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It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drugs for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP, indicating that the Chinese drugs for tonifying the kidney can regulate gene expression of the hypothalamic GnRH, pituitary FSH, LH, and osteoblastic BGP, which is possibly one of the main mechanisms of the Chinese drug for tonifying the kidney, regulating ephebic development process andimproving skeletal development in sexual precocity children.

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Immunoreactive gonadotropin-releasing hormone (ir-GnRH) was detected in brain extracts of newborn and 10-day-old rats and in adult guinea pigs; it was also present in extracts of the neural ganglion and gland of a protochordate. Radioimmunoassay (RIA) using different GnRH antisera after high-performance liquid chromatography (HPLC) revealed that the dominant form of GnRH is the mammalian form (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) both during ontogenesis in the rat and in the adult guinea pig known to have variant forms of other peptide hormones. None of the other forms of GnRH identified in nonmammalian species to date appear to be present in the rat or guinea pig. A small amount of an unidentified HPLC early eluting form of GnRH is present, but detection by antiserum B-6 implies that it is also mammalian GnRH, with the possibility of changes in positions 2-4. The molecular form of GnRH in a protochordate, the sea squirt Chelyosoma productum, is distinct from salmon and mammalian GnRHs. Cross-reactivity with the sea squirt GnRH-like molecule was highest with an antiserum made against lamprey GnRH; the same antiserum was used to stain nerve fibers in the neural ganglion and some of its roots. This is the first report using RIA, HPLC, and immunocytochemistry to show that protochordates have GnRH-like material. The results suggest that GnRH may have been present at the transition between the invertebrates and vertebrates.

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Although there have been several reports indicating the existence of GnRH or GnRH-like substance in the rat ovary, its synthesis as well as its physiological role in the ovary still remains unknown. In the present study, GnRH mRNA in the rat ovary was examined by using a cRNA probe complementary to the gene of the rat GnRH. The hybridizing band of 700bp was found in the extract from the rat ovary by northern blotting with 32P-labeled GnRH cRNA. In situ hybridization with 35S-labeled cRNA revealed that the site of synthesis of GnRH was granulosa cells and its synthesis was stimulated by the administration of PMSG. These results suggest that GnRH is synthesized in the rat ovary and may play a role in the local regulation of ovarian function.

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Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-D2Nal1,D4-Cl-Phe2,D-Trp3,D-Arg6, D-Ala10]- GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P less than 0.01) from 1.7 ng/ml at 0 h to less than 0.5 ng/ml during 4-32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 +/- 8 to 56 +/- 5 ng/ml (P less than 0.05) at 32 h. In males FSH was low (5.7 +/- 0.5 ng/ml) at 0 h and did not change significantly. To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P less than 0.01) to 0.2-0.4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P less than 0.001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P less than 0.05), but not on Day 24. Treatment of females with the GnRH antagonist did not influence (P greater than 0.10) oestradiol-17 beta concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17 beta values (P less than 0.01) and reduced testicular weight (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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The DNAs from two independent pancreatic cancers (tumors 1 and 2) in a patient with multiple endocrine neoplasia type 1 were analyzed. No amplification or gross rearrangement of 19 protooncogenes was observed. However, Southern blot analysis using polymorphic DNA probes revealed loss of heterozygosity at loci on chromosome 11p in both tumors. In tumor 1, an extensive region including the HRAS1, PTH, CALCA, and D11S151 loci was deleted, while in tumor 2 loss of heterozygosity was limited at the HRAS1 and D11S151 loci. Because loss of heterozygosity at other chromosomal loci in the two tumors was quite rare, loss of genes on 11p might be nonrandom. It is noteworthy that the same allele at the HRAS1 locus and also the same allele at the D11S151 locus were lost in the two independent tumors. These results suggest that loss of genes at the HRAS1 and/or D11S151 loci plays an important role unmasking the remaining sequences probably having a recessive mutation.

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Experiments were carried out to investigate the effects of ovariectomy on gonadotropin-releasing hormone (GnRH) messenger RNA (mRNA), proGnRH and GnRH peptide levels in the hypothalamus of female rats. Intact proestrous female rats and female rats, which had been ovariectomized for 2 weeks, were sacrificed at 9.00 h and the preoptic area (POA) and basal hypothalamus (BH) were dissected out and frozen on dry ice. One group of tissues from proestrous control and ovariectomized females were extracted in acetic acid, centrifuged at 13,000 g and the supernatant purified on a C18 column. The purified extract was then radioimmunoassayed for proGnRH, using a specific antiserum to rat proGnRH (ARK-2), and for GnRH using the E1-14 antiserum. Total cellular RNA was isolated from another group of tissues and prepared as Northern blots. Hybridization with 32P-labeled GnRH cRNA was used to detect GnRH mRNA. A third group of proestrous and ovariectomized female rats were perfused, and 50 microns vibratome sections were cut. These were immunostained with proGnRH or GnRH antiserum, followed by in situ hybridization with 35S-labeled GnRH cRNA to detect GnRH mRNA. Based on the histochemical staining, mRNA was colocalized to the cell soma of neurons containing proGnRH and GnRH throughout the POA and BH. Based on the radioimmunoassay, proGnRH levels were 2 times higher in the POA versus the BH, but GnRH levels were 6-7 times higher in the BH. Ovariectomy significantly decreased proGnRH levels in both the POA and BH, while GnRH decreased in the BH. In contrast, quantitative Northern blot analysis demonstrated that ovariectomy had no effect on mRNA levels in the POA and BH. These data indicate that the effects of ovariectomy on proGnRH and GnRH levels are a result of altered translation, posttranslational processing and/or secretion of GnRH.

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Pituitary gonadotrophin function is controlled to a great extent by the central nervous system and by the feedback action (positive and negative) of sex steroids. Neural structures involved in this mechanism may be divided into two levels. The first level is represented by the nervous structures releasing gonadotrophin-releasing hormone (GnRH) in a pulsatile manner into the portal circulation. The gene encoding the precursor protein for GnRH has been described recently. The precursor protein appears to be composed of 92 amino acids, in which the GnRH decapeptide is preceded by a signal peptide and followed by a peptide termed GAP for GnRH-associated peptide. The GnRH-synthesizing neurones and the nervous structures synchronizing the GnRH discharge (pulse generator) in the monkey, and probably also in the human, reside in the medial basal hypothalamus, which appears to have a highly integrated structure. The GnRH pulse generator is influenced by nervous structures outside the medial basal hypothalamus (second level of control) as well as by ovarian and other hormones. These influences probably impinge directly or indirectly on the hypothalamic oscillator. Concerning the chemical nature of the substances mediating the action, a large number of neurotransmitters and neuropeptides have been reported to influence GnRH secretion.

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A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.

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Normal human placenta and amnion from the 20th week of pregnancy were transplanted into nude mice. The transplants retained the histological and immunohistochemical picture of the original tissue and maintained synthesis of human chorionic gonadotropin (hCG), pregnancy-specific beta 1-glycoprotein (SP1), GnRH and placental proteins PP5, PP11 and PP12 for 5 weeks. This experimental model has a wide potential application for studies on placental protein synthesis and the hormonal influence of human placenta on other simultaneously transplanted human tissues.

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The challenge to define the hypothalamic-like hormones and to determine where they are produced in the placenta and how they control the production of other hormones such as human chorionic gonadotropin, progesterone, and estrogen, is an important current topic of investigation. This article reviews the existing data that have indicated or established the existence of placental hypothalamic-like peptide hormones.

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Although the mechanism of GnRH synthesis has not yet been elucidated, many experimental data argue in favour of a ribosomal pathway resulting in the biosynthesis of a precursor with a size larger than the decapeptide. The existence of high mol. wt (HMW) immunoreactive forms of GnRH in hypothalamic extracts has been reported but without any evidence that they are precursors of the hypothalamic hormone. Some results obtained with immunocytological methods also suggest that at least a part of immunoreactive GnRH in perikarya might be extended on one or both its terminal ends. In our laboratory, we first investigated the process of GnRH biosynthesis in vivo by infusing tritiated amino acids into the third ventricle of rats. The results, along with the demonstration of the existence of HMW GnRH, support a ribosomal mechanism of GnRH synthesis. Also, preliminary data from our laboratory demonstrate that poly (A+) enriched RNA preparations from rat hypothalami contain compounds capable of hybridizing to a synthetic oligodeoxynucleotide probe which is complementary to a part of the GnRH sequence.

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Hypogonadism in the mutant hpg mouse is characterized by a deficiency of hypothalamic gonadotropin releasing hormone (GnRH). Affected male mice exhibit immature reproductive organs, small abdominal testes and low pituitary and plasma gonadotropin concentrations. Recent studies have demonstrated the potential of fetal brain transplants to establish functional connections with host tissues. We therefore sought to use this approach to correct the hpg deficit. Fetal preoptic area (POA) (a site of GnRH production) from unaffected animals of the hpg strain was transplanted into the anterior third ventricle of adult hpg mice. We report that in such implanted animals, killed 2 months post-implantation, the POA grafts contained GnRH neurones, from which GnRH-positive fibres could be traced to capillaries of the median eminence. Hypothalamic GnRH and pituitary and plasma gonadotropin concentrations were increased compared with levels in untreated (hpg) animals. The testes were enlarged and had descended into the scrotum. Evidence of full spermatogenesis and interstitial cell development was present in testicular sections. No such effects were seen with transplants of cortical tissue.

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Normal female and ovariectomized rats were infused into the 3rd ventricle with [3H]glycine or [3H]alanine. Some rats were pretreated with cycloheximide. Hypothalamus, anterior pituitary and fragments of cortex were excised, homogenized, extracted and treated with specific antiserum to GnRH, bound to Sepharose. The radioactivity of immuno-absorbed products was counted either immediately of after extraction and thin-layer chromatography by using two different solvent systems. With the two systems, the location of the immuno-absorbed radioactivity always coincided with the spot of synthetic GnRH. Our results show that [3H]glycine was incorporated, as a function of time, into GnRH isolated from rat hypothalami. The amount was incorporated, as a function of time, into GnRH isolated from rat hypothalami. The amount of radioactivity incorporated into hypothalami from diestrous-I rats was similar to that of ovariectomized rats and twice as high as in late proestrous rats. Only minute amounts of radioactivity were incorporated into the immuno-absorbed product. Cycloheximide inhibited incorporation of [3H]glycine into the immuno-absorbed product to the same extent as its incorporation into the total protein from the hypothalamus. Our experimental results support the hypothesis of ribosomal mechanisms being involved in the biosynthesis of GnRH. They also suggest that the rate of accumulation of newly synthesized labeled GnRH is of the same order in the hypothalamus of ovariectomized rats as in diestrous-I rats.

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Nuclear and nucleolar volumes of the cells of some hypothalamic centers (SON, PVN, SCN, VMN, AN) were estimated in control rats and in those with deafferented medial basal hypothalamus. There is sex difference in measured values among parvocellular formations of control animals: nuclei and nucleoli of the neurons in AN and in VMN and nucleoli of the neurons in SCN are larger in females than in males. Deafferentation led to an increase in the nuclear volume, especially in the formations outside the isolated area. It was more pronounced in rats with persistent estrus. The difference between estrous and diestrous rats is statistically significant for volumes of nuclei and nucleoli of the neurons in SCN and not for those in AN, inspite of the impressive diversity of the pituitary LH content. The suggestion is made that gonadoliberins are not secreted by the cells of AN, and that SCN might control the stage of the estrous cycle.

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In insulin-dependent diabetics high GH values are usually observed. The OGTT does not modify these levels. It was observed that CB 154 (2-Br-alpha ergocryptine) seems capable of lowering GH in acromegalics. We thought it pertinent to check whether or not CB 154 could reduce GH also in diabetics. OGTT was performed in insulin-dependent diabetic patients; 5 days later the same test was repeated after pretreatment with 2.5 mg of CB 154. Serum IRI, GH, hPRL levels and blood glucose values were checked during the test. Significant GH and hPRL variations were not observed after OGTT in the diabetic subjects examined; IRI was always absent, and glycemia followed the model of the diabetic curve. On the contrary, after pretreatment with CB 154, we observed: 1) hPRL reduction; 2) GH increase, sometimes as early as 30 min after OGTT; 3) IRI was never present and blood glucose levels were lower than the values observed with glucose alone. Contrary to what is observed in acromegalics, CB 154 is unable to lower the increased GH levels normally present in insulin-dependent diabetics. After pretreatment with CB 154, OGTT seems to release GH, which is not observed after glucose alone. This can be accounted for only by postulating an antagonism at the hypothalamic level between the adrenergic and serotoninergic pathways of GH stimulation.

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