Hypothalamic aromatase activity in male and female rats during juvenile peripubertal development

Evidence exists that testosterone (T) regulates brain aromatase activity in adult rats. It is not known, however, whether the activity and/or its regulation by androgens change during the time of puberty. In the present study, we examined the change in basal aromatase activity associated with puberty in both male and female rats. We also assessed the influence of castration and treatment with a nonaromatizable androgen, dihydrotestosterone (DHT), on the hypothalamic aromatase system during juvenile and peripubertal development of male rats. Aromatase activity was estimated by both quantifying the 3H2O released from [1 beta-3H]T and by isolating the estrogen product(s) by thin-layer chromatography (TLC) after incubations with [1,2,6,7-3H]T. 5 alpha-Reductase activity was determined simultaneously in the male hypothalamus by TLC using [1 alpha-3H]T as the substrate. Aromatase activity was linear with time of incubation and amount of tissue used. It was detected at similar levels in both tissue fragments and acutely dispersed cell preparations. Expression in the latter, but not the former required the addition of NADPH. Intracellular rates of both aromatase and 5 alpha-reductase activities were highest in the mitochondrial-microsomal fraction. In both males and females the time of puberty was associated with a decrease in hypothalamic aromatase activity. In females, this drop was found to occur between the days of first proestrus and first estrus. In males, it occurred between 48 and 68 days of age (i.e., after the animals had reached puberty, as assessed by the presence of free sperm in the seminiferous tubules).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypothalamic, pituitary and testicular function during sexual maturation of the male rat.

Hypothalamic content of gonadotrophin-releasing hormone (GnRH), serum LH and FSH, capacity of the testis to synthesize testosterone in vitro, and testicular 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase and 17 beta-hydroxysteroid dehydrogenase were measured in groups of rats at approximately 5 day intervals from birth to day 64 and at days 74 and 89. The capacity of the testes to synthesize testosterone in vitro was measured in the presence of a saturating dose of rat LH. Gonadotrophin-releasing hormone increased steadily from 0-17 ng per hypothalamus at birth to a maximum of 7 ng at day 52 and then remained constant. LH concentrations were highly variable and often exceeded adult values between days 10 and 32. After day 32 a steady rise was observed which reached adult values between days 37 and 42. FSH concentrations markedly increased from 255 ng/ml observed at birth and day 10 to a peak value of 1000 ng/ml at day 32. Subsequently there was a steady decline in FSH values until day 74 when the concentration returned to values found at birth. 5-ene-3 beta-Hydroxysteroid dehydrogenase-isomerase activity exhibited a rapid increase between days 12 and 19 followed by an even greater rate of increase between days 19 and 32 when adult levels were attained. 17 beta-Hydroxysteroid dehydrogenase activity was very low between birth and day 22. Enzyme activity began to increase at day 22 with a rapid increase in activity observed between days 37 and 58. The increase in capacity to synthesize testosterone closely followed the increase in 17 beta-hydroxysteroid dehydrogenase activity. The study demonstrates that during sexual maturation in the male rat, changes in serum LH and FSH do not reflect changes in hypothalamic GnRH. The appearance of Leydig cells as monitored by 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity precedes by approximately 20 days the increase in testicular capacity to synthesize testosterone in vitro. The latter coincides with the increase in 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that 17 beta-hydroxysteroid dehydrogenase is a limiting factor in the ability of the testis to respond to LH stimulation.     

Hypothalamic dopamine and catechol oestrogens in rats with spontaneous pituitary tumours

Dopamine concentration within the hypothalamus and its depletion after the administration of alpha-methyl-para-tyrosine were measured in young rats and compared with values obtained in aged animals with and without spontaneously occurring pituitary tumours. Old rats had significantly reduced hypothalamic dopamine concentrations and there was less depletion of dopamine compared with young animals but there were no differences between tumorous and non-tumorous animals. Hyperprolactinaemia induced in young animals caused a much greater depletion of hypothalamic dopamine than in old tumorous rats with comparable plasma prolactin concentrations. The catechol oestrogen 2-hydroxyoestradiol inhibited the release of prolactin from normal rat pituitary glands in vitro but measurement of catechol oestrogens in the hypothalamus showed no differences between young and old tumorous or non-tumorous rats. It is concluded that reduced dopamine concentration and an impaired response to hyperprolactinaemia in old rats may facilitate the growth of prolactin-secreting tumours arising in the pituitary gland.     

Hypothalamic luteinizing hormone releasing factor and corticotrophin releasing activity in relation to pituitary and plasma hormone levels in male and female rats.

Hypothalamic corticotrophin releasing (CR) activity and LH-releasing factor (RF) content, and pituitary and plasma LH, FSH and ACTH were measured in adult male and female Wistar rats maintained under 14 h light per day. Hypothalamic LH-RF and pituitary and plasma hormones were estimated by radioimmunoassay while CR-activity was assessed by the amount of ACTH released from hemipituitaries in vitro. Two experiments were carried out on male animals. In the first, some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting regime, hypothalamic LH-RF content and pituitary gonadotrophin concentrations were significantly lower than the values in male rats kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there was no significant differences between the LH-RF contents measured at 3-4 h intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly increased at 12.00 h. There was little differences between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations (determined by competitive protein-binding assay) with the peak occurring between 15.00 and 18.00 h. The profiles of plasma and pituitary ACTH were similar to that of plasma corticosterone. Corticotrophin releasing activity dropped significantly between 12.00 and 16.00 h, but remained steady at the other times. In female rats there were no significant differences between hypothalamic LH-RF content throughout the 4-day cycle. During pro-oestrus the mean LH-RF content rose to teach a high level at 18.00 h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the preovulatory surge. Plasma FSH concentration also rose significantly between 15.00 and 18.00 h of pro-oestrus. At metoestrus and dioestrus, plasma FSH levels were lower in the morning than in the evening. These results suggest that (1) there is no diurnal rhythm in hypothalamic LH-RF, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of LH-RF does occur on the afternoon of pro-oestrus, the rate of LH-RF synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than daylength. The results provide further evidence for the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophysial control.     

Implication of pituitary vasoactive intestinal peptide in dopaminergic inhibition of estrogen-induced pituitary hyperplasia and vascular endothelial growth factor expression

We have shown that pituitary vasoactive intestinal peptide (VIP) mediates the effects of estrogen on lactotrope hyperplasia, angiogenesis and hyperprolactinemia, and reduces the pituitary content of transforming growth factor beta beta1 (TGF-beta1, an inhibitor of lactotrope proliferation). Dopamine agonists reverse lactotrope hyperplasia and hyperprolactinemia and also reduce the pituitary VIP content in hyperestrogenized rats. To elucidate the interaction of bromocriptine (BC) and pituitary VIP, a VIP receptor antagonist (VA), BC, or both drugs were administered for 5 days to F344 rats treated with diethylstilbestrol (DES). Both BC and VA similarly blocked the effects of DES on pituitary weight and pituitary content of prolactin (PRL), proliferating cell nuclear antigen, and vascular endothelial growth factor, without evidence of synergism. The estrogen effect on pituitary TGF-beta1 was completely inhibited by VA, but only partially by BC. On the contrary, serum PRL was close to the normal levels in the BC group 2 h after the first dose, while VA only reduced serum PRL after 5 days. DES increased VIP and VIP mRNA levels specifically at the pituitary, this effect being partially blocked by BC. These data suggest that the dopamine agonists inhibit lactotrope proliferation and angiogenesis by blocking the autocrine/paracrine action of VIP. On the other hand, the dopamine agonists inhibit the estrogen-induced hyperprolactinemia by acting through different pathways than those implicated in the proliferative process.     

Inhibition of estrogen-induced anterior pituitary enlargement and arteriogenesis by bromocriptine in Fischer 344 rats

The interrelationship between dopamine (DA) regulation and changes in the blood supply of the anterior pituitary lobe (AP) in the etiology of estradiol (E2)-induced proliferation of pituitary cells was studied in Fischer 344 rats. Rats were implanted with E2-filled or empty Silastic capsules for 21 days alone or in conjunction with pellets of the potent DA agonist bromocriptine (CB-154). Changes in vascularization of the AP, median eminence DA content, and responsiveness to DA of cultured AP cells were measured. Development of a direct arterial blood supply was assessed by the injection of 15-microns microspheres that can only reach the AP by newly formed arteries (arteriogenesis). APs were enzymatically dispersed and cultured for 3 days before challenges with increasing concentrations of DA for 3 h. DA content was measured by radioenzymatic assay, and serum PRL was determined by RIA. E2 treatment increased the weight of the pituitary gland, serum PRL levels, and the number of microspheres in the AP 4.5-, 173-, and 142-fold, respectively, over control values. Median eminence DA content was decreased 71% by E2 treatment, while the ability of DA to suppress PRL secretion in vitro decreased from a maximum of 70% to 40% with no change in the ED50. Simultaneous treatment with CB-154 dramatically decreased the effect of E2 on arteriogenesis, pituitary weight, serum PRL levels, and median eminence DA content. Blockade of E2-induced AP enlargement by increased dopaminergic stimulation was closely correlated with inhibition of arteriogenesis, which further suggests an important role for vascular changes in lactotroph proliferation.     

Effects of estrogen-induced hyperprolactinemia on endocrine and sexual functions in adult male rats

Chronic estrogen treatment can lead to development of prolactin (PRL) secreting pituitary tumors. We have tested the ability of diethylstilbestrol (DES) to produce persistent hyperprolactinemia (hyperPRL) in adult male rats and examined the effects of this treatment on hypothalamic-pituitary-testicular function, adenohypophyseal structure, copulatory behavior and fertility. Silastic capsules containing approximately 5 mg DES were subcutaneously implanted into adult male CDF (F-344)/CrlBR rats and removed 15 or 20 weeks later. Extreme hyperPRL, as well as suppression of plasma LH and FSH levels, persisted after DES capsules were removed. In contrast, plasma testosterone levels increased rapidly after removal of DES capsules and reached normal levels within 4-6 weeks. Copulatory behavior was assessed on two occasions between 7 and 14 weeks after removal of the DES capsules and was found to be suppressed in DES-treated rats, as evidenced by significant increases in latencies to mount, to intromit and to ejaculate. Moreover, when the animals were placed with normal females, the interval until conception was significantly greater in DES-treated than in control males. In spite of these differences in copulatory behavior, 10 of 11 DES-treated males were fertile. At autopsy, 44 weeks after capsule implantation (i.e. 24 or 29 weeks after capsule removal), DES-treated rats had marked enlargement of the anterior pituitary, increased weights of the lateral prostate and the adrenals, increased levels of testicular hCG-binding sites, reduced concentration of dopamine and norepinephrine in the median eminence and increased concentration of LHRH in the preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kallikrein gene expression in estrogen-induced pituitary tumors

Anterior pituitary kallkrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.     

Hypothalamic pituitary complications in Kabuki syndrome

Kabuki syndrome is characterized by distinctive facial features, multiple anomalies and mental retardation. In this syndrome, structural CNS abnormalities are commonly observed, but congenital abnormalities in the pituitary gland or hypothalamus have rarely been reported. We searched the published medical literature on the complications in hypothalamic pituitary axis in this syndrome. As a result, only nine patients with Kabuki syndrome had been reported to have complications in hypothalamic pituitary axis in previous papers. Among the nine reported patients and one presented case in this report, GH deficiency was the most frequent complication and found in six patients. Precocious puberty and central diabetes insipidus (DI) was identified in two cases, respectively, and ACTH deficiency was found in one. One case had combination of GH deficiency and central DI. Three of the 10 patients demonstrated abnormal pituitary findings in MRI study. Two of the six patients with GH deficiency were accompanied with premature thelarche. This review highlights that patients with Kabuki syndrome could present various clinical manifestations due to abnormalities in hypothalamic pituitary axis.     

Hypothalamic LH-releasing activity in young and aged intact and gonadectomized rats

Hypothalamic LH-releasing activity content was measured in young (3-5 mo) and aged (22-26 mo) intact and gonadectomized male and female rats. Hypothalamic extracts (0.25, 0.5 and 1.0 hypothalamus equivalents) from young and aged rats were incubated with untreated hemisectioned rat pituitaries in medium 199. All hypothalamic extract treatments stimulated LH release from the incubated pituitaries. Increased amounts of hypothalamic extracts added to to the incubation medium proportionally increased LH release. There were no differences in LH release stimulated by young or aged hypothalamic extracts from either the intact or gonadectomized groups. In addition serum testosterone concentrations were reduced in the aged male rats and serum LH was lower in aged male and female rats than in the young groups. Although serum LH was increased after gonadectomy in all groups, the increase was of smaller magnitude in the aged rats. These data indicate significant alterations in the responsiveness of the hypothalamus to steroid feedback in the aged rat. Although the hypothalamus contains sufficient LH-releasing activity to stimulate higher levels of pituitary and gonadal endocrine function, aging effects on the neuroendocrine control mechanisms inhibit hypothalamic hormone function.     

Hypothalamic lh-releasing activity in young and aged intact and gonadectomized rats

Hypothalamic LH-releasing activity content was measured in young (3–5 mo) and aged (22–26 mo) intact and gonadectomized male and female rats. Hypothalamic extracts (0.25, 0.5 and 1.0 hypothalamic equivalents) from young and aged rats were incubated with untreated hemisectioned rat pituitaries in medium 199. All hypothalamic extract treatments stimulated LH release from the incubated pituitaries. Increased amounts of hypothalamic extracts added to to the incubation medium proportionally increased LH release. There were no differences in LH release stimulated by young or aged hypothalamic extracts from either the intact or gonadectomized groups. In addition serum testosterone concentrations were reduced in the aged male rats and serum LH was lower in aged male and female rats than in the young groups. Although serum LH was increased after gonadectomy in all groups, the increase was of smaller magnitude in the aged rats. These data indicate significant alterations in fee responsiveness of the hypothalamus to steroid feedback in the aged rat. Although the hypothalamus contains sufficient LH-releasing activity to stimulate higher levels of pituitary and gonadal endocrine function, aging effects on the neuroendocrine control mechanisms inhibit hypothalamic hormone function.     

Interactions of photoperiod and ectopic pituitary grafts on hypothalamic and pituitary function in male hamsters

Exposure of adult male hamsters to short days (less than 12.5 light/day) leads to suppression of gonadal function which is secondary to reductions in gonadotropin and prolactin (PRL) secretion. PRL secretion is decreased in short days despite a reduction of dopaminergic (DA) input from the hypothalamus, suggesting that the pituitary may become more sensitive to the inhibitory effects of DA. Although hypothalamic DA metabolism is altered by short-day exposure, it is not known whether the DA system can respond to PRL feedback or whether these changes in DA or PRL levels are responsible for the observed changes in gonadotropin secretion. To address these questions, the effects of PRL-secreting ectopic pituitary grafts on hypothalamic catecholamine metabolism and the effects of experimental manipulations of catecholamine metabolism on PRL and gonadotropin secretion were evaluated in adult male hamsters exposed to a 14 h light: 10 h dark (14L:10D) or a 5L:19D photoperiod. Short-photoperiod exposure led to expected reductions in testes weight, plasma PRL levels, and in vitro PRL secretion, but circulating levels of luteinizing hormone or follicle-stimulating hormone were not affected. Norepinephrine and DA turnover in the median eminence and in the medial basal hypothalamus was also reduced in the 5L:19D as compared to the 14L:10D animals. Pituitary grafts elevated PRL levels and hypothalamic DA turnover in animals from either photoperiod, but in vitro PRL secretion was reduced only from the pituitaries of 14L:10D hamsters. Short-photoperiod exposure increased the ability of DA to suppress PRL secretion, and this effect could be reversed by the presence of an ectopic pituitary graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of pituitary FSH sialylation in male rats

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galβ1,3GlcNAcα2,3-sialyltransferase (ST3Gal III) and Galβ1,4GlcNAcα2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing α2,6-linked sialic acid is hormonally regulated in male rats.     

Daily illuminance levels affect pituitary prolactin in male rats

The 24-h patterns of melatonin, PRL, and gonadotropins in male rats maintained under natural lighting conditions have been found to differ from the patterns in rats kept under artificial lighting. In the present experiments we studied the role of different daily illuminances as a possible causative factor for the variation of the hormonal patterns. Three groups of male rats were kept under artificial lighting conditions (12 h on/12 h off), where the daily illuminance was 550, 110 or 25 lux. After a 7-day adaptation period the pineal content of melatonin, the serum levels of LH, FSH and PRL, and the pituitary content of these hormones were measured by RIAs in samples taken at 10.00, 13.00, 22.00 and 01.00 h. The patterns of pineal melatonin were equal in all three groups. The variation of daily illuminance did not change the serum levels of LH, FSH and PRL or the pituitary content of the gonadotropins. However, the pituitary content of PRL during the light phase was inversely related to the illuminance. The results suggest that the intensity of daily lighting in the studied range does not affect the patterns of melatonin or gonadotropins, but the synthesis of prolactin may be significantly regulated by the daily illuminance level.