Potent action of RFamide-related peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion.

We identified a gene in the ovine hypothalamus encoding for RFamide-related peptide-3 (RFRP-3), and tested the hypothesis that this system produces a hypophysiotropic hormone that inhibits the function of pituitary gonadotropes. The RFRP-3 gene encodes for a peptide that appears identical to human RFRP-3 homolog. Using an antiserum raised against RFRP-3, cells were localized to the dorsomedial hypothalamic nucleus/paraventricular nucleus of the ovine brain and shown to project to the neurosecretory zone of the ovine median eminence, predicating a role for this peptide in the regulation of anterior pituitary gland function. Ovine RFRP-3 peptide was tested for biological activity in vitro and in vivo, and was shown to reduce LH and FSH secretion in a specific manner. RFRP-3 potently inhibited GnRH-stimulated mobilization of intracellular calcium in gonadotropes. These data indicate that RFRP-3 is a specific and potent mammalian gonadotropin-inhibiting hormone, and that it acts upon pituitary gonadotropes to reduce GnRH-stimulated gonadotropin secretion.

Regulative actions of the Chinese drugs for tonifying the kidney on gene expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP.

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.

Transgenic studies on the regulation of the anterior pituitary gland function by the hypothalamus.

The anterior pituitary gland is composed of five different cell types secreting hormones whose functions include the regulation of post-natal growth (growth hormone, GH), lactation (prolactin, PRL), reproduction (luteinising hormone, LH, and follicle stimulating hormone, FSH), metabolism (thyroid stimulating hormone, TSH), and stress (adrenocorticotrophic hormone, ACTH). The synthesis and secretion of the anterior pituitary hormones is under the control of neuropeptides released from the hypothalamus into a capillary portal plexus which flows through the external zone of the median eminence to the anterior lobe. This review describes the ways that gene transfer technologies have been applied to whole animals in order to study the regulation of anterior pituitary function by the hypothalamus. The extensive studies on these neuronal systems, within the context of the physiological integrity of the intact organism, not only exemplify the successful application of transgenic technologies to neuroendocrine systems, but also illustrate the problems that have been encountered, and the challenges that lie ahead.

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis.

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

Isolation and characterization of the human gonadotropin-releasing hormone gene in the hypothalamus and placenta.

The GnRH gene has been cloned in several species, but the location of the promoter and the exact start of transcription have not previously been determined. To characterize the low abundance human GnRH mRNA in the hypothalamus and placenta, we have employed the polymerase chain reaction. The hypothalamus was found to have a 61-base pair first exon, and its transcriptional start site was determined. The human hypothalamic GnRH cDNAs isolated thus far have all contained a short 5' untranslated region which would correspond to this start site. However, all human placental GnRH cDNAs reported to date have a long 5' untranslated region, which extends more than 140-base pairs 5' to this start site in the hypothalamus, suggesting the utilization of an alternative promoter in the placenta. In addition, the human GnRH gene undergoes differential splicing in these tissues. The first intron is removed from the hypothalamic, but retained in the placental, GnRH mRNA. Thus, the placenta has a very long first exon, while the hypothalamus has a comparatively short first exon, followed by a long first intron. This characterization of the human GnRH gene will now allow hormonal regulatory studies to be performed using gene transfer techniques.

[Gene expression of gonadotropin releasing hormone in the rat ovary].

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.

The gene encoding GnRH and its associated peptide GAP: some insights into hypogonadism.

The hypogonadal (hpg) mouse represents a unique animal model for hypogonadism. In this mutant the truncation of the gene encoding GnRH and its associated peptide GAP leads to drastically lowered gonadotropin levels and increased circulating prolactin. This deficiency in turn leads to a failure of testes and ovaries to develop normally. Using gene therapy we have restored the reproductive functions of the hpg mouse. The success of this therapy uniquely underscores the importance of the gene encoding the GnRH precursor and lends credence to the hypothesis that no other gene in mammals can replace it. As a consequence, defects in the control and/or structural properties of the human GnRH are expected to result in hypogonadism in humans.

[GnRH and its analogs--structure, mechanism of action and therapeutic applications]. / La GnRH et ses analogues--structure, mecanisme d'action et applications thérapeutiques.

In the last decade, much has been learned about the physiology and cellular biology of GnRH. In addition more than 2000 analogs agonists and antagonists have been synthesized. The GnRH precursor cDNA has been cloned from human placenta and hypothalamus mRNA's. The GnRH gene is located on the short arm of chromosome 8. The mechanism of action of GnRH requires Ca and its two intracellular receptors calmodulin and protein kinase C. The physiological effect of GnRH is to induce the release of both gonadotropins and to increase the alpha and beta subunit mRNA. In addition, GnRH stimulates terminal glycosylation of LH and FSH. Pulsatile GnRH exposure induces an up regulation of the receptors. In contrast continuous GnRH or GnRH agonist administration induces a receptor loss and a pituitary desensitization. The process of desensitization is unclear and requires some post receptor events which remain to be elucidated. Changes in the aminoacid composition of GnRH result in 2 classes of analogs agonists and antagonists. Both are used to induce a reversible medical castration. GnRH agonists lead to an initial rise in gonadotropins and gonadal steroid secretion. They are not able to suppress bioactive FSH. In contrast, GnRH antagonists compete with GnRH for its receptors and have an immediate and sustained suppressive effect on LH and FSH secretion. GnRH analogs are useful tools to study the gonadotropin regulation.

The rat gonadotropin-releasing hormone: SH locus: structure and hypothalamic expression.

The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5'-sequences spliced to a common 3'-region.

Pubertal changes in gonadotropin-releasing hormone and proopiomelanocortin gene expression in the brain of the male rat.

Pubertal development in mammals is in part attributable to a brain-dependent process, whereby increased pulsatile GnRH secretion leads to the awakening of the entire reproductive system. However, the brain mechanisms controlling this event are unknown. The apparent increase in GnRH secretion at puberty could reflect an autonomous change in the activity of GnRH neurons themselves or in the afferent networks leading to GnRH neurons. If there were a significant increase in the secretion of GnRH with puberty onset, we hypothesized that there would be a commensurate increase in the biosynthetic capacity of GnRH neurons to meet the increasing demand. We tested this hypothesis by comparing the level of cellular prepro-GnRH mRNA (GnRH mRNA) observed between prepubertal (25-day-old; n = 5) and adult (75-day-old; n = 4) male rats by in situ hybridization. We detected no significant change with puberty in GnRH mRNA signal levels in any of the anatomical areas examined, which included the vertical limb of the diagonal band of Broca, medial septum, lateral preoptic area, and medial preoptic area. Given the variance of our analytical technique, we determined that there was a greater than 90% probability that we would have detected a 20% increase in GnRH mRNA had there been one. Endogenous opioid peptides have been implicated in timing the onset of puberty in the rat, with the argument being that a loss in opioid tone could effect a disinhibition of GnRH secretion. One opioid peptide, beta-endorphin, is among several peptides cleaved from the precursor POMC. We hypothesized that with puberty, POMC neurons in the arcuate nucleus would have an attenuated capacity to produce beta-endorphin. We tested this hypothesis by comparing cellular pre-POMC mRNA (POMC mRNA) levels in the arcuate nuclei of prepubertal (n = 6) and adult (n = 7) male rats with in situ hybridization. We observed an increase in POMC mRNA levels with puberty; prepubertal rats had relative POMC mRNA signal levels of 119 +/- 10 grains/cell, while adult rats contained 167 +/- 12 grains/cell (P less than 0.02). This increase in cellular POMC mRNA was confined to the rostral portion of the arcuate nucleus. We conclude that the GnRH gene is fully expressed well before the time of normal puberty onset and that the increase in POMC mRNA that occurs with the onset of puberty may be important for the development of pulsatile GnRH secretion.

Combined immunohistochemistry for gonadotropin-releasing hormone (GnRH) and pro-GnRH, and in situ hybridization for GnRH messenger ribonucleic acid in rat brain.

Experiments were performed to explore the distribution of neurons containing pro-GnRH and GnRH in the rat brain and to determine the correspondence of immunoreactive peptides and pro-GnRH mRNA. Using avidin-biotin immunohistochemistry on free floating vibratome sections it was found that pro-GnRH- and GnRH-containing cells exhibited a similar distribution within the preoptic area-basal hypothalamus region. Within individual neurons pro-GnRH was primarily detected in the cell soma and proximal fibers, whereas the decapeptide was present in cells, fibers, and nerve terminals. Combined avidin-biotin immunohistochemistry for GnRH or pro-GnRH peptides and in situ hybridization for pro-GnRH mRNA using a cRNA probe revealed that the peptides and mRNA could be detected in the same cells. In both male and female rats pro-GnRH mRNA was localized primarily in GnRH-containing cells; however, not all immunoreactive GnRH neurons contained detectable levels of pro-GnRH mRNA, and not all neurons containing pro-GnRH mRNA contained GnRH peptides. In proestrous females a close correlation existed between the total number of neurons containing GnRH and those containing pro-GnRH mRNA (r = 0.84-0.9), while in intact male rats the correlation was not as high (r = 0.56). These results document the distribution of pro-GnRH and GnRH in the rat preoptic area-basal hypothalamus and describe the extent of colocalization of GnRH peptide and pro-GnRH mRNA in proestrous females and intact male rats. Further work will determine how each of the molecular components is regulated during different reproductive states.

Effects of ovariectomy on GnRH mRNA, proGnRH and GnRH levels in the preoptic hypothalamus of the female rat.

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.

Testing of Arg-8-gonadotropin-releasing hormone-directed antisera by immunological and immunocytochemical methods for use in comparative studies.

Three polyclonal antisera raised in rabbits against the mammalian molecular form of gonadotropin-releasing hormone (GnRH) were tested in enzyme-linked immunosorbent assays for crossreactivity with naturally occurring GnRHs and with GnRH analogues. Antisera were then tested immunocytochemically in order (i) to identify amino acids essential for the binding of each antiserum, and (ii) to evaluate the specificity of the immunocytochemical reaction in brain sections from various species of cyclostomes, amphibians, reptiles, and birds. Antiserum GnRH 80/1, recognizing mainly a discontinuous determinant including the NH2- and COOH-termini, crossreacts with GnRHs the molecular bending of which enables the spatial approach of both terminal amino acid residues. Antiserum GnRH 80/2, by requiring the COOH-terminus for binding and not tolerating substitutions by aromatic amino acids in the middle region of the molecule, recognizes chicken I GnRH, however, not the salmon form. The use of this antiserum is appropriate in species synthesizing the mammalian and/or the chicken I form of GnRH. GnRH antiserum 81/1 is specific mostly for mammalian GnRH. The results obtained by ELISAs are confirmed by immunocytochemical studies. A comparison between the results obtained in ELISA and in immunocytochemistry involving mammalian-, chicken I-, chicken II-, salmon-, and lamprey-directed GnRH antisera resulted in the following conclusions: (1) An antiserum recognizing the discontinuous antigen determinant including both NH2- and COOH-termini may be reactive in most vertebrate brain sections thus being appropriate for phylogenetically directed immunocytochemical studies. (2) Moreover, this discontinuous determinant seems to be immunocytochemically reactive in all parts of the neurons in the GnRH system, whereas, in some species, determinants located in the middle region of the molecule(s) tend to become reactive only during the axonal transport. (3) A crossreaction between tissue-bound antigen and antibodies recognizing the above cited discontinuous determinant indicates an appropriate bending of the molecule even in case of severe molecular differences, e.g., in lamprey form of GnRH. (4) It follows that in phylogenetic studies, an immunologically well characterized antiserum can be substituted for a species-directed antiserum.

[Gene expression of gonadotropin releasing hormone in the rat brain--an in situ hybridization technic using an oligoRNA probe].

We studied the localization of GnRH cell bodies in the rat brain by using an in situ hybridization technique. We used radiolabeled single stranded oligoRNA probe (62-mer) complementary to the part of rat GnRH mRNA which included the coding sequence for the decapeptide. Only a single hybridizing band was observed on Northern blotting of mRNA extracts of the rat brain. This probe detects GnRH mRNA in individual cells in sections of the rat brain. In situ hybridization revealed the presence of GnRH cell bodies in the preoptic area and diagonal bands of Broca. We found that the oligoRNA probe was useful because it was easy to prepare and accessible to cellular mRNA.

Quantitative analysis of synaptic input to gonadotropin-releasing hormone neurons in normal mice and hpg mice with preoptic area grafts.

Mutant hypogonadal (hpg) mice with a truncated gene for the precursor to gonadotropin-releasing hormone (GnRH) show certain aspects of recovery of reproductive function after receiving grafts of normal preoptic area into the third ventricle. We have previously shown that GnRH neurons from within the grafts can innervate the appropriate neural-hemal target in the host. To determine if in turn these exogenously derived neurons receive a synaptic input comparable to the GnRH neurons in the normal animal we have now carried out a quantitative ultrastructural analysis to compare the synaptic input to GnRH neurons in the normal preoptic area and in the grafts. In almost all cases GnRH cells or dendrites in normal brains and within the grafts received a synaptic input. In normal animals, input to GnRH dendritic profiles was significantly greater (P less than 0.001) than to the somatic plasma membrane and this trend was also observed within the grafts though the difference was not statistically significant. In addition, no statistically significant difference was found between the input to GnRH structures within the grafts and in normal preoptic area. However, a substantial variability in input among grafted animals was evident which was not observed in normal animals. The sources of variability within the grafts are discussed and we suggest that the deficiencies and differences that exist in regulation of gonadotropin secretion among grafted hpg animals may be reflected in aberrant synaptic input.

Male transmission of the gene for isolated gonadotropin-releasing hormone deficiency.

Three black women, daughters of the same father but three unrelated mothers, presented with isolated gonadotropin deficiency (IGD). Clinically, the patients had no midline defects and intact smell and taste senses. Biochemically, the essential feature was very low unstimulated and stimulated follicle-stimulating hormone and luteinizing hormone levels, even after priming with gonadotropin-releasing hormone over a 5-day period. Growth hormone response to insulin-induced hypoglycemia was somewhat blunted, but prolactin, cortisol, and thyroid-stimulating hormone responses were quite normal. All three patients had the 46,XX karyotype; clinical or biochemical aberrations could not be demonstrated in any of the remaining family members. The disorder was, apparently, transmitted by the deceased father, who manifestly did not have an IGD deficiency nor any of the midline stigmata associated with IGD. The mode of inheritance seems most likely to be autosomal dominant with variable penetrance.