Kiss1和GPR54基因多态性与多囊卵巢综合征相关性分析

本研究旨在探讨Kiss1和GPR54基因多态性与多囊卵巢综合征的相关性。利用超声检查卵巢体积、血清睾酮、游离雄激素指数情况;临床评估患者身高(cm)和体重(kg)、BMI、静息血压、痤疮和黑棘皮病的分布;ELISA酶联免疫法检测血清中的kisspeptin和睾酮水平,使用Next generation sequencing方法(LGC group, Germany)对基因(Kiss1, GPR54)进行测序。结果显示,PCOS患者比对照组女性具有更高的BMI和mFG评分,PCOS患者血清Kisspeptin和睾酮浓度显著提高,且LH浓度也显著高于对照组(p<0.05)。GPR54和Kiss1 2个基因在患者体内存在多态性;测序分析结果显示GPR54基因存在的2个新的SNP位点(chr19:918686, A→G和chr19:918735, A→G),这2个新的多态性位于内含子区域(内含子2),Kiss1基因也存在两个SNP,位于非翻译变体5的末端(rs5780218)和外显子3 (rs4889),即GPR54基因存在A→G多态性,Kiss1基因为CTT→CT/G→C多态性,且相关性关联分析结果表明,GPR54基因型多态性(Chr19:918735)与PCOS风险增加相关(p<0.05);而Kiss1 SNP的基因型与PCOS风险之间没有关联。此外,PCOS与GPR54和Kiss1基因的单倍型没有显著关联。本研究推论对PCOS发生风险的遗传影响可能不仅是通过直接改变Kiss1/GPR54相互作用,而且还可能通过改变个体与环境因素的相互作用。     

Design and synthesis of fluorescent probes for GPR54

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin–GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments.     

Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat

Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.     

Association Between Sexual Precocity and Alleles of KISS-1 and GPR54 Genes in Goats

KISS-1 and GPR54 were regarded as key regulators for the puberty onset and fundamental gatekeepers of sexual maturation in mammals. To explore the possible association between variations in KISS-1 and GPR54 with sexual precocity, mutation screening of exon 1 of KISS-1 and exon 1, exon 3, and partial exon 5 of GPR54 was performed in a sexual precocious breed (Jining Grey goats) and sexual late-maturing breeds (Inner Mongolia Cashmere, Angora, and Boer goats) by PCR-SSCP. The results showed that five novel mutations were identified in exon 1 and partial exon 5 of GPR54 including C96 T, T173C, G176A, G825A, and C981 T. The Jining Grey goats with genotype BB or AB had 1.07 (P < 0.05) or 0.40 (P < 0.05) kids more than those with AA. The Jining Grey goats with genotype DD or CD had 1.80 (P < 0.05) or 0.55 (P < 0.05) kids more than CC, respectively. The present study preliminarily showed an association between alleles B and D of GPR54 with high litter size and sexual precocity in Jining Grey goats.     

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.     

Chk1 is essential for the development of murine epidermal melanocytes

Embryonic deletion of mouse Chk1 is lethal; however, whether Chk1 is essential in all individual tissues is unknown. By breeding C57Bl/ 6 mice homozygous for a conditional allele of Chk1 (Chk1fl/fl) and bearing melanocyte‐specific Tyr::Cre and DCT:: LacZ transgenes, we investigated the consequences of Chk1 deletion in the melanocytic lineage. We show that adult Tyr::Cre; Chk1fl/fl mice lack coat pigmentation and epidermal melanocytes in the hair follicles, but retain eye pigmentation in the retinal pigmented epithelium (RPE). Melanoblasts formed normally during embryogenesis in Tyr::Cre; Chk1fl/fl mice at early times (embryonic day 10.5; E10.5) but were completely absent by stage E13.5, most probably as a consequence of spontaneous DNA damage and apoptosis. By contrast, melanoblast numbers were only slightly reduced in heterozygous Tyr::Cre; Chk1fl/ + embryos, and these mice exhibited normal coat pigmentation as adults. Thus, Chk1 is essential for the developmental formation of murine epidermal melanocytes but hemizygosity has little, if any, permanent developmental consequence in this cell type.     

The essential requirement for Runx1 in the development of the sternum

Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1^Re/Re embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1^Re/Re embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.     

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.     

Syntaxin 1A is required for normal in utero development

We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.     

Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling

The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.     

Coxsackievirus and adenovirus receptor is essential for cardiomyocyte development

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that is known to be a site of viral attachment and entry, but its physiologic functions are undefined. CAR expression is maximal in neonates and wanes rapidly after birth in organs such as heart, muscle, and brain, suggesting that CAR plays a role in the development of these tissues. Here, we show that CAR deficiency resulted in an embryonic lethal condition associated with cardiac defects. Specifically, commencing approximately 10.5 days postconception (dpc), CAR-/- cardiomyocytes exhibited regional apoptosis evidenced by both histopathologic features of cell death and positive staining for the apoptotic marker cleaved caspase 3. CAR-/- fetuses invariably suffered from degeneration of the myocardial wall and thoracic hemorrhaging, leading to death by 11.5 dpc. These findings are consistent with the view that CAR provides positive survival signals to cardiomyocytes that are essential for normal heart development.     

The murine stanniocalcin 1 gene is not essential for growth and development

The stanniocalcin 1 (STC1) gene is expressed in a wide variety of tissues, including the kidney, prostate, thyroid, bone, and ovary. STC1 protein is considered to have roles in many physiological processes, including bone development, reproduction, wound healing, angiogenesis, and modulation of inflammatory response. In fish, STC1 is a hormone that is secreted by the corpuscles of Stannius and is involved in calcium and phosphate homeostasis. To determine the role of STC1 in mammals, we generated Stc1-null mice by gene targeting. The number of Stc1-/- mice obtained was in accordance with Mendelian ratios, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues. Our results demonstrated that Stc1 function is not essential for growth or reproduction in the mouse.     

干预GPR1通路对实验性小鼠脂肪累积的影响

一直以来,肥胖是令人担忧和烦恼的健康问题,可导致包括2型糖尿病在内的代谢综合征发生.与肥胖相关疾病的发病机制是多因子影响的结果,但是,越来越多的证据表明,脂肪组织分泌的细胞因子(脂联素、瘦素、TNF-α等)的改变,以及局部的炎症反应对于这些疾病的发生具有重要作用.Chemerin(也被称为他扎罗汀诱导基因2或者视黄酸受体反应子2),是近年来发现的一种脂肪细胞因子,是G蛋白偶联受体1(GPR1)的配体,在调节代谢、先天免疫等方面具有重要的作用.为了研究Chemerin及其受体GPR1对小鼠脂肪累积的影响,本课题组通过高脂饲料喂养,成功建立小鼠肥胖模型,利用si RNA干扰技术沉默小鼠和分化前3T3-L1细胞中Chemerin或GPR1基因的表达发现:a.Chemerin及其受体GPR1在高脂饲料喂养小鼠的腹股沟脂肪以及肩胛下脂肪中的表达高于正常饲料组;b.沉默C57BL/6小鼠体内Chemerin或GPR1基因的表达后,肝脏以及腹股沟脂肪组织中脂质的累积受到抑制;c.3T3-L1细胞在体外分化成熟过程中,Chemerin和GPR1也呈高表达的趋势,沉默分化前3T3-L1细胞中Chemerin或GPR1基因的表达后,3T3-L1细胞向脂肪细胞的分化受到影响,降低了脂肪细胞中脂质的累积以及与脂质代谢相关基因的表达,改变了成熟脂肪细胞中新陈代谢功能.这些结果提示,Chemerin及其受体GPR1可能在小鼠脂肪累积中具有调控作用.综上所述,Chemerin/GPR1可能是一种调节脂肪组织中脂质累积的潜在信号通路,为肥胖症等代谢紊乱疾病的治疗提供了可能的作用靶点.     

Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.     

Foxn1 is essential for vascularization of the murine thymus anlage

We addressed whether vascularization of the thymus anlage depends on Foxn1 expression. In the thymus anlagen of wild-type mice, CD31⁺ endothelial cells are initially observed between epithelial cells on embryonic day (Ed)12.5 and form luminal structure on Ed13. VEGF are produced in epithelial cells and mesenchymal cells which invaginate in the epithelial region of the anlagen on Ed13. However, in the nude thymus anlagen, neither CD31⁺ cells nor VEGF producing mesenchymal cells is detected in the epithelial region. The present results indicate that Foxn1 dependent epithelial development is essential for vascularization of the thymus anlagen.     

The targeted disruption of the MYPT1 gene results in embryonic lethality

Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.     

The essential roles of TGFB1 in reproduction

Transforming growth factor beta 1 (TGFB1) is implicated as a key regulator of the development and cyclic remodelling characteristic of reproductive tissues. The physiological significance of TGFB1 in reproductive biology and fertility has been extensively examined in Tgfb1 null mutant mice. Genetic deficiency in TGFB1 causes perturbed functioning of the hypothalamic–pituitary–gonadal axis, inhibiting luteinising hormone (LH) synthesis and leading to downstream effects on testosterone production in males and estrous cycle abnormalities in females. Oocyte developmental incompetence, accompanied by early embryo arrest as well as altered pubertal mammary gland morphogenesis are observed. In addition to LH and testosterone deficiency, male Tgfb1 null mice demonstrate complete inability to mate with females, associated with failure to initiate and/or sustain successful penile intromission or ejaculation. These studies demonstrate the profound significance of TGFB1 in male and female reproductive physiology, and provide a foundation for exploring the significance of this cytokine in human infertility and sexual dysfunction.     

Discovery of N-arylpyrroles as agonists of GPR120 for the treatment of type II diabetes

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.