牛黄体中促黄体素受体剪接异构体的表达研究

利用RT-PCR结合测序技术,对牛黄体中促黄体素受体(Luteinizing hormone receptor,LHR)在转录后通过选择性剪接而形成的剪接异构体进行鉴定,并比较不同剪接异构体编码蛋白质氨基酸序列的差异,利用RT-PCR对黄体中的LHR剪接异构体进行定量分析,为研究LHR剪接异构体在黄体中的功能奠定基础.结果表明,牛黄体中LHR通过选择性剪接产生了包括全长mRNA在内的6种剪接异构体,其中第11外显子5'端的266个碱基的缺失会使mRNA因发生移码突变而使翻译提前终止.实时定量PCR结果显示,发生外显子缺失的mRNA的表述量总量是全长mRNA表达量的1.94倍.     

促卵泡素受体和促黄体素受体在猪子宫中的定位研究

为探明2种促性腺激素受体（FSHR、LHR）在猪子宫中的位置分布,本试验运用免疫组化ABC法对卵泡期、黄体期猪的子宫FSHR、LHR分别进行定位染色。结果显示,FSHR阳性细胞主要见于子宫内膜上皮细胞、血管平滑肌细胞。LHR阳性细胞主要见于子宫内膜上皮细胞、子宫肌层平滑肌细胞及血管平滑肌细胞;动情周期不同,FSHR、LHR的表达量也不同。卵泡期子宫内膜上皮细胞FSHR的表达量高于黄体期,血管平滑肌FSHR的表达量低于黄体期;卵泡期子宫血管平滑肌细胞LHR的的表达量高于黄体期,而子宫内膜上皮细胞、平滑肌细胞LHR的表达量均低于黄体期。     

猪黄体不同发育时期活性氧水平及多种抗氧化酶mRNA表达量变化规律的研究

试验旨在研究活性氧(ROS)及抗氧化酶基因表达在猪黄体组织发育及退化过程中的变化规律,为诠释猪黄体抗氧化机制补充理论基础并提供新的思路。试验所用卵巢采自延吉屠宰场,将黄体从卵巢剥离后,通过其形态大小将黄体初步分为初期、中期、后期以及白体,然后通过检测孕酮水平准确区分中期及后期黄体;通过冰冻切片及DHE荧光染色技术检测各时期黄体ROS水平,通过实时荧光定量PCR对各时期黄体内锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)以及谷胱甘肽过氧化氢酶(GPx)的mRNA表达量进行检测。结果表明:猪黄体内ROS水平随猪黄体周期性发育进程出现规律性升高,即后期ROS水平显著高于其他时期(P<0.05);中期显著高于初期与白体(P<0.05);而初期与白体间差异不显著(P>0.05)。Mn-SOD、GPx1及GPx4基因的表达水平随黄体的发育进程则出现规律性的下降:初期及白体中这几个基因的表达水平显著高于中期与后期(P<0.05),中期显著高于后期(P<0.05)。对于GPx3基因,其在白体的表达量显著高于其他时期(P<0.05),初期表达水平显著高于中期与后期(P<0.05),中期显著高于后期(P<0.05)。CAT基因表达量不随黄体的发育而变化(P>0.05)。综合上述试验结果,ROS、Mn-SOD及GPx与猪黄体发育及退化有关;而ROS水平与Mn-SOD及GPx的表达水平呈现出相反的规律。     

受体牛膘情与黄体发育关系的探讨

奶牛胚胎移植对受体牛的膘情选择比较严格,必须达到中等以上。2003年青海省畜牧兽医总站承担了“良种奶牛胚胎移植技术推广项目”,对5770头受体牛进行检查,结果膘情的好与差与黄体发育有着密切关系,营养水平较差的受体牛,其黄体发育普遍不理想。     

B超诊断技术在胚胎移植受体牛黄体鉴定中的应用

本试验选用150头育成牛进行同期发情,采用人工直肠检查法和超声波法检测黄体级别,对检查合格的受体进行单枚克隆胚胎移植。结果显示,同期发情处理牛112头有发情表现,直肠检测黄体合格牛89头,胚胎移植后51头妊娠,受胎率57.3%;超声波检测112头发情牛,其中26头符合要求,移植后24头妊娠,受胎率92.3%;二者差异显著(P<0.05)。因此,B超诊断可作为胚胎移植受体牛黄体鉴定中的一项辅助技术,能够显著提高受胎率。     

发情周期不同阶段牦牛子宫中黄体生成素受体的表达变化

为研究发情周期不同阶段牦牛子宫中黄体生成索受体(LHR)的定位及表达变化,笔者利用免疫组织化学SP法分别检测发情期、发情后期、间情期和发情前期牦牛子宫中LHR的表达,并进行光密度值分析.结果表明,LHR免疫阳性产物在牦牛子宫腺上皮细胞、基质细胞、血管内皮细胞、血管平滑肌细胞和肌层平滑肌细胞中均有表达;腺上皮细胞、基质细胞和肌层平滑肌细胞中LHR在发情前期和发情期表达最弱,发情后期表达增加,间情期表达最强(P＜0.05);子宫内膜血管平滑肌细胞中LHR的表达在发情期最强,间情期最弱(P＜0.05);血管内皮中LHR在发情期和发情前期表达很强,发情后期和间情期显著下降(P＜0.05).结果表明LHR参与了发情周期不同阶段牦牛子宫功能变化的调控.     

青年荷斯坦牛黄体发育状况对胚胎移植妊娠率的影响

本文阐述了青年荷斯坦牛胚胎移植受体的选择以及黄体鉴定标准，并针对黄体发育状况和月龄对移植妊娠率的影响进行了试验研究。研究表明：A、B级黄体均为可移植黄体，移植妊娠率差异不显著（P〉0．05）；C级黄体移植妊娠率为27．27％，显著低于A、B级黄体（P〈0．05）；D级黄体为不可用黄体。15月龄以下受体牛的可用率极显著低于16月龄以上受体牛（P〈0．01），但各月龄的可用受体牛的移植妊娠率差异不显著（P〉0．05）。     

实时荧光PCR检测牛边缘无浆体方法的建立及应用

根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血（奶牛和肉牛）,其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。     

家蚕丝蛋白基因在不同发育时期表达量的变化

本文采用半定量RT-PCR方法,检测了家蚕4～5龄期丝腺中5个丝蛋白基因(Fib-H、Fib-L、Ser-2短片段、Ser-2长片段、Ser-3)在不同发育时期表达量的变化。结果表明,Fib-H、FIB-L及Ser-3基因的表达规律相似,总体上4龄期表达量明显低于5龄,但4龄眠期均有明显表达,从5龄起蚕以后表达量逐渐上升,至熟蚕或熟蚕后2d达到高峰,此后迅速下降;Ser-2基因两个转录本的表达规律基本一致,与以上3个基因相比,Ser-2基因在4龄期存在相对较高的表达量,但在4龄眠期表达量明显降低,从5龄起蚕以后表达量逐渐上升,Ser-2短片段和长片段分别从5龄96h和144h之后表达量开始降低,到熟蚕期基本达到最低值。     

Nitric oxide in bovine corpus luteum: Possible mechanisms of action in luteolysis

Although prostaglandin (PG) F_2α is considered as the principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intraovarian factors. Among them, nitric oxide (NO) seems to play a mandatory role in luteolysis. In this article we review the background and current status of work on possible roles of NO in the CL function, based on available information and our own experimental data. NO is produced in all three main types of bovine CL cells: steroidogenic, endothelial and immune cells. PGF_2α and some luteolytic cytokines (tumor necrosis factor, interferon) increase NO production and stimulate NO synthase expression in the bovine CL. NO inhibits progesterone production, stimulates the secretion of PGF_2α and leukotriene C4, reduces the number of viable luteal cells and, finally, participates in functional luteolysis. NO induces the apoptotic death of CL cells by the modulation of bcl‐2 family gene expression and the stimulation of caspase‐3 gene expression and activity. However, this simple molecule shows both luteolytic and luteotropic actions during the estrous cycle in ruminants. The aim of this overview is to present and discuss the recent findings crucial for understanding NO role in the process of CL regression in cattle.     

Age‐related changes in the bovine corpus luteum function and progesterone secretion

Decreased fertility associated with maternal ageing is a well‐known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age‐associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age‐dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β‐HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation‐ and senescence‐related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age‐dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.     

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary

The aim of this study was to characterize expression patterns of hypoxia‐inducible factor‐1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol‐17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.     

Roles of reactive oxygen species in the corpus luteum

Cells living under aerobic conditions always face the oxygen paradox. Oxygen is necessary for cells to maintain their lives. However, reactive oxygen species such as superoxide radicals, hydroxyl radicals and hydrogen peroxide are generated from oxygen and damage cells. Oxidative stress occurs as a consequence of the excessive production of reactive oxygen species and impaired antioxidant defense systems. Antioxidant enzymes include superoxide dismutase (SOD), which is a specific enzyme to scavenge superoxide radicals; copper‐zinc SOD, located in the cytosol and Mn‐SOD, located in the mitochondria. Both types of SOD belong to the first enzymatic step to scavenge superoxide radicals. It has been reported that a number of local factors such as cytokines, growth factors and eicosanoids are involved in the regulation of the corpus luteum (CL) function in addition to gonadotropins. Since reactive oxygen species are generated and SOD is expressed in the CL, there is a possibility that reactive oxygen species and SOD work as local regulators of the CL function. The present review reports that reactive oxygen species and their scavenging systems play important roles in the regulation of the CL function.     

Relationships among the corpus luteum,follicles and conceptus in sheep

This study aimed to investigate the intra- and interovarian relationships among the corpus luteum (CL), the largest follicle (LF) and follicular population in non-pregnant and between the conceptus and ovarian structures in pregnant ewes. In experiment 1, the follicular and luteal structures were examined in 538 reproductive systems of non-pregnant Awassi ewes. The follicular population was categorised into small (SF), medium (MF) and large (LF) groups. Inter-relationships between CL and follicular population and between LF and subordinate follicles were determined. In experiment 2, the location and number of conceptuses were identified and correlated with the ovarian structures in 58 reproductive systems of pregnant ewes. Effects of pregnancy status, stage of pregnancy, pregnancy side and conceptual number on follicular population were determined. The results showed that the right ovary was more active than the left ovary. CL had intraovarian positive effect on the number of medium and large follicles. LF had no local suppressive effect on the subordinate follicles. Side and stage of pregnancy and the conceptual number did not affect the follicular population. Accordingly, it can be concluded that the LF has no local suppressive effect on the subordinate follicles. The CL has intraovarian positive effect on the follicular population. Follicular population does not show remarkable changes during the first term of pregnancy. The present study probably provides information which may help in the understanding of the ovarian dynamics during pregnancy in sheep.     

山羊黄体弥散性神经内分泌细胞的分布

为了探查山羊卵巢黄体中是否存在弥散性神经内分泌细胞,采用免疫组化链霉素抗生物素蛋白-过氧化物酶法对12~18月龄妊娠奶山羊卵巢中弥散性神经内分泌细胞标志物的分布进行了研究。结果显示:黄体组织中各区均含有催产素、神经元特异性烯醇化酶、S-100蛋白、突触素和5-羟色胺样免疫反应阳性细胞。这些细胞散在或成团分布,多呈圆形、卵圆形和三角形,一些细胞具有明显的突起。阳性细胞数由多至少顺序为:催产素、突触素、S-100蛋白、神经元特异性烯醇化酶、5-羟色胺。鉴于神经元特异性烯醇化酶、S-100蛋白、突触素和5-羟色胺是弥散性神经内分泌系统广谱的、共同的细胞标记物,结果提示山羊卵巢黄体中存在弥散性神经内分泌细胞。     

The effects of luteinizing hormone as a suppression factor for apoptosis in bovine luteal cells in vitro

The fate of the corpus luteum, a transient endocrine gland formed and degraded during an oestrous cycle, is decided by various physiological factors, such as luteinizing hormone (LH). As a stimulator of progesterone, LH is known to maintain corpus luteum functional and structural integrity by inhibiting apoptosis, a programmed cell death. Therefore, we aim to investigate its action during the mid-luteal phase hypothesized that LH suppresses the death mechanism of bovine luteal steroidogenic cells (LSC) by analysing the expression of proteins involved. Cultured bovine LSC obtained from corpus luteum were treated for 24 hr with recombinant TNF and IFNG in the presence or absence of LH. The result showed that LH proved to have a protective effect by increased cell viability (p < .05) and prevented DNA fragmentation (p < .05), as demonstrated by the WST-1 colorimetric assay and TUNEL assay. Expression analysis of mRNA and protein levels showed that LH altered the expression of BCL2 (p < .05), CASP3 (p < .05), FAS (p < .05), and BAX (p < .05) to support cell survival. In conclusion, our study suggests that LH prolongs the corpus luteum life span through the anti-apoptotic mechanism by increasing cell viability and suppressing apoptosis-related genes and protein expression.     

FSHR与LHR的研究进展

本文重点论述了FSHR与LHR的结构及在卵母细胞体外成熟过程中的表达和调控,并阐述了FSHR与LHR的研究现状及存在的问题。