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I. J. Clarke

Summary. Pregnant ewes were implanted with 1 g testosterone between Days 30–80, 50–100, 70–120 or 90–140 of gestation. Treatments which began on Days 30, 50 or 70 resulted in the birth of androgenized females which failed to show regular oestrous cycles in adult life, but which exhibited patterns of male-like behaviour. This was most marked in the Day 50–100 and 70–120 groups, whereas complete masculinization of the external genitalia was confined to the Day 30–80 group. Animals in the Day 90–140 group had regular oestrous cycles although they showed slight enhancement of masculine behaviour compared to the control ewes.

These results demonstrate that androgenization involves both a suppression of female behavioural patterns, and the development of male patterns; these are not mutually exclusive.

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I. J. Clarke and R. J. Scaramuzzi

Summary. The behavioural and endocrine responses to single injections of 50 or 500 μg oestradiol-17β or 5 mg testosterone were recorded in spayed (control) ewes and in spayed ewes exposed to testosterone between Days 30 and 80 or Days 50 and 100 of prenatal life, The control ewes showed oestrus after injections on 17/18 occasions. The androgenized ewes showed poorer oestrous responses to each hormone although rams showed interest in the ewes. Masculine sexual and aggressive behaviour was shown by the androgenized ewes given either steroid. Both steroids caused a reduction in the plasma LH levels of all the ewes (negative feedback), followed by a preovulatory-type surge (positive feedback). The peak LH values were significantly lower (P < 0·05) in the Day 50–100 androgenized ewes than in the controls.

It is concluded that prenatal androgenization causes a qualitative shift in the sexual behaviour of ewes from the female type to the male type and affects the sensitivity of the brain to 'positive feedback' by steroids.

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I. J. Clarke and J. T. Cummins

Summary. We have measured the size of the releasable pools of LH and FSH in the pituitary glands of ovariectomized ewes in which the pituitary was isolated surgically from the hypothalamus. The ewes were given GnRH pulses (250 ng) every hour (N = 3) or every 2 h (N = 3) for 1 week and then given a high dose GnRH infusion (0·5 μg/min) for 4 h. Blood samples were collected to characterize the LH and FSH responses to the GnRH pulses and infusion. The LH, but not FSH, responses to the individual GnRH pulses were pulsatile and the amplitudes of the LH pulses were greater in the sheep receiving pulses every 2 h. The sheep receiving hourly pulses showed lower LH responses to the high-dose infusions than did the sheep receiving pulses every 2 h.

These data indicate a relationship between the amplitude of LH pulses and the size of the releasable pool of LH in the pituitary gland. As the frequency of GnRH pulses is decreased the amplitude of the LH responses is increased in direct proportion to the size of the releasable LH pool.

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I. J. Clarke, J.W. Funder and J. K. Findlay

Summary. Ovariectomized ewes were given a single injection (i.v.) of 100 μg oestradiol-17β. Nuclear oestrogen receptor values in the pituitary, as a function of total receptor concentrations, were 3·0 ± 1·0% in controls, 56·0 ± 5·4% at 1 h (P < 0·001) and 6·5 ± 2·1% at 6 h (P > 0·05) after oestradiol injection. There was a fall in plasma LH values from 5·7 ± 1·0 (preinjection) to 2·1 ± 0·3 ng/ml (P < 0·01) 4–6 h after oestradiol. At 13–21 h after injection plasma levels increased to 37 ± 8 ng/ml (P < 0·001). Plasma FSH levels declined from 840 ± 18 to 506 ± 48 ng/ml after 20–22 h (P < 0·001). Plasma prolactin concentrations fell from 90±16 ng/ml before injection to 29 ± 9 ng/ml at 1 h (P > 0·05), and then rose to a maximum of 1537 ± 24 ng/ml (P < 0·001) 11–14 h later. These results show that transient nuclear compartmentalization of oestrogen receptors after a bolus injection of oestradiol was associated with the feedback effects of oestradiol on LH, FSH and prolactin release.

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R. J. E. Horton, J. T. Cummins and I. J. Clarke

Summary. Ewes were sampled during the mid-late luteal phase of the oestrous cycle. Hypophysial portal and jugular venous blood samples were collected at 5–10 min intervals for a minimum of 3 h, before i.v. infusions of saline (12 ml/h; N = 6) or naloxone (40 mg/h; N = 6) for 2 h. During the 2-h saline infusion 2/6 sheep exhibited a GnRH/LH pulse; 3/6 saline infused ewes did not show a pulse during the 6–8-h portal blood sampling period. In contrast, large amplitude GnRH/LH pulses were observed during naloxone treatment in 5/6 ewes. The mean (±s.e.m.) amplitude of the LH secretory episodes during the naloxone infusion (1·07 ± 0·11 ng/ml) was significantly (P < 0·05) greater than that before the infusion in the same sheep (0·54 ± 0·15 ng/ml). Naloxone significantly (P < 0·005) increased the mean GnRH pulse amplitude in the 5/6 responding ewes from a pre-infusion value of 0·99 ± 0·22 pg/min to 4·39 ± 1·10 pg/min during infusion. This episodic GnRH secretory rate during naloxone treatment was also significantly (P < 0·05) greater than in the saline-infused sheep (1·53 ± 0·28 pg/min). Plasma FSH and prolactin concentrations did not change in response to the opiate antagonist.

Perturbation of the endogenous opioid peptide system in the ewe by naloxone therefore increases the secretion of hypothalamic GnRH into the hypophysial portal vasculature. The response is characterized by a large-amplitude GnRH pulse which, in turn, causes a large-amplitude pulse of LH to be released by the pituitary gland.

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J. K. Findlay, D. M. Robertson and I. J. Clarke

Summary. Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of M r 31 000. Various doses (0·25, 0·5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1·0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420·4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of M r 31 000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.

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H. M. Fraser, I. J. Clarke and A. S. McNeilly

Summary. Endogenous LH-RH in ewes was inhibited by active immunization or by injection of LH-RH antiserum. Plasma levels of LH and FSH were elevated in 3 ovariectomized control ewes but low in 3 LH-RH immunized ovariectomized ewes. Oestradiol benzoate (50 μg i.m.) caused a marked rise in LH concentrations in control ewes but not in the immunized ewes. In the immunized ewes the low plasma levels of FSH decreased even further 8–36 h after injection of oestrogen, indicating a direct inhibitory action of the steroid on the pituitary. Both groups responded to the oestrogen injection by a rise in plasma levels of prolactin and by exhibiting normal oestrous behaviour.

When the control ewes were again challenged with oestradiol benzoate and, after 10 h, given an i.v. injection of 75 ml antiserum to LH-RH, the LH surge was abolished in one animal and reduced in another. These experiments indicate that the continued presence of LH-RH is necessary for the occurrence of the oestrogen-induced LH surge in the ewe.

Administration of a stimulatory analogue of LH-RH released LH and FSH in control and immunized ewes but the responsiveness to further injections at intervals of 3 h decreased, particularly for FSH.

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R. C. Fry, I. J. Clarke and L. P. Cahill

Summary. Ewes were unilaterally ovariectomized and/or hypophysectomized and treated with PMSG and hCG. For a given gonadotrophin treatment the ovulation rate per ewe was maintained, i.e. the ovulation rate of the remaining ovary was significantly increased (P < 0·05), after the removal of one ovary in hypophysectomized and in pituitary-intact ewes. It is concluded that compensation of ovulation rate in the remaining ovary after unilateral ovariectomy in the sheep may be independent of feedback from the ovary and the release of gonadotrophins from the pituitary gland.

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M. I. Hossain, C. S. Lee, I. J. Clarke and J. D. O'Shea

Summary. Ovarian and luteal blood flow rates were studied using radioactive microspheres in guinea-pigs between Day 6 of the oestrous cycle and Day 1 of the following cycle. Peripheral plasma progesterone levels were measured by radioimmunoassay on the same days of the oestrous cycle.

Ovarian blood flow was greatest between Days 9 and 12 and had fallen by Day 16 both in absolute (ml·min−1) and relative (ml·min−1·g−1) terms. Luteal weight and blood flow were also greatest between Days 9 and 12 and had fallen sharply by Day 16. The highest mean (±s.d.) luteal flows measured were 0·10 ± 0·04 ml · min−1 per corpus luteum, and 24·26 ± 9·3 ml · min−1 · g−1 luteal tissue on Day 10 of the cycle. Mean peripheral plasma progesterone levels reached a maximum of 3·66 ± 1·1 ng/ml at Day 12 of the cycle and fell thereafter, reaching 0·74 ± 0·5 ng/ml by Day 1 of the following cycle. Plasma progesterone levels declined significantly between Days 12 and 14 of the cycle, whereas no significant drop in luteal blood flow was demonstrable until after Day 14. These data do not support the idea that declining luteal blood flow is an initiating mechanism in luteal regression in the guinea-pig.

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I. J. Clarke, J. K. Findlay, J. T. Cummins and W. J. Ewens

Summary. Ovariectomized ewes were given 2 ml s.c. injections of ovine follicular fluid (oFF) (N = 3) or serum (N = 3) and blood samples were collected each day for 3 days. Follicular fluid caused a significant (P < 0·005) reduction in FSH within 1 day, but did not affect mean LH values. Two groups of 3 ewes were treated as above but sampled intensively (each 10 min for 6h) on Days 1 (before treatment) and 4; mean plasma FSH concentration and plasma LH pulse frequency and amplitude were ascertained. Significant (P < 0·005) reduction of FSH concentration was seen in the oFF-treated ewes. A non-specific reduction in LH pulse amplitude, but not pulse frequency, was noted in the control ewes. This experiment was repeated with 2 groups of 4 ewes that were conditioned to the experimental environment and effects on LH secretion were not observed in the controls given serum. Treatment with oFF caused a 70% reduction (P < 0·005) in plasma FSH and a small (30%) but significant (P < 0·005) reduction in mean LH concentrations. The latter was probably associated with a reduction in LH pulse amplitude in 3/4 animals (N.S.) with no change in LH pulse frequency. Treatment with oFF, as in Exp. 1, caused a 95% reduction in FSH values and significant (P < 0·01) reduction (32%) of LH pulse amplitude in ovariectomized ewes that had been subjected to hypothalamo-pituitary disconnection and in which gonadotrophin secretion was reinstated with pulses of 250 ng GnRH every 2 h.

These results suggest that proteins from the sheep follicular fluid, including inhibin, act at the pituitary level to inhibit FSH secretion and may have some effects on LH pulse amplitude.