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《中国兽医学报》2019,(8):1628-1634
为探讨卵泡期卵泡颗粒细胞促卵泡素特异性受体(FSHR)差异剪接体表达的变化,选择30只道寒杂交一代育成母羊肌注PG+埋植CIDR+肌注PG进行同期发情处理,选取9只发情母羊屠宰,取两侧卵巢组织,按小卵泡(≤3.0 mm),中等卵泡(3.0～5.0 mm)和大卵泡(≥5.0 mm)级别分别收集卵泡液和颗粒细胞,采用放射性免疫法测定类固醇激素(E_2和P_4)和FSH浓度,实时定量PCR检测不同直径卵泡颗粒细胞FSHR差异剪接体表达水平。结果表明,小卵泡和中等卵泡中FSHR1和FSHR3 mRNA的表达量极显著高于FSHR2和FSHR4(P0.01),在中等卵泡中FSHR3 mRNA的表达量显著高于FSHR1(P0.01);大卵泡中FSHR2 mRNA的表达量显著高于FSHR1、FSHR3和FSHR4(P0.01),FSHR1、FSHR3和FSHR4 mRNA的表达量差异不显著(P0.05)。大卵泡中雌激素浓度极显著高于小卵泡和中等卵泡(P0.01),孕酮浓度无显著性差异(P0.05),E_2/P_4比值与大卵泡中FSHR3 mRNA的表达量呈极显著负相关(P0.01)。由结果可知,卵泡发育过程中FSHR1和FSHR3可能是小卵泡和中等卵泡颗粒细胞增殖的2种主要剪接形式。 相似文献
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体外成熟培养前后山羊卵泡发育相关基因及其受体表达特性的研究 总被引：1，自引：0，他引：1
为研究山羊卵母细胞体外成熟培养前后部分转移生长因子β基因及其受体表达特性,本试验以6~8周龄晋岚绒山羊母羔超数排卵获取的卵丘细胞-卵母细胞复合体为研究对象,应用Real time PCR技术检测成熟培养前后BMP15、GDF9、BMP6及其受体基因BMPRⅡ、TGFβRI和BMPRIB的表达差异。结果显示:山羊卵母细胞体外成熟培养后,卵泡发育相关的基因及受体基因中,BMP6 mRNA表达量显著增加(P0.05),BMPRⅡ、TGFβRI和BMPRIB的mRNA表达量极显著上调(P0.01),Ptgs2基因的表达量极显著下调(P0.01),BMP15和GDF9基因差异不显著(P0.05)。 相似文献
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《畜牧与兽医》2017,(12):60-65
本试验旨在研究淫羊藿苷(ICA)对猪卵泡体外培养过程中颗粒细胞凋亡的影响。设置不同浓度ICA(0、01、1、10μg/mL)添加组,体外培养猪3~5 mm健康有腔卵泡12 h和24 h后,分别收集各组卵泡颗粒细胞,流式细胞仪检测细胞凋亡率,RT-PCR检测凋亡相关基因Caspase-3、Bcl-2和Bax mRNA相对表达水平。结果表明,猪卵泡体外培养12 h和24 h,添加1μg/mL ICA组颗粒细胞凋亡率较对照组显著降低(P005),1μg/mL组的Caspase-3、Bax mRNA表达量最低,Bcl-2 mRNA表达量最高,且显著高于对照组(P005)。研究结果提示,ICA在卵泡闭锁过程中可明显抑制颗粒细胞凋亡,这一作用可能主要是通过抑制线粒体凋亡途径而实现的。 相似文献
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旨在检测p21基因在水牛卵母细胞体外成熟过程中的表达变化,并克隆摩拉水牛p21基因CDs序列,构建真核表达载体。收集体外成熟培养不同时期(0、6、12、24h)和不同形态[有第1极体(first polar body,PB1)PB1和无PB1]的水牛卵母细胞,通过RT-qPCR检测其p21mRNA相对表达量。同时利用RTPCR技术从摩拉水牛卵巢颗粒细胞中扩增出p21基因编码序列,插入pEGFP-N1真核表达载体中,构建出重组质粒。将重组质粒pEGFP-N1-p21转染水牛颗粒细胞,并于培养24h和48h后观察转染细胞的荧光表达情况。收集转染48h后的颗粒细胞,通过RT-qPCR检测p21mRNA相对表达量。结果显示,成熟培养6h的卵母细胞p21mRNA表达量显著高于成熟培养0、12、24h的表达量(P0.05),有PB1的卵母细胞中p21mRNA的表达量显著高于无PB1的卵母细胞(P0.05);重组质粒pEGFP-N1-p21转染颗粒细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-p21转染组的p21mRNA表达量显著升高。结果表明,在体外成熟培养过程中均能检测到水牛卵母细胞p21mRNA表达量,成功构建了摩拉水牛p21基因真核表达载体,为下一步研究p21基因在水牛卵母细胞成熟和胚胎发育过程中的功能和调控奠定了基础。 相似文献
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旨在在猪卵母细胞体外成熟培养液中添加N-乙酰半胱氨酸(NAC),揭示NAC对不同直径卵泡来源卵母细胞体外成熟效果的影响，解析其对氧化还原平衡的调控作用。本研究收集猪卵巢上大腔(4～6 mm)、中腔(2～4 mm)、小腔(1～2 mm)卵泡中的卵母细胞，在以上3种卵泡来源猪卵母细胞的体外成熟培养液中，均分别添加0、1、2、4 mmol·L-1浓度的NAC处理，评价卵丘扩展指数、第一极体排出率等成熟指标，检测卵母细胞中ROS水平、谷胱甘肽(GSH)含量和抗氧化基因的表达水平。结果表明，NAC处理对大腔卵泡来源卵母细胞的卵丘扩展指数和ROS水平无显著影响(P>0.05),对大、中腔卵泡来源卵母细胞的第一极体排出率无显著作用(P>0.05);而适量浓度的NAC(2 mmol·L-1)处理可显著提高中、小腔卵泡来源卵母细胞的卵丘扩展指数(P<0.05),降低ROS水平(P<0.05),提升小腔卵泡来源卵母细胞的第一极体排出率(P<0.05)。同时，NAC处理对大、中腔卵泡来源卵母细胞中的GSH含量和抗氧化基因SOD、CAT... 相似文献
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猪卵泡卵母细胞体外成熟与冷冻保存 总被引：1，自引：0，他引：1
研究了激素、猪卵泡液、不同类型血清对猪卵泡卵母细胞体外成熟的影响 ;比较了卵泡直径小于 2 mm、2～ 5 mm和大于 5 mm的卵母细胞体外成熟能力的差异 ,并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。结果表明 :猪卵母细胞体外培养 4 8h时 ,培养的前 2 4 h培养液中加入激素 ,后 2 4 h去掉激素 ,卵母细胞的 A级成熟率 (5 1.73% )和总成熟率 (83.2 5 % )最高 ,极显著高于前 2 4 h不加激素 ,后 2 4 h添加激素培养的成熟率 (P<0 .0 1) ;也显著高于不含激素的培养液连续培养 4 8h的成熟率 (P<0 .0 5 ) ;但与添加激素连续培养 4 8h的成熟率差异不显著 (P>0 .0 5 )。在体外成熟培养液中 ,添加 10 % (体积分数 ) ECS的成熟率 (72 .86 % )显著高于添加 10 % (体积分数 ) NCS的成熟率(6 2 .2 1% ) (P<0 .0 5 ) ,而添加 10 % p FF则抑制卵母细胞的体外成熟。随着卵泡直径的增大 ,卵母细胞体外成熟能力逐渐增强。采用程序冷冻保存方法 ,成熟卵母细胞的成活率 (35 .5 9% )显著高于培养前 (2 4 .6 4 % )和培养 2 4 h(2 3.36 % ) (P<0 .0 5 )。培养 2 4 h(77.2 2 % )和培养成熟 (72 .81% )的卵母细胞解冻后的形态完整率均显著高于培养前(5 3.2 4 % ) (P<0 .0 5 ) 相似文献
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本试验旨在探讨不同外源激素组合的添加、胰岛素—转铁蛋白—亚硒酸钠(ITS)的添加、不同培养皿、石蜡油的添加对卵母细胞体外成熟培养(IVM)的影响。结果显示:①孕马血清促性腺激素+人绒毛膜促性腺激素+促卵泡素(PMSG+HCG+FSH)组高于促卵泡素+促黄体素(FSH+LH)组和尿促性腺激素(hMG)组,相互之间差异显著(80.1%、68.3%、53.3%,P<0.05);②添加1% ITS到猪卵母细胞培养液中对卵母细胞成熟率无显著提高(P>0.05),但显著提高了孤雌激活后孤雌胚胎的卵裂率和囊胚率(63.3%、55.1%,18.7%、12.1%,P<0.05);③凹槽皿组猪卵母细胞成熟率显著高于四孔板和30 mm塑料皿组(73.3%、68.0%、68.3%,P<0.05);④石蜡油的添加对卵丘细胞扩展和卵母细胞体外成熟率均有显著提高(84.8%、69.9%,P<0.05)。结果表明培养液中添加PMSG+HCG+FSH和ITS及选用凹槽皿、成熟培养液上覆石蜡油可提高猪卵母细胞IVM效果。 相似文献
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In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage. 总被引：2，自引：0，他引：2
C Bishonga Y Takahashi S Katagiri M Nagano A Ishikawa 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2001,63(6):619-624
Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage. 相似文献
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培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响 总被引：29，自引：0，他引：29
A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。 相似文献
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Brito IR Saraiva MV Araújo VR Celestino JJ Magalhães-Padilha DM Lima IM Van den Hurk R Figueiredo JR Silva JR 《Research in veterinary science》2012,93(2):729-732
This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture. 相似文献
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在卵母细胞体外成熟培养过程中,培养基中添加激素与否及其激素添加的先后顺序是影响猪卵母细胞核成熟和质成熟的一个重要因素.本试验将猪卵母细胞分别在FSH→不含激素、FSH→LH、FSH LH不含激素中培养48 h(培养第20～22 h后换液),并于成熟培养的第24 h(未换液)、48 h将卵母细胞进行荧光染色,观察其生发泡内染色质构型及卵母细胞核成熟情况.实验表明:(1)在IVM的前24 h,添加FSH LH组的GVIV期卵母细胞比例低于只添加FSH组,但差异不显著(8.99%比17.19%,P＞0.05);(2)在FSH存在的情况下,IVM的前期和后期添加LH能促进卵母细胞发生GVBD;(3)FSH LH培养24 h后转入不含激素培养基组,卵母细胞的核成熟比率显著高于添加FSH组和先添加FSH培养24 h后转入添加LH组(P＜0.05). 相似文献
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试验旨在探讨不同发育时期卵泡上四跨膜蛋白CD9的表达,以及体外成熟和超低温冷冻对绵羊卵母细胞CD9的影响。采用免疫组织化学技术检测CD9蛋白在卵泡上的表达部位,实时荧光定量PCR技术检测CD9 mRNA的表达量,Western blotting技术检测CD9蛋白的含量。免疫组织化学结果发现,在绵羊原始卵泡上就开始检测到CD9荧光信号,且随着卵泡的成熟荧光信号逐渐增强,成熟卵泡时荧光信号达到最强;实时荧光定量PCR检测新鲜MⅡ期卵母细胞中CD9 mRNA的表达量显著增高（P<0.05）,冷冻GV期卵母细胞CD9 mRNA的表达量最少;Western blotting检测得出新鲜MⅡ期卵母细胞中CD9蛋白表达最多,冷冻GV期卵母细胞上表达最少,与实时荧光定量PCR结果一致。卵母细胞的冷冻保存要比胚胎保存应用前景广泛,而四跨膜蛋白CD9在精卵融合中更有着重要的作用,以上结果均显示,冷冻保存降低卵母细胞四跨膜蛋白CD9含量,而CD9的损伤是受精率下降的重要原因之一。 相似文献
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试验根据直径将猪卵泡分为2组:G1组(4~7 mm)和G2组(2~4 mm),对2组卵泡内获取的卵母细胞体外成熟率和发育潜能进行了比较,利用相对定量PCR检测了卵丘细胞中卵丘扩展相关基因Has2、Ptgs2、Ptx31及Pgr的表达水平,应用绝对定量PCR检测了成熟培养前后卵母细胞线粒体拷贝数,并利用5,5'-二巯基-2-硝基苯酸(DTNB)酶循环法检测了体外成熟培养过程中卵母细胞谷胱甘肽(GSH)的含量。结果显示,G1组和G2组卵母细胞体外成熟率分别为95.06%和68.19%,G1和G2组排出第一极体的成熟卵母细胞孤雌激活后的囊胚率分别为51.47%和29.44%,2组卵母细胞在体外成熟率和孤雌发育率上均差异显著(P<0.05)。G1组卵丘细胞在体外成熟培养过程中的扩展程度明显高于G2组,G1组卵母细胞对应的卵丘细胞扩展相关基因Has2、Ptgs2、Ptx31、Pgr的表达水平高于G2组(Has2基因在卵母细胞成熟培养0、24 h除外);G1组卵母细胞线粒体数、谷胱甘肽含量均高于G2组。以上结果表明,大卵泡来源的卵母细胞体外成熟能力和发育潜力优于小卵泡来源的卵母细胞,这可能与卵母细胞成熟过程中卵丘扩展程度、卵丘扩展相关基因表达激活情况、卵胞质内谷胱甘肽含量和线粒体拷贝数有关。 相似文献
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本研究旨在探究Fox O1在绵羊卵巢卵泡的表达模式。屠宰场取卵巢获得卵泡,分为小卵泡(直径≤3 mm)、中卵泡(3 mm<直径<5 mm)、大卵泡(直径≥5 mm)3组。利用免疫组化技术对不同直径卵泡的FoxO1进行定位分析,通过机械分离法分离卵泡颗粒细胞,qRT-PCR和Western blotting技术检测FoxO1在小、中、大卵泡颗粒细胞的表达量。结果显示:在绵羊卵泡中,FoxO1表达于颗粒细胞层;中卵泡的FoxO1 mRNA表达量高于小卵泡和大卵泡(P<0.01),小卵泡FoxO1mRNA表达量高于大卵泡(P<0.01);FoxO1蛋白在大卵泡的表达量低于小卵泡和中卵泡(P<0.01),但小卵泡和中卵泡表达量差异不显著。综上表明,FoxO1在绵羊卵泡颗粒细胞层表达,且在大卵泡中的表达量极显著低于小卵泡和中卵泡。 相似文献
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In vitro Development of Buffalo Oocytes in Media-containing Fluids from Different Size Class Follicles 总被引：1，自引：0，他引：1
S Nandi HM Raghu BM Ravindranatha PSP Gupta PV Sarma 《Reproduction in domestic animals》2004,39(1):33-38
Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro. 相似文献
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M Durlej K Knapczyk‐Stwora M Duda J Galas M Slomczynska 《Reproduction in domestic animals》2011,46(3):377-384
This study was designed to reveal the FSHR mRNA and protein expression in the neonatal porcine ovary and to determine whether maternal administration of antiandrogen flutamide may affect FSHR expression in the ovary of newborn piglets using real‐time PCR, immunohistochemistry and Western blot analysis. Pregnant sows were injected with flutamide at a dose of 50 mg/kg body weight, given five times, every second day, starting at day 20 post‐coitum (p.c.) or day 80 p.c., and ovaries were obtained from neonatal pigs. The FSHR mRNA expression was significantly decreased after flutamide administration. Furthermore, higher down‐regulation was observed following exposure to antiandrogen at day 20 than at day 80 p.c. Immunohistochemistry showed the positive immunostaining for FSHR in the oocytes, granulosa cells of primary follicles and the surface epithelium of the ovaries from both control and flutamide‐treated pigs. However, oocytes and granulosa cells of primary follicles in the ovaries exposed in utero to flutamide were weakly immunostained when compared to those in the control ones. The presence of FSHR protein in all investigated ovaries was confirmed by Western blot analysis. Based on our findings, we suggest that FSHR may be involved in the early follicle formation in pigs, which begins during prenatal life. Furthermore, the regulation of FSHR mRNA and protein expression in neonatal porcine ovaries after maternal exposure to flutamide confirms that androgens play a crucial role in porcine folliculogenesis at the early stages. 相似文献