Restricting feeding to the active phase in middle-aged mice attenuates adverse metabolic effects of a high-fat diet.

Time-restricted feeding ameliorates the deleterious effects of a high-fat diet on body weight and metabolism in young adult mice. Because obesity is highly prevalent in the middle-aged population, this study tested the hypothesis that time-restricted feeding alleviates the adverse effects of a high-fat diet in male middle-aged (12months) mice. C57BL6/J mice were fed one of three diets for 21-25weeks: 1) high-fat diet (60% total calories from fat) ad-libitum (HFD-AL), 2) HFD, time-restricted feeding (HFD-TRF), and 3) low-fat diet (10% total calories from fat) ad-libitum (LFD-AL) (n=15 each). HFD-TRF mice only had food access for 8h/day during their active period. HFD-TRF mice gained significantly less weight than HFD-AL mice (~20% vs 55% of initial weight, respectively). Caloric intake differed between these groups only during the first 8weeks and accounted for most but not all of their body weight difference during this time. TRF of a HFD lowered glucose tolerance in terms of incremental area under the curve (iAUC) (p<0.02) to that of LFD-AL mice. TRF of a HFD lowered liver weight (p<0.0001), but not retroperitoneal or epididymal fat pad weight, to that of LFD-AL mice. Neither HFD-AL nor HFD-TRF had any effect on performance in the novel object recognition or object location memory tests. Circulating corticosterone levels either before or after restraint stress were not affected by diet. In conclusion, TRF without caloric restriction is an effective strategy in middle-aged mice for alleviating the negative effects of a HFD on body weight, liver weight, and glucose tolerance.

Effect of arousing stimuli on circulating corticosterone and the circadian rhythms of luteinizing hormone (LH) surges and locomotor activity in estradiol-treated ovariectomized (ovx+EB) Syrian hamsters.

In most proestrous hamsters, novel wheel exposure phase advances activity rhythms and blocks the preovulatory LH surge, which occurs 2h earlier the next day. Because wheel immobilization does not prevent these effects we hypothesized that arousal alone blocks and phase advances the LH surge. Ovariectomized (ovx) hamsters received a jugular vein cannula and estradiol benzoate (EB) or vehicle was injected sc. The next day (Day 1), at zeitgeber time (ZT) 4-5 (ZT 12 = lights off), after obtaining a blood sample, each hamster was exposed to constant darkness (DD), and either remained in her home cage or was transferred to a new cage and exposed to a running wheel or a 2-hour arousal paradigm. Blood samples were obtained in dim red light and activity was recorded hourly until ~ZT 10-11 on Days 1 and 2. For the next 1-2 weeks, activity was monitored in DD. Plasma LH and corticosterone were assessed by RIA. Novel wheel exposure or arousal at ZT 4 greatly attenuated the Day 1 LH surge in ovx+EB hamsters, and phase advanced the Day 2 LH surge by about 2h. In proestrous hamsters, novel wheel exposure led to a prolonged (>2h) increase in corticosterone levels only when LH surges were blocked. Phase advances in activity rhythms were enhanced by estradiol and arousal. The results suggest that estradiol modulates the effectiveness of non-photic stimuli. The role of the increased activity of the hypothalamic-pituitary-adrenal axis associated with novel wheel-induced attenuation of LH surges in ovx+EB hamsters remains to be determined.

Novel wheel running blocks the preovulatory luteinizing hormone surge and advances the hamster circadian pacemaker.

In rodents, the preovulatory luteinizing hormone (LH) surge is timed by a circadian rhythm. We recently reported that a phenobarbital-induced delay of the estrous cycle in Syrian hamsters is associated with an approximately 2-h phase advance in both the circadian locomotor activity rhythm and the timing of the LH surge. The following study tests the hypothesis that a >2-h nonpharmacological phase advance in the circadian pacemaker that delays the estrous cycle by a day will also phase advance the LH surge by approximately 2 h. Activity rhythms were continuously monitored in regularly cycling hamsters using running wheels or infrared detectors for about 10 days prior to jugular cannulation. The next day, on proestrus, hamsters were transferred to the laboratory for 1 of 3 treatments: transfer to a "new cage" (and wheel) from zeitgeber time (ZT) 4 to 8 (with ZT12 defined as time of lights-off), or exposure to a "novel wheel" at ZT5 or ZT1. All animals were then placed in constant dark (DD). Blood samples were obtained just before onset of DD and hourly for the next 6 h, on that day and the next day for determination of plasma LH concentrations. Running activity was monitored in DD for about 10 more days. Transfer to a novel wheel at either ZT5 or ZT1 delayed the LH surge to day 2 in most hamsters, whereas exposure to a new cage did not. Only the delayed LH surges were phase advanced at least 2.5 h on average in all 3 groups. However, wheel-running activity was similarly phase advanced in all 3 groups regardless of the timing of the LH surge; thus, the phase advances in circadian activity rhythms were not associated with the 1-day delay of the LH surge. Interestingly, the number of wheel revolutions was closely associated with the 1-day delay of LH surges following exposure to a novel wheel at either ZT1 or ZT5. These results suggest that the intensity of wheel running (or an associated stimulus) plays an important role in the circadian timing mechanism for the LH surge.

Estrogen and progesterone do not activate Fos in AVPV or LHRH neurons in male rats.

In rodents, females but not males, in response to escalating levels of estrogen, express a luteinizing hormone (LH) surge that is prompted by a surge in luteinizing hormone-releasing hormone (LHRH). It cannot take place if estrogen-sensitive afferents located in the anteroventral periventricular nucleus (AVPV) are either absent or disabled. Males appear to lack the ability to exhibit an LH surge, but it is unclear what level of the CNS contributes to this dimorphic response. This study was conducted to determine whether estrogen followed by progesterone treatment (E + P) of gonadectomized males evokes Fos activation in LHRH and AVPV neurons as it does in females. The results indicated that, consistent with the males' inability to express an LH surge in response to E + P treatment, LHRH and AVPV neurons in males failed to show increased Fos activation. Examination of neuron nuclear antigen (NeuN, a neuron-specific marker), estrogen receptor (ERalpha) and progesterone receptor (PR) neurons in AVPV neurons indicated that, while essentially all the neurons of the caudal AVPV in males and females are steroid responsive, the male possessed half the number of steroid responsive neurons within the caudal AVPV (where activation of Fos is maximal in females) compared to the female. Together, these data indicate that the male lacks a substantial population of steroid receptive AVPV neurons and is unable to respond to the presence of E and P and activate either AVPV or LHRH neurons.

Oestrogen receptor-alpha and -beta immunoreactivity in gonadotropin-releasing hormone neurones after ovariectomy and chronic exposure to oestradiol.

Oestradiol exerts negative- and positive-feedback actions on luteinizing hormone (LH) secretion by modulating gonadotropin-releasing hormone (GnRH) release. Furthermore, a chronic increase in circulating oestradiol in either young ovariectomized (OVX) rats, or in middle-aged persistent oestrous (PE) rats, causes a gradual attenuation of LH surges until the positive-feedback action of oestradiol disappears. Based on these findings, and on the equivocal evidence regarding a direct action of oestradiol on GnRH neurones, we tested the hypothesis that chronic oestradiol abolishes LH surges by decreasing the proportion of GnRH neurones containing oestrogen receptor (ER)alpha or beta. Regularly cycling rats were ovariectomized, and half immediately received oestradiol. Three days, or 2 or 4 weeks later, rats were perfused at 18.00 h, and GnRH was colocalized with ERalpha or ERbeta by immunocytochemistry. ERbeta was expressed in 76% of GnRH neurones, whereas virtually no GnRH cells were immunopositive for ERalpha. The proportion of GnRH cells expressing ERalpha or beta in OVX rats was not altered by oestradiol or time after OVX, and this was the case regardless of their medial to lateral, or rostral to caudal location. The results indicate that the mechanisms for the positive-feedback action of oestradiol, and the loss of LH surges in OVX rats after chronic oestradiol, are not mediated by changes in the proportion of oestrogen-receptor containing GnRH neurones.

Chronic elevation of estradiol in young ovariectomized rats causes aging-like loss of steroid-induced luteinizing hormone surges.

This study was designed to test the hypothesis that the loss of LH surges in response to the stimulatory actions of estradiol and progesterone in middle-aged, persistent-estrous (PE) rats may be caused by chronic elevations in circulating estradiol. Five groups of regularly cycling young rats received an s.c. estradiol implant immediately after ovariectomy (Day 0). For determination of LH surges, blood samples were collected hourly between 1200-1900 h from each of the five groups at one of the following times: 3 days, or 1, 2, 4, or 8 wk later. On the next day, either progesterone (0.5 mg/100 g BW) or corn oil was injected s.c. at 1200 h, and samples were obtained as before. Incidence and amplitude of estradiol-induced LH surges decreased during the first 2 wk of estradiol treatment, after which no surges occurred. Progesterone enhanced the incidence and amplitude of estradiol-induced LH surges thus delaying their disappearance. These results support our hypothesis and demonstrate that the stimulatory actions of estradiol and progesterone on the LH surge sequentially diminish with time after exposure to estradiol in young rats. Thus, young rats chronically treated with estradiol may be a useful model for studying the mechanisms whereby LH surges are abolished in middle age during the hyperestrogenic state of PE.

Multiple-format sessions for teaching endocrine physiology.

The University of Kentucky medical curriculum was revised in 1994 to implement a more interactive approach. The Endocrine Physiology section of the new physiology course, Human Function, was modified from its former daily lecture and weekly laboratory format to eight daily 3 1/2-h sessions. Each session is composed of four components: a didactic lecture, a whole class discussion session, a quiz, and a patient presentation. These components are presented in a staggered format over the course of 2 days, i.e., the lecture is presented on the first day, and the remaining three components take place on the second day. This allows students to assimilate the new lecture material before participating in the discussion session, quiz, and patient presentation, which are more interactive. This format has been received favorably by the students because of its variety, and it is easier to keep up with the material.

Suppression of tonic luteinizing hormone secretion and norepinephrine release near the GnRH neurons by estradiol in ovariectomized rats.

One of the major neurotransmitters that controls pulsatile luteinizing hormone (LH) secretion is norepinephrine (NE). NE pulses detected in the median eminence of ovariectomized rhesus monkeys are highly correlated with both GnRH and LH pulses. In contrast, previous reports suggest that this is not the case in rats, thus it remains to be determined whether NE stimulates LH release on a pulse-by-pulse basis in that species. Further, a variety of indirect evidence supports the hypothesis that in rats, estradiol exerts its negative feedback action on LH secretion in part by inhibiting noradrenergic neurotransmission that is stimulatory to LH release, but there is no direct evidence to support this hypothesis. Therefore the following study was designed to test the hypothesis that estradiol suppresses NE release in the vicinity of the GnRH neurons after ovariectomy. In addition, we examined whether episodes of NE release are correlated with LH pulses in ovariectomized rats. Blood samples and microdialysates of the diagonal band of Broca/medial preoptic area (DBB/MPOA) were collected every 5 min from 09:00 to 14:00 h from untreated or estradiol-treated (4-5 days), long-term ovariectomized (1-4 months) rats for determination of plasma LH by RIA and NE release by HPLC. The results indicate that in both untreated and estradiol-treated ovariectomized rats, LH pulses are not correlated with episodes of NE. Thus, NE may play a permissive role in the control of pulsatile LH secretion in rats. Further, estradiol treatment leads to a suppression of both plasma LH levels and NE release in the DBB/MPOA, supporting the hypothesis that a decrease in NE neurotransmission that is stimulatory to LH release mediates the negative feedback action of estradiol on tonic LH secretion.

Patterns of circulating gonadotropins and ovarian steroids during the first periovulatory period in the developing sheep.

This report provides evidence that an increment in serum gonadotropin levels occurs at puberty in the sheep and that this reflects the critical hormonal event culminating in first ovulation in this species. Blood samples were collected from 6 female lambs at 4-h intervals for a period of approximately 2 mo around the expected time of puberty (32 wk of age) until behavioral estrus was observed and ovulation was verified by assay of serum progesterone. Patterns of circulating LH, FSH, progesterone, and estradiol concentrations were characterized during the peripubertal period for each lamb. A rise in serum levels of both LH and FSH began approximately 7-10 days before the first preovulatory surge of gonadotropins. Although the increase in gonadotropin levels occurred gradually over several days, serum estradiol levels rose only during the final 40-60 h prior to the preovulatory surge of gonadotropin. Serum progesterone profiles revealed, however, that normal (14-16-day) luteal phases were induced in only 2 of 6 females as a result of the first surge. In four lambs, a short luteal phase of 2.5 days' duration occurred, which was followed by another estradiol rise and a preovulatory surge that then resulted in a full luteal phase of 14 days' duration. These data demonstrate clearly that the precipitating event at puberty in the female sheep is an increase in circulating gonadotropin levels and that the estradiol secreted from the newly stimulated follicle provides the signal for the first preovulatory surge.

Progesterone ensures full-length luteal phases during the breeding season in ewes.

To test the hypothesis that each luteal-phase increase in the serum concentration of progesterone throughout the breeding season prevents a short luteal phase in the next cycle, 22 ewes were treated with an i.v. injection of 10 micrograms gonadotrophin-releasing hormone (GnRH) agonist every 12 h for 33 days beginning on day 12 of a cycle synchronized with prostaglandin F2 alpha. Six days after the last injection of GnRH agonist, ten of the ewes were treated s.c. for 14 days with progesterone-containing silicone elastomer implants to generate luteal-phase serum concentrations. Twenty ewes stopped cycling during GnRH agonist treatment and 16 of these, eight controls and eight treated with progesterone, resumed cycling after the end of treatment. In the control ewes, oestrous cycles began 25.0 +/- 7.5 (S.E.M.) days after the end of GnRH agonist administration, a short luteal phase preceding initiation of cycles in six ewes. In contrast, all eight progesterone-treated ewes resumed cycling synchronously 22.0 +/- 0.2 days after the end of GnRH agonist treatment and all began with full-length luteal phases. These results support the hypothesis that each luteal-phase increment in the serum concentration of progesterone throughout the breeding season prevents a short luteal phase in the next cycle.

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.

Does the first LH surge of the breeding season initiate the first full-length cycle in the ewe?

To determine whether the first LH surge of the breeding season initiates a transient rise in progesterone in most ewes, serum progesterone (daily) and LH (every 4 h) concentrations were measured in samples collected from 7 ewes between 19 July and first oestrus or 8 September, whichever came first. In 6 of the 7 ewes, the first LH surge of the breeding season was followed within 5 days by a transient, 2-day rise in progesterone. Within less than 5 (N = 4), or 9 (N = 1) or 10 (N = 1) days later, a second LH surge occurred, which was similar in maximum amplitude and duration to the first surge, and which initiated the first full-length luteal phase of the breeding season. In the remaining ewe, the first LH surge of the breeding season induced an abbreviated (9 days) and insufficient (maximum progesterone, 0.94 ng/ml) luteal phase. These results demonstrate that most ewes have more than one LH surge before the first full-length luteal phase, the first surge inducing a transient rise in progesterone. Therefore, although the seasonal decrease in response to oestradiol negative feedback is sufficient for initiation of the first LH surge of the breeding season, additional endocrine mechanisms may be necessary to induce the first full-length luteal phase.

Changes in LH pulse frequency and serum progesterone concentrations during the transition to breeding season in ewes.

To characterize the changes in LH pulse frequency during the transition to breeding season. LH pulse patterns and serum progesterone profiles were determined in 8 intact ewes from mid-anoestrus to the early breeding season. Overall, 8 increases in LH pulse frequency were observed and these were restricted to 5 ewes. Of the 8 increases, 7 occurred during the 4 weeks before the first cycle, 5 of them within 1 week after a pulse frequency typical of anoestrus (0-2 per 8 h). Six of them occurred less than 1 week before either a full-length luteal phase (n = 2) or a 1-3-day increment in progesterone (n = 4). Seven of these brief progesterone increases were observed in 6 ewes, 5 of them immediately preceding the first full-length luteal phase. These results are consistent with the hypothesis that the seasonal decrease in response to oestradiol negative feedback at the beginning of the breeding season causes an increase in GnRH, and thereby LH pulse frequency. In addition, they demonstrate that the first increase in tonic LH secretion occurs in less than 1 week and, in most ewes, initiates either the first full-length cycle or a transient increase in progesterone, the latter occurring more often.

Delayed puberty in lambs chronically treated with oestradiol.

Intact female lambs were chronically treated with low levels of oestradiol by Silastic implant from 20 weeks of age. Reproductive cycles were initiated in only 33% of these lambs (3 of 9) compared to 80% of untreated females (11 of 14) by 45 weeks when the study was terminated. Moreover, in the 3 oestradiol-treated lambs which began cycles, the age at first oestrus was delayed 3 weeks (37 +/- 1 weeks of age vs 34 +/- 1 weeks of age for untreated controls). Retardation of the pubertal process was not due to absence of the pubertal rise in circulating LH. At about 32 weeks of age, chronic oestradiol treatment was no longer able to suppress tonic LH secretion and serum LH increased in intact, oestradiol-treated lambs. These results indicate that a maturational decrease in responsiveness to oestradiol inhibition of tonic LH secretion can be demonstrated in the intact female, as in the ovariectomized female. However, chronic oestradiol suppression of prepubertal LH secretion also delays onset of reproductive cycles. This finding raises the possibility that low tonic LH secretion, presumably in the form of slow pulses, is necessary for development or maintenance of ovarian function before puberty. In the absence of LH during the last part of sexual maturation, the ability of the ovary to respond to the high frequency LH pulses during the pubertal gonadotrophin rise may be delayed.

Importance of short luteal phases in the endocrine mechanism controlling initiation of estrous cycles in anestrous ewes.

A transient increase in serum progesterone concentrations (to 1 ng/ml for 1-2 days) is observed in the majority of ewes before the first estrous cycle of the breeding season. To determine whether such a brief antecedent rise in progesterone ensures initiation of a full-length cycle by the next LH surge, synthetic GnRH was administered for 3 days to 24 anestrous ewes in a pulsatile fashion designed to mimic the pattern of LH secretion during the preovulatory period of the breeding season. Six ewes received no further treatment, and 6 ewes were treated sc with Silastic implants containing progesterone for 3 days before injecting GnRH. The remaining 12 ewes were treated with additional injections of GnRH every 4 h for the next 5 days. Four of these ewes received a second increase in GnRH pulse frequency, every 2 h and hourly on the subsequent 2 days. An LH surge was stimulated by each regimen of increasing GnRH pulse frequency in all ewes; progesterone pretreatment had no effect on its time of onset, duration, or amplitude. The LH surges induced full-length luteal phases in 10 of 10 ewes when preceded by either an exogenous (n = 6) or an endogenous (n = 4) progesterone increment, but in only 8 of 18 ewes not pretreated with progesterone. These results indicate that a transient increase in progesterone ensures that an ensuing LH surge will initiate an estrous cycle and suggest that progesterone may play an important role in the endocrine mechanisms governing transitions from acyclic to cyclic states.

Changes in patterns of luteinizing hormone secretion before and after the first ovulation in the postpartum mare.

To determine whether luteinizing hormone (LH) secretion during the first estrous cycle postpartum is characterized by pulsatile release, circulating LH concentrations were measured in 8 postpartum mares, 4 of which had been treated with 150 mg progesterone and 10 mg estradiol daily for 20 days after foaling to delay ovulation. Blood samples were collected every 15 min for 8 h on 4 occasions: 3 times during the follicular phase (Days 2-4, 5-7, and 8-11 after either foaling or end of steroid treatment), and once during the luteal phase (Days 5-8 after ovulation). Ovulation occurred in 4 mares 13.2 +/- 0.6 days postpartum and in 3 of 4 mares 12.0 +/- 1.1 days post-treatment. Before ovulation, low-amplitude LH pulses (approximately 1 ng/ml) were observed in 3 mares; such LH pulses occurred irregularly (1-2/8 h) and were unrelated to mean circulating LH levels, which gradually increased from less than 1 ng/ml at foaling or end of steroid treatment to maximum levels (12.3 ng/ml) within 48 h after ovulation. In contrast, 1-3 high-amplitude LH pulses (3.7 +/- 0.7 ng/ml) were observed in 6 of 7 mares during an 8-h period of the luteal phase. The results suggest that in postpartum mares LH release is pulsatile during the luteal phase of the estrous cycle, whereas before ovulation LH pulses cannot be readily identified.

Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency?

To test the hypothesis that the anestrous increase in estradiol negative feedback prevents estrous cycles by suppressing hypothalamic gonadotropin-releasing hormone (GnRH) pulse frequency, a variety of regimens of increasing GnRH pulse frequency were administered to anestrous ewes for 3 days. A luteinizing hormone (LH) surge was induced in 45 of 46 ewes regardless of amplitude or frequency of GnRH pulses, but only 19 had luteal phases. Estradiol administration induced LH surges in 6 of 6 ewes, only 3 having luteal phases. Anestrous luteal phase progesterone profiles were similar in incidence, time course, and amplitude to those of the first luteal phases of the breeding season, which in turn had lower progesterone maxima than late breeding season luteal phases. In the remaining ewes, progesterone increased briefly or not at all, the increases being similar to the transient rises in progesterone occurring in most ewes at the onset of the breeding season. These results demonstrate that increasing GnRH pulse frequency induces LH surges in anestrus and that the subsequent events are similar to those at the beginning of the breeding season. Finally, they support the hypothesis that the negative feedback action of estradiol prevents cycles in anestrus by suppressing the frequency of the hypothalamic pulse generator.

Can the transition into anoestrus in the ewe be accounted for solely by insufficient tonic LH secretion?

It has been proposed that a seasonal increase in oestradiol negative feedback elicits anoestrus by preventing a key step in the preovulatory sequence of endocrine events, namely a sustained increase in tonic LH secretion. In the present study we compared the patterns of serum LH, FSH, oestradiol and progesterone after regression of the last corpus luteum of the breeding season, with their respective patterns during an ovulatory cycle in the late breeding season (samples obtained every 4 h from eight ewes). After regression of the last corpus luteum of the breeding season, serum LH and oestradiol showed distinct deviations from their respective late breeding season patterns. The rise in tonic LH secretion was curtailed. Further, there were no marked increases in oestradiol, despite a distinct, although brief, tonic LH rise; thus there were no gonadotrophin surges. If the hypothesis that the transition into anoestrus is caused solely by insufficient tonic LH secretion were correct, the brief increase in LH should have induced a transient rise in oestradiol. Since this was not the case, these results suggest that a decreased ovarian response to LH may also contribute to the termination of oestrous cyclicity at the transition to anoestrus.

Changes in pulsatile LH secretion and the positive and negative feedback actions of oestradiol after administration of a GnRH agonist in the ovariectomized ewe.

Administration of a GnRH agonist (5 micrograms) every 12 h to long-term ovariectomized ewes for 5 or 10 days during the breeding season suppressed mean LH levels from around 6 to 1 ng/ml on Days 1 and 4 after treatment; on Day 1 after treatment LH pulse frequency and amplitude were lower than pretreatment values. On Day 4 after treatment LH pulse frequency was restored to pretreatment levels (1 per h) whereas LH pulse amplitude had only slightly increased from 0.5 to 1 ng/ml, a value 25% of that before treatment. This increase in amplitude was greater the shorter the duration of treatment. Ovariectomized ewes treated with the agonist for 5 days exhibited both negative and positive feedback actions after implantation of a capsule containing oestradiol; however, compared to control ewes treated with oestradiol only, the positive and negative feedback actions of oestradiol were blunted. These results suggest that the recovery of tonic LH concentrations after GnRH agonist-induced suppression is limited primarily by changes in LH pulse amplitude. The results also demonstrate that the feedback actions of oestradiol are attenuated, but not blocked, by GnRH agonist treatment.