MTNR1a和MTNR1b基因在繁殖期与非繁殖期雄性高原鼢鼠HPG轴上的表达

高原鼢鼠 (Eospalax baileyi) 终年营地下生活,感光受洞道限制,但褪黑素 (Melatonin) 分泌水平仍存有季节差异,为探明褪黑素对高原鼢鼠季节性繁殖的调控作用,研究利用q?PCR技术检测雄性高原鼢鼠繁殖期 (5月) 和非繁殖期 (9月) 下丘脑、垂体及睾丸中褪黑素受体1a (Melatonin receptor 1a, MTNR1a) 和褪黑素受体1b (Melatonin receptor 1b, MTNR1b) 基因mRNA的相对表达量,通过免疫组织化学技术对MTNR1a和MTNR1b在睾丸中定位,并采用Image Pro Plus软件进行免疫组化阳性评价。结果发现,高原鼢鼠繁殖期下丘脑和垂体中MTNR1a基因的相对表达量显著高于非繁殖期的相对表达量 (P < 0.05),MTNR1b基因的相对表达量在不同时期无显著差异 (P > 0.05),但非繁殖期睾丸中MTNR1a和MTNR1b基因的相对表达量均显著高于繁殖期 (P < 0.01);繁殖期除长形精子外的所有类型细胞以及非繁殖期的间质细胞、支持细胞和精原细胞中均观察到MTNR1a的阳性信号,繁殖期除精原细胞和长形精子细胞外的所有类型细胞,以及非繁殖期间质细胞和支持细胞中均观察到MTNR1b的阳性信号,且非繁殖期MTNR1a和MTNR1b的平均光密度值均显著高于繁殖期 (P < 0.01)。MTNR1a和MTNR1b基因在雄性高原鼢鼠HPG轴上的表达模式,提示了褪黑素在其季节性繁殖调控中的潜在作用。     

光周期—褪黑素信号调控高原鼠兔精原细胞分化和季节性精子发生

高原鼠兔是青藏高原特有的小型哺乳动物,其繁殖活动呈现明显的季节性。成年雄性高原鼠兔在繁殖期睾丸重量显著增加,精子发生正常进行,而在非繁殖期睾丸退化,精子发生阻断在未分化精原细胞阶段。光周期控制实验显示,长光照（16h∶8h）诱导非繁殖期高原鼠兔重新启动精原细胞分化和精子发生;而短光照（8h∶16h）显著抑制繁殖期高原鼠兔精子发生。酶联免疫分析发现,褪黑素分泌水平在长日照条件下降低而在短日照条件下升高。非繁殖期高原鼠兔连续注射褪黑素拮抗剂能诱导生殖细胞发育和精子发生恢复。促性腺激素释放激素(GnRH)与黄体生成素(LH)在繁殖期鼠兔下丘脑垂体显著升高,促卵泡素（FSH）水平无显著差异。注射GnRH可以促进非繁殖期高原鼠兔精原细胞分化和精子发生,而褪黑素注射后抑制GnRH的分泌进而负调控性腺轴。综上,高原鼠兔季节性精子发生受光周期-褪黑素信号控制,后者主要通过控制GnRH、LH水平影响精原细胞分化。本研究对理解季节性动物精子发生的调控机制有重要借鉴意义。     

MTNR1a和MTNR1b在牦牛不同组织中的表达及其在睾丸中的定位

褪黑素对调节季节性繁殖哺乳动物的生殖具有重要调节作用。其受体MTNR1a（Melatonin receptor 1a,褪黑素受体1a）主要参与昼夜节律和生殖调控,MTNR1b（Melatonin receptor 1b,褪黑素受体1b）与多种疾病发生密切相关。为了探讨褪黑素受体基因的生物学功能,本实验对牦牛不同组织中MTNR1a、MTNR1b基因的表达与定位情况进行了研究。采用qRT-PCR (Quantitative Real-Time PCR, qRT-PCR) 检测成年雄性牦牛各组织及不同发育阶段（30日龄,2岁、4岁、6岁和8岁龄）牦牛睾丸组织中MTNR1a、MTNR1b mRNA的表达规律,并运用免疫组化技术对不同年龄牦牛睾丸中MTNR1a、MTNR1b蛋白进行了定位研究。结果发现,MTNR1a mRNA在松果体组织中表达量最高,肺脏、肌肉和睾丸次之;随着年龄增加,MTNR1a mRNA在睾丸中的表达量逐渐升高,到4岁后表达量趋于平稳;MTNR1a蛋白在不同发育阶段牦牛睾丸组织中均有表达,圆形精子呈现较强的免疫阳性,其次为初级精母细胞;MTNR1b mRNA在松果体表达量最高（P<0.05）,肾脏、肝脏和下丘脑次之;在不同年龄牦牛睾丸中MTNR1b mRNA均有表达,且随着年龄的增加表达量逐渐增加,在8岁时表达量最高;MTNR1b蛋白主要定位在圆形精子细胞中。MTNR1a、MTNR1b基因在牦牛不同组织及不同发育阶段睾丸中的广泛表达,揭示了其在雄性牦牛生殖等生理过程中的重要作用。     

高原鼢鼠精子发生的形态学特征和关键调控因子探究

季节性繁殖是动物在长期进化中为适应环境变化而形成的生活史特征,受光周期和下丘脑-垂体-性腺轴的严密调控。高原鼢鼠（Eospalax baileyi）是青藏高原特有的地下啮齿类动物,其繁殖活动表现出明显的季节性。然而,地下啮齿类动物精子发生的形态特征和关键调控因子尚不明确。本研究以成年高原鼢鼠为研究对象,发现繁殖期成年雄性睾丸曲精小管内有各级生殖细胞,附睾内有长形精子,生精上皮可分为10个期;而非繁殖期睾丸重量显著下降,曲精小管内仅见精原细胞和支持细胞。激素水平检测结果显示,与非繁殖期相比,繁殖期褪黑素水平显著降低（P < 0.05）,促性腺激素释放激素、促黄体生成素和睾酮水平显著升高（P < 0.05）,而卵泡刺激素水平无显著差异。进一步研究发现,精原细胞分化的关键诱导因子维甲酸水平和其调控基因表达均呈季节性变化,且外源维甲酸注射能够诱导非繁殖期高原鼢鼠重启精子发生。综上,高原鼢鼠虽为地下动物,但其精子发生与下丘脑-垂体-性腺轴激素水平明显相关,且受睾酮和维甲酸信号的调控。本研究首次揭示了高原鼢鼠精子发生的形态学特征和关键调控因子,为理解季节性繁殖动物尤其是地下啮齿类动物生殖生理的调控机制提供了重要参考。     

重复制动应激对雌性大鼠下丘脑-垂体-卵巢轴的影响

目的: 探索重复制动应激对雌性大鼠下丘脑-垂体-卵巢轴的影响。方法: 40只SD雌鼠随机分为两组(n=20),对照组和实验组,一组正常饲养,一组采取递增负荷束缚应激,每天置于束缚器内制动应激一次(从上午9:00开始),第1日制动2 h,以后采用递增负荷,每日增加0.5 h,持续两周,通过检测体重、脏器系数、动情周期、性激素、病理和相关基因的表达探索其对下丘脑-垂体-卵巢轴的危害。结果: 重复制动应激使雌性大鼠体重下降、动情周期延长,卵巢和子宫的脏器系数和形态发生改变,利用qPCR技术对其相关基因检测,发现下丘脑促性腺激素释放激素、垂体促性腺激素释放激素受体、促卵泡生成素和促黄体生成素mRNA的表达显著下降,卵巢促卵泡生成素和黄体生成素受体 mRNA的表达显著上升,卵巢和子宫雌激素受体mRNA的表达显著下降。结论: 重复制动应激可能通过干扰下丘脑-垂体-卵巢轴的内分泌调节作用,使动情周期紊乱,从而损伤雌性动物的性腺和生殖内分泌功能。     

蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体表达的影响

目的:探讨蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体的影响。方法:选取40只健康雌性未孕SD大鼠,随机分为空白组、对照组、乌力吉-18高、低2个剂量组,每组10只。空白组灌胃等体积蒸馏水,对照组灌胃逍遥丸,高、低剂量组分别灌胃2.0 g·kg^-1·d^-1、1.0 g·kg^-1·d^-1乌力吉-18,连续给药31学艺术d。采用酶联免疫吸附法测定血清促性腺激素释放激素(GnRH)、促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E₂)及孕酮(PROG)的含量;免疫组化法检测下丘脑组织促性腺激素释放激素(GnRH)、垂体组织促性腺激素释放激素受体(GnRHR)的表达;以蛋白免疫印迹技术检测卵巢组织促卵泡生成素受体(FSHR)、黄体生成素受体(LHR)蛋白表达量。以实时荧光定量PCR检测卵巢组织中FSHR、LHR基因表达量。结果:与空白组比较,乌力吉-18低剂量组可明显升高血清LH含量(P<0.05),上调下丘脑组织GnRH、垂体组织GnRHR表达及卵巢组织FSHR、LHR蛋白表达(P<0.05);乌力吉-18高剂量组可显著升高血清FSH、LH、E₂含量(P<0.05),上调下丘脑组织GnRH表达及卵巢组织FSHR表达量(P<0.05),并可显著升高卵巢组织中FSHR、LHR基因表达量(P<0.05);对照组可明显升高血清E₂含量(P<0.05)。结论:蒙药乌力吉-18可明显升高血清FSH、LH及E₂的含量,促进下丘脑组织GnRH、垂体组织GnRHR及卵巢组织中FSHR、LHR的表达,表明乌力吉-18能够对下丘脑-垂体-卵巢轴相关激素及受体表达产生影响。     

抚仙金线鲃kisspeptin基因的克隆及组织表达特性

抚仙金线鲃Sinocyclocheilus tingi是云南抚仙湖的特有种,虽已突破其人工繁殖技术,但在塘养环境下不能自然繁衍。kiss1基因编码的神经多肽kisspeptin被认为是下丘脑-垂体-性腺轴的重要调节因子,通过调控促性腺激素释放激素、促性腺激素等激素的分泌参与到生殖调控中。本研究采用反转录PCR和c DNA末端快速扩增(RACE)技术在抚仙金线鲃中克隆出编码kisspeptin的2个基因kiss1和kiss2的c DNA,其中kiss1的开放阅读框长度为351 bp,编码116个氨基酸;kiss2的开放阅读框长度为369 bp,编码122个氨基酸。反转录PCR结果显示,kiss1在抚仙金线鲃雌雄个体中均是肠道的表达量最高;kiss2表达量最高的组织在雄性和雌性中不同,雄性为肠道,雌性则是下丘脑和全脑。为进一步探究kiss1和kiss2在中枢神经系统及垂体中的分布,本研究中将抚仙金线鲃的脑部分为8个部分进行反转录PCR,结果显示,雄性脊髓和垂体中的kiss1表达量较高,雌性垂体中的kiss1表达量最高,其次是视顶盖;kiss2在雌性和雄性中均在下丘脑的表达量最高。本研究的结果为抚仙金线鲃人工繁殖提供了更多探索的方向。     

大鼠运动病敏感性性别差异与血浆和垂体中AVP水平及垂体V1b受体表达水平的关系

目的：观察不同性别大鼠旋转前后不同时间点血浆和垂体精氨酸加压素（AVP）的含量以及垂体AvP—V1b受体阳性神经元数目和受体表达量,探讨AVP与运动病性别差异间的联系,为进一步认识运动病的发病机制提供实验依据。方法：采用条件性厌食症作为运动病模型。98只SD大鼠,雌雄各半,分别用放射免疫分析法、免疫组化及Western—blot法测定血浆、垂体AVP含量和垂体V1b受体表达水平。结果：旋转刺激后雌性大鼠糖精水（0．15％）饮用量的减少程度高于雄性大鼠。雌性大鼠血浆AVP含量在基础状态下高于雄性大鼠,旋转刺激后下降,而雄性大鼠无显著性变化。雌性大鼠垂体AVP含量在基础状态下也高于雄性大鼠,旋转刺激后8h下降。24h降低有显著性;雄性大鼠旋转后8h垂体AVP含量较旋转前明显下降,但降幅不及雌性大鼠,旋转后24h已近恢复。与应激反应密切相关的垂体V1b受体表达为阳性的神经元数目及V1b受体表达水平,在基础状态下,雌性大鼠显著高于雄性;旋转刺激后,雌性大鼠V1b受体表达为阳性的神经元数目和表达水平均明显降低,而雄性大鼠则无显著性改变。结论：运动病诱发刺激后,雌雄性大鼠血浆和垂体中AVP含量及垂体V1b受体表达均有差异,提示AVP的内分泌状态与运动病敏感性性别差异可能有某种关联。     

Rspo1对雌性脊椎动物性别分化的功能

长期以来雌性脊椎动物的性别分化被认为是一个“默认”的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

Rspo1在雌性脊椎动物性别分化中的功能

长期以来雌性脊椎动物的性别分化被认为是一个"默认"的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

MTNR1A基因对大白猪和长白猪产仔数的影响

根据MTNR1A基因在GenBank中的已知DNA序列设计了2对引物,采用PCR-SSCP技术在一个大白猪和长白猪群体中进行单核苷酸多态性（single nucleotide polymorphism, SNP）检测,发现了一个SNP位点,并对其不同基因型个体PCR回收产物进行测序。测序结果发现该SNP是由于在+159碱基处（GenBank中序列）发生了G→A的同义突变而引起的。并对该SNP与产仔数进行了关联分析,结果表明该SNP对产仔数有影响。     

Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality

The aim of this study was to examine the polymorphism in MTNR1A gene and its relation to reproductive seasonality in Mediterranean Italian buffaloes reared in Sardinia. The mating period and calving of 100 multiparous buffalo-cows were recorded for three years (2005-2008). Genomic DNA was subjected to PCR for the amplification of the exon II, then 40 amplicons were sequenced. The obtained sequence was deposited in GeneBank database (accession number GU817415). PCR products were checked for the presence of HpaI restriction sites and assigned to genotypes “C/C”, “C/T” or “T/T”. Allelic frequency of C and T alleles was 0.44 and 0.56 and genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T. In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January and their calving mainly occurred from August to September. On the other hand animals with T/T genotype showed mating mostly in the semester between February and July and calving occurred largely from March to May in all the three years. Heterozygous, in all the three years, showed about the same number of animals mated within each six-month period. The results of the present study provide for the first time a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from buffaloes. Moreover our data showed an association between Single Nucleotide Polymorphism and seasonal reproductive activity in these animals.     

Effect of genotype at the MTNR1A locus and melatonin treatment on first conception in Sarda ewe lambs

A sample of 423 Sarda ewe lambs from three different farms was used to evaluate the effect of one or two melatonin implants on the time of first conception. On each farm, 141 animals were divided into three groups. On June 30 these animals received either no treatment (Group C), 18 mg melatonin (Group M1, one implant), or 18 + 18 mg melatonin (Group M2, two implants). Thirty-five days after treatment, rams were introduced in the ewe lambs flock and subsequently removed after 40 days. Lambing dates were recorded between 150 and 190 days from the first day of male introduction. Genotyping and sequencing of the MT1 exon 2 were carried out to analyze the structure and the possible influence of the MT1 receptor gene on reproductive response to melatonin treatment. Melatonin-treated animals had a higher rate of pregnancy (P < 0.05) and lambed earlier (P < 0.05) compared with untreated animals. Single nucleotide polymorphisms were found in exon II of MT1 gene at positions C606T and G612A leading to genotypes C/C, C/T or T/T and +/+, +/− and −/−, respectively. Melatonin-treated animals of +/+ genotype showed a higher number of pregnancies (P < 0.05) and lambed earlier (P < 0.05) compared to untreated animals of the same genotype. Melatonin treatment did not affect reproductive activity in any other genotype analyzed. No correlation between genotype and the time of first conception was found in untreated animals. Concluding data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that +/+ genotype is able to influence reproductive response to melatonin treatment.     

Analysis of MTNR1B gene polymorphisms in relationship with IRS2 gene variants,epicardial fat thickness,glucose homeostasis and cognitive performance in the elderly

Genome-wide association studies pinpointed common variants in or near the MTNR1B gene encoding MT2 melatonin receptor to be strongly associated with fasting glucose levels. IRS2 gene polymorphisms impact insulin resistance and epicardial fat (EF) thickness, which in turn is correlated with visceral adiposity, cognitive ability and risk for metabolic plus cardiovascular disease. We aimed to discover the interactions between MTNR1B and IRS2 gene polymorphisms, insulin sensitivity, EF thickness and cognitive performance in the elderly. In 60 subjects aged 60 years and older, we evaluated five single nucleotide polymorphisms (SNPs) within the MTNR1B locus (rs10830962, rs4753426, rs12804291, rs10830963, rs3781638), the Gly1057Asp variant of IRS2 gene (rs1805097), biochemical parameters, cognitive performance by the Mini Mental State Examination (MMSE) and EF thickness by transthoracic echocardiography. We found that MTNR1B and IRS2 gene variants impacted EF thickness, lipid profile and glucose homeostasis. IRS2 but not MTNR1B variants impacted MMSE scores. In conclusion, MTNR1B SNPs interact with IRS2 gene variant, correlate with the amount of epicardial adipose tissue and impact glucose homeostasis and lipid profile influencing cardiometabolic risk.     

The variant red coat colour phenotype of Holstein cattle maps to BTA27

The variant red phenotype in Holstein cattle is indistinguishable from the traditional e/e recessive red phenotype caused by a mutation in melanocortin 1 receptor, but is inherited as a dominant trait in relation to black. Co-segregation analysis in four half-sib families segregating for variant red was conducted, excluding melanocortin 1 receptor , agouti signalling protein , attractin and melatonin receptor 1A as causative genes. However, variant red co-segregated with markers in a region of BTA27 that includes beta-defensin 103 ( DEFB103 ). Two newly identified microsatellites and 5 SNPs 5' of DEFB103 were used for linkage mapping in four segregating families (LOD = 3.26). One haplotype was inherited in VR cattle in a 6-generation pedigree.     

人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化

本研究探讨Noah信号通路在人骨髓间充质干细胞（human mesenchymal stem cells,hMSCs）体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂（β-ME,DMSO,BHA）作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶（neuron-specific enolase,NSE）和尼氏体的表达以确定诱导效果：用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1（JAG1）、调节蛋白活化相关物早老素1（presenilin 1,PS1）、靶基因hairy and enhancer of split1（HES1）信号分子表达的变化。结果显示：诱导前,处于G0/G1期的hMSCs占58．5％,S＋G2/M期的细胞占41．5％;诱导后,G0/G1期细胞比例升高,而S＋G2/M期细胞比例下降,NSE阳性细胞率达（77±0．35）％,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PSl和HES1 mRNA表达量较诱导前明显降低（均P〈0．05）。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。