovulatory activity in ewes. This hormonal pattern reflects an increase in the intensity of the negative feedback exerted by oestradiol under long days. Neuropharmacological studies have shown that this inhibition of LH secretion involves activation of catecholaminergic systems from preoptic and mediobasal hypothalamus (MBH) by oestradiol during anoestrus, and that 5-hydroxytryptamine inputs may also play a role. Within the MBH, the most important structures appear to be the retrochiasmatic region of the hypothalamus, which contains the A15 dopaminergic nucleus, and the median eminence, which contains the axon terminals of the GnRH cells controlling the pulsatile release of LH. In ovariectomized ewes in which oestradiol tonically inhibits LH secretion during the anoestrous season, LH pulse frequency is increased when the cells of the A15 nucleus are destroyed. The median eminence and other mediobasal structures contain more catecholamines and their metabolites under long days than under short days. Microdialysis of the A15 nucleus in vivo during long days revealed increased catecholaminergic activity under oestradiol treatment due to stimulation of tyrosine hydroxylase, the rate-limiting enzyme in the pathway of catecholaminergic synthesis. Tyrosine hydroxylase activity within the median eminence is increased under the various photoperiodic regimens that inhibit LH secretion. Neurochemical changes in the A15 nucleus and median eminence, in response to photoperiodic or oestradiol treatments, suggest a functional relationship which acts at the level of the GnRH axon terminals.

Estradiol increases tyrosine hydroxylase activity of the A15 nucleus dopaminergic neurons during long days in the ewe.

In the ewe, the inhibition of LH secretion during long days results from increased negative feedback by estradiol. This effect depends on aminergic systems, mainly in the lateral retrochiasmatic area, a region that includes the dopaminergic A15 nucleus. Recently we demonstrated that the inhibition of LH secretion by estradiol under long days is accompanied by an increase in extracellular levels of amine metabolites (3,4-dihydroxyphenylacetic acid [DOPAC], homovanillic acid [HVA], and 5-hydroxyindolacetic acid [5-HIAA]) in the lateral retrochiasmatic area of the hypothalamus. The increase in catecholamine metabolite levels could be due to stimulation of tyrosine hydroxylase (TH) activity in the catecholaminergic systems of this region. In the present study, we tested this hypothesis by incorporating in vivo measurement of TH activity in the brain into our microdialysis model. We used this approach to first examine the effect of estradiol on TH activity in both the lateral retrochiasmatic area and the caudate nucleus during long days (experiment 1). Two dialysis sessions were carried out on each of six ewes: one after a 10-day estradiol treatment (s.c. implant) and the other after 10 days without estradiol treatment. Estradiol treatment reduced pulsatile LH secretion without affecting the secretion of prolactin. The steroid also significantly increased TH activity in the lateral retrochiasmatic area as assessed by the formation of L-3-4 dihydroxyphenylalanine (L-DOPA). There was no variation in the TH activity of the catecholaminergic systems of the caudate nucleus, suggesting that the effect of estradiol is specifically hypothalamic. In experiment 2, we estimated the relative contribution to the accumulation of L-DOPA in the lateral retrochiasmatic area by dopamine (DA) synthesis in the dopaminergic neurons and noradrenaline (NA) synthesis in the noradrenergic afferents of the A15 nucleus. This study also allowed us to test whether DOPAC and HVA molecules released in this medium originate from DA metabolism in dopaminergic cells of the A15 nucleus or in its noradrenergic afferents. We monitored TH activity as well as amine metabolite concentrations in the lateral retrochiasmatic area after a partial destruction of noradrenergic nerves in the brain using intracerebroventricular (i.c.v.) injections of nomifensine followed by 6-hydroxydopamine (6-OHDA) injections. We compared the responses in four experimental ewes with those in four control ewes receiving a single i.c.v. injection of nomifensine only. Decreases in extracellular concentrations of 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG) in the left lateral retrochiasmatic area and the tissue content of NA in the contralateral structure confirmed the effectiveness of the lesion of the noradrenergic endings.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-day effects of melatonin on luteinizing hormone secretion in the ewe: evidence for central sites of action in the mediobasal hypothalamus.

Experiments were designed to localize the central sites of action of melatonin involved in the control of seasonal reproduction. Ewes were exposed to long days and received microimplants of melatonin in the preoptic area (n = 9), anterior hypothalamus (n = 4), dorsolateral hypothalamus (n = 4), or mediobasal hypothalamus (n = 12). The effects of implants were determined by comparison with control ewes (untreated or sham-operated, n = 10) and with ewes treated with an s.c. implant of melatonin (n = 8) or ewes subjected to short days (n = 8). All ewes were ovariectomized and treated s.c. with a silastic capsule of estradiol. Melatonin released in the preoptic area as well as in the anterior and lateral hypothalamus did not cause any difference as compared with the controls (no stimulation of LH secretion and no inhibition of prolactin secretion). In contrast, melatonin implanted in the mediobasal hypothalamus caused an increase in LH secretion in 7 of the 12 ewes on Day 53.0 +/- 4.2 after implantation (mean +/- SEM). Their response was not different compared with that of ewes treated s.c. with melatonin or exposed to short days either in terms of timing (Day 56.3 +/- 6.2 and 59.5 +/- 3.1, respectively, for controls) or of amplitude of the LH response. Similarly, melatonin caused only a reduction of prolactin secretion in the mediobasal, s.c., and short-day groups. It is concluded that the mediobasal hypothalamus or the surrounding tissues could be the sites of action of melatonin involved in the control of seasonal reproduction.

Oestradiol increases the extracellular levels of amine metabolites in the ewe hypothalamus during anoestrus: a microdialysis study.

Giving a subcutaneous oestradiol implant during anoestrus to ovariectomized ewes inhibits pulsatile LH secretion. This effect results from an increased negative feedback of oestradiol and depends on the synthesis of biogenic amines, mainly from the mediobasal hypothalamus. In the present study, we examined the effect of oestradiol on the extracellular levels of amines and their metabolites. Eight ewes were sampled by microdialysis from the lateral retrochiasmatic area, including the dopaminergic A15 nucleus, during inhibition of LH secretion by long days. Two dialysis sessions were carried out on each ewe; one after a 10-day oestradiol treatment and the other one after 10 days without oestradiol treatment. Half of the ewes were first oestradiol-treated then untreated, the other half received the treatment in the reverse order. Oestradiol caused a decline in pulsatile LH secretion without affecting the secretion of prolactin. This steroid also led to a significant increase in the levels of amine metabolites: 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in the extracellular medium. These results demonstrate the effect of oestradiol on aminergic activity as related to changes in hormonal secretions during long days (16 h of light per 24 h). Thus our data support the hypothesis that amines inhibit gonadotrophic secretion during anoestrus in the ewe and suggest that there is an activation of the aminergic neurones from the retrochiasmatic area in this regulatory mechanism.

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)

LHRH-immunoreactive structures in the sheep brain.

Neural 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.

Hypothalamic multiunit activity and LH secretion in conscious sheep.

The relationship between the pulsatile secretion of luteinizing hormone (LH) and multiunit activity (MUA) in the median eminence and retrochiasmatic area (RCh-ME) of the hypothalamus was investigated within 13 conscious, ovariectomized ewes during the anoestrous season. Eight of the ewes had been treated with oestradiol to reduce their LH pulse frequency. To test whether the activity had been recorded from sites involved in the control of GnRH release, we electrically stimulated the sites studied with the recording electrode and used the LH responses to classify the animals retrospectively for analysis of the MUA data. Following stimulation, LH secretion was either stimulated (Group STIM, n = 5 ewes), inhibited (Group INHIB, n = 4) or showed no response (Group NR, n = 4). Statistical analysis of the MUA data revealed that the onset of LH pulses was associated with an increase in cell activity in STIM ewes and a decrease in activity in both Group INHIB and Group NR ewes. Histological examination revealed that the electrodes were located near the midline in Group STIM ewes, but 1.25 or 2 mm lateral of the midline in Groups NR and INHIB, respectively. We concluded that the MUA in the RCh-ME probably reflects the activity of cells with fibres or terminals involved in the control of GnRH release. Activity which increased at the onset of LH pulses was detected in medial areas of the RCh-ME and may reflect the activity of GnRH neurones. The activity which decreased at the onset of LH pulses in lateral areas of the RCh-ME suggests that both stimulatory and inhibitory inputs may be involved in the release of GnRH pulses.

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.

Basic neuroendocrine events before puberty in cattle, sheep and pigs.

Neuroendocrine events before puberty are compared in male and female cattle, sheep and pigs. The patterns of secretion of gonadotrophin, the age-related LH responses to castration or LH-RH administration and the effects of prolonged steroid treatment give information about the maturity of the hypothalamic-pituitary axis. It appears that in all three species the mechanisms involved in the expression of puberty are progressive rather than abrupt events. Some original contributions reported here, such as age-related changes in plasma levels and pulses of LH in the gilt, and the variations in pituitary cytosol receptors to 5 alpha-dihydrotestosterone and oestradiol-17 beta in the male lamb support this contention. Finally, this brief review of the neuroendocrine events known to occur before puberty in three domestic mammals underlines numerous missing links in our understanding of the prepubertal processes.

Effects of anterior hypothalamic deafferentation on LH, FSH, prolactin and sexual behavior in the ovariectomized ewe.

PIP: The effects of anterior hypothalamic deafferentation on luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), and sexual behavior in the ovariectomized ewe was investigated. Response to injection of estradiol benzoate (50 mcg) was measured before and after surgery. In all 4 cases the estradiol-induced release of LH and PRL observed prior to surgery was eliminated after deafferentation. FSH and basal-PRL levels were also affected. After 3 months, 2 ewes showed partial recovery. Sexual behavior was eliminated in 2 cases. In the ewe, the anterior dorsal inputs from the preoptic anterior hypothalamus are required for the positive feedback of estrogens on LH and maintenance of FSH levels.^ieng

[Electrical activity of the medio-basal cells of the hypothalamus of the ewe after estrogen injection]. / Etude de l'activité électriqu- des cellules de l'hypothalamus médio-basal chez la Brebis à suite d'injections d'aestrogènes

Single intravenous injections of estradiol benzoate in castrated, progesteron primed ewes, appeared to activate 34% of the cells located in the hypothalamus, namely in infundibular and ventromedial nuclei. 53% of the activated cells exhibited a typical bursting pattern of discharge. The increase in the mean firing rate could be transitory or durable.