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M. Wilkinson and R. Bhanot


Ovariectomy of prepubertal rats (9 days of age) eliminates the ability of the opiate peptide FK 33-824 to inhibit LH secretion when tested 19 days later. We have investigated whether this removal of opiate inhibition would modify the LH/FSH response to stimulation with oestradiol benzoate/progesterone priming. Ovariectomy of rats during infancy (9 days after birth) amplifies the stimulatory effects of these steroids on LH/FSH secretion when tested 19 days later. This amplification was not seen in rats ovariectomized before (day 24) or after puberty (day 43) and tested 19 days later. The pituitary content of LH/FSH does not appear to contribute to this phenomenon, though increased responsiveness to injected gonadotrophin-releasing hormone (GnRH) is clearly involved; ovariectomy at day 9 is considerably more effective than ovariectomy at day 24 of life in enhancing the response to GnRH.

We conclude that infantile ovariectomy either removes, or prevents the development of, a hypothalamic inhibitory mechanism which normally modulates the responsiveness of the pituitary to stimulation with GnRH.

J. Endocr. (1985) 106, 133–139

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R. Bhanot and M. Wilkinson

We have investigated the effects of repeated flurothyl-induced seizures on reproductive function in the female rat. This treatment rapidly induced a state of pseudopregnancy in intact cyclic rats. Prolactin is clearly implicated in this response since treatment with bromocriptine readily counteracted the influence of the convulsions. The mechanism of action of repeated seizures was further characterized in experiments on ovariectomized rats. Thus, 11 daily convulsions, but not a single acute seizure, were able to inhibit the positive feedback effect of progesterone on LH and FSH release in oestrogen-primed animals. In this model also the pituitary gland response to gonadotrophin releasing hormone in vitro was significantly reduced. However, the convulsions had no effect on basal serum or basal in-vitro secretion of LH and FSH in ovariectomized or oestrogen-treated ovariectomized rats. Thus, repeated seizures modified the hypothalamo-pituitary axis in such a way as to prevent it from responding to stimulation.

Our results indicate that normal reproductive function in the female rat is very sensitive to repeated seizures and suggest that similar effects may be evident in women subjected to electroconvulsive shock therapy. The successful use of bromocriptine in reversing the influence of seizures in the rat suggests its use in man also.

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R. Bhanot and M. Wilkinson


We have attempted to clarify the physiological involvement of endogenous opiates in the steroid-mediated control of gonadotrophin release. Our studies showed that there was an acute reduction in the inhibitory effects of endogenous opiates on LH and FSH release following gonadectomy in the rat. This was indicated by a significant reduction in the ability of naloxone to stimulate serum LH/FSH levels (sampled at 15 min) in 26-day-old female rats 48 h after ovariectomy. Luteinizing hormone was highly sensitive to the inhibitory effects of the synthetic met-enkephalin analogue, FK 33-824, at this time (sampled at 90 min). An unexpected observation was that long-term absence of gonadal steroids also disrupted the ability of exogenous opiates, FK 33-824 and morphine, to influence LH release. This was seen as an inability of FK 33-824 (1·0 or 3·0 mg/kg) to inhibit LH secretion. The effects of gonadectomy on opiate control of LH occurred at all developmental stages and were not due to a disruption of sexual maturation. Opiate involvement in prolactin secretion did not appear to be adversely affected by an absence of gonadal steroids. Another novel aspect of this work was that the opiatergic component in the control of gonadotrophin secretion could be reinstated in long-term gonadectomized rats by treatment with oestradiol benzoate or testosterone propionate. Similarly, priming with increasing dosages of oestradiol benzoate which resulted in progressively lower LH levels gave larger naloxone responses. This steroid–opiate interdependency suggests that the negative feedback influence of gonadal steroids on LH secretion is conveyed, in part, by hypothalamic opiate peptides. Our results therefore provide a neurochemical basis for gonadal steroid negative feedback.

J. Endocr. (1984) 102, 133–141

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M. C. MacDonald and M. Wilkinson


We have investigated the consequences of chronic morphine or fentanyl exposure on the timing of puberty in female Sprague–Dawley rats. The μ-receptor agonists morphine and fentanyl were either added to the drinking water or, in the case of fentanyl, in osmotic minipumps. Morphine and fentanyl treatment, beginning at postnatal day 22, delayed the time of the first ovulation/vaginal opening (VO). For example, morphine included in the drinking water (800 mg/l) delayed VO by 5 days. Fentanyl gave similar results but at lower concentrations (30 mg/l). Surprisingly, these treatments delayed but did not prevent VO; i.e. in the face of continued opiate treatment most rats ovulated normally. In contrast, identical doses of morphine blocked oestrous cyclicity in drug-naive adults rats. However, morphine-dependent rats, which have reached VO (and first ovulation) then became briefly acyclic before beginning regular cycles even though opiate treatment was continued. The effects of fentanyl on immature rats were identical with those of morphine, i.e. VO was delayed but ovulation occurred in spite of continued drug treatment. On the other hand, fentanyl did not prevent cyclicity subsequent to VO.

Our observation that immature female rats can reach first ovulation despite chronic opiate treatment suggests that some degree of tolerance to opiates may develop. Such a mechanism could operate in normal drug-free rats, via endogenous opioid peptides, in the timing of puberty.

Journal of Endocrinology (1991) 129, 253–259

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K. M. Landymore and M. Wilkinson


Hypothalamic opioid peptides have been implicated in the timing of sexual maturation in several species. We have examined the effects of neonatal opioid blockade on the timing of puberty in the female rat and have compared these with the effects of neonatal GnRH injection. Intermittent naloxone (2·5 mg/kg) or GnRH (200 ng/100 g body wt) injected s.c. at 6-h intervals for the first 10 days of life only slightly advanced the mean day of vaginal opening (VO). However, the degree of precocity was significantly more marked in a subgroup of drug-injected rats. In contrast, injections of the long-acting opioid antagonist naltrexone (50 mg/kg) had no effect on the timing of VO.

The results suggested that the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effect. Therefore, additional studies were performed to compare receptor occupancy of naloxone and naltrexone in 9-day-old rat pups. An exvivo binding assay was utilized to determine the availability of hypothalamic opioid-binding sites at various intervals following a single s.c. injection of antagonist. The time-course of inhibition of tritium labelled [d-Ala2-N-Me-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding (μ-opioid sites) revealed that naloxone occupies the μ-receptor for a relatively short period of time. Naloxone (2·5 and 50 mg/kg) produced extensive inhibition of [3H]DAGO binding at 30 min following injection but binding was 100% of control at 1 h and 3·5 h respectively. In contrast, hypothalamic binding in naltrexone-treated (50 mg/kg) pups did not reach 50% of control values until 12 h and 100% of control values at 20 h after injection. The results are consistent with naltrexone's role as a long-acting antagonist but indicate that this blockade is not sustained for 24 h as previously indicated by indirect assessment with opiate challenge tests. Our data demonstrate that the duration of μ-opioid receptor occupancy is an important consideration in attempting to understand why intermittent, but not more continuous, blockade of opioid receptors induces some degree of sexual precocity in female rats. Also of interest is the observation that anterior pituitary stimulation with GnRH, during the first 10 days of life, can modify the timing of sexual maturation.

J. Endocr. (1988) 119, 447–452

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R. C. Carson and M. Wilkinson


We have shown that pre- and post-pubertal female rats are sensitive to seizures. For example, daily convulsions commencing at 24 days of age delay puberty. Here we examine the effect of seizures at various ages. In addition, because opioid peptides are implicated in regulating the onset of puberty and are activated by convulsions, we also investigate the effect of opioid antagonists in the seizure-induced delay of puberty.

A single daily electroconvulsive shock (ECS) was given for 10 days to neonatal (days 2–11), infantile (days 15–24) and juvenile (days 22–31) rats. The treatment delayed vaginal opening (VO) in juvenile rats. Neonatal and infantile rats were unaffected. VO was also delayed by daily ECS for only 5 days in the late juvenile (days 27–31) period. The opioid receptor antagonists naloxone, naltrexone and nalmefene injected before and after single daily ECS were unable to block this effect of ECS on VO. To examine whether the effect of ECS is related to stress, we examined several stressors known to induce opioid-mediated alterations in gonadotrophin secretion. Footshock, immobilization and ether stress administered in the juvenile period (days 27–31) did not affect the timing of VO. In addition, rats anaesthetized with halothane, and then given ECS, still showed a delay of VO. These data demonstrate that rats in the late juvenile stage of development are most sensitive to convulsions. We also suggest that opioids are not critical to the mechanism by which the ECS disturbs puberty, and that ECS elicits its effect seemingly independently of the convulsive stress.

Journal of Endocrinology (1989) 121, 229–238

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M Campbell-Thompson, KK Reyher and LB Wilkinson

A sexual dimorphism in gastric acid secretion has been known for many years, with women secreting less acid ( approximately 40%) than men. The mechanisms mediating this sex difference are unknown, but a role for estrogens is suggested from animal models. Two estrogen receptor (ER) subtypes, ER alpha and ER beta, mediate genomic effects of estrogens, and mRNA for both subtypes has been detected in the rat stomach. The objective of this study was to determine the cellular distribution of ER alpha and ER beta proteins in the rat stomach. ER alpha and ER beta proteins were detected in nuclei of fundic parietal cells and epithelial cells in the progenitor zone. In the antrum, several cells were immunoreactive for ER beta in regions containing stem and neuroendocrine cell types but ER alpha protein was not detected in antral glands. Both ER alpha and ER beta proteins were expressed in enteric neurons within the nucleus and cytoplasm, with specific punctate staining for ER alpha in cell bodies and fibers. These studies are the first to show differences between ER alpha and ER beta proteins in the epithelial cellular distribution in the fundus and antrum and to detect co-expression in enteric neurons. These results suggest that estrogens may inhibit gastric acid secretion via genomic effects in fundic parietal cells through either ER subtype and in antral neuroendocrine cells via ER beta. Moreover, co-expression of ER alpha and ER beta in enteric neurons indicates that estrogenic effects could also be mediated through neurogenic reflexes. Our findings imply that direct regulation of multiple cell types by estrogens may contribute to the modulation of gastric functions that have been recognized during the estrous cycle and between the sexes.

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The effects of oestrogen priming on the sensitivity of the anterior pituitary gland to stimulation with gonadotrophin releasing hormone (GnRH) was investigated in immature female rats using a new organ culture technique. Hemipituitary glands obtained from animals primed with a single dose of oestradiol benzoate (OB; 20 μg/100 g body weight) released significantly more LH when pulsed with GnRH (4 nmol/l) than did control hemipituitary glands. This potentiating effect was detectable as early as 5 days after birth. After a second stimulation, LH secretion remained high. These results were compared with those obtained from animals treated to induce increased levels of endogenous oestrogen on day 26 of life. Thus, hemipituitary glands were obtained from animals given two injections of OB, an injection of pregnant mare serum gonadotrophin (PMSG) or a unilateral brain lesion placed in the basal hypothalamus. Pituitary tissue was stimulated as before with a pulse of GnRH. Two injections of OB enhanced the sensitivity to stimulation. Conversely, both PMSG and lesion treatment severely reduced the sensitivity to GnRH, although PMSG-treated and lesioned animals have been used as models for the study of ovulation.

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An investigation of pituitary sensitivity, assessed in terms of increments in plasma LH and FSH concentrations, to stimulation with one or two injections of gonadotrophin releasing hormone (GnRH) was carried out on 26-day-old immature female rats which had received one of the following priming treatments: 10 μg oestradiol benzoate (OB) as a single injection on day 23 or day 25, or on both days; 10 i.u. pregnant mare serum gonadotrophin (PMSG) on day 24; an electrochemical brain lesion placed in the mediobasal hypothalamus on day 23; control animals received either vehicle alone or a sham lesion.

Pituitary sensitivity assessed at 10.00 h on day 26, after one or two injections of GnRH (100 ng/100 g body weight, s.c.), was enhanced to a similar degree in the three groups treated with OB in terms of LH (P < 0-01). The FSH response also increased after OB treatment but was not statistically significant. In contrast, 48 h after the injection of PMSG (i.e. when the rats were in a 'pro-oestrous-like' condition) pituitary sensitivity in terms of both LH and FSH dropped sharply (P < 0·001). In lesioned animals, pituitary sensitivity to one injection of GnRH was unchanged. A second GnRH injection administered after a 60 min interval induced a slightly larger LH response in control animals. In contrast, the ratio of the second response to the first increased in animals treated with PMSG, despite the state of overall decrease in sensitivity, being 4·5:1 in PMSG-treated rats versus 1·4:1 in controls.

In a second set of experiments, we investigated the variation of pituitary sensitivity in conjunction with an experimentally induced gonadotrophin surge. In animals treated with OB on day 23 and with 1 mg progesterone at 12·00 h on day 26, pituitary sensitivity was increased at both 14.00 and 17.00 h as compared with that in the day 23 OB-treated group at 10.00 h. The PMSG-treated animals maintained their state of decreased responsiveness at 14.00 h, but exhibited increased pituitary sensitivity at the time of the gonadotrophin surge (17.00 h).

These results show that OB increases pituitary sensitivity to GnRH in 26-day-old female rats and that the induction of a gonadotrophin surge further increases this sensitivity. In contrast, PMSG-treated rats displayed a state of decreased responsiveness 48 and 52 h, but not 55 h, after the injection. Pituitary sensitivity on the second day after PMSG treatment thus clearly differs from that observed during pro-oestrus in the adult cyclic female rat.

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Département de Physiologie, Université de Genève, Ecole de Médecine, 1211 Genève 4, Switzerland

(Received 23 February 1976)

The sensitivity of the anterior pituitary gland towards gonadotrophin-releasing hormone (Gn-RH) varies throughout the oestrous cycle of the rat (Aiyer, Fink & Greig, 1974). Oestrogen appears to be partially responsible for the increase in sensitivity observed at pro-oestrus (Aiyer & Fink, 1974). Prior exposure of ovariectomized heifers or rats to oestrogen results in an increased response, in terms of luteinizing hormone (LH) release, to Gn-RH in vitro (Hobson & Hansel, 1974; Apfelbaum & Taleisnik, 1976).

The present study was designed to investigate the ontogeny of this modulatory effect of oestrogen on the pituitary response to Gn-RH. Our results show that the pituitaries of immature female rats, primed with a single injected dose of oestradiol benzoate (OB), respond to synthetic Gn-RH in vitro with an enhanced release of LH. This sensitizing effect of OB