蔗糖预处理去核:一种可靠的无损害小鼠卵母细胞MII期核去除法

小鼠是发育生物学中最为有用的实验动物模型 ,特别是在核移植方面。自从 1 983年 Mc-Grath等 [1]报道成功地进行小鼠的核移植以来 ,已采用小鼠的 4-8细胞期的早期胚胎细胞 [2 ] 、桑椹胚 [3] 、内细胞团和滋养层细胞 [4 ] 以及体细胞 [5] 进行了核移植 ,并获得了后代。作为核移植的受体 ,可采用去核的合子 [1] ,2 -细胞期胚胎 [6 ,7] 和去核卵母细胞 [8] 。其中以去核卵母细胞使用最为广泛 ,它可接受各个时期以及不同类型的供体细胞[9] 。但 MII期小鼠卵母细胞的核(纺锤体 )不能在相差显微镜下观察到[10 ] 。而且 ,小鼠卵母细胞第一极体的…     

胞质完整的无透明带卵母细胞临床利用价值探讨

目的探讨胞质完整的无透明带卵母细胞(ZFO)的临床利用价值。方法选取本中心2016年1月至12月接受ICSI治疗的123例不孕症患者,根据卵母细胞有无透明带分为2组:36个ICSI周期中的36枚ZFO作为实验组,87个ICSI周期中749枚透明带完整的成熟卵母细胞作为对照组。比较两组患者一般临床特征、促性腺激素(Gn)用量及天数、HCG日血清雌二醇(E_2)水平及平均获卵数,同时对两组的受精率、双原核(2PN)受精率、异常受精率、卵裂率、囊胚形成率及优质囊胚形成率进行比较分析。结果两组患者的年龄、不孕年限、体重指数(BMI)、基础FSH水平、Gn用量及使用天数、HCG日E_2水平及平均获卵数差异均无统计学意义(P0.05);实验组与对照组ICSI后的受精率、2PN受精率、异常受精率、卵裂率、囊胚形成率及优质囊胚形成率相比较,差异均无统计学意义(P0.05)。结论无透明带卵母细胞可以正常受精、卵裂并发育为囊胚;若患者在无可利用胚胎的前提下,临床上可以考虑将其利用,以增加患者的累积妊娠率。     

蜡样透明带卵母细胞患者ICSI助孕活产1例病例报道并文献复习

蜡样透明带的卵母细胞发育潜力低于正常形态卵母细胞,蜡样透明带影响精卵正常结合、从而影响受精率和胚胎发育。采用卵胞浆内单精子注射(ICSI)是最佳治疗策略,使得具有蜡样透明带的卵母细胞能够正常发育并最终分娩健康的后代,获得理想的临床妊娠结局。本文报道病例体外受精助孕共4个周期所获卵母细胞均见蜡样透明带,行ICSI后获可利用胚胎,冻融胚胎移植后获临床妊娠,并分娩活婴。另外,本文就蜡样透明带卵母细胞不孕患者的临床资料进行总结分析,并对相关文献进行系统性回顾。     

不同融合参数对小鼠圆形精子细胞与卵母细胞融合胚胎发育的影响

目的利用手工克隆(handmade cloning,HMC)技术对小鼠圆形精子细胞与去透明带卵母细胞进行电融合,以研究电融合参数对融合胚胎发育潜能的影响。方法选取549枚卵母细胞与圆形精子细胞融合,根据电融合参数分为三组:A组(n=186)采用电场强度60V/mm、脉宽30μs、脉冲次数为2次的直流电脉冲;B组(n=188)采用电场强度80V/mm、脉宽20μs、脉冲次数为2次的直流电脉冲;C组(n=175)采用电场强度100V/mm、脉宽10μs、脉冲次数为2次的直流电脉冲。观察记录不同电融合参数下圆形精子细胞与无透明带卵母细胞的融合率、融合胚胎卵裂率以及囊胚形成率。结果(1)A组圆形精子细胞与去透明带卵母细胞的平均融合率为(84.87±3.84)%,与其他两组[(74.93±5.46)%、(73.60±5.48)%]相比平均融合率显著升高(P0.05);(2)A组融合胚胎的平均卵裂率为(77.37±3.97)%,与B组、C组融合胚胎的卵裂率[(67.74±7.32)%、(66.17±9.87)%]相比显著升高(P0.05);(3)A组的平均囊胚形成率为(41.28±6.50)%,显著高于C组[(31.03±6.09)%](P0.05)。结论 A组采用电场强度60V/mm、脉宽30μs、脉冲次数为2的直流电脉冲是圆形精子细胞与去透明带卵母细胞融合的最佳方案,有利于提高胚胎的发育潜能。     

小鼠透明带结合蛋白sp38在大肠杆菌中的融合表达和纯化

目的　表达及纯化小鼠透明带结合蛋白sp3 8。方法　从BALB/C小鼠睾丸组织提取总RNA ,利用RT PCR技术扩增sp3 8基因编码区序列 ,并将其重组到含麦芽糖结合蛋白 (MBP)的融合蛋白原核表达载体 pMAL c2x中 ,在大肠杆菌中进行表达。结果　序列测定表明克隆获得的sp3 8全长与基因库所登录的序列一致。经IPTG诱导表达出的MBP sp3 8融合蛋白分子量约 88kD ,经Westernblot分析证实。经直链淀粉琼脂糖凝胶 (amyloseresin)亲和层析和SephadexG 10 0分子筛层析纯化后 ,MBP sp3 8纯度达 90 %。 结论　成功地获得sp3 8蛋白 ,为进一步研究其生物学性能和用于免疫避孕的可能性打下了基础     

卵母细胞透明带透亮致密患者妊娠结局的影响因素分析

目的探讨年龄、移植策略、受精方式和不同促排卵方案对卵母细胞透明带透亮致密患者妊娠结局的影响。方法回顾性分析23个透明带透亮致密的移植周期,根据患者年龄分为30岁组和30~37岁组,依据移植策略分为新鲜周期移植和冻融周期移植,依据受精方式分为IVF组、ICSI组和早补救ICSI组,根据促排卵方案不同分为常规促排卵组和微刺激组,比较其对临床妊娠率、种植率、流产率和抱婴率的影响。结果两个年龄组的临床妊娠率、种植率、流产率和抱婴率均无统计学差异(P0.05);新鲜周期移植除临床妊娠率显著高于冻融周期移植外(70.0%vs.23.1%,P0.05),种植率、流产率和抱婴率均无统计学差异(P0.05);IVF组、早补救ICSI组和ICSI组的临床妊娠率、种植率、流产率和抱婴率均无统计学差异(P0.05);常规促排卵方案组和微刺激组的临床妊娠率、种植率、流产率和抱婴率均无统计学差异(P0.05)。结论透明带透亮、致密、折光性强的患者行早补救ICSI可获得满意的临床结局;冻融胚胎移植人工辅助孵化减薄透明带并不能改善这部分患者的妊娠结局。     

透明带与男性生育

卵透明带(ZP)在精卵识别、结合、穿透以及阻止多精受精和保护着床前胚胎等方面起关键作用,与男性生育关系密切。本文就透明带的组成、分子结构、生物学功能及其在男性生育领域的应用进行综述。     

镍对小鼠卵母细胞成熟和体外受精的影响

为了研究镍的生殖毒性 ,采用卵母细胞体外培养、体外受精的方法研究镍对小鼠卵母细胞的成熟和体外受精率的影响。结果 :氯化镍对卵母细胞生发泡破裂没有影响 ,但抑制卵母细胞第一极体的排放 ,降低体外受精率 ,并降低卵母细胞的存活率。实验结果提示 ,氯化镍破坏小鼠卵母细胞的减数分裂 ,影响小鼠的生殖功能。     

两种不同玻璃化冷冻方案对小鼠卵母细胞纺锤体的影响

目的:探讨两种不同的玻璃化冷冻方案对小鼠第二次减数分裂中期卵母细胞纺锤体的影响。方法:实验分3组:方案A组、方案B组和对照组(新鲜卵母细胞)。均以冷冻环为载体,方案A组用乙二醇(EG)作为冷冻保护剂,方案B组用乙二醇联合二甲亚砜(DMSO)作为冷冻保护剂,用直接投液氮高速制冷冷冻小鼠卵母细胞;解冻后3h小鼠卵母细胞经固定并用间接免疫荧光法染色微管及染色体,观察纺锤体及染色体的形态。结果:两种玻璃化冷冻方案中,卵母细胞解冻后的存活率差异无显著性(80.3%vs87.5%,P>0.05);经玻璃化冷冻方案A冷冻的卵母细胞解冻后具有完整纺锤体结构的卵母细胞数显著低于对照组和方案B组(15.2%vs78.7%,15.2%vs77.5%,P<0.05),后两者差异无显著性(78.7%vs77.5%,P>0.05);方案A组解冻后具有正常染色体的卵母细胞数显著低于对照组和方案B组(17.4%vs76.6%,17.4%vs72.5%,P<0.05),后两者差异无显著性(76.6%vs72.5%,P>0.05);方案A组异常染色体数显著高于对照组和方案B组(82.6%vs19.1%,82.6%vs27.5%,P<0.05),后两者差异无显著性(19.1%vs27.5%,P>0.05)。结论:改变玻璃化冷冻方案有利于保持解冻后小鼠卵母细胞纺锤体结构完整性。     

A method for mouse oocyte enucleation assisted with zona pellucida dilating

<正>Objective: To study the reliability of a new enucleation method, zona pellucida Dilating (ZPD) assisted enucleation method, for mouse oocyte enucleation. Methods: In ZPD enucleation method, the zona pellucida was dilating with the help of outside pression in enucleation needle after breakthrough by needle tip and the nucleus was aspirated by the needle entering the perivitelline space for enucleation. This method was employed and was compared with three other methods in this experiment. The efficiency of enucleation of mouse oocyte was examed. Result:ZPD assisted enucleation method is better than three others at the survival rate after enucleation and the simplicity of micromanipulation. The rate of forming pronucleus of reconstructed embryo from enucleated oocyte by ZPD methods is as high as 62. 8% and the reconstructed embryo at 4-cell stage was obtained. Conclusion: ZPD assisted method is a highly efficient and simple enucleation method with the advantage of saving manipulating time and it does less damage to the oocyte.     

人胚胎透明带厚度及其变量与体外受精-胚胎移植妊娠结局的关系

目的　探讨透明带厚度 (ZPT)及透明带厚度变量 (ZPTV)与体外受精 胚胎移植 (IVF ET)妊娠结局的关系。　方法　选择 2 0 0 2年 4～ 8月间连续进行的培养 3d新鲜的用于移植、形态摄像清楚的 81个周期 2 4 6个胚胎进行分析 ,用激光系统附带软件测量移植胚胎ZPT ,计算ZPTV。　结果　 (1)ZPT、ZPTV随患者年龄的增加而减小 ,尤其≥ 35岁组ZPTV(16 .2 3± 4 .78) %显著小于 <30～ 34岁组 (2 0 .14± 5 .91) % (P <0 .0 0 1) ;(2 )妊娠组 (87个胚胎 )ZPT(16 .71± 2 .10 ) μm小于未妊娠组 (15 9个胚胎 ) (17.0 6± 1.75 ) μm ,但无显著差异 ;妊娠组ZPTV(2 1.73± 5 .6 1) %显著大于未妊娠组 (18.6 1± 4 .90 ) %(P <0 .0 0 5 )。移植胚胎平均ZPTV值≥ 2 0 %组的妊娠率 (5 0 .0 % )高于 <2 0 %组 (17.1% ) ,P <0 .0 5 ;(3)优质胚胎组 (2 4 6个胚胎 )ZPT(16 .84± 1.99) μm与劣质胚胎组 (12 7个胚胎 )ZPT(17.11± 2 .0 4 ) μm相比无显著差异 ;但优质胚胎组ZPTV(19.71± 5 .39) %大于劣质胚胎组 (16 .6 0± 5 .4 2 ) % ,P <0 .0 0 1。　结论　ZPTV是评价胚胎质量的一个有效指标 ,优质胚胎的ZPTV高于劣质胚胎 ,年龄≥ 35岁者ZPTV显著减小 ;移植胚胎平均ZPTV值≥ 2 0 %者妊娠率明显提高。在临床挑选移植胚胎时应?     

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.     

Sperm penetration through the human zona pellucida as a predictor of in vitro fertilization

The aim of this study was to determine the predictive value of sperm penetration into the perivitelline space of human cadaveric oocytes on in vitro fertilization outcome. Forty-two patients with tubal infertility undergoing ovarian stimulation with gonadotropin for in vitro fertilization and embryo transfer participated in the study. The number of spermatozoa bound to the human zona pellucida, the percentage of cadaveric oocytes with one or more spermatozoa in the perivitelline space, and the in vitro fertilization outcome were evaluated. Spermatozoa from 37 of 42 patients were able to penetrate the perivitelline space of cadaveric oocytes as well as to fertilize human oocytes in vitro. In three individuals, no penetration of the perivitelline space of cadaveric oocytes was observed and no in vitro fertilization was detected. Only two patients were able to fertilize the couple's oocytes without penetration of the cadaveric oocytes. Based on these results the specificity and the sensitivity of the assay to predict in vitro fertilization was 100% and 94.1%, respectively. Accordingly, these results suggest that sperm-zona penetration is a useful bioassay to predict male fertility potential in IVF outcome.     

碳酸氢盐在小鼠精子体外获能和顶体反应中的作用

检验了精子获能和透明带（ＺＰ）及孕酮激发顶体反应是否需要ＨＣＯ３／ＣＯ２。小鼠精子分别在改良的Ｔｙｒｏｄｅ（ｍＴ－Ｂ２５，含２５ｍｍｏｌ／ＬＨＣＯ３／ＣＯ２）或ｍＴ－Ｈ（无ＨＣＯ３Ｈ／ＣＯ２，含２０ｍｍｏｌ／ＬＨｅｐｅｓ）中预培养９０ｍｉｎ后，以２步７０％和３５％ｐｅｒｃｏｌｌ／ｍＴ－Ｈ洗涤精子，并将精子重新悬浮于ｍＴ－Ｂ２５，ｍＴ－Ｂ１５ｍＴ－ＢＨ和ｍＴ－Ｈ中，用ＩＺＰ／μｌ或１５μｍｏｌ／Ｌ孕酮激发精子顶体反应。在上述ｍＴ诸培养基中，“Ｂ”精子（以ＣＴＣ荧光染色法确定）发生率为６１％～６７％；顶体反应均可达到４１．０士１．４％～４８．０±１．４％。表示精子一旦在ＨＣＯ３／Ｃ０２环境中获能，顶体反应就可在无ＨＣＯ３／ＣＯ２环境中发生。然而，精子预先在ｍＴ－Ｈ中培养，与上述相同方法处理精子，“Ｂ”型精子（２２％～３３％）明显低于前者，对ＺＰ或孕酮的刺激不起反应（１４．Ｏ士３．６～２４．７士０．６％），甚至将精子重新悬浮于ｍＴ－Ｂ２５中也不发生反应（２２．０±９．５％），表示这些精子并未获能。上述结果表明小鼠精子获能依赖于ＨＣＯ３／ＣＯ２，但顶体反应则否。     

体外受精-胚胎移植中移植胚胎透明带厚度变量与治疗结果的相关性研究

目的 :观察移植胚胎透明带厚度变量 ( ZPTV)对体外受精 -胚胎移植 ( IVF-ET)治疗结果的影响。方法 :在 2 3 2对夫妇进行 2 51个 IVF-ET治疗周期 ,电脑记录移植胚胎图象 ,应用胚胎测量软件进行透明带厚度的测量 ,计算 ZPTV。结果 :2 51个周期共移植 747个新鲜胚胎 ,获临床妊娠 1 0 9例 ( 43 % )。妊娠周期与非妊娠周期的平均 ZPTV值分别为 1 8.4± 6.0 ,1 6.0± 5.6,差异显著 ( P<0 .0 0 5)。并根据移植胚胎的平均 ZPTV值>2 0 %胚胎数目 0、1、≥ 2个将治疗周期分为 3组 ,其临床妊娠率分别为 2 7%、51 %、55%( P<0 .0 0 5) ,胚胎种植率分别为 1 3 %、2 5%、3 0 % ( P<0 .0 0 1 ) ,均随移植胚胎数增加而显著升高。年龄 <3 5岁组 ,移植周期的多胎妊娠率随平均 ZPTV值 >2 0 %胚胎移植数 ( 0、1、≥ 2 )的增加而升高 ( 2 9%、3 4%、62 % ) ,差异有显著性 ( P<0 .0 5) ;但年龄≥ 3 5岁组 ( 3 3 %、2 0 %、2 9% ) ,无显著差异 ( P<0 .0 5) ,移植胚胎形态等级和各年龄段妇女中移植胚胎平均ZPTV值均无显著差异 ( P>0 .0 5)。结论 :移植胚胎的平均 ZPTV值与胚胎种植率、临床妊娠率和多胎妊娠率密切相关 ,平均 ZPTV是衡量胚胎质量的又一重要指标。     

The influence of sperm/zona pellucida collision rates on zona binding

Summary. The satisfying success rates reported with intra-cytoplasmic sperm injection (ICSI) urged clinicians and scientists to re-address the emphasis in the management of the male factor patient towards gamete manipulation in order to circumvent the underlying problem causing fertilization failure. We have designed a study to (i) calculate the collision rate of a sperm population with the human zona pellucida, using a mathematical hypothesis and (ii) use the calculated collision rate to evaluate subsequent zona binding results obtained under hemizona assay conditions. Microdroplets were used to co-incubate sperm and human oocytes in order to evaluate zona binding. Using microvolumes, the track followed by sperm, as well as the maximum distance travelled were employed to calculate the collision rate of sperm and zona pellucida. The sperm concentrations of swim up samples were adjusted to 4 × 10⁶ and 0.8 × 10⁶ sperm ml⁻¹. Five separate droplets each of 20 μl containing 4 × 10⁶ sperm ml⁻¹ (80000 motile sperm) and 200 μl containing 0.8 × 10⁶ sperm ml⁻¹ (160000 motile sperm), respectively, were prepared. Both volumes were incubated for 18 h at 37°C. The mean (±SD) number of spermatozoa tightly bound to hemizona, incubated in 20 μl and 200 μl sperm droplets, was 2444±612 and 548±315, respectively ( P = 0.0001). The results can be used as a guideline to calculate the optimum insemination concentration needed for a specific sperm population to ensure the maximum collision rates with the oocyte.     

激光削薄透明带对小鼠囊胚形成和孵出的影响

目的探索激光削薄透明带对体外培养昆明小鼠囊胚形成和孵出的影响。方法激光孵化组为应用1．48μm二极管激光对186枚2细胞期昆明小白鼠胚胎实施透明带削薄处理;对照组不削薄透明带,在离胚胎一个直径处发射激光。比较两组囊胚形成率、孵出率、完全孵出率、透明带厚度和胚胎直径。结果激光组和对照组的囊胚形成率分别为73．7％（137／186）和71．6％（53／74）,两组比较差异无统计学意义。激光组的孵出率（86．9％,119／137）明显高于对照组（71．7％,38／53,P〈0．05）;囊胚嵌顿率（74．8％,89／119）明显高于对照组（23．1％,9／38,P〈0．001）。结论激光辅助孵化对体外培养昆明小鼠囊胚形成无不良影响且可以提高囊胚孵出率,但囊胚嵌顿率显著升高。     

Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell‐to‐cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl‐ and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA‐Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA‐Gal) binding activity was similar to that of BSA‐Fuc, but was weaker. In acrosome‐reacted spermatozoa treated with the Ca²⁺ ionophore A23187, BSA‐zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA‐Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml^?1 BSA‐Fuc, in vitro sperm–ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm–egg ZP interactions. At the same concentration, BSA‐Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm–ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.