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L. Pinilla, M. Tena-Sempere and E. Aguilar

Administration of sex steroids to neonatal female rats resulted in anovulation and absence of positive and negative feedback between oestradiol and LH secretion. In the present experiments, the role of excitatory amino acids in the control of gonadotrophin secretion in anovulatory adult rats sterilized by neonatal administration of oestradiol benzoate or testosterone propionate (100 mg or 1.25 mg on the day of birth, respectively) was studied. Cyclic females in metoestrus were used as controls. Serum LH and FSH concentrations were measured at different times after i.p. administration of N-methyl-d-aspartic acid (NMDA), kainic acid (agonists of NMDA and kainate receptors, respectively), MK-801 or 6,7-dinitroquinoxaline-2,3-dione (DNQX) (antagonists of NMDA and kainate receptors, respectively). Experiments were also performed in control and sterilized females 1 week after ovariectomy. It was found that: (1) the effectiveness of NMDA and kainic acid in stimulating LH secretion was significantly higher in sterilized than in cyclic females; (2) ovariectomy increased LH secretion only in control females; (3) the stimulatory effect of NMDA and kainic acid on LH secretion after ovariectomy was observed only in sterilized females; (4) MK-801 and DNQX selectively decreased LH secretion in sterilized females; and (5) FSH secretion remained unaffected after NMDA or kainic acid administration in both control and sterilized females. In conclusion, the results obtained in sterilized females showed both a tonic release of endogenous excitatory amino acids and a greater responsiveness to NMDA and kainic acid than in controls.

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L. Pinilla, M. Tena-Sempere and E. Aguilar

Glutamate stimulates LH secretion in adult female rats after activation of N-methyl-d-aspartic acid (NMDA), kainate, and 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid receptors. In contrast to the positive role of kainate receptors in the control of LH secretion in adult females, neither activation nor antagonization of kainate receptors in immature rats modified the onset of puberty. The present experiments were carried out to establish why, if kainate stimulates LH release in adult rats, it fails to advance puberty in immature rats, and to determine whether the role of kainate receptors is sexually dimorphic around puberty. In Expt 1, 4-, 8-, 12-, 16-, 20- and 30-day-old females were investigated 15 min after administration of vehicle or kainic acid, a kainate receptor agonist (2.5 mg kg−1). In Expt 2, 30-day-old female rats were studied 2, 5 and 10 min after administration of vehicle, 2.5 mg kainic acid kg−1 or NMDA, an NMDA receptor agonist (15 mg kg−1). In Expt 3, female and male rats were gonadectomized or sham-gonadectomized on day 23 and investigated on day 30 after injection of vehicle, kainic acid (2.5 mg kg−1 at −15 min) or 6,7 dinitroquinoxaline-2,3 dione (DNQX), a kainate antagonist (2 mg kg−1 in two injections at −120 and −60 min). Finally, 30-day-old female and male rats were investigated 15 min after injection of vehicle or NMDA (15 mg kg−1) or 60 min after administration of different doses of 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), an NMDA antagonist (0.1, 0.25 or 0.50 mg kg−1). The results indicate that the role of kainate receptors in the control of gonadotrophin secretion is sexually dimorphic around puberty, since: (a) LH secretion was stimulated by kainic acid in male rats but inhibited in females; (b) FSH secretion was inhibited by kainic acid in ovariectomized females, but not in orchidectomized males; and (c) DNQX inhibited LH secretion in males but not in females. These differences were specific for kainate receptors since, in both sexes, NMDA stimulated and MK-801 inhibited LH secretion. It may be concluded that the secretion of gonadotrophins is modulated differently by kainate receptors in prepubertal male and female rats.

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C. Bellido, L. Pinilla, R. Aguilar, F. Gaytan and E. Aguilar

Summary. Rats were treated neonatally with oestrogen (500 μg oestradiol benzoate injected on Day 1 of life). Treatment with FSH and LH (80 μg/100 g body wt and 40 μg/100 g body wt respectively) during the early post-natal period (Days 1–10) abolished the effects of oestradiol on the morphological and functional development of the testes and on the regulation of prolactin secretion, but had no action on the effects of oestradiol on the development of the sex accessory glands. Treatment with prolactin (100 μg/100 g body wt) during the early post-natal period did not affect the integrity of the reproductive system in adult life. These results suggest that neonatal oestradiol acts indirectly, through an inhibition of gonadotrophin secretion on testicular development, and directly on the development of the sex accessory glands.

Keywords: neonate; oestrogen; gonadotrophins; testis; sex accessory glands; rat

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LC Gonzalez, L Pinilla, M Tena-Sempere, C Dieguez, FF Casanueva and E Aguilar

Recent data indicate that leptin is involved in the control of reproductive function. Experiments were carried out to analyse the role of endogenous leptin in the regulation of LH and prolactin secretion during the afternoon of pro-oestrus and that induced by ovarian steroids in ovariectomized rats. In the first experiment, cyclic female rats were implanted with intra-auricular and intracerebroventricular (i.c.v.) cannulae and, at pro-oestrus, were injected (i.c.v.) with 10 microliters normal rabbit serum or leptin antiserum (at 13:00 and 14:00 h). Blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the second experiment, female rats in pro-oestrus were injected with normal rabbit serum or leptin antiserum at 16:00 and 18:00 h and blood samples were taken every 10 min between 18:00 and 20:00 h. In the third experiment, adult female rats that had been ovariectomized 2 weeks before were implanted with intra-auricular and i.c.v. cannulae and treated with oestradiol benzoate (30 micrograms s.c.) at 10:00 h and progesterone (2 mg s.c.) 48 h later. Normal rabbit serum (10 microliters) or leptin antiserum (10 microliters) were injected (i.c.v.) at 13:00 and 14:00 h, and blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the fourth experiment, hemipituitaries from ovariectomized steroid-treated female rats were incubated in the presence of leptin116-130 (an active fragment of the native molecule), GnRH or leptin + GnRH. Prolactin and LH secretion during the afternoon of pro-oestrus in females treated with leptin antiserum was similar to that observed in animals injected with normal rabbit serum. In ovariectomized female rats, the steroid-induced LH surge increased slightly after administration of leptin antiserum, whereas the prolactin surge remained unchanged. In vitro, leptin116-130 (10(-5) to 10(-8) mol l-1) inhibited LH secretion and modulated the effect of GnRH on LH release, depending on the concentration of GnRH: leptin116-130 (10(-6) mol l-1) reduced the effectiveness of 10(-7) mol GnRH l-1 and increased that of 10(-9) mol GnRH l-1. In conclusion, these experiments indicate that acute immunoneutralization of endogenous leptin does not interfere with spontaneous or steroid-induced LH and prolactin surges. In addition, the finding that leptin116-130 inhibited LH release and modulated the effectiveness of GnRH in vitro provides evidence of the direct modulatory role of leptin on LH secretion acting at the pituitary.

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L Pinilla, LC Gonzalez, F Gaytan, M Tena-Sempere and E Aguilar

Selective oestrogen receptor modulators constitute a family of drugs that are used increasingly in the management of oestrogen-associated pathology. Raloxifene is a selective oestrogen receptor modulator that is used to treat and prevent osteoporosis in post-menopausal women. The actions of raloxifene on bone, breast, uterus and serum cholesterol concentrations have been widely analysed, but very few studies have investigated the possible actions of this drug on the central nervous system. The central nervous system of the newborn rat is very sensitive to oestrogen action. In this study a series of experiments was conducted to analyse the effects of different doses of raloxifene (50, 100, 250 or 500 microg per rat per day) administered to neonatal rats on days 1-5 of age. Female rats treated with raloxifene showed decreased gonadotrophin secretion, hyperprolactinaemia, advanced vaginal opening, decreased body weight, persistent presence of cornified epithelial cells in vaginal smears, anovulation, inhibition of positive feedback between oestradiol and LH, and infertility. Male rats showed delayed balanopreputial separation, reduced body weight and hyperprolactinaemia. All these changes resemble those obtained after neonatal administration of oestradiol benzoate, thus indicating, for the first time, that raloxifene exerts an oestrogenic action on the hypothalamic-pituitary structures controlling reproductive function in rats.

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L. Pinilla, E. Trimiño, P. Garnelo, C. Bellido, R. Aguilar, F. Gaytán and E. Aguilar

The following experiments were performed: (i) concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin in plasma were measured at 2, 5, 8, 10 and 15 days in female Wistar rats treated on the first day of life with 100 μg oestradiol benzoate or vehicle; (ii) females injected on day 1 with 100 μg of oestradiol benzoate or 1 mg of testosterone propionate and from day 1 to day 10 or 15 with FSH and LH were killed on day 90; (iii) females injected from day 1 to day 10 or 15 with prolactin or vehicle were killed on day 90; (iv) females injected on day 1 with oestradiol benzoate and from day 1 to day 15 with a luteinizing-hormone-releasing hormone (LHRH) agonist were killed on day 90; (v) groups of females injected on days 1, 4, 7, 10, 13 and 16 with an LHRH antagonist were killed on day 90. Onset of puberty, vaginal cycles, organ weights and hormonal plasma concentrations were measured. Females treated on the first day of life with 100 μg oestradiol showed inhibition of gonadotrophin secretion and stimulation of prolactin secretion during the neonatal period. Females injected on the first day of life with oestradiol benzoate or testosterone propionate showed, in adulthood, anovulation, ovarian atrophy, reduced FSH plasma concentrations, increased prolactin plasma concentrations and reduced pituitary prolactin content. These alterations were due neither to blocked gonadotrophin secretion nor to stimulated prolactin secretion observed immediately after steroid injection, since: (i) development of the anovulatory syndrome was not blocked by the administration of exogenous gonadotrophins or LHRH-agonist; and (ii) blockade of gonadotrophin secretion immediately after birth with an LHRH antagonist or neonatal injection of prolactin did not induce the anovulatory syndrome. It is concluded that anovulation induced by administration of neonatal steroid was mediated neither by the early inhibition of gonadotrophin secretion nor by the stimulation of prolactin secretion.