Microdialysis measurement of neurochemical changes in the mediobasal hypothalamus of ovariectomized ewes during oestrus

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.     

The Role of Oxytocin Release in the Paraventricular Nucleus in the Control of Maternal Behaviour in the Sheep

Oxytocin (OT) release within the brain is thought to play a major role in inducing maternal behaviour in a number of mammalian species but little is known about the sites of release which are important in this respect. We have investigated whether the paraventricular nucleus of the hypothalamus (PVN) is a site of OT action on maternal behaviour in the sheep. In vivo microdialysis and retrodialysis was used to determine whether OT is released in the region of the PVN during the post-partum induction of maternal behaviour and if its release at this site can stimulate maternal behaviour in non-pregnant animals. In vivo sampling showed that OT concentrations increased significantly in the region of PVN at birth. When OT was retrodialysed bilaterally into the PVN (1 or 10 μM) of multiparous ewes treated with progesterone and oestradiol to stimulate lactation, maternal behaviour was induced in a significant number of animals (1 μM, 6/8 and 10 μM, 5/8) compared with controls (0/8 ewes). Similar infusions of the ring structure of OT, tocinoic acid (TOC—10 μM), also induced maternal behaviour in a significant proportion of animals (5/6 ewes) as did intracerebroventricular (ICV) OT (6/8 ewes) and artificial stimulation of the vagina and cervix (VCS, 8/9 ewes). On the other hand, vasopressin (AVP) 1 μM did not induce maternal behaviour in any ewes and a 10 μM dose only induced it in 2/8 animals. The neurochemical changes accompanying the above treatments were also investigated. Noradrenaline concentrations increased in the PVN after the retrodialysis administration of OT 1 μM and 10 μM, TOC 10 μM and AVP 1 μM, OT ICV and VCS. Dopamine concentrations were also increased by OT 10 μM, TOC 10 μM, AVP 1 μM and OT ICV. Aspartate and glutamate concentrations were significantly reduced by retrodialysis infusions of OT 1 μM and AVP 1 and 10 μM but not by any other treatment. Finally, the retrodialysis infusions of OT and TOC, as well as ICV OT, significantly increased plasma OT release whereas AVP infusions did not. These results provide evidence that OT is released in the PVN during parturition and is important for the induction of maternal behaviour. It seems probable that OT release at this site has a positive feedback effect on both parvocellular and magnocellular OT neurones to facilitate co-ordinated OT release both in central OT terminal regions (to facilitate maternal behaviour) and peripherally into the blood (to facilitate uterine contractions/milk let down). The potential functional roles for the actions of OT on monoamine and amino acid transmitter release in the PVN are discussed.     

Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe

Most of the literature suggests that in sheep as in rodents nervous structures involved in female sexual behaviour are not necessarily identical to those involved in the LH surge. In rodents, oestradiol triggers female sexual behaviour by acting on a restricted area of the mediobasal hypothalamus whereas the concomitant induction of the preovulatory LH surge is at least partially under the control of more anterior structures. The central sites of oestradiol action, however, remained poorly defined in sheep. To provide this definition, 37 ovariectomized ewes were stereotaxically implanted unilaterally or bilaterally with a guide cannula in preoptic area (POA), anterior, mediobasal, lateral, or posterior hypothalamus (AH, MBH, LHT, PH). Experiments were made during the breeding season (Br) and the anoestrous period (An: unilat only) and females were primed with a peripheral treatment of progesterone and a dose of 17 beta-oestradiol subthreshold for both the LH surge and sexual behaviour. Intracranial implants (i.d. = 0.45 mm) of crystalline E2 were lowered 16 h after progesterone removal and left in the brain for 48 h. Whereas POA implants never had any significant effects on either the behaviour or the LH surge, all MBH implants caused receptivity (11 bilat, 5 unilat Br and 5 unilat An). Bilateral MBH implants also induced proceptivity in 9 of 11 ewes and increased the LH levels in 7 of them. These proportions do not differ significantly from those observed after a 25 microgram peripheral injection of E2. Unilateral MBH implants had no significant effect on proceptivity and LH increase but oestrous behaviour was induced by some implants placed laterally to the MBH (25 recept and 3/5 procept).(ABSTRACT TRUNCATED AT 250 WORDS)     

Male Faces and Odours Evoke Differential Patterns of Neurochemical Release in the Mediobasal Hypothalamus of the Ewe During Oestrus: An Insight Into Sexual Motivation?

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.     

Inhibition of luteinizing hormone secretion in the ewe by progesterone: associated changes in the release of gamma-aminobutyric Acid and noradrenaline in the preoptic area as measured by intracranial microdialysis

Progesterone inhibits the pulsatile release of luteinizing hormone (LH) in sheep by an action in the brain to suppress the release of LH-releasing hormone (LHRH). In addition, progesterone blocks the preovulatory surge of LH in this species. The neural basis of this inhibitory action is unknown, but as LHRH cells do not appear to contain progestin receptors other neural systems must mediate the action of this ovarian steroid on LH release. This study focuses on a possible role for the inhibitory amino-acid GABA and the monoamines (noradrenaline, adrenaline, dopamine and serotonin). The technique of microdialysis was used to monitor changes in these substances in the vicinity of the LHRH cell bodies (in the preoptic area) both before and following the administration of progesterone. Levels of this steroid, similar to those measured during the mid-luteal phase of the oestrous cycle, inhibited LH release and this was associated with significant alterations in the release of GABA and noradrenaline (but not adrenaline, dopamine or serotonin). Specifically, progesterone augmented GABA while noradrenaline release was depressed. Whether steroid actions on these neurotransmitters were mediated by opioids was also investigated. This possibility arises because of the reported involvement of opioids in progesterone negative feedback in the ewe. The long-acting opioid antagonist, naltrexone, was administered and GABA and noradrenaline release monitored for a further period both in the presence and absence of progesterone. Naltrexone significantly depressed GABA release in steroid-treated (but not untreated) ewes suggesting that the actions of progesterone on GABA are mediated by the endogenous opioid peptides. However, noradrenaline release was unaltered. In an earlier study we demonstrated that GABA release fell prior to the LH surge while noradrenaline release increased. These data, in conjunction with those from the present study, suggest that the mechanism by which progesterone is able to inhibit the preovulatory surge of LH in the ewe is by enhancing GABA and depressing noradrenaline release in the vicinity of the LHRH cell bodies. As opioid tone is also reported to fall prior to the surge, the interaction between opiate and GABAergic systems in the regulation of gonadotrophin secretion warrants further investigation.     

Inhibition of sexual behaviour and the luteinizing hormone surge by intracerebral progesterone implants in the female sheep

In female sheep, progesterone blocks the induction by oestradiol of both sexual behaviour and the pre-ovulatory surges of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH). However, the central sites of action of progesterone remain poorly defined, so we attempted to locate them by implanting progesterone intracerebrally in ovariectomised ewes treated with exogenous steroids to induce oestrous behaviour and the LH surge. Single bilateral implants or a double bilateral implants filled with progesterone or cholesterol were placed in the ventromedial hypothalamus (VMH) or the preoptic area (POA). Control ewes were not implanted. To determine the inhibitory capacity of the central progesterone implants, ewes received an injection (i.m.) of 8 μg or 16 μg of oestradiol. The single bilateral implants of progesterone failed to block oestrous behaviour and the LH surge induced by 8 μg of oestradiol. Double bilateral progesterone implants in the VMH blocked the sexual behaviour (P < 0.05) and the LH surge (P < 0.05), but implants in the POA blocked only sexual receptivity (P < 0.05). No changes were observed after central implantation of cholesterol. Our results support the hypothesis that progesterone acts centrally in the VMH and the POA to inhibit the induction of LH surge and sexual behaviour by oestradiol.     

A dopamine antagonist blocks vaginocervical stimulation-induced neuronal responses in the rat forebrain

During mating in rats, the male provides vaginocervical stimulation (VCS) to the female via intromissions. VCS, provided manually, mimics many aspects of mating, including facilitation of lordosis, induction of sexual receptivity, abbreviation of the period of sexual receptivity, and induction of twice-daily prolactin surges, which result in pseudopregnancy. VCS also induces the expression of Fos, the protein product of the immediate early gene c-fos, which has been used as a marker for neurons that are responsive to mating stimuli. Because VCS induces the release of dopamine in the forebrain, as well as phosphorylation of DARPP-32, a phosphoprotein associated with activation of the D(1) subtype of dopamine receptor, we tested the hypothesis that VCS induces Fos expression by acting on the D(1) class of dopamine receptors. Injection of SCH 23390, an antagonist of the D(1) class of dopamine receptors, virtually eliminated VCS-induced Fos expression without affecting constitutive levels of Fos-Immunoreactivity (Fos-IR) in all brain areas in which VCS induced Fos expression. In a follow-up experiment, expression of a second immediate early protein, egr-1, was blocked as well, suggesting that these results are not specific to Fos. Therefore, the results are consistent with the idea that VCS induces dopamine release, causing activation of D(1) dopamine receptors, which in turn, results in neuronal response, as seen by both Fos and egr-1 expression.     

Effects of oestrogen and androgen on the sexual behaviour responses of the ovariectomized ewe

The secretion of gonadal steroid hormones that stimulate sexual behavior differs between males and females in 2 respects: the hormones are chemically different, estrogen and progesterone on 1 hand and androgen on the other, and their pattern of release into the blood stream differs according to the sex of the animal. Those produced by the female are released during a limited period whereas testosterone exhibits little day-to-day variation. 12 ovariectomized Ile-de-France ewes were injected either with 50 mg of estradiol benzoate of 10 mg or testosterone propionate 48 hours after the last of 5 daily injections of 25 mg of progesterone. In both cases the experimental females exhibited normal female sexual behavior. In a 2nd experiment, the ewes were injected daily for 4 weeks either with 50 mg of estradiol benzoate or with 10 mg of testosterone propionate. In both cases male patterns of sexual behavior appeared, but more intensely with androgen than with estrogen, and simultaneously, the ewes became receptive. However, receptivity declined rapidly after a few days of estrogen treatment. This decline did not occur with androgen.     

Involvement of serotonin and γ-aminobutyric acid in the timing of estrous receptivity in the cyclic female rat

The involvement of serotonin (5HT) and γ-aminobutyric acid (GABA) in the control of estrous sexual receptivity has been investigated in the cyclic female rat. (1) When injected at 1300 hr on proestrus parachlorophenylalanine (pCPA) specifically increased sexual receptivity as expressed by the number of receptive females between 1800 and 1900 hr and simultaneously depressed 5HT turnover. (2) A specific decrease in blood progesterone (P) concentration was observed on proestrus at 1530 hr in females treated with pCPA on proestrus at 1300 hr. (3) When injected on proestrus at 1730 hr dipropylacetate (DPA) induced a decrease in sexual receptivity between 1830 and 1900 hr in both intact and bulbectomized females but it depressed the lordosis quotient only in unoperated females. (4) An increase in the amount of γ-aminobutyric acid (GABA) in the olfactory bulbs and in the hypothalamus was noted at 1845 hr in DPA treated females. A significant decrease in the olfactory bulb GABA content occurred from 1845 to 2245 hr on proestrus. (5) The results are in favor of an involvement of 5HT and GABA in the control of the timing of sexual receptivity by the olfactory bulbs in the rat. They also suggest that interactions between P and 5HT are implicated in the control of sexual receptivity in the cyclic female rat.     

Interactions between oestradiol and the progesterone antagonist RU-486 in establishing and maintaining female rats' sexual responsiveness: central versus peripheral effects.

Progesterone is well known to contribute specifically to the emergence of the female rats' sexual behaviour by the establishment of 'proceptivity'. Analysis of the mechanism of progesterone action benefits from the availability of highly effective anti-progestagenic compounds. However, results obtained during the study of female rats' sexual behaviour, including such compounds into the experimental protocol, appear equivocal. The present experiments were designed to further examine the possible effects of the antiprogestagenic compound RU-486 (Mifepristone) on the female rats' sexual responsiveness as elicited through exposure of the animals to oestradiol alone. The experimental design aimed to distinguish between receptivity (defined as response to sexually active males) and proceptivity (defined as female-initiated sexual behaviour). Mifepristone advanced the onset of receptivity after the injection of oestradiol benzoate (OB). Upon further investigation a steady level of receptivity was reached during prolonged treatment with OB and this level appeared unaltered through concurrent treatment with Mifepristone. OB alone was insufficient to induce full proceptivity as revealed by observations of sexual behaviour with tethered males. Such defined proceptivity was significantly further inhibited by Mifepristone. It thus appears that, dependent upon the time and type of female sexual behavioural analysis, Mifepristone either enhances, inhibits, or does not affect sexual responsiveness. After the observation period, autopsy revealed the presence of copulatory plugs and infections in the uterus of OB + Mifepristone-treated rats. This unexpected finding could result from effects of the compound on the uterine cervical musculature. Uterine infections might result in painful, aversive, intra-abdominal sensations, especially during intravaginal penile intromissions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A site for estradiol priming of progesterone-facilitated sexual receptivity in the ventrolateral hypothalamus of female guinea pigs.

The induction of progestin receptors by estradiol in the mediobasal hypothalamus (MBH) has been correlated with progesterone facilitation of sexual receptivity in female guinea pigs. We used an immunocytochemical technique to visualize the induction of progestin receptors by estradiol in a population of neurons correlated with estradiol-induced responsiveness to progesterone. Progestin receptor-immunoreactivity (PR-IR) and responsiveness to progesterone-facilitation of sexual behavior were induced by intracranial implantation of small bilateral cannulae (28- or 33-gauge) containing 1 or 10% estradiol diluted with cholesterol. In the first experiment, 28-gauge cannulae containing estradiol were effective only when implanted in the rostral part of the MBH. In the second experiment in which 33-gauge cannulae were used, behavioral responsiveness to progesterone was induced only when the cannulae were localized bilaterally in the rostro-ventral part of the ventrolateral hypothalamus (VLH). The presence of estradiol-induced PR-IR within the rostro-ventral VLH was correlated with the ability of progesterone to facilitate sexual behavior. These data suggest that neurons containing estrogen-induced progestin receptors specifically within the rostro-ventral VLH are involved in progesterone facilitation of sexual receptivity in estradiol-primed guinea pigs.     

Reproductively-Relevant Stimuli Induce Fos-Immunoreactivity within Progestin Receptor-Containing Neurons in Localized Regions of Female Rat Forebrain

An experiment was conducted to determine if neurons that respond to stimuli associated with mating in female brain also contain progestin receptors. We found that a portion of the neurons that respond to stimuli associated with mating also contains progestin receptors. While the appropriate hormonal conditions are important for sexual receptivity, somatosensory information provided by the male also influences sexual behavior. One important stimulus provided by the male during copulation is vaginal-cervical stimulation (VCS). VCS has been shown to elicit many different behavioral and endocrine changes in female rats, such as increases in lordosis, pseudopregnancy, and termination of sexual receptivity. VCS also increases the expression of the immediate early gene product, Fos, in areas associated with reproduction. A portion of the neurons responding to VCS with increased Fos-immunoreactivity (Fos-IR) in female rat forebrain also contains estrogen receptors, illustrating that hormonal and mating-stimuli converge in a population of cells. As progesterone also plays an important role in female sex behavior, it is important to determine if some of the neurons also integrate information concerning serum progesterone levels and social interactions. Thus, we used a dual immunofluorescent technique to label both Fos-IR and progestin receptor-immunoreactivity (PR-IR) in the brains of estradiol-primed, ovariectomized female rats following VCS manually applied by the experimenter. Many of the neurons that respond to VCS with increased Fos-IR within the medial preoptic area, the arcuate nucleus, and the progestin receptor-rich areas of the rostral and caudal ventromedial nucleus of the hypothalamus also contain PR-IR.     

Vaginocervical stimulation-induced release of classical neurotransmitters and nitric oxide in the nucleus of the solitary tract varies as a function of the oestrus cycle

The effects of vaginocervical stimulation (VCS) on glutamate (GLU), aspartate (ASP), gamma-aminobutyric acid (GABA), noradrenaline (NA), arginine (ARG) and nitric oxide (NO) (citrulline) release in the nucleus of the solitary tract (nTS) were measured in anaesthetised female rats as a function of the oestrus cycle. During pro-oestrus/oestrus (P/E), but not during met-oestrus/di-oestrus (M/D), VCS significantly increased concentrations of NA, ASP, GLU, NO (citrulline) and GABA, but not ARG. Basal NA concentrations were also increased in P/E. These effects were prevented by bilateral section of either the vagus nerve or pelvic and hypogastric nerves. Vagotomy also significantly decreased basal NO concentrations in M/D and P/E while pelvic and hypogastric nerve section significantly increased GABA concentrations. Our results therefore confirm that the nTS is a relay structure for the visceral afferents sending information from the uterus into the central nervous system. The ability of VCS to trigger classical transmitter release and NO in the female is influenced by the stage of the oestrous cycle and is routed both via the vagus and pelvic/hypogastric nerves.     

Noradrenaline Involvement in the Negative-Feedback Effects of Ovarian Steroids on Luteinising Hormone Secretion

Noradrenaline has been shown to modulate the ovarian-steroid feedback on luteinising-hormone (LH) release. However, despite the high amount of evidence accumulated over many years, the role of noradrenaline in LH regulation is still not clearly understood. The present study aimed to further investigate the involvement of noradrenaline in the negative-feedback effect of oestradiol and progesterone on basal LH secretion. In experiment 1, ovariectomised (OVX) rats received a single injection of oil, oestradiol, or progesterone at 09.00–10.00 h and were decapitated 30 or 60 min later. Levels of noradrenaline and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), were determined in microdissections of the preoptic area (POA) and medial basal hypothalamus-median eminence (MBH-ME) and correlated with LH secretion. Basal LH levels were decreased 30 and 60 min after oestradiol or progesterone injection, and this hormonal response was significantly correlated with a reduction in POA MHPG levels, which reflect noradrenaline release. In addition, noradrenaline levels in the POA were increased, whereas noradrenaline turnover (MHPG/noradrenaline ratio) was decreased 60 min after the injection of both hormones. No effect was found in the MBH-ME. In experiment 2, i.c.v. administration of noradrenaline (60 nmol), performed 15 min before oestradiol or progesterone injection in jugular vein-cannulated OVX rats, completely prevented the ovarian steroid-induced inhibition of LH secretion. The data obtained provide direct evidence that LH secretion in OVX rats is positively regulated by basal noradrenergic activity in the POA, and its reduction appears to play a role in the negative-feedback effect of ovarian steroids on LH secretion in vivo .     

Steroid priming promotes oxytocin-induced norepinephrine release in the ventromedial hypothalamus of female rats

In vivo microdialysis was used to detect norepinephrine (NE) release in the ventromedial hypothalamus of estradiol (E₂)- or E₂ plus progesterone (P)-treated female rats injected with 1.0 IU of oxytocin (OXY). Dialysates were collected before and after OXY administration on 3 consecutive days and analyzed for NE content by high performance liquid chromatography with electrochemical detection. After the last sample was collected on day 1, animals were injected with 3 μg E₂ benzoate or oil. On day 3, E₂-primed animals received 200 μg of P and control females received oil prior to OXY administration. OXY administration did not induce NE release on day 1. When OXY was administered to animals that received E₂ approximately 20 h earlier, increased release of NE was not consistently seen. In contrast, E₂-primed animals that received P on day 3 displayed significant increases in the release of NE after OXY administration compared to their own basal levels and to NE levels in control animals. To distinguish whether E₂ priming is sufficient to promote OXY-induced release of NE without the addition of P, NE content of VMH dialysates in a second group of animals was examined following exposure to vehicle or E₂ alone. When OXY was administered 24 or 48 h after estrogen priming, only 1 of 4 E₂-primed females had modestly elevated dialysate NE levels. To evaluate the interactions between OXY and NE in the regulation of reproductive behavior, lordosis responses were observed in hormone-primed female rats receiving systemic injections of OXY, the ₁-adrenoceptor antagonist prazosin, or both OXY and prazosin. OXY enhanced lordosis behavior in females primed with subthreshold doses of E₂ and P. Prazosin abolished lordosis behavior in rats primed with behaviorally effective doses of E₂ and P and significantly inhibited lordosis in steroid-primed females given OXY. These data suggest that after priming with both E₂ and P together, but not with E₂ alone, OXY may facilitate lordosis behavior through activation of NE transmission.     

Ovarian steroids and sexual interaction alter oxytocinergic content and distribution in the basal forebrain

The immunoreactive levels of oxytocin (OXY) were assessed by radioimmunoassay (RIA) and immunohistology after treatments with estradiol or estradiol and progesterone in ovariectomized rats that were mounted or not mounted by males. In micropunches from the medial preoptic area (MPOA) OXY immunoreactive levels increased significantly in estrogen-progesterone-treated receptive animals (mean lordosis quotient (LQ) = 82.9 +/- 4.4) that were mounted by males over levels in estrogen-treated unreceptive animals (mean LQ = 2.3 +/- 1.3). No other areas demonstrated significant changes in OXY levels across these treatments, although the paraventricular nucleus also had elevated OXY levels in receptive mounted females. In other animals, immunocytochemistry revealed that the number of oxytocinergic perikarya in the MPOA decreased in receptive animals (LQ = 86.7 +/- 4.9) that were mounted. The number of oxytocinergic perikarya per 50 microns vibratome section in the lateral subcommissural nucleus was lower in all estrogen-treated groups than in oil vehicle-treated controls. We suggest that the combination of increasing immunoreactive levels of OXY in the MPOA with decreasing numbers of oxytocinergic perikarya indicates that preoptic OXY is moved out of cell bodies, in receptive rats that are mounted by males, to a state that is more accessible to the RIA.     

Changes in Oxytocin Immunoreactivity and mRNA Expression in the Sheep Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone

The effects of pregnancy, parturition and lactation and exogenous treatments with oestradiol and progesterone on oxytocin (OXY) immunoreactivity and gene expression in the sheep brain were investigated. Immunocytochemistry was used to demonstrate that increased OXY-immunoreactivity occurred in cells of the paraventricular (PVN) and supraoptic nuclei (SON), the bed nucleus of the stria terminalis (BNST), the anterior commissural nuclei (ACN) and the periventricular part of the medial preoptic area (PvMP). Oxytocin immunoreactive terminals were also seen in the accessory olfactory nucleus, the glomerular and peri-glomerular layers of the olfactory bulb, the lateral septum, the zona incerta and the pars compacta of the substantia nigra. Compared to ovariectomized and late pregnant animals, the intensity of immunoreactivity was increased in all of these oxytocinergic elements at parturition, during lactation and following exogenous treatment with oestradiol. The OXY-immunoreactivity was also more intense in late pregnant animals compared to ovariectomized ones. Quantitative in situ hybridization histochemistry showed that cells in the PVN, SON, BNST and PvMP all showed significantly increased expression of OXY mRNA in animals at parturition and during lactation compared to late pregnant or ovariectomized animals. Expression levels in late pregnant animals were also significantly higher than in ovariectomized ones. Progesterone treatment significantly increased OXY mRNA in the PVN, SON, BNST and PvMP whereas oestradiol treatment was only effective in the PVN, BNST and PvMP. Combined treatment with these steroids did not significantly increase OXY mRNA levels in comparison with their administration alone. These results show that OXY-immunoreactivity and mRNA expression are at their highest in the sheep brain when maternal behaviour is induced. The increased synthesis/storage of the peptide at parturition may be due to changes in circulating concentrations of both progesterone and oestradiol during late pregnancy.     

Progesterone stimulates sexual behaviour in female rats by increasing 5-HT activity on 5-HT2 receptors

We have investigated the possibility that there is a correlation between female sexual receptivity and CNS serotonergic activity. In ovariectomized rats primed with a submaximal steroid regime of 2 micrograms oestradiol benzoate (OB) plus 0.2 mg progesterone (P), so that only a proportion were receptive, the 5-hydroxyindoleacetic acid:5-hydroxytryptamine (5-HT) ratio in the hypothalamus was greater in the receptive compared to the non-receptive rats. When rats were primed with 10 micrograms OB alone, there was no difference in 5-HT activity in the receptive and non-receptive animals. Depletion of hypothalamic 5-HT levels by p-chlorophenylalanine methyl ester (PCPA) inhibited behaviour normally induced by OB plus P and this could be reversed by 5-hydroxytryptophan. PCPA had no effect in animals made receptive by OB alone. This indicates that P, but not OB, exerts its stimulatory effect on sexual behaviour via increasing 5-HT activity. Administration of 5-HT into the lateral, but not 3rd ventricle stimulated sexual behaviour and systemic injection of the selective 5-HT1 and 5-HT2 agonists, Ru 24969 and MK 212, inhibited and stimulated behaviour, respectively. It is suggested that 5-HT has a dual effect on female sexual receptivity acting via different systems, the inhibitory tract acting on 5-HT1 and the stimulatory tract on 5-HT2 receptors.     

Sexual responsiveness and its relationship to vaginal stimulation-produced analgesia in the rat

Increasing amounts of pressure applied to the cervix produce a dose-response-like elevation of pain threshold in rats. This vaginal stimulation-produced analgesia (VSPA) is facilitated in animals given estrogen (E) doses sufficient to induce high levels of sexual receptivity. It has been proposed that enhancement of VSPA may serve to decrease any noxious input associated with multiple intromissions by the male. In this study, the anti-nociceptive effect of VSPA was compared in animals given E doses insufficient to increase receptivity with animals made receptive using subthreshold E levels + progesterone (P) in an attempt to determine if enhancement of VSPA is associated with the receptive state of the animal or the dose of E used. Tail flick latencies and tail shock vocalization thresholds were measured in groups of E, E + P and oil-treated rats during application of 0, 100 and 200 g of force on the cervix. Within oil, E and E + P-treated animals, significant increases in tail flick latencies were observed at 100 and 200 g with respect to baseline (0 g). Moreover, at 100 g of force E treated animals displayed a significant increase in tail flick latency over oil and E + P treated rats. In contrast, tail shock vocalization was increased at 100 and 200 g levels of probing in oil and E + P groups but was not facilitated by E. In the present study, as in previous work, VSPA was potentiated by E; however, this potentiation was not correlated with steroid-induced receptivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoamine Content of the Stalk-Median Eminence and Hypothalamus in Adult Female Sheep as Affected by Daylength

In the ewe, plasma luteinizing hormone and prolactin concentrations exhibit seasonal variations. During long days, inhibition of pulsatile luteinizing hormone secretion is mediated by monoamines. In a model of ovariectomized ewes bearing a subcutaneous oestradiol implant, we previously showed that the steroid-dependent inhibition of luteinizing hormone involves the A15 dopaminergic nucleus of the retrochiasmatic area. In the present work, we compared the aminergic activities of tele-diencephalic structures in groups of ovariectomized ewes under artificial illumination for short versus long days (8 versus 16 h/day of light, respectively). Half the animals in each group were bearing a subcutaneous oestradiol implant. Using high-performance liquid chromatography and electrochemical detection, we measured the levels of amines and amine metabolites in ‘punches’ of tissues from regions containing luteinizing hormone-releasing hormone axon terminals or cell bodies and catecholaminergic structures. Concurrently, we checked the pulsatile luteinizing hormone release and plasma prolactin concentration to assess the ability of our model to mimic seasonal changes in the hormonal status. As expected, ovariectomized ewes with a subcutaneous oestradiol implant showed an inhibition of the pulsatile luteinizing hormone release under long days. A higher concentration of plasma prolactin was also observed under long days, without any effect of the steroid treatment. Under this light regimen, statistically significant higher contents of dopamine than under short days were found in the stalk-median eminence. Larger contents of homovanillic acid, a dopamine metabolite, and 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), a noradrenaline metabolite were observed in the infundibular nucleus, while the catechola-mines themselves remained unchanged. Furthermore, oestradiol also significantly increased the content of MHPG in the latter structure. During long days, animals without oestradiol treatment exhibited a significant lower content of noradrenaline in the A15 nucleus, without any alteration of the dopamine content. Daylength or oestradiol treatment had no significant effects on the levels of amines or amine metabolites in the preoptic or septal areas. Thus, our results in the ewe underline the role played by the medial basal hypothalamus in the catecholaminergic regulation of seasonal changes in hormone release and suggest modifications in the turnover of the neurotransmitters in some structures.