The stimulatory effect of alpha-melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin-1beta and by tumour necrosis factor-alpha.

AIM: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide alpha-melanotropin and the cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro-oestrous rats. We also investigate an interaction between these cytokines and alpha-melanotropin in the modulation of progesterone secretion. METHODS: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of alpha-melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. RESULTS: The addition of alpha-melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin-1beta or TNF-alpha were added to the cell cultures. However, when interleukin-1beta or TNF-alpha were added simultaneously with 1 microm alpha-melanotropin, a significant reduction (P < 0.01 for interleukin-1beta and P < 0.05 for TNF-alpha) of the steroid release was found with respect to the alpha-melanotropin-treated group. CONCLUSIONS: These results lead us to suggest that, although alpha-melanotropin stimulates progesterone release in pre-ovulatory GC, this effect is blocked by the presence of interleukin-1beta or TNF-alpha.

Effects of alpha-MSH on progesterone and nitric oxide release by cultured ovarian granulosa cells in experimental rat autoimmune oophoritis.

The peptide alpha-melanocyte-stimulating hormone (alpha-MSH) occurs within the pituitary, brain, skin, ovary and other tissues, and has potent anti-inflammatory activity. For this reason, we examined its effects on an autoimmune disease: the experimental autoimmune-oophoritis (EAO). We analyzed the effect of the peptide on the release of nitric oxide (NO) and progesterone from cultured ovarian granulosa (GL) cells at 0, 7, 14, 21 and 28 days after sensitization of the rats. On day 0 the progesterone levels were higher in estrous rats than those in proestrus and diestrus. The NO amount did not differ among the diverse days of the cycles. The administration of alpha-MSH induced a decrease of NO in estrus and diestrus, but did not affect progesterone release. The EAO rats showed a period of constant diestrus ranging from about 7 to 14 days after immunization. At the onset (day 7) and the end of this period (day 14), the NO significantly increased in estrous rats which was correlated with a reduction in progesterone concentration. This effect was reverted by alpha-MSH. At 21 and 28 days, progesterone release increased only when the rats were in proestrus, while NO production was similar to that on day 0. Administration of alpha-MSH reduced progesterone release when the rats were in proestrus and these results were correlated with an increase in NO only at day 14. The results obtained suggest that alpha-MSH could act as a modulator of EAO, specially when the rats are in estrus.

Effect of neuropeptide-EI on the binding of [3H]SCH 23390 to the dopamine D1 receptor in rat striatal membranes.

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D1 receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D1 receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-3H] (R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D1 receptor in a concentration-dependent manner.

Correlation of increased grooming behavior and motor activity with alterations in nigrostriatal and mesolimbic catecholamines after alpha-melanotropin and neuropeptide glutamine-isoleucine injection in the rat ventral tegmental area.

1. We wished to further study the behavioral effects of alpha-melanotropin (alpha-MSH), melanin-concentrating hormone (MCH), and neuropeptide glutamine-isoleucine (NEI). 2. To this effect we administered alpha-MSH, MCH, and NEI in the ventral tegmental area of the rat, a structure where these neuropeptides are highly concentrated. To further elucidate the biochemical mechanisms of the behavioral effect of these neuropeptides, we determined the degree of grooming behavior and the levels of catecholamines. after neuropeptide administration. 3. We preselected those animals responding to the central injection of alpha-MSH with excessive grooming behavior. We administered the neuropeptides at the dose of 1 microg/0.5 microL, in each side of the ventral tegmental area, bilaterally. We studied grooming behavior, locomotor activity, and total behavior scores, 30 and 65 min after administration of the peptides. 4. Three groups of animals were decapitated immediately after the injection of the neuropeptides, and 30 or 65 min after injection. We measured dopamine (DA), noradrenaline (NA), and the dopac/dopamine ratio (DOPAC/DA) to determine steady state levels of catecholamines and an indirect measure of DA release and metabolism, respectively. 5. Injections of alpha-MSH produced significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with significant decreases in DA content, increases in DOPAC content, and increases in the DOPAC/DA ratio. In the caudate putamen, changes in catecholamines occurred both at 30 and 65 min after injection. In the nucleus accumbens, changes were present at 65 min after injection. Conversely, there were no alterations in NA content, either in the caudate putamen or in the nucleus accumbens, at any time after the injection. 6. Injections of NEI resulted in significant elevations in grooming behavior, locomotor activity, and total behavior scores, both 30 and 65 min after peptide administration. This was correlated with increased DOPAC/DA ratio in the nucleus caudatus but not in the nucleus accumbens. Conversely, NEI produced increased NA concentrations in the nucleus accumbens, but not in the nucleus caudatus. 7. Injections of MCH did not produce significant changes in behavior or significant changes in nucleus caudatus or nucleus accumbens catecholamines. 8. Our results indicate (a) There is a correlation with alterations in behavior as induced for the neuropeptides injected here, and changes in extrapyramidal catecholamines. (b) There is a correlation between alterations in behavior and increases in DOPAC/DA ratio in the nucleus caudatus. (c) There is a correlation between alterations in behavior and alterations in catecholamines in the nucleus accumbens. In the nucleus accumbens, DOPAC/DA ratio is changed after alpha-MSH, and NA ratio is changed after NEI injection. (d) Absence of alterations in extrapyramidal catecholamines, and in particular in catecholamines in the nucleus accumbens, correlates with absence of behavioral alterations after neuropeptide administration to the ventral tegmental area. 9. In conclusion, the behavioral effect of exogenous administration of neuropeptides in the ventral tegmental area is peptide-specific, and is probably associated with alterations in catecholamine metabolism and release in the nucleus caudatus and the nucleus accumbens. Both alpha-MSH and NEI seem to stimulate the nigrostriatal DA system. While alpha-MSH appears to stimulate the mesolimbic DA system as well, NEI may exert its actions not through the DA, but through the NA mesolimbic system. The precise contribution of DA and NA, and the relative role of the nucleus caudatus and nucleus accumbens in these behaviors remain to be elucidated.

The role of melanocortin receptors in sexual behavior in female rats.

Earlier data have indicated that alpha-MSH may play a role for sexual behavior in rats. In this study we investigated the effects of MSH peptides on sexual receptivity in ovariectomized-adrenalectomized female rats, pre-treated with benzoate of estradiol, in presence of vigorous male rats. The results show that alpha-MSH significantly increases lordosis behavior in female rats after injections into the ventromedial nucleus. Interestingly, we have for the first time shown that gamma-MSH also causes significant increase in lordosis in female rats. Furthermore, we show that HS014, an antagonist for the central MC receptors, in dose dependent manner blocks the effect of alpha-MSH on lordosis. The results indicate that the effects of MSH peptides on female sexual behaviour are mediated through a specific MC receptor, which could be the MC3 receptor.

Evidence that alpha-MSH induced grooming is not primarily mediated by any of the cloned melanocortin receptors.

It is well established that melanocortic peptides, such as melanocyte-stimulating hormone (MSH) and adrenocorticotropin, induce grooming behavior. The MC3 and MC4 receptors are the MC receptors which are most abundantly expressed in the brain. gamma-MSH, a peptide with preference to the MC3 receptor, however, does not induce grooming. Recent studies have shown that MC4 receptor antagonists are very effective in inhibiting alpha-MSH induced grooming. These data have indicated that grooming behavior in rodents may be mediated by the MC4 receptor. In this study we investigated if the recently developed MC1 receptor selective agonist MS05 was able to induce grooming in comparison with alpha-MSH. The results show that MS05 is effective in inducing grooming after either intracerebroventricular or ventral tegmental area administration in rats. Central administration of either MS05 or alpha-MSH besides grooming also induced stretching, yawning, rearing and locomotion. The results indicate that the earlier hypothesis that the MC4 receptor is the main mediator of grooming behavior has to be modified. Moreover, as this behaviour does not pharmacologically correlate to the profile of any of the five cloned MC receptors, we suggest that alpha-MSH induced grooming may not primarily be mediated by any of these receptors.

Interaction between alpha-MSH and acetylcholinergic system upon striatal cAMP and IP(3) levels.

The interaction between the neuropeptide alpha-MSH and the acetylcholinergic system as reflected by changes in cAMP and inositol 1-3-5 triphosphate(IP(3))production was investigated in an in vitro model of striatal slices. The possible involvement of D(1) receptors in cholinergic and alpha-MSH- stimulated cAMP and IP(3) production in slices of rat striatum was also examined, because it has been demonstrated that acetylcholinergic drugs induce endogenous dopamine release in the striatum. alpha-MSH, pilocarpine(PL) and the selective muscarinic M1 agonist McN-A-343 increased cAMP and IP(3) striatal levels, effects blocked by the D(1) antagonist SCH-23390, except for the effects of alpha-MSH on IP(3). The muscarinic M(2) antagonist gallamine (GL) brought about an increase in cAMP levels, an effect blocked by SCH-23390. The M(1) antagonist pirenzepine (Pz) induced a decrease both in cAMP and IP(3) content, and the nicotinic antagonist di-hydro-beta-eritroidine(DBE) only diminished cAMP production. When alpha-MSH and cholinergic agents were simultaneously added, cAMP and IP(3) levels were modified with respect to the values reached when these agents were added alone. An interaction between the acetylcholinergic system and alpha-MSH through M(1) and nicotinic receptors was also observed. These results suggest that the intracellular signaling pathways related to cAMP and IP(3) production gated by alpha-MSH and these cholinergic receptors are probably related. alpha-MSH striatum cAMP IP(3) muscarinic and nicotinic receptors an in vitro model.

Interaction of alpha-melanotropin (alpha-MSH) and noradrenaline in the median eminence in the control of female sexual behavior.

In the present study, we examined the effects of the injection of alpha-melanotropin (alpha-MSH), noradrenaline (NA), and dopamine in the median eminence of ovariectomized-adrenalectomized rats on female sexual behavior. The animals were primed with l0 microg of estradiol benzoate, and 52-54 h later they were injected into the median eminence with either 1 microl of artificial cerebrospinal fluid, 1 microg/rat alpha-MSH, 200 ng/rat NA, 200 ng or 2 microg/rat dopamine, in 1 microl of artificial cerebrospinal fluid. Both alpha-MSH and NA significantly stimulated sexual behavior. This effect was antagonized by two beta-adrenergic antagonists: propranolol (500 ng/rat) and metoprolol (400 ng/rat) applied 15 min before the alpha-MSH or NA. The alpha-adrenergic antagonist prazosine (500 ng/rat) was ineffective in reducing the effect of alpha-MSH. The vehicle and dopamine at both doses had no effect on sexual activity. These results indicate that alpha-MSH and NA in the median eminence stimulate female sexual behavior and that NA mediates the action of alpha-MSH via beta-receptors.

Effects and interactions between alpha-MSH and MCH/NEI upon striatal cAMP levels.

It is known that alpha-MSH augments cAMP levels in rat brain slices containing accumbens and caudate-putamen nuclei. In this study we examined: a) the effect of other neuropeptides: MCH and NEI, on this cyclic nucleotide; b) if the effects of alpha-MSH on cAMP production can be modulated by addition of MCH or NEI to the incubation medium. Both MCH and NEI (3.6 microM) increased the production of cAMP, whereas at doses of 0.6 microM exerted no effects. When alpha-MSH 0.6 microM was added with NEI or MCH (0.6 microM), only MCH blocked the increase in the cAMP induced by alpha-MSH. Neither MCH nor NEI at the highest dose used (3.6 microM) had any additive effect on AMPc when added together with alpha-MSH. We conclude that, at a high concentration, (MCH/NEI)-like peptides can use the intracellular signal transduction linked to cyclic nucleotides in the CNS.

Age-related changes in grooming behavior and motor activity in female rats.

The influence of hormonal status and the age of the rat on the expression of grooming behavior and motor activity were studied. Grooming, locomotion, and rearing were measured in young (4-months-old), adult (6-8-months-old), and old (18-months-old) female rats, during the estrous cycle. These behavioral performances were influenced by the hormonal changes that occur in young and adult female rats during the estrous cycle. In old rats there were no significant differences among the different days of the estrous cycle. A significant age-related decrease in grooming behavior and motor activity was also found. Locomotion and rearing were the parameters most affected by age. These findings could be related to: (a) the gonadal hormonal status, which appears to be able to modulate behavioral responses; and (b) the age-related changes, which may affect the normal display of these behaviors. The possible role of central peptidergic, cholinergic, and dopaminergic neural systems is discussed.

The inter-relationship between gonadal steroids and POMC peptides, beta-endorphin and alpha-MSH, in the control of sexual behavior in the female rat.

Estradiol benzoate (10 microg EB) given to ovariectomized-adrenalectomized rats induced sexual receptivity in half the animals and increased alpha-MSH in the preoptic area, ventromedial nucleus (VMN) and arcuate nucleus (ARC), in all the animals, although levels were significantly higher in the VMN and ARC of the receptive (R) subgroup. EB also raised levels of beta-endorphin in the VMN and ARC in the R rats only. POMC expression was not altered. EB did not affect alphaMSH in extra-hypothalamic areas, but addition of progesterone, raised levels in the septum, amygdala, hippocampus and caudate putamen. Only in the VMN, ARC and septum were the steroid-induced increases correlated with onset of sexual behavior.

In vitro effect of alpha-MSH administration on steroidogenesis of prepubertal ovaries.

The effects of alpha-MSH on ovarian progesterone (P) and estradiol (E2) release were investigated. To this purpose, different doses of alpha-MSH were administered to prepubertal ovaries incubated under the following conditions: ten 5 min-pulses of perfusion medium (PM) plus LH at 160 ng/ml each one followed by a 25-min period of PM. The medium of the treated group contained alpha-MSH (0.1, 1, 5, or 10 microg/ml, in saline solution) and the control group saline solution. PM was assayed for P and E2 release. The area under the curve, pulse amplitude and width, and basal hormonal release were calculated for each hormone profile. With respect to P release, an increase in the areas under the curve could be observed in ovaries treated with 1, 5 and 10 microg alpha-MSH/ml. This increment generally coincided with an increase in P pulse amplitude and/or basal P release. The P pulse width was not modified. No significant variations were observed in the parameters under analysis with regard to E2 release. It is concluded that alpha-MSH administered to prepubertal ovaries could exert a physiological role through stimulation of P release.

Interaction between alpha-MSH and gabaergic agents upon striatal cAMP levels: an in vitro model.

We have tried to investigate the possible interaction between the gabaergic system and alpha-MSH at a cellular level in an in vitro model of male albino rats tissue slices containing accumbens and caudate-putamen nuclei. Alpha-MSH alone increases cAMP levels, as does diazepam and phaclofen; however, these effects were blocked by SCH-23390. Both flumazenil and baclofen induced a decrease in the cAMP content. When both alpha-MSH and gabaergic agents were incubated together, cAMP levels were modified. It can be assumed that cAMP production by the neuropeptide and the gabaergic agents could be linked to the activation of dopaminergic D1 receptors. The latter receptors had no prominent effect on the interaction between alpha-MSH and the GABA agonists and antagonists. In summary, our results suggested that alpha-MSH and GABA system could be biochemically linked to produce a cellular effect.

Alpha-melanotropin hormone inhibits the binding of [3H]SCH 23390 to the dopamine D1 receptor in vitro.

We have previously demonstrated that the simultaneous presence of alpha-melanocyte stimulating hormone (alpha-MSH) and dopamine resulted in a reduction in cyclic AMP (cAMP) levels in slices containing caudate putamen and accumbens nuclei as compared to those treated only with dopamine or alpha-MSH. This study was carried out to explore if the interaction between alpha-MSH and dopamine could be explained on the basis of a direct interaction between alpha-MSH and the dopamine D1 receptor. Saturation curves for [n-methyl-3 H](R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-o] hemimaleate ([3H]SCH 23390) binding in the presence of increasing concentrations of alpha-MSH were performed. Nonlinear regression in the presence of alpha-MSH revealed an increased dissociation constant (Kd). The binding capacity (Bmax) was not affected by the peptide. These data suggest an apparent competitive interaction between alpha-MSH and [3H]SCH 23390 in striatal membranes on the dopamine D1 receptor; (Ki = 1.2 X 10(-7) M). The present data show that alpha-MSH could interact with the dopamine D1 receptor modulating allosterically the affinity of [3H]SCH 23390 for the receptor or by causing a change in the lipid environment of the dopamine receptor, resulting in an inhibition of the ligand binding to it.

Interaction of alpha-MSH and substance P with interfaces containing gangliosides.

In the present work we studied the interaction of alpha-MSH and substance P neuropeptides with gangliosides using lipid monolayers, fluorescence spectroscopy, and differential scanning calorimetry techniques. The positively charged weak amphiphilic neuropeptides did not show surface activity in the range of concentrations tested (0.1-0.3 muM), but they were preferentially able to penetrate monolayers formed by acidic lipids, showing the best interaction with the more complex gangliosides. The general order of interaction found for both peptides is GTIh > GDIa = GMI > DLPA > sulphatide. Neither neuropeptide interacted with phosphatidylcholine monolayers above 10 mN.m-1. The binding of alpha-MSH to GMI micelles followed by changes in the fluorescence of its tryptophan residue takes place with an increase in the hydrophobic environment of the neuropeptide. An apparent dissociation constant of 13 muM was estimated for this process. Similar result was found with GMI:DMPC vesicles (1:10 molar ratio). The thermotropic profile of GMI micelles is modified in the presence of the neuropeptides. The calorimetric enthalpy of GMI transition increased 21% and 37% in the presence of alpha-MSH and substance P, respectively. Both neuropeptides induced the same increment in the transition temperature Tm from 19 to 20.5 degrees C. The basic physicochemical studies herein indicated that both positively charged neuropeptides, alpha-MSH and substance P, interact with interfaces containing gangliosides in a mainly electrostatic form, whereas the hydrophobic interaction seems to play a secondary role.

Synergic action of gangliosides on alpha-MSH-induced cyclic AMP levels in rat brain slices.

Gangliosides are particularly enriched in neuron cell membranes and they were postulated to be involved in the modulation of membrane-mediated transduction of information. In this study we explore the possibility that the increase in cAMP tissular levels induced by alpha-MSH may be modulated by the action of exogenously added gangliosides. We measured the level of cAMP in both tissues and medium in response to the alpha-MSH in slices previously incubated with total bovine brain gangliosides (TBG). When slices were exposed to TBG, the effect of alpha-MSH on inducing an increase in the content of cAMP was practically twice compared to the changes induced by alpha-MSH or TBG alone. We conclude that the presence of gangliosides may facilitate the alpha-MSH interaction with its receptor, increasing the cAMP levels in slices containing the CP and Acc nuclei.

Effects of alpha-MSH and cholinergic agents on cGMP and IP3 levels in rat brain slices.

On one hand, it has been demonstrated that the exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH brings about an increase in cAMP. This increase is affected when dopamine is present in the incubation medium. On the other hand, an interaction of melanotropinergic-like peptides with acetylcholinergic drugs has been showed to be similar to the one observed with dopamine. In this study we have intended to measure cGMP or IP3 in response to alpha-MSH, and also to study the interaction with cholinergic drugs by measuring the second messengers recently mentioned. cGMP and IP3 have been measured in tissues and medium in their response to the effect of alpha-MSH alone or in the presence of the peptide plus pilocarpine (selective muscarinic agonist) or atropine (selective muscarinic antagonist). None of them modified the cGMP levels when compared with the control group. The exposure of rat brain slices containing CP and Acc nuclei to alpha-MSH resulted in an increase in IP3 levels. Pilocarpine by itself brought about an increase of IP3 only when the highest doses was used. Atropine did not modify the IP3 content. However, when slices were exposured to both alpha-MSH and pilocarpine, IP3 content was similar to control values. The blockage of the muscarinic receptor with atropine blocked the IP3 increase induced by alpha-MSH as well. Therefore, we assume that alpha-MSH does not induce changes in cGMP but it does change the IP3 levels, probably acting at the muscarinic receptor level.

alpha-MSH changes cyclic AMP levels in rat brain slices by an interaction with the D1 dopamine receptor.

The exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH or dopamine (DA) results in an increase in cyclic AMP (cAMP) levels. When tissues are compared with those containing both alpha-MSH and DA, a reduction in the cyclic nucleotide is observable. This study was carried out to determine whether variations in tissular cAMP levels induced by alpha-MSH might be explained by an interaction between the peptide and some dopaminergic receptors. Therefore, we measured cAMP in tissues and medium in response to alpha-MSH in the presence of haloperidol, the selective D1 (SCH 23390) or D2 (sulpiride) antagonists, or the selective D1 (SKF 38393) or D2 (bromocriptine) agonists. Haloperidol by itself induced no changes either in the cAMP content or in the cAMP efflux to the medium. When slices were exposed to alpha-MSH and haloperidol, the latter blocked the alpha-MSH effect of inducing an increase in the content of cAMP. None of the specific antagonists (at the administered doses) induced changes in the content of cAMP when compared with the control group. The presence of SCH 23390 in the incubation medium together with alpha-MSH yielded a reduction in cAMP levels compared with those incubated with alpha-MSH. A slight stimulatory effect on cAMP formation was observed when the dopaminergic agonists (SKF 38393 10 microM) were used. We conclude that alpha-MSH interacts with the D1 dopamine receptor, changing the cAMP levels in striatum and accumbens nuclei.

alpha-MSH-induced behavior: changes after diazepam and baclofen administration related with cyclic AMP levels.

The present work was performed to evaluate the participation of the benzodiacepinic GABAA and GABAB components upon excessive grooming, locomotion, rearing, and stretching/yawning syndrome induced by the intracerebroventricularly alpha-MSH administration by using GABAA and GABAB agonists. It also aims at evaluating possible relation between changes in cAMP levels in caudate-putamen and accumbens nuclei and the behavioral responses. Injection of diazepam or baclofen reduced the total behavioral scores in a dose-related manner as well as the cAMP levels with respect to the control values (animals treated with artificial cerebrospinal fluid). When diazepam was tested in animals simultaneously injected with alpha-MSH, behavioral scores decreased with respect to those treated with the peptide alone. Cyclic AMP also decreased after combined treatment (MSH + diazepam).

Possible mechanism by which alpha-melanotropin advances the time of vaginal opening.

The aim of this work was to determine whether the acute administration of alpha-melanotropin (alpha-MSH) advances the time of vaginal opening (VO) by modifying the secretion of luteinizing hormone (LH), prolactin, growth hormone (GH), progesterone (P), estradiol (E2) and/or testosterone (T). Female rats received a subcutaneous injection of estradiol benzoate (EB; 10 micrograms/100 g body weight) on day 26 and 27 postnatal (PN). On day 28 PN, a third dose of EB was given together with either intraperitoneal alpha-MSH (treated group) or 0.1 ml saline (control group). On day 32 PN, the animals were inspected for VO and groups of rats were decapitated at hourly intervals between 12:00 and 18:00 hours. Serum concentrations of the hormones listed above were measured by radioimmunoassay. Acute administration of alpha-MSH significantly advances (P < 0.001) the day of VO compared to the control group, as well as increased serum LH levels (P < 0.001) in the period under study, in comparison to controls, but had no significant effect on any of the others hormones analyzed. LH profiles were determined in more detail beginning on the day after alpha-MSH administration (day 29 PN) to VO (day 33 PN). Blood samples were collected from the cannulated external jugular vein at 20 minutes intervals daily between 14:00 and 18:00 hours. alpha-MSH treatment did not significantly modify plasma LH levels on days 29 and 30 PN, although treated animals showed a sustained increase in LH on day 31 PN. On this day, mean LH levels of treated animals was significantly (P < 0.05) higher than the control group.(ABSTRACT TRUNCATED AT 250 WORDS)