Effect of Insulin on Development oflCR Mouse Embryos in Vitro

Objective To study the effects and the appropriate concentration of insulin on the development of mouse embryos in vitro, and the effects of insulin on the development of different stages of embryos.
Methods Mouse embryos were cultured in vitro in the mKSOM media supplemented with insulin at different concentrations and with insulin during different stages of embryos. The blastoeyst rates and the cell numbers were counted.
Results Additions of insulin significantly increased the rates of blastocyst and the total cell numbers. The concentrations of 0.005 μg/ml and 0.05 μg/ml insulin caused a significant increase in the total cell numbers compared with the control and experimental groups. Addition of insulin from 2-cell to 4-cell stage or from the 4-cell to morula stage, significantly increased the blastocyst rates compared with control and experi- mental groups.
Conclusion Insulin can promote the development of mouse embryos in vitro. The appropriate concentration of insulin added in mKSOM was 0.005 μg/ml and 0.05μg/ml. Exposure of embryos to insulin, beginning at the 2-cell and extending to the 4-cell stage or beginning at the 4-cell and extending to the morula stage, is important for the development of lCR mouse embryos in vitro.     

胰岛素对ICR小鼠胚胎体外发育的影响

目的研究ICR小鼠胚胎体外培养时,添加胰岛素的作用、浓度及所需阶段．方法用添加不同浓度胰岛素的mKSOM培养基对小鼠胚胎进行体外培养及选择最适浓度对不同阶段鼠胚进行体外培养,观察囊胚发育率和囊胚细胞数的变化．结果所有添加胰岛素的浓度组,囊胚发育率均高于对照组,其中0．005μg／mL和0．05μg／mL浓度组囊胚细胞数显著高于对照组和其他各浓度组．2-细胞至4-细胞、4-细胞至桑椹胚前添加胰岛素囊胚率显著高于对照组和其他实验组．结论在ICR小鼠胚胎体外培养时添加胰岛素能够促进胚胎发育;胰岛素浓度增至μg／mL对在ICR小鼠胚胎无毒性作用;在ICR小鼠胚胎体外培养的适宜胰岛素浓度为0．005μG／mL和0．05μG／mL;2-细胞至4-细胞、4-细胞至桑椹胚前添加胰岛素更加重要．     

Effect of an Improved Mechanical Method for Assisted Hatching on the in vitro Development of Mouse Embryos

Implantation of embryo occurs after its escape from the zona pellucida (ZP), the pro- cess of which is called hatching. In vivo hatching is viewed as a prerequisite for successfuldialogue between the embryo and the endometrium. It has been speculated that…     

胰岛素对不同时期ICR小鼠胚胎体外发育的影响

目的研究胰岛素对不同时期小鼠胚胎体外发育的影响.方法收集1-细胞、2-细胞、4-细胞及桑椹胚期鼠胚,分别在含0（对照组）、0.005、0.05、0.5、5、10μg/mL胰岛素的KSOM中培养.结果在1-细胞及2-细胞阶段添加系列浓度时,0.005μg/mL及0.05μg/mL胰岛素组不仅提高囊胚率、孵化率,而且提高囊胚细胞数.4-细胞阶段添加系列浓度时0.05μg/ml胰岛素组能够提高囊胚率、孵化率以及囊胚细胞数;而至桑椹胚阶段再添加系列浓度胰岛素时,囊胚率、孵化率以及囊胚细胞数均无统计学意义（P〉0.05）.结论在ICR小鼠胚胎体外培养时适宜的胰岛素浓度为0.05μg/mL;桑椹胚之前添加胰岛素对小鼠胚胎发育是必要的.在一定发育阶段内,随着胚胎的生长对胰岛素的需求也相应增加.     

Effect of Different High CO2 Concentrations on the Development of 2-cell Mouse Embryos in vitro

Objective To investigate effects of differen high CO2 concentrations on the develop- ment of 2-cell mouse embryos in vitro Methods At levels of 5 % CO2 (control group), 5.7 % CO2, 6.0% CO2 and 15% CO2,embryos were incubated in drops with CZB medium, respectively, and the drops were covered by paraffin oil which was treated with three-distilled water. In addition, at the level of 15 % CO2, there were another two groups, in which paraffin oil was treated with phosibhate-buffered saline (PBS ) solution or the drops were uncovered. The development of embryos in all stages was noted. Results The developmental rates of blastocysts in five experimental groups were significantly lower than that of the control group (P＜0. 01). At the level of 5.7% CO2, the developmental rate of blastocysts was 4. 3%, and those of other experimental groups were O. At the levels of 5.7 % and 6.0 % CO2, embryos were blocked in the 2cell or the 4-cell stage, and no significant difference was showed between the two groups (P＞0. 05). At the level of 15% CO2, 15% embryos developed in the 4cell stage with irregular blastomere and degenerated quickly in the group which paraffin oil was treated with distilled water~ 2.2 % embryos developed in the 4-cell stage in the group which paraffin oil was treated with PBS and the rest stagnated in the 2-cell stage. Conclusions High CO2 concentrations had toxice effect on the vitro development of 2-cell mouse embryos, and was responsible for teh inhibition of thedevelopment of the embryos. It isimportant for the development of embryos in vitro to detect strictly CO2 concentration.     

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

Clenbuterol hydrochloride is a strongβ2 receptor agitator,and it was used toprevent and treat bronchial asthma.However,it is seldom used today because of itsstrong side-effect to the heart[1 ] .Clenbuterol hydrochloride also helps in the forma-tion of bo…     

人卵泡液对体外培养鼠胚发育的影响

昆明种雌鼠经孕马血清促性腺激素（ＰＳＭＧ）和人绒毛膜促性腺激素（ＨＣＧ）超数排卵，与雄鼠交配后在注射ＨＣＧ４２ｈ获得晚期２－细胞鼠胚．置Ｈａｍ′ｓＦ~（１０）为主的培养液中，发育到囊胚的平均比率为６２．０％。实验组培养液中加入５％，１０％，１５％（Ｖ／Ｖ）人卵泡液，囊胚比率为２３．７％（Ｐ＜０．０５），１９．５％（Ｐ＜０．０５），１７．８％（Ｐ＜０．０５）。不同浓度卵泡液组间无显著性差异（Ｐ＞０．０５）。结果表明：（１）２－细胞晚期鼠胚体外培养结果稳定，适宜作人胚胎及卵子体外培养条件的质量监控。（２）人卵泡液抑制鼠胚体外发育，且这种抑制与卵泡液浓度无关。     

小鼠胚胎原始生殖细胞的体外培养

采用细胞生物学方法和组织培养技术研究小鼠胚胎原始生殖细胞(PGCs)的一般形态学特征及生物学特点.在体外培养条件下,PGCs 按体积大小可分大、中、小3类。本研究发现,中、小型原始生殖细胞在培养的第2、3 d 增多,形态似大型原始生殖细胞。培养中的 PGCs 不贴壁而悬浮生长于培养液中,培养到第6～7 d时开始死亡。体细胞(间充质细胞或间皮细胞)贴壁生长,生长旺盛,与PGCs 同时死亡。     

昆明小鼠体外受精胚冷冻保存的研究

在PBS 20?S 0.5mol/L蔗糖中加2.0和3.0mol/L甘油组的胚胎发育率分别为51.4%和44.4%,冷冻效果明显好于其它浓度甘油;在PBS 20?S十2.0mol/L甘油中,加0.5mol/L蔗糖组的胚胎发育率为47.7%,冷冻效果好于其它浓度蔗糖;慢速降温冷冻组的胚胎发育率为48.8%,效果明显好于快速冷冻法;在各期胚胎冷冻后的发育率中,2细胞胚最低,桑椹胚的存活率明显高于4细胞胚和早期囊胚;冷冻保存后体外受精胚的发育率显著低于体内受精胚:112枚经冷冻保存的体外受精胚移植给受体鼠,2/9妊娠。     

黄芪多糖对小鼠1-细胞胚胎体外发育效果的初步研究

目的：探讨黄芪多糖对小鼠1-细胞胚胎体外发育的作用。方法：以mCZB添加抗生素为对照组，以添加不同浓度黄芪多糖（Astragulus Polysacharin，Aps）为试验组，观察胚胎体外发育率、孵化胚胎细胞数目及切割取样后桑椹胚发育率和细胞数目。结果：体外培养96h，试验组囊胚发育率（86．0％、85．8％、89．5％）均显著高于对照组（75．8％）（P〈0．05），各试验组间差异无显著性（P〉0．05）；体外培养144h，试验组胚胎孵化率（28．9％、31．0％、25．4％）均高于对照组（22．6％），以Aps2（100μg／mL）组孵化率为最高（31．0％）。孵化胚胎细胞数试验各组（76．00±6．25、81．73±7．01、75．89±4．54）均极显著高于对照组（69．06±5．15）（P〈0．01）。切割取样（≤5或〉5个）后桑椹胚体外培养72h，试验组发育率和细胞数分别为（≤5组为50％和44±2．7、50％和53±2．7、44．4％和55±2．7；〉5组为23．1％和42±1．6、20．0％和44±1．0、14．3％和41±1．0）均高于对照组（33．3％和41±1．7；0％和0）。结论：中药有效成分黄芪多糖能促进小鼠早期胚胎的体外发育及胚胎细胞增殖，并对切割取样后胚胎的体外培养有一定促进作用，且切割样本数≤5对胚胎发育比较有利。     

Effects of Malachite Green on the Development of Mouse Embryo In Vitro

Objective To observe effects of malachite green （MG） on the development of mouse embryo in vitro.Methods Two-cell mouse embryos were obtained and exposed to different concentrations of MG （0 ng/ml, group A, the control; 10 ng/ml, group B; 100 ng/ml, group C; 1 000 ng/ml, group D）. The 2-cell mouse embryo assay was used to determine the 2-cell embryo development to the blastocyst. In addition, at the same concentrations of MG, the human sperm motility assay was used to evaluate the influence of MG on sperm viability.Results At the 8-cell stage, groups C and D showed inhibitory effects when compared with the control （P〈0.01）. Rate of the blastocyst in groups C and D was lower than that of the control （P〈O.O1）. In the sperm motility assay, significant changes in motility after 24 h of incubation in groups C and D were observed compared with the control （P〈0. 01）. However, no differences were found on 24 h sperm motility between group B and the control （P〉0.05）. Conclusion MG would have deleterious effects on the development of mouse embryo, and the effects were dose-related. The sperm motility assay could also detect high levels of MG in culture medium.     

雌激素、孕激素对小鼠胚卵体外发育及胚泡着床的影响

目的:研究雌、孕激素在小鼠胚卵体外发育及胚泡着床过程中的作用.方法:在小鼠桑椹胚与人蜕膜细胞共培养体系中分别加入雌激素(E2组)、孕激素(P组)、雌激素和孕激素(E2 P4组),观察各组胚卵发育以及胚泡粘附和铺展情况.结果:E2组桑椹胚向胚泡的发育率高于对照组,E2 P4组胚泡的粘附率和滋养层细胞的铺展率均较对照组高.结论:单独用雌激素能够促进小鼠桑椹胚发育成胚泡,雌激素和孕激素联合应用能够促进胚泡的粘附和铺展.     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

A Preliminary Observation on the Development of Mouse Embryos Co－cultured with Human Oviductal Tissue or Conditioned Medium i

ＡＰｒｅｌｉｍｉｎａｒｙＯｂｓｅｒｖａｔｉｏｎｏｎｔｈｅＤｅｖｅｌｏｐｍｅｎｔｏｆＭｏｕｓｅＥｍｂｒｙｏｓＣｏ－ｃｕｌｔｕｒｅｄｗｉｔｈＨｕｍａｎＯｖｉｄｕｃｔａｌＴｉｓｓｕｅｏｒＣｏｎｄｉｔｉｏｎｅｄＭｅｄｉｕｍｉｎＶｉｔｒｏＺｈｏｎｇＹｕ；Ｚｈａ...     

关于统计学符号的书写方法

为探讨输卵管上皮细胞对小鼠雌雄原核受精卵（2PN受精卵）体外发育的影响,首选建立了羊输卵管上皮细胞共培养体制,再将小鼠2PN受精卵分别放在M16培养液（对照组）中和羊输卵管上皮细胞＋M16培养液（实验组）中培养,结果表明：在对照组中,只有50％的2－细胞胚胎能克服体外发育的阻断而达到4－细胞阶段,发育到囊胚遥比率仅为28．2％。实验组中,有86．75％的2－细胞胚胎可以克服体外发育的阻断而达到4－细胞阶段,发育到囊胚期的比率为45．7％。对照组与实验组相比,统计学上有显著差异（P＜0．01）。这提示：输卵管上皮细胞有助于小鼠胚胎克服体外发育阻断,并增加发育囊胚期的胚胎数。     

LIF对体外培养小鼠孕早期胚胎发育及ICAM-1表达的影响

目的研究白血病抑制因子（ＬＩＦ）在胚胎发育中的作用并探讨其参与胚泡着床的机制。方法收集昆明种小鼠的单细胞受精卵,连续培养１２０ｈ,观察不同浓度ＬＩＦ对胚胎发育的影响;用免疫印迹法测定培养液中细胞间粘附分子（ＩＣＡＭ－１的表达水平。结果ＬＩＦ在０．１～１０ｎｇ／ｍｌ浓度之间对胚胎发育有促进作用,其作用在桑椹胚及胚泡期最明显（Ｐ＜０．０５）;ＬＩＦ增加胚胎ＩＣＡＭ－１的表达,其作用在０．１ｎｇ／ｍｌ浓度时最强,并随着浓度的增加而减弱。结论适应浓度的ＬＩＦ可促进胚胎发育,增加ＩＣＡＭ－１的表达,从而参与胚胎着床过程。     

鼠胚实验在新建胚胎培养实验室质量控制中的作用

目的采用鼠胚实验(mouse embryo assay,MEA)检测新建的胚胎培养实验室的培养体系是否符合人类胚胎体外培养的要求。方法采用配子体外授精法和收集2细胞胚胎培养法进行实验。在胚胎体外培养的过程中定时对胚胎的发育状况进行观察并拍照记录。结果配子体外受精法的囊胚形成率为90.84%±7.09%。收集2细胞胚胎培养法的囊胚形成率为91.17%±6.97%。两种方法的囊胚形成率之间差异无显著性。结论配子体外授精法和收集2细胞胚胎培养法均可作为新建胚胎培养实验室的质量控制方法。     

瘦素对小鼠着床前胚胎发育影响的体外研究

目的　探讨瘦素在体外对小鼠着床前胚胎发育的影响。方法　 1收集小鼠 2 -细胞胚胎 ,在不同剂量瘦素的 CZB培养液中进行体外培养 ,观察胚胎发育情况 ,并进行胚胎移植 ,观察着床率 ;2收集小鼠 2 -细胞胚胎在空白 CZB培养液中体外培养至桑葚胚期 ,再分别置入不同剂量瘦素的 CZB培养液中进行体外培养 ,观察胚胎进一步发育情况。结果　瘦素能提高 2 -细胞胚胎体外发育的质量、发育率和胚胎着床率。当瘦素剂量为 10 ng/ ml时平均胚胎形态学评分 (AES)为 16 .0 8± 0 .37,剂量为 5 0 ng/ ml时 AES为 17.5 7± 0 .4 2 ,两者间差别有统计学意义(P<0 .0 5 )。但剂量进一步增加 ,促进作用不再递增。瘦素对桑葚胚体外进一步发育无显著影响。结论　瘦素参与了小鼠着床前胚胎的发育 ,能提高胚胎发育质量、发育率 ,有利于胚胎的进一步着床。     

苯对小鼠胚胎发育的毒性作用研究

目的探讨苯对小鼠胚胎体内发育的毒性。方法随机选择健康性成熟的昆明种小鼠,雌雄合笼产生孕鼠,孕鼠被随机分成5组（溶剂对照组、高、中、低剂量苯染毒组和环磷酰胺染毒组）,每组8只,分别在小鼠妊娠第6-15天用25（低剂量）、100（中剂量）、400 mg/kg.d（高剂量）的苯进行灌胃染毒,每天一次,同时设植物油溶剂对照和环磷酰胺（10 mg/kg体重）阳性对照。观察孕鼠的体重增长情况;于妊娠第18天处死孕鼠取出胚胎,观察胚胎的生长发育状况、死胎和吸收胎鼠数及畸形发生情况。结果与植物油对照组比较,中、高剂量苯染毒可降低孕鼠体重的增长率（P〈0.05）,高剂量苯染毒孕鼠能使胚胎吸收和死亡发生率增加（P〈0.05）,并可抑制胎盘和胎仔生长发育,中、高剂量苯染毒导致胎鼠畸形发生率增加（P〈0.05）,但对孕鼠和胎仔的毒作用影响均弱于环磷酰胺的毒性作用（P〈0.05）。结论苯在体内对小鼠胚胎具有发育毒作用和致畸形作用。     

ICR小鼠胚胎及生后不同阶段睾丸内生殖细胞的发育

目的 :观察ICR小鼠胚胎及出生后的睾丸内生殖细胞在不同阶段的发育情况 ,了解雄性生殖细胞分化、发育和成熟的过程。方法 :采用半薄切片甲苯胺蓝染色法 ,对雄性胚胎第 13,16 ,18d和出生后第 1,3,7,10 ,15 ,19d、4 ,6周及成年 (8周 )睾丸生殖细胞进行观察。结果 :原始生殖细胞向精原细胞的演化经历了一个分散 居中 分散靠边的过程。出生后第 7d ,支持细胞达到一个增殖高峰 ;出生后第 10d ,原始生殖细胞出现一个增殖高峰 ;出生后第 10d ,间质细胞达到一个增殖高峰 ;出生后第 15d ,出现初级精母细胞 ;出生后第 4周 ,产生精子细胞 ,同时间质细胞出现另一个增殖高峰并保持在一个高水平 ;出生后第 6周 ,开始有精子出现。结论 :当精原细胞大量增殖和生成精子细胞与精子时 ,生殖细胞可能需间质细胞提供大量激素相辅助 ;支持细胞在支持生精细胞、促进生精细胞转位及精子生成过程中起了一定作用。