Biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in hypothalamic-pituitary unit of anoestrous and cyclic ewes.

This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states.

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal's physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

The Central Effect of ß-Endorphin and Naloxone on The Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The Hypothalamic-Pituitary Unit of Follicular-Phase Ewes.

The effects of prolonged, intermittent infusion of ß-endorphin or naloxone into the third cerebral ventricle of follicular-phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme-linked immunoabsorbent assay. Activation or blockade of µ-opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with ß-endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that ß-endorphinergic system in the hypothalamus of follicular-phase ewes affects directly or via ß-endorphin-sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic-pituitary axis.

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.

The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes.

The effect of prolonged intermittent infusion of beta-endorphin or naloxone into the third cerebral ventricle in ewes during the follicular phase of the estrous cycle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined by Real time-PCR. Activation of micro opioid receptors decreased GnRH mRNA levels in the hypothalamus and led to complex changes in GnRH-R mRNA: an increase of GnRH-R mRNA in the preoptic area, no change in the anterior hypothalamus and decrease in the ventromedial hypothalamus and stalk/median eminence. In beta-endorphin treated ewes the levels of GnRH-R mRNA in the anterior pituitary gland also decreased significantly. These complex changes in the levels of GnRH mRNA and GnRH-R mRNA were reflected in the decrease of LH secretion. Blockade of micro opioid receptors affected neither GnRH mRNA and GnRH-R mRNA nor LH levels secretion. These results indicate that beta-endorphin displays a suppressive effect on the expression of the GnRH gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland, but affects GnRH-R gene expression in a specific manner in the various parts of hypothalamus; altogether these events lead to the decrease in GnRH/LH secretion.

The role of GabaA receptors in the neural systems of the ventromedial hypothalamus-nucleus infundibular region in the control of GnRH release in ewes during follicular phase.

To examine the role of the GABAA receptor mediating systems in the control of gonadotropin releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) of the hypothalamus of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin (B-END), noradrenaline (NE), dopamine (DA), and their metabolites MHPG, DOPAC and concentration of luteinizing hormone (LH) in blood plasma were quantified during local stimulation or blockade of GABAA receptors with muscimol and bicuculline, respectively. Stimulation of GABAA receptors attenuated GnRH and LH release, increased beta-endorphin outflow and dopaminergic activity but had no evident effect on noradrenergic activity. Blockade of GABAA receptors decreased beta-endorphin release but had no evident effect on the extracellular concentration of GnRH, LH levels in the blood and catecholaminergic activity. It is suggested that suppression of GnRH/LH release under muscimol treatment may result from activation of GABAA receptors on GnRH nerve terminals and through GABAA receptor mechanism activated beta-endorphinergic and dopaminergic neurons in the VEN/NI. Lack of changes in NE and MHPG concentration during stimulation or blockade of GABAA receptors suggests, that during the follicular phase of the estrous cycle the noradrenergic system in the VEN/NI is not involved in the control of GnRH/LH release by GABA.

The role of GABA(A) receptors in the neural systems of the medial preoptic area in the control of GnRH release in ewes during follicular phase.

To examine the role of gamma-aminobutyric acid (GABA)(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the medial preoptic area (MPOA) of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenyl-glycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during the local infusion of muscimol and bicuculline (agonist and antagonist of GABA(A) receptors, respectively) to this structure. Stimulation of GABA(A) receptors markedly attenuated GnRH release, increased beta-endorphin release and noradrenergic system activity in the MPOA. The decrease of the luteinizing hormone (LH) concentration in blood plasma and LH pulse amplitude suggests that a GABA(A) receptor agonist in the MPOA also suppresses GnRH release from the GnRH axon terminals in the ventromedial hypothalamus/nucleus infundibularis region (VEN/NI). Blockade of GABA(A) receptors had no evident effect on GnRH/LH secretion but decreased beta-endorphin release and increased the extracellular DOPAC concentration. The suppressive influence of muscimol in the MPOA on GnRH release might be considered a net result of its direct inhibitory effect on GnRH release, indirect inhibitory influence on GnRH release through activation of the beta-endorphinergic system, and facilitation of GnRH neurons by increasing noradrenaline release. The results obtained during bicuculline perfusion on these systems' activity are not sufficiently consistent to provide a clear understanding of the lack of changes in the GnRH/LH release under blockade of GABA(A) receptors. We conclude that the MPOA in ewes during the follicular phase is an important regulatory site where stimulation of GABA(A) receptors both decreases GnRH secretion and increases beta-endorphin release.

The Involvement of GABAA receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the preoptic area in anestrous ewes.

This study examined role of GABA A receptors in the control of GnRH, beta-endorphin release and catecholaminergic system activity in the preoptic area and LH secretion in anestrous ewes. Stimulation of GABA A receptors in the medial preoptic area (MPOA) by muscimol attenuated GnRH release and dopaminergic system activity and increased extracellular noradrenaline (NE) and MHPG concentration. Muscimol has no evident effect on the extracellular concentration of beta-endorphin-like immunoreactivity (B-END-LI) in the MPOA. The decrease of LH pulse frequency and concentration of this hormone in blood plasma suggests that GABA A receptor agonist applied in the MPOA suppresses GnRH release from the GnRH axon terminals in the ventromedial hypothalamus-nucleus infundibularis region (VEN/NI) into the hypophyseal vascular system. Blockade of GABA A receptors with bicuculline did not change GnRH release, catecholaminergic activity, B-END-LI concentration in the MPOA, and LH release. The presented data indicate that activation of GABA A receptors in the MPOA decreases extracellular concentration of GnRH in this structure and LH level in the blood plasma thus suggesting that GABA may act in the MPOA to inhibit GnRH release in the VEN/NI. These results suggest that suppression of GnRH/LH release during muscimol treatment may result from activation of GABA A receptors on the GnRH perikarya and/or through GABA A receptor mechanism on the dopaminergic and noradrenergic system in the MPOA. Lack of changes in B-END-LI concentration during stimulation or blocking GABA A receptors suggests, that beta-endorphinergic system in the MPOA does not participate in the GABA A receptors mechanism modulating GnRH release.

The role of GABA(A) receptors in the neural systems of the ventromedial-infundibular region of the hypothalamus in the control of gonadotropin release during the luteal phase in ewes.

To examine the role of GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) in ewes during luteal phase, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), and their metabolites: MHPG and DOPAC were quantified by local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline, respectively. Stimulation of GABA(A) receptors in the VEN/NI did not affect GnRH, beta-endorphin release or catecholaminergic system activity. Blockade of GABA(A) receptors decreased beta-endorphinergic and dopaminergic activity, and lowered the extracellular concentration of MHPG. It did not affect GnRH release or luteinizing hormone (LH) secretion. It is suggested that progesterone-induced GABAergic activity during the luteal phase may desensitize GABA(A) receptors to muscimol. Lack of changes in GnRH/LH secretion with concomitant depressed beta-endorphinergic activity corroborated the conclusion that beta-endorphin does not inhibit GnRH release from the VEN/NI during the luteal phase. The physiological significance of changes in the catecholaminergic system activity under GABA(A) receptor blockade in the control of GnRH secretion awaits to be established.

The involvement of GABA(A) receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the ventromedial-infundibular region of hypothalamus in anestrous ewes.

To examine the role of the GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/IN) of anestrous ewes, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline respectively. In most animals stimulation of GABA(A) receptors significantly attenuates GnRH release with concomitant increase of beta-endorphin and DA release, and MHPG and DOPAC levels. Blockade of the GABA(A) receptors generally did not affect GnRH and NE release but inhibited in most animals beta-endorphin release and decreased dopaminergic activity. These results suggest, that GABA may suppress GnRH release directly by GABA(A) receptor mechanism on the axon terminal of GnRH neurons or indirectly by GABA(A) receptor processes activating beta-endorphin-ergic and dopaminergic neurons in the VEN/NI. On the basis of these results in could not be distinguish between these two events. The decrease in extracellular beta-endorphin and dopamine concentration without evident changes in the GnRH level during GABA(A) receptor blockade may suggest that other neuronal systems are involved in this effect.

Changes in extracellular LHRH and beta-endorphin-like immunoreactivity in the nucleus infundibularis-median eminence of anestrous ewes under stress condition.

To clarify the effect of beta-endorphin released under stress condition on LHRH secretion, the concentration of LHRH and beta-endorphin-like-immunoreactivity (beta-END-LI) were analysed in perfusates from the nucleus infundibularis-median eminence of anestrous ewes subjected to footshocks stimulation. The dynamics of LHRH and beta-END-LI release during time-course of footshocks stimuli was altered. A brief facilitatory influence of stress on LHRH and beta-END-LI release was observed at the beginning of the stimulation on the first and third day; however the peaks of beta-END-LI were delayed about half an hour in relation to LHRH. Than on the first day of the stimulation LHRH returned to the control value but on the third day it declined below the control levels. Prolonged stress had stimulatory effect on beta-END-LI secretion during first and third day. The presented results indicate that: 1) short stress activates LHRH and beta-END-LI release, 2) prolonged stress has stimulatory effect on beta-END-LI release but leads to suppression of LHRH release. It is suggested that stress-induced beta-endorphin release may be one of the factors responsible for suppression of LHRH activity.

Extracellular monoamines and their metabolites in the mediobasal hypothalamus--median eminence of anestrous and estrous ewes during CRF treatment.

In order to clarify the effect of exogenous corticotropin-releasing factor (CRF) on catecholaminergic and serotoninergic system activity in the mediobasal hypothalamus-median eminence (MBH-ME) of ewes the changes in extracellular levels of noradrenaline (NA) and serotonin (5HT), and main metabolites of monoamines, 4-hydroxy-3-methoxyphenylglycol (MHPG), 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxy-indolo-3-acetic acid (5-HIAA) were quantified in the perfusates collected from MBH-ME. NA, 5-HT and monoamine metabolites in the perfusates were analyzed using high performance liquid chromatography with electrochemical detection. CRF induced a rise in extracellular concentration of NA and 5-HT only in the estrous ewes prior to a preovulatory LH surge. CRF treatment caused a heterogenous effect on extra-cellular concentrations of 5-HT in ewes during the preovulatory LH surge. Except for DOPAC and HVA in some estrous ewes during the preovulatory LH surge, CRF caused an increase in monoamine metabolites levels in the MBH-ME in anestrous and estrous animals. These results indicate that CRF facilities NA release in the MBH-ME during the presurge LH period in ewes, and that CRF increases metabolic activities of the monoaminergic systems in this structure in the anestrous and estrous ewes, except dopaminergic system in the ewes during the preovulatory LH surge. It is suggested that: 1) the responses of monoaminergic systems activity in the MBH-ME to CRF in large degree is dependent upon physiological state of ewes and 2) in some endocrinological phases CRF may affect LHRH and other hypothalamic hormone secretion indirectly by altering monoaminergic system activity in the MBH-ME.

Release of luteinizing hormone-releasing hormone, beta-endorphin and noradrenaline by the nucleus infundibularis/median eminence during periovulatory period in the sheep.

The relationships between the release of LHRH, beta-endorphin (beta-END) and noradrenaline (NA) from the hypothalamic infundibular nuclei/median eminence (NI/ME) during the periovulatory period in the ewe was studied. Neurohormone release was assayed in perfusates collected from the NI/ME via push-pull cannulae. LHRH concentrations in perfusates ranged from below detectable values (5 pg) to 50 pg and from 15 to 240 pg/20 min perfusate on the days of proestrus and estrus, respectively. beta-END concentrations in perfusates ranged from 320 to 6,000 pg on the day of proestrus and fell to a range between 100 and 380 pg/20 min perfusate on the day of estrus. The NA content of perfusates ranged from an undetectable level to 0.9 x 10(4) pg/perfusate during proestrus, and rose from 1.0 x 10(4) to 6.6 x 10(4) pg/perfusate shortly before the preovulatory release of LHRH and LH. On the basis of the present observations, the following sequence of events leading to the massive LH ovulatory surge in the sheep is suggested: (1) increased secretion of beta-END in the NI/ME on the day of proestrus generates an increase in the releasable pool of LHRH through inhibition of LHRH release; (2) on the day of estrus a decreased release of beta-END allows the expression of NA activity in the NI/ME and the augmentation of NA tone facilitates the release of newly accumulated LHRH; (3) the resultant intensified LHRH output with its significantly changing pattern of release triggers the preovulatory surge of LH.

Suppressive effect of beta-endorphin and naloxone on the secretion of cortisol under stress conditions in sheep.

The effect of an infusion of beta-endorphin and naloxone into the 3rd cerebral ventricle on the blood serum concentration of cortisol in non-stressed and stressed anoestrous sheep was studied. The infusion of naloxone alone did not alter the secretion of cortisol in non-stressed animals but the response to footshock was inhibited in that the cortisol values were significantly lower (P less than 0.05) when compared with those during the infusion of saline and footshocking. This opiate antagonist also suppressed the cortisol response to an infusion of beta-endorphin (P less than 0.001 and P less than 0.05). When the sheep infused with beta-endorphin were additionally exposed to footshock there was less enhancement of the secretion of cortisol (P less than 0.001 and P less than 0.05). The depressive action of beta-endorphin upon cortisol secretion in stressed sheep is matched by corresponding observations on man and it seems that endogenous opioids may inhibit or attenuate some hormonal responses to stress.

The effect of prolonged stress on the hypothalamic luteinizing hormone-releasing hormone (LHRH) in the anoestrous ewe.

A study was performed to examine the effect of the intermittent long-term (20 min/h daily during 3 consecutive days) electric stress (footshocks) on the hypothalamic LHRH in anoestrous ewe. On the third day, immediately after the last stimulation, the animals were decapitated and the hypothalami and pituitaries were taken for analysis of LHRH and LH by immunocytochemistry; LHRH was also assayed by radioimmunoassay. In the stressed animals the concentration of immunoreactive LHRH (IR-LHRH) and LHRH analysed by radioimmunoassay (RI-LHRH) increased significantly in the median eminence (ME) and the medial preoptic area (MPOA) when compared with the controls. Additionally, IR-LHRH perikarya appeared in the MPOA of stressed animals, whereas they were not seen in the controls. RI-LHRH in the medial basal hypothalamus (MBH) of stressed ewes was markedly lower than in control ones. No significant differences in RI-LHRH concentration were found in the septum between stressed and control ewes. The present study clearly indicates that prolonged stressful stimuli modify LHRH concentration in the discrete areas of the hypothalamus of the anoestrous ewe. It is suggested that these changes are attributed mainly to an inhibition of LHRH release from the ME and a suppression of LHRH transport along neural fibres within the hypothalamus.

Differential behavioural and hormonal responses to two different stressors (footshocking and immobilization) in sheep.

Two different stressors footshocking and immobilization applied for 3 days induced in sheep differential behavioural and hormonal responses in cortisol and prolactin secretion. Immobilization drastically disturbed rumen motoric activity (significantly attenuated its contractions), caused loss of appetite and severe general depression. Footshocking did not induce any of these symptoms. Immobilization induced 2-3 fold higher rise of plasma cortisol mesors on the days of stressing as compared to footshocking. On the poststressing days after footshocking as well as after immobilization plasma cortisol mesors fell to 50% of the prestimulatory values. The rise of plasma prolactin mesors in footshocked and in immobilized animals on the days of stressing was nearly of the same magnitude. However, a significant difference in the response of prolactin secretion between footshocked and immobilized animals occurred in the circadian rhythmicity of the hormone secretion. Footshocking induced circadian rhythmicity with characteristic acrophases, while immobilization did not induce the circadian pattern of prolactin secretion. An attempt of interpretation of the described phenomena has been undertaken.

The effect of 6-hydroxydopamine infusion into the third cerebral ventricle on the plasma prolactin levels in anoestrous ewes subjected to prolonged and repeated stress.

The effect of 6-hydroxydopamine (6-OHDA) infusion into the third cerebral ventricle on the secretion pattern of prolactin in anoestrous sheep, subjected to repeated and prolonged stress-stimuli was followed. On the 12-15 th day after the infusion of 6-OHDA into the third cerebral ventricle and after having tested the effect of this infusion on plasma prolactin concentration, the animals were subjected to mild electric footshocks 9 hrs daily (9 a.m.-6 p.m.) for 3 consecutive days. Infusion of 6-OHDA induced a very high rise of plasma prolactin values by about 3 times as compared to those found before infusion (from 118.2 +/- 39.5 to 338.9 +/- 81.7 ng/ml mesor 24 h). On the other hand, the response of the animals pretreated with 6-OHDA to electric stimulation was characterized by the lack of changes in plasma prolactin concentration; their plasma hormone concentrations were neither raised during stimulation nor decreased after the stimulation and were maintained during all these days nearly at the same level as after the infusion of 6-OHDA. Central mechanisms which could account for the so high and prolonged elevation of plasma prolactin concentration after the infusion of 6-OHDA and for the lack of its rise under conditions of prolonged stress are discussed.

Effect of repeated and prolonged stress stimuli on the plasma prolactin concentration in sheep.

The effect of repeated and prolonged stress-stimuli applied during 3 days on the plasma prolactin concentration in sheep, in anoestrous, prooestrous and pregnant animals were studied. The following results were obtained: Repeated and prolonged weak electric stimuli applied in the form of footshocks in sheep during 3 days elicited a rise of mesor plasma concentration. The magnitude and the onset of this rise was not associated and dependent on the physiological state of animals. The mesor values of plasma prolactin concentration in all groups of animals except prooestrous ewes on the day following stimulation returned to their prestressing levels. Repeated and prolonged stimuli disturbed circadian rhythm of prolactin secretion existing only in prooestrous ewes, i.e. they caused its disappearance on the days during stimulation and its reappearance on the day following stimulation but at an accelerated and inverse time of the day to that found before stimulation. The induced rise of plasma prolactin concentration in anoestrous and pregnant ewes on the days during electric stimulation showed, according to Cosinor analysis, all features characteristic for acrophase of the circadian rhythm. This phenomenon may be considered as induced acrophase in stress condition.

Changes in circadian rhythm and suppression of the plasma cortisol level after prolonged stress in the sheep.

Diurnal variations in the plasma cortisol level were studied in anoestrous, pro-oestrous and pregnant ewes subjected to weak electric stimulation of the fore-limbs 9 h daily for 3 consecutive days. In non-pregnant ewes the cortisol level rose on each of the 3 days when the stimulation was applied and then decreased on the day following the stimulation. A similar decrease in plasma cortisol concentrations in pregnant ewes appeared on the second day of footshocking. The acrophase of the circadian rhythm on electrostimulation days was synchronous with the time of application of footshocks; therefore, in stimulated ewes it was significantly accelerated compared to the prestimulatory day. A decrease in the plasma cortisol level in pro-oestrous and pregnant ewes was accompanied by disappearance of its normal rhythmicity. Since a normal plasma cortisol response to exogenous corticotrophin was noted after 3 days of footshocking it seems unlikely that the decrease in the cortisol level after prolonged stress was caused by exhaustion of the adrenal cortex. Some central mechanisms which could account for the biphasic changes in the plasma cortisol level and for disturbances of the hormone diurnal rhythmicity under conditions of prolonged stress are discussed.