A developmental increase in the expression of messenger ribonucleic acid encoding a second form of gonadotropin-releasing hormone in the rhesus macaque hypothalamus

GnRH-I is thought to represent the primary neuroendocrine link between the brain and the reproductive axis. Recently, however, a second molecular form of this decapeptide (GnRH-II) was found to be highly expressed in the brains of humans and nonhuman primates. In this study, in situ hybridization was used to examine the regional expression of GnRH-II messenger ribonucleic acid in the hypothalamus of immature (0.6 yr) and adult (10-15 yr) male and female rhesus macaques (Macaca mulatta). Overall, no sex-related differences were observed. In all of the animals (n = 3 animals/group), intense hybridization of a monkey GnRH-II riboprobe was evident in the paraventricular nucleus and supraoptic nucleus and to a lesser extent in the suprachiasmatic nucleus, but no age- or sex-related differences were apparent. Intense hybridization of the riboprobe also occurred in the mediobasal hypothalamus, and this was markedly greater in the adults than in the immature animals. These data show that the expression of GnRH-II messenger ribonucleic acid increases developmentally in a key neuroendocrine center of the brain. Moreover, because GnRH-II can stimulate LH release in vivo, it is plausible that changes in its gene expression represent an important component of the mechanism by which the hypothalamus controls reproductive function.     

A second gene for gonadotropin-releasing hormone: cDNA and expression pattern in the brain.

In vertebrates, the gonadotropin-releasing hormone(GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulatereproduction via control of synthesis and release of pituitary gonadotropins.Only one GnRH peptide has been found in mammals, with one exception, althoughnumerous other vertebrate species express more than one of the eight knowndecapeptide forms as shown by immunocytochemical labeling of distinct cellgroups in the brain. However, neither the functional nor the evolutionaryrelationships among these GnRH forms are clear, because only one preprohormonegene sequence from any species has been reported. The most ubiquitousalternative form of GnRH is [His5,Trp7,Tyr8]GnRH (also referred to aschicken-II), which differs from the mammalian sequence at amino acids 5, 7, and8. This peptide has been shown to have the most potent releasing-hormoneactivity, although immunocytochemical staining has suggested it is synthesizedonly in the mesencephalon. Here we report the cloning and expression pattern ofthe gene for the precursor of this form from the teleost fish Haplochromisburtoni. This is the second GnRH-encoding gene to be characterized in thisspecies. The newly discovered preprohormone gene differs from that previouslyreported in two ways. First, whereas the original gene predicts only a singleassociated peptide, this one predicts two associated peptides, both of whichappear to be unique. Second, the gene for [His5,Trp7,Tyr8]GnRH is expressed inonly one cell group in the mesencephalon. In contrast, the previously reportedgene is expressed only in the terminal nerve. The striking differences betweenthe preprohormone structure and localization suggest that the genes coding forthe two known GnRH forms in H. burtoni did not arise from a recent duplicationevent. Interestingly, neither of the two genes found to date in this species isexpressed in cells which project from the hypothalamus to the pituitary,suggesting that yet a third gene coding for GnRH may exist.     

Expression and antiproliferative effect of a second form of gonadotropin-releasing hormone in normal and neoplastic ovarian surface epithelial cells

Epithelial ovarian carcinomas are the most common cause of death from gynecological malignancies and appear to arise from ovarian surface epithelium (OSE), but the exact mechanism of ovarian tumorigenesis has not been elucidated. Recent cloning of a second form of gonadotropin-releasing hormone (GnRH-II) has been reported in various human tissues including the ovary. However, the expression and role of GnRH-II in human OSE and ovarian carcinomas is not known. In the present study, we demonstrated that in addition to the GnRH receptor (GnRH-R), GnRH-II mRNA is expressed in normal OSE, immortalized OSE (IOSE) cells, primary cultures of ovarian tumors and ovarian cancer cell lines. Treatments with increasing doses (10(-9)-10(-7) M) of GnRH-I and -II resulted in a growth-inhibition in both non-tumorigenic IOSE-29 and tumorigenic IOSE-29EC cells. These results indicate for the first time the expression and potential anti-proliferative effect of GnRH-II, suggesting that GnRH-II, similar to GnRH-I, may have a growth-regulatory effect in normal and neoplastic OSE cells.     

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.     

Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins.

The purpose of this study was to determine the expression of genes encoding various sarcoplasmic reticulum components that are functionally coupled with calcium release, uptake, and storage function during cardiac hypertrophy induced by thyroid hormone. Hyperthyroidism was induced in two groups of rabbits by the injection of 200 micrograms/kg L-thyroxine (T4) daily for 4 days (T4-4-day group) and 8 days (T4-8-day group). Hypothyroidism was induced in another group of rabbits by adding 0.8 mg/ml propylthiouracil to the drinking water for 4 weeks. The relative expression level of mRNA encoding different sarcoplasmic reticulum proteins was determined by RNA slot blot and Northern blot analysis. In hyperthyroid hearts, the steady-state level of cardiac ryanodine receptor mRNA and sarcoplasmic reticulum cardiac/slow-twitch Ca(2+)-ATPase mRNA were both increased to 147% (T4-4-day group) and 186% (T4-8-day group) of control, respectively, but decreased to 71% and 75%, respectively, in hypothyroid ventricles. The mRNA level for phospholamban was decreased in both hyperthyroidism (T4-8-day group, 72%) and hypothyroidism (77%) in these hearts. On the other hand, calsequestrin mRNA levels did not change in hyperthyroid and hypothyroid ventricles. In accord with the changes in Ca(2+)-ATPase mRNA levels, the Ca(2+)-ATPase protein was increased to 199% (T4-8-day group) in hyperthyroid ventricles and decreased to 86% of control in hypothyroid ventricles. The expression levels of ryanodine receptor, Ca(2+)-ATPase, phospholamban, and calsequestrin mRNAs were similarly altered in skeletal muscle tissues from hyperthyroid and hypothyroid rabbits. These results indicate that the mRNA levels of sarcoplasmic reticulum proteins responsible for calcium release and calcium uptake are coordinately regulated in response to changes in thyroid hormone level in both heart and skeletal muscle. These changes in mRNA level should lead to changes in protein levels and thus to altered calcium release and uptake in the chronic stages of hyperthyroidism and hypothyroidism.     

Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.     

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.     

Differential brain distribution of gonadotropin-releasing hormone receptors in the goldfish

The present study describes the differential distributions in the brain of the two goldfish gonadotropin-releasing hormone (GnRH) receptors, using both immunohistochemistry and in situ hybridization approaches. The goldfish GnRH GfA and GfB receptors are variant forms of the same receptor subtype, although with distinct differences in ligand binding characteristics, and differential distributions in the pituitary and body tissues [Proc. Natl. Acad. Sci. USA 96 (1999) 2526]. The goldfish GnRH GfA receptor was found to be widespread throughout the brain, with neurons showing immunoreactivity in the olfactory bulbs, telencephalon, preoptic region, ventro-basal hypothalamus, thalamus, midbrain, motor neurons of the fifth, seventh, and tenth cranial nerves, reticular formation, cerebellum, and motor zone of the vagal lobes. The tracts in the posterior commissure, optic tectum, and motor zone of the vagal lobes also demonstrated immunoreactivity. While the brain was not systematically surveyed for in situ hybridization, hybridization was found in similar locations in the telencephalon, preoptic region, ventro-basal hypothalamus, cerebellum, and optic tectum. Hybridization was additionally found in the medial hypothalamus. The goldfish GnRH GfB receptor was found to have a more restricted distribution in the brain, with neurons showing immunoreactivity in the telencephalon, preoptic region, and ventro-basal hypothalamus. In situ hybridization demonstrated a somewhat wider distribution of expression of the receptor, with hybridization occurring in the preoptic region, ventro-basal and medial hypothalamus, as well as in the thalamus, epithalamus, and optic tectum. The widespread distribution of GnRH GfA receptor, and in particular its localization in the midbrain tegmentum in the region of the GnRH-II neurons, suggests that this receptor may be involved in the behavioral actions of GnRH peptides in the goldfish.     

Primary structure of a novel gonadotropin-releasing hormone in the brain of a teleost, Pejerrey

The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.     

In situ hybridization histochemistry for messenger ribonucleic acid (mRNA) encoding gonadotropin-releasing hormone (GnRH): effect of estrogen on cellular levels of GnRH mRNA in female rat brain

Using in situ hybridization histochemistry, we have detected perikarya containing mRNA encoding GnRH and GnRH-associated peptide (GAP) in rat brains. Synthetic DNA oligomers with sequences complementary to the rat cDNA encoding the GnRH structural region and the GAP structural region were hybridized to formaldehyde-fixed coronal sections. The distribution and number of cells containing GnRH/GAP mRNA were similar to those shown by immunocytochemical studies. The areas in which GnRH mRNA perikarya were shown included the medial septal area, the diagonal band of Broca, the preoptic area, and the anterior hypothalamus. Up to 55 cells were detected in a single 12-micron section containing the diagonal band and organum vasculosum lamina terminalis (OVLT) whereas cell numbers diminished in more caudal regions. In addition, both probes labeled the same cells contained within adjacent sections. We used this technique to examine the effect of estrogen on GnRH mRNA levels in the area of the OVLT of normal and androgen-sterilized female rats, using an estrogen treatment paradigm previously characterized in studies investigating the hypothalamic regulation of negative and positive estrogen feedback. We found that 7 days after ovariectomy, 2 days of estrogen treatment resulted in a significant reduction in the average cellular level of GnRH mRNA in both normal and androgen-sterilized females. Analysis of histograms relating the intensity of labeling to the abundance of cells suggested that a small population of GnRH cells responded to the estrogen treatment. However, we found no evidence for a discrete neuroanatomical segregation of such a subpopulation of GnRH-responsive cells within the area of the OVLT.     

Nonmammalian gonadotropin-releasing hormone molecules in the brain of promoter transgenic rats

Mammalian gonadotropin-releasing hormone (GnRH1) and nonmammalian immunoreactive GnRH subtypes were examined in transgenic rats carrying an enhanced GFP (EGFP) reporter gene driven by a rat GnRH1 promoter. Double-label immunocytochemistry was performed on EGFP(+)/GnRH1 brain sections by using antisera against GnRH1, GnRH2 (chicken II), GnRH3 (salmon), or seabream GnRH. EGFP(+)/GnRH1 neurons were in the septal-preoptic hypothalamus but not in the midbrain, consistent with GnRH1-immunopositive neurons in WT rats. Apparent coexpression of EGFP(+)/GnRH1 with other GnRH subtypes was observed. All EGFP(+) neurons in the septal-preoptic hypothalamus were GnRH1-immunopositive. However, only approximately 80% of GnRH1-immunopositive neurons were EGFP(+), which awaits further elucidation. GnRH subtypes-immunopositive fibers and EGFP(+)/GnRH1 fibers were conspicuous in the organum vasculosum of the lamina terminalis, median eminence, and surrounding the ependymal walls of the third ventricle and the aqueduct in the midbrain. These results demonstrate that the expression of the EGFP-GnRH1 transgene is restricted to the bona fide GnRH1 population and provide clear morphological evidence supporting the existence of GnRH1 neuronal subpopulations in the septal-preoptic hypothalamus, which might be driven by different segments of the GnRH promoter. This genetic construct permits analyses of promoter usage in GnRH neurons, and our histochemical approaches open questions about functional relations among isoforms of this peptide, which regulates reproductive physiology in its behavioral and endocrine aspects.     

Glucocorticoids affect gonadotropin-releasing hormone immunoreactivity in musk shrew brain

Multiple interactions between the hypothalamic-pituitary-adrenal and the hypothalamic-pituitary-gonadal systems exist. In this study, we asked if glucocorticoid administration affected gonadotropin-releasing hormone (GnRH) immunoreactivity. We found that musk shrews treated with dexamethasone (DEX), a synthetic glucocorticoid, had more GnRH-immunoreactive (ir) cells in the forebrain than did cortisol- or control-treated animals. The effects of DEX were noted rapidly, within 15 min, after administration. These effects were observed in the forebrain as a whole and also in specific subpopulations of GnRH-ir cells located in the medial septum/diagonal band and the hypothalamus.     

Critical role for estrogen receptor alpha in negative feedback regulation of gonadotropin-releasing hormone mRNA expression in the female mouse

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor alpha (ERalpha) or ERbeta or both ERs is presently unclear. Using female mice with targeted disruptions of ERalpha and ERbeta (alphaERKO and betaERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact alphaERKO females and increased modestly (p < 0.05) in intact betaERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and betaERKO (p < 0.05) mice but not changed in alphaERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact alphaERKO mice compared with intact wild-type and betaERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and betaERKO (p < 0.05) females but not alphaERKO mice. These data demonstrate that both ERalpha and ERbeta are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERalpha appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.     

Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel

Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.     

Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus

The present study was undertaken to determine whether T3, estrogen, and 11-ketotestosterone could alter a specific population of GnRH-containing neurons, as indicated by a change in messenger RNA (mRNA) levels in sexually immature male tilapia, Oreochromis niloticus. Two weeks after castration, fish were assigned to four treatment groups. One group served as the control (sesame oil); a single ip injection of (T3; 5 microg/g), estradiol benzoate (EB; 5 microg/g), or 11-ketotestosterone (KT; 5 microg/g) was administered to the remaining three groups. Twenty-four hours after the injection, brains were collected and processed for in situ hybridization histochemistry using 35S-labeled 30-mer antisense oligonucleotide probes complementary to the GnRH-coding region of chicken II, salmon, and seabream GnRH. Computerized image analysis was performed to quantify mRNA concentrations, neuronal numbers, and neuronal size of the terminal nerve-nucleus olfactoretinalis, preoptic, and midbrain GnRH neurons. KT had no effect on any of the above neuronal parameters examined for salmon or seabream GnRH. Neither T3, EB, nor KT was effective to induce changes in midbrain chicken GnRH II mRNA concentrations, neuronal numbers, and neuronal size, indicating that an as yet unknown regulatory mechanism may operate midbrain GnRH neurons. T3 specifically suppressed the concentration of terminal nerve salmon GnRH mRNA, and EB significantly increased preoptic seabream GnRH neuronal numbers. These results are consistent with the hypothesis that thyroid hormone, by suppressing terminal nerve GnRH expression, promotes inhibition of sexual maturation. Furthermore, the failure of KT, a nonaromatizable androgen, to influence preoptic GnRH neurons emphasizes that an estrogenic pathway, at the onset of sexual maturation, is responsible for the recruitment of additional preoptic GnRH neurons that are fundamental to reproduction and behavior.     

Effects of pinealectomy and melatonin on gonadotropin-releasing hormone (GnRH) gene expression in the male rat brain

Melatonin, a pineal hormone, is known to be an important neurohormonal factor involved in the timing of reproductive events which occur seasonally in various mammalian species. In order to evaluate the influence of melatonin on neurons which are producing gonadotropin-releasing hormone (GnRH), we studied the effect of light-dark cycle as well as pinealectomy and melatonin administration on GnRH gene expression in the adult male rat medial preoptic area (MPOA) using quantitativein situ hybridization. The animals were kept under artificial light (light on 6:00 h–20:00 h). In animals which were sacrificed at 24:00 h (when endogenous melatonin levels are high), the hybridization signal was higher than that detected in animals sacrificed at 20:00 h (before the onset of darkness). Administration of melatonin during the light period (16:00 h) induced a 15% increase in the amount of GnRH mRNA after 4 h. Three weeks after pinealectomy mRNA levels were decreased by 35%. Injection of melatonin to pinealectomized rats 4 h before sacrifice increase the amount of GnRH mRNA, completely reversing the decrease in mRNA induced by pinealectomy. These results strongly suggest that melatonin produced by the pineal gland exerts a positive influence on GnRH neuronal activity in the male rat.