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M. D. Gonzalez, F. López, and E. Aguilar

ABSTRACT

Pimozide (1 mg/kg per day), bromocriptine (1 mg/kg per day) or domperidone (0·1 mg/kg per day) administered daily to rats from day 21 did not change the age at which vaginal opening occurred, nor did they affect the body weight at that age. Therefore the evolution of prolactin levels was different in these three groups. The pimozide-treated group showed high prolactin levels measured on day 23, at vaginal opening and at first oestrus. In the bromocriptine-treated group, levels were undetectable on the day of vaginal opening. Chronic treatment with domperidone failed to increase prolactin levels on day 23 and at vaginal opening. Nevertheless, large increases were observed after a single injection of domperidone at both 21 and 30 days of age.

A significant increase in LH observed on day 23 in the pimozide-treated group was the only effect on gonadotrophin levels which was detected. Ovarian weights were unaffected by the treatments, whereas adrenal weight was increased in the bromocriptine-treated group and decreased in the pimozide- and domperidone-treated groups.

Female rats grafted on day 21 with one additional pituitary gland from adult (90 days) or young (21 days) donors showed a similar advancement in the time of vaginal opening, although the animals bearing an adult pituitary gland showed higher prolactin levels than those observed in animals grafted with young pituitary glands.

This study suggested that the onset of puberty is not closely linked with the evolution of prolactin levels and that the hormone itself is not indispensible for the process.

J. Endocr. (1984) 101, 63–68

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L Gonzalez, AI Sotelo, A Bartke, and D Turyn

To study the effects of homologous mouse GH (mGH) on the presence and characteristics of serum GH-binding protein (GHBP) we have used transgenic mice expressing GH-releasing hormone (GHRH) as a model. Chromatographic techniques allowed the characterization of GHBP bioactivity, and immunological techniques were used to determine its concentration and molecular components. Chromatographic separation of labeled human GH or mGH cross-linked to serum GHBPs showed two GH-binding serum fractions in normal as well as in transgenic mice serum. SDS-PAGE of this material revealed a specific band of 66 kDa and another higher molecular weight broad band, which, in the presence of 2-mercapto-ethanol, is converted into the 66 kDa fraction.Since normal mice serum has an mGH concentration of 0. 40+/-0.06 nM and a GHBP concentration of 5.7+/-1.1 nM, while the high-affinity site for mGH has a K(d)</+/-27 nM, only a small percentage (2.9%) of total serum mGH is bound to GHBP in the sera of these mice. In transgenic mice serum, where the mGH concentration is 60 times higher (23+/-2.7 nM), 22.5% of total serum mGH is bound to serum GHBP. These values agree with the experimental data (4+/-2% and 17+/-4% for normal and transgenic mice serum respectively).The concentration of GHBP in GHRH transgenic mice was found to be increased four- to tenfold, depending on the technique used. This increment closely resembles the increased concentration of GHBP in the serum of transgenic bovine GH (bGH) mice, in which peripheral bGH levels are grossly elevated. Our results support the idea that the circulating levels of mGH in normal mouse serum are capable of influencing the levels of GHBP in peripheral circulation in a way similar to that of bGH, in spite of the different affinities of these two hormones. The fact that the up-regulation of GHBP occurs, even though a small percentage of mGH is bound in these animals, strongly suggests the existence of a physiological function for GHBP. These results also question some of the assigned or attributed physiological roles of GHBP, at least in the mouse, since only a negligible percentage of total mGH would be prevented from degradation and/or renal filtration by binding to GHBP. This small percentage of bound mGH also invalidates its role as a reservoir or a buffer of mGH concentration during pulses of GH release or rapid changes of mGH levels. Our results also demonstrate the presence of high molecular weight forms of GH-GHBP complexes that could be dissociated by dilution or in the presence of 2-mercapto-ethanol.

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C Bellido, D Gonzalez, R Aguilar, and JE Sanchez-Criado

We have previously shown that administration of antiprogestin (AP) type II RU486 to ovariectomized (OVX) rats on the morning of pro-oestrus decreases the magnitude of preovulatory gonadotrophin surge. This suggests that the effect of RU486 on LHRH-dependent gonadotrophin release may be independent of its ability to block progesterone actions. The aim of the present research was to study the possible site of RU486 action and to determine whether the gonadotrophin suppressive effect of APs RU486 and ZK299 is dependent on the oestrogen background. Intact or OVX rats in the morning of pro-oestrus were injected s.c. with 4 mg of RU486 or ZK299 (AP type I) at 0900 h on pro-oestrus. At 1830 h, serum concentration of FSH and LH and median eminence (ME) content of LHRH were determined. In the second experiment, the effect of RU486 and ZK299 on pituitary responsiveness to LHRH (100 ng, i.p.) and ME content of LHRH at 1830 h pentobarbital-blocked intact or OVX rats was evaluated. In the last study, the anterior pituitary release of FSH and LH from pro-oestrus or metoestrus donors incubated with or without LHRH (1, 10 or 100 nM) in the presence or absence of APs (20 nM) was evaluated. Both APs reduced serum FSH and LH levels at 1830 h on pro-oestrus in intact and OVX rats. The suppressive effect on gonadotrophin release brought about by AP treatment was also evidenced in PB-blocked intact and OVX rats. This suggested that the inhibitory effect of APs occurred, at least in part, at pituitary level. Furthermore, in the absence of the natural ligand, APs significantly reduced basal and LHRH-stimulated FSH and LH release from pro-oestrous but not from metoestrus pituitaries. In conclusion, these experiments have shown, both 'in vivo' and 'in vitro', that APs RU486 and ZK299 have suppressive effects at pituitary level on basal and LHRH-stimulated FSH and LH secretion, regardless of their antiprogestagenic activity, in pro-oestrus but not in metoestrus.

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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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A Logan, C Smith, G P Becks, A M Gonzalez, I D Phillips, and D J Hill

Abstract

Transforming growth factor-β1 (TGF-β1) has been reported to influence the growth rate and iodine uptake and organification in vitro by isolated thyrocytes. We have determined changes in the expression and presence of TGF-β1 within the rat thyroid during goitre induction, and subsequent involution following goitrogen withdrawal. Hyperplastic goitres were induced in adult rats by administration of methimazole together with a low iodine diet for up to 12 weeks. Goitrogen-treated rats quickly became hypothyroid compared with controls, and exhibited thyroid hyperplasia and hypertrophy assessed by thyroid weight, and DNA and protein content (control: total serum thyroxine (T4) 66 ± 4 nmol/l, thyroid weight 5 ± 1 mg/100 g body weight, mean ± s.d., n = 10; 2 weeks goitrogen: T4 undetectable, thyroid weight 27 ± 4 mg/100 g, n = 10). Thyroid growth rate slowed subsequently between 2 and 10 weeks. Messenger RNA for TGF-β1 was compared in the thyroids and livers of control and goitrous rats by ribonuclease protection assay. Low levels of mRNA for TGF-β1 were detected in thyroids from control rats at all time-points, while TGF-β1 mRNA was barely detectable in liver. Thyroid TGF-β1 mRNA levels substantially and progressively increased at 1 and 2 weeks of goitrogen treatment respectively, and remained above control levels at 4 and 10 weeks. As thyroid involution occurred 4 weeks following goitrogen withdrawal, so thyroid TGF-β1 mRNA levels declined. In control animals, the cellular localization of TGF-β1 mRNA, determined by in situ hybridization, was found to be a subpopulation of follicular epithelial cells, and immunohistochemical co-localization of TGF-β1 and calcitonin identified these tentatively as parafollicular or C-cells. During goitre formation, abundant TGF-β1 mRNA and peptide were found to be widely distributed within the entire follicular epithelium. While this ubiquitous distribution had largely disappeared in the involuting gland, TGF-β1 peptide was retained within the parafollicular cells, which appeared more abundant than in thyroids from control animals. These results suggest that an increased local expression of TGF-β1, a putative growth inhibitor, during thyroid hyperplasia may contribute to the temporal stabilization of goitre size.

Journal of Endocrinology (1994) 141, 45–57

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A. López-Calderón, C. Ariznavarreta, M. D. Calderón, J. A. F. Tresguerres, and M. I. Gonzalez-Quijano

ABSTRACT

The response of prolactin to chronic stress in intact, adrenalectomized and adrenomedullectomized male rats was studied. Immobilization stress in intact animals induced a significant increase in plasma concentrations of prolactin after 20 and 45 min and a significant decrease when the rats were submitted to chronic restraint (6 h daily for 4 days). Five weeks after adrenomedullectomy, plasma prolactin and corticosterone responses to chronic stress were not modified. In contrast, the inhibitory effect of chronic stress on prolactin secretion was totally suppressed by adrenalectomy. When treated with dexamethasone during the 4 days of restraint, adrenalectomized stressed rats showed similar plasma concentrations of prolactin to the intact stressed rats. These data indicate that the adrenal cortex is able to play an inhibitory role on prolactin secretion during stress only through a prolonged release of glucocorticoids.

Journal of Endocrinology (1989) 120, 269–273

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JE Sanchez-Criado, C Bellido, M Tebar, A Ruiz, and D Gonzalez

Administration of 4 mg of the antisteroid RU486 over 8 consecutive days to adult male rats dissociated in vivo and in vitro gonadotrophin secretion, increasing FSH and decreasing LH secretion. In subsequent experiments we evaluated the involvement of testicular or adrenal secretory products, as well as hypothalamic LHRH, in the effects of 4 consecutive days of RU486 treatment on the secretion of gonadotrophins. The first day of RU486 injection was designated day 1, subsequent days being numbered consecutively. Groups of rats injected with oil (0.2 ml) or RU486 (4 mg) were: (i) injected s.c. from day 1 to day 4 with the antiandrogen flutamide (10 mg/kg); (ii) bilateral orchidectomized (ORCH) on day 1; and (iii) bilateral adrenalectomized (ADX) on day 1. Controls were given flutamide vehicle or were sham operated. To ascertain whether the secretion of LHRH is involved in the effects of RU486 on gonadotrophin secretion, we measured the LHRH secretion into the pituitary stalk blood vessels at 1100 h on day 5 in oil- or RU486-treated rats. Additional oil- and RU486-treated rats were injected i.p. with 100 ng LHRH at 1000 h on day 5, or s.c. with 1 mg LHRH antagonist (LHRH-ANT) at 1000 h on days 2 and 4. Controls were given saline. All animals were decapitated at 1100 h on day 5, trunk blood collected and serum stored frozen until FSH, LH and testosterone assays.%While ADX had no effect on FSH and LH secretion in either oil- or RU486-treated rats, the removal of androgen negative feedback with flutamide treatment or by ORCH substantially increased serum levels of FSH and LH in both oil- and RU486-treated rats, and thus annulled the effects of RU486. No differences in pituitary stalk plasma LHRH concentrations were found between oil- and RU486-treated rats. Injection of LHRH increased serum FSH and LH concentrations in oil-treated rats but only, and to a lesser extent, LH concentrations in RU486-treated rats. Treatment with LHRH-ANT decreased serum concentrations of FSH and LH in both oil- and RU486-treated rats. These results suggest that RU486 inhibited LHRH-stimulated LH secretion at the pituitary level, and that FSH secretion increased in response to a reduction in the negative feedback of androgen.

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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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J E Sánchez-Criado, G Hernandez, C Bellido, D Gonzalez, M Tébar, M A Diaz-Cruz, and R Alonso

Abstract

The antiprogesterone RU486 injected on the morning of pro-oestrus blunts the preovulatory secretion of LH and FSH and abolishes the secondary secretion of FSH during oestrus without affecting ovulation in the rat. To ascertain whether the secretion of LHRH is involved in these effects, we studied the effects of RU486 (4 mg/0·2 ml oil), given s.c. at 0800 h on pro-oestrus, on LHRH secretion into the pituitary stalk blood vessels and on peripheral plasma concentrations of LH and FSH at 1800 h on pro-oestrus and 0200 h on oestrus. Furthermore, we determined the effects of an s.c. injection of 1 mg of an LHRH antagonist (LHRH-A; ORG30276) at 2000 h on pro-oestrus and those of an i.p. injection of 100 ng LHRH (Peninsula 7201) at 0100 h on oestrus on serum concentrations of LH, FSH and oestradiol at 0200 h on oestrus in oil- and RU486-treated rats.

RU486 decreased LHRH secretion at 1800 h on prooestrus while this was increased at 0200 h on oestrus. While the reduction of preovulatory LHRH secretion in RU486-treated rats coincided with a reduction in both LH and FSH surges during the evening of pro-oestrus, the increased LHRH secretion during the early hours of oestrus was only accompanied by an increased concentration of LH. An injection of LHRH stimulated, while that of LHRH-A inhibited serum concentrations of LH at 0200 h on oestrus in both oil- and RU486-treated rats. An injection of LHRH-A had no effect on FSH concentration at 0200 h on oestrus in either oil- or RU486-treated rats. On the contrary, exogenous LHRH increased FSH concentration at 0200 h on oestrus only in oil-treated rats.

The results indicate that, in the rat, progesterone secretion during the afternoon and evening of pro-oestrus enhances preovulatory LHRH and suppresses LHRH release during early oestrus into the pituitary stalk blood vessels on the afternoon of pro-oestrus and during early oestrus respectively. While the secretion of LH during early oestrus is blunted by progesterone and entirely coupled to LHRH secretion, the secondary secretion of FSH during oestrus is not dependent on endogenous LHRH and at the same time is completely dependent on the actions (direct and/or indirect) of progesterone.

Journal of Endocrinology (1994) 141, 7–14

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N Zmora, D Gonzalez-Martinez, JA Munoz-Cueto, T Madigou, E Mananos-Sanchez, SZ Doste, Y Zohar, O Kah, and A Elizur

The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.