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L Pinilla, M Tena-Sempere and E Aguilar

Abstract

It is well known that in the rat neonatal manipulation of the sex steroid environment results in altered hypothalamic-pituitary function in adulthood which implies an abnormal prolactin secretion and gonadotrophin response to orchidectomy. The present paper analyses the involvement of excitatory amino acid pathways in the disturbed gonadotrophin and prolactin secretion in male rats neonatally injected with oestradiol benzoate (500 μg on the day of birth). In the first experiment, 60-day-old control and oestrogenized male rats (intact or orchidectomized a week before) were killed 15 min after injection of vehicle, N-methyl-d-aspartic acid (NMDA; 15 mg/kg) or kainic acid (KA; 15 mg/kg). In the second experiment, prepubertal males were killed 15 min after injection of vehicle or NMDA. In the third experiment 30-, 45-, 60-and 90-day-old intact control and oestrogenized males were killed 15 min after injection of vehicle or KA. In the fourth experiment, control and oestrogenized males were sham-orchidectomized, orchidectomized or orchidectomized and implanted with Silastic capsules at 30 days of age and killed on day 90, 15 min after vehicle or KA injection.

We found that (1) as previously reported, oestrogenized males showed hyperprolactinaemia and absence of LH and FSH responses after orchidectomy, (2) the stimulatory effect of NMDA on LH secretion was observed in prepubertal control and in prepubertal and adult oestrogenized males, (3) KA stimulated LH secretion only in prepubertal (30-day-old) and peripubertal (45-day-old) control males, whereas stimulation was observed in oestrogenized males at all ages studied, except when they were implanted with testosterone, (4) FSH secretion was only stimulated in oestrogenized orchidectomized males, and (5) prolactin secretion was inhibited by NMDA and KA in control and oestrogenized males.

The greater effectiveness of NMDA and KA on gonadotrophin secretion in oestrogenized males, a finding especially noticeable after orchidectomy, may reflect the existence of a greater pool of releasable GnRH, a consequence of the absence of response to orchidectomy. The decrease in prolactin secretion after NMDA or KA administration in oestrogenized males may be mediated by an increase in dopaminergic tone, presumably reduced in the hyperprolactinaemic oestrogenized males. Finally, the effectiveness of KA on prolactin and LH secretion was maintained in adult oestrogenized males, perhaps because of the permanent decrease in testosterone secretion.

Journal of Endocrinology (1995) 147, 51–57

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

The secretion of PRL is controlled by different hypothalamic signals. Depending on the experimental model, PRL secretion increases or decreases after activation of N-methyl-d -aspartic acid and kainate receptors. Recently we have described that activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors inhibits PRL secretion in prepubertal male rats. The aim of present study was to examine (1) the physiological relevance of this finding, (2) the possible age-related changes observed after activation or blockade of AMPA receptors, (3) the specificity of the AMPA effect, (4) the hypothalamic and/or pituitary localization of AMPA action, and (5) the mechanism(s) of action of AMPA agonists. In a first set of experiments, neonatal males (5 and 10 days old) and prepubertal (23 days old) male rats were injected with AMPA (1, 2.5 or 5 mg/kg) or the antagonist of AMPA receptors 1,2,3, 4-tetrahydro-6-nitro-2,3-dioxo-! benzo (f) quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg). Serum PRL concentrations decreased significantly 15 and 30 min after i.p. administration of AMPA in prepubertal male rats, while the inhibitory effect of AMPA was not observed in 5- and 10-day-old males. The effect of AMPA was abolished by NBQX but not by MK-801 (a selective antagonist of NMDA receptors). NBQX alone (0.25 or 0.50 mg/kg) had no effect on PRL release. In vitro, AMPA slightly stimulated PRL secretion by hemipituitaries from prepubertal males, suggesting that the hypothalamus is likely the site of action for the reported inhibitory action of AMPA on PRL release. In this sense, the blockade of AMPA effects in animals pretreated with domperidone (a dopaminergic antagonist) or alpha-methyl-p-tyrosine (an inhibitor of dopamine synthesis) suggests that an increase in the release of hypothalamic dopamine is probably the mechanism i! nvolved in the effect of AMPA. In a second set of experiments, the effects of AMPA (2.5 mg/kg i.p.) and NBQX (0.5 mg/kg i.p. and 20 or 40 nmol i.c.v.) were tested in freely moving adult male rats sampled during periods of 2, 3 or 6 h. In contrast with data obtained in prepubertal rats, neither AMPA nor NBQX affected PRL secretion. In conclusion, these data indicate that activation of AMPA receptors inhibits PRL secretion in prepubertal male rats. This effect probably involves the release of dopamine from the hypothalamus and disappears in adulthood.

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

Excitatory amino acids, such as glutamate, constitute a major transmitter system in the control of hypothalamic-pituitary secretion. Different subtypes of glutamate receptors, such as NMDA (N-methyl-d-aspartic acid) and KA (kainate) receptors, are involved in the control of anterior pituitary secretion. Other receptor subtypes, such as AMPA (activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and metabotropic receptors, have been identified, although their role in the control of neuroendocrine function remains largely unknown. Recent reports have demonstrated the involvement of AMPA receptors in the control of the steroid-induced luteinizing hormone (LH) surge in female and growth hormone (GH) secretion in male rats. The aim of this study was to assess the potential role of AMPA receptors in the control of GH, prolactin (PRL), LH and follicle-stimulating hormone (FSH) secretion in prepubertal 23-day-old rats. To this end, prepubertal female rats were injected with AMPA (2.5 or 5 mg/kg i.p.) or the antagonist of AMPA receptors 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo (f) quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg i.p.). Serum LH and FSH concentrations and hypothalamic LH-releasing hormone (LHRH) content remained unchanged after AMPA or NBQX administration. In contrast, serum PRL levels significantly decreased 15, 30 and 60 min after i.p. administration of AMPA and increased 120 min after NBQX treatment, whereas serum GH levels increased after AMPA treatment and decreased after NBQX administration. Considering that AMPA has been shown to activate a subset of kainate receptors, its effects were compared with those elicited by 2.5 mg/kg KA in prepubertal female rats. At this age, however, KA was unable to reproduce the effects of AMPA on PRL and GH secretion, thus suggesting that the actions observed after AMPA administration were carried out specifically through AMPA receptors. In addition, as the effects of AMPA on LH secretion in adult females have been proved to be steroid-dependent, the effects of AMPA (2.5 mg/kg) and NBQX (0.5 mg/kg) were tested in prepubertal animals with different gonadal backgrounds, i.e. intact males, and intact and ovariectomized (OVX) females. The effects of AMPA in prepubertal females appeared to be modulated by ovarian secretion, as the inhibition of PRL secretion disappeared and LH secretion was partially suppressed by AMPA in OVX animals whereas the stimulatory effect on GH release was enhanced by ovariectomy. Furthermore, in male rats, AMPA administration significantly decreased PRL secretion and increased serum GH levels, the amplitude of the GH response being higher than in prepubertal females. To ascertain the pituitary component for the reported actions of AMPA, hemi-pituitaries of male rats were incubated in the presence of AMPA (10(-8)-10(-6) M). The results obtained showed no effect of AMPA on PRL, GH and gonadotropin secretion in vitro. Finally, we investigated the involvement of the dopaminergic (DA) system in the inhibitory action of AMPA on PRL secretion. Pre-treatment of prepubertal female rats with a dopamine receptor antagonist (domperidone: 1 mg/kg) resulted in the blockage of AMPA-mediated inhibition of PRL secretion, thus suggesting that this action is probably mediated by an increase in DA activity. In conclusion, we provide evidence for the physiological role of AMPA receptors in the control of PRL and GH secretion in prepubertal rats. In contrast, our data cast doubts on the involvement of AMPA receptors in the regulation of gonadotropin secretion at this age. The effects of AMPA reported herein were not mediated through activation of kainate receptors and were probably exerted at the hypothalamic or suprahypothalamic levels. In addition, we show that ovarian secretion actively modulates the effects of AMPA receptor activation on anterior pituitary secretion in prepubertal female rats.

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L Pinilla, M Tena-Sempere, D Gonzalez and E Aguilar

Abstract

It is well known that the control of LH secretion depends on the steroid milieu during the postnatal period. In this study LH secretion was analysed in adult male rats injected neonatally with 500 μg oestradiol benzoate (1) after orchidectomy, (2) after selective elimination of androgens by destruction of Leydig cells with ethylene dimethane sulphonate (EDS), and (3) after removal in orchidectomized animals of Silastic capsules containing testosterone. In addition, (4) in vivo and in vitro LH secretion in response to LHRH agonist and antagonists, (5) the hypothalamic LHRH content, (6) the basal and stimulated in vitro LHRH release, and (7) the LH responses after administration of naloxone (2 mg/kg), α-methyl-p-tyrosine (α-MPT; 250 mg/kg), N-methyl-d-aspartic acid (NMDA, 15 mg/kg) or kainic acid (KA; 15 mg/kg) were also examined. Our data indicated that (1) the LH response after orchidectomy, after EDS administration and after removal of Silastic capsules containing testosterone was diminished in oestrogenized male rats, (2) the pituitaries from oestrogenized males retained responsiveness to LHRH, (3) hypothalamic LHRH content was reduced in oestrogenized males, but the hypothalamus from oestrogenized males released more LHRH than those of control groups both under basal conditions or after depolarization, (4) α-MPT decreased LH secretion only in oestrogenized males, and (5) NMDA and KA stimulated LH only in oestrogenized males. We conclude that in oestrogenized male rats the loss of sensitivity to the negative feedback action of testosterone on LH secretion was not due to decreased pituitary responsiveness to LHRH stimulation or to the inherent damage of LHRH neurones. In contrast, changes in the mechanisms governing LHRH release seem to be involved. A lack of activation of the excitatory noradrenergic and aminoacidergic systems seems to be part of the neurochemical basis of altered gonadotrophin secretion in neonatally oestrogenized male rats.

Journal of Endocrinology (1995) 147, 43–50

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M Tena-Sempere, J Kero, A Rannikko and I Huhtaniemi

In the rat, the cytotoxic drug ethylene dimethane sulfonate (EDS) selectively eliminates mature Leydig cells (LCs) from testicular interstitium, activating a complex process of proliferation and differentiation of pre-existing LC precursors. We observed previously that after EDS treatment, the early LC precursors persistently express a truncated 1.8 kb form of LH receptor (LHR) mRNA. This prompted us to study whether experimental cryptorchidism, known to alter the process of LC repopulation, can influence the pattern of testicular LHR mRNA expression after EDS administration. EDS treatment completely eliminated mature LCs both in control and unilaterally cryptorchid (UC) rats. This response was followed by gradual reappearance of newly formed, functionally active LCs, as evidenced by the recovery in testicular LHR content and plasma testosterone levels in both experimental groups. Noteworthy, the rate of LC repopulation was higher in the abdominal testes of UC rats, in keeping with previous findings. Interestingly, the 1.8 kb LHR transcript was persistently expressed in scrotal testes at all time-points, but undetectable upon Northern hybridization in abdominal testes at early stages after EDS administration, when low levels of expression of truncated LHR transcripts could only be detected by semi-quantitative RT-PCR analysis. In addition, the faster LC repopulation in cryptorchid testes was associated with precocious recovery of the complete array of LHR mRNA transcripts, including the 1.8 kb species. These changes appeared acutely and irreversibly, as unilateral positioning of scrotal testes into the abdomen resulted in a rapid loss of expression of the 1.8 kb LHR transcript, whereas scrotal relocation of the UC testes failed to alter the pattern of LHR gene expression. In conclusion, experimental cryptorchidism changes the pattern of LHR mRNA expression in rat testis after selective LC destruction by EDS. This change, i.e. repression of the 1.8 kb LHR transcript after EDS administration, is acute and irreversible, and likely related to the impairment of testicular microenvironment following cryptorchidism. However, even though at low levels, the expression of truncated forms of LHR mRNA appears to be a universal feature of proliferating LC precursors. The UC testis may represent a good model for analysis of the regulatory signals involved in the control of LHR gene expression.

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L Pinilla, P Garnelo, M Tena-Sempere, F Gaytan and E Aguilar

Abstract

It is well known that males injected neonatally with oestradiol or antiserum or antagonists (ANT) against gonadotrophin-releasing hormone (GnRH) show multiple reproductive disorders. In the present work, in males treated neonatally with GnRH-ANT, we have analysed: (1) whether the impairment of reproductive function can be blocked by simultaneous treatment with gonadotrophins, (2) the possible differences in the effects of GnRH-ANT injected before or after the proliferation of Sertoli cells which takes place between days 1 and 15 of age, and (3) the mechanism(s) for the increased FSH secretion observed in adulthood. Experimental designs included: administration of GnRH-ANT between days 1 and 16 or 15 and 30 of age, simultaneous administration of gonadotrophins and GnRH-ANT to neonatal males, and measurement of FSH secretion after orchidectomy or specific destruction of Leydig cells with ethylene dimethane sulphonate (EDS) in adult males treated neonatally with GnRH-ANT.

The principal new data presented in our studies are the following: (1) delayed puberty was observed not only in males injected neonatally with GnRH-ANT, but also in those injected with gonadotrophins or with GnRH-ANT and gonadotrophins, (2) the decreased fertility and increased FSH secretion observed in adult males treated neonatally with GnRH-ANT were normalized by simultaneous administration of GnRH-ANT and gonadotrophins, and (3) the increased FSH secretion in adult males treated neonatally with GnRH-ANT remained after EDS or orchidectomy, suggesting that mechanisms other than decreased inhibin secretion were involved in the increased secretion of FSH.

Journal of Endocrinology (1994) 142, 517–525

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M Tena-Sempere, J Navarro, L Pinilla, LC Gonzalez, I Huhtaniemi and E Aguilar

The biological actions of estrogens on target cells are mediated by two nuclear receptors: the estrogen receptor (ER) alpha and the recently characterized ER beta. In the male rat, the physiological role of estrogens involves multiple actions, from masculinization of brain areas related to reproductive function and sexual behavior to regulation of testicular development and function. Paradoxically, however, administration of high doses of estrogen during the critical period of neonatal differentiation results in an array of defects in the reproductive axis that permanently disrupt male fertility. The focus of this study was to characterize the effects and mechanism(s) of action of neonatal estrogenization on the pattern of testicular ER alpha and beta gene expression during postnatal development. To this end, groups of male rats were treated at day 1 of age with estradiol benzoate (500 microg/rat), and testicular ER alpha and ER beta mRNA levels were assayed by semi-quantitative RT-PCR from the neonatal period until puberty (days 1-45 of age). Furthermore, the expression of androgen receptor (AR) mRNA was evaluated, given the partially overlapping pattern of tissue distribution of ER alpha, ER beta and AR messages in the developing rat testis. In addition, potential mechanisms for neonatal estrogen action were explored. Thus, to discriminate between direct effects and indirect actions through estrogen-induced suppression of serum gonadotropins, the effects of neonatal estrogenization were compared with those induced by blockade of gonadotropin secretion with a potent LHRH antagonist in the neonatal period. Our results indicate that neonatal exposure to estrogen differentially alters testicular expression of alpha and beta ER messages: ER alpha mRNA levels, as well as those of AR, were significantly decreased, whereas relative and total expression levels of ER beta mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by LHRH antagonist treatment. It is concluded that the effect of estrogen on the expression levels of ER alpha and beta mRNAs probably involves a direct action on the developing testis, and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period.

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L Pinilla, D Gonzalez, M Tena-Sempere, R Aguilar and E Aguilar

Abstract

Activation of excitatory N-methyl-d-aspartate and kainate receptors evokes multiple and diverse neuroendocrine changes. We have previously shown that kainic acid (KA), an agonist of kainate receptors, inhibits prolactin (PRL) secretion in male rats when given systemically. In the present studies we have characterized this inhibitory action. KA inhibited in vivo PRL secretion in neonatal, prepubertal and adult male rats. This inhibition was independent of gonadal secretion and was evident in male rats whether intact, orchidectomized, or orchidectomized and treated with testosterone. In addition, KA inhibited PRL secretion in male rats rendered hyperprolactinaemic by neonatal administration of oestradiol benzoate. The decrease in serum PRL levels after KA administration was accompanied by an increase in pituitary concentrations of dopamine, and the KA effect on PRL disappeared in males pretreated with domperidone, an antagonist of dopaminergic receptors. These findings strongly suggest that an increase in dopamine release was involved in the effects of KA. Also, KA inhibited in vitro PRL secretion by adenohypophysial dispersed cells and this effect was blocked by 6,7-dinitroquinoxaline, a kainate receptor antagonist, which indicates that the pituitary is also a possible site of action of KA. Nw-nitro-l-arginine-methyl ester, a blocker of nitric oxide synthase, reduced the effects of KA in vivo and slightly stimulated PRL release in vitro.

We conclude that the inhibitory action of KA is independent of the age of the animal, the gonadal status and the prevailing PRL levels. The action of KA is probably mediated by an increase in dopamine secretion and by a direct effect at the pituitary level. Finally, the effect of KA on PRL secretion is partially dependent on endogenous nitric oxide.

Journal of Endocrinology (1996) 151, 159–167

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

Leptin, the product of the ob gene, has emerged recently as a pivotal signal in the regulation of fertility. Although the actions of leptin in the control of reproductive function are thought to be exerted mainly at the hypothalamic level, the potential direct effects of leptin at the pituitary and gonadal level have been poorly characterised. In the present study, we first assessed the ability of leptin to regulate testicular testosterone secretion in vitro. Secondly, we aimed to evaluate whether leptin can modulate basal gonadotrophin and prolactin (PRL) release by incubated hemi-pituitaries from fasted male rats. To attain the first goal, testicular slices from prepubertal and adult rats were incubated with increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Assuming that in vitro testicular responsiveness to leptin may be dependent on the background leptin levels, testicular tissue from both food-deprived and normally-fed animals was used. Furthermore, leptin modulation of stimulated testosterone secretion was evaluated by incubation of testicular samples with different doses of leptin in the presence of 10 IU human chorionic gonadotrophin (hCG). In addition, analysis of leptin actions on pituitary function was carried out using hemi-pituitaries from fasted adult male rats incubated in the presence of increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Serum testosterone levels, and basal and hCG-stimulated testosterone secretion by incubated testicular tissue were significantly decreased by fasting in prepubertal and adult male rats. However, a significant reduction in circulating LH levels was only evident in adult fasted rats. Doses of 10(-9)-10(-7) M leptin had no effect on basal or hCG-stimulated testosterone secretion by testes from prepubertal rats, regardless of the nutritional state of the donor animal. In contrast, leptin significantly decreased basal and hCG-induced testosterone secretion by testes from fasted and fed adult rats. In addition, 10(-9) M leptin inhibited LH and FSH secretion by incubated hemi-pituitaries from fasted adult males, whereas, at all doses tested, it was ineffective in modulating PRL release. Our results show that leptin, depending on the state of sexual maturation, is able to inhibit testosterone secretion acting at the testicular level. Furthermore, the present data suggest that the actions of leptin on the reproductive system are complex and are probably carried out at different levels of the hypothalamic-pituitary-gonadal axis.

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L Pinilla, ML Barreiro, LC Gonzalez, M Tena-Sempere and E Aguilar

Hypothalamic differentiation in the female rat during the neonatal period is critically dependent on the steroid milieu, as permanent changes in reproductive function are observed after administration of oestradiol and testosterone during such a critical stage. Selective oestrogen modulators (SERMs) constitute a family of drugs that, depending on the tissue, are able to exert oestrogenic or antioestrogenic actions. The present experiments were conducted to analyse whether the SERMs, tamoxifen and raloxifene, can cause oestrogenic actions during the hypothalamic differentiation period. Postnatal female rats were injected between days 1 and 5 with 100 microg/day tamoxifen, raloxifene or ICI 182,780 (a pure antioestrogen). Other groups of animals were injected on day 1 of age with 100 microg oestradiol benzoate (OeB) or 1.25 mg testosterone propionate (TP) alone or in combination with raloxifene (500 microg/day between days 1 and 5). In all experimental groups, the age, body weight and concentrations of serum gonadotrophins at vaginal opening were recorded, whereas vaginal cyclicity and the negative and positive feedback between oestradiol and LH were monitored in adulthood. The results obtained confirmed the ability of high doses of OeB or TP to alter the normal differentiation of the brain permanently. They also reinforced the hypothesis that oestrogens are also necessary for normal brain differentiation in female rats because administration of a pure antioestrogen, such as ICI 182,780 permanently altered the function of the reproductive axis. In addition, our data provided evidence for different actions of the two SERMs under analysis (raloxifene and tamoxifen) upon peripheral targets, as raloxifene advanced vaginal opening whereas tamoxifen did not. In contrast, their actions on brain differentiation appeared similar and analogous to those obtained after neonatal administration of oestradiol, as evidenced by vaginal acyclicity, ovarian atrophy, sterility and abolition of negative and positive feedback between oestradiol and LH, thus suggesting an oestrogenic action of these SERMs on hypothalamic differentiation. Moreover, the oestrogenic activity of raloxifene was supported by its inability to block the effects of OeB and TP administered neonatally. In conclusion, the present results indicated that the SERMs, tamoxifen and raloxifene, exert an oestrogen-like effect upon hypothalamic differentiation of the neonatal female rat.