Regulation of gonadotropin-releasing hormone (GnRH) receptor gene expression in sheep: interaction of GnRH and estradiol

GnRH and estradiol are important regulators of GnRH receptors. When delivered to the anterior pituitary gland continuously, GnRH decreases numbers of GnRH receptors on gonadotropes. Treatment with estradiol consistently increases numbers of GnRH receptors. Because estradiol acts via intracellular receptors while GnRH exerts its effects through a membrane receptor, it is likely that these hormones influence GnRH receptor expression via different mechanisms. In this experiment, we tested two hypotheses: 1) continuous infusion of GnRH will decrease expression of the GnRH receptor gene; and 2) estradiol will override the negative effects of continuous infusion of GnRH on GnRH receptor expression. Ovariectomized ewes were administered either GnRH (10 microg/h, n = 10) or saline (n = 10) continuously for 136 h. At 124 h, 5 ewes in each group were administered estradiol (25 microg i.m.) and anterior pituitary glands were collected 12 h later. Treatment with GnRH caused an abrupt increase in circulating concentrations of LH, and the maximal mean concentration was observed 4 h after the start of GnRH infusion. Following this increase, concentrations of LH in GnRH-treated ewes declined and were similar to those in saline-treated ewes from 8 h to 124 h. After injection of estradiol at 124 h, circulating concentrations of LH increased in both GnRH- and saline-treated ewes. However, this response occurred within 6 h in ewes treated with GnRH compared with 9 h in ewes treated with saline (P < 0.05). Compared with saline-treated controls, treatment with GnRH decreased mean steady-state amount of GnRH receptor messenger RNA (mRNA) (P < 0.01) and concentration of GnRH receptors (P < 0.05). Treatment with estradiol caused an increase in concentrations of GnRH receptor mRNA (P < 0.05) and GnRH receptors (P < 0.01). Amounts of GnRH receptor mRNA and numbers of GnRH receptors in ewes treated with both GnRH and estradiol were not different from those in the control group but were higher (P < 0.002) relative to ewes treated with GnRH alone. Treatment with GnRH and estradiol also influenced the expression of genes encoding the LHbeta and FSHbeta subunits. Compared with saline-treated controls, treatment with GnRH reduced steady-state amounts of mRNA encoding LHbeta subunit (P < 0.005) and FSHbeta subunit (P < 0.05). Treatment with estradiol caused a decrease in concentrations of FSHbeta subunit mRNA (P < 0.01) but did not affect amounts of LHbeta subunit mRNA. The combined treatment of GnRH and estradiol reduced concentrations of mRNA encoding LHbeta subunit (P < 0.01) and FSHbeta subunit (P < 0.005). From these data we conclude that 1) reduced numbers of GnRH receptors during continuous infusion of GnRH are mediated in part by decreased expression of the GnRH receptor gene; and 2) estradiol is able to override the negative effect of GnRH by stimulating an increase in GnRH receptor gene expression and GnRH receptor concentrations. Therefore, although the gonadotrope becomes refractory to GnRH during homologous desensitization, this desensitization does not affect the cell's ability to respond to estradiol.     

Embryonic development of the gonadotropin-releasing hormone (GnRH) system in the chick: a spatio-temporal analysis of GnRH neuronal generation, site of origin, and migration

We present a quantitative immunocytochemical study of GnRH migration by developmental stage. GnRH peptide was detected in cells of the olfactory epithelium at stage 19. Migration was initiated a few hours later at stage 20. Of interest is the observation that GnRH neurons paused at the central nervous system border for 3 days, entering the brain at stage 29. The major expansions of the GnRH population occurred at two points; stages 26 and 42. In one animal a third population expansion occurred after hatching, with the number of GnRH cells reaching 6600. To determine the site of origin of GnRH cells, embryos were exposed to tritiated thymidine and killed 5 h later. Most GnRH cells incorporated label in the olfactory epithelium; however, some autoradiographically labeled GnRH cells, possessing a neuronal morphology, were found in the olfactory nerve and the forebrain, suggesting that some GnRH neurons divide as they migrate. A cumulative labeling method employing tritiated thymidine was used to examine the birth date of GnRH neurons. Postmitotic GnRH cells were first detected at stages 19-21. At stage 24, a peak in GnRH neurogenesis preceded the increase in GnRH neurons expressing their peptide at stage 26. After stage 24, there was a gradual addition of postmitotic cells to the population through stage 35. A pulse-chase paradigm indicated that birth date did not influence the final GnRH cell distribution. Injections at stage 29, when 10% of the GnRH neurons are born, generated double labeled cells in all locations where placode-derived GnRH neurons reside.     

Inhibition of growth and proliferation of EcRG293 cell line expressing high-affinity gonadotropin-releasing hormone (GnRH) receptor under the control of an inducible promoter by GnRH agonist (D-Lys6)GnRH and antagonist (Antide)

The mechanism by which gonadotropin-releasing hormone (GnRH) agonists and antagonists inhibit tumor cell growth and proliferation is controversial. Direct mediation of the antitumor effects through the high-affinity GnRH receptors has been questioned because of the low level of expression of receptors on the tumor cells. We have developed a human kidney embryonic cell line (EcRG293) that expresses high-affinity GnRH receptor under the control of an inducible promoter activated by muristerone A. Treatment of this cell line with either GnRH agonist (D-Lys6)GnRH or GnRH antagonist (Antide) resulted in a significant, time-dependent decrease in cell proliferation in muristerone A-induced cells but not in the uninduced cells, which do not express the GnRH receptor. These data suggest strongly that the antitumor effect of GnRH agonists and antagonist is specific, direct, and mediated through high-affinity GnRH receptors present on the cell membranes of tumor cells.     

Importance of the gonadotropin-releasing hormone (GnRH) surge for induction of the preovulatory luteinizing hormone surge of the ewe: dose-response relationship and excess of GnRH

The preovulatory LH surge in the ewe is stimulated by a large sustained surge of GnRH. We have previously demonstrated that the duration of this GnRH signal exceeds that necessary to initiate and sustain the LH surge. The objective of the present study was to determine whether a similar excess exists for amplitude of the GnRH surge. Experiments were performed using an animal model in which GnRH secretion was blocked by progesterone, which in itself does not block the pituitary response to GnRH. To assess the amplitude of the GnRH surge needed to induce the LH surge, we introduced artificial GnRH surges of normal contour and duration but varying amplitudes. Twelve ewes were run through 3 successive artificial follicular phases (total of 36). Six of these artificial follicular phases were positive controls, in which progesterone was removed, the estradiol stimulus was provided, and vehicle was infused. In these control cycles, animals generated endogenous LH surges. In the remaining artificial follicular phases, progesterone was not withdrawn, the estradiol stimulus was provided, and either vehicle (negative control) or GnRH solutions of varying concentrations (experimental) were infused. The circulating GnRH concentrations achieved by infusion were monitored. No LH surges were observed in negative controls, whereas LH surges were induced in experimental cycles provided a sufficient dose of GnRH was infused. A highly significant dose-response relationship was observed between the amplitude of the GnRH surge and both the amplitude of the LH surge and the area under the curve describing the LH response, but no such relationship existed between the amplitude of the GnRH surge and the duration of the LH response. In numerous cases, LH surges similar to those in the positive control animals resulted from infusion of amounts of GnRH estimated to be considerably less than those delivered to the pituitary during the endogenously generated GnRH/LH surge. These findings indicate that, in the ewe, increased GnRH secretion drives the preovulatory LH surge in a dose-dependent fashion, and they provide evidence that the amplitude of the GnRH surge may exceed that needed to generate the LH surge.     

Photoperiodic effects on gonadotropin-releasing hormone (GnRH) content and the GnRH-immunoreactive neuronal system of male Siberian hamsters

Despite profound photoperiodic differences in circulating gonadotropin levels, consistent differences in the GnRH system have not been observed in Siberian hamsters (Phodopus sungorus) housed chronically in short or long days. During the transition from short to long days, however, male hamsters exhibit a transient increase in the number of cells expressing prepro-GnRH mRNA on the morning of the second long day. Here, we present two experiments designed to examine whether or not this change in mRNA level is associated with changes in GnRH protein synthesis. In the first experiment, we used RIA to measure GnRH content in preoptic area-mediobasal hypothalamic homogenates. We observed a significant increase in GnRH protein levels on the morning of the second long day relative to short- and long-day controls. The latter two groups did not differ from one another. In the second experiment, we used immunocytochemistry to quantify GnRH cell number in the various treatment groups. GnRH-immunoreactive (-ir) cell number did not increase significantly after long-day transfer relative to that in short-day controls; however, both of these groups had significantly more GnRH-ir neurons than long-day controls. We hypothesize that during the transition from short to long days, male Siberian hamsters experience a transient increase in GnRH production in a stable population of neurons. When GnRH secretion subsequently increases on long days, peptide content within neuronal cell bodies declines, leading to a decrease in the number of immunoreactive neurons detected. The rapid response of the hypothalamo-pituitary-gonadal axis in Siberian hamsters to a change in day length defines a narrow temporal window in which to identify the physiological, cellular, and molecular mechanisms mediating the photoperiodic regulation of reproduction.     

Gonadotropin-releasing hormone (GnRH) in ancient teleosts, the bonytongue fishes: putative origin of salmon GnRH

PURPOSE: To measure the compressive forces of the haptics of 28 intraocular lens (IOL) models for different modes of compression and compare the results of two types of measurements. SETTING: Department of Ophthalmology, Central Hospital of Central Finland, Jyv?skyl?, Finland. METHODS: The haptics of 28 types of IOLs were compressed to a diameter of 9.0 mm between curved anvils. The compression forces in the plane of compression (i.e., in the plane of the optics) were measured at 0.5 mm intervals. During compression, the optics and the haptics were free to rotate with respect to the anvils. The results were compared with those of earlier measurements in which the optics were held fixed during compression. Perpendicular forces were measured at 0.4 mm intervals. RESULTS: The measured forces in the plane of the optics varied between 114 and 659 mg at a diameter of 10.0 mm and 192 and 1047 mg at a diameter of 9.0 mm. When compressed to 10.0 mm in diameter, the forces were 1 to 75% lower than when lens rotation was not possible. The forces perpendicular to the optic varied between 0 and 96 mg at a 10.0 mm diameter and correlated with the forces in the plane of the optic. CONCLUSION: The compression forces of the lens haptics were generally lower when the lenses were allowed to rotate during compression. The orders of stiffness of the haptics in these two measurements were similar. The perpendicular forces were generally small and correlated significantly with the forces measured in the plane of the optic.     

Effects of bovine follicular fluid and passive immunization against gonadotropin-releasing hormone (GnRH) on messenger ribonucleic acid for GnRH receptor and gonadotropin subunits in ovariectomized ewes

In cultured ovine pituitary cells, inhibin increases concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. The objective of this study was to test the hypothesis that inhibin increases concentrations of ovine GnRH receptor mRNA in vivo. Ovariectomized ewes were used to eliminate effects of endogenous ovarian hormones, and passive immunization against GnRH was employed to avoid possible confounding influences of GnRH on GnRH receptor gene expression. Two groups of ewes (n = 5/group) were treated with 50 ml GnRH antiserum on Days 0 and 3 of the experiment. One group of immunized ewes received 10 ml charcoal-extracted bovine follicular fluid (bFF) as a source of inhibin every 8 h for 48 h on Days 4-6 of the experiment. A third group of ewes was not passively immunized and was treated only with bFF, and control ewes received no treatments. Anterior pituitary glands were collected from all ewes on Day 6. Passive immunization against GnRH, alone or in combination with treatment with bFF, decreased mean concentrations of LH (p < 0.01) and LH pulse amplitude (p < 0.001). In ewes treated only with GnRH antiserum, number of LH pulses was also reduced (p < 0.03). Circulating concentrations of FSH tended to be lower (p = 0.06) in passively immunized ewes compared to controls. Treatment with bFF, alone or in combination with GnRH antiserum, reduced circulating concentrations of FSH (p < 0.02) and amounts of FSHbeta subunit mRNA (p < 0.001) to less than 30% and 10% of control values, respectively. Despite effects of bFF on concentrations of FSHbeta mRNA and secretion of FSH, concentrations of GnRH receptor mRNA were similar among controls, ewes treated with bFF alone, and passively immunized ewes treated with bFF. Passive immunization against GnRH did not affect concentrations of GnRH receptor mRNA but resulted in a reduction (p < 0.05) in amount of LHbeta mRNA. Treatment with bFF did not affect amounts of either alpha subunit or LHbeta subunit mRNA except when combined with treatment with antiserum, when amounts of both alpha and LHbeta subunit mRNA were reduced (p < 0.05). These results do not support the hypothesis that inhibin increases concentrations of GnRH receptor mRNA in the ewe, and they provide evidence that inhibin is not an acute regulator of ovine GnRH receptor gene expression in vivo.     

Gonadotropin-I and -II subunit gene expression of male striped bass (Morone saxatilis) after gonadotropin-releasing hormone analogue injection: quantitation using an optimized ribonuclease protection assay

We have demonstrated that sodium butyrate induces differentiation in human hepatoma cells; however, recent studies have shown that this agent causes apoptosis in some types of cancer cells. In this study, we examined whether sodium butyrate causes apoptosis in the human hepatoma cell lines, HCC-M and HCC-T. The growth of human hepatoma cells was dose-dependently reduced by sodium butyrate. Flow cytometric analysis showed cell-cycle arrest at the G1 phase in the sodium butyrate-treated cells. Apoptotic change was never found in treated cells at concentration levels of less than 5 mmol/L. Sodium butyrate decreased p53 expression and increased p21WAF-1 expression in HCC-T and HCC-M cells having the wild-type p53 gene. Western blot analysis showed that Bcl-2 was expressed in the HCC-T and HCC-M cells, and its expression was increased after exposure to sodium butyrate. Antisense oligodeoxynucleotide against bcl-2 easily caused apoptosis. These results indicate that sodium butyrate hardly induces apoptotic change in the human hepatoma cell lines, HCC-T and HCC-M, with the increase of Bcl-2 expression. Cell-cycle arrest in the G1 phase caused by sodium butyrate was suggested to be induced by the increase in p21WAF-1 expression, but this change did not link with the p53 increase.     

Effects of long-term testosterone, gonadotropin-releasing hormone agonist, and pimozide treatments on gonadotropin II levels and ovarian development in juvenile female striped bass (Morone saxatilis)

The ability of the juvenile female reproductive axis to respond to hormonal stimulation was investigated in a Perciform fish, the striped bass (Morone saxatilis) using various combinations of testosterone (T), GnRH agonist (GnRHa), and pimozide. A long-term treatment with T alone, or T in combination with GnRHa, increased pituitary gonadotropin II (GtH II) levels 2- and 3-fold, respectively, suggesting that T and GnRHa each stimulate GtH II accumulation. Release of the accumulated GtH II could be induced only by high doses of GnRHa in combination with T, indicating that GtH II synthesis and release require different levels of GnRH stimulation. The addition of the dopamine antagonist pimozide did not affect pituitary and plasma GtH II levels but, in response to an additional acute GnRHa challenge, inhibited the release of GtH II. Although ovarian development was slightly stimulated by a combined T and GnRHa treatment, vitellogenesis was generally not initiated. The present study demonstrated that the juvenile striped bass pituitary is responsive to hormonal stimulation, resulting in elevated levels of GtH II in the pituitary and plasma. However, increased plasma levels of GtH II did not result in precocious puberty, suggesting that additional factors are required for the initiation of ovarian development in this teleost.     

GnRH molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes). Immunological and chromatographic evidence for the presence of a novel molecular variant

Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.     

Analysis of the uncoupling protein-1 (UCP1) gene in obese and lean subjects: identification of four amino acid variants

We report the case of a patient with fatal obstructive lung disease after an HLA-haploidentical sibling cord blood transplant (CBT), with severe acute GVHD. A 2-year-old girl developed expiratory air trapping gradually with acute and chronic GVHD after CBT for the treatment of ALL. Anti-CMV and immunosuppressive therapy were ineffective, and the patient died of progressive respiratory acidosis. Necropsy of the lung revealed severe bronchiolitis obliterans with cytomegalic inclusion cells in the granulation tissues of the bronchiolitis. Thus, immunologic and GVHD problems can occur even in CBT.     

High-resolution GTG-banding and nucleolar organizer regions of chromosomes of two vole species: Microtus rossiaemeridonionalis and M. transcaspicus (Rodentia, Arvicolidae)

Two lines of White Leghorns that had undergone long-term selection for high (HH) or low (LL) antibody response to sheep red blood cell antigen(s) formed the nuclear lines for this experiment. Matings were made in a full diallel cross to produce in a single hatch from age-contemporary breeders the parental lines, reciprocal F1 and F2 crosses, and backcrosses for 16 progeny types. For males and females, there were parental line differences in BW to 42 d of age, after which there was decline between lines for males. Differences in BW between reciprocal F1 crosses and maternal heterosis declined with age, primarily reflecting dissipation of effects of egg weight. Heterosis of BW was dependent on the particular F1 cross and recombination effects were not important. At 50 d of age chicks were inoculated with either a 1 or 10% suspension of spleen extract from chickens infected with marble spleen disease virus (MSDV). A third group served as uninjected controls. Response to MSDV was evaluated by spleen weight 6 d after inoculation. Spleen weights relative to BW of control chicks were heavier for the HH than LL line with evidence from the crosses of sexlinkage and negative heterosis. Line LL chicks were more resistant to MSDV than Line HH chicks was F1 crosses intermediate to and different from either parental line with no evidence of heterosis.     

Cytogenetic of nucleolar organizer regions (NOR) of human chromosomes: identification of four morphofunctional variants of NOR, their inter-individual and inter-chromosomal distribution

Cytogenetic characters of the nucleolus organizer regions (NORs) located on the short arms of five acrocentric chromosomes were studied in chromosome preparations obtained from cultured blood cells of 17 donors. In situ hybridization to a 3H-labeled probe was used to estimate the relative copy number of ribosomal genes (RGs) in all NORs of chromosomes identified by G-banding in each sample. The relative amount of potentially active RGs (0 to 4 arbitrary units) in each NOR was estimated from the size of AgNOR selectively stained with silver nitrate. Linear regression analysis revealed clusters of silent RGs (CSRGs) in 24 out of 170 NORs (14%). Based on the presence or absence of active and inactive RG clusters, NORs of human chromosomes were classified into four morphological functional variants (MFVs): (1) Ag-/CSRG-, (2) Ag-/CSRG+, (3) Ag+/CSRG-, and (4) Ag+/CSRG+. These variants were observed in 7.65%, 2.35%, 11.8%, and 78.2% of 170 analyzed NORs, respectively. NORs with CSRGs (MFV 2 and 3) were absent in 5 out of 17 donors. One, two, and three NORs with CSRGs were observed in four donors each. Analysis of the chromosome distribution of NOR MFVs showed that their frequencies remained almost the same in group-D and group-G acrocentric chromosomes. Although the tested samples were small (34 chromosomes for each pair), two observations were made with regard to individual chromosomes. First, almost half MFV-1 NORs (6 out of 13) were detected on chromosome 15. Second, the frequency of CSRGs was higher in chromosome 21 (29%) than in the other chromosomes (10%).     

I&R (identification and registration) system cattle: an analysis of its use during a foot-and-mouth-disease outbreak in The Netherlands

The performance of the Dutch cattle I&R system was analysed, with emphasis on the potential use of the system for the control of an outbreak of foot and mouth disease (FMD). The analysis showed that not all mutations were centrally registered within the three working days that are allowed to pass between the actual change and the obligatory report. In particular, young calves and cattle heading for slaughter were reported too late. During an outbreak of FMD, the best strategy to trace animals off a farm seems to be first to identify all animals on the farm, using a preprocessed list of the animals that should be present on the farm according to the I&R system. Then all the animals that have left the farm during the last month can be traced in the central I&R computer or by asking the person to whom the farmer sold the cattle (the next owner). In the present study, 54% of the animals were localized within one day, and 94% within one week. Finally, some suggestions for improvement for the system are given.