The product of X-linked Kallmann's syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons

X-linked Kallmann's syndrome (KS) is a genetic disease characterized by anosmia and hypogonadism due to impairment in the development of olfactory axons and in the migration of gonadotropin-releasing hormone (GnRH)-producing neurons. Deletions or point mutations of a gene located at Xp22.3 (KAL1) are responsible for the disease. This gene encodes for a secreted heparin-binding protein (KAL or anosmin-1) which exhibits similarities with cell-adhesion molecules. In the present study, we show for the first time a direct action of anosmin-1 on the migratory activity of GnRH neurons. Specifically, we exposed immortalized migrating GnRH neurons (GN11 cells) to conditioned media (CM) of COS or CHO cells transiently transfected with human KAL1 gene in microchemotaxis and collagen gel assays. We found that anosmin-1-enriched media produced a cell-specific chemotactic response of GN11 cells. None of the CM enriched on three forms of anosmin-1 carrying different missense mutations (N267K, E514K and F517L) found in patients affected by X-linked KS affected the chemomigration of GN11 cells. Anosmin binds to the GN11 cell surface by interacting with the heparan sulphate proteoglycans, and the chemotactic effect of anosmin-1-enriched CM can be specifically blocked by heparin or by heparitinase pretreatment. These results strongly suggest an involvement of anosmin-1 in the control of the migratory behaviour of GnRH neurons and provide novel information on the pathogenesis of KS.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the fetal and early postnatal mouse brain

The objectives were to (a) determine the age in development when GnRH is first detectable in the brain and (b) observe the distribution of GnRH throughout the fetal and early postnatal period. GnRH was localized immunohistochemically in fetal (15, 16, 17 and 19 days of gestation) and early postnatal (1- and 7-day-old) mice with the peroxidase-antiperoxidase (PAP) method of Sternberger. In the organum vasculosum of the lamina terminalis (OVLT) and in the median eminence of the fetus, GnRH was first detected at 17 days of gestation. In the OVLT, GnRH was found ventral to the preoptic recess of the third ventricle near the ventral surface of the brain. In addition, GnRH was located adjacent to the superficial portal capillaries near the surface of the median eminence. At 19 days of gestation, the distribution of GnRH was similar to that observed at 17 days and there was a marked increase in amount. In the newborn mouse, GnRH was undetectable in the OVLT and its content in the median eminence was decreased as compared to that observed in the fetus. By the seventh postnatal day, a considerable accumulation of GnRH had occurred in the OVLT and median eminence. In the OVLT, it was associated with capillaries ventral to the preoptic recess, and its distribution in the median eminence was similar to that in the adult mouse. In both the OVLT and median eminence of the fetal and early postnatal mouse GnRH appeared to be stored in axons and axon endings, but was not detectable in nerve cell bodies or ependymal cells. These observations suggest that the potential for neuroendocrine control of gonadotropin secretion exists in the fetal mouse as early as 17 days of gestation.     

Ultrastructural characterization of gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve cells in the dwarf gourami

Recently we have been studying gonadotropin-releasing hormone (GnRH) cells of the terminal nerve (TN) in the dwarf gourami, which may serve as a good model system for the study of neuromodulator functions. Here we report on the ultrastructural characterization of TN-GnRH cells using postembedding immunoelectron microscopy. The GnRH immunoreactivities could be demonstrated on dense-cored vesicles (DCVs) in cell bodies, fibers and varicosities. However, we could find no evidence of GnRH-immunoreactive synapses which are characterized by active zones. This may suggest that GnRH is released non-synaptically from DCV-containing fiber varicosities and that it exerts its modulatory action on GnRH receptors located on nearby as well as distant target neurons.     

Expression of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor in sheep spinal cord

Consistent with its neuroendocrine role, gonadotropin-releasing hormone (GnRH) is located principally within the hypothalamus, although extra-hypothalamic expression has been reported. The present study characterized the expression of GnRH and GnRH receptor (GnRH-R) in sheep spinal cord using real-time PCR and immunocytochemistry. Both GnRH and GnRH-R mRNA were detected in sheep spinal cord. Expression of GnRH peptide was localized to discrete locations in the spinal cord, including lamina X (the area surrounding the central canal) and motoneurons in the ventral horn. Although there is no known functional role for GnRH in spinal cord, a role as a potential neurotransmitter/neuromodulator is supported by the expression of both GnRH and GnRH-R in this tissue.     

GnRH及其受体与肿瘤关系的研究进展

GnRH最初是从哺乳类下丘脑分离出的一种十肽激素 ,其生理功能是调节垂体前叶促性腺激素的分泌 ,从而对生殖轴起作用。GnRH对垂体外的多种外周组织的生理功能亦有调节作用。近年的研究发现 ,卵巢、子宫内膜、前列腺、垂体、胰腺和肝脏发生的肿瘤细胞上有GnRH结合位点 ,GnRH类似物可抑制这些肿瘤细胞生长。GnRH的肿瘤抑制效应是通过一种独立于垂体促性腺激素释放之外的机制实现的。目前已有很多学者开始对GnRH与肿瘤作用机理方面进行研究。阐明这些作用机理将具有重要的理论意义和临床实用价值     

Ontogeny and distribution of gonadotropin-releasing hormone (GnRH) neuronal systems in the brain of the cichlid fish Cichlasoma dimerus

Using immunocytochemistry we have described the distribution and ontogeny of three distinct gonadotropin-releasing hormone (GnRH) neural systems, emphasizing the analysis during the period of sex differentiation in the South American cichlid fish Cichlasoma dimerus. In the forebrain a group of neurones immunoreactive to salmon GnRH that formed clusters in the nucleus olfacto retinalis (NOR), was located at the junction of the olfactory bulb and the telencephalon. These neurones differentiated 3 days after fertilization from the olfactory placodes. GnRH immunoreactive neurones along the olfactory nerves through the rostrobasal olfactory bulb were observed on day 4 and at the NOR on day 10. A group of neurones immunoreactive to chicken GnRH II was seen in the dorsal midbrain tegmentum. They originate from the ventricular ependyma between days 5 and 6. These neurones remained close to blood vessels throughout development. Between days 22 and 30 a group of neurones immunoreactive to seabream GnRH was detected in the anterior basal preoptic area. GnRH innervation of the pituitary was detected after the differentiation of preoptic neurones and in coincidence with gonadal differentiation. We hypothesize that the GnRH neural systems have three distinct embryonic origins. Furthermore, we show that the NOR and the midbrain GnRH neurones might have functions other than gonadal development, whereas the preoptic GnRH neurones in C. dimerus might be associated with gonadal sex differentiation.     

GnRH在宫颈鳞状细胞癌中的表达及意义

目的:从蛋白质及核酸水平研究GnRH(促性腺激素释放激素)在宫颈鳞状细胞癌中的表达,以探讨其存在的临床意义。方法:采用免疫组化和原位杂交两种实验方法,分别从蛋白质和核酸水平检测45例宫颈鳞状细胞癌及20例慢性宫颈炎中GnRH的表达。结果:在20例慢性宫颈炎宫颈上皮组织中GnRH及GnRH mRNA表达均为阴性,而在45例宫颈鳞状细胞癌组织中,GnRH阳性表达率为80%,GnRH mRNA阳性表达率为64.44%,即无论是在蛋白质水平还是在核酸水平比较两者均显示差异有显著性;定性及定量比较GnRH及GnRH mRNA在不同临床期别的宫颈癌组织中的表达情况,结果为GnRH定性表达率Ⅰ期50%(6/12),Ⅱa期85.7%(18/21),Ⅱb-期100%(12/12);GnRH mRNA定性表达率,Ⅰb期33.3%(4/12),Ⅱa期71.4%(15/21),Ⅱb-期83.3%(10/12);GnRH及GnRH mRNA在定性上表达有显著差别(P<0.05)。结论:宫颈鳞状细胞癌组织存在较高的GnRH,其有可能参与宫颈鳞状细胞癌发生、发展,为进一步研究通过抑制GnRH的产生,减少宫颈癌的发生及治疗宫颈癌提供了一定的理论基础。     

Dose-finding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention of premature luteinizing hormone surges in IVF/ICSI patients: antide and hormone levels

BACKGROUND: The aim of this study was to define the minimal effective dose of antide (Iturelix) to prevent premature luteinizing hormone (LH) surges in in vitro fertilization (IVF) patients. METHODS: In a prospective, single centre study, 144 IVF/ICSI patients were stimulated with r-hFSH from cycle day 2 and from cycle day 6 onwards, cotreated with daily 2 mg/2 ml (n=30), 1 mg/ml (n=30), 0.5 mg/ml (n=31), 0.5 mg/0.5 ml (n=23) and 0.25 mg/ml (n=30) GnRH antagonist (antide). Serum samples were taken three times daily during antide administration to assess antide and hormone levels. The minimal effective dose was defined as the lowest dose group with <2 LH surges (LH >12.4 IU/l and progesterone >2 ng/ml). RESULTS: Serum antide levels, mean LH and E2 levels per day and their area under the curves were dose-related to antide. The bioavailability of antide almost doubled after dilution in larger volumes. Pre-injection LH levels gradually increased during GnRH antagonist treatment. LH surges occurred in the lowest dose groups 0.5 mg/ml (3.2%), 0.5 mg/0.5 ml (6.7%) and 0.25 mg/ml (13.3%). Hence, 0.5 mg/ml is considered to be the minimal effective dose. Antide was overall well tolerated and safe. CONCLUSIONS: 0.5 mg/ml antide is the minimal effective dose to prevent an untimely LH surge in IVF patients stimulated with r-hFSH.     

子宫内膜癌患者孕激素受体B基因甲基化状态分析

目的探讨子宫内膜癌患者孕激素受体B基因CpG岛甲基化状态改变及其与肿瘤发生的关系。方法采用甲基化特异性PCR技术检测32例子宫内膜癌患及28例正常对照石蜡包埋标本孕激素受体B基因CpG岛甲基化状态。结果中国子宫内膜癌患者孕激素受体B基因异常甲基化发生率达66.7%,而28例正常对照标本没有发现甲基化异常。结论孕激素受体B基因甲基化可能与子宫内膜癌发生发展密切相关。     

Gonadotropin-releasing hormone (GnRH) physiology in men and women

The combined approach used in studies of GnRH secretion provided a complimentary array of techniques with which to establish the program of amplitude (dose) and frequency of GnRH secretion in the physiologic state. Normative data in men and women were useful in formulating frequency estimates, which could then be applied to the task of replacement of GnRH in deficient (IHH) individuals. Comparison of the results of therapy with these 'ablation-replacement' models then allowed to arrive closer to the true amplitude or dose of exogenous GnRH required to duplicate the physiologic ideal. In addition to providing insight into the neuroendocrine control of reproduction, these applications provided treatment of various reproductive disorders in men and women. Further expansion of the efforts into other potential defects of endogenous GnRH secretion will ultimately uncover those disorders amenable to therapy.     

Expression of glycodelin and cyclooxygenase-2 in human endometrial tissue following three-dimensional culture

PROBLEM: Our previous study showed that in vitro culture of human endometrial tissue in a three-dimensional (3D) fibrin matrix could mimic the early stages of endometriosis with invasion, gland and stroma formation and sprouting of new vessels. The objective of the present study was to evaluate the expression of glycodelin (Gd) and cyclooxygenase-2 (COX-2), two angiogenic factors, to further validate the 3D culture model of endometriosis. METHOD OF STUDY: Human endometrial fragments were obtained from endometrial biopsies and placed in a 3D fibrin matrix culture. Immunohistochemistry with specific antibodies to Gd and COX-2 was used to examine endometrial epithelium and blood vessels, and 4, 6-diamidino-2-phenylindole staining was used for nuclear identification. RESULTS: Three-dimensional culture of human endometrial tissue in the fibrin matrix resulted in the proliferation of endometrial stromal cells, glandular epithelium and angiogenesis. Gd positive glandular epithelium was seen in 85% of wells with developing endometrial glands and COX-2 positive new vessels were seen in 80% of wells with angiogenesis-like structures after 4 weeks of culture. CONCLUSION: Our findings confirm that angiogenesis occurs following the culture of endometrial tissue in the 3D fibrin matrix, and suggests that Gd and COX-2 might play important roles in promoting neovascularization and cell proliferation in the establishment of endometriosis.     

The gonadotropin-releasing hormone (Gnrh) gene maps to mouse chromosome 14 and identifies a homologous region on human chromosome 8

The murine gonadotropin-releasing hormone (Gnrh) locus has been mapped to mouse chromosome 14 using a mouse × Chinese hamster somatic cell hybrid panel. The equivalent human locus, known as luteinizing hormone-releasing hormone (LHRH), has been previously mapped to 8p21-8p11.2. Four other loci mapping to the human chromosome 8 short arm have been mapped to mouse chromosome 8; two of these (PLAT, GSR) lie proximal toLHRH, and two (LPL, DEF1) lie distal toLHRH. The localization ofGnrh, the murine homolog ofLHRH, to mouse chromosome 14 therefore defines a hitherto unrecognized block of homology between man and mouse. Furthermore, it indicates that the region of homology between the human chromosome 8 short arm and mouse chromosome 8 is composed of two separate blocks.     

Synaptic integration in hypothalamic gonadotropin releasing hormone (GnRH) neurons

The impact of the A-type GABA (GABA-A) receptor in gonadotropin releasing hormone (GnRH) neurons is controversial. In adult GnRH neurons, the GABA-A receptor conductance has been reported to either hyperpolarize or depolarize GnRH neurons. Regardless of whether GABA is inhibitory or excitatory in GnRH neurons, GABAergic input would be integrated with post-synaptic potentials generated by other synaptic inputs. We used dynamic current clamping and compartmental computer modeling to examine the integration of AMPA-type glutamatergic input and GABA-mediated input in both the hyperpolarizing (inhibitory) and depolarizing (excitatory) modes in GnRH neurons from transgenic mice (Mus Musculus) generated on a C57BL6 background. In both living and model neurons, action potentials were most likely a few ms after a maximum in AMPA conductance coincided with a minimum in inhibitory GABA. Excitatory GABA interacted differently with AMPA, with spikes most likely, in both dynamic clamping of living neurons and in model neurons, when a maximum in AMPA coincided with the decay from peak of a maximum in GABA. Distributing synapses along the dendrite maximized the temporal relationship between AMPA and GABA conductances and therefore, the potential for spiking. Thus, these two dominant neurotransmitters could interact in multiple frames to generate action potentials in GnRH neurons.     

Expression of c-kit (CD117) in benign and malignant human endometrial epithelium

BACKGROUND: The proto-oncogene c-kit encodes a tyrosine kinase receptor (CD117) with a molecular weight of 145 kd. Previous studies, predominantly utilizing immunohistochemistry, have led to contradictory findings regarding the expression of CD117 in the endometrium. To help resolve this issue, we analyzed a series of benign and malignant endometrial tissues using both immunohistochemistry and Western blot analysis. OBJECTIVE: To examine the expression of CD117 in benign and malignant human endometrial tissues. METHODS: The expression of CD117 in 35 benign endometrial tissues (7 hyperplastic, 14 proliferative, 14 secretory) and 10 endometrioid carcinomas was investigated by immunohistochemistry (clone K45 monoclonal antibody). Immunoprecipitation (clone K69 monoclonal antibody) followed by Western blotting (clone K45 monoclonal antibody and clone 1.D9.3D6 monoclonal antibody) was performed to confirm CD117 expression. RESULTS: Fifty-seven percent of the hyperplasias, 93% of proliferative endometria, and 79% of secretory endometria immunostained positively for CD117. In benign endometria, epithelial staining tended to be more intense in the hyperplastic and proliferative endometria as compared to the secretory endometria, whereas endometrial stromal cells were not immunoreactive. Of the 10 frozen endometrial tissues analyzed by immunohistochemistry, 4 of 9 endometrioid carcinomas and a single case of an endometrioid polyp developing in association with a carcinoma expressed CD117. Immunoprecipitation followed by Western blot analysis confirmed expression of full-length CD117 in an endometrial polyp and carcinoma, and revealed a correlation between levels of immunoprecipitated CD117 and immunohistochemical staining intensity. CONCLUSIONS: Benign and malignant endometrial tissues express CD117. Our data suggest (a) a possible relationship between estrogen and CD117 expression in benign endometrium and (b) potential involvement of this growth factor receptor in endometrial carcinogenesis.     

Half-dose,long-acting gonadotropin-releasing hormone agonist (Diphereline) is comparable with daily injections of short-acting gonadotropin-releasing hormone agonist (Suprefact) in IVF/ICSI cycles

Introduction

The aim of the study was to compare the efficacy of half-dose, long-acting GnRH analogue (Diphereline) with Suprefact in IVF/ICSI (in vitro fertilization/intracytoplasmic sperm injection) cycles.

Material and methods

In this randomized clinical trial performed in Royan Institute, 126 infertile women who were first time candidates for IVF/ICSI were enrolled. Patients were randomly divided into two groups by using a random number table. In one group, 62 patients received a single half dose, 1.87 mg Diphereline, in mid-luteal phase. In the other group, 64 cases were treated with buserelin from the previous mid-luteal phase. P value less than 0.05 was considered significant.

Results

The mean age of patients in the Diphereline and Suprefact groups was 27.9 ±3.6 and 29.6 ±3.5 years, respectively (p = 0.01). In the Diphereline group, the mean number of used gonadotropins was 25.6 ±12.1 ampoules, while in the second group it was 25.9 ±8.5 ampoules. Numbers of retrieved and MII oocytes were significantly higher in the Diphereline group (12.1 ±6.3 and 9.6 ±5.5) in comparison to the Suprefact group (9.4 ±6.4 and 7.2 ±5.1). Although the number of developed embryos in the Diphereline group was statistically higher than in the Suprefact group (6.1 ±3.9 vs. 4.7 ±3.4, p = 0.04) there was no significant difference in pregnancy rate (37.1%, 95% CI [26.16-49.54] vs. 37.5%, 95% CI [26.67-49.75]).

Conclusions

A half-dose, long-acting GnRH agonist can be successfully used in ovarian stimulation and produces a higher number of MII oocytes and embryos. The pregnancy rates with this method are acceptable.     

Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.)

Using specific antibodies for the two molecular forms of gonadotropin-releasing hormone (GnRH) present in the European eel, Anguilla anguilla, (mammalian GnRH, mGnRH, and chicken GnRH II, cGnRH-II), we employed immunocytochemistry to determine the distribution of these two peptides in the brain and in the pituitary. The results indicate that mGnRH and cGnRH-II are localized in different neurons: mGnRH-immunoreactive (ir) perikaria were observed in the olfactory bulbs, the junction between olfactory bulbs and telencephalon (nucleus olfactoretinalis), the telencephalon, the preoptic region and the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. Mammalian GnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, optic tectum, mesencephalon) and in the pituitary. Chicken GnRH-II-ir cell bodies were detected in the nucleus of the medial longitudinal fasciculus of the midbrain tegmentum, but only scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II-ir fibers, contrasting with an extensive mGnRH innervation. These results are in agreement with our previous data obtained in the same species using specific radioimmunoassays for mGnRH and cGnRH-II. They demonstrate a differential distribution of the two forms of GnRH in the brain of the eel, as in the brain of some other vertebrate species, and suggest differential physiological roles for the two GnRH forms in the eel. They also provide information concerning the evolution of the GnRH systems in vertebrates.