Didier Chesneau

Rhythmic Secretion of Melatonin by the Superfused Pike Pineal Organ: Thermo- and Photoperiod Interaction

In the pineal organ of the pike (Esox lucius, teleost), the rhythmic production of melatonin by the photoreceptor cells is governed by a population of endogenous circadian oscillators, synchronized and entrained by the light/dark (L/D) cycle. Production of melatonin is inhibited by light and highly increased in the dark. In the present study, isolated and superfused pike pineals were exposed to a 24-hour temperature cycle of high (10 degrees C) or low (4 degrees C) amplitude, either under L/D, or under constant darkness. Under L/D, photoperiod is the dominant entraining stimulus for the melatonin secretion rhythm. It was high during the dark phase and low during the light phase, either under cold light/warm dark or under warm light/cold dark conditions. Under a warm light/cold dark cycle, the amplitude of the melatonin rhythm was reduced. In pineals cultured at 10 degrees C, a 20 degrees C temperature pulse potentiated or reduced the night-induced rise in melatonin production when applied, during the dark or during the preceding light phase, respectively. Under constant darkness, high- or low-amplitude temperature cycles could synchronize the rhythmic production of melatonin, which peaked with the high temperature. However, the shape of the oscillation could appear modified under warm subjective-L/cold subjective-D, depending on the experimental conditions. Finally, the rhythmic release of melatonin synchronized by a temperature cycle under constant darkness was no longer observed after removal of the external zeitgeber. The present study provides direct evidence that the pineal acts as a photothermotransducer.

Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes

Our aim was to study the role of the olfactory amygdala (medial and cortical nuclei) and the ventromedial nucleus of the hypothalamus (VMN) in the ability of the male odour or live males to induce a release of luteinizing hormone in anoestrus ewes. To achieve this, we temporarily blocked the activity of these structures by localized retrodialysis administration of the anaesthetic lidocaine. The effect of ram odour on the secretion of luteinizing hormone was completely blocked by inactivation of the cortical nucleus of the amygdala. In contrast, inactivation of part of the accessory olfactory system (the medial nucleus of the amygdala or the VMN) had no effect. In the presence of the male, lidocaine never impaired the endocrine response of the ewes. These results show that modulation of reproduction by the sexual partner even through pheromonal cues does not occur via the direct circuit of the accessory system. On the contrary, the cortical nucleus of the amygdala is absolutely necessary for the treatment of and/or the response to the male olfactory signal but this structure can be bypassed when other sensory cues are available.

Biphasic role of dopamine on female sexual behaviour via D2 receptors in the mediobasal hypothalamus.

Dopamine has been implicated in the control of sexual behaviour, but its role seems quite complex and controversial. The aim of the present experiments was to investigate the effects of dopamine (DA) acting on D2 receptors in the mediobasal hypothalamus (MBH) on sexual behaviour in female sheep. To achieve this, the D2 agonist, quinpirole, was administered bilaterally via microdialysis probes into the MBH of ovariectomized ewes either before or after oestradiol (E2) administration. Quinpirole (100 ng/ml) infused for 6 h just before E2 hastened the onset of oestrus behaviour and the luteinizing hormone surge, whereas the same treatment given 6-12 h or 18-21 h after E2 decreased the intensity of sexual receptivity without affecting LH or prolactin secretion. We then tested the hypothesis that E2 stimulates the onset of oestrus partly by decreasing DA activation of D2 receptors. In this case the D2 antagonists pimozide or spiperone (100 ng/ml) were infused into the MBH via microdialysis probes for 11 h in the absence of E2 administration. A significant number of ewes showed induction of receptivity with both antagonists, although its intensity was significantly lower than that induced by E2. These treatments generally did not significantly alter extracellular concentrations of monoamines or aminoacids although quinpirole modulated the ability of sexual interactions to increase noradrenaline release. These experiments show that DA acts via D2 receptors in the MBH to control female sexual behaviour in a biphasic manner: the onset of sexual motivation and receptivity requiring an initial increase in activation followed by a decrease. This dual action could explain some of the controversies concerning DA action on sexual behaviour.

The Interaction Between Photoperiod and Nutrition and Its Effects on Seasonal Rhythms of Reproduction in the Ewe

ABSTRACT In sheep, the seasonal patterns of reproductive activity are driven primarily by the annual photoperiodic cycle, but can also respond to other environmental factors, such as nutrition, yet little is known about the mechanisms underlying this interaction. This study was designed to define the interaction between photoperiodic and nutritional cues on seasonal patterns of ovarian activity, and to determine if there is a central interaction between these cues. Groups of Ile-de-France ewes were maintained in two nutritional states (restricted and well fed) under a simulated annual photoperiod of 8–16 h of light per day over two breeding seasons. At the end of the first breeding season, half of the animals of each group were ovariectomized (OVX) and fitted subcutaneously with estradiol implants. Low nutritional status shortened the season of ovarian activity, determined from the pattern of progesterone concentrations, by modifying the timing of seasonal transitions between periods of ovarian activity and anestrus. The same results were observed for the seasonal rhythm of neuroendocrine activity, assessed in the OVX ewes, from the pattern of luteinizing hormone concentrations. These results were then confirmed for neuroendocrine activity induced by a photoperiodic treatment. We conclude that nutrition centrally modulates the interpretation of photoperiod to affect seasonal reproductive transitions. The mechanisms of this interaction are discussed in the paper.

Rapid effects of melatonin on hormonal and behavioral stressful responses in ewes

Sheep are gregarious mammals with complex social interactions. As such, they are very sensitive to social isolation and constitute a relevant animal model to study specifically the biological consequences of social stress. We examined previously the behavioral and endocrine responses in ewes isolated socially in the familiar conspecific withdrawal model (FCW) and showed that stressful responses increased and maintenance behaviors decreased, confirming that social isolation is a strong stressor in sheep. Melatonin synchronizes seasonal and circadian rhythms; and several studies reported its implication in cognitive processes as emotion. Here we investigated its role in the modulation of social stressful responses. Firstly, we studied ewes in the FCW model during the day (characterized by low melatonin levels) and the night (characterized by high melatonin levels). We found lower stressful responses (significant lower levels of cortisol plasma, number of foot pawings, of circling attempts) during the night as compared to the day. To investigate whether these effects were due to melatonin or to darkness, we submitted ewes to FCW during the night with lights on, a condition that suppresses melatonin secretion. Ewes infused with melatonin under these conditions showed decreased stressful responses (significant lower levels cortisol plasma, number of vocalizations, time spent with the head out of the cage) as compared to ewes infused with saline. These findings demonstrate that melatonin diminishes the endocrine and behavioral impact of social isolation in ewes and support the idea that melatonin has a calming effect in socially stressful situations.

The effect of an intracerebroventricular injection of metformin or AICAR on the plasma concentrations of melatonin in the ewe: potential involvement of AMPK?

BackgroundIt is now widely accepted that AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis. Recently, it has been shown to regulate circadian clocks. In seasonal breeding species such as sheep, the circadian clock controls the secretion of an endogenous rhythm of melatonin and, as a consequence, is probably involved in the generation of seasonal rhythms of reproduction. Considering this, we identified the presence of the subunits of AMPK in different hypothalamic nuclei involved in the pre- and post-pineal pathways that control seasonality of reproduction in the ewe and we investigated if the intracerebroventricular (i.c.v.) injection of two activators of AMPK, metformin and AICAR, affected the circadian rhythm of melatonin in ewes that were housed in constant darkness. In parallel the secretion of insulin was monitored as a peripheral metabolic marker. We also investigated the effects of i.c.v. AICAR on the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in brain structures along the photoneuroendocrine pathway to the pineal gland.ResultsAll the subunits of AMPK that we studied were identified in all brain areas that were dissected but with some differences in their level of expression among structures. Metformin and AICAR both reduced (p < 0.001 and p < 0.01 respectively) the amplitude of the circadian rhythm of melatonin secretion independently of insulin secretion. The i.c.v. injection of AICAR only tended (p = 0.1) to increase the levels of phosphorylated AMPK in the paraventricular nucleus but significantly increased the levels of phosphorylated ACC in the paraventricular nucleus (p < 0.001) and in the pineal gland (p < 0.05).ConclusionsTaken together, these results suggest a potential role for AMPK on the secretion of melatonin probably acting trough the paraventricular nucleus and/or directly in the pineal gland. We conclude that AMPK may act as a metabolic cue to modulate the rhythm of melatonin secretion.

Behavioral and endocrine evaluation of the stressfulness of single-pen housing compared to group-housing and social isolation conditions

Regulation of neuroendocrine responses is often studied in animals housed indoors in individual contiguous pens. In sheep, these housing conditions are used to control the environment, facilitate biological sampling and limit social stress. However, this type of housing also prevents exploratory behaviors and could induce stereotypies, non-compliant with welfare and possibly associated with a state of stress. In this context, we investigated the impact of housing in a single-pen, with other familiar conspecifics, on emotional state by evaluating behavioral, hormonal and neuronal measures in adult ewes. We hypothesized that emotional state would be more negative in animals housed in a single-pen for one week (Pen) than in freely moving animals (Free) but less negative than in socially isolated subjects (Isol). We tested our hypothesis in ovariectomized ewes to avoid the interaction with sexual steroid variations. Our behavioral, endocrine and neuronal (Fos activation of the corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus) measures confirmed that withdrawing familiar conspecifics was sufficient to induce strong stress responses in Isol ewes, but there was no indication that Pen ewes were stressed. However, the latter showed less mastication activity than Free ewes, probably due to limited accessibility to straw. The highest plasma prolactin levels were observed in Isol and Free animals, which might result from stress and physical activity, respectively. In Free ewes, plasma dopamine was low, consistent with its inhibitory control of prolactin. However, Isol animals had both high levels of prolactin and dopamine, suggesting a dysregulated balance in socially stressed ewes. As in other species, we suggest that the regulation of prolactin by dopamine varies with stress and/or social context. Overall, this study shows that the impact of housing conditions on different neuroendocrine systems should be considered more in the future.

The impact of thyroid hormone in seasonal breeding has a restricted transcriptional signature

Thyroid hormone (TH) directs seasonal breeding through reciprocal regulation of TH deiodinase (Dio2/Dio3) gene expression in tanycytes in the ependymal zone of the medio-basal hypothalamus (MBH). Thyrotropin secretion by the pars tuberalis (PT) is a major photoperiod-dependent upstream regulator of Dio2/Dio3 gene expression. Long days enhance thyrotropin production, which increases Dio2 expression and suppresses Dio3 expression, thereby heightening TH signaling in the MBH. Short days appear to exert the converse effect. Here, we combined endocrine profiling and transcriptomics to understand how photoperiod and TH control the ovine reproductive status through effects on hypothalamic function. Almost 3000 genes showed altered hypothalamic expression between the breeding- and non-breeding seasons, showing gene ontology enrichment for cell signaling, epigenetics and neural plasticity. In contrast, acute switching from a short (SP) to a long photoperiod (LP) affected the expression of a much smaller core of 134 LP-responsive genes, including a canonical group previously linked to photoperiodic synchronization. Reproductive switch-off at the end of the winter breeding season was completely blocked by thyroidectomy (THX), despite a very modest effect on the hypothalamic transcriptome. Only 49 genes displayed altered expression between intact and THX ewes, including less than 10% of the LP-induced gene set. Neuroanatomical mapping showed that many LP-induced genes were expressed in the PT, independently of the TH status. In contrast, TH-sensitive seasonal genes were principally expressed in the ependymal zone. These data highlight the distinctions between seasonal remodeling effects, which appear to be largely independent of TH, and TH-dependent localised effects which are permissive for transition to the non-breeding state.

Acute injection and chronic perfusion of kisspeptin elicit gonadotropins release but fail to trigger ovulation in the mare.

ABSTRACT Kisspeptin has emerged as the most potent gonadotropin-releasing hormone (GnRH) secretagogue and appears to represent the penultimate step in the central control of reproduction. In the sheep, we showed that kisspeptin could be used to manipulate gonadotropin secretion and control ovulation. Prompted by these results, we decided to investigate whether kisspeptin could be used as an ovulation-inducing agent in another photoperiodic domestic mammal, the horse. Equine kisspeptin-10 (eKp10) was administered intravenously as bolus injections or short- to long-term perfusions to Welsh pony mares, either during the anestrus season or at various stages of the cycle during the breeding season. In all the experimental conditions, eKp10 reliably increased peripheral concentrations of both luteinizing hormone and follicle-stimulating hormone. The nature of the response to eKp10 was consistent across experimental conditions and physiological states: the increase in gonadotropins was always rapid and essentially transient even when eKp10 was perfused for prolonged periods. Furthermore, eKp10 consistently failed to induce ovulation in the mare. To gain insights into the underlying mechanisms, we used acute injections or perfusions of GnRH. We also cloned the equine orthologues of the kisspeptin precursor and Kiss1r; this was justified by the facts that the current equine genome assembly predicted an amino acid difference between eKp10 and Kp10 in other species while an equine orthologue for Kiss1r was missing altogether. In light of these findings, potential reasons for the divergence in the response to kisspeptin between ewe and mare are discussed. Our data highlight that kisspeptin is not a universal ovulation-inducing agent.

Melatonin deprival modifies follicular and corpus luteal growth dynamics in a sheep model

This study assessed the effect of melatonin deprival on ovarian status and function in sheep. Experimental procedures were carried out within two consecutive breeding seasons. Animals were divided into two groups: pinealectomised (n=6) and sham-operated (n=6). The completeness of the pineal gland removal was confirmed by the plasma concentration of melatonin. Ovarian status was monitored by ovarian ultrasonography for 1 year to study reproductive seasonality. Follicular and corpus luteal growth dynamics were assessed during an induced oestrous cycle. As the effects of melatonin on the ovary may also be mediated by its antioxidant properties, plasma Trolox equivalent antioxidant capacity (TEAC) was determined monthly for 1 year. Pinealectomy significantly extended the breeding season (310±24.7 vs 217.5±24.7 days in controls; P<0.05). Both pinealectomised and sham-operated ewes showed a well-defined wave-like pattern of follicle dynamics; however, melatonin deficiency caused fewer waves during the oestrous cycle (4.3±0.2 vs 5.2±0.2; P<0.05), because waves were 1 day longer when compared with the controls (7.2±0.3 vs 6.1±0.3; P<0.05). The mean area of the corpora lutea (105.4±5.9 vs 65.4±5.9 mm(2); P<0.05) and plasma progesterone levels (7.1±0.7 vs 4.9±0.6 ng/ml; P<0.05) were significantly higher in sham-operated ewes compared with pinealectomised ewes. In addition, TEAC values were significantly lower in pinealectomised ewes compared with control ones. These data suggest that melatonin, besides exerting its well-known role in the synchronisation of seasonal reproductive fluctuations, influences the growth pattern of the follicles and the steroidogenic capacity of the corpus luteum.