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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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E. Aguilar, M. L. Rodríguez-Padilla, and L. Pinilla

ABSTRACT

Prolactin has been involved in different types of hypertension both in man and in rats. In an attempt to substantiate this hypothesis, we have analysed the correlation between plasma concentrations of prolactin and systolic blood pressure (SBP) in female and male rats from spontaneously hypertensive (SH) and normotensive Wistar–Kyoto strains (30, 60 and 90 days old), as well as in adult female Wistar rats rendered hyperprolactinaemic by the administration of 100 μg testosterone propionate on day 1 of life, or adult males with low plasma concentrations of prolactin after administration of bromocriptine (4 mg/kg per day) over 15 days. Our results indicate a lack of correlation between plasma concentrations of prolactin and SBP since plasma concentrations of prolactin were normal in male and female SH rats and hyper- and hypoprolactinaemia did not affect SBP.

In spite of these normal plasma concentrations of prolactin, SH rats showed subtle changes in the secretion of this hormone in vitro and in vivo in response to exogenous serotonin administration and to immobilization.

Journal of Endocrinology (1990) 125, 359–364

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F Gaytan, L Pinilla, J L Romero, and E Aguilar

Abstract

Neonatal and prepubertal male rats were treated with human chorionic gonadotropin (hCG, 5 IU/g body weight per day) on days 2–4 or 20–22. Depending on the date of treatment, different groups of rats were sacrificed at 5, 23, 30 and 100 days of age, in order to study the short-and long-term effects of the treatment with hCG on the development of the testes and sex accessory organs. Rats treated with hCG on days 2–4 showed increased number and size of foetal Leydig cells at 5 days of age. However, long-term effects include decreased numbers of adult-type Leydig cells, decreased weight of the testes and sex accessory organs, decreased basal and hCG-stimulated testosterone secretion, and delayed balano-preputial separation. In contrast, animals treated with hCG on days 20–22 showed similar short- and long-term effects, consisting of increased number of adult-type Leydig cells and macrophages, increased weight of the testes and sex accessory organs and advanced balano-preputial separation. In adulthood, both groups showed normal reproductive function.

These results seem to indicate that the effects of hCG treatment in prepubertal rats are dependent on the type of Leydig cell stimulated, and suggest that foetal Leydig cells play a regulatory role in the early postnatal testicular development.

Journal of Endocrinology (1994) 142, 527–534

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M. Rodriguez-Padilla, C. Bellido, L. Pinilla, and E. Aguilar

ABSTRACT

Weights of testes, seminal vesicles, ventral prostate and pituitary, plasma testosterone and LH concentrations, pituitary LH content and concentration, the LH in-vivo response after LHRH administration (1 μg), and basal and LHRH-stimulated secretion in vitro were analysed in adult male spontaneously hypertensive (SH) and normotensive control (WKY) rats.

Spontaneously hypertensive rats showed: (1) testis and pituitary hypertrophy; (2) seminal vesicle and ventral prostate atrophy; (3) increased plasma testosterone and LH concentrations; (4) increased pituitary LH content and concentration; (5) unchanged net increase of plasma concentrations of LH 15 and 45 min after administration of 1 μg LHRH; and (6) increased basal LH secretion in vitro with a normal response to LHRH stimulation.

These results provide evidence that SH rats show increased LH secretion with a normal response to LHRH stimulation. The coexistence of high plasma concentrations of testosterone with seminal vesicle and ventral prostate atrophy suggest a reduction in the effectiveness of testosterone in these structures.

J. Endocr. (1987) 113, 255–260

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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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P. Garnelo, L. Pinilla, F. Gaytan, and E. Aguilar

ABSTRACT

Acute and long-term effects of neonatal and prepubertal treatments with an LH-releasing hormone agonist (LHRH-A) were studied in Wistar male rats. Animals injected with d-Ala6-d-Gly10-LHRH ethylamide (2 μg/kg per day) or vehicle from days 1 to 15 or from days 16 to 29 were killed at different ages. Treatment between days 1 and 15 induced a decrease in both pituitary FSH and LH content as well as a reduction in plasma FSH and blockade of the response to LHRH. These effects were apparent on day 16 after treatment. Basal and human chorionic gonadotrophin (hCG)-stimulated progesterone and testosterone secretion in vitro was similar in testes from male rats treated with LHRH-A or vehicle. Reduced testicular weight was observed until day 90, whereas puberty, spermatogenesis and fertility were unaffected. The decrease in plasma FSH concentrations after neonatal treatment with LHRH-A was also found in groups of animals killed on day 10 and was possibly the cause of reduced testicular weight, since treatment with FSH from day 1 to day 15 blocked the effect of LHRH-A. Likewise, treatment with LHRH-A from day 1 to day 15 also reduced FSH and LH secretion in males orchidectomized on day 1 of life.

Animals injected with LHRH-A from day 15 to day 29 exhibited, at the end of the treatment period, reduced testicular weight, and decreased pituitary gonadotrophin content and plasma FSH concentrations, whereas LH plasma concentrations were normal. In adulthood, the pituitary-testis function did not vary from normal. Our results demonstrate that: (1) administration of LHRH-A from day 1 to day 15 of life desensitized the gonadotrophs, which in turn lowered plasma gonadotrophin concentrations and caused a long-term reduction in testis weight without changes in the quality of spermatogenesis or reproductive activity in adulthood; (2) chronic treatment with LHRH-A during the neonatal period did not result in a steroidogenic lesion; and (3) administration of LHRH-A from day 15 to day 29 produced only a transient reduction in testicular weight and in spermatogenesis.

Journal of Endocrinology (1992) 134, 269–277

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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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L. Pinilla, P. Garnelo, F. Gaytan, and E. Aguilar

ABSTRACT

Neonatal oestrogen administration to male rats permanently impaired the function of the pituitary-testicular axis possibly by inhibiting neonatal gonadotrophin secretion. To analyse the hypothalamus and/or pituitary involvement in this inhibition, pituitary responsiveness to acute stimulation with LH-releasing hormone (LHRH) was studied in vivo and in vitro in Wistar male rats injected on day 1 of age with oestradiol benzoate (OB) or olive oil. FSH and LH pituitary content and plasma concentrations were reduced in oestrogenized male rats at days 10 and 16 of age. Likewise, the in-vivo increase in gonadotrophin plasma concentrations after acute stimulation with LHRH was almost completely suppressed in 10-and 16-day-old oestrogenized males. In vitro, the increased secretion of FSH after LHRH stimulation was abolished and the LH response strongly reduced in pituitaries from oestrogenized males. Finally, the effects of neonatal oestrogenization were not abolished by treatment from day 1 to day 15 with an LHRH agonist (0·01 μg/kg per 12 h). We conclude that in male rats the effects of oestrogenization are due to both a reduction in LHRH endogenous secretion and a decrease in the pituitary responsiveness to LHRH.

Journal of Endocrinology (1992) 134, 279–286

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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.