Growth activity of bovid herpesvirus 1 in bovine follicular oocytes with cumulus cells.

Bovine follicular oocytes collected from bovine ovaries were exposed to bovid herpesvirus 1 (BHV-1). After washings, these oocytes were cultured to mature. As a result BHV-1 could not be removed from the oocytes and could replicate in the oocytes with cumulus cells, but not in the oocytes without the cells. Moreover, the specific fluorescence for BHV-1 was detected in the cumulus cells by a indirect immunofluorescent technique. Therefore these findings suggested BHV-1 could be absorbed in the oocytes but the replication of BHV-1 was done in the cumulus cells.     

牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。     

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the correlation between the degree of cumulus expansion and in vitro development of porcine cumulus-oocytes complexes (COCs) matured and fertilized in vitro. The COCs were matured in the maturation medium (IVMM) supplemented with 15% or 5% of porcine follicular fluid (PFF) from small, medium and large follicles (<2 mm, 2-5 mm and >5 mm, respectively). COCs cultured in IVMM with PFF for 48 h displayed less expansion than those cultured in IVMM alone (P<0.05), irrespective of follicle size. After culture for 24 h in IVMM with PFF and for another 24 h in IVMM alone, the degree of cumulus expansion was more prominent than culture in the presence of PFF for the entire 48 h period (P<0.05), but the percentages of oocytes with PB I showed no significant difference between the control and experimental groups (P>0.05). After in vitro fertilization, the oocytes failed to develop to the morula/blastocyst stages except for those matured in IVMM supplemented with 15% or 5% PFF obtained from >5 mm follicles for the first 24 h and followed by in IVMM alone for the second 24 h (12.5% and 11.1% of the embryos developed to morulae and blastocysts, respectively). The expanded cumulus areas of COCs were significantly positively correlated with their in vitro development (p=0.0058, 0.0001 and 0.0348 for the percentages of embryos developed to 2-4 cell, beyond 4 cell and morula and blastocyst stages, respectively). In conclusion, PFF had an inhibiting effect on cumulus expansion, and the inhibitory effect decreased progressively with the increase in size of follicles from which PFF was obtained, and the action of PFF on cumulus expansion was affected by the PFF culture time. The areas of the expanded cumulus mass may be used as a parameter to predict development of porcine oocytes matured and fertilized in vitro.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

Pregnancies from bovine oocytes matured and fertilized in vitro

Production of transgenic animals and embryo cloning are only a few examples of new biotechnological methods applied to animal embryos. All these techniques require large amounts of oocytes and early embryos. In many laboratory animals, embryos matured and fertilized in vivo are easily obtained, but with larger domestic species it requires laborious surgical procedures and the number of embryos obtained remains relatively small (Bracken et al. 1982). The in vitro maturation of follicular oocytes derived from slaughterhouse ovaries and their in vitro fertilization provides large numbers of oocytes and embryos with considerably less effort. The final proof of the success in the in vitro maturation and fertilization procedure is the birth of healthy progeny. Also the normal preimplantation development of the embryos gives useful information about the efficiency of the method employed.     

绵羊卵母细胞和卵丘细胞体外成熟过程中ghrelin mRNA的表达

运用实时定量RT-PCR技术检测了不同成熟培养时间绵羊卵母细胞和卵丘细胞ghrelin mRNA的相对表达量。结果表明,卵母细胞ghrelin mRNA相对表达量在16、24h呈现显著升高,而卵丘细胞ghrelin mRNA则随着成熟时间的延长表现显著降低。绵羊卵母细胞和卵丘细胞ghrelin mRNA的持续表达以及动力学变化提示这一新型分子在生殖功能上有潜在调控作用。     

水牛卵母细胞玻璃化冷冻保存

以水牛MII期的卵母细胞为材料,利用玻璃化冷冻液EDS33(16.5%EG+16.5%DMSO+sucrose)对水牛MII期的卵母细胞进行两步法(玻璃毛细管(GMP)和拉细的开口塑料细管(OPS))玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG+7.5%DMSO+sucrose)中平衡3 min,再移入玻璃化冷冻液中30 s后装管直接投入液氮.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞孤雌激活,通过囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP和OPS法冷冻保存的水牛卵母细胞解冻后的存活率(分别为96.80%和97.41%)与对照组卵母细胞的存活率(100%)3者之间差异均不显著(P>0.05).GMP法和OPS法冷冻的水牛卵母细胞激活后的胚胎分裂率和囊胚发育率2者均明显低于对照组(分别为30.58%和28.32% vs50.94%,10.81%和9.38% vs 29.63%,P<0.05),而这2种方法冷冻的水牛卵母细胞激活后的分裂率和囊胚发育率差异均不显著(P>0.05).这表明GMP和OPS玻璃化冷冻方法可以用于水牛卵母细胞的冷冻,并且玻璃化冷冻的卵母细胞能继续分裂并发育到囊胚.     

罗同丘细胞对小鼠卵母细胞体外成熟及其后续发育的影响

观察了卵丘细胞共培养对小鼠生发泡期部分裸露（PNO）和裸卵（NO）成熟和发育能力的影响；分别用小鼠、大鼠、猪的卵丘细胞与小鼠PNO和NO进行了共培养。检测了小鼠PNO和NO的减数分裂能力、生发泡构型、受精和胚胎发育能力。结果，PNO、NO的减数分裂能力显著（P〈0．05）低于卵丘完整复合体（COCs）的减数分裂能力。COCs中SN型卵母细胞比率显著高于PNO和NO中SN型卵母细胞比率（P〈0．05），大部分PNO和NO的卵母细胞核型为NSN型。与对照组相比，与小鼠或大鼠卵丘细胞共培养的小鼠PNO和NO的减数分裂能力没有显著提高，但是与猪卵丘细胞共培养却可以提高小鼠PNO和NO的减数分裂能力。结果表明，猪卵丘细胞能促进小鼠卵母细胞的体外成熟，但并不影响小鼠卵母细胞的受精和胚胎发育。     

Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响

本研究的目的是探讨Ghrelin对水牛体外受精和孤雌激活胚胎体外发育的影响.体外成熟的水牛卵母细胞经体外受精或离子霉素孤雌激活后.分别在舍0,0.5,5,50和500 μg/L Ghrelin的培养液中进行体外培养,观察各组胚胎的卵裂率和囊胚率.结果显示,在培养液中添加不同浓度的Ghrelin对体外受精和孤雌激活胚胎的卵裂率均无显著影响(P>0.05),但添加500 μg/L的Ghrelin显著提高体外受精胚胎的囊胚发育率(33.5% vs 13.7%,P<0.05),50 μg/L或500 μg/L的Ghrelin均显著提高孤雌激活胚胎的囊胚发育率(32.4%和34.6% vs 14.5%,P<0.05).结果表明,培养液中添加Ghrelin对胚胎的早期卵裂没有影响,但可促进水牛体外受精和孤雌激活胚胎囊胚的形成.     

L-肉碱对水牛卵母细胞体外成熟的影响

以水牛为模型,通过探讨L-肉碱(L-carnitine)对水牛卵母细胞体外成熟的影响,同时深入研究添加L-肉碱后卵母细胞抗氧化能力的变化情况,以进一步阐明L-肉碱影响卵母细胞成熟的作用机制。水牛卵丘-卵母细胞复合体(COCs)在添加有不同质量浓度的L-肉碱(0,1,10,100 mg/L即对照组、Ⅰ组、Ⅱ组和Ⅲ组)的成熟液中体外成熟培养24h后,统计卵母细胞第一极体排出率。结果显示:Ⅰ组、Ⅱ组和Ⅲ组卵母细胞第一极体排出率分别为(64.44±1.93)%、(66.17±2.76)%和(63.27±1.19)%,与对照组(56.60±1.56)%相比差异显著(P<0.05)。免疫荧光染色观察显示:添加L-肉碱的各处理组的卵母细胞中活性氧的含量显著低于对照组(P<0.05)。同时,利用酶联免疫法测定卵母细胞中脂质过氧化物丙二醛的含量,结果显示Ⅰ组、Ⅱ组和Ⅲ组的卵母细胞中脂质过氧化物丙二醛的含量分别为0.125 797,0.125 217,0.124 155μmol/L,极显著低于对照组(0.142 609μmol/L)(P<0.01)。此外,采用实时荧光定量PCR方法检测卵母细胞周围的卵丘细胞中卵丘扩展相关基因ptx3、has2以及卵母细胞抗氧化相关基因gpx4的表达情况,分析结果显示:Ⅰ组、Ⅱ组和Ⅲ组卵丘细胞中has2的表达量均显著高于对照组的表达量(P<0.05),其中以Ⅱ组的最高;而Ⅱ组和Ⅲ组的卵丘细胞中ptx3基因和卵母细胞中gpx4基因的表达量显著高于对照组(P<0.05)。综上可知,水牛卵母细胞体外成熟液中添加适合质量浓度的L-肉碱能显著提高卵母细胞体外成熟率,推测L-肉碱可以通过提高卵母细胞抗氧化能力来促进卵母细胞的体外成熟。     

In vitro fertilization and development of in vitro matured bovine follicular oocytes

Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。     

牛卵泡卵母细胞的体外成熟和冷冻保存

在简化牛卵母细胞体外成熟的基础上,观察了保护剂、平衡温度、预平衡方法、解冻方法以及冷冻方法对牛卵泡卵母细胞冷冻的影响。结果表明:在体外成熟培养液中添加26.2 mmol/L的NaHCO3和对屠宰场卵巢进行选择更能促进牛卵母细胞的体外成熟;无论是程序冷冻还是玻璃化冷冻,乙二醇(EG)与甘油(GLY)相比更能促进牛卵泡卵母细胞的冷冻效果;在程序冷冻中,冷冻液中添加0.1 mol/L蔗糖比添加0.3mol/L的蔗糖更能促进牛卵泡卵母细胞的冷冻;在玻璃化冷冻中,37℃和25℃的平衡温度比4℃更适合牛卵泡卵母细胞的冷冻;在10%、5%、1%的预平衡浓度之间,5%的预平衡浓度即可达到预平衡的效果;在解冻时,多步脱除保护剂更能保护冷冻的卵母细胞;比较了几种最小样本量(minimum size sample,MSS)玻璃化冷冻方法,解冻成熟培养后的成熟率,拉细毛细玻璃管冷冻法为(41.67±3.19)%,极显著高于未拉细毛细玻璃管冷冻法(glassmicropipette,GMP)的(30.19±1.93)%和固体表面冷冻法(solid surface vitrification,SSV)的(28.33±2.89)%(P<0.01)。     

单层细胞共培养对猪去卵丘卵母细胞体外成熟和孤雌发育的影响

本试验比较观察第一极体(The first polar body,PbⅠ)、Oosight imaging system观察和hocchst33342染色法对第2次减数分裂中期(Metaphase Ⅱ,MⅡ)卵母细胞判定结果的相关性分析,并探讨卵巢皮质细胞(porcine ovarian cortex cells,pOCCs)、猪输卵管上皮细胞(porcine oviductal epithelial cells,pOECs)和猪卵丘颗粒细胞(porcine cumulus cells,pCCs)等3种单层细胞体外共培养体系对猪去卵丘卵母细胞(cumulus cells denuded oocytes,Dos)体外成熟(in vitro maturation,IVM)和孤雌发育的影响。结果显示:(1)Oosight imaging system判定卵母细胞成熟的结果与hocchst33342染色法判定的结果有很强相关性(R=0.973,P<0.01,N=90);(2)pOCCs单层细胞共培养体系中猪去卵丘卵母细胞成熟率显著高于pOECs((52.5±0.30)%vs(43.8±2.18)%,P<0.05),且...     

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm

The objective of this study was to evaluate fertility and full‐term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co‐cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full‐term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.