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International audience; Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10(-6)M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10(-4)M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge.

International audience; In seasonally anoestrous ewes of many breeds, the introduction of rams triggers an increase in gonadotrophin secretion that induces ovulation, a phenomenon known as the 'ram effect'. The ram effect is a practical method for mating ewes outside the natural breeding season, and also can provide synchronised lambing, but the variability of the response, especially in young animals, reduces its potential for widespread application. The aim of our study was to assess two factors that are thought to contribute to the variability in young ewes: temperament and sexual experience. We used anovulatory ewes from a flock that had been genetically selected for 'calm' or 'nervous' temperament and compared the endocrine and ovarian responses to the ram effect in four groups (each n = 15): 'calm' and parous (3-6 years old); 'calm' and nulliparous (2 years old); 'nervous' and parous; and 'nervous' and nulliparous. Parous ewes, independently of their temperament, exhibited a faster endocrine response and a higher proportion of females cycling after ram introduction than nulliparous ewes. 'Nervous' ewes exhibited a higher proportion of females cycling after ram introduction than calm ewes, but only in the nulliparous group. We conclude that temperament exerts little influence on the response to the ram effect in sexually experienced ewes, and that females of 'nervous' temperament appear to respond better when sexually 'naive'. Both sexual experience and temperament need to be taken into consideration when flock management involves the ram effect. Finally, some ewes were cyclic at ram introduction, yet exhibited an increase in LH secretion even in the presence of high concentrations of progesterone. The mechanism by which the inhibitory effect of progesterone on LH secretion was bypassed needs to be clarified.

Similarities as well as differences across species in the control of sexual behavior are helping to fully understand the subtle relations between physiology and eco-ethological constraints and how the brain integrates such information. We will illustrate this with sexual behavior in domestic ruminants and especially ewes. Females of these species like humans, but unlike rodents, have a long luteal phase. A prolonged exposure to progesterone (Pg) before the preovulatory estradiol rise is necessary for estrous behavior to be displayed. Estradiol action and receptor localization is very similar to that observed in other species. But not too surprisingly, the role of Pg is rather different with a priming effect not observed in rodents. However, as in rodents, Pg also has an inhibitory effect, is necessary for the display of proceptivity and is responsible for the timing of the different periovulatory events. These steroids act on the central nervous system in similar areas across mammalian species to regulate estrous behavior. Steroid fluctuations during the estrous cycle cause changes in catecholaminergic activity in the hypothalamus. Interestingly, these neurotransmitters seem to have very similar effects in ewes and rats as illustrated by the norepinephrine rise after male-female interactions observed in both species. Similar comparisons can be made regarding the action of some neuropeptides, including oxytocin and GnRH, and more integrative processes like sexual differentiation and modulation of reproduction by social interactions. Data on sheep, goats and cows will be compared with those of rodents.

Remerciements : INRA, 0868-Systèmes d'Elevage Méditerranéens et Tropicaux (SELMET), Campus Supagro Montpellier 2 place Viala 34060 Montpellier ; INRA, 0332-Domaine Expérimental Bourges-La Sapinière, 18390 OSMOY; The proportion of anoestrous ewes ovulating after exposure to a sexually active ram is variable mainly due to whether an LH surge is induced. The aim of this study was to determine the role of oestradiol (E2) in the ram-induced LH surge. In one study, we measured the plasma concentrations of E2 in ewes of different breeds before and after the 'ram effect' and related these patterns to the presence and latency of the LH surge, while another compared ovarian responses with the 'ram effect' following exposure to rams for 2 or 12 h. In all ewes, the concentration of E2 increased 2-4 h after rams were introduced and remained elevated for 14.5±0.86 h. The quantity of E2 secreted before the LH surge varied among breeds as did the mean concentration of E2. The granulosa cells of IF ewes collected after 12 h exposure to rams secreted more E2 and progesterone and had higher levels of StAR than the 2 h group but in MV ewes there was no differences between these groups for any of these parameters. These results demonstrate that the LH surge induced by the rams is a result of increased E2 secretion associated with increased levels of STAR in granulosa cells and that these responses varied among breeds. The results suggest that the variable occurrence of a LH surge and ovulation may be the result of variable ovarian responses to the 'ram effect' and insensitivity of the hypothalamus to the E2-positive feedback signal.; La proportion des brebis en anoestrus qui ovulent après exposition à un bélier sexuellement actif est variable et ceci est du à l’induction ou non d’un pic de LH. Le but de cette étude était de déterminer le rôle de l’oestradiol dans l’induction par le bélier d’un pic de LH. Dans une étude nous avons mesuré les concentrations plasmatiques d’oestradiol chez des brebis dedifférentes races avant et après l’effet bélier et relié ces profilesà la présence et la latence des pics de LH. Dans une autre étude nous avons comparé les réponses ovariennes à l’exposition à un bélier pendant 2 ou 12 h. Chez toutes les brebis les concentrations d’oestradiolaugmentent 2 à 4 h après l’introduction des béliers et restent élevées pendant 14.5 ± 0.86 h. La quantité d’oestradiol secrétée avant le pic de LH et la concentration moyenne d’oestradiol varient selon les races. Les cellules de la granulosa collectées après exposition au bélier pendant 12 h sécrètent plus d’oestradiol et de progestéroneen culture et ont des niveaux de mRNA de STAR (Steroidogenic acute regulatory protein) plus élevés qu’après exposition au bélier pendant 2 h chez les brebis de race Ile de France qui ovulent fréquemment après effet mâle que chez les brebis Mouton Vendéen. Ces résultats montrent que le pic de LH induit par le bélier est lié à une augmentation de la sécrétion d’oestradiol associée à une augmentationdes niveaux de STAR dans les cellules de la granulosa et que ces réponses varient suivant les races. Ces résultats suggèrent que la variabilité dans l’apparition des pics de LH et del’ovulation est due suivant les races à la réponse ovarienne à l’introduction des béliers et à lasensibilité de l’hypothalamus au feedback positif de l’oestradiol.

During the non-breeding season, anestrous ewes do not experience ovarian cycles but exposure to a ram or its odor results in the activation of the luteinizing hormone secretion leading to ovulation. The aim of our work was to identify the neural pathways involved in this phenomenon. Using Fos immunocytochemistry, we examined the brain areas activated by the male or its fleece, in comparison with ewes exposed to the female fleece or the testing room (control group). In comparison with the control group, the male or its odor significantly increases Fos neuronal expression in the main and accessory olfactory bulbs, anterior olfactory nucleus, cortical and basal amygdala, dentate gyrus, ventromedial nucleus of the hypothalamus, piriform and orbitofrontal cortices. The main olfactory bulb, the cortical amygdala and the dentate gyrus are specifically more activated by the male odor than the female odor. Using a procedure of double labeling for Fos and gonadotropin-releasing hormone, we also compared the number of gonadotropin-releasing hormone neurons activated in the four groups of females. The male or its odor significantly increases the number and the proportion of gonadotropin-releasing hormone cells expressing Fos-immunoreactivity in the preoptic area and the organum vasculosum of the lamina terminalis, whereas no such induction of Fos-immunoreactivity was found in gonadotropin-releasing hormone neurons of ewes exposed to the female odor or the testing room. These findings emphasize the role of the main olfactory system in the detection and the integration of the ram odor, and also suggest the participation of the accessory olfactory system. Numerous structures widely distributed seem involved in the processing of the male olfactory cue to reach the gonadotropin-releasing hormone neurons.

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.

Remerciements : Laboratoire Phénotypage - Endocrinologie INRA, UMR Physiologie de la Reproduction et des Comportements, Centre Val de Loire; In sheep, the ‘ram effect’ induces out-of-season fertility and good nutrition increases prolificacy. This experiment determined if fatness or short-term nutritional supplementation modified the response to the ‘ram effect’. A group of 48 Île-de-France ewes were fed diets that produced groups with body-condition scores (BCS) of >3.0 and

Anoestrous ewes can be induced to ovulate by the socio-sexual, ‘ram effect’. However, in some ewes, the induced ovulation is followed by an abnormally short luteal phase causing a so-called ‘short cycle’. The defect responsible for this luteal dysfunction has not been identified. In this study, we investigated ovarian and uterine factors implicated in male-induced short cycles in anoestrous ewes using a combined endocrine and molecular strategy. Before ovulation, we were able to detect a moderate loss of thecal expression of steroid acute regulatory protein (STAR) in ewes that had not received progesterone priming (which prevents short cycles). At and following ovulation, we were able to identify a significant loss of expression of genes coding key proteins involved in the biosynthesis of progesterone (STAR,CYP11A1andHSD3B1(HSD3B)) as well as genes coding proteins critical for vascular development during early luteal development (VEGFAandKDR(VEGFR2)), suggesting dysfunction in at least two pathways critical for normal luteal function. Furthermore, these changes were associated with a significant reduction of progesterone production and luteal weight. Additionally, we cast doubt on the proposed uterus-mediated effect of prostaglandin F2α (PGF2α) as a cause of short cycles by demonstrating the dysregulation of luteal expression of the PGF receptor, which mediates the luteal effects of PGF2α, and by finding no significant changes in the circulating concentrations of PGFM, the principal metabolite of PGF2α in ewes with short cycles. This study is the first of its kind to examine concurrently the endocrine and molecular events in the follicular and early luteal stages of the short cycle.Free French abstractA French translation of this abstract is freely available athttp://www.reproduction-online.org/content/147/3/357/suppl/DC1

Differential activation of neural substrates was investigated in female sheep exposed to a male when they were in oestrus, and sexually receptive and attracted to males, as opposed to anoestrus when they were not. Changes in neuronal activation were visualized in ovariectomized, hormone-treated ewes by quantifying changes in cellular expression of c- fos messenger RNA by in situ hybridization histochemistry. Results showed that, while oestrus induction had no significant effects on c- fos expression per se , a 5 min exposure to a male significantly increased it in a number of primary and association cortical regions (the mitral and granule cell layers of the olfactory bulb, visual, somatosensory, orbitofrontal, piriform, cingulate and temporal cortices), the limbic system (CA1 region of the hippocampus, subiculum, lateral septum, lateral and basolateral amygdala, bed nucleus of the stria terminalis) and hypothalamus (mediobasal hypothalamus, medial preoptic area and paraventricular nucleus) as well as the nucleus accumbens and mediodorsal thalamus. Intromissions did not contribute significantly to these c- fos changes however. In anoestrus females, exposure to a male only produced a small significant increase in c- fos messenger RNA expression in the temporal cortex inspite of receiving similar amounts of visual and olfactory cues from him and a number of mating attempts. These results clearly demonstrate that changes in sexual motivation markedly alter the neural processing of sensory cues from males. They also show that the hormonal induction of sexual attraction to males cues and the resultant stimulation of sexual behaviour is due not only to altered responsiveness of oestrogen-sensitive brain regions involved in mediating behavioural responses towards the male, but also to changes in primary and secondary/tertiary somatosensory, olfactory and visual processing regions which relay sensory information to them.

Exposure of anoestrous ewes to rams induces oestrous cycles (the 'ram effect'). This response is poor in young-sexually naive females, possibly because they lack sexual experience and are sensitive to stress. Firstly, we assessed the conditions required for the acquisition of sexual experience. We exposed naive females at 5 or 9 months of age to rams and subsequently assessed their response at I year of age, to the ram effect. Unexpectedly, 2 weeks pre-exposure at 5 months and under some conditions at 9 months, inhibited the ram effect at I year of age, suggesting that early contact with rams was stressful and had long-term negative effects on reproduction. Consequently, we assessed the combined effect of stress and sexual experience on responses to the ram effect. At 9 months of age, we pre-exposed naive females using sexually active rams to achieve 'positive' (voluntary contact) or 'negative' (forced contact) experiences, and assessed responses to the ram effect at a year of age. In parallel, we subjected naive females to stressful conditions at the time of the male effect. During pre-exposure, females with 'negative' contact exhibited more vigilance and stress cues and had higher levels of cortisol than 'positive' contact ewes. Regardless of pre-exposure or stress, the response to the ram effect at a year of age was inhibited. These experiments confirm that contact with sexual partners can be stressful to young ewes and highlight the need to consider their emotional state when conditioning them to the presence of rams and when managing breeding programs. (C) 2009 Elsevier Inc. All rights reserved.

International audience; Exposure of anestrous females to a ram or his odour induces rapid secretion of LH that can lead to ovulation. This response is mediated by the main olfactory system (MOS) in sexually experienced ewes. The accessory olfactory system (AOS) has a minor, but unclear, role in this response. In sexually naive ewes, male odour is less effective than in experienced ewes, but the neural pathway involved is not known. In our experiment, we investigated the brain regions activated by the male or his odour in young and sexually naive anestrous ewes using immunohistochemistry for Fos alone, Fos double-labeled with Gonadotropin-Releasing Hormone (GnRH) or with Tyrosine Hydroxylase (TH). Ram odour caused neural activation in the main olfactory bulb (MOB) and not the AOS. Exposure to a male induced significant activation in the MOB, the cortical and medial nuclei of the annygdala and the ventromedial hypothalamus but not in cortical areas, or in GnRH or TH positive neurons. These results confirm the predominant role of the MOS in the detection of olfactory signals in sheep and underline the importance of learning processes. (C) 2009 Elsevier B.V. All rights reserved.

Adult ewes were studied after prenatal treatment with androgen. Although seven of the eight androgenized ewes (ANDR) when intact presented signs of cyclic ovarian activity at least once, none of them showed cyclic female receptivity when intact or after ovariectomy and progesterone (P) + estradiol (E2) treatments, whereas all ten control (CONT) ewes did. Receptivity also was induced in CONT, but not in ANDR, ewes by long term E2 or testosterone (T) treatments. Increases in LH levels, with latency and duration similar to the LH surges of the CONT ewes, were observed in six (P + low E2 cycle) and five (P + high E2 cycle) of the eight ANDR ewes. The maximum LH level was lower in the ANDR than in the CONT ewes after high E2 treatment (p less than 0.002). Progesterone had a clear inhibitory effect on the induction of the LH surge by E2 only in the CONT ewes. Male-type mounting and nudging were observed more often in the ANDR than in the CONT ewes when intact (p less than 0.05). However, there was no facilitation of male sexual behaviour induced by long term E2 or T treatment in the ANDR compared to the CONT ewes. Progesterone inhibited male behaviour induced by T in the CONT but not in the ANDR ewes. This study shows that prenatal androgens defeminize LH secretion and sexual behaviour of ewes in a dissociated manner. It also suggests that prenatal androgen does not really masculinize the females but decreases their sensitivity to the inhibitory effect of P, and thus extends the "juvenile state" in which male-like patterns appear independently of the hormone milieu.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

In sheep, exposure of seasonally anestrous females to the male or its fleece results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. The study of the neural pathways involved in this phenomenon, commonly named "male effect", show that the main olfactory system plays a critical role in the detection and the integration of the male odor. The accessory olfactory system participates in the perception of the ram odor but does not seem necessary for the endocrine response. According to the hypothesis that the neuroanatomical differences between the two olfactory systems could be associated with different functional roles, we investigated the importance of sexual experience and learning processes in the male effect. Our results showed that female responses depend on previous sexual experience. We also demonstrated that the LH response to male odor could result from an associative learning process. The aim of the present report was to summarize our current knowledge concerning the "male effect" and in particular to clarify the role of sexual experience and learning in the processes involved in this effect.

Exposure of anestrous ewes to a ram or its odor results in the activation of the luteinizing hormone (LH) secretion leading to reinstatement of cyclicity in most females. Sexual experience and learning have been suggested as important factors to explain the variability of the female responses. In experiment 1, we compared the behavioral and endocrine responses of four groups of anestrous females that differed in age (young or adult) and previous exposure to males [naive (no exposure) or experienced (courtship behavior for young and numerous mating for adults)]. Age did not seem to affect the LH response to males or their odor. In contrast, sexual experience was a critical factor: the proportion of females exhibiting an LH response to male odor was significantly higher in experienced than in naive ewes. Sexual experience affected the response to male odor, but did not have an effect on responses to the male himself. A second experiment investigated whether the LH response to male odor could result from an associative learning process. Accordingly, we tested the effectiveness of a conditioned stimulus (lavender odor) previously associated with the male, in inducing the endocrine response. The results indicate that the odor of lavender activated LH secretion only in ewes that have been previously exposed to scented males. This demonstrates that ewes are able to learn the association between a neutral odor and their sexual partner.

In sheep and goats, exposure of seasonally anestrous females to sexually active males results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. This phenomenon is named "the male effect" and seems to constitute a major factor in the control of reproductive events. This effect depends mostly on olfactory cues and is largely mimicked by exposure to male fleece only. In sheep, preventing the vomeronasal organ (VNO) from functioning does not affect the female responses to male odor suggesting that, unlike in rodents, the accessory olfactory system does not play the major role in the perception of this pheromonal cue. Female responses also seem to depend on previous experience, an effect that is not common for pheromones and renders this model of special interest. The aim of the present report is to summarize our current knowledge concerning the "male effect" and in particular to clarify the respective roles of the two olfactory systems in the processes involved in this effect.

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.

In sheep, essentially all the neurokinin B (NKB) neurones of the infundibular nucleus express oestradiol receptor alpha, and analysis of female and male brains has revealed an exceptionally marked female-dominant sex difference in the numbers of NKB neurones in the infundibular nucleus. This neuronal population is located in an oestradiol-sensitive brain area involved in the control of gonadotropin-releasing hormone (GnRH) secretion and oestrous behaviour, but its physiological role is poorly documented. The aim of the present study was to analyse NKB mRNA expression at a crucial time when the steroid has stimulated the pathways leading to the induction of these two events. After cloning a specific ovine NKB antisense riboprobe, we examined the effects of a short oestradiol treatment (4 h subcutaneously) on the expression of NKB mRNA in the caudal part of the infundibular nucleus of progesterone-primed ovariectomized ewes. We demonstrated that oestradiol decreased both the level of NKB mRNA expression (34%) and the number of cells containing NKB mRNA (43%). Oestradiol acts strongly on these NKB cells in the short term. We suggest that this early change in NKB mRNA expression during the preovulatory period might be involved in the control of the induction of GnRH secretion or oestrous behaviour.

We assessed the role of learning in the expression of female sexual behavior and evaluated the relative importance of age versus experience. Two studies were conducted with ovariectomized ewes submitted to steroid treatment that mimicked an estrus cycle. We compared behavioral (experiments 1 and 2), neurochemical (experiment 1), and endocrine (experiment 2) responses of sexually naive young and adult ewes versus sexually experienced adults when exposed to males. In a third study, we compared their performance in an instrumental learning test and the extent to which it was affected by stress. These experiments showed that proceptivity is affected both by age and sexual experience. In experiment 1 only experienced adults were proceptive and displayed an increase in hypothalamic norepinephrine. By the second estrus cycle (experiment 2) naive adults performed similarly to experienced adults but proceptive behavior was still inferior in young ewes. Receptivity was also different between groups but affected more by age than by sexual experience. All ewes mated during the first interaction with a male, although males' latency to ejaculation was shorter with experienced females than naive adults or naive young. Young ewes found food as readily as adults in experiment 3 but were more affected by stress. Together, these experiments show that both experience and age influence sexual activity and that sensitivity to stress may also be involved. This may contribute to the deficient reproductive performance that is often observed in young female mammals.

In sheep, the mediobasal hypothalamus (MBH) has been shown to be the primary central site of estradiol (E2) action that induces both the preovulatory surge and sexual behaviour. However, the nature of the neurotransmitters or neuromodulators synthesized in the MBH during E2 stimulation remains to be clearly defined. After the cloning of the ovine cDNA sequences and using in situ hybridization, hypothalamic proopiomelanocortin (POMC), and preproneuropeptide Y (preproNPY) mRNA expression was studied in ovariectomized ewes that received a sequential treatment of progesterone and E2. As we showed that an exposition to E2 only for 4h well in advance on the LH surge onset is sufficient to induce the preovulatory surge and estrous behaviour, mRNA expression was evaluated in ewes treated with 6x30-mm E2 implants (experimental group) or with empty implants (control group) and slaughtered 4h after the start of the E2 treatment. Our results demonstrate that this short E2 treatment significantly decreased both the mean number of silver grains per POMC-containing cell (35%) and the mean number of POMC-cells (38%) in the ovine infundibular nucleus, whereas the treatment had no effect on preproNPY mRNA expression. These observations suggest that a reduction of POMC gene transcription could participate to the early neural mechanism of E2 feedback.

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.

During behavioural oestrus female sheep, like females of many species, become both attracted to and sexually receptive towards males, whereas at other times they will avoid them. The mediobasal hypothalamus is the main site for the feedback action of sex steroids to induce sexual behaviour in the sheep and in previous studies we have shown that noradrenaline and serotonin are released in this region during sexual interactions with males. The current study investigated whether such changes are specific to interactions with males and if visual or olfactory cues or somatosensory stimulation during mating are critical. In vivo microdialysis sampling was carried out in the mediobasal hypothalamus of ovariectomized ewes submitted to artificial oestrous cycles. Release of monoamines and amino acid transmitters was first measured in animals during and after oestrus when they were exposed to interactions with either males or females or presentation of food. Noradrenaline concentrations only increased significantly when the females were in oestrus and interacted with males irrespective of whether intromissions were permitted. Females were then exposed to visual (faces) or odour (a home pen) cues from males or to the males themselves. Slide images of male faces increased concentrations of amines, glutamate and GABA during early oestrus, when females spent most time looking at them. During late oestrus noradrenaline, glutamate and GABA concentrations also increased in response to the male faces but no transmitter changes were seen during the luteal phase or at any time where the females were exposed to female faces, or inverted male faces. Exposure to male odour produced a lower increase in noradrenaline concentrations when females were in early oestrus but marked increases 20 and 30 min after exposure to male odours in late oestrus. No other transmitters were affected. Exposure to a male and mating with him when females were in early or late oestrus produced increased noradrenaline concentrations similar to those seen with face stimuli alone although other neurotransmitters were unaffected. These results show that noradrenaline, and to a lesser extent dopamine, serotonin, glutamate and GABA release in the mediobasal hypothalamus, can be modulated specifically in the oestrous female by sensory information coming from the male during oestrus. The differential effects of male cues during early and late oestrus suggest their involvement in (i) proceptive or anticipatory sexual responses shown by the female to male cues, and (ii) receptive sexual responses, and suggest that the mediobasal hypothalamus plays a key role in the integration of hormonal action on sexual motivation and processing of sensory information during oestrus.

The preovulatory surge of LH, or positive feedback response to oestradiol, is related to the steroid's direct stimulating action on the pituitary gland, but we have demonstrated that, in sheep, the central nervous system plays an essential role in its action: a preovulatory GnRH surge. Since GnRH neurons appear to contain few, if any receptors for oestradiol the question is raised: Where in the central nervous system does oestradiol act to stimulate GnRH secretion? A series of experiments were conducted with a combination of techniques for sampling pituitary portal blood and stereotaxic brain micro-implantations of oestradiol. Castrated Ile-de-France breed ewes were treated during the pseudo follicular phase of successive artificial cycles, receiving either peripheral oestradiol implants or brain implants containing oestradiol or cholesterol. Administration of oestradiol implants into the ventromedial region of the hypothalamus induced a preovulatory GnRH surge in 5 out of 10 animals. The interval between estradiol and the GnRH surge was comparable with that observed in animals treated with a peripheral implant although the amplitude of the surge was smaller. No GnRH surges were observed in animals treated with cholesterol. Finally, there was an inverse correlation between the response intensity (amplitude of the GnRH/LH surges) and the distance separating implants from cells in the region carrying oestradiol receptors. These results show that the mediobasal hypothalamus is a site of action for the oestradiol-induced preovulatory GnRH surge in the ewe. Immunohistochemical tests should identify the nature of the cells forming the relay for the steroid action.

Oestrus behaviour and the luteinizing hormone (LH) surge are induced in ovariectomized ewes by oestradiol (E2) after a period of progesterone priming with a low level of E2 (Pge2) and we have previously shown that these effects are primarily mediated through their action on the mediobasal hypothalamus (MBH). The aim of the present study was to assess what neurochemical changes in the MBH are induced by these steroids that might mediate their action on oestrus behaviour and LH release. Eight ovariectomized ewes were implanted with microdialysis probes in the MBH and submitted to three artificial cycles, so that they exhibited either both oestrus behaviour and an LH surge (Pge2 + E2), and LH surge alone (E2 alone) or neither oestrus behaviour nor an LH surge (Pge2 alone). Microdialysis and blood samples were collected every 30 min from 4 h before the end of Pge2 treatment until the end of oestrus. Behavioural tests with a ram were made to assess receptivity. Dopamine (DA) levels were found to increase significantly at the termination of Pge2 treatment after both Pge2 + E2 and Pge2 treatments. When the ewes received E2 after a Pge2 + low estradiol priming (Pge2 + E2), DA levels decreased 16 h later (4 h after E2) whereas they did not change after E2 or Pge2 alone. By contrast, serotonin (5HT) levels did not change significantly during the first 24 h but then increased when ewes received E2 alone and decreased when they were treated with Pge2 + E2. γ-Aminobutyric acid (GABA) concentrations decreased significantly at the beginning of the sampling period after all treatments but this decrease lasted longer after Pge2 + E2 and was most pronounced at the beginning of receptivity. No significant long term effects of these steroid treatments were found on noradrenaline (NA), aspartate, glutamate, glycine and taurine levels. However, E2 administration was followed during the next few hours by a significant increase in glycine and to a smaller extent in glutamate and GABA. More importantly, when ewes were treated with Pge2 + E2, NA levels increased signicantly following the behavioural interactions with a ram when the ewes were sexually receptive. In contrast to this, DA levels only increased during interactions with the ram when the ewes were not receptive. 5HT levels increased after tests where the ewe was either receptive or unreceptive to the male. GABA, aspartate and glycine levels increased in the sample just preceding the test and then decreased during it. These results show that a number of neurotransmitter changes occur in the MBH during hormonal induction of oestrus. Changes mainly occured in DA, NA, 5HT and GABA concentrations during oestrus when animals were treated with Pge2 + E2 and suggest that they may be potentially involved in the control of female sexual behaviour. The changes occuring when animals received E2 alone and showed only prolactin and LH surges, but no behavioural oestrus, are much more limited. Furthermore, they do not correspond to changes after the E2 injection in the Pge2 + E2 treatment, so it is unlikely that the neurochemical changes we observed are directly related to these endocrine events.

Oestrogen plays a key role in the regulation of the endocrine and behavioural events associated with the oestrous cycle. It is important, therefore, to know the location of neurones receptive to this steroid and to know whether their distribution varies with the oestrous cycle. We have undertaken experiments to identify the location of oestrogen receptors (ER) within the preoptico-hypothalamic continuum of ovariectomized ewes submitted to a variety of different hormone replacement regimes which mimic the different stages of the oestrous cycle. We used a monoclonal antibody to ER and detected receptors with immunohistological methods in the non-vascular part of the organum vasculosum of the lamina terminalis, the lateral septum, the medial preoptic area, the supraoptic, suprachiasmatic and arcuate (ARC) nuclei, the ventromedial hypothalamus (HVM) and in the region close to the mamillari recess. ER neurones were scarce or absent from the anterior hypothalamus and the paraventricular nucleus. The density of ER staining in the HVM, but in no other localization, was found to be higher, and in a more lateral position, during the induced luteal phase (progesterone treatment) than during the follicular phase (7 days of progesterone treatment followed by oestradiol) or in the ovariectomized female. In all areas studied, except for the ARC, the apparent surface area of the nucleus in ER immunoreactive cells varied with hormonal treatment. These data, and especially those in the HVM, contribute towards our understanding of how steroids may act in the ovine to control sexual behaviour.

Dose-response relationships have been suggested for the role of estradiol (E2) in the induction of both the LH surge and estrous behavior in the ewe. However, it is not always clear whether it is the amount of E2 or the length of time that E2 is present that is important. Furthermore, in previous studies, the intensity of sexual behavior has rarely been studied quantitatively. Our aims in this work were to analyze in more detail the effects of two injected doses of estradiol benzoate (25 or 50 micrograms) for the induction of both the LH surge and estrous behavior (experiment I) and to dissociate the roles of quantity and duration of presence of E2 as administered by implants (experiment II). Increasing the dose of E2 in both experiment I and experiment II decreased the latency to onset of both the LH surge and the receptivity. The duration of E2 presence marginally affected the latency of both responses and the duration of the LH surge. By contrast, the duration of receptivity depended largely on the duration of E2 presence (experiment II: r = 0.569, P0.01). This result explains the dose-duration effect mentioned previously in the literature and found in experiment I. The maximum response increased with dose in both experiment I and experiment II for receptivity and only in experiment I for the LH surge. We conclude that, at estrus, E2 acts in a similar fashion to induce receptive behavior and the LH surge but controls the maximum levels and the duration of these endocrine and behavioral responses differently.

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 μM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA. Finally, in a fourth experiment is was shown that multiple intromissions significantly reduced sexual receptivity. These results show that OXY release in the MBH is increased by natural or artificial VCS and that this is modulated by sex steroids. The inhibitory action of OXY infusions into the MBH on sexual receptivity suggests that a functional role of this peptide within this brain region might be to mediate decreases in sexual receptivity which we observed following multiple intromissions. It is probable that the behavioural effects of this peptide are primarily mediated directly rather than through a modulation of noradrenaline and GABA release.

The inhibitory effect of cyproterone acetate on sexual behavior has been investigated in adult ovariectomized ewes treated with either testosterone propionate (10 mg/day) or estradiol benzoate (200 micrograms/day) to induce male activity and female receptivity simultaneously. Intra-muscular injections of 100 mg cyproterone acetate/day resulted in a rapid decrease in some of the male sexual responses after both testosterone and estradiol treatments, whereas the female reactions were eliminated by cyproterone acetate only when they had been induced by testosterone.

Ovariectomized adult Ewes daily treated with I/M injections of testosterone propionate (10 mg/day) or oestradiol benzoate (200 microgram/day) show male-like sexual behaviour and simultaneously a permanent female receptivity. Additional daily treatment with 100 mg of progresterone results in a rapid decrease in male sexual responses both when Ewes are treated with testosterone and with oestradiol. No effect is observed on the female receptivity. Cyproterone acetate (100 mg/day) given to Ewes in addition to daily injection of testosterone propionate (10 mg/day) has effects similar to those of progesterone. These results show that progesterone has antiandrogen and antioestrogen properties and that this hormone administered at high doses is able to inhibit male activity.

Ovariectomized Ewes, treated daily with 100 micrograms of oestradiol benzoate (OB) show simultaneously female and male sexual behaviour. Male sexual behaviour is never seen in intact females. When given simultaneous with OB, progesterone prevents the action of the oestrogen. When given before the start of oestrogen treatment, progesterone also inhibits the effects of OB on male sexual behaviour for several weeks. By contrast, progesterone pretreatment has both inhibitory and facilitatory effect on the induction of female sexual behaviour. At first, it delays the appearance of female behaviour but then increases the response to oestrogen. These results suggest that progesterone could be involved in the dissociation between male and female behaviour normally seen in Sheep.

Adult ovariectomized ewes, daily injected with testosterone propionate (2 to 22 mg), showed male-like sexual behaviour, never seen in untreated females, but very similar to that in normal rams. After a few days of latency (3 days minimum), this behaviour gradually appeared and increased in frequency until a plateau was reached. The time-course in which the plateau was reached depended on the androgen dose and on previous experience with male sexual behaviour. Some male-like behaviour might have persisted after the end of the treatment. The results have been discussed in terms of possible mechanisms of action on the central nervous system.

Herds of grazing mammals characteristically produce precocial offspring in synchrony, and it is therefore important for the mother to form a rapid recognition of her own offspring to distinguish them from others. In sheep, the ewe forms such a selective bond with her lamb within 2–4 hours of parturition, a bond which is primarily dependent on olfactory sensory recognition. Here we report that the neuronal mechanism whereby the olfactory “imprint” is made is dependent on the centrifugal noradrenergic projections to the olfactory bulbs. Lesioning of this neural pathway prevents the ewe from forming a selective bond with her own lamb, enabling her to adopt alien lambs.

All motor patterns of male sexual behaviour can be observed in young lambs from the first days of the life. They considerably increase on weeks 2 to 5 when they decrease dramatically and remain stable up to over 6 months of age. This evolution has no direct relation with that of the physiological events. The adult female has a complete bisexual potentiality. The development of the sexual behaviour during ontogeny appears as a continuous process rather than to be limited to the period of puberty or sexual differentiation.

The effects of sophrology and epidural analgesia on early relationship between the mother and her child were studied on a simple of 190 deliveries. The mothers were observed during and just after delivery. Mothers who had been separated from their child before the end of the observation were excluded from the study. The patients had the choice between epidural analgesia or prenatal care with sophrology. Participation to prenatal courses has statistically a positive effect on the relation between the mother and her child (p less than 0.01). Instead, epidural analgesia and posture have very limited effect on this factor. However, a trend to more interaction is found in multipari and patients who didn't choose epidural analgesia.

Remerciements : INRA, Domaine de la Sapinière,18390 OSMOY, Centre Val de Loire; During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the “ram effect” in ewes that had a “precocious” LH surge (starting within 6 hours), a “normal” surge (between 6 and 28h) and “late» surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. “Precocious” LH surges were not preceded by a large increase in E2 unlike “normal” surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the “precocious” LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the “ram effect”. Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.