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I. J. Clarke


Anoestrous ewes were studied to determine the pattern of secretion of gonadotrophin-releasing hormone (GnRH) in the resting state and following a single i.m. injection of 50 μg oestradiol benzoate. In three out of four untreated ewes, two or three GnRH pulses were observed over a 6-h sampling period. In the fourth sheep the GnRH pulse frequency was higher (six pulses/6 h), but GnRH pulse amplitudes were lower. Following oestrogen treatment, GnRH pulses continued until the occurrence of an LH surge 12 h later. In five out of six sheep sampled during the oestrogen-induced LH surge a marked rise in GnRH secretion was seen. In the sixth ewe a large pulse of GnRH was seen at the start of the LH surge followed by increased GnRH secretion.

It is concluded that GnRH pulse frequency is lower, generally, during anoestrus than during the mating season, and that oestrogen treatment of anoestrous ewes causes a surge in GnRH secretion unlike that seen in similarly treated ovariectomized ewes or the natural cyclic preovulatory changes in GnRH secretion.

J. Endocr. (1988) 117, 355–360

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I. J. Clarke

The effects of tamoxifen on peripheral plasma concentrations of gonadotrophins were studied in ovariectomized ewes. First, ovariectomized ewes were injected (i.m.) with 10 mg tamoxifen citrate/day for 4 days which caused a significant reduction in plasma LH concentrations within 4 days and plasma FSH concentrations within 1 day of the commencement of treatment. Further groups of ovariectomized ewes were then injected (i.m.) with two injections of 10 mg tamoxifen citrate 6 h apart or 20 μg oestradiol benzoate (OB) or tamoxifen citrate plus OB or oil. Tamoxifen treatment caused a reduction in plasma LH and FSH concentrations within 6 h. In four of our ewes receiving OB, a surge in LH secretion was observed; a similar response was observed in two out of four ewes given the combination of tamoxifen citrate and OB. No LH surge was seen in ovariectomized ewes given tamoxifen alone.

These results show that tamoxifen reduces plasma gonadotrophin levels in ovariectomized ewes suggesting it is an oestrogen agonist in the sheep pituitary gland. A partial oestrogen antagonist action of tamoxifen is similarly suggested by its ability to block the oestrogen-induced LH surge in some ovariectomized ewes. Since tamoxifen consistently lowers plasma gonadotrophin levels in ovariectomized ewes this could result from action via oestrogen receptors or by central nervous system, non-oestrogen receptor-mediated effects.

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I J Clarke


Experiments were performed to test the hypothesis that there is a negative feedback 'clamp' of ovarian hormones on the hypothalamus and pituitary gland during the follicular phase of the oestrous cycle that limits the secretion of GnRH and LH. GnRH secretion was monitored by sampling the hypophysial portal blood of ewes during the luteal phase of the oestrous cycle and either 24 h or 48 h after the induction of luteolysis by the injection of cloprostenol, a prostaglandin analogue. There was an increase in GnRH pulse frequency in the transition from the luteal to the follicular phase of the cycle. A reduction in the amplitude of GnRH pulses did not occur until 48 h after cloprostenol, suggestive of negative feedback at the level of the hypothalamus that is more profound in the latter part of the follicular phase.

The responsivity of the pituitary gland to GnRH was monitored in ewes during the luteal phase of the oestrous cycle and 24 h or 48 h after cloprostenol. Injections of 250 ng or 1000 ng GnRH were given (i.v.) to ewes that had been anaesthetised to suppress endogenous secretion of GnRH and LH. Using the lower dose, the responses 48 h after cloprostenol were not significantly different from those in the luteal phase. With the higher dose of GnRH, a significant (P<0·05) increase in mean responsivity was seen 48 h after cloprostenol. There was, however, a marked variation in response, with some ewes showing profound increases in LH secretion in response to GnRH and others showing responses that were similar to those obtained during the luteal phase of the cycle. These data are interpreted to mean that the secretion of LH is 'clamped' during the follicular phase of the oestrous cycle and the 'clamp' is only released near the time of the preovulatory LH surge.

To test whether or not a rise in GnRH input to the pituitary gland could over-ride the 'clamp' on the pituitary secretion of LH in the late follicular phase of the cycle, sheep were treated 40 h after cloprostenol with either a bolus injection of 500 ng GnRH or four pulses of 125 ng GnRH given at 10-min intervals. These treatments caused small elevations in LH secretion but did not always cause preovulatory LH surges. In some cases, a small rise in LH secretion was induced by GnRH treatments and levels of LH in plasma returned to baseline with the preovulatory LH surge occurring a few hours later. In one clear case, a bolus injection of GnRH induced an LH surge. The overall data from the GnRH-treated groups, however, indicated a significant delay in the onset of the LH surge which may have been due to perturbation of the subcellular mechanisms in the gonadotrophs. These data were interpreted to mean that the secretion of LH from the pituitary gland is inhibited up to very soon before the onset of the LH surge. The inhibitory factor could be oestrogen but could also be some other pituitary feedback hormone such as gonadotrophin surge-attenuating factor.

It is concluded that the increase in the secretion of GnRH at the time of the onset of the LH surge is closely linked to an increase in the responsivity of the gonadotrophs to GnRH. The latter is not caused by the increase in the secretion of GnRH.

Journal of Endocrinology (1995) 145, 271–282

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R. J. E. Horton and I. J. Clarke


To determine whether opioid mechanisms modulate the positive feedback effect of oestrogen on LH secretion, anoestrous ewes were given a single injection of 50 μg oestradiol benzoate (OB), followed by infusions of morphine or naloxone. All sheep were injected i.m. with 50 μg OB at 00.00 h. In experiment 1, sheep were given i.v. infusions of the following: group 1, 12 ml saline/h from 09.00 to 15.00 h (n=12); group 2, 40 mg naloxone/h from 09.00 to 12.00 h (n = 5); group 3, 40 mg naloxone/h from 10.00 to 14.00 h (n = 5); group 4, 10 mg morphine/h from 09.00 to 15.00 h (n = 5); and group 5, 20 mg morphine/h from 09.00 to 15.00 h (n = 5). Jugular blood samples were taken at 30-min intervals to monitor LH surges, which commenced 13.0 ± 0·6 h after injection of OB in control (OB plus saline) ewes. The infusions of naloxone or morphine did not affect the timing or magnitude of the oestrogen-induced LH surge.

To examine the possibility that opioidergic regulation of the LH surge occurred earlier than the infusion regimens in experiment 1, sheep were infused from the time of the OB injection (00.00 h) until 15.00 h. In this experiment (experiment 2), sheep were given i.v. infusions of the following: group 1, 4·2 ml saline/h (n=5); group 2, 20 mg naloxone/h (n=5); and group 3, 20 mg morphine/h (n=5). As in experiment 1, treatment with neither the opioid agonist or antagonist was able to alter the positive feedback response of OB.

These results suggest that neither the timing of the LH surge or the peak concentrations of LH achieved in plasma are influenced by opiates in this model. This suggests that in contrast to the rat, the mechanisms responsible for generating the oestrogen-induced preovulatory-like LH surge in the anoestrous ewe do not involve any endogenous opioid peptide mechanisms.

J. Endocr. (1988) 119, 89–93

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M.R.C. Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EHI 2QW

(Received 31 October 1977)

Exposure to testosterone during development masculinizes the genitalia and behaviour of ewes (Clarke, Scaramuzzi & Short, 1976a; Clarke, 1977) and causes ovulatory failure (Clarke, Scaramuzzi & Short, 1977). Androgenized ewes do not release luteinizing hormone (LH) after oestrogen treatment during anoestrus (Clarke, Scaramuzzi & Short, 1976b). These experiments were performed to determine the site of action (hypothalamus or anterior pituitary gland) of prenatally administered androgens in blocking the preovulatory release of LH.

Finnish Landrace × Dorset Horn ewes were used in their second breeding season after exposure to testosterone between days 30 and 80 (D30–80, n = 6), 50 and 100 (D50–100, n = 1), 70 and 120 (D70–120, n = 6) or 90 and 140 (D90–140, n = 5) of prenatal life (Clarke et al. 1976a). Eight normal ewes served as controls. To facilitate

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The binding of three catechol oestrogens, 2-OH-oestradiol-17β, 4-OH-oestrone and 2-OH-oestrone, to the ovine pituitary oestrogen receptor was measured in vitro to establish doses for the assessment of the effects of catechol oestrogens in vivo. Relative to oestradiol (100%) the compounds had receptor affinities of 30, 20 and 5% respectively. A dose of oestradiol sufficient to cause negative-feedback effects on the secretion of LH and FSH in ovariectomized ewes was established by intracarotid (i.c.) injections of 0·625–5·0 μg/dose (n = 3), and by measuring plasma levels of gonadotrophins in jugular venous samples taken at intervals of 20 min from 3 h before until 4 h after injection. A dose-dependent relationship (r = 0·88, P<0·001) was found for oestradiol and plasma LH levels. Plasma FSH was slightly (12–25%) but significantly (P<0·05) reduced by doses of 1·25–5·0 μg oestradiol, but no dose–response relationship was observed.

Ovariectomized ewes (n = 4/group) were given 2·5 μg oestradiol (i.c.) simultaneously with 83 μg 2-OH-oestradiol, 125 μg 4-OH-oestrone or 500 μg 2-OH-oestrone. These doses of catechol oestrogens were chosen as being ten times that of oestradiol, with the relative affinities for oestrogen receptor taken into account. Concurrent administration of such doses of catechol oestrogens had no effect on the negative-feedback action of oestradiol in vivo. We have concluded that catechol oestrogens in the circulation probably do not modulate the action of oestradiol on release of LH or FSH; this does not preclude a possible role for them as locally produced regulators of oestrogen action.

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D. J. Phillips and I. J. Clarke


Glucocorticoids have been found to inhibit reproductive function in most domestic species studied but, in the ewe, preliminary reports suggest that glucocorticoids may have little or no effect. This study investigated the effects of the synthetic glucocorticoid dexamethasone on oestrus and ovulation rate in ewes during the breeding season and gonadotrophin secretion in the breeding and non-breeding seasons. In cyclic ewes, dexamethasone treatment at rates of up to 2 mg/day did not affect the natural or pregnant mare serum gonadotrophin-stimulated ovulation rate, or the timing and incidence of behavioural oestrus (P>0·05). Dexamethasone administration (2 mg/day) had no effect on LH secretion or the plasma LH response to a 1 μg injection of gonadotrophin-releasing hormone (GnRH) in ovariectomized ewes in the breeding and non-breeding seasons, and did not compromise the inhibition of plasma LH levels during chronic treatment with oestrogen. Similarly, dexamethasone had no effect on plasma FSH concentrations, but significantly (P<0·05) reduced the plasma FSH response to a 1 μg GnRH injection during chronic negative treatment with oestrogen in ovariectomized ewes. Collectively, these data show that in these experiments dexamethasone did not significantly modify reproductive function in the ewe, a finding that is in contrast to that found in other domestic species.

Journal of Endocrinology (1990) 126, 289–295

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P. J. Wright and I. J. Clarke


The nature of the gonadotrophin-releasing hormone (GnRH) stimulus of the pituitary necessary for the oestrogen-induced plasma LH surge was studied in ovariectomized ewes. The sheep were treated with oestradiol benzoate (50 μg i.m.) at 0 h, and the hypothalamic contribution to the LH surge was blocked by pentobarbitone anaesthesia over the time during which the surge was expected (11–31 h). Pituitary responsiveness to exogenous GnRH (100 ng) administered i.v. in a pulsatile mode (once per hour or once per 20 min) over the period 15–30 h was assessed from plasma concentrations of LH. Neither of the GnRH treatments induced patterns of LH secretion similar to those seen in conscious ovariectomized ewes given oestrogen only. Plasma LH secretion in response to hourly GnRH pulses was less (P<0·01) than that associated with oestrogen-induced plasma LH surges in conscious control ewes. With pulses of GnRH administered every 20 min the amount of LH released was greater (P<0·05) than that in oestrogen-treated conscious control ewes. In contrast to the single surge induced by oestradiol in conscious ewes, GnRH pulses given every 20 min elicited phasic patterns of LH secretion consisting of two or three distinct surges. The failure of GnRH treatment to elicit an LH surge similar to an oestrogen-induced surge could reflect inappropriate GnRH treatment regimens, and/or inadequate priming of the pituitary with GnRH after induction of anaesthesia but before GnRH treatment.

J. Endocr. (1988) 116, 143–148

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I. J. Clarke and J. T. Cummins


A series of experiments was conducted to ascertain the significance of 'small' pulses of gonadotrophin-releasing hormone (GnRH). In the first experiment, ovariectomized hypothalamo-pituitary disconnected (HPD) ewes were given 250 ng pulses of GnRH every 2 h for 1 week, 25 ng pulses every 2 h for 24 h, 25 ng pulses hourly for 24 h and then alternating hourly pulses of 25 and 250 ng. During the 25 ng pulses, LH was not detectable in plasma and FSH concentrations declined after 2 days. Following the 25 ng pulses, the resumption of 250 ng pulses led to exaggerated LH responses (mean ± s.e.m. pulse amplitude 18·7 ± 1·7 vs 10·2 ± 1·2 μg/l in the first week). In a second experiment, ovariectomized–HPD ewes were maintained on 250 ng GnRH pulses every 2 h for 1 week and were then given three 25 ng pulses mid-way between the 250 ng pulses. Samples of blood were taken over three 250 ng pulses without 25 ng insertions and over three pulses with insertions. The insertion of 25 ng GnRH pulses did not cause LH pulses in their own right and did not alter the LH responses to the 250 ng pulses. In a third experiment, 50 ng GnRH pulses were inserted between the 250 ng GnRH pulses, as in experiment 2; these 50 ng pulses caused small LH pulses and led to a reduction in the response of the LH pulse amplitude to the 250 ng pulses. The 'small' LH pulses which occurred in response to 50 ng GnRH compensated for the reduced responses to the 250 ng pulses. Hence, the integrated area under the LH curve and between successive 250 ng pulses remained the same, irrespective of the 50 ng insertions.

From these data we conclude that 'small' GnRH pulses alone can sustain ongoing LH synthesis without release, leading to an accumulation of releasable LH, and that the insertion of 'small' GnRH pulses may modify the pattern of pituitary responsiveness to 'large' GnRH pulses.

J. Endocr. (1987) 113, 413–418

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I. J. Clarke and B. W. Doughton

Effects of various anaesthetics on plasma LH, FSH and prolactin levels were studied in ovariectomized ewes. In the first experimental series, conducted between June and November (late breeding season, early anoestrous season), the following treatments were given: saline (i.v.) (n = 7); single thiopentone injection (i.v.) (n = 4); induction of anaesthesia for 2 h with thiopentone (n = 5), ketamine/thiopentone mixture (n = 6), Alphathesin (n = 6) or induction with thiopentone and maintenance with halothane (n = 6). The major findings were: (1) halothane anaesthesia reduced mean plasma LH levels by preventing pulsatile secretion of LH; (2) Alphathesin had the least effect on tonic LH concentration; (3) a single thiopentone injection did not affect LH levels; (4) continuous thiopentone anaesthesia increased LH pulse amplitude; (5) plasma FSH concentration was not affected by any of the treatments; (6) ketamine/thiopentone-induced and Alphathesin-induced anaesthesia increased plasma prolactin levels.

In a second experimental series four ovariectomized ewes were anaesthetized with thiopentone for 3 h in January. In contrast to the results obtained with thiopentone in August, treatment in January reduced plasma LH pulse amplitude and mean plasma LH levels. These latter results support the hypothesis that there may be seasonal variation in responses to barbiturate anaesthesia.