LH Secretion In Ewes Research Articles

Light-induced sexually active rams provoke LH preovulatory surges and enhances LH concentrations in ewes after progestagen treatment.

The effect of the introduction of sexually active rams in spring on LH secretion in ewes after progestagen treatment has been studied. Two rams were induced into a sexually active state by exposure to 2 months of long days (16 h of light/d) from 1 January (SAR), and another 2 rams were exposed to the natural photoperiod, so that they were not sexually activated in spring (control; C). At the end of the long-day period, rams were returned to natural photoperiod conditions. Fifteen ewes synchronized in estrus by intravaginal sponges were assigned to three groups at sponge withdrawal (hour 0): SAR (n = 5), exposed to SAR rams; C (n = 5), exposed to C rams, and ISO (n = 5), kept isolated from rams. Twenty-four hours after pessary removal (hour 0), rams were introduced into the SAR and C groups. Three SAR ewes presented preovulatory LH surges; the proportion was significantly (P < 0.05) higher in this group (3/5) than in the others (C: 0/5, ISO: 0/5). SAR introduction induced a more marked (P < 0.05) increase in mean LH plasma concentrations (before rams: 1.34 ± 0.19; after rams: 6.94 ± 2.66 ng/ml) than C (before: 0.96 ± 0.29; after: 3.60 ± 1.44) or ISO (before: 1.26 ± 0.42; after: 2.14 ± 1.36) groups, and significantly (P < 0.05) higher plasma LH levels after ram introduction. In conclusion, only light-treated sexually activated rams induced LH preovulatory surges in ewes in the seasonal anestrus, when ewes are synchronized with progestagen treatment in the absence of eCG.

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

No Evidence That RFamide-Related Peptide 3 Directly Modulates LH Secretion in the Ewe.

The neuropeptide RFamide-related peptide 3 (RFRP-3) has been implicated in the control of gonadotropin secretion in both birds and mammals. However, in mammals, depending on species, sex and photoperiod, inhibitory, excitatory, or no effect of RFRP-3 on the plasma concentration of LH has been reported. In the ewe, treatment with RFRP-3 either reduced LH concentration or had no effect, and treatment with an RFRP-3 receptor antagonist (ie, RF9) resulted in increased concentration of plasma LH. To clarify these conflicting results in the present study, a set of experiments was performed in ewes. Multiple iv injections of RFRP-3 (6 × 50 μg) in ovariectomized ewes had no effect on plasma LH pulsatility. In intact ewes a bolus injection (500 μg) or an injection (250, 500, or 1000 μg) followed by a 4-hour perfusion (250, 500, or 1000 μg · h(-1)) of RFRP-3 had no effect on the LH pulse induced by kisspeptin (6.5 μg). In ovariectomized, estrogen-replaced ewes, the LH surge induced by estradiol benzoate was not modified by a 24-hour perfusion of RFRP-3 (500 μg h(-1)). Finally, although treatment with RF9 induced a robust release of LH, treatment with a more selective RFRP-3 receptor antagonist, GJ14, resulted in no evident increase of LH. In contrast to the inhibitory effect previously suggested, our data are more consistent with the concept that RFRP-3 has no direct effect on LH secretion in ewes and that RF9 effect on LH release is likely not RFRP-3 receptor mediated. Hence, RFRP-3 probably has a minor role on the control of LH secretion in the ewe.

Somatostatin Inhibition of GnRH Neuronal Activity and the Morphological Relationship between GnRH and Somatostatin Neurons in Rats

In rodents, GnRH neurons are diffusely distributed from the medial septum through to the medial preoptic area and control gonadal functions through the pituitary. The activity of GnRH neurons is regulated by a variety of bioactive substances, including the inhibitory peptide somatostatin. In the present study, we focused on somatostatin because intracerebroventricular injection of somatostatin inhibits the LH surge in rats and reduces LH secretion in ewes. Somatostatin also decreases GnRH release from rat hypothalamic slices. In mice, somatostatin is also thought to suppress GnRH neuronal activity through contact on the soma of GnRH neurons. However, similar data are missing in rats. Moreover, rat GnRH neurons receive only a few synaptic inputs. In this study, we assessed the morphological relationship between GnRH and somatostatin neurons. Confocal microscopy on the sections from the medial septum through medial preoptic area revealed about 35 close contacts per rat between the GnRH and somatostatin neuronal fibers in the organum vasculosum of the lamina terminalis region. No contact of somatostatin fibers on the GnRH neuronal somata was observed. Multicell RT-PCR for somatostatin receptor mRNA in rat GnRH neurons was also performed, which revealed moderate expression of somatostatin receptor subtypes 1-5. In addition, patch clamp experiments were carried out in acute slice preparations. Somatostatin suppressed neuronal firing in cells recorded in a cell-attached configuration and also induced whole-cell outward currents in GnRH neurons. These findings suggest that somatostatin directly inhibits the activity of rat GnRH neurons through volume transmission in the organum vasculosum of the lamina terminalis region.

Neurokinin B Acts via the Neurokinin-3 Receptor in the Retrochiasmatic Area to Stimulate Luteinizing Hormone Secretion in Sheep

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Effect of stress on the expression of GnRH and GnRH receptor (GnRH-R) genes in the preoptic area-hypothalamus and GnRH-R gene in the stalk/median eminence and anterior pituitary gland in ewes during follicular phase of the estrous cycle

The RT-PCR (reverse transcription polymerase chain reaction) technique was used to analyze GnRH mRNA and GnRH-R mRNA in the preoptic area, anterior and ventromedial hypothalamus, and GnRH-R mRNA in the stalk/median eminence and anterior pituitary gland of follicular ewes subjected to short (3 h during one day) or prolonged (5 h daily during four consecutive days) footshock stimulation. To analyze relationship between expression of GnRH and GnRH-R genes with LH secretion the blood samples were collected at 10 min intervals to determine LH levels in control and stressed animals. The concentration of GnRH mRNA increased significantly in the preoptic area, anterior and ventromedial hypothalamus of ewes subjected to short stress. The prolonged stressful stimuli significantly decreased GnRH mRNA levels in all analyzed structures. In short stressed ewes the significant augmentation of mRNA encoding GnRH-R was detected in the preoptic area, entire hypothalamus, stalk/median eminence and anterior pituitary gland. The GnRH-R mRNA was significantly reduced in all tested structures of animals subjected to prolonged footshocking except for the preoptic area, where GnRH-R mRNA did not differ from control values. The changes in GnRH mRNA and GnRH-R mRNA levels under short or prolonged stress were associated with an increase or decrease of LH concentration in blood plasma, suggesting the existence of a direct relationship between GnRH mRNA and GnRH-R mRNA expression with LH secretion. The results indicate that the expression of both GnRH gene and GnRH-R gene, as well as LH secretion in ewes during the follicular phase of the estrous cycle, are dependent upon the kind of stress. Correspondence should be addressed to M. Ciechanowska, Email: m.ciechanowska@ifzz.pan.pl

Effect of prolactin infused into the third ventricle on LH secretion in follicular-phase and ovariectomized ewes

This study tested a hypothesis that an acute enhancement of prolactin concentration within the central nervous system (CNS) would affect the LH secretion in ewes, depending on the level of endogenous estrogens in the organism. A 3-h long intracerebroventricular (icv.) infusion of ovine prolactin was made in late follicular-phase ewes, experiment 1, and in ovariectomized (OVX) ewes (experiment 2). No significant differences were found in mean LH concentrations and LH peak number before, during and after prolactin administration (50 μg/100 μl/h) in intact cyclic ewes. No diurnal rhythm in LH was detected in prolactin-infused ewes. From the two doses of prolactin used in OVX ewes (25 and 50 μg/100 μl/h) only the lower dose suppressed significantly the mean plasma LH concentration after the infusion, compared to those noted before ( P<0.01) and during ( P<0.001) prolactin treatment. Prolactin had no effect on LH pulse frequency in OVX ewes, however, a tendency to decrease in LH peak number was observed after administration of a lower dose. Plasma prolactin levels decreased significantly ( P<0.01 and P<0.001) after the icv. infusion in all groups, indicating a high degree of effectiveness for exogenous prolactin at the level of the CNS.

Effect of melatonin on daily LH secretion in intact and ovariectomized ewes during the breeding season

This study was conducted to find out whether daily LH secretion in ewes may be modulated by melatonin during the breeding season, when the secretion of both hormones is raised. Patterns of plasma LH were determined in luteal-phase ewes infused intracerebroventricularly (icv.) with Ringer–Locke solution (control) and with melatonin (100 μg/100 μl/h). Response in LH secretion to melatonin was also defined in ovariectomized (OVX) ewes without and after estradiol treatment (OVX+E 2). Basal LH concentrations by themselves did not differ significantly before, during and after both control and melatonin infusions in intact, luteal-phase ewes. However, single significant ( P<0.05) increases in LH concentration were noted during the early dark phase in the control and 1 h after start of infusion in melatonin treated ewes. In both OVX and OVX+E 2 ewes, melatonin decreased significantly ( P<0.01, P<0.05, respectively) mean plasma LH concentrations as compared to the levels noted before the infusions. In OVX+E 2 ewes, a single significant ( P<0.05) increase in LH occurred 1 h after start of melatonin treatment, similarly as in luteal-phase ewes. No significant differences in the frequencies of LH pulses before, during and after melatonin infusion were found in all treatments groups. In conclusion, melatonin may exert a modulatory effect on daily LH secretion in ewes during the breeding season, stimulating the release of this gonadotropin in the presence of estradiol feedback and inhibiting it during steroid deprivation. Thus, estradiol seems to be positively linked with the action of melatonin on reproductive activity in ewes.

Effects of Sexually Activated Rams or Ewes on Pulsatile LH Secretion in Anoestrous Sheep

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Daily GnRH and LH secretion in ewes is not modified by exogenous melatonin during seasonal anestrus

The effect of central, short-term melatonin administration on daily GnRH and LH secretion was studied in ewes during seasonal anestrus. Melatonin, in a total dose of 32 mmg and the vehicle were perfused for 4 hours into the mediobasal hypothalamus/median eminence (MBH/ME). The mean GnRH concentration during perfusion with melatonin decreased significantly (P

A note on the effect of individual housing conditions on LH secretion in ewes after exposure to a ram

To determine whether it is feasible to use the stimulus of exposure to a ram to induce sexual activity in individually housed ewes, 33 adult anoestrous ewes were randomly allocated to two treatments: Group 1, n=20, housed in individual pens, with a ram’s pen situated between every two ewes (exposed to rams through the pen’s bars) and Group 2, n=13, housed in a communal pen with five rams (full contact with the rams). The day before the introduction of rams, blood samples were collected from Group 1 ewes, for 6 h at 15 min intervals commencing 10:00 h. Rams were introduced in both groups the following day at 8:00 h. Two hours later, blood samples were collected from Group 1 ewes for 6 h at 15 min intervals. Thereafter, ewes and rams were moved to a communal pen and blood samples collected every 3 h until 10:00 h the next day. In Group 2, blood samples were collected every 3 h for 24 h starting at 10:00 h, 2 h after the rams were introduced. From the samples collected at 15 min intervals, no differences between mean LH concentrations, pulse frequencies and amplitudes before or after the introduction of rams were observed in Group 1. The percentage of ewes presenting a pre-ovulatory surge of LH in the first 24 h after the introduction of rams was similar in both groups (Group 1: 50%, 10 ewes; Group 2: 61.5%, 8 ewes). The pre-ovulatory LH surges began approximately 7 h later ( P=0.05) in Group 1 than in Group 2 (18.5±2.2 h versus 11.8±2.6 h, respectively). Mean plasma LH concentrations were significantly higher in Group 2, during the first 8 h after the introduction of rams, these differences disappeared when all the animals were moved to the communal pen. In conclusion, individual housing together with frequent blood sampling conditions do not induce a significant increase of LH secretion of anoestrous ewes after a ram effect, as compared with that in rams and ewes in communal pens. Further experiments are necessary to separate, on one hand, the effect of housing and on the other hand, the effect of sampling frequency on LH secretion after a ram effect.

Effect of cerebroventricular infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in diet-restricted ewes☆

The objective was to determine the effect of central infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in the ewe. Twenty-two ovariectomized ewes (32 wk of age) were fitted with two lateral cerebroventricular (LCV) cannulae and fed 33% of NRC requirements for 8 wk. Ewes ( n ≥ 5/group) were then infused, via LCV cannulae, with artificial cerebrospinal fluid (aCSF) or aCSF containing physiological concentrations of insulin (INS), glucose (GLU), or INS + GLU; the mass of each increasing linearly from Day 0 (mass = 0 units/h) to Day 8 (mass of INS = 80 mIU/hr and GLU = 10 mg/hr). Jugular serum was collected every 12 min for 4 hr on Days 0, 2, and 4. Ewes treated with INS or INS + GLU had greater (P < 0.06) mean concentrations of LH than aCSF treated ewes on Day 2 (13.8 ± 1.8 and 12.5 ± 1.3 > 8.0 ± 3.3 ng/ml). Furthermore, on Day 4, concentrations of LH in INS treated ewes exceeded that (P < 0.07) of aCSF treated ewes (14.8 ± 2.0 > 7.4 ± 3.0 ng/ml). Expression of NPY mRNA did not differ between treatments (P = 0.87). Leptin receptor mRNA expression was dramatically reduced (P < 0.0002) in INS+GLU versus aCSF treated ewes. These data provide evidence to suggest that insulin may be an important component of hypothalamic mechanisms regulating secretion of LH and expression of leptin receptors in undernourished ruminants.

Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season.

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 +/- 0.9 h versus 48.3 +/- 0.8 h, P < 0.02; ovariectomized model: 40.8 +/- 0.6 h versus 42.6 +/- 0.5 h, P < 0.02). Disruption of the hypothalamus-pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 +/- 4 ng ml-1 versus intact transported 22 +/- 5 ng ml-1, P < 0.02; ovariectomized model controls: 35 +/- 7 ng ml-1 versus model transported 15 +/- 2 ng ml-1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 +/- 0.3 versus 0.3 +/- 0.02 ng ml-1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 +/- 0.4 versus 1.7 +/- 0.6 pulses per 2 h). In conclusion, activation of the hypothalamus-pituitary-adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.

Reproductive response and LH secretion in ewes treated with melatonin implants and induced to ovulate with the ram effect

Abstract Two experiments were conducted to examine the effects of treating seasonally anoestrous ewes with melatonin before ram introduction on reproductive response, and on LH secretion in anoestrous ewes induced to ovulate by rams. In Experiment 1, a total of 667 ewes from three flocks involving Merino (Flock 1, N = 149), Merino entrefino (Flock 2, N = 325) and Rasa Aragonesa (Flock 3, N = 203) breeds were used. Within each flock, ewes isolated from rams since the previous lambing were assigned at random to receive melatonin implants of Regulin (75, 175 and 105 in Merino, Merino entrefino and Rasa Aragonesa flocks, respectively) or to serve as untreated controls (74 in Merino, 150 in Merino entrefino and 98 in Rasa Aragonesa flocks). Fertile rams were introduced into all flocks 5 weeks after implantation in March (Flocks 1 and 2) or April (Flock 3), and remained with the ewes for a 50 day mating period. Percentage of ewes with luteal activity at ram introduction did not differ between melatonin treated and control ewes in any flock. There were no significant differences in either the mean interval from ram introduction to lambing or the distribution of lambing. Implantation with melatonin resulted in an improvement of prolificacy in all three flocks, although this only reached statistical significance in the Merino flock (1.15 vs. 1.03 in treated and control ewes, respectively, P In Experiment 2, two trials were undertaken utilizing a total of 63 ewes. Trial 1 involved 24 mature Manchega ewes and Trial 2 involved 39 Merino ewe lambs. Half of the animals in each trial received a Regulin implant on 28 February (Trial 1) or 12 March (Trial 2) and the remaining half acted as controls. Rams were introduced 5 weeks after implantation and remained with the ewes for a 25 day period. In both trials, anoestrous ewes at ram introduction were bled at 20 min intervals for 3 h before and 5 h after ram introduction and then at 3 h intervals over the next 24 h for assessment of plasma concentrations of LH. Secretion of LH before or following introduction of rams was not affected by melatonin. Both treated and control anoestrous ewes in each trial responded to introduction of rams with an increase in the frequency of the LH pulses (P Results from this study show that: (1) melatonin implants administered during early seasonal anoestrus have the potential to improve reproductive performance in Spanish breeds of sheep, but the response is conditioned by breed, management system and environmental factors; (2) melatonin did not modify the secretion of LH in anoestrous ewes induced to ovulate by the ram effect under our experimental conditions.

FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist

The study investigated the relationship between the plasma concentration of FSH and the stimulation of preovulatory follicle growth in vivo in ewes chronically treated with the gonadotrophin-releasing hormone (GnRH) agonist buserelin (HOE 766). Welsh Mountain ewes with regular oestrous cycles were treated for 6 weeks with two discs implants placed s.c., each containing 5 mg of the agonist in a matrix of polyhydroxybutyric acid. Treatment with the agonist for 35 days produced a sustained suppression of the plasma concentration of FSH, stopped the pulsatile release of LH and prevented follicular development beyond 2.5 mm diameter. There was no difference between the total number of follicles greater than 1.0 mm diameter present in the ovaries of GnRH agonist-treated ewes and day 8 luteal phase control ewes. During the sixth week of agonist treatment ewes were infused with ovine FSH (6 micrograms NIADDK-oFSH-16/h) in the presence of only basal concentrations of LH. After 24, 48, 72 or 120 h of FSH infusion, the mean number of follicles greater than 1.0 mm diameters per ewe was not significantly different between treated and control animals. Infusion of FSH caused a time-dependent increase in (1) the number of follicles per ovary greater than 2.5 mm, (2) the mean diameter of these follicles and (3) the proportion of the large follicles which could be classified as oestrogenic (greater than 3.7 nmol oestradiol/follicle per h in vitro). Injection of human chorionic gonadotrophin (750 IU i.m.) after 120 h of FSH infusion caused the majority of these large follicles to ovulate and form apparently normal corpora lutea. These results indicate that, in the absence of pulsatile LH, FSH stimulates the growth of normal large oestrogenic follicles which, when stimulated, ovulate to produce viable corpora lutea.

Effects of domperidone and thyrotropin-releasing hormone on secretion of luteinizing hormone and prolactin during the luteal phase and following induction of luteal regression in sheep

Effects of domperidone, a peripheral dopamine receptor antagonist, on secretion of LH and prolactin were studied during the luteal phase and following administration of PGF 2α. Since hyperprolactinemia has been reported to inhibit secretion of LH in ewes, effects of thyrotropin-releasing hormone (TRH) also were examined. Ewes 8–10 days post-estrus were assigned to be treated with: 1) vehicle (n=5); 2) 0.3 mg domperidone (n=6); 3) 1.0 mg domperidone (n=6); 4) 3 μg TRH (n=6); or 5) 10μg TRH (n=6) every 4 hours for 60 hr. Luteal regression was induced with PGF 2α at 12 hr after initiation of treatments. During the luteal phase, pulses of LH were more frequent (P<.05) and the amplitudes of these were higher (P<.05) in ewes treated with domperidone or TRH than in control ewes. These changes in LH occured even though each treatment elevated markedly concentrations of prolactin in plasma. After induction of luteal regression, mean of LH and frequency of LH discharges were similar in all groups. However, in ewes treated with the 1.0 mg/4 hr dose of domperidone the pulse amplitude was greater than in the other groups (2.3 vs 1.1 ng/ml). Dose-response relationships and the magnitude of the prolactin release following domperidone or TRH varied with time. Treatments did not affect the timing of the LH surge or the increase in progesterone associated with the subsequent cycle. Based on these data we conclude that: 1) endogenous hypothalamic dopamine mediates some of the inhibitory effects of progesterone on secretion of LH and prolactin; 2) dopamine limits the extent of the usual follicular phase increase in prolactin; 3) acute hyperprolactinemia does not inhibit the secretion of LH; and 4) administration of TRH can increase the frequency and amplitude of LH pulse frequency and amplitude in sheep in the luteal phase of LH in sheep.

Changes in pulsatile LH secretion after ovariectomy in Ile-de-France ewes in two seasons.

Two experiments were conducted in Ile-de-France ewes to study changes in pulsatile LH secretion in ewes ovariectomized during anoestrus or during the midluteal phase of the oestrous cycle. In Exp. 1, blood samples were taken every 20 min for 12 h the day before ovariectomy (Day 0). After ovariectomy, samples were taken every 10 min for 6 h (10 ewes per group), on Days 1, 3, 7 and 15. In Exp. 2 samples were taken every 10 min for 6 h (10 ewes per group) on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy. Further samples were taken (5 ewes per group) at 9 and 12 months after ovariectomy. There were significant interactions between season and day of sampling for the interval between LH pulses in both experiments. LH pulse frequency increased within 1 day of ovariectomy and the increase was more rapid during the breeding season. There were clear seasonal differences in pulse frequency in Exp. 2. Compared with ewes ovariectomized in anoestrus, pulse frequency was significantly higher for ewes ovariectomized in the breeding season, from Day 7 until Day 120. Once pulse frequency had increased in ewes about the time of the normal breeding season, pulse frequency remained high and subsequent seasonal changes were greatly reduced. Pulse amplitude increased immediately after ovariectomy to reach a maximum on Day 7 and there were no differences between season of ovariectomy in the initial changes in amplitude. In Exp. 2, changes in amplitude followed changes in pulse interval and there was a significant interaction between season and day of sampling. There were no significant effects of season on nadir LH concentrations which increased throughout the duration of the experiments. These results show that, in ovariectomized ewes, LH pulse frequency observed on a given day depends on time after ovariectomy, season at the time of sampling and on previous exposure of ewes to stimulatory effects of season. The direct effects of season on LH pulse frequency and seasonal changes in sensitivity to steroid feedback may contribute to control of the breeding season and their relative contributions to the beginning and end of the breeding season may differ.