应用反转录聚合酶链式反应检测鸡Th1样淋巴因子mRNA表？…

应用反转录聚合酶链式反应（ＲＴ－ＰＣＲ）和限制性酶切分析技术对鸡脾细胞体外受到有丝分裂原ＣonＡ刺激的情况下,其Ｔh1样淋巴因子mＲＮＡ的表达水平进行了研究。实验结 果表明,ＣonＡ诱导培养３个小时的鸡脾细胞表达α－干扰素（ＣhＩＦＮ－α）、γ－干扰素（ＣhＩＦＮ－γ）和白细胞介素－２（ＣhＩＬ－２）等鸡的Ｔh1样淋巴因子mＲＮＡ 。未诱导但培养了３小时的鸡脾细胞可表达ＣhＩＦＮ－α和ＣhＩＦＮ－γmＲＮＡ,而未     

限制性内切酶片段长度多态性在几个绵羊品种中的研究

本文利用羊的促卵泡激素（ＦＳＨ）ｃＤＮＡ，胸腺（Ｔｈｙｍｕｓ）基因组ＤＮＡ及卵泡抑止素（Ｆｏｌｌｉｓｔａｔｉｎ）ｃＤＮＡ等３种探针与泰国长尾羊，喀麦隆羊（Ｃａｍｅｒｏｏｎ）及它们Ｆ１代杂种的基因组ＤＮＡ进行分子杂交，结果在ＥｃｏＲＩ，ＨｉｎｄⅢ和ＴａｑⅠ酶切的ＤＮＡ片段与ＦＳＨｃＤＮＡ杂交的图谱上，可观察到ＲＦＬＰ的存在，并可根据某些特殊区带的存在与否区别两个不同种的羊。用ＥｃｏＲＩ酶切的ＤＮＡ片段与胸腺基因组ＤＮＡ探针杂交，也发现了ＲＦＬＰ，而其它３种酶的酶切片段与此探针杂交，个体间的图谱是一致的。利用本实验所采用的４种限制性内切酶，在所测定品种及杂交种的卵泡抑止素位点没有发现变异。     

限制性内切酶片段长度多态性有几个绵羊品种中的研究

本文利用羊的促卵泡激素（ＦＳＨ）ｃＤＮＡ，胸腺基因组ＤＮＡ及卵泡抑止素ｃＤＮＡ等３种探针与泰国长尾羊，咯麦隆羊及它们Ｆ１代杂种的基因组ＤＮＡ进行分子杂交，结果在ＥｃｏＲＩ，ＨｉｎｄⅢ和ＴａｑⅠ酶切的ＤＮＡ片段与ＦＳＨ ｃＤＮＡ杂交的图谱上，可观察到ＲＦＬＰ的存在，并可根据某些特殊区带的存在与否区别两个不同种的羊。用ＥｃｏＲⅠ酶切的ＤＮＡ片段与胸腺基因组ＤＮＡ探针杂交，也发现了ＲＦＬＰ，而其它３种酶     

应用生物素标记的NDV—cDNA探针检测感染尿囊液中DNV—RNA的初…

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＤＮＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体－Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐ ｃＤＮＡ片段经光敏生物素标记后，即成ＤＮＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＤＮＶ强毒株和疫苗毒株的基因组ＲＮＡ，而不与ＩＢＤＶ－ｄｓＲＮＡ，ＡＩＢＶ－ｓｓＲＮＡ，ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ０ｄｓＤＮＡ、Ｆ     

新城疫病毒F48E9株及东北地区流行株F基因遗传变异分析

采用异硫氰酸胍／酚／氯仿抽提一步法,提取新城疫病毒（ＮＤＶ）Ｆ４８Ｅ９株及２个东北地区分离株ＤＢ３、ＤＢ５基因组ＲＮＡ,并以其为模板经反转录合成Ｆ基因ｃＤＮＡ第１链,再利用ＰＣＲ技术分段增出Ｆ基因的ｃＤＮＡ。Ｆ４８Ｅ９、ＤＢ３和ＤＢ５株Ｆ基因的ｃＤＮＡ经酶切修饰后,插入ｐＵＣ１９的多克隆位点中,转化感受态Ｅ。ｃｄｉ ＤＨ５ａ,经相对分子质量比较、酶切分析及ＰＣＲ等鉴定方法筛选出Ｆ４８Ｅ９、ＤＢ３和     

杜大长猪IGF-I基因的克隆及其序列分析

采用ＴＲＩzol法从杜大长杂交猪的肝脏中提出总ＲＮＡ,将其纯化后作为ＰＣＲ扩增模板 ,以设计的Ｐ１（５′－ＣＴＡＣＡＴＴＣＴＧＴＡＧＴＴＣＴＴＧＴＴＴＣＣ－３′）为引物合成ＩＧＦ－Ｉ基因cＤＮＡ的第１链,再以Ｐ１和Ｐ２（５′－ＡＴＧＧＣＣＣＴＧＴＧＣＴＴＧＣＴＣＴＣＣＴＴ－３′）为引物扩增到大小约为３６０bp的产和,并将其达隆至pＧＥＭ－Ｔ载体上。经筛选、酶切、我分析,表明该片段为ＩＧＦ－Ｉ基因的cＤＮＡ     

同源GnRH对无血清培养鸡卵泡颗粒细胞孕酮分泌的作用

选用产蛋规律的鸡，在一个产蛋序列中，排卵前１～３ｈ剖腹收集各级卵泡，分离颗粒层建立卵泡颗粒细胞无血清单层贴壁培养模型。在此基础上，用不同剂量鸡促性腺激素释放激素（ＧｎＲＨ－Ⅱ）单独处理或与羊ＬＨ（ｏＬＨ）协同处理，并使用ＧｎＲＨ－Ａｎｔａｇｏｎｉｓｔ（ＧＡ），以观察ＧｎＲＨ－Ⅱ对颗粒细胞孕酮分泌的影响。研究获得了以下结果：（１）ＧｎＲＨ－Ⅱ对鸡不同卵抱（Ｆ１、Ｆ３、Ｆ５）颗粒细胞孕酮分泌均有促进作用，并呈现剂量－反应关系；（２）ＧｎＲＨ－对ｏＬＨ促鸡卵泡颗粒细胞孕酮的分泌有明显的协同作用；（３）ＧｎＲＨ拮抗物使ＧｎＲＨ－Ⅱ的促卵泡颗粒细胞孕酮分泌作用受到阻断。     

应用生物素标记的NDV－cDNA探针检测感染尿囊液中NDV－RNA的初步研究

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＮＤＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体──Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐｃＤＮＡ片段经光敏生物素标记后,即成ＮＤＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＮＤＶ强毒株和疫苗毒株的基因组ＲＮＡ,而不与ＩＢＤｖ－ｄｓＲＮＡ、ＡＩＢｖ－ｓｓＲＮＡ、ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ－ｄｓＤＮＡ,ＦＰＶ－ｄｓＲＮＡ及ＡＩＬＶ－ｄｓＤＮＡ发生交叉杂交反应。试验结果表明：尽管该探钎含有编码Ｆｏ蛋白酶切位点序列的碱基顺序,但它还是不能把ＮＤＶ的强、弱毒株区分开。这说明ＮＤＶ强、弱毒株比区域内的碱基存在着相当大的同源性。不过,此探针对ＮＤＶ来说具有特异性,这就为ＮＤＶ的诊断技术开创了基因水平检测的新途径。     

套式反转录—聚合酶链反应检测恙螨及鼠肺内肾综合征出血热病毒RNA

选择覆盖肾综合征出血热病毒（ＨＦＲＳＶ）膜蛋白（ＭＰ）基因的保守区核苷酸序列合成２对引物,采用异硫氰酸胍一步抽提ＲＮＡ,建立了ＲＴ－ＰＣＲ检测鼠体恙螨及游离恙螨体内ＨＦＲＳＶ－ＲＮＡ的方法,扩增产物经凝胶电泳及斑点印迹杂交证实具有特异性。结果显示,ＨＦＲＳＶ抗原阳性鼠体恙螨５０只组、１０只组、游离螨５０只组,ＨＦＲＳＶ抗原阳性鼠肺１０００ｍｇ、５００ｍｇ组经ＲＴ－ＰＣＲ检测为阳性;ＨＦＲＳＶ抗原阳性鼠体恙螨５只组,ＨＦＲＳＶ抗原阴性鼠体恙螨５０只和１０只组,游离螨１０只和５只组,鼠肺ＨＦＲＳＶ抗原阳性１００ｍｇ组均未见有明显扩增带。进一步用套式反转录－聚合酶链反应（ＮｅｓｔｅｄＲＴ－ＰＣＲ）检测,在ＲＴ－ＰＣＲ未测出ＨＦＲＳＶ－ＲＮＡ的各组中均检测有ＨＦＲＳＶ－ＲＮＡ。结果表明ＮｅｓｔｅｄＲＴ－ＰＣＲ具有高特异、高敏感的特点,可用于检测恙螨体内微量ＨＦＲＳＶ－ＲＮＡ,为确认恙螨作为ＨＦＲＳＶ的传播媒介提供了分子生物学证据。     

鸡新城疫病毒B95株融合蛋白基因的克隆与序列分析

以鸡新城疫病毒（ＮＤＶ）澳大利亚布里斯班分离株Ｒ９５的 ＲＮＡ为模板,运用的ＲＴ－ＰＣＲ技术扩增其融合蛋白（Ｆ０）基因的ｃＤＮＡ。将扩增的Ｆ基因克隆到质粒ＰＵＣ１９中,转化入大肠杆菌ＪＭ１０９中,经ＡＩＸ平板筛选,ＰＣＲ等方法鉴定出Ｆ基因的阳性重组质粒,同时测定了Ｆ基因５’端７０９个核苷酸及３’端７６８个核苷酸的序列,对相应的氨基酸序列的研究表明,Ｂ９５株属弱毒株,含有Ｆ０中保守的疏水功能区和可糖基化位点,Ｂ９５与强毒株Ｆ４８Ｅ８株、Ｍｉｙａｄｅｒａ株和中等毒力的Ｂｅａｕｄｅｔｔｅ Ｃ株的氨基酸序列的同源性在９０％左右。     

大豆黄酮对伊莎鸡卵泡发育及其P450arom基因表达的影响

实验探讨了大豆黄酮(DAI)对伊莎鸡卵泡发育及其芳香化酶(P450arom)mRNA表达的影响。实验选取16只产蛋后期伊莎鸡,等分为对照组和DAI处理组。对照组饲喂基础日粮,实验组在基础日粮中添加10 mg/kgDAI。实验持续7周后,分离排卵前卵泡(F1、F2、F3……)的颗粒层及小黄卵泡和大白卵泡,通过RT-PCR法检测P450arom mRNA表达的相对丰度。结果表明:DAI明显提高了伊莎鸡小黄卵泡和大白卵泡的数量,P450arommRNA在伊莎鸡不同发育阶段卵泡中的表达存在差异,部分卵泡P450arom mRNA表达的相对丰度显著增加。因此,在产蛋后期伊莎鸡基础日粮中添加DAI可增加不同发育阶段卵泡的数目,上调部分卵泡中与发育相关的基因表达以促进卵泡发育。     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Expression of aquaporin 4 in the chicken ovary in relation to follicle development

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1–4 mm), yellowish (4–8 mm), small yellow and the three largest yellow pre‐ovulatory follicles F3‐F1 (F3 < F2 < F1; 20–36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre‐ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real‐time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre‐ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Comparison of aryl hydrocarbon receptor gene expression in laser dissected granulosa cell layers of immature rat ovaries

In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more β-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.     

Selected Ovarian Ultrasonographic Characteristics During Vernal Transition are Useful to Estimate Time of First Ovulation of the Year

It is important to get mares pregnant as early as possible after vernal transition and thus, identification signs of impending 1st ovulation of the year are warranted. To identify clinical indicators of an approaching first ovulation of the year, mares were teased with a stallion for oestrous detection starting January 3 and subjected to ultrasonographic examination. Day of first appearance of uterus oedema, follicular wall invagination, intrafollicular echogenicity, double contour of the follicle wall, increase in granulosa thickness, follicular wall hyperechogenicity and appearance of pear‐shaped follicles was registered, as well as follicle diameter and number. Seventy per cent of the mares had anovulatory oestrous periods of 4.6 ± 3.6 days, with an interoestroual interval of 12.5 ± 12.2 days. Number of anovulatory oestruses per mare was 2.4 ± 2.3. Uterine oedema occurred in 77% of the mares, 32.4 ± 25.6 days before ovulation. Invagination of the follicular wall appeared in 44.4% of the animals, 24.5 ± 18.4 days before ovulation. Intrafollicular echogenicity was seen in all mares and double contour of the follicle was seen in 77% of the animals. Both last two characteristics appeared 1–72 days before ovulation. Increased thickness of the granulosa occurred in 66% of the mares, 1–19 days before ovulation. Pear‐shaped follicles and follicular wall hyperechogenicity were detected 3 or less days before the first ovulation, in 44.4% and 55.5% of mares, respectively. Mean number of follicles >15 mm decreased at least 16 days before ovulation. We concluded that no isolated characteristic was a reliable indicator. However, increase in granulosa thickness, formation of a pear‐shaped follicle and follicular wall hyperechogenicity, associated with the reduction of the number of follicles >15 mm in diameter to <3, resulted in the first ovulation of the year in 44–67% of the transitional mares, 1–19 days after the characteristics appeared.     

Ovarian follicular dynamics in lines of sheep (Finn, Merinos) selected on ovulation rate

Ewes from selected lines of sheep from each of two breeds, Finns (high ovulation rate, low ovulation rate and control lines with respective ovulation rates of 5.4, 2.7 and 3.3) and Merinos (T Merinos selected for increased ovulation rate and control Merinos with respective ovulation rates of 1.9 and 1.2) were used to examine how selection to alter ovulation rate had altered follicle development. Ovarian antral follicles were counted, measured, classified as nonatretic or atretic (more than five pyknotic bodies). The growth of ovulatory follicles in vivo, followed by repeated follicle ink marking, also was compared in the three lines of Finns. Regardless of breed, ewes selected for high ovulation rate had a similar number of antral follicles and a similar extent of atresia compared with their controls. Alterations induced by selection were located in the last stages of folliculogenesis. T Merinos exhibited a lower proportion of atretic follicles among follicles greater than 3 mm and a larger diameter of the largest healthy follicle when preovulatory follicles were excluded. High-line Finn ewes recruited more follicles, which produced smaller preovulatory follicles, each containing a smaller number of granulosa cells compared with either the low- or control-line ewes. Hence, physiological selection for high ovulation rate raised it by different methods in Merino than in Finn ewes.