VEGF在绵羊生殖管道中的表达规律

以不同发情周期雌性绵羊子宫、输卵管为研究对象,采用免疫组织化学技术,针对血管内皮生长因子（VEGF）在绵羊子宫、输卵管的表达、定位和变化规律进行了检测,同时应用相关图像分析软件对抗原染色强度进行了定量分析。结果表明：输卵管在发情0～15d,VEGF表达量在第9天达到峰值后经历波动逐渐下降过程,输卵管内膜上皮细胞是VEGF抗原的主要靶细胞;而子宫角在发情0～15d,VEGF表达量在第5天达到峰值后经历波动逐渐下降过程,子宫内膜固有层及腺体周围细胞为VEGF抗原的主要靶细胞。该研究结果为绵羊生产中进一步提高受胎率和妊娠率及频密产羔等技术的应用提供了科学依据。     

绵羊发情周期中VEGF在卵巢中的表达

应用免疫组化方法结合计算机图像分析技术,分析同期发情后0、5、9、12、15d的绵羊卵巢中血管内皮生长因子（Vascular endothelial growth factor,VEGF）表达强度变化,以期了解VEGF在绵羊卵巢发情周期不同时期的表达规律。结果显示：VEGF阳性目标主要出现于卵泡膜与颗粒细胞。原始卵泡、初级卵泡、次级卵泡VEGF表达依次增强（P〈0．05）。发情周期0～5d,大窦腔卵泡（颗粒细胞4～8层）VEGF表达量骤然上升（P〈0．05）,而9d开始显著下降,与5d比较差异显著（P〈0．05）。12d继续下降（P〈0．05）且为最低值,15d又明显上升（P〈0．05）。VEGF在卵巢间质呈弱表达,各个时期之间差异不显著（P〉0．05）。结果表明,绵羊卵巢存在着血管周期性新生的变化特点,而VEGF在这种周期性血管新生过程中起着重要的调控作用。     

绵羊发情周期卵巢微血管密度的观察

采用S-P免疫组织化学法通过CD34抗体标记血管内皮细胞,测定绵羊发情周期的0、5、9、12、15d的卵巢内原始卵泡、初级卵泡、次级卵泡、近成熟卵泡、卵巢间质微血管的分布,并分析微血管密度（MVD）的变化规律,探讨绵羊发情周期不同时期各级卵泡微血管生成状态及卵泡微血管生成与卵泡发育的关系。结果表明,绵羊的原始卵泡周围无独立血管网,而初级卵泡的卵泡膜附近开始出现微血管,其MVD值为3.60±0.89,次级卵泡周围微血管密度值显著增高（P〈0.05）,MVD值为6.80±0.84。大窦腔卵泡0、5、9、12、15dMVD分别为15.80±0.84、23.00±2.30、22.40±2.41、21.20±2.28、34.80±2.39。0～5dMVD值显著升高（P〈0.05）,而5、9、12dMVD值差异均不显著（P〉0.05）,12～15dMVD值又明显升高（P〈0.05）。卵巢间质MVD值在各个发情周期各个时期差异均不显著（P〉0.05）。说明绵羊在发情周期内卵泡的血管新生是从初级卵泡开始的,并且卵巢的血管新生主要发生在卵泡上,而卵巢间质无血管新生现象。     

胰岛素样生长因子及其受体mRNAs在绵羊生殖系统中的表达和分布

为检测胰岛素样生长因子（IGFs）及其受体（IGFR）mRNAs在绵羊发情周期早期卵巢、子宫和输卵管中的表达,探讨绵羊胚胎早期发育过程中其发育环境——生殖道中生长因子的表达、分泌及其作用,取绵羊发情周期早期卵巢、子宫和输卵管,经固定、切片、免疫染色,观察IGFs mRNAs的表达和分布情况。同时用RT-PCR技术研究了各组织中IGF-Ⅰ、IGF-Ⅱ、IGF-ⅠR、IGF-ⅡR mRNAs的表达情况。结果表明,IGFs mRNAs在绵羊发情周期早期的卵巢、子宫和输卵管中都有表达,4种因子表达模式相似：在卵巢中,IGFs主要定位于卵泡颗粒细胞,间质细胞亦有少量表达。在输卵管中,上皮细胞免疫染色呈阳性;在子宫中,腺细胞及上皮细胞的阳性信号强于固有层。RT-PCR检测表明IGFs mRNAs在3种组织中均有表达。     

瘦素对绵羊子宫内膜上皮细胞增殖、迁移和凋亡的影响

试验旨在明确瘦素在妊娠和非妊娠绵羊子宫内膜组织中的定位特征,探究其对绵羊子宫内膜上皮细胞中胚胎附植的影响。使用免疫组化法检测瘦素及其受体在妊娠和未妊娠绵羊的子宫内膜上皮组织的表达及定位;利用组织块分离培养原代绵羊子宫内膜上皮细胞,CCK8法确定瘦素最适浓度,RT-qPCR和Western blot检测瘦素对绵羊子宫内膜上皮细胞凋亡的作用,细胞划痕试验检测瘦素对绵羊子宫内膜上皮细胞迁移的作用,RT-qPCR检测瘦素对胚胎附植相关因子血管内皮生长因子（VEGF）、白血病抑制因子（LIF）、白细胞介素-6(IL-6)、子宫内膜附植位点黏蛋白（Muc1）、基质金属蛋白9(MMP9)、白细胞介素-1β(IL-1β)、骨桥蛋白（OPN）、泛素样修饰因子（ISG15）等基因mRNA的表达情况。结果显示,瘦素蛋白主要表达在未妊娠绵羊子宫内膜的基质和上皮组织中,主要在妊娠绵羊子宫内膜上皮组织中表达,瘦素受体表达无明显变化;组织块法可成功获得具有典型上皮样细胞特征并表达角蛋白CK18的原代绵羊子宫内膜上皮细胞。250 mg/L瘦素促进绵羊子宫内膜上皮细胞增殖和迁移,并显著上调Bcl-2,下调BAX、Cas...     

妊娠期绵羊子宫内膜Fit-1基因表达的RT—PCR检测

以β-actin基因作为内源性内标，采用RT—PCR方法检测了Flt-1基因mRNA在东北细毛羊、小尾寒羊妊娠期不同阶段子宫内膜中的表达量。结果显示，绵羊妊娠期在不同阶段的子宫内膜中Flt-1mRNA均强烈表达，且随着妊娠期的延长，Flt—1mRNA的表达量有逐渐增强的趋势（P〈0．05）；在相同的妊娠阶段小尾寒羊子宫内膜中的Flt-1mRNA表达量显著地高于东北细毛羊的Flt-1mRNA表达量（P〈0．05）。     

雌性哺乳动物子宫血管新生及其调控机制

雌性哺乳动物在进入初情期（青春期）之后,其子宫内膜随着发情周期（月经周期）及妊娠建立等机体生理机能的转换,发生一系列周期性组织结构上的变化,而这种周期性变化主要与其本身血管周期性新生密切相关,血管新生则主要受血管内皮生长因子（VEGF）的调控。本文对雌性哺乳动物子宫内膜在发情周期、胚胎植入过程及胚胎发育过程及胎盘形成等的血管新生特点及VEGF的表达规律进行综述,以便系统地了解雌性动物子宫内膜的血管新生过程及调控机制,为提高雌性动物繁殖效率提供参考依据。     

发情周期中山羊子宫和输卵管内肥大细胞及组织胺含量的变化

应用改良甲苯胺蓝染色法和透射电镜技术观察了山羊发情周期4个阶段肥大细胞（MC）在子宫和输卵管内的分布、形态及数量变化规律,用荧光测定改良法检测了子宫和输卵管组织中组织胺（HA）含量。结果显示：MC呈圆形、椭圆形或梭形,在子宫内主要分布在肌层,子宫外膜中零星散在,内膜中散在分布于固有层的子宫腺和血管周围,子宫阜和内膜上皮中未发现。输卵管内MC在黏膜皱襞、肌层和外膜中均有分布。MC在发情后期有明显的脱颗粒现象。发情周期中子宫及输卵管MC数量变化为：MC（发情后期）〉MC（发情前期）〉MC（间情期）〉MC（发情期）,子宫肌层MC数量各组间差异极显著（P〈0．01）,子宫固有层和输卵管内无显著变化。子宫角内HA含量在发情后期与其它时期有显著变化（P〈0．05）,子宫体、子宫颈和输卵管的变化不明显（P〉0．05）。     

绵羊妊娠期生殖组织VEGF基因的克隆及其组织表达量的检测

从雌性绵羊妊娠期不同阶段的输卵管、子宫内膜、黄体组织中提取总RNA,根据已经发表的绵羊的Flt-Ⅰ基因的cDNA序列设计合成引物,采用RT-PCR扩增出绵羊VEGF基因;将扩增产物克隆于裁体后进行序列分析,以基因为内参,对扩增的VEGF基因进行琼脂糖凝胶电泳后,应用,推断出不同组织中Flt-Ⅰ基因的表达量.结果表明:从雌性绵羊妊娠期不同阶段生殖道各组织上皮均获得82 bp的VEGF基因的扩增片段,且VEGF基因在雌性绵羊生殖道各组织内的表达量不同.说明VEGF基因对绵羊的妊娠维持起着重要的作用.     

绵羊妊娠识别、建立和维持的信号系统

妊娠的建立和维持归因于胚体的信号和黄体对孕酮的产生,在反刍动物,胚体滋养层分泌的激素抑制子宫内膜产生前列腺素PGF2α。在发情周期的绵羊,孕酮下调子宫内膜腔上皮和子宫腺上皮孕酮受体基因的表达,并伴随着上皮雌激素受体和催产素受体的增加。在催产素的作用下,子宫开始分泌溶黄体作用的前列腺素F2α。在妊娠绵羊,孕体滋养层产生的干扰素可作用于子宫内膜而直接抑制雌激素受体基因和催产素受体基因的转录。孕酮、干扰素、胎盘催乳素和生殖激素共同组成了一个"作用网络"来调节子宫的功能分化和子宫腺的形态发生,从而维持绵羊妊娠。本文综述了绵羊妊娠识别、建立和维持的信号系统。     

Effect of the uterus on subnormal luteal function in anestrous beef cows

The effect of the uterus on luteal lifespan and pattern of secretion of progesterone following early weaning of calves from anestrous beef cows was studied. Calves were weaned from 15 anestrous beef cows 23 to 33 d postpartum, and cows were allotted to a control (sham surgery, n = 8) or a hysterectomy (n = 7) group, with surgery performed at weaning. Cows in the hysterectomy group were injected (im) with 25 mg prostaglandin F2 alpha (PGF2 alpha) approximately 20 d after first estrus (d 0). The interval from weaning to estrus was longer (P less than .05) for the hysterectomy group (10.4 +/- 1.6 d) than the control group (6.2 +/- .5 d). In the control group, the first estrous cycle (8.8 +/- .3 d) was shorter (P less than .01) than the second estrous cycle (20.2 +/- .5 d). Following first estrus in the hysterectomy group, cows were not detected in estrus until after injection of PGF2 alpha and did not return to estrus. From d 0 to 5, mean concentrations of plasma progesterone were similar (P greater than .05) between groups for both estrous cycles; after d 5 of estrous cycle 1, concentrations of plasma progesterone decreased in the control group. Within the hysterectomy group, the pattern of secretion of progesterone from d 0 to 16 was similar after the first and second estrus. Furthermore, there was no difference in the pattern of secretion of progesterone from d 0 to 16 between hysterectomy (first or second estrous cycles) and control (second estrous cycle) groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle stimulating hormone pattern and luteal function in ewes receiving bovine follicular fluid during three stages of the estrous cycle

The objectives of this study were to determine 1) the ability of charcoal-extracted bovine follicular fluid (bFF) to suppress endogenous follicle stimulating hormone (FSH) at various stages of the estrous cycle and 2) the effects of suppression of FSH on luteal function and lengths of the current and subsequent estrous cycles. Twenty-six mature ewes were assigned randomly to receive 5 ml of either bFF or saline, subcutaneously, at 8-h intervals on d 1 through 5 (bFF n = 6; saline n = 3), d 6 through 10 (bFF n = 6; saline n = 3) or d 11 through 15 (bFF n = 6; saline n = 2) of the estrous cycle (d 0 = estrus). Blood was collected daily beginning at estrus and continued until the third estrus (two estrous cycles) or 40 d; more frequent samples were collected 2 h prior to initiation of treatment (0600), hourly for the first 8 h of treatment, then every 4 h until 0800 on the first day after treatment, and finally at 1600 and 2400 on that day. Plasma concentrations of FSH were lower (P less than .001) in bFF-treated than in saline-treated ewes. Treatment with bFF reduced (P less than .05) plasma concentrations of progesterone during the current but not during the subsequent estrous cycle. Treatment with bFF did not affect plasma concentrations of estradiol-17 beta. Administration of bFF on d 11 through 15 of the estrous cycle lengthened the interval from the decline in progesterone to estrus and the inter-estrous interval by approximately 3 and 4 d, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

杜蒙杂交母羊的繁殖规律研究

旨在研究杜蒙杂交母羊的繁殖规律。对内蒙古自治区乌兰察布市四子王旗范围内养殖的杜蒙杂交母羊（n=668）的首次发情时间、妊娠期、产羔率、产双羔率等指标数据进行整理;采用公羊试情方法,观察并记录杜蒙杂交母羊（n=302）的发情周期和发情持续时间。对上述指标数据进行分析,探究该地区杜蒙杂交母羊的繁殖规律。结果表明：杜蒙杂交母羊平均首次发情时间为8.5月龄（最早7.0月龄、最晚10.7月龄）,平均发情周期为16.0 d（最短13.5 d、最长18.5 d）,平均发情持续时间为24.0 h（最短20.0 h、最长27.5 h）,平均妊娠期为146.0 d（最短143.0 d、最长149.0 d）,产羔率为119.7%,产双羔率为19.7%。研究结果为掌握杜蒙杂交母羊自然发情规律,以及准确把握最佳配种时间提供了依据。     

Effect of termination of pregnancy or long-term progestogen exposure on subsequent estrous cycle length and concentration of progesterone in plasma of heifers

An experiment was conducted to determine whether short estrous cycles following abortion of heifers between 70 and 75 d of gestation are due to factors associated with the previous presence of a conceptus or long-term exposure of the uterus and(or) ovaries to a progestogen. Fifty crossbred heifers were randomly allotted at estrus (d 0) to five groups: control (n = 10), pregnant (Preg.; n = 14), progestogen (norgestomet) implant (Norg.; n = 9), progesterone-releasing intravaginal device (PRID; n = 9), or hysterectomy (Hyst.; n = 8). Control heifers were injected during the mid-luteal phase of an estrous cycle with 25 mg prostaglandin F2 alpha (PGF2 alpha) and length of the subsequent estrous cycle was determined. Beginning 6 to 8 d after estrus, heifers in the Norg. or PRID groups were given norgestomet ear implants or intravaginal coils, respectively, every 10 d for 70 d. Heifers were hysterectomized 5 to 8 d after estrus. Seventy to 75 d after conception, progestogen treatment or hysterectomy, heifers were injected (i.m.) with 25 mg PGF2 alpha and the last norgestomet ear implants or PRIDs were removed. Interval from PGF2 alpha injection to first estrus (means +/- SE) ranged from 2.5 +/- .2 to 4.4 +/- .7 d (P greater than .05). Length of the first estrous cycle means +/- SE) following PGF2 alpha-induced luteolysis or progestogen withdrawal was shorter (P less than .01) for the Preg. group (8.2 +/- .4 d) than for the control, Norg. and PRID groups (21.5 +/- .6 d; 19.3 +/- 1.4 d; and 18.2 +/- 1.3 d, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism of glucose by bovine reproductive tissues obtained during the estrous cycle and after calving.

Two experiments were conducted to determine the effect of physiological state (stage of the estrous cycle or time after calving) on the in vitro metabolism of glucose by reproductive tissues. In Exp. 1, the corpus luteum and ipsilateral uterine horn were collected at surgery from 15 cows at early (d 6 to 7), middle (d 10 to 13), or late (d 17 to 19) stages of the estrous cycle. Luteal or endometrial tissues (45 mg) were incubated (4 h, 37 degrees C) in metabolic flasks containing Nutrient Mixture F-10 (3 mL), increasing concentrations of glucose (1, 2, 5, 10, or 15 mM), and 1 microCi of [U-14C]glucose. Luteal tissue collected at the middle stage of the estrous cycle had greater (P less than .04) rates of glucose uptake (17.8 vs 12.1 ng/mg of wet tissue per min) and oxidation (138.6 vs 67.7 pg/mg of wet tissue per min) and a lower (P less than .02) rate of metabolism of glucose to lactate (10.5 vs 13.3 ng/mg of wet tissue per min) than tissue collected at the late stage. Compared to tissue collected at early and late stages, endometrial tissue collected at the middle stage of the estrous cycle had a similar (P greater than .5) rate of glucose uptake (29.6 vs 27.8 and 26.1 ng/mg of wet tissue per min), a lower (P less than .02) rate of metabolism of glucose to lactate (13.8 vs 16.6 and 16.4 ng/mg of wet tissue per min), and a greater (P less than .1) rate of oxidation of glucose (84.1 vs 65.8 and 72.3 pg/mg of wet tissue per min). In Exp. 2, the previously gravid uterine horn was collected at surgery on d 20 (n = 8) or 30 (n = 7) after calving, or the uterine horn ipsilateral to the preovulatory follicle was collected at first observed estrus after calving (n = 8). Uterine tissue obtained at first estrus after calving had a greater (P less than .003) rate of glucose uptake and oxidation (88.8 vs 77.3 and 75.5 pg/mg of tissue per min) than d-20 or d-30 endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of day of the estrous cycle at the initiation of a timed artificial insemination protocol on reproductive responses in dairy heifers

Our objectives were to identify stages of the estrous cycle at which initiation of a timed artificial insemination (Ovsynch/TAI) protocol may reduce pregnancy rates and to monitor ovarian follicle dynamics and corpus luteum development after initiation of the Ovsynch/TAI protocol at different stages of the cycle. Cycling Holstein heifers (n = 24) were injected twice with prostaglandin F2alpha to induce estrus and were scanned by ovarian ultrasonography to determine the day of ovulation (d 0). Heifers were assigned to initiate the Ovsynch/TAI protocol at d 2 (n = 5), 5 (n = 5), 10 (n = 4), 15 (n = 5), or 18 (n = 5) of the cycle. The Ovsynch/TAI was initiated with an injection of gonadotropin-releasing hormone agonist followed 7 d later with an injection of prostaglandin F2alpha. At 36 h after injection of prostaglandin F2alpha, heifers were injected with gonadotropin-releasing hormone agonist and inseminated 16 h later. Heifers were scanned daily during the Ovsynch/TAI protocol and every other day after insemination until 16 d later. Blood samples were collected daily starting at the 1st day heifers were scanned and continued until 16 d after insemination. Initiation of the Ovsynch/TAI protocol at d 15 of the estrous cycle caused heifers to ovulate prior to insemination. A shortened return to estrus (< 16 d) was caused by ovulation failure to the second gonadotropin-releasing hormone injection, by incomplete regression of the corpus luteum, and by short life-span of the induced corpus luteum. Day of the cycle in which the Ovsynch/TAI protocol is initiated affects dynamics of follicular development, plasma progesterone profiles, and occurrence of premature ovulation. Size of the pre-ovulatory follicle was associated positively with subsequent progesterone concentrations following insemination.