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F. Gaytan and E. Aguilar

Summary. On Day 1 of age rats were treated with 500 μg oestradiol benzoate. Oestrogen-treated rats had increased numbers of Sertoli cells per reference area or volume, whereas the total number of cells per testis was unchanged. The mean nuclear size was significantly smaller in oestrogen-treated rats than in control rats, at 22 and 45 days of age. The volume density of the heterochromatin clumps decreased from 22 to 45 days of age in control rats (68% fall), the decrease being slower in oestrogenized animals (30% fall) during the same period. The differences were significant at 45 days of age only. The relative volume occupied by the nuclear membrane infoldings was significantly less in oestrogenized rats than in control ones at the two ages considered. Nucleolar development was delayed in oestrogen-treated rats, which had lower numbers of nuclear sections showing nucleoli, as well as a decrease in the nucleolar diameter. We suggest that these Sertoli cell alterations are due to the altered gonadotrophin and testosterone concentrations induced by the steroid treatment rather than to a direct effect of oestrogen.

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R. Aguilar, C. Bellido, J. E. Sánchez-Criado and E. Aguilar

Summary. Male rats were grafted on Day 21 of age with 'young' (21 days old) or 'adult' (90 days old) pituitary glands and then treated daily with 4 mg bromocriptine/kg or vehicle. Plasma samples were obtained on Days 21, 25 and 35 and when balano-preputial separation occurred. Both types of grafts advanced the age at which balano-preputial separation occurred and increased prolactin concentrations. Bromocriptine treatment reduced the prolactin values in both grafted groups, but did not block the advancement of puberty in rats treated with 'young' pituitary grafts.

These results suggest the existence of two possible mechanisms in precocious puberty induced by pituitary grafts: one is prolactin-dependent (when 'adult' pituitary glands were used) and the other not directly related to prolactin (when 'young' pituitary glands were used).

Keywords: balano-preputial separation; pituitary graft; prolactin; bromocriptine; testosterone; rat

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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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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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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, 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.

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F. Gaytan, C. Bellido, E. Aguilar and N. van Rooijen

Testicular macrophages in rats were selectively depleted by an intratesticular injection of liposomes containing dichloromethylene diphosphonate into the right testis to study the possible role of these macrophages during the prepubertal development of Leydig cells. The contralateral testes were injected with 0.9% NaCl and served as controls. The animals were injected with the liposomes and NaCl at 5, 10, 15, 20 or 25 days of age. In macrophage-depleted testes, Leydig cell development was inhibited in the animals injected at 5, 10 or 15 days of age. At 35 days of age, the testis was repopulated with macrophages and Leydig cells also developed. Rats treated at 20 or 25 days of age, when Leydig cells were already present in low numbers, did not show any further increases in the number of Leydig cells up to 35 days of age. To study whether the effects of gonadotrophins on Leydig cell development require the presence of macrophages, 21-day-old rats, injected 3 days before with liposomes (right testis) and NaCl (left testis), were treated with 75 iu human FSH kg−1 bodymass day−1, 10 iu hCG per rat day−1, combined hFSH and hCG, or vehicle (PBS with 0.5% BSA) for 6 days. Treatment with hCG induced a sevenfold increase in the number of Leydig cells in the left (macrophage-containing) testis, whereas no increase was found in the right (macrophage-depleted) testis. These results indicate that macrophages are needed for Leydig cell development and for the Leydig cell response to hCG during postnatal maturation.

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F. Gaytán, C. Bellido, C. Morales, E. Aguilar and J. E. Sánchez-Criado

Adult cyclic rats were studied from 16:00 h on pro-oestrus to 07:00 h on oestrus to relate the cyclic hormonal changes to the proliferative activity and growth pattern of growing follicles. The proliferative activity was studied by 5-bromodeoxyuridine (BrdU) labelling and by the presence of mitoses. Small growing follicles (less than 275 μ in diameter) were divided into five classes: multilaminar classes a (Ma, up to 75 μm in diameter), b (Mb, 76–150 μm), c (Mc, 151–200 μm) and d (Md, 201-274 μm) and follicles measuring ≥275 μm in diameter were considered as ≥ class 1, following previous classifications. LH concentrations were maximal at 18:30 h on pro-oestrus, and this was coincident with an increase in FSH, prolactin and progesterone concentrations, whereas oestradiol and testosterone concentrations were decreased. From 02:00 h on oestrus the concentrations of all hormones, except those of FSH, were decreased. The number of Ma, Mb and Mc follicles did not change during pro-oestrus-oestrus, whereas an increase in the number of follicles ≥ class 1 was found at 07:00 h on oestrus. This appears to be a consequence of the increased proliferative activity of Md follicles, evidenced by the increase in the BrdU labelling and mitotic index of this follicle class, found from 02:00 to 07:00 h on oestrus, together with a decrease in the percentage of early atretic follicles ≥ class 1 at 07:00 h on oestrus. This study provides an improved classification of small growing follicles into discrete classes and delineates a size class of follicles (Md follicles) that is responsive to the cyclic hormonal changes on early oestrus.

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F. Gaytan, C. Bellido, C. Morales, C. Reymundo, E. Aguilar and N. van Rooijen

Testicular macrophages were selectively eliminated with dichloromethylene diphosphonate-containing liposomes (Cl2MDP-lp) to study the role of these cells in the repopulation of Leydig cells after treatment with ethylene dimethane sulfonate (EDS). Right testes were injected with Cl2MDP-lp to deplete macrophages and left testes were injected with sodium chloride and served as controls. Injection of Cl2MDP-lp produced a 97% reduction in the number of macrophages 10 days after treatment. Twenty-one days after destruction of the existing Leydig cells with EDS, abundant differentiating Leydig cells were present in the left (macrophage-containing) testes. On the contrary, in the right (macrophage-depleted) testes, differentiating Leydig cells were scarce, and was 3% of that found in the control testes. The inhibition of Leydig cell repopulation in macrophage-depleted testes was more evident at 30 days after EDS treatment, when the number of Leydig cells in the right testes was 1% of that found in control testes. The lack of Leydig cell development was also indirectly shown by the lower mass and more atrophic seminiferous epithelium of the right testes, as well as the decreased weight of the ipsilateral epididymis compared with the left testes. These results indicate that testicular macrophages are central to the proliferation and differentiation of new Leydig cells after EDS treatment, and point out the significance of paracrine regulatory mechanisms in rat testes.

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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.