细胞周期检查点激酶1的表达对猪卵母细胞体外成熟的影响

细胞周期检查点激酶1(Chk1)作为DNA复制检查点和DNA损伤反应的重要成员,在有丝分裂和减数分裂中都具有重要作用。为探究Chk1对猪卵母细胞减数分裂的影响,本研究首先采用免疫荧光染色和实时荧光定量PCR技术检测了Chk1在猪卵母细胞不同时期的表达和定位,随后利用chir-124抑制Chk1的表达,研究其对猪卵母细胞减数分裂的影响,以及相关基因的表达和纺锤体的变化。结果表明,Chk1在4个时期(GV、GVBD、MI、MII)均有表达,GV期主要定位在生发泡内,GVBD期主要定位在核周围,MI期和MII期主要定位在纺锤体上;用不同浓度的chir-124处理卵母细胞来抑制Chk1,发现0.5μmol/L的chir-124对卵母细胞生发泡破裂(GVBD)的影响最大,可显著提高猪卵母细胞处于GV-IV期的比例(P0.05),并且可显著提高卵母细胞达到GVBD期的比率(P0.05),但对后续的成熟没有显著影响;随后对MII期卵母细胞的基因表达进行检测发现,Chk1的抑制可调节cdk1、cdc25C、cyclin b1基因的表达,并使gwl基因的表达显著下降(P0.05),从而调节卵母细胞恢复减数分裂的进程;Chk1的表达会激活纺锤体组装检查点(SAC),阻止同源染色体分离,将猪卵母细胞定位在MI期,而减少Chk1的表达后,猪卵母细胞可渡过MI期,进入MII期。综上,在GV期使用chir-124抑制Chk1的表达可促进猪卵母细胞恢复减数分裂,并促进后续的成熟。     

胰岛素对犬卵母细胞体外成熟的影响

研究不同浓度胰岛素及不同培养时间对犬卵母细胞体外成熟率的影响,为改善犬卵母细胞体外培养体系提供参考,采用切割法收集卵巢表面卵丘—卵母细胞复合体(cumulus oocyte complexes, COCs),在含有0.6%葡萄糖的TCM199中添加不同浓度的胰岛素（0、3、6、9 IU/mL）,38.5 ℃、5% CO₂培养箱内成熟培养,观察卵丘扩散程度,剥离卵丘细胞获得裸卵后,室温下固定15 min;Hoechst 33342染色,压片,荧光显微镜下观察核形态,用SPSS 14.0软件统计试验数据。不同浓度胰岛素培养48 h后,各组卵丘细胞扩散效果都不明显;卵母细胞核成熟期没有达到减数分裂中期（MⅡ）,但是6 IU/mL胰岛素组生发泡破裂期（GVBD）比率（35.88%±14.63%）显著高于对照组（11.25%±9.75%）;6 IU/mL胰岛素组延长培养时间至72和96 h后,MⅠ-MⅡ期卵母细胞成熟率分别为13.33%±1.5%、20.8%±1.9%。以上结果表明,犬体外成熟培养基中添加胰岛素既没有提高犬卵母细胞核成熟到MⅡ期,也没有改善犬卵母细胞卵丘扩散效果。但是,在6 IU/mL浓度下延长培养时间,相对增加了成熟率。     

次黄嘌呤维持小鼠卵母细胞减数分裂阻滞的时效性和可逆性研究

为提高体外成熟卵母细胞的发育能力,本实验采用卵泡内存在的减数分裂抑制剂次黄嘌呤(Hypoxanthine,HX)在体外维持小鼠GV期卵母细胞减数分裂阻滞,探讨了次黄嘌呤对卵母细胞减数分裂抑制作用的时效性、可逆性以及对卵丘扩展和解除抑制后的发育能力的影响。结果表明(1)4mmol/LHX维持减数分裂阻滞的作用具有时效性,GV%在18h时显著下降。(2)HX处理时间短于24h时,解除抑制后再成熟时卵母细胞的成熟率不受影响,抑制24h再成熟14h时成熟率仍可达86%。(3)HX处理会抑制卵丘扩展,解除抑制后再成熟时卵丘扩展程度跟抑制时间长短有关。(4)HX处理6h后,卵母细胞的孤雌激活率上升(42%vs20%,P<0.05),但胚胎的发育能力下降。这证明HX维持小鼠卵母细胞减数分裂阻滞的作用具有时效性和可逆性的特点,为建立提高体外成熟卵母细胞的发育能力的培养体系打下了基础。     

在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟及体外受精后早期胚胎发育的影响

研究目的在于探讨在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟、卵丘细胞扩散及体外受精后早期胚胎发育的影响。卵母细胞·卵丘细胞复合体在含FSH和LH的以下处理组的成熟液中成熟培养 2 3～ 2 4h :(1)对照组-改良TCM - 199+0 .1%PVA ;(2 )试验组 1-改良TCM - 199+10 %新生牛血清 ;(3)试验组 2 -改良TCM - 199+10 %猪卵泡液 ,再移至无FSH和LH的不同处理组的成熟液中成熟培养 2 3～ 34h。试验 1中 ,卵母细胞在 4 6～ 4 8h成熟培养后 ,观察卵丘细胞扩散情况 ,并对卵母细胞进行固定和染色 ,鉴定卵母细胞减数分裂情况 :试验 2中 ,对在不同处理组的成熟液中成熟培养 4 6～ 4 8h的卵母细胞进行体外受精 ,再培养 8d。受精后第 2天检查分裂率、第 6天检查桑椹胚 /囊胚率、第 8天检查囊胚率。 4 6～ 4 8h成熟培养后试验组 1和试验组 2的大部分卵母细胞 -卵丘细胞复合体的卵丘细胞完全扩散 ,而对照组的卵丘细胞只有 5 0 %扩散。试验组 1和试验组 2的卵母细胞核成熟率分别为 39.9% (77/ 193)和 4 4 .3% (93/ 2 10 ) ,与对照组的卵母细胞核成熟率 4 8.1% (99/ 2 0 6 )相比没有显著差异 (P <0 .0 5 )。卵母细胞分裂率试验组 1(5 0 .0± 1.8) %和试验组 2 (49.9± 2 .6 ) %与对照组的卵母细胞分裂率 (49.0± 2     

注射hCG后不同时间猪所排卵泡内COC的形态和卵母细胞减数分裂进程

研究了超排处理注射 h CG后不同时间猪卵丘卵母细胞复合体 (COC)的回收率和形态、卵母细胞减数分裂进程以及 COC形态与生发泡 (GV)染色质构型之间的关系。结果表明 :(1)注射 h CG后 4 h COC的回收率为 5 3.1% ,明显低于注射 h CG后 18、2 2、2 4 h(71.2 %、76 .5 %、70 .0 % ,P<0 .0 5 ) ;(2 )注射 h CG后 18、2 2、2 4 h收集的 COC分别有98.9%、98.0 %、91.4 %的卵丘已经发生扩展 ,而注射 h CG后 4 h收集的 COC则无一发生卵丘扩展 ;(3)注射 h CG后 4h,有少量卵母细胞开始恢复减数分裂 ,至 18h左右开始发生生发泡破裂 (GVBD) ,到 2 2～ 2 4 h有 5 8.4 %～ 6 0 .0 %的卵母细胞发生 GVBD;(4)外层卵丘扩展、放射冠轻微扩展的卵母细胞处于 GV- 1期的比例 (6 9.6 % )明显高于卵丘完全扩展的卵母细胞 (47.8% )和放射冠部分扩展、卵丘已经脱落的卵母细胞 (2 9.3% ) ,而后 2者发生 GVBD的比例(40 %、4 4 % )则略高于前者 (2 7% )。     

uPA对体外成熟牛卵母细胞生发泡破裂的影响

为了探明尿激酶型纤溶酶原激活剂(uPA)对体外成熟牛卵母细胞核成熟的可能作用,试验根据uPA及其特异性抑制剂(amiloride)的有效浓度分成4组,即对照组、uPA(0.5 ng/mL)组、amiloride(1μg/mL)组及联合添加(uPA+amiloride)组,然后采用地衣红染色方法分别研究体外成熟培养6 h与12 h后uPA对牛卵母细胞生发泡破裂(GVBD)的影响,采用ELISA方法研究体外成熟培养6 h后uPA对牛卵丘-卵母细胞复合体(COCs)中cAMP水平变化的可能调节作用。结果表明:减数分裂不同时期(GV期、GVBD期及MⅠ~MⅡ期)卵母细胞染色质(体)形态呈现不同特征。卵母细胞在体外成熟培养6 h后各组细胞均处于GV~GVBD期,其中uPA组GVBD期卵母细胞率(12.72%)显著低于对照组(24.83%)与联合添加组(30.42%,P0.05);体外成熟培养12 h后,uPA组GVBD期卵母细胞率(54.81%)显著高于对照组(37.49%)与联合添加组(25.64%,P0.05),uPA组MⅠ~MⅡ期卵母细胞率(44.85%)显著低于对照组(62.51%)与联合添加组(74.36%,P0.05);体外成熟培养6 h后,uPA组cAMP水平显著高于对照组与联合添加组(P0.05)。说明uPA通过上调cAMP水平阻滞牛卵母细胞的减数分裂进程。     

不同培养液及不同培养时间对犬卵母细胞体外成熟的影响

试验采集犬卵巢,用切割法回收卵泡中的卵母细胞,选取胞质均匀、2层或2层以上颗粒细胞、直径≥110μm的卵母细胞移入不同的培养液中:(A)M199+10%FBS+0.5μg/mL FSH+10 IU/mL hCG,(B)M199+0.1%PVA进行体外成熟培养。在培养不同时间(0,48,72 h)用1%醋酸地衣红染色,在显微镜下观察卵母细胞核成熟情况,探索适宜犬卵母细胞体外成熟的培养体系。结果表明:卵母细胞在A液中培养48 h MII期比例(7.84%)高于B液中的比例(P<0.01);在培养72 h MII期比例也是A液中(8.00%)高于B液,虽然二者差异不显著(P>0.05),但72 h卵母细胞退化比例高于48 h(P<0.01)。因此培养液中添加FBS,FSH和hCG有利于犬卵母细胞体外成熟,且在此培养液中培养48 h为最佳成熟时间。     

GMP玻璃化冷冻未成熟牛卵母细胞核成熟及冷冻损伤试验

本试验通过荧光染色的方法建立了未成熟牛卵母细胞在体外培养过程中第1次减数分裂的各个阶段的参考判定图谱;根据这个标准来观察毛细玻璃管(GMP)玻璃化冷冻对卵母细胞核成熟和冷冻损伤的影响。结果表明,从屠宰场废弃的卵巢表面卵泡内抽取的COCs,70%处于生发泡期,12.5%生发泡开始破裂,7.5%已开始浓缩,这说明从屠宰场获得的COCs有较高的异质性;卵母细胞在成熟培养22h时收获排出第一极体的卵母细胞,可得到丰度较高的极体-胞质染色体对称、紧密相邻的成熟卵母细胞;GMP玻璃化冷冻损伤主要有2种表现形式,首先,直接影响膜结构的完整性,包括细胞膜和核膜,这可从退化的细胞比例看出(8～24h,有21.9%～27.2%的细胞处于该阶段),其次,影响CONDENSED向MⅠ期的过渡,这可从处于CONDENSED卵母细胞的比例看出(8～24h,有24.1%～34.3%的细胞处于该阶段)。     

微滴单培养犬卵母细胞的体外成熟能力及直径、透明带变化规律的研究

为了完善犬卵母细胞体外培养体系,试验以犬卵巢为材料,研究微滴单培养犬卵母细胞的体外成熟能力及卵母细胞直径与透明带厚度的变化规律。结果表明:体外培养48 h后,卵泡期与黄体期犬卵母细胞达到第1次减数分裂中期-第2次减数分裂中期(MⅠ-MⅡ期)比率分别为(15.9±8.0)%、(17.1±4.9)%,两者差异不显著(P>0.05),但均显著高于乏情期[(6.1±5.5)%](P<0.05),可见微滴单培养犬卵母细胞具有一定的体外成熟能力;处于生发泡期(GV期)、生发泡破裂期(GVBD期)、MⅠ期和MⅡ期的犬卵母细胞直径分别为155.42,171.12,183.50,155.96μm,透明带厚度分别为29.20,33.70,33.91,30.31μm,均无显著性差异(P>0.05),但均存在由小变大再逐渐变小的趋势。     

KDM2B基因克隆及其在牦牛组织和卵母细胞减数分裂过程中的表达研究

本研究旨在克隆牦牛组蛋白去甲基化酶2B(Lysine-specific histone demethylase 2B,KDM2B)基因,检测其在牦牛不同组织及其在卵母细胞减数分裂过程中的表达水平,从而为研究KDM2B基因在牦牛减数分裂中的作用机制提供试验依据。牦牛屠宰后,采集心、肝、脾、肺、肾、脑、小肠、胃、肌肉、卵巢、睾丸和子宫组织样品,分别提取各个样本的总RNA。另选择3~5周岁的健康牦牛,采集卵巢后,抽取卵丘-卵母细胞复合体(Cumulus-oocyte complex,COCs),透明质酸酶作用后,获得卵母细胞和颗粒细胞,利用RT-PCR扩增得到KDM2B基因CDS区序列,实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)检测KDM2B基因在不同组织中的表达水平。体外培养获得GV、MI、MII 3个时期COCs,用qRT-PCR法检测KDM2B基因在卵母细胞减数分裂过程中的表达规律。结果表明,KDM2B基因CDS区长为3 930bp,编码1 309个氨基酸,与牦牛已有预测序列相比,属于长型转录本。与牛、绵羊、山羊的同源性较高,与斑马鱼和鸡的同源性较低。KDM2B基因在牦牛的各个组织中均有表达,其中脾、子宫、睾丸和卵巢的表达量较高。在MI期卵母细胞中,KDM2B基因的表达水平显著高于GV期和MII期(P0.01)。在卵丘颗粒细胞中,KDM2B基因的表达水平随减数分裂的进行而显著增加(P0.01)。本研究为进一步研究牦牛卵母细胞减数分裂机制及改善牦牛繁殖效率提供基础资料。     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

马卵母细胞胞质内精子注射后体外发育能力的研究

本研究在非繁殖季节评估卵丘形态(松散型、致密型)、成熟培养体系(TCM 199、NCSU 23)、体外成熟时间(34、38 h)和离子霉素结合6-DMAP激活对马卵母细胞胞质内精子注射(ICSI)后体外发育能力的影响。从屠宰场采集马卵巢,获得的卵母细胞进行体外成熟,然后注射马冷冻解冻精液,统计分裂情况。试验结果表明,①马松散型卵母细胞成熟率显著高于致密型卵母细胞(P<0.05),分别为61.09%和41.24%,但ICSI后36 h分裂率无显著差异(P>0.05),分别为47.34%和44.92%;②两种培养体系对马松散型或致密型卵母细胞成熟率及ICSI后36 h分裂率无显著影响(P>0.05),但相同成熟体系培养松散型卵母细胞成熟率均显著高于致密型卵母细胞(P<0.05),然而ICSI后36 h分裂率差异不显著(P>0.05);③松散型或致密型卵母细胞在TCM 199或NCSU 23中成熟38 h成熟率均高于34 h成熟率,分别为44.43%～68.87%和34.52%～58.90%,松散型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组或对照组的分裂率显著高于成熟38 h、ICSI后激活组的分裂率(P<0.05),以及致密型卵母细胞在TCM 199体系中成熟34 h、ICSI后激活组的分裂率(P<0.05),而且显著高于松散型卵母细胞在NCSU 23体系中成熟38 h、ICSI后对照组的分裂率(P<0.05);④ICSI后用离子霉素结合6-DMAP激活对马卵母细胞ICSI后36 h分裂无显著影响(P>0.05)。因此,马松散型和致密型卵母细胞的成熟能力存在差异,TCM 199和NCSU 23成熟体系对这2种类型卵母细胞的发育能力无显著影响(P>0.05),马卵母细胞成熟38 h成熟率高于34 h成熟率,TCM 199成熟体系培养松散型卵母细胞34 h进行ICSI后的分裂率最高。离子霉素结合6-DMAP激活对TCM 199或NCSU 23体系成熟马卵母细胞ICSI后的体外发育能力无显著影响(P>0.05)。     

Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.     

Quality and Developmental Ability of Dromedary (Camelus dromedarius) Embryos Obtained by IVM/IVF, In Vivo Matured/IVF or In Vivo Matured/Fertilized Oocytes

The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro fertilized oocytes whether they were matured in vitro (Group 1; 35% and 32%) or in vivo (Group 2; 32% and 45%). Pregnancy rates were not significantly different amongst all groups for the three first months following embryo transfer. All pregnancies were lost after day 90 follow transfer except for in vivo matured and in vivo matured/fertilized groups. Only in vivo matured/in vitro fertilized and in vivo matured/fertilized produced embryos continued normal development until term and resulted in the birth of normal and healthy live calves. Six claves (29%; 6/21) were born from Group 3 and one (8%; 1/13) calf was born from Group 2. This study shows that the IVC system used is able to support camel embryo development. However, developmental competence and viability of dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

G蛋白偶联受体50在牦牛卵母细胞体外成熟过程中的表达与亚细胞定位研究

试验旨在研究G蛋白偶联受体50（G protein-coupled receptor 50,GPR50）在牦牛卵母细胞体外成熟过程中的表达与定位规律,为进一步解析卵母细胞成熟的分子机制及理解牦牛繁殖的特异性提供依据。通过牦牛卵母细胞体外成熟培养,利用免疫荧光染色监测不同时间点（0~24 h）纺锤丝形态和核相的变化,确定牦牛卵母细胞减数分裂4个时期,包括生发泡期（germinal vesicle,GV）、生发泡破裂期（germinal vesicle break down,GVBD）、第一次减数分裂中期（metaphase Ⅰ,MⅠ）与第二次减数分裂中期（metaphase Ⅱ,MⅡ）的时间点。在此基础上,通过实时荧光定量PCR检测GPR50基因在牦牛卵母细胞成熟过程中的动态表达量,免疫荧光染色检测GPR50蛋白在卵母细胞成熟过程中的的亚细胞动态定位情况。结果表明,牦牛卵母细胞体外成熟0 h时90%处于GV期,6 h时94%处于GVBD期,16 h时92%细胞处于MⅠ期,24 h时94%处于MⅡ期。实时荧光定量PCR结果表明,GPR50基因在牦牛卵母细胞GV期即有表达,并在GVBD、MⅠ、MⅡ期成熟过程中逐渐升高,在MⅡ期达到顶峰,且极显著高于GV与GVBD期（P<0.01）。GPR50蛋白在牦牛卵母细胞GV期时集中在膜上表达,并随着成熟进程的发展在细胞质和细胞膜均大量表达,在MⅡ期高亮度弥散表达。以上结果表明,GPR50基因参与牦牛卵母细胞减数分裂过程并发挥重要作用,为研究GPR50在牦牛卵母细胞成熟过程中的作用及机制提供了依据。     

In vitro maturation and fertilization in the Nilgai (Boselaphus tragocamelus) using oocytes and spermatozoa recovered post-mortem from animals that had died because of foot and mouth disease outbreak

The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the developmental competence of gametes recovered from nilgai that had died because of foot and mouth disease outbreak. Oocytes collected from the ovaries of seven dead nilgais were allowed to mature in vitro and were tested for developmental potential by in vitro fertilization (IVF) with epididymal spermatozoa collected also post-mortem. The average number of oocytes (n = 517) recovered per ovary was 36.9, and the side (right or left), size and weight of the ovaries had no significant effect on the number and quality of oocytes recovered. In vitro maturation studies indicated that the proportion of matured oocytes (MII stage) at 18, 24 and 30 h was 55.6%, 63.4% and 63.6%, respectively. Furthermore, 43% of the matured oocytes cleaved following in vitro fertilization and 12% of the cleaved oocytes (6/49) developed to the 4-8 cell stage. These findings suggest that the gametes recovered from nilgai post-mortem could be utilized for in vitro production of embryos.     

In vitro Fertilization of Bovine Oocytes Exposed to Bovine Herpesvirus 1 (BHV-1)

Contents: Cumulus-oocyte complexes were matured for 24 hours in the presence or absence of bovine herpes virus-1 (BHV-1, 10⁶ TCID₅₀/ml), fertilized in vitro and then co-cultured on oviductal cells to the blastocyst stage. The percentages of oocytes which cleaved were 48% (n = 905) for oocytes matured in the presence of BHV-1, and 51% (n = 1004) for the control oocytes. Comparing BHV-1-treated to control, the percentages of cleaved oocytes which developed to the blastocyst stage were 29% and 31%, respectively (p > 0.05). Embryos produced from the experimental group of oocytes tested positive for BHV-1.     

体外成熟对牛卵母细胞孤雌激活后发育潜力的影响

本试验在卵母细胞体外成熟的研究基础上,研究了培养用水、卵泡液和成熟时间对卵母细胞体外成熟及孤雌激活后发育潜力的影响.结果表明(1)将卵母细胞分别于蒸馏水和Milli-Q超纯水配制的成熟培养液中培养22～24 h, 孤雌激活后, 卵裂率无显著差异(P>0.05); 但孤雌卵在超纯水配制的胚胎培养液中培养,囊胚发育率为22.3%, 明显高于在蒸馏水配制的培养液中的囊胚发育率14.1%(P<0.05).(2)在成熟培养液中添加10%、20%卵泡液,成熟卵母细胞孤雌激活后的囊胚发育率(32.3%, 30.9%)明显高于添加5%和0%卵泡液组的囊胚发育率(21.8%, 22.7%,P<0.05).卵母细胞在添加20%卵泡液的培养液中成熟培养,孤雌激活后囊胚孵化率明显低于添加0、5%、10%卵泡液组的囊胚孵化率(P<0.05).(3)成熟28 h或30 h的卵母细胞孤雌激活后,其囊胚发育率(30.5%或29.4%)明显高于成熟24 h或26 h组的囊胚发育率(20.5%或22.1%,P<0.05).