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

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

Effect of a two-week treatment with low dose of ortho-substituted polychlorinated biphenyls (PCB104 and PCB153) on VEGF-receptor system expression in the choroid plexus in adult ewes.

Ortho-substituted polychlorinated biphenyl (PCB) congeners, which constitute a large part of PCB residues found in the environment and in animal tissues, are known to exert potent vascular effects and can activate endothelial cells in the periphery and in the brain. The choroid plexus (CP) is responsible for cerebrospinal fluid (CSF) production and its epithelial cell layer is responsible for structure and functions of the blood-CSF barrier. The aims of this study were: 1) to investigate if environmentally relevant doses of PCB153 and similar doses of PCB104 caused changes in the expression of vascular endothelial growth factor (VEGF)--receptor system, which maintains CP function, and 2) to determine the level of both congeners in blood plasma after their oral administration. Studies of both congeners were performed on ovariectomized ewes treated per os with low doses (0.1 mg/kg, three times a week for two weeks) of PCB153 (n = 4) or PCB104 (n = 4) and vehicle (control, n = 4). The effects of PCB153 and PCB104 treatment on mRNA expression of two isoforms of VEGF (VEGF120 and VEGF164) and their receptors Flt-1 and KDR were determined using real-time PCR. Plasma concentration of PCBs was measured using high resolution chromatography/tandem mass spectrometry (HRGC/MS-MS). We observed that neither PCB153 nor PCB104 significantly altered the mRNA of the VEGF-receptor system in the CP. In PCB treated animals plasma concentration of PCB153 (1.425 +/- 0.16 ng/g of dry mass, DM) was about 150 times higher than PCB104 (0.009 +/- 0.007 ng/g DM). In control animals the PCB153 level was 0.14 +/- 0.031 ng/g DM, while the PCB104 level was below detection level. This indicates that increase in plasma PCB153 concentration to levels similar to those reported in humans and of PCB104 concentration to levels 100 times higher than those found in human plasma did not affect the VEGF-receptor system in the CP in adult ewes. The significantly lower increase of PCB104 than PCB153 concentration in blood after oral administration suggests different absorption of both congeners from the digestive tract.

The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent.

Seasonal reproduction is grounded in several mechanisms, among which are plasticity in both hormone synthesis and neuronal networks. Increased daylength on long days (LD) translates into local tri-iodothyronin (T3) production in the mediobasal hypothalamus that will enable the transition to the anoestrus season in sheep. The photoperiod also strongly affects the content of kisspeptin (Kiss), a hypothalamic neuropeptide exerting a potent stimulatory effect on gonadotrophin-releasing hormone release. Our hypothesis was that T3 directly inhibits Kiss release during LD. Using double immunocytochemistry, we first searched for coexpression of thyroid hormone receptor (THR)α in Kiss neurones in ewes with an active or inactive gonadotrophic axis. In both the preoptic area and the arcuate nucleus, most Kiss neurones were labelled by THR antibody under both physiological/photoperiodic conditions. These results suggest thyroid hormones may affect Kiss synthesis and release all through the year. We then attempted to assess the influence of T3 on Kiss content in hypothalamic explants sampled from ewes with an active gonadotrophic axis. Kiss produced by hypothalamic explants cultured with different doses of T3 (300 or 600 pg) and subjected to different times of incubation (2 or 24 h) was measured. No significant effects of T3 on Kiss tissular content were observed for the two doses of T3 and for the two incubation times. In light of these findings, potential reasons for the divergent effects of thyroid hormones on Kiss content are discussed. Our data emphasise that the effects of thyroid hormone on Kiss synthesis are not one-sided and may affect a wide range of functions.

No Evidence That RFamide-Related Peptide 3 Directly Modulates LH Secretion in the Ewe.

The neuropeptide RFamide-related peptide 3 (RFRP-3) has been implicated in the control of gonadotropin secretion in both birds and mammals. However, in mammals, depending on species, sex and photoperiod, inhibitory, excitatory, or no effect of RFRP-3 on the plasma concentration of LH has been reported. In the ewe, treatment with RFRP-3 either reduced LH concentration or had no effect, and treatment with an RFRP-3 receptor antagonist (ie, RF9) resulted in increased concentration of plasma LH. To clarify these conflicting results in the present study, a set of experiments was performed in ewes. Multiple iv injections of RFRP-3 (6 × 50 µg) in ovariectomized ewes had no effect on plasma LH pulsatility. In intact ewes a bolus injection (500 µg) or an injection (250, 500, or 1000 µg) followed by a 4-hour perfusion (250, 500, or 1000 µg · h(-1)) of RFRP-3 had no effect on the LH pulse induced by kisspeptin (6.5 µg). In ovariectomized, estrogen-replaced ewes, the LH surge induced by estradiol benzoate was not modified by a 24-hour perfusion of RFRP-3 (500 µg h(-1)). Finally, although treatment with RF9 induced a robust release of LH, treatment with a more selective RFRP-3 receptor antagonist, GJ14, resulted in no evident increase of LH. In contrast to the inhibitory effect previously suggested, our data are more consistent with the concept that RFRP-3 has no direct effect on LH secretion in ewes and that RF9 effect on LH release is likely not RFRP-3 receptor mediated. Hence, RFRP-3 probably has a minor role on the control of LH secretion in the ewe.

Oestrogen receptor beta-immunoreactive neurones in the ovine hypothalamus: distribution and colocalisation with gonadotropin-releasing hormone.

Oestrogen powerfully affects the secretion of gonadotropin-releasing hormone (GnRH) from the brain in all species investigated, including sheep. Until recently, it was hypothesised that such regulation occurs indirectly because few or no GnRH neurones were found to express oestrogen receptor (ER) alpha. The discovery of a second oestrogen receptor, ERbeta, and its subsequent localisation in numerous GnRH neurones in the rat, led to a reconsideration of this hypothesis. However, colocalisation of immunoreactive ERbeta protein in GnRH neurones has only been demonstrated in the rat, raising the possibility that such putative direct regulation of GnRH neurones by oestrogen may be peculiar to this species. We have previously shown that steroid receptors in the sheep brain are acutely sensitive to fixation and the full complement of immunoreactive cells can only be visualised after antigen retrieval. The aims of this study were therefore to map immunocytochemically the distribution of ERbeta neurones in the ewe brain, and to determine which proportion of GnRH neurones express ERbeta. Brain sections (20 microm) from four ewes killed in anestrus were subjected to high temperature antigen retrieval and immunocytochemistry. Numerous ERbeta-immunoreactive cells were located throughout the hypothalamus and, following dual-label immunocytochemistry, over 50% of the GnRH neurones were found to express immunoreactive ERbeta. The functional significance of these ERbeta-expressing GnRH neurones in the ovine brain remains to be determined.

Photoperiod affects the cerebrospinal fluid proteome: a comparison between short day- and long day-treated ewes.

Photoperiod is the main physical synchronizer of seasonal functions and a key factor in the modulation of molecule access to cerebrospinal fluid (CSF) in animals. Previous work has shown that photoperiod affects the transfer rate of steroids and protein hormones from blood to CSF and modulates choroid plexus tight junction protein content. We hypothesized that the CSF proteome would also be modified by photoperiod. We tested this hypothesis by comparing CSF obtained from the third ventricle of mature, ovariectomized, estradiol-replaced ewes exposed to long day length (LD) or short day length (SD). Variations in CSF protein expression between SD- or LD-treated ewes were studied in pools of CSF collected for 48 h. Proteins were precipitated, concentrated, and included in a polyacrylamide gel without protein fractionation. After in-gel tryptic digestion of total protein samples, we analyzed the resulting peptides by nanoliquid chromatography coupled with high-resolution tandem mass spectrometry (GeLC-MS/MS). Quantitative analysis was performed using 2 methods based on spectral counting and extracted ion chromatograms. Among 103 identified proteins, 41 were differentially expressed between LD and SD ewes (with P < 0.05 and at least a 1.5-fold difference). Of the 41 differentially expressed proteins, 22 were identified by both methods and 19 using extracted ion chromatograms only. Eighteen proteins were more abundant in LD ewes and 23 were more abundant in SD ewes. These proteins are involved in numerous functions including hormone transport, immune system activity, metabolism, and angiogenesis. To confirm proteomic results, 2 proteins, pigment epithelium-derived factor (PEDF) and gelsolin, for each individual sample of CSF collected under SD or LD were analyzed with Western blots. These results suggest an important photoperiod-dependent change in CSF proteome composition. Nevertheless, additional studies are required to assess the role of each protein in seasonal functions.

Multiple peptides infrequently coexist in progesterone receptor-containing neurons in the ventrolateral hypothalamic nucleus of the guinea-pig: an immunocytochemical triple-label analysis of somatostatin, neurotensin and substance P.

Progesterone plays an important role in regulating reproductive behaviour in guinea-pigs through actions exerted at the ventrolateral nucleus (VL), an area of the brain that contains progesterone receptors (PR) and neuroactive peptides, somatostatin (SOM), neurotensin (NT) and substance P (SP). Previous double-label analyses provided evidence that a substantial proportion of these neuropeptidergic cells contain PR. By means of triple-label immunofluorescence histochemistry, we examined whether PR are colocalized with two neuropeptides (SOM + NT or SP + SOM or SP + NT) within the same neurons in the VL. Ovariectomized guinea-pigs were primed with estradiol to induce PR immunoreactivity, and treated with colchicine to visualize immunoreactive (IR) neuropeptidergic cells. Both monoclonal mouse PR and polyclonal rabbit neuropeptide antibodies were used in double staining and in elution-restaining experiments. In the whole VL, the proportion of each coexisting peptide with PR obtained after double immunofluorescence appeared in decreasing order as: SOM (34%)>NT (25%)>SP (20%). Occasional colocalization was seen between PR and two neuropeptides throughout the rostrocaudal extent of the VL. Combining our various quantitative observations, we found that, of the total population of PR-IR neurons containing any combination of SOM, NT and SP, only about 1.5% contained SOM and NT, 2% contained SP and SOM and 1.6% contained SP and NT. These results indicate that while many PR-IR neurons also contain SOM or NT or SP in the guinea-pig VL, there may be very few PR-IR neurons that express more than one of these three peptides.

Immunoreactive galanin expression in ovine gonadotropin-releasing hormone neurones: no effects of gender or reproductive status.

The neuropeptide, galanin, has been implicated to play a significant role in numerous physiological functions, including reproduction. Studies on several species have shown that galanin enhances gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone secretion. In rodents, a subset of GnRH neurones expresses galanin in a sexually dimorphic manner and it has been suggested that this may underpin the differences in GnRH secretion observed between the sexes. However, there are few data available for other species. Previous studies in sheep have shown that the distribution of GnRH neurones overlaps with galanin cells. The primary objectives of our study were to determine whether GnRH and galanin coexist in the sheep brain and, importantly, if a sex difference is apparent in the colocalization of these two peptides. Using immunocytochemistry coupled to high temperature antigen retrieval, we found that all GnRH neurones in the ovine brain colocalize with galanin. There is also a distinct population of galanin neurones that do not secrete GnRH. In addition, the distribution of galanin-immunoreactive cells was similar to that previously reported for colchicine treated ewes and, in agreement with earlier studies, the number of GnRH neurones did not differ between rams and ewes or between ewes killed at different stages of the oestrous cycle. These results suggest that, in sheep, GnRH and galanin may be cosecreted but the functional significance of this coexpression and possible cosecretion remains to be elucidated.

Perinatal malnutrition programs sympathoadrenal and hypothalamic-pituitary-adrenal axis responsiveness to restraint stress in adult male rats.

In humans, an altered control of cortisol secretion was reported in adult men born with a low birth weight making the hypothalamic-pituitary-adrenal (HPA) axis a possible primary target of early life programming. In rats, we have recently shown that maternal food restriction during late pregnancy induces both an intrauterine growth retardation and an overexposure of fetuses to maternal corticosterone, which disturb the development of the HPA axis in offspring. The first aim of this work was to investigate, in adult male rats, whether perinatal malnutrition has long-lasting effects on the HPA axis activity during both basal and stressful conditions. Moreover, as the HPA axis and sympathetic nervous system are both activated by stress, the second aim of this work was to investigate, in these rats, the adrenomedullary catecholaminergic system under basal and stressful conditions. This study was conducted on 4-month-old male rats malnourished during their perinatal life and on age-matched control animals. Under basal conditions, perinatal malnutrition reduced body weight and plasma corticosteroid-binding globulin (CBG) level but increased mineralocorticoid receptor (MR) gene expression in CA1 hippocampal area. After 30 min of restraint, perinatally malnourished (PM) rats showed increased plasma noradrenaline, adrenocorticotropin hormone (ACTH) and corticosterone concentrations similarly as controls, but calculated plasma-free corticosterone concentration was significantly higher and adrenaline level lower than controls. During the phase of recovery, PM rats showed a rapid return of plasma ACTH and corticosterone concentrations to baseline levels in comparison with controls. These data suggest that in PM rats, an elevation of basal concentrations of corticosterone, in face of reduced CBG and probably increased hippocampal MR lead to a much larger impact of corticosterone on target cells that mediate the negative-feedback mechanism on the activities of both the HPA axis and sympathoadrenal one.

Quantitative studies of progesterone receptor and nitric oxide synthase colocalization with somatostatin, or neurotensin, or substance P in neurons of the guinea pig ventrolateral hypothalamic nucleus: an immunocytochemical triple-label analysis.

Previous double-label studies have demonstrated that progesterone receptors (PR) may coexist with nitric oxide synthase (NOS) and also with neuroactive peptides such as somatostatin (SOM), neurotensin (NT) and substance P (SP) in neurons of the ventrolateral hypothalamic nucleus (VL) of the guinea pig. In the present study, triple-label immunofluorescence histochemistry was used to determine whether neurons co-expressing both PR and NOS also contain one neuropeptide (SOM, NT or SP) and to quantify these colocalization relationships. Ovariectomized guinea pigs were primed with estradiol to induce PR immunoreactivity and treated with colchicine to visualize immunoreactive (IR) peptidergic neurons. Using three primary antibodies raised in different species and labeled with three different markers, it is possible to recognize which type(s) of immunoreactivity each cell contains on the same sections. We observed that PR, NOS and SOM co-occurred extensively, whereas PR, NOS and NT or PR, NOS and SP were colocalized infrequently in neurons of the VL. Combining our various quantitative observations, we found that SOM immunoreactivity appeared in 33% of the PR/NOS-IR neurons in the caudal aspect of the nucleus and that nearly all neurons containing SOM and NOS contained PR. The occasional localization between PR, NOS and SP seen in the rostral subdivision of the VL represented a very low proportion of the PR/NOS-IR neurons but 48% of NOS/SP-IR neurons. The relatively high proportion of PR/NOS-IR cells expressing SOM in the VL suggests that these neurons are part of a neural circuitry involved in a variety of steroid-dependent functions.

Estrogen modulation of neuropeptides: somatostatin, neurotensin and substance P, in the ventrolateral and arcuate nuclei of the female guinea pig.

In the guinea pig, steroid target cells reside in the ventrolateral hypothalamic nucleus (VLH), an important site in the mediation of female receptive behavior, and in the arcuate nucleus (AR), a structure essential for stimulation effects of ovarian hormones on gonadotropin secretion. However, the mechanisms by which these steroid-dependent reproductive neuroendocrine processes occur are only partially understood. Estrogen is known to affect the hypothalamus content of certain neuropeptides. In the present study, we investigated the effects of estradiol benzoate (EB) on immunoreactivity of neurons containing one of three following neuropeptides: somatostatin (SOM), neurotensin (NT) and substance P (SP) in VLH and AR. The number of immunoreactive (IR)-neurons was quantified in anatomically matched sections through VLH and AR of ovariectomized (OVX), OVX + EB and OVX + oil-treated guinea pigs. Analysis of variance revealed that the number of SOM-IR and SP-IR neurons significantly increased in all regions of VLH of OVX + EB-treated guinea pigs as compared to OVX or OVX + oil-treated animals (P < 0.01) but showed no EB effect on the number of NT-IR neurons. Although the number of SOM-IR and NT-IR neurons slightly increased following treatment with EB in AR, analysis of variance revealed no significant change. The present results provide additional information relevant to possible involvement of these neuropeptides in facilitation of female typical sexual behavior.

Sex differences in estradiol-induced progestin receptor immunoreactivity in the guinea pig preoptic area and hypothalamus.

Progesterone exerts on the central nervous system a number of effects, some of which are estrogen dependent mostly in the preoptic area and the mediobasal hypothalamus. In these regions, an immunocytochemical study was used to evaluate differences in progesterone receptor (PR) immunoreactivity between the male and the female guinea pig in response to 10 micrograms/day estradiol benzoate (EB) for 5 consecutive days. Compared to EB-treated females, EB-treated males showed a slightly lower number of PR-immunoreactive cells in the preoptic area whereas PR-immunoreactivity appeared in more cells in the anterior part of the ventrolateral nucleus. The numbers of PR-immunoreactive cells in the arcuate nucleus did not differ significantly between males and females. These results show that regionally localized sex differences exist in the induced PR system after 5 days exposure to EB.

Differential effects of colchicine on the induction of nitric oxide synthase in neurons containing progesterone receptors of the guinea pig hypothalamus.

Using nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPHd) histochemistry, we analyzed the effects of an intracerebroventricular injection of colchicine on the activity of neuronal nitric oxide synthase in the hypothalamic nuclei of intact and ovariectomized estradiol-primed guinea pigs. We also examined the effects of colchicine on the immunocytochemical colocalization of nitric oxide synthase with the progesterone receptor in the ventrolateral nucleus, a key region in the control of sexual behavior. Treatment with colchicine resulted in a significant increase in the number of NADPHd-positive neurons in the ventrolateral nucleus in intact as well as in ovariectomized estradiol-primed animals, whereas in the other hypothalamic regions analyzed (preoptic area, paraventricular nucleus and posterior arcuate nucleus), the enzymatic activity remained unchanged. Quantitative analysis showed a significantly greater number of NADPHd-positive cells in the medial and the posterior aspects of the ventrolateral nucleus of colchicine-treated guinea pigs compared to the control group. In the caudal subdivision of this nucleus, colchicine induced nitric oxide synthase in the target cells for progesterone. These results suggest that neuronal nitric oxide synthase activity in the hypothalamus is affected by colchicine in a region-specific manner and especially in the ventrolateral nucleus, which is involved in progesterone-facilitated lordosis.

Relationship between polysialylated neural cell adhesion molecule and beta-endorphin- or gonadotropin releasing hormone-containing neurons during activation of the gonadotrope axis in short daylength in the ewe.

Morphological plasticity has been demonstrated between breeding and anestrous seasons in the ewe hypothalamus, particularly for the gonadotropin-releasing hormone (GnRH) system. We sought to determine the impact of a photoperiodic transition, from long days (LD, 16 h light/24 h) to short days (SD; 8 h light/24 h), on the association between a marker of cerebral plasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM), and two diencephalic populations: the GnRH and beta-endorphin (beta-END) neurons, the latter being potent inhibitors of GnRH neuronal activity. We also estimated the number of contacts on GnRH neurons after the passage to SD, using synaptophysin as a marker for synaptic buttons. Those parameters were evaluated in ovariectomized estradiol-replaced ewes using double immunocytochemistry and confocal microscopy at different times after the transition to SD: day 0 (D0), D30, D45, D60 and D112. Luteinizing hormone (LH) secretion was recorded throughout the experiment. High LH levels were observed only at D112. Significantly more PSA-NCAM was found in the GnRH neuron perimeters in the D112 group than in the other groups. This increase was not associated with any change in the number of synaptophysin-immunoreactive contacts on GnRH neurons. The beta-END peri-neuronal space was affected negatively by the transition to SD: the percentage of PSA-NCAM on beta-END neurons decreased between D45 and D112 in the posterior two thirds of the arcuate nucleus (ARC). These results suggest that photoperiod may reorganize cell interactions in different hypothalamic areas, ultimately reactivating GnRH neurons, in our model of ovariectomized-estradiol replaced ewes.

Effects of photoperiod on kisspeptin neuronal populations of the ewe diencephalon in connection with reproductive function.

Kisspeptin (Kiss) is a key regulator of reproductive function in both prepubertal and adult mammals. Its expression appears to vary throughout the year in seasonal species. We aimed to determine the impact of a change of photoperiod on the size of Kiss neuronal populations found in the preoptic area (POA) and arcuate nucleus (ARC) of the ewe brain. Using immunocytochemistry, we first examined the proportion of neurones expressing Kiss, using HuC/D as a neuronal marker, at different time-points after transition from long days (LD; 16 : 8 h light/dark cycle) to short days (SD; 8 : 16 h light/dark cycle). Luteinising hormone (LH) secretion was measured in ovariectomised oestradiol replaced ewes from the month preceding the transition to SD until the sacrifice of the animals at days 0, 45 and 112 from this photoperiodic transition. High LH levels were only observed in animals killed at day 112. The number of Kiss neurones/mm(2) doubled in the caudal ARC at day 112. The percentage of neurones showing Kiss immunoreactivity increased significantly in both the POA and ARC in the day 112 group. In a second experiment, ewes kept in LD received an i.c.v. injection of colchicine 20 h before sacrifice. Colchicine treatment increased the number and the percentage of neurones with Kiss in both the POA and caudal ARC. The data obtained suggest that the increase in Kiss neurones detected in the POA and caudal ARC after transition to SD stemmed from an increase in Kiss synthesis. This up-regulation of Kiss content under the shorter day condition appears to be a late event within the cascade activated by a longer secretion of melatonin, which is a critical factor in switching gonadotrophin-releasing hormone secretion to a breeding season profile.

Projections from ventrolateral hypothalamic neurons containing progesterone receptors and somatostatin to the midbrain central gray in the female guinea pig.

The goal of this study was to determine, by combining retrograde fluorescent tract tracing with double immunocytochemistry, whether neurons immunoreactive (IR) for both progesterone receptors (PR) and somatostatin (SOM) in the guinea-pig ventrolateral nucleus (VL) send their axons directly to the midbrain. Unilateral microinjections of true blue (TB) fluorescent dye were made into the midbrain central gray (MCG) of ovariectomized animals primed with estradiol to induce PR and injected intracerebroventricularly with colchicine to visualize SOM-IR neurons. The highest number of PR/SOM-IR neurons in the VL that projected to the midbrain was found when the fluorescent retrograde tracer was confined to the lateral region of the MCG at the mid-rostrocaudal level. More specifically, 29% of the progesterone target neurons containing SOM detected in the VL projected directly to the midbrain, accounting for 7% of the PR-IR neurons and for 9% of the SOM-IR neurons. These triple-labeled cells were found throughout the extent of the VL. Double-labeled cells also sent axons to the MCG and were either PR-IR (14%) or SOM-IR (12%). These results provide morphological evidence that a subset of the PR-IR neurons expressing SOM in the VL have long projections to the midbrain and suggest that the SOM system may modulate neural circuits involved in the regulation of steroid-influenced behaviors and neuroendocrine functions.

Colocalization of progesterone receptor and somatostatin immunoreactivities in the hypothalamus of the male and female guinea pig.

A double-label immunofluorescence technique was used to determine whether progesterone receptor (PR)-containing neurons in the preoptic area and hypothalamus also contain somatostatin (SOM) in both the male and female guinea pig. Animals were gonadectomized, primed by estradiol to induce PR and injected intracerebroventricularly with colchicine to visualize SOM-immunoreactive (SOM-IR) neurons. The only sites of significant overlap between the two immunoreactivities were the medial preoptic nucleus, the periventricular preoptic and hypothalamic regions, the arcuate nucleus (Ar) and the ventrolateral nucleus (VL). No sex differences were detected at this level. In the preoptic area and the periventricular regions, no SOM-IR neurons were shown to have PR. In the Ar, only very few SOM-IR perikarya were found to be also PR-IR. SOM varicosities appeared in close proximity to neurons with PR-containing nuclei. Within the VL, in the female as well as in the male, many SOM-IR cells were also IR for PR. This colocalization persisted throughout the rostrocaudal extent of the nucleus but our quantification revealed a significant sex difference in the percentage of PR-IR neurons with SOM in the caudal VL. These results provide neuroanatomical evidence that progesterone may exert its effect directly upon more than one third of SOM-synthesizing cells in the medial and caudal regions of VL, a site which plays a key role in the control of sexual behavior.