A high salt diet inhibits obesity and delays puberty in the female rat.

BACKGROUND/OBJECTIVES: Processed foods are considered major contributors to the worldwide obesity epidemic. In addition to high sugar and fat contents, processed foods contain large amounts of salt. Owing to the correlations with rising adiposity, salt has recently been proposed to be obesogenic. This study investigated three hypotheses: (i) high salt contributes to weight gain and adiposity in juvenile female rats, (ii) puberty onset would be altered because salt is known to affect neuronal systems involved in activating the reproductive system, and (iii) enhanced adiposity will act synergistically with salt to drive early puberty onset. DESIGN: Female weanling rats (post-natal day 21, n=105) were fed a low fat/low salt diet, low fat/high salt diet, high fat/low salt diet or a high salt/high fat diet for 24 days. Metabolic measures, including weight gain, food intake, fecal output, activity and temperature were recorded in subsets of animals. RESULTS: Body weight, retroperitoneal and perirenal fat pad weight, and adipocyte size were all lower in animals fed high fat/high salt compared with animals fed high fat alone. Leptin levels were reduced in high fat/high salt fed animals compared with high fat/low salt-fed animals. Daily calorie intake was higher initially but declined with adjusted food intake and was not different among groups after 5 days. Osmolality and corticosterone were not different among groups. Fecal analysis showed excess fat excretion and a decreased digestive efficiency in animals fed high fat/low salt but not in animals fed high fat/high salt. Although respiratory exchange ratio was reduced by high dietary fat or salt, aerobic-resting metabolic rate was not affected by the diet. High salt delayed puberty onset, regardless of dietary fat content. CONCLUSIONS: Salt delays puberty and prevents the obesogenic effect of a high fat diet. The reduced weight gain evident in high salt-fed animals is not due to differences in food intake or digestive efficiency.

Predominant suppression of follicle-stimulating hormone ß-immunoreactivity after long-term treatment of intact and castrate adult male rats with the gonadotrophin-releasing hormone agonist deslorelin.

Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHß and LHß, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHß-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHß-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHß-ir cell population, although the loss of FSHß in bi-hormonal cells increased the proportion of mono-hormonal LHß-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHß is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.

The heart: a novel gonadotrophin-releasing hormone target.

Gonadotrophin-releasing hormone (GnRH) is a hypothalamic hormone transported by the hypophyseal portal bloodstream to the pituitary gland, where it binds to GnRH receptors. However, GnRH receptors are expressed in multiple extrapituitary tissues, although their physiological relevance is not fully understood. GnRH agonists are employed extensively in steroid deprivation therapy, especially to suppress testosterone in prostate cancer. Because GnRH agonist treatment is associated with increased coronary heart disease and myocardial infarction, we investigated the impact of GnRH on cardiomyocyte contractile function. Cardiomyocytes were isolated from mouse hearts and mechanical and intracellular Ca(2+) properties were evaluated, including peak shortening amplitude (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90) ), maximal velocity of shortening/relengthening (± dLdt), electrically-stimulated rise in Fura-2 fluorescence intensity (ΔFFI) and Ca(2+) decay. GnRH (1 ng/ml) increased PS, ± dL/dt, resting FFI and ΔFFI, and prolonged TPS, TR(90) and Ca(2+) decay time, whereas 1 pg/ml GnRH affected all these cardiomyocyte variables, except TPS, resting FFI and ΔFFI. A concentration of 1 fg/ml GnRH and the GnRH cleavage product, GnRH-[1-5] (300 pg/ml), had no effect on any cardiomyocyte parameter. The 1 pg/ml GnRH-elicited responses were attenuated by the GnRH receptor antagonist cetrorelix (10 µm), the protein kinase A (PKA) inhibitor H89 (1 µm) but not the protein kinase C inhibitor chelerythrine chloride (1 µm). These data revealed that GnRH is capable of regulating cardiac contractile function via a GnRH receptor/PKA-dependent mechanism. If present in the human heart, dysfunction of such a system may play an important role in cardiac pathology observed in men treated with GnRH agonists for prostate cancer.

Effects of gonadotrophin-releasing hormone outside the hypothalamic-pituitary-reproductive axis.

Gonadotrophin-releasing hormone (GnRH) is a hypothalamic decapeptide with an undisputed role as a primary regulator of gonadal function. It exerts this regulation by controlling the release of gonadotrophins. However, it is becoming apparent that GnRH may have a variety of other vital roles in normal physiology. A reconsideration of the potential widespread action that this traditional reproductive hormone exerts may lead to the generation of novel therapies and provide insight into seemingly incongruent outcomes from current treatments using GnRH analogues to combat diseases such as prostate cancer.

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.

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.

Prolactin release during the estradiol-induced LH surge in ewes: modulation by progesterone but no evidence for prolactin-releasing peptide involvement.

An estradiol-induced prolactin surge accompanies the LH surge in several species, including sheep. However, the neural mechanisms underlying this surge remain poorly understood. A first study on estradiol- and progesterone-treated ovariectomized ewes examined whether the prolactin surge, like the LH surge, is sensitive to progesterone. Our data clearly showed that the estradiol-induced prolactin surge in the ewe is blocked by continuous exposure to progesterone and, importantly, that this blockade is overcome by pretreatment with the progesterone receptor antagonist, RU486. In a second study, we established that the generation of the prolactin surge is not dependent on the co-secretion of a prolactin-releasing peptide in the hypophyseal portal blood or cerebrospinal fluid. The neuronal pathways targeted by estradiol and progesterone to modulate prolactin secretion at the time of the LH surge remain to be identified. Importantly, it has not been established whether there is any overlap in the neuronal systems generating the gonadotropin-releasing hormone and prolactin surges.

Effect of ram introduction on the oestrous cycle of springbok ewes (Antidorcas marsupialis).

Springbok are aseasonally breeding wild ungulates that inhabit arid environments, and interest has been shown in domesticating them for agricultural purposes. The present study was conducted for husbandry purposes to determine the effect of introducing a vasectomized ram to an isolated herd of springbok ewes (n = 9). Blood was collected from ewes every third day, before and after introduction of a vasectomized ram. Ewes were subjected to the ram for 42 days. Plasma progesterone was measured by radioimmunoassay and was used to establish the stage of the oestrous cycle. After introduction of the ram, the variation in the timing of the follicular phase between ewes was clearly reduced, compressing the spread of oestrus in the springbok ewes from 11 to 3 days. In seven of the nine ewes, the ram was introduced during the luteal phase of the oestrous cycle, causing this cycle to be significantly longer in duration (P < 0.05) and to have a higher maximum concentration of progesterone (P < 0.001) than cycles before and after introduction of the ram. This finding implies that the mechanism of synchronization operates through a luteotrophic effect. These results indicate that rams may be used successfully to synchronize breeding in springbok.

Measurement and possible function of GnRH in cerebrospinal fluid in ewes.

Large mammal models are unique in that it is possible to analyse the real-time release of neural factors over several hours or even days in a conscious unstressed state. Until recently, hypophyseal portal blood sampling was the only reliable method available for this purpose. However, development of an alternative approach, in which cerebrospinal fluid (CSF) is collected from specific sites within the cerebroventricular system, has provided another route by which hypothalamic activity can be investigated. Use of this approach, in combination with other methods, such as intracerebroventricular infusion or simultaneous hypophyseal portal blood collection, has yielded exciting novel data that challenge long-held dogma on the pathways of communication in the brain. It is clear that factors in the CSF are released site-specifically and, thus, this fluid is not homogeneous; the concentration of a factor in lumbar CSF may bear no relation to its ventricular concentration. Data also indicate that there is little, if any, transfer of factors between the CSF and the hypophyseal portal system. In addition, there is mounting evidence indicating that factors in CSF may serve as part of a non-synaptic communication system in the brain. Establishing an unequivocal function for CSF-borne factors may prove technically difficult, if not impossible. However, we believe that there is strong evidence supporting a role for one such factor, GnRH in CSF, in sexual behaviour.

Neuroendocrine control of pulsatile GnRH secretion during the ovarian cycle: evidence from the ewe.

This article reviews the neuroendocrine control of episodic GnRH secretion during the ovine oestrous cycle. There is general agreement that endogenous opioid peptides (EOPs) mediate the negative feedback action of progesterone on GnRH pulse frequency during the luteal phase of the ovarian cycle and recent preliminary data have implicated the dynorphin-kappa-receptor system in this effect of progesterone. Progesterone also acutely inhibits GnRH pulse frequency via a non-EOP mechanism, as naloxone does not block the rapid effects of this steroid. The effects of bicuculline, 3alpha-hydroxy-5alpha-pregnan-20-one and RU486 consistently indicated that the gamma-aminobutyric acid A (GABA-A) receptor is also not involved in the acute actions of progesterone. Thus, the neural system mediating this effect remains to be determined. Oestradiol has several actions on episodic GnRH secretion. The most well characterized action is inhibition of GnRH pulse amplitude, which is probably mediated by noradrenergic neurones. Oestradiol also increases the response to progesterone negative feedback, alters GnRH pulse shape and increases GnRH pulse frequency. The first two of these actions may involve EOPs, whereas the mechanisms underlying GnRH pulse frequency are currently unknown. Finally, there is also evidence that EOPs play a physiological role in synchronizing the firing of the GnRH neurones responsible for episodic release. Specifically, the effects of naloxone on the GnRH pulse shape lead to the hypothesis that EOP tone contributes to the termination of each GnRH pulse and prevents random firing of these GnRH neurones between pulses. Thus, it appears that EOPs play an important role in controlling several different aspects of pulsatile GnRH release during the ovine oestrous cycle.

Neuroendocrine mechanisms underlying the effects of progesterone on the oestradiol-induced GnRH/LH surge.

Oestradiol provides the drive to reproductive cyclicity in female mammals through its ability to stimulate the GnRH surge. In contrast, progesterone can be seen as the 'clutch and brakes' within reproductive cycles, as it can modify the response of the GnRH neurosecretory system to oestradiol. In this regard, progesterone has multiple and sometimes opposing effects on the GnRH neurosecretory system. For example, dependent upon the timing of exposure, progesterone enhances the amplitude of the oestradiol-induced LH (rats) and GnRH surge (within cerebrospinal fluid in sheep, mRNA concentrations in rats), but can also inhibit pulsatile GnRH secretion, and delay or even block expression of the surge (monkeys, rats and sheep). Investigations of the mechanisms of action of progesterone are complicated further by the fact that some of the observed effects of progesterone, such as the ability to block the oestradiol-induced surge, appear to be mediated via several different routes. Consequently, a variety of approaches are needed to advance our understanding of this fundamental reproductive neuroendocrine system. In this context, large animal neuroendocrine models have provided important information about the mechanisms of progesterone action and provide many exciting opportunities for future research.

Melatonin and seasonal reproduction: understanding the neuroendocrine mechanisms using the sheep as a model.

The mechanisms by which melatonin controls seasonal reproduction are poorly understood. The use of a large animal model, namely the sheep, has allowed progress in the understanding of these mechanisms, and is the subject of this review. Firstly, the contribution made by large animal models to demonstrating that melatonin acts in the hypothalamus and the identification of this hypothalamic target is reviewed. Secondly, the way in which large animal models have facilitated the demonstration of a specific mechanism of release of melatonin in the cerebrospinal fluid and, thus, raised the question of the route used by melatonin to reach its central targets is discussed. Finally, the human and agricultural relevance of the data presented is considered.

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.

Annual ovarian cycles in an aseasonal breeder, the springbok (Antidorcas marsupialis).

The springbok is an arid-adapted antelope inhabiting the desert and semidesert regions of southern Africa. Because it thrives in these sparsely vegetated areas, the springbok is of potential agricultural importance and the prospect of domestication has been speculated for many years. However, apart from observational studies on its breeding in the wild, suggesting it is an aseasonal breeder, little is known about the underlying reproductive endocrinology of this species. In this study, biweekly peripheral blood samples were collected from eight captive springbok ewes from October 1995 until September 1998 and analyzed for progesterone. At the start of the study, six ewes were prepubertal and cycling commenced spontaneously between November 1995 and June 1996. Cycling had already commenced in two ewes. At the end of November 1996, estrous cycles ceased abruptly in all ewes and restarted in April 1997. Cycling ceased again between December 1997 and February 1998 and restarted in June 1998 in six ewes; there was no cessation of estrous cycles in two ewes. Thus, although some individuals cycle continuously, there is a clear endocrine anestrus of between 4 and 5 mo in springbok, the timing and duration of which is synchronized between some individuals but the time of onset and cessation is variable from year to year. To ensure that the fluctuations we observed in progesterone levels were reliable indicators of changes in the estrous cycle, blood samples were collected every 6 h for 16 days in August 1998. A surge in LH secretion was observed in all ewes 55 +/- 5 h after the fall in progesterone. Progesterone levels increased again 45 +/- 8 h after the surge. A final study showed that the pattern of melatonin release in springbok exhibits a normal day/night profile, and thus photoperiodic information is transformed into an endocrine code to springbok but does not appear to affect reproduction. Rather, our data raise the possibility that the prevailing ambient temperature may influence the onset of ovarian activity in this species.

Unmasking the progesterone receptor in the preoptic area and hypothalamus of the ewe: no colocalization with gonadotropin-releasing neurons.

Progesterone powerfully inhibits GnRH secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Visualization of the neural ovine progesterone receptor has proved elusive but, using a high temperature antigen unmasking technique, the progesterone receptor was revealed in the ewe brain. Progesterone receptors were located in the preoptic-hypothalamic continuum, especially in the preoptic area, ventrolateral region of the ventromedial nucleus and the arcuate nucleus. This study also suggests that the inhibitory action of progesterone on GnRH release is not transduced directly through the GnRH neurons as a single GnRH perikaryon of 732 was immunoreactive for the progesterone receptor.

Neuroendocrine effects of progesterone.

Progesterone (P) is secreted by the corpus luteum under the control of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH). Progesterone (P) is essential for reproduction because: (1) it induces in the endometrium the transcription of specific genes involved in the implantation of the blastocyst, (2) it modulates GnRH/LH secretion by decreasing GnRH pulse frequency, which in turn enriches the gonadotroph cells in FSH and avoids a second LH surge. Using the ewe as a model, we investigated the immediate GnRH and LH responses to acute changes of circulating P levels. Our results show that P changes cause dramatic modifications in GnRH pulse frequency: P removal induces an acceleration of the pulse generator, while P administration slows the pulse frequency. LH secretion was modified in parallel to the changes in GnRH. Other experiments proved that these neuroendocrine effects of P are mediated by P itself, not by its hydroxylated metabolites, and occur at the level of P receptors. Finally, these effects require priming by estradiol. Additionally, in the final stage of the follicular phase, P plays a role in the triggering of the LH surge. This has been shown in rodents, non-human primates, and in women. Such a phenomenon is not observed in ewes, although in these species luteal P modulates the amplitude of the estradiol-induced LH surge.

Duration and amplitude of the luteal phase progesterone increment times the estradiol-induced luteinizing hormone surge in ewes.

Progesterone (P) powerfully inhibits the neuroendocrine reproductive axis, but the mechanisms and site or sites of action of this steroid remain poorly understood. Progesterone exposure during the luteal phase also alters the responsiveness of the hypothalamus to increased concentrations of estrogen (E) during the follicular phase. Using an ovariectomized ovine follicular phase model, we investigated whether the amplitude and duration of the luteal phase increase in circulating P affects the E-induced surge in LH. Treatment of ewes for 10 days with two, one, or half an intravaginal P-releasing implant or with an empty implant demonstrated that P concentrations significantly (P: < 0.0001) delayed the time to surge onset upon exposure to an equal concentration of E. This delay was not due to a time-related difference in responsiveness to E after P clearance because the time of surge onset was not different when E treatment began 6, 12, or 24 h after the withdrawal of two P implants that had been present for 10 days. The final study demonstrated that the duration of P before treatment (5, 10, or 30 days) significantly (P: < 0.0001) delayed the responsiveness of the estradiol-dependent surge-generating system. There was no effect on surge amplitude or duration in any experiment. Thus, the amplitude and duration of exposure to luteal phase P significantly affect the neural elements targeted by E to induce the preovulatory LH surge.

The negative feedback action of progesterone on luteinizing hormone release is not associated with changes in GnRH mRNA expression in the Ewe.

Progesterone is the ovarian hormone that times events in the ovine reproductive cycle. When elevated, this ovarian hormone acts centrally to inhibit both the tonic and surge modes of gonadotrophin releasing hormone (GnRH) release. Two studies were performed to address the underlying neural mechanisms. The first tested the hypothesis that the rapid rise in GnRH release, that results from an acute fall in progesterone concentrations (such as occurs following luteolysis), is temporally associated with a rapid rise in the cellular content of GnRH mRNA. Three groups of ovariectomised (OVX) ewes were treated with exogenous progesterone for 10 days, while one remained steroid free (OVX, n=7). To determine the effects of acute progesterone (P) withdrawal, ewes were killed on day 10 while implants were still in place (OVX+P, n=6) or 4 (OVX-P4, n=7) or 12 h (OVX-P12, n=7) after progesterone removal. Coronal sections through the rostral portion of the medial preoptic area (rPOA) were processed for cellular in-situ hybridization for GnRH mRNA. An increase in progesterone concentrations markedly suppressed luteinizing hormone (LH) release, while removal of the implants caused progesterone concentrations to fall (P<0.01) within 1 h and LH pulse frequency to increase (P<0.05) within 4 h. Despite these progesterone-induced changes in LH/GnRH release there were no differences in the cellular content of GnRH mRNA among the four groups. In the second study, three groups of ovariectomised ewes were used to determined whether the inhibitory actions of early (EL; n=8) and mid-luteal (ML; n=8) phase concentrations of progesterone on LH release are accompanied by a decrease in GnRH mRNA expression. P inhibited the secretion of LH in a dose dependant manner; pulses of LH were virtually absent in the ML group. Despite this marked inhibitory steroid action, there was no significant difference in the cellular content of GnRH mRNA among the OVX, OVX (EL) and OVX (ML) groups. Thus, both the negative feedback actions of physiological concentrations of progesterone on GnRH release and the rapid escape from progesterone-inhibition are independent of changes in the cellular content of GnRH mRNA. These data suggest that the mechanism by which progesterone controls the timing of events in the ovine oestrous cycle is primarily by altering the secretion of GnRH rather than GnRH biosynthesis.

High melatonin concentrations in third ventricular cerebrospinal fluid are not due to Galen vein blood recirculating through the choroid plexus.

Melatonin has been implicated in several neurotropic effects, but few studies have investigated the bioavailability of melatonin in the brain. The discovery of periventricular sites of action adjacent to the third ventricle forced us to investigate the dynamics of cerebrospinal fluid (CSF) melatonin release and the source of this melatonin. Our first study demonstrated unequivocally that third ventricle CSF melatonin, like jugular plasma melatonin, accurately reflects the duration of the night and is rapidly suppressed by light. However, third ventricle CSF melatonin levels are 20-fold higher than nocturnal plasma concentrations. A further study showed that melatonin increased in plasma before third ventricle CSF, raising the possibility that melatonin is taken up from the blood after recirculation through the Galen vein. However, a final experiment suggested strongly that CSF melatonin is released directly into the third ventricle, as melatonin levels in the lateral ventricle were 7-fold lower than those in the third ventricle. Our study raises the possibility that there may be two compartments of melatonin affecting physiological functioning: the first in plasma acting on peripheral organs, and the second in the CSF affecting neurally mediated functions at a much higher concentration of this pineal indoleamine.

The progesterone blockade of the luteinizing hormone surge is overcome by RU486.

Progesterone can prevent the oestrogen-induced and spontaneous preovulatory luteinizing hormone (LH) surges but the mechanisms underlying this effect remain poorly understood. Using a follicular phase ovariectomised (OVX) ewe model and by elevating progesterone in the presence of oestrogen to inhibit the LH surge, we investigated whether the progesterone receptor antagonist, RU486, could block the inhibitory effects of progesterone. Accordingly, intravaginal progesterone implants were inserted into OVX Ile-de-France ewes (n = 18), bearing 10 mm Silastic 17beta-oestradiol implants. Ten days later, the progesterone implants were removed, whereupon new implants were inserted immediately into 12 ewes: six of which were also injected with 100 mg RU486 dissolved in 10 mL vehicle (10% alcohol in peanut oil) and six received vehicle only. The remaining six ewes were injected with vehicle only. RU486 and vehicle injections were made again 12 and 24 h later. After the last injection, oestrogen concentrations were raised to peak follicular phase levels in all ewes by subcutaneous insertion of four 3-cm 17beta-oestradiol implants. Blood samples were collected every 2 h for 40 h starting 9 h after the insertion of the oestrogen implants. As expected, the six ewes treated only with oestradiol had a LH surge whereas no ewes given the implants in the presence of progesterone surged. RU486 completely blocked the inhibitory effect of progesterone. There were no differences in the time of LH surge onset, duration over which LH levels remained above their half-maximal concentration or magnitude of the LH surge between the two groups showing surges. Our study suggests strongly that the progesterone-mediated blockade of the ovine oestrogen-induced LH surge is not through allopregnanolone activation of the GABA(A) receptor. Rather, our study demonstrates that this effect is transduced by the classic nuclear progesterone receptor.