Polymorphism of Follicle-Stimulating Hormone Receptor Gene in Ningxia Tan Sheep

[ Objective] To study the polymorphism of follicle-stimulating hormone receptor （FSHR） gene in Ningxia Tan sheep and thus to provide a theoretical basis for breeding. I Methodl Genotypes of 111 healthy Ningxia Tan sheep were examined by polymerase chain reaction and restriction fragment length polymorphism （PCR-RFLP）. [ResultS] A 306-bp fragment was amplified. The PCR products digested with restriction enzyme Alu I showed polymorphism with three genotypes, L e., GG, CG and CC. The genotypic frequencies of GG, CG and CC were 0.135 1 （ 15 individuals）, 0.666 7 （74 individuals） and 0.198 2 （22 individuals）, respectively. The allele frequencies of G and C were 0.468 5 and 0.531 5, respectively.[ Conclusion] FSHR aene is Dolvmomhic in Ninaxia Tan Sheeo.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Cloning and Transcriptional Activity of Follicle-Stimulating Hormone Receptor Promoter in the Jintang Black Goat

[ Objective] To clone follicle-stimulating hormone receptor （FSHR） promoter in the Jintang black goat, study its transcriptional activity, and provide a basis for alternative splicing of FSHR gene. [Method] The total DNA were extracted from the womb of Jintang black goat, and one pair of primers were designed for amplification of FSHR promoter fragments, then the sequences and homology were analyzed. The FSHR promoter fragment was inserted into the pcFSHRB1 expression vector to substitute the CMV promoter and construct the pcFSHRB2 expression vector. The pcFSHRB1 and pcFSHRB2 expression vectors were transformed into HEK293 cells, respectively. Then these cells were collected after 24 and 48 h treatment with 2 mlU/ml follicle-stimulating hormone （FSH）, and the cAMP levels were detected. [Result] The FSHR promoter sequence of Jin- tang black goat had 34.2% homology to that of chicken and 41.6% to that of rat, respectively. The transcription initial site of FSHR was at -576 bp and its upstream sequences contained two TATA-boxes, four CAAT-boxes, one E-box and one Wl-box. After treating for 24 and 48 h, the cAMP levels of pcFSHRB2 were respectively 299.581 3 and 125.528 1 pmol/L; and that of pcFSHRB1 were respectively 120.057 1 and 109.940 7 pmoVL. [Conclusion] The FSHR promoter of Jintang black goat is a typical type 2 eukaryotic promoter, and it is also a strong promoter.

Role of follicle-stimulating hormone and estradiol benzoate in recovering spermatogenesis in tamoxifen-injured rats

Objective:To evaluate the effects of follicle-stimulating hormone (FSH) and estradiol benzoate (EB) on the recovery of spermatogenesis, histology, sexual hormones levels and testicular gene expression in testes of tamoxifen-injured rats.Methods:Forty adult rats were divided into eight groups in a factorial arrangement of tamoxifen and hormonal treatments. Half of the groups orally received 0.6 mg/kg tamoxifen, and 30 d later tamoxifen and no-tamoxifen groups (controls) were paired and assigned into four hormonal treatments with daily intramuscular injections for 10 consecutive days: 1 mL saline (control);7.5 IU FSH;12 μg/kg EB;and 7.5 IU FSH+12 μg/kg EB. One day after the last treatment, spermatozoa were recovered from epididymis, blood was processed for sex hormones concentration (testosterone, FSH and luteinizing hormone) and testes were processed for histology and RNA extraction for expression of genes related to apoptosis [caspase 3, inducible nitric oxide synthase (iNOS) and B-cell lymphoma-2 (Bcl-2)].Results: Control groups did not show significant changes in most parameters, but hormonal treatments decreased caspase 3 and iNOS and increased Bcl-2 expression. Tamoxifen significantly decreased counts, motility and viability of spermatozoa, Bcl-2 expression and sex hormones. It increased intertubular space, caspase 3 and iNOS expression, and induced seminiferous tubular atrophy. The hormonal treatments reverted spermatogenesis, hormonal levels and histology compared with controls, however not attaining the same sperm quality as controls.Conclusions:Tamoxifen is clearly detrimental to spermatogenesis and overall testicular structure and function, whereas hormonal therapy with FSH and EB can improve testicular function and revert tamoxifen-induced azoospermia.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

Androgen receptor gene polymorphism and sex hormones in elderly men:the Tromsøstudy

The aim of this study was to examine whether CAG/GGN repeats are significant modulators of serum concentrations of total and free testosterone(T)as well as of luteinizing hormone(LH)in elderly men.Sixty-nine 60-to 80-year-old men with subnormal T levels(≤11.0 nmol L^(-1))and 104 men with normal T levels taking part in a nested case-control study were used for these analyses.Sex hormones were measured and free T was calculated.The CAG and GGN polymorphisms in the androgen receptor gene were determined by polymerase chain reaction and subsequent direct sequencing.There were no differences in the CAG and GGN repeat lengths between the groups.In cross-sectional analyses of the whole cohort,total and free T were positively associated with CAG length(all P<0.05)before,but not after,waist circumference or body mass index was added to the model.CAG repeat lengths were weakly,but not independently,associated with total and free T.These findings indicate that when clinically evaluating T and LH levels in elderly men,the CAG and GGN repeat lengths do not need to be taken into consideration.

Fluoride Exposure,Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women

The effects of fluoride exposure on thefunctions of reproductive and endocrine systemshave attracted widespread attention in academiccircle nowadays. However, it is unclear whether thegene-environment interaction may modify thesecretion and activity of hypothalamus-pituitary-ovarian （HPO） axis hormones. Thus, the aim of thisstudy was to explore the influence of fluorideexposure and follicle stimulating hormone receptor（FSHR） gene polymorphism on reproductivehormones in Chinese women. A cross sectionalstudy was conducted in seven villages of HenanProvince, China during 2010-2011. A total of 679women aged 18-48 years were recruited throughcluster sampling and divided into three groups, i.e.endemic fluorosis group （EFG）, defluoridationproject group （DFPG）, and control group （CG） basedon the local fluoride concentration in drinkingwater. The serum levels of gonadotropin releasinghormone （GnRH）, follicle stimulating hormone（FSH）, luteinizing hormone （LH）, and estradiol （E2）were determined respectively and the FSHRpolymorphism was detected by real time PCR assay.The results provided the preliminary evidenceindicating the gene-environment interaction onHPO axishormones in women.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

The Determination and Evaluation of the Biological Activities for the Commercialization of Recombinant Follicle-Stimulating Hormone in Vitro

Follicle-stimulating hormone (FSH) plays a central role in mammals reproduction, with the actions of FSH mediated by follicle-stimulating hormone receptors (FSHRs) on the surface of target cells. The purposes of this study were to determine and evaluate the biological activities for the commercialization of recombinant follicle-stimulating hormone (rFSH) in vitro through the cellular internalization using cloned 293T-FSHR cell lines as target. Using imaging approaches we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus by endocytosis. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization, but the rFSH have promoted the internalization of the fluorescent, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.

Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor

The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR Promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFkB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase- dependent manner.

siRNA-targeted inhibition of growth hormone receptor in human colon cancer SW480 cells

AIM:To determine the effects of RNAi-mediated inhibition of the growth hormone receptor(GHR)gene on tumors and colon cancer cells in vivo.METHODS:Construction of a eukaryotic vector for human GHR expression,the pcDNA 6.2-GW/EmGFPsmall interfering RNAs(siRNAs)-GHR plasmid,was used to inhibit GHR expression.Thirty-six BALB/c nude mice were randomly divided into groups and treated with normal saline(NS),recombinant plasmid(G2),growth hormone(GH),5-fluorouracil(FU),G2+FU or G2+FU+GH.Each nude mouse was subcutaneously inoculated with 1×107human colon cancer SW480 cells;the nude mice were weighed before inoculation and on the 2nd,5th,8th,11th,14thand 17thday after inoculation.All nude mice were sacrificed after 17 d.Each subcutaneous tumor was removed and studied.Tumor volume was measured on the 5th,8th,11th,14thand 17thday after inoculation.The expression of GHR protein in the tumor tissue was detected by Western blotting analysis,and the differences in GHR mRNA expression in the tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction.RESULTS:Compared to the control group,the weights of the inoculated nude mice on the 17thday after inoculation were:G2:21.60±0.71 g,GH:21.64±0.45 g,FU:18.94±0.47 g,FU+G2:19.40±0.60 g,G2+FU+GH:21.04±0.78 g vs NS:20.68±0.66 g,P<0.05;the tumor volumes after the subcutaneous inoculation were:G2:9.71±3.82 mm3,FU:11.54±2.42mm3,FU+G2:11.42±1.11 mm3,G2+FU+GH:10.47±1.02 mm3vs NS:116.81±10.61 mm3,P<0.05.Compared to the GH group,the tumor volumes were significantly decreased in the experimental groups.The GHR protein expression(G2:0.39±0.02,FU:0.40±0.02,FU+G2:0.38±0.01,G2+FU+GH:0.39±0.01 vs NS:0.94±0.02,P<0.05)and the GHR mRNA expression(G2:14.12±0.10,FU:15.15±0.44,FU+G2:16.46±0.27,G2+FU+GH:15.37±0.57 vs NS:12.63±0.14,P<0.05)were significantly decreased and increased,respectively,in the experimental groups.CONCLUSION:Inhibition of GHR in human colon cancer SW480 cells resulted in anti-tumor effects in nude mice.

Thyroid hormone regulation of apoptotic tissue remodeling during anuran metamorphosis

Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval or- gans/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apop- tosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, which are conserved at both sequence and function levels in different animal species. A major challenge for future research lies at determining the signaling pathways leading to the activation of apoptotic processes and whether different death genes are involved in the regulation of apoptosis in different tissues/organs to effect tissue-specific transformations.

卵泡刺激素受体基因多态性与多囊卵巢综合征关系的研究进展

多囊卵巢综合征(PCOS)是一种高度异质性生殖内分泌紊乱性疾病,对女性生活产生极大影响。卵泡刺激素(FSH)通过与分布在性腺上的特异性卵泡刺激素受体(FSHR)结合,起到促进和维持正常的性腺发育和生殖功能,FSHR是PCOS易感基因的位点之一。近年来,FSHR基因多态性相关研究较多,FSHR基因突变可降低FSH敏感性,雄激素水平升高,导致排卵功能障碍,影响辅助生殖等。本文对FSHR基因多态性及其对PCOS的影响进行综述,为PCOS发病风险的评估及其遗传学相关研究提供一定的理论依据。

Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of humanFSH receptor promoter

<abstract>Aim: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. Methods: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5' flanking region fragment of its promoter. Results: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic litter mates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5 '-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

Developmental Changes of GHR Gene Expression in Multiple Tissues of Mink

[Objective] The paper was to explore the changes of GHR gene expression in different tissues of mink.[Method] With American mink as the research object, the expression volumes of GHR gene in heart, liver, spleen, lung, kidney, intestine and skin tissues at different growth stages(45, 90, 120, 150 and 180 days of age) were detected by real-time PCR, and comparative analysis was performed.[Result] The GHR gene expressions in heart, liver and spleen tissues at 180 days of age were extremely higher than those at other days of age( P<0.01). The GHR gene expression in lung tissue at 120 days of age was extremely higher than those at other days of age( P<0.01). The GHR gene expressions in intestine tissue at 45 and 120 days of age were extremely higher than those at other days of age(P<0.01), but no significant difference was observed between 45 and 120 days of age(P>0.05). The GHR gene expression in kidney tissue at 150 days of age was significantly higher than those at other days of age( P<0.05).The GHR gene expression in skin tissue was extremely higher than that those at other days of age( P<0.01). The GHR gene expressions in intestinal tissue at 45 and 120 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in skin tissue at 90 days of age was extremely higher than those in other tissues(P<0.01). The GHR gene expressions in intestine, spleen and kidney tissues at 150 days of age were extremely higher than those in other tissues(P<0.01). The GHR gene expressions in spleen tissue at 180 days of age was extremely higher than those in other tissues(P<0.01).[Conclusion] The expression of GHR gene in mink showed obvious spatio-temporal specificity.

Sequencing and Analysis of G HR Gene from Genomic DNA Pools of Mink

In order to study the single nucleotide polymorphisms （SNPs） of growth hormone receptor （GHR） gene in mink populations, genomic DNA pools of Minghua black minks and silver-blue minks were constructed, and the 10^th exon of GHR gene was PCR amplified from the two DNA pools and sequenced. The results showed that two SNPs were found at position 209 （T/C） and position 533 （C/A） of the 10^th exon of GHR gene in the two mink populations.

Analysis of thyroid stimulating hormone receptor gene mutation in children with hyperthyroidism

Objective To explore the characterization of thyroidstimulating hormone receptor ( TSHR) gene mutationalspectrum in children with hyperthyroidism from Guangzhou.Methods Ninety children were diagnosed with hyperthyroidismfrom July 2009 to July 2014 in our institute.Their median age at diagnosis was ( 7. 5 ± 3. 4 )years,and there were 28 males and 62 females. Mutationalanalysis were performed by performing polymerasechain reaction(PCR) and DNA direct sequencing of exon10 of TSHR gene. TSHR gene mutations from 50 unrelatedhealthy children were served as controls. The correlationbetween TSHR gene and hyperthyroidism in childrenwas explored. Results A total of 3 mutations were identifiedin ninety children who were diagnosed with hyperthyroidism,one synonymous mutations(p. V614V),andtwo missense mutations ( p. R707W and p. D727E).

CRHR1基因rs242941位点多态性与哮喘儿童ICS 长期安全用药的关系

目的研究促肾上腺皮质激素释放激素受体1(CRHR1)基因rs242941位点的多态性与哮喘儿童吸入性糖皮质激素(ICS)长期安全用药的关系。方法选取2018年10月至2020年10月该院收治的100例哮喘儿童,均接受ICS治疗及常规对症治疗。采用TaqMan法检测CRHR1基因rs242941位点多态性,根据基因型分为GG、GT+TT,比较不同基因型患儿的肺功能、哮喘控制测试评分及不良反应发生情况。采用多因素Logistic回归分析影响哮喘儿童ICS安全用药的危险因素。结果CRHR1基因rs242941位点存在3种基因型:GG(72例)、GT(20例)、TT(8例),G和T等位基因频率分别为82%和18%。治疗12周后不同基因型患儿的哮喘控制测试评分、第1秒用力呼气容积(FEV1)、FEV1/用力肺活量(FVC)比值与治疗前比较均明显提升,其中GG基因型患儿改善情况优于GT+TT基因型,GG基因型患儿的症状及体征完全缓解时间明显短于GT+TT基因型,差异有统计学意义(P<0.05)。GG基因型不良反应发生率低于GT+TT基因型,差异有统计学意义(P<0.05)。单因素、多因素Logistic回归分析显示,rs242941位点基因型多态性是影响患者用药安全的独立危险因素(P<0.05)。结论CRHR1基因rs242941位点多态性与哮喘儿童ICS的疗效及长期用药安全性密切关联,其中TT基因型患儿具有更高的ICS不良反应发生风险。

骨质疏松受体基因研究方向

骨质疏松症(osteoporosis)是一种以骨量低下、骨组织微结构损坏,导致骨脆性增加,易发生骨折为特征的全身性骨病。骨质疏松是多因素、多基因疾病,是遗传和环境因素交互作用的结果。笔者论述了降钙素受体基因、甲状旁腺素受体基因、雌激素受体基因、肿瘤坏死因子基因、胰岛素样生长因子1受体基因、骨保护素基因、转化生长因子基因、维生素D受体基因、Ⅰ型胶原蛋白基因的生物学特征及其与骨密度、骨质疏松、骨质疏松性骨折的关联,为骨质疏松的发病机制、预防治疗、新药开发研究提供分子生物学依据。

miR-9-5p在乳腺癌组织中的表达与生物信息学分析

目的探讨miR-9-5p在乳腺癌组织中的表达及其与临床病理特征的关系,预测miR-9-5p靶基因并分析其在乳腺癌进展中的作用。方法通过实时荧光定量PCR检测83例乳腺癌和癌旁正常组织中miR-9-5p的相对表达量。分析miR-9-5p表达水平与乳腺癌患者临床病理特征的关系。使用生物信息学方法预测miR-9-5p的靶基因并筛选关键节点基因,并对靶基因进行富集分析。结果miR-9-5p在乳腺癌组织中的表达高于癌旁正常组织[1.45(0.41,4.72)vs 1.01(0.35,3.68),P<0.01],其表达水平与乳腺癌组织学级别、肿瘤大小、雌激素受体状态、孕激素受体状态、Ki67增殖指数和分子分型有关(P<0.05),与年龄、肿瘤部位、TNM分期、淋巴结转移情况和人表皮生长因子受体2状态无关(P>0.05)。miR-9-5p在80.95%(17/21)的三阴性乳腺癌、32.56%(14/43)的管腔型乳腺癌呈高表达,差异具有统计学意义(P<0.05)。生物信息学方法预测的靶基因有138个,并筛选出SIRT1、BCL6、FOXP1等关键节点基因。miR-9-5p靶基因功能集中于细胞对激素刺激的反应、雄激素受体信号通路负调控、生长调控等方面。结论miR-9-5p过表达与乳腺癌进展关系密切,参与激素受体相关的信号通路调控可能是重要的作用机制。