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

Treatment with non-steroidal anti-inflammatory drugs, either non-selective or selective cyclooxygenase-2 (COX-2) inhibitors, consistently impairs ovulation, indicating the essential role of COX-2/prostaglandins in the ovulatory process. Indomethacin, a potent inhibitor of both COX-1 and COX-2, induced several ovulatory alterations, consisting of a decrease in the number of oocytes effectively ovulated, trapping of oocytes inside the luteinized follicle, as well as abnormal follicle rupture at the basolateral sides, with release of the oocyte and follicular fluid to the interstitium. Yet, the precise role of prostaglandins in ovulation and whether some of the ovulatory defects induced by indomethacin are due to interference with additional components of the ovulatory cascade, beyond prostaglandin synthesis, are not completely understood. We have used gonadotrophin-primed immature rats to analyse whether, compared to indomethacin, selective inhibition of COX-2, with or without concomitant inhibition of COX-1, or selective inhibition of the lipooxygenase (LOX) pathway, induce similar ovulatory alterations. Immature rats (27 days of age) were injected PMSG (10 IU), and 48 h later hCG (10 IU) subcutaneously, and different anti-inflammatory drugs. Animals were killed at 21 h after hCG injection. Rats treated with the selective COX-2 inhibitor NS398 (10 mg/kg body weight, (bw)) showed alterations in follicle rupture as those treated with indomethacin (0.5 mg/rat), albeit affecting a lower number of follicles, irrespective of the concomitant inhibition of COX-1 with the selective inhibitor SC560 (10 mg/kg bw). Rats treated with the LOX inhibitor NDGA (300 mg/kg bw) did not show ovulatory alterations. These data indicate that the characteristic alterations of follicle rupture induced by indomethacin, are also induced by selective COX-2 inhibitors, strengthening the contention that prostaglandins play a crucial role in the spatial targeting of follicle rupture at the apex.

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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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J. E. Sánchez-Criado, M. Tébar, A. Sánchez and F. Gaytán

Administration of antiprogesterone RU486 to female cyclic rats results in blockade of ovulation associated with both a decreased ovulatory release of LH and an increased rate of follicular atresia. These rats also exhibit increased LH:FSH and testosterone:oestradiol ratios in serum during the period of follicular development as well as an increase in serum concentrations of prolactin that can be suppressed by a dopamine agonist. The increase in either prolactin or testosterone concentrations as well as the relative deficiency in FSH might be responsible for the increase in follicular atresia. The present work evaluated the involvement of LH, FSH, prolactin and testosterone in follicular atresia and in blockade of ovulation induced by RU486 in the cyclic rat. Although bromocriptine treatment did not modify the blockade of ovulation induced by RU486, unilateral ovariectomy at metoestrus and antiandrogen flutamide treatment reversed, in part, the effects of RU486 on both follicular development and ovulation. The combined increase in FSH serum concentration during dioestrus induced by unilateral ovariectomy and the treatment with flutamide had no additive effects. Furthermore, treatment with a superovulatory amount of hFSH did not reverse the effects of RU486. Moreover, unilateral ovariectomy halved testosterone serum concentrations and flutamide treatment had no effect on LH and FSH concentrations in RU486-treated rats. It was therefore concluded that androgens play a role, at least in part, in the process of follicular atresia induced by RU486.

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F Gaytan, C Bellido, C Morales and JE Sanchez-Criado

In cyclic rats, apoptosis of luteal cells during structural luteolysis occurs cyclically at the transition from pro-oestrus to oestrus in response to the preovulatory prolactin surge. This finding indicates that cyclic changes in apoptosis during luteolysis are dependent on prolactin surge cyclicity. In this study, the effects of prolactin on structural luteolysis were studied under different experimental conditions in relation to the phase of the oestrous cycle. In rats treated with prolactin at metoestrus and dioestrus, apoptosis did not occur in regressing corpora lutea, whereas in rats treated with prolactin on the morning of pro-oestrus, a 12.3-fold and 3.4-fold increase were observed in the number of apoptotic cells in regressing corpora lutea of the current and previous oestrous cycles, respectively. However, when the preovulatory prolactin surge and hence the subsequent apoptotic burst were blocked, prolactin treatment at the dioestrus phase induced a 13-fold increase in the number of apoptotic cells and significant changes in the volume of the corpus luteum (38% decrease) and the number of steroidogenic cells per corpus luteum (70% decrease). The results of this study indicate that the responsiveness of the regressing corpus luteum to the pro-apoptotic effects of prolactin are dependent on the phase of the oestrous cycle and on the presence or absence of an apoptotic burst in response to the preovulatory prolactin surge on the evening of pro-oestrus. Steroidogenic cells surviving to the apoptotic burst during the transition from pro-oestrus to oestrus became refractory to the lytic effect of prolactin. Furthermore, these cells also responded to the luteotrophic effects of prolactin, reaching full morphological luteinization, as indicated by the rescue of regressing cyclic corpora lutea during pregnancy.

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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 Gaytan, E Tarradas, C Morales, C Bellido and JE Sanchez-Criado

The ovulatory process in cyclic rats was studied after prostanoid synthesis was blocked using indomethacin. Animals were injected at 12:00 h in pro-oestrus with 1.0 mg indomethacin or vehicle (olive oil) and killed at 18:30 h in pro-oestrus, at 02:00, 09:00 and 19:00 h in oestrus and at 09:00 h in metoestrus. Additional rats injected with 0.5 or 4.0 mg indomethacin were killed at 09:00 h in oestrus. No differences in either morphology or serum LH concentrations were found between vehicle or indomethacin-treated rats at 18:30 h in pro-oestrus. However, from 02:00 h in oestrus onward, the process of follicle rupture was altered considerably in indomethacin-treated rats, irrespective of the dose. Early vascular changes, observed in control rats at the apex of the follicle, were absent in indomethacin-treated rats. In some follicles, disruption of the theca layers, invasion of the perifollicular tissue by granulosa cells and follicular fluid, and release of the oocyte to the ovarian interstitium were observed at 02:00 h in oestrus. A small number of follicles ruptured at the ovarian surface. Furthermore, invasion of interstitial tissue, rupture of blood vessel walls, production of emboli of granulosa cells and follicular fluid, and inflammatory reactions were observed in oestrus and metoestrus. The results of the present study demonstrate uncontrolled proteolytic activity, and indicate that abnormal follicle rupture (but not inhibition of follicle rupture) is responsible for ovulation failure in indomethacin-treated rats.

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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, 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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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.