Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor

Neuroscience 140 (2006) 791– 800

NEURAL PATHWAYS INVOLVED IN THE ENDOCRINE RESPONSE OF ANESTROUS EWES TO THE MALE OR ITS ODOR H. GELEZ* AND C. FABRE-NYS

roendocrine processes controlling puberty (Vandenbergh, 1969), testicular growth (Lawton and Whitsett, 1979), estrous cycle (Whitten, 1956), and pregnancy (Bruce, 1959). According to their effects, chemosensory cues have been classically classified as releaser or primer pheromones (Keverne, 1983). In rodents, pheromones are often detected by sensory neurons located in the vomeronasal organ (VNO) which send axons to the accessory olfactory bulb (AOB) which in turn project to the medial nucleus of the amygdala (MeA), the bed nucleus of the stria terminalis (BNST) and the preoptic area (POA). Lesional approaches have demonstrated the critical role of the VNO and AOB in the detection of chemosignals in rat, mice and hamster (Johns et al., 1978; Coquelin et al., 1984; Johnston et al., 1987). In these species, a general view of the pathway involved in the process of biologically relevant odors has been monitored by studies using immunohistochemical detection of the Fos protein. In the brain, basal expression of c-fos is low and increases following various kinds of behavioral, sensory and pharmacological stimulation (Morgan et al., 1987). The Fos protein can act as a transcription factor regulating early cellular responses and is commonly used as a neuronal marker of cerebral activation (Hoffman et al., 1993; Hoffman and Lyo, 2002). In rodents, chemosensory signals increase nuclear Fos protein essentially in the structures belonging to the accessory olfactory pathway including the VNO epithelium, AOB, BNST, MeA and POA (Fernandez-Fewell and Meredith, 1994; Bressler and Baum, 1996; Tubbiola and Wysocki, 1997). These findings suggest that the accessory olfactory system is specialized in the detection of chemosignals involved in social communication. On the other hand, the main olfactory system is considered as a general analyzer of environmental odors. However, the respective role of the two olfactory systems remains controversial and is still a subject of debate (recently reviewed in a special issue of Hormones and Behavior, 2004). Considerably less research on pheromonal communication has been carried out in non-rodent mammals. In these species, the main olfactory system seems implicated in responses to biologically relevant odors. Indeed, the VNO is not required for the detection of social chemical signals such as the mother pheromone inducing nipple search in newborn rabbits (Hudson and Distel, 1986), the boar saliva eliciting standing posture (Dorries et al., 1997), the odor of sexual partners in ferret (Kelliher and Baum, 2001). In female sheep, during the anestrous period, the ram or its odor elicits an immediate increase of luteinizing hormone (LH), followed by ovulation if the contact between partners is maintained (Knight et al., 1983; Martin et al., 1986). First studies showed that the destruction of the neu-

Station de Physiologie de la Reproduction et des Comportements, UMR 6175 INRA/CNRS, Université de Tours, Haras Nationaux 37380 Nouzilly, France

Abstract—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. © 2006 Published by Elsevier Ltd on behalf of IBRO. Key words: main and accessory olfactory systems, amygdala, fos, preoptic area, GnRH, LH.

In most mammals, olfactory cues are critical for successful copulation and the associated physiological events. Olfactory stimuli induce behavioral changes leading to locating a mate and identifying a sexual partner, and affect also neu*Corresponding author. Tel: ⫹33-2-47-42-79-75; fax: ⫹33-2-47-42-77-43. E-mail address: [email protected] (H. Gelez). Abbreviations: AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; BNST, bed nucleus of the stria terminalis; BSA, bovine serum albumin; CoA, cortical nucleus of the amygdala; Fos-IR, Fos immunoreactivity; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; MeA, medial nucleus of the amygdala; MOB, main olfactory bulb; OVLT, organum vasculosum of the lamina terminalis; PBS, phosphate-buffered saline; POA, preoptic area; TA, triton sodium azide; VMN, ventromedial nucleus of the hypothalamus; VNO, vomeronasal organ. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2006.02.066

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H. Gelez and C. Fabre-Nys / Neuroscience 140 (2006) 791– 800

roreceptors of the olfactory epithelium impairs the ability of ewes to respond to the ram odor, whereas the elimination of the VNO has no effect (Cohen-Tanoudji et al., 1989; Gelez and Fabre-Nys, 2004). This set of data emphasizes the role of the main olfactory system in the processing of the male odor but does not exclude the participation of the accessory system. Furthermore, bulbectomized ewes are still able to exhibit an endocrine response when they are exposed to males (Cohen-Tanoudji et al., 1986). This result shows the importance of sensory information other than olfaction and suggests the existence of a widespread neural pathway to integrate the male cues and trigger the gonadotropinreleasing hormone (GnRH) neurons controlling the LH response. However, in sheep and other non-rodents species, the neurobiological mechanisms and the different brain areas involved in the responses to social chemosignals remain unknown. Our study was designed to investigate the brain regions activated by the male or its odor in anestrous ewes in order to identify the neural pathways directing the male cues to the structures initiating the activation of LH secretion. The choice of the structures to analyze was established according to preliminary data showing all the brain areas activated in a female exposed to male in comparison with a control ewe. Our second aim was to define the population of GnRH neurons involved in the LH endocrine response. It has been postulated that the GnRH neurons could be localized in anatomically and functionally distinct populations. In hamster, the GnRH neurons in the caudal POA control the LH pulses whereas the GnRH neurons in the medial septum and the rostral POA would be recruited during the preovulatory surge (Berriman et al., 1992). In anestrous ewes, which GnRH neurons control the LH increase in response to the ram or its odor, remains an open question. So, using a procedure of double-labeling for Fos and GnRH, we examined the GnRH neurons activated by the male or its odor.

efforts were made to minimize the number of animals used and their suffering.

Experimental protocol During the anestrous period, all females were isolated from males for at least 1 month and were habituated to manipulation and bleeding during two weeks. They received in their jugular vein a catheter that permits the collection of blood samples. Afterward, blood samples were collected every 15 min for 5 h (control period). The day after, the females were divided in four groups and were randomly exposed for 1 h 30 min to: ● ● ● ●

the testing environment: control group (n⫽5) female odor (n⫽5): some female fleece was introduced in the females’ pens male odor (n⫽6): some male fleece was introduced in the females’ pens male (n⫽6): two sexually experienced males were introduced in the females’ pens

Blood samples were collected during the last hour (to verify the endocrine response to the stimulation) and, immediately after, the females were decapitated by a licensed butcher. The heads were immediately perfused through both carotid arteries with 2 l of 1% sodium nitrite solution (dissolved in phosphate buffer 0.1 M, pH 7.4) and 4 l of cold 4% paraformaldehyde (in the same phosphate buffer). The brains were removed, cut into three blocks, postfixed for 24 h in 4% paraformaldehyde and cryoprotected in 30% sucrose and 0.1% sodium azide in phosphate buffer 0.1 M, pH 7.4. The ovaries were dissected to verify the absence of corpus luteum.

LH assay Blood samples were centrifuged and plasmas were stored at ⫺20 °C. Concentrations of LH were measured in duplicate samples of 100 ␮l plasma by the radioimmunoassay method of Pelletier et al. (1968) as modified by Montgomery et al. (1985). The sensitivity of the assay was 0.16⫾0.05 ng/ml (four assays) standard 1051-CY-LH (i.e. 0.31 ng/ml NIH LH-S1). The intra-assay and inter-assay coefficients of variation were 3.9% and 9.6%, respectively.

Fos immunocytochemistry

EXPERIMENTAL PROCEDURES Animals The experiment was performed in spring, during the anestrus season, at the INRA station in Nouzilly. We used 22 adult (2–5 years-old) sexually experienced, Ile-de-France or Romanov⫻IleDe-France anestrous ewes. The females were housed with a stimulus female, in indoor pens which had not previously contained rams, under a natural photoperiod. They were fed daily with a constant diet of straw, maize, Lucerne pellets and mineral supplements and had free access to water. The ewes were diagnosed as seasonally anovulatory by persistent low concentrations of circulating progesterone (⬍1 ng/ml) in weekly progesterone assays, indicating the absence of a functional corpus luteum. These assays were performed with a method adapted from Terqui and Thimonier (1974). Four sexually experienced adult, Ile-de-France rams, were used as stimuli. Fleece of 10 other Ile-de-France or Romanov rams and 10 Ile-de-France ewes was collected during the breeding season and stored at ⫺20 °C. All experimental procedures were performed in accordance with the European Community Council Directive (86/609/ECC) and with the local animal regulation (Authorisation No. 006259 of the French Ministry of Agriculture) on animal experimentation. All

Free-floating frontal sections (40 ␮m-thick) were cut on a freezing microtome (Leica, Paris, France) and kept in cryoprotectant (NaCl 9% polyvinyl pyrrolidone 10%, saccharose 30%, ethylene glycol 30%, phosphate buffer 0.1 M 50%) at 4 °C. One out of 10 sections was mounted and stained with Cresyl Violet to allow histological identification and delimitation of the brain areas. For each brain structure, three representative sections from anterior, median and posterior part of the structure were selected. These sections were rinsed in phosphate-buffered saline (PBS, 0.1 M pH 7.4) and successively incubated with 1% H2O2 (1 h, 4 °C), PBS containing 0.3% Triton X-100, 0.1% sodium azide and 2% bovine serum albumin (BSA, Roche Diagnostics Corporation, Indianapolis, IN, USA) (1 h, 4 °C), then with an affinity-purified rabbit polyclonal antibody raised against the Fos protein (Ab-2, PC38, Oncogene Research Products, Calbiochem, San Diego, CA, USA, diluted 1:60,000 in PBS–triton sodium azide (TA)–BSA, 3 days, 4 °C). Sections were rinsed in PBS, incubated with a sheep anti-rabbit immunoglobulin serum (diluted 1:400 in PBS, 3 h, 4 °C), rinsed and incubated overnight with a rat peroxidase–antiperoxidase complex solution (Jackson Immunoresearch, West Grove, PA, USA, diluted 1:1000 in PBS). The peroxidase complex was visualized after incubation in a 0.04% 3-3=diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO, USA), 0.3% nickel ammonium sulfate and 0.01% H2O2 solution in Tris–HCl buffer (0.05 M, pH 7.4), producing a black precipitate. The reaction was

H. Gelez and C. Fabre-Nys / Neuroscience 140 (2006) 791– 800 terminated by several washes in Tris–HCl. The sections were mounted on gelatin-chrome alum coated slices, left to dry overnight and counterstained with 0.05% Cresyl Violet to facilitate histological identification of structures according to the atlas of Richard (1967) and the neuroanatomical data of Jansen et al. (1998). Finally, they were dehydrated in an ascending ethanol series and toluene, and coverslipped with Depex® (BDH Laboratory Supplies, Poole, UK). The anti-Fos was raised against residues 4 –17 of the human peptide gene product, and the specificity of the labeling was tested on adjacent sections incubated with 1 ml of diluted (1:60,000) anti-Fos preincubated overnight at 4 °C with 1 ng of Fos peptide (PP10, Oncogene). The effect of omission of the secondary antibody (sheep serum anti-rabbit IgG) or the peroxidase–antiperoxidase complex was also tested.

GnRH immunocytochemistry For each animal, three sections in the organum vasculosum of the lamina terminalis (OVLT) and the POA were selected for the double labeling. These sections were first stained for the Fos protein as for single labeling and then incubated with an affinitypurified rabbit polyclonal antibody raised against GnRH (a generous gift from Dr Y. Tillet of INRA Nouzilly, diluted 1:20,000 in PBS–TA containing 2% human serum albumin, 4 days, 4 °C). Sections were rinsed in PBS, incubated with a CY3-conjugated anti-rabbit immunoglobulin made in mouse (Jackson, diluted 1:1000 in PBS, overnight, 4 °C). The sections were rinsed in PBS, mounted in gelatin-chrome alum coated slices, left to dry overnight and coverslipped with a drop of glycine–PBS (80%–20%).

Analysis of immunostaining Quantification of Fos immunoreactivity (Fos-IR) positive cells was performed with an image analysis system equipped with a cellcount analysis software (Biocom, Paris, France). A Zeiss light microscope with a motorized stage and a video camera was connected to a PC computer and color monitor (640⫻400 resolution) for cell counting. Images (⫻10) were projected onto the monitor and a semi-automatic program selected Fos-IR positive cells (Gelez and Fabre-Nys, 2001). This semi-automatic program was elaborated to establish the parameters that ensured a reliable detection of positive cells and to define a gray level threshold characterizing a Fos-IR positive nucleus. For each brain section, the average background gray level and illumination were automatically estimated and subtracted from the original picture. Objects (stained nuclei) were defined by segmentation of the image using a predetermined value as gray level threshold. The computer recorded the number of positives cells in each field. Objects ⬍25 ␮m2 or ⬎200 ␮m2 were eliminated to discard artifacts as much possible. Each field were also visually checked, suspect objects were erased and clusters of nuclei disjointed manually. The density of Fos-IR cells was defined as the total number of labeled nuclei divided by the total area where they were distributed. GnRH-IR neurons were counted manually with the same image analysis system. Images (⫻10) were projected onto the monitor and an observer marked each GnRH-IR cell on the screen.

Preliminary screening To validate the immunocytochemistry technique and to select the structures to analyze, we first examined the Fos neuronal expression in the entire brains of two females: one exposed to the male and one exposed to the testing room. Exposure to the male increases Fos neuronal activation in most of the olfactory structures belonging to both olfactory systems, but also in the OVLT, the medial POA, the ventromedial nucleus of the hypothalamus (VMN), the basolateral amygdala, the dentate gyrus, the somato-

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sensory and visual cortices. The results of this screening have been used in the final experiment to complete the list of the structures to analyze.

Statistical analysis LH levels were analyzed by a t-test comparing each ewe LH secretion before and during the stimulation. Mean number of Fos-IR cells and GnRH-IR neurons was calculated for each animal in each region and then an overall mean and standard error was calculated for each treatment group. Because these variables were not normally distributed, statistical comparisons between treatment groups were carried out using nonparametric tests. We used the nonparametric Kruskall-Wallis test to do an overall analysis of the four groups of females. If the result of this test reached significance, we then conducted pairwise comparisons using the Mann-Whitney test.

RESULTS LH secretion All females (six of six) exposed to the male and five of the six females exposed to the male odor exhibited a significant increase in LH secretion (P⬍0.05, t-test). None of the females in the group exposed to the female fleece or in the control group (except for one) displayed a significant change of LH level. Fos immunocytochemistry According to the results of the screening of two entire brains of a female exposed to the male and a control ewe, Fos-IR cells were quantified in 21 brain regions across the olfactory bulbs, the limbic system, the hypothalamus and different cortical areas (summarized in Table 1). The Fos labeling was characterized by a dense black precipitate in cell nuclei (Fig. 1a). The control tests prove the specificity of the labeling showing that the staining disappeared after preincubation of the primary Fos antiserum with the Oncogene Fos peptide or in the absence of the secondary antibody or the peroxidase– antiperoxidase complex. In most structures analyzed, ewes exposed to the male exhibit a density of Fos-IR cells significantly higher than control ewes, both in the accessory (AOB, MeA) and the main olfactory systems [main olfactory bulb (MOB), anterior olfactory nucleus (AON), cortical amygdala (CoA)], the basal amygdala, the dentate gyrus, the POA, the VMN and numerous cortical areas (piriform, entorhinal, orbitofrontal, frontomedial) (Figs. 2– 4). Exposure to the male elicits a significantly higher Fos neuronal activation than its odor alone in the CoA, the basal amygdala, the POA and the VMN (Figs. 2 and 3). In comparison with the control group, the male odor also elicits a significantly higher Fos neuronal activation both in the accessory (AOB) and the main olfactory systems (mitral layer of the MOB, AON, CoA), the basal amygdala, the dentate gyrus, the VMN and the piriform and orbitofrontal cortices (Figs. 2– 4). In contrast, ewes exposed to the female fleece exhibit a significant higher number of Fos-IR than control females only in the CoA, the basal amygdala and the piriform and orbitofrontal cortices (Figs. 2 and 3).

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Table 1. Density of Fos-IR neurons in anestrous ewes exposed to different stimuli Structure

Main olfactory system MOB total Granular layer Mitral layer Glomerular layer AON Cortical amygdala total C1 C2 C3 Basal amygdala Anterior piriform cortex Posterior piriform cortex Entorhinal cortex Orbitofrontal cortex Frontomedial cortex Ventral hippocampus Dorsal hippocampus Dentate gyrus Accessory olfactory system AOB total Granula mitral layers Glomerular layer Medial amygdala OVLT Medial preoptic area Lateral preoptic area VMN Limbic system Lateral septum Lateral amygdala Central amygdala Basal ganglia Nucleus accumbens Hypothalamus Anterior hypothalamic area Paraventricular nucleus Suprachiasmatic nucleus Supraoptic nucleus Cortex Visual cortex V1 Visual cortex V2 Cingulate cortex

Testing room (n⫽5)

Female fleece (n⫽5)

Male fleece (n⫽6)

Male (n⫽6)

4.2⫾1.8 5.6⫾2.5 8.8⫾3.7 1.2⫾0.6 3.3⫾1.1 3.2⫾0.7 4.2⫾0.4 2.6⫾0.7 3.7⫾2 2.3⫾0.5 5.8⫾1.7 2.7⫾0.8 3.2⫾0.8 0.7⫾0.3 0.8⫾0.3 0.38⫾0.15 0.12⫾0.04 0.5⫾0.1

6⫾1.5 9.7⫾2.4 9.3⫾2.4 1.2⫾0.3 5.1⫾1 6.8⫾0.5a 6.8⫾1.1 7.0⫾1a 6.2⫾2.6 4.7⫾0.8a 10.6⫾2.1a 2.6⫾0.8 3.6⫾0.8 2.1⫾0.4a 1.3⫾0.3 0.56⫾0.20 0.12⫾0.04 0.4⫾0.2

7.2⫾1.4 13.6⫾3.4 18.8⫾2.9a,b 1.2⫾0.2 10.7⫾3.5a 10.5⫾1.4a,b 11.3⫾2.7a 10.0⫾1.2a 9.2⫾2.3 4.1⫾0.7a 13.0⫾3.0a 4.7⫾1.7 6.2⫾1.1a 2.0⫾0.4a 1.7⫾0.2a 0.26⫾0.07 0.10⫾0.06 1.1⫾0.2a,b

11.2⫾2.5a 20.2⫾4.8a 26.9⫾7.3a,b 3.1⫾0.9 11.6⫾3.3a,b 20.8⫾3.2a,b,c 33.2⫾6.8a,b,c 18.1⫾3.4a,b,c 16.4⫾2a,b,c 8.7⫾0.9a,b,c 19.1⫾3.1a 9.3⫾2.0a,b 8.9⫾1.2a,b 1.6⫾0.3a 2.1⫾0.5a 0.50⫾0.20 0.09⫾0.03 1.5⫾0.5a,b

2.5⫾0.8 7.1⫾2.5 0.4⫾0.3 6.3⫾1.6 5⫾1.6 4.1⫾1 0.9⫾0.3 8.6⫾2

4.6⫾0.9 12.3⫾1.9 0.5⫾0.2 13.4⫾4.1 5.3⫾1.1 4.6⫾0.7 1.3⫾0.3 15.2⫾4.2

5.4⫾0.5a 14.7⫾1.3 0.4⫾0.2 14.1⫾6.5 5.6⫾1.6 6.2⫾1.4 1.1⫾0.5 15.6⫾2.4a

7.7⫾1.1a 20.2⫾3.8a 0.3⫾0.2 29.8⫾3.9a,b 8.1⫾1.4 14.8⫾1.2a,b,c 3.3⫾0.3a,b,c 36⫾6.4a,b,c

1.4⫾0.5 0.9⫾0.2 1.4⫾0.4

3.9⫾0.9 1.2⫾0.2 1.4⫾0.4

2.2⫾0.6 1.0⫾0.2 1.3⫾0.3

2.7⫾0.8 1.6⫾0.3 2.0⫾0.3

1.9⫾0.8

4.9⫾1.5

1.9⫾0.8 8.7⫾1.7 9.0⫾3.0 2.2⫾0.8 0.2⫾0.1

9.8⫾2.2 15.8⫾4.3 4.0⫾1.1 0.3⫾0.1

8.9⫾1.4 11.0⫾4.0 2.7⫾0.8 0.2⫾0.1

11.4⫾1.7 13.7⫾3.8 4.7⫾1.0 0.2⫾0.1

1.4⫾0.4 1.1⫾0.4 10.9⫾1.8

3.9⫾1.2 2.7⫾0.8 6.8⫾3.6

2.8⫾0.9 1.5⫾0.4 11.7⫾3.9

2.3⫾0.5 1.5⫾0.3 6.6⫾1.7

Data are expressed in density of Fos-IR cells (number of Fos-IR cells/mm2): mean⫾SEM. Different from control group: P⬍0.05 (Mann-Whitney test). b Different from female fleece: P⬍0.05 (Mann-Whitney test). c Different from male fleece: P⬍0.05 (Mann-Whitney test). a

Three structures: the MOB (mitral layer), the CoA and the dentate gyrus are specifically more activated by the male odor than the female odor (Fig. 2 and 3). In the cingulate cortex, control ewes exhibit a density of Fos-IR that tends to be higher than in females exposed to the male but the difference does not reach significance. No differences in the density of Fos-IR between the four groups of females were observed in the lateral septum, the central and lateral nuclei of amygdala, the hippocampus, the OVLT, the anterior hypothalamic area, the supraoptic

nucleus, the suprachiasmatic nucleus, the nucleus accumbens and the visual cortex. GnRH and GnRH/Fos immunocytochemistry The GnRH neurons in the POA and the OVLT have a similar size and most of them have a long shape (Fig. 1b). No significant differences in the number of GnRH-IR were observed between the four groups of females. In ewes exposed to the male odor, the number of GnRH-IR in the OVLT tends to be superior to that of the ewes exposed to the

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Fig. 1. Photomicrographs showing Fos-immunoreactivity (A), GnRH neuron that does not express Fos protein (B) and GnRH neuron expressing Fos protein (C).

female odor but the difference does not reach significance (P⫽0.06, Table 2). Double-staining GnRH/Fos neurons appeared like a white cytoplasm (GnRH) with a black reaction product in the cell nuclei (Fos) (Fig. 1c). In each group, very few GnRH-IR neurons were double stained for the Fos protein. However, in both the POA and the OVLT, the male or its odor increases significantly the number and the proportion of GnRH cells expressing Fos-IR, whereas no such induction of Fos-IR in GnRH neurons occurred in

ewes exposed to the female odor or the testing room (P⬍0.05 for all comparisons, except in the POA for the comparison between female odor/male odor: P⫽0.08, Table 2). It is noteworthy that the GnRH-IR and the GnRH cells expressing Fos-IR have not been analyzed in other brain areas than the OVLT and the POA because the number of double-stained GnRH/Fos neurons was very low and no differences between groups were observed, even in the MBH.

Fig. 2. Density of Fos-IR neurons in anestrous ewes exposed to different stimuli (testing room⫽control, female fleece, male fleece or male). Data are expressed in density of Fos-IR cells (number of Fos-IR cells/mm2): mean⫾S.E.M. a⫽Different from control group (Mann-Whitney test): P⬍0.05; b⫽different from female (Mann-Whitney test): P⬍0.05; c⫽different from male fleece (Mann-Whitney test): P⬍0.05.

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Fig. 3. Photomicrographs illustrating Fos-immunoreactivity in the AOB and the MOB in four groups of anestrous ewes exposed to different stimuli (testing room⫽control, female fleece, male fleece or male).

DISCUSSION Neuronal activation elicited by the male odor Our results give a map of the neural pathways involved in the effect of the ram fleece on the LH secretion in

anestrous ewes, underlying the critical role of the main olfactory system. Indeed, the MOB (mitral layer) and the CoA are specifically more activated by the male than the female odor and seem therefore to be primarily involved in the detection and the integration of the ram odor. Mitral

Fig. 4. Photomicrographs illustrating Fos-immunoreactivity in the MeA, the CoA, the VMN and the piriform cortex (Piri Cx) in four groups of anestrous ewes exposed to different stimuli (testing room⫽control, female fleece, male fleece or male).

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Table 2. Number of GnRH-IR and GnRH/Fos-IR in anestrous ewes exposed to different stimuli Structure

GnRH

Testing room (n⫽5)

Female fleece (n⫽5)

Male fleece (n⫽6)

Male (n⫽6)

OVLT

GnRH-IR GnRH/Fos-IR GnRH/Fos-IR % GnRH-IR GnRH/Fos-IR GnRH/Fos-IR %

36.8⫾3.4 0.3⫾0.1 0.7 4.9⫾0.8 0.05⫾0.03 1

28.2⫾4.4 0.2⫾0.1 0.9 5.7⫾0.8 0.2⫾0.1 2.8

37.6⫾4.5 2.1⫾0.4a,b 5.5a,b 4.2⫾0.8 0.5⫾0.2a 12.3a,b

39.6⫾3.3 1.6⫾0.3a,b 4.1a,b 6.2⫾1.2 0.9⫾0.2a,b 13.3a,b

POA

Data are expressed in number of GnRH-IR neurons and GnRH/Fos-IR neurons: mean⫾SEM. Different from control group: P⬍0.05 (Mann-Whitney test). b Different from female fleece: P⬍0.05 (Mann-Whitney test). a

cells provide the output from the olfactory bulb to the rest of the brain (Halasz and Sheperd, 1983). The neuronal Fos activation localized in the mitral layer of the MOB suggests that the role of the MOB is firstly to convey the male olfactory cue to the areas with those it is connected. The male odor increases Fos expression in other structures belonging to the main olfactory system: the AON, piriform and entorhinal cortices, but also in the AOB. This result shows that the accessory olfactory system participates in the detection of the ram odor. However, its involvement seems limited and much less important than that of the main olfactory system, since a significant difference of neuronal Fos activation is observed only in the AOB and not in other structures belonging to the accessory olfactory system. Furthermore, in this structure, both male and female odors induce a similar density of Fos-IR. This set of data, emphasizing a critical role of the main olfactory system and a minor role of the accessory system, is consistent with our previous findings demonstrating that the destruction of the neuroreceptors of the olfactory epithelium or the inactivation of the CoA completely blocks the LH response to male odor, whereas destruction of the VNO or inactivation of the MeA has no effect (Gelez et al., 2004; Gelez and Fabre-Nys, 2004; Cohen-Tanoudji et al., 1989). In rodents, biologically relevant odors are detected by the VNO and subsequently processed by the accessory olfactory system. This has been demonstrated by different approaches including lesioning techniques and Fos imaging. A large number of studies showed that in rodents, exposure to olfactory cues eliciting physiological changes induces Fos-IR essentially in the structures belonging to the accessory olfactory pathway including the VNO, AOB, BNST, MeA and POA (rat: Bressler and Baum, 1996; Inamura et al., 1999; Dudley and Moss, 1999; hamster: Fernandez-Fewell and Meredith, 1994; prairie vole: Moffatt et al., 1995; Tubbiola and Wysocki, 1997). Our results show that the mechanisms involved in the process of primer pheromones can profoundly differ between species. In sheep, the roles of the VNO and the accessory olfactory system remain unclear. Some studies suggested that the VNO is not required for sex odor discrimination but may be used to prolong contact with the chemosignals (mice: Pankevich et al., 2004; guinea-pig: Petrulis et al., 1999; hamster: Beauchamp et al., 1982; ferret: Woodley et al., 2004).

The accessory olfactory system might play a similar role in ewes, enhancing investigations of the male fleece. In this view, the accessory olfactory system could facilitate and maximize the response to the male odor. The dentate gyrus is the third structure specifically more activated by the male than the female odor. This tends to confirm the role of this structure in the treatment of olfactory cues, as previously suggested (Vanderwolf, 2001). Even if the male odor elicits a higher Fos neuronal activation only in three structures (MOB, CoA and dentate gyrus), in comparison with the female odor, it triggers the LH response and increases the percentage of GnRH cells expressing FOS-IR. The GnRH/fos-IR cells were homogenously distributed through the OVLT and POA and not in a limited anatomical area. However, this does not exclude that only a precise neuronal population controls the endocrine response. The role of the OVLT is restricted to endocrine and physiological processes whereas the POA is involved in the control of female appetitive behavior and motivational processes (Whitney, 1986; Hoshina et al., 1994). The higher percentage of double stained GnRH/Fos neurons in the POA than in the OVLT could be related to a change of arousal or motivation in ewes exposed to the male fleece. Furthermore, this result is the first example that sex odors alone can increase Fos expression within GnRH neurons. This has not been observed in other species (rat: Rajendren et al., 1993; ferret: Wersinger and Baum, 1997; Bakker et al., 2001). This might reflect the major role of sexual interactions in modulating reproduction in sheep. It has been suggested that separate populations of GnRH-IR neurons are associated with pulsatile and surge modes of LH secretion. In female hamster, the LH pulses would be controlled by the GnRH neurons localized in the rostral part of the POA whereas the preovulatory surge involves the GnRH neurons of the caudal POA (Berriman et al., 1992). In sheep, the GnRH neurons of the MBH are responsible for the pulsatile LH secretion whereas the preovulatory surge of LH requires the GnRH neurons through the OVLT, POA and MBH (Moenter et al., 1993; Boukhliq et al., 1999). Our results do not confirm this hypothesis. This discrepancy can be explained by the difference of the models used, since Boukhliq et al. (1999) used a pharmacological agent, an opioid antagonist, to stimulate the episodic LH secretion in luteal phase ewes.

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In our experiment, anestrous ewes exhibit an increase of the LH pulses in response to a natural stimulus. Our data allow us to establish the hypothetical pathway supporting the action of the male odor and suggest the male olfactory cues reach the GnRH neurons via a neural pathway more complex than the direct AOB–MeA–POA pathway involved in the processing of social chemosignals in rodents. In the anterior part of the piriform cortex and the basal nucleus of amygdala, the male and the female odors induce a similar neuronal Fos-IR activation that is significantly higher than in the control group. The piriform cortex and the basal amygdala play a critical role in the acquisition and the expression of associative learning (Gallagher, 2000; Haberly, 2001). They are involved in the processing of olfactory stimuli that have acquired a meaning through association with natural incentives or rewards, like ejaculation in males rats (Kippin et al., 2003). In our study, these two structures are similarly activated by the male and the female odors. The LH responses prove that ewes discriminate male and female odors. The piriform cortex and the basal amygdala could be involved in this process of discrimination. They could act differently according to the olfactory stimulus and subsequently direct the information in different neural pathways leading the activation of the GnRH neurons in response only to the male odor. The orbitofrontal and the frontomedial cortices can also be involved in this process of discrimination, since in these structures the male odor greatly increases the neuronal Fos activation compared with control ewes. According to the anatomical connections between the MOB and the CoA, the piriform, orbitofrontal and frontomedial cortices, and the projections from the piriform cortex and the CoA to the basal nucleus of the amygdala, it can postulated that all these structures are involved in the process of the male olfactory cue to reach the GnRH neurons, even if the connections between the olfactory systems and the GnRH neurons remain not clearly identified. Neuronal activation elicited by the male The male augments the neuronal Fos expression significantly more than the control situation in almost all the structures analyzed, and significantly more than its odor alone in the POA, VMN, MeA, CoA and the basal amygdala. This widespread neuronal activation can be induced by an increase of the intensity and/or the quality of the olfactory stimulation with the male body odors or its urine, but also by other sensory cues confirming that the response to the male effect is multisensorial. This has been demonstrated in previous studies showing that bulbectomized ewes are still able to exhibit an LH response during exposure to males (Cohen-Tanoudji et al., 1986). The complex neural pathway recruited for the response to the male can be related to the greater efficiency of the male as compared with its odor to activate the LH secretion. Exposure to the male does not increase the neuronal Fos-IR activation in the visual cortex. Although this result is surprising it is in accordance with the data of Ohkura et al. (1997) who found no difference in the expression of c-fos

mRNA in the areas V1 and V2 of the visual cortex between anestrus ewes exposed to males and estrus ewes exposed only to the testing environment. The c-fos mRNA expression increases only in estrus ewes. This suggests that male visual cues have only a minor role during the anestrous period. The neuronal Fos-IR activation elicited by exposure to the male reinforces our view of the hypothetical neural pathway involved in the processing of the male olfactory cue. Indeed, in the mitral layer of the MOB, the AON and the CoA, females exposed to the male, like those exposed to the male odor, exhibited a significantly higher number of Fos-IR than ewes exposed to the female odor or the testing environment. This confirms the preponderant role of the main olfactory system in the detection of the ram odor. The neuronal Fos-IR activation detected in the AOB, the MeA after exposure to males confirms the participation of the accessory olfactory system and also the probable involvement of the basal amygdala, the VMN and the piriform cortex in the processing of the male olfactory cue. Our results give a general overview of the neural pathways involved in the male effect. They confirm the critical role of the main olfactory system in the detection of primer pheromones in other species than rodents and reveal the participation of the accessory system in this process, even if its role seems limited. The activation of the GnRH neurons recruited to induce the LH response can involve numerous structures widely distributed such as the dentate gyrus, the basal amygdala and the piriform cortex. Our data emphasize that even if olfactory cues can induce similar physiological changes across species, the neural mechanisms supporting their action can be profoundly different. Acknowledgments—The authors thank Yves Tillet for his gift of the GnRH antibody, Didier Chesneau for his help with the LH assays, Daniel Tanguy for his help with the system of image analysis and Odile Moulin for her help with the illustrations.

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(Accepted 27 February 2006) (Available online 2 May 2006)