8型解脲支原体对小鼠早期胚胎发育的致病力研究

目的观察不同浓度的8型解脲支原体对小鼠早期胚胎发育的影响,以探讨解脲支原体对早期胚胎发育的致病作用。方法取小鼠2-细胞期胚胎随机分为实验组与对照组,对照组：组1,2-细胞期鼠胚在不含8型UU的HTF培养液中培养;实验组：组2、组3、组4、组5,2-细胞期鼠胚分别在含102CCU/ml～105CCU/ml浓度的8型UU的HTF培养液中培养,在37℃、5%CO2的培养箱中培养72h,每24h在Nikon倒置显微镜下观察记录,通过统计各组的D2评分、4-细胞期胚胎形成率、桑椹期胚胎形成率、囊胚形成率,评价早期胚胎的发育情况。以观察不同浓度8型UU对小鼠早期胚胎发育的影响。结果 8型UU在培养液中的终浓度为102CCU/ml组2-细胞胚胎以后的D2评分、4-细胞期胚胎形成率、桑椹期胚胎形成率、囊胚形成率等各阶段的胚胎发育指标与对照组比较差异均无统计学意义（P〉0.05）,终浓度分别为104CCU/ml、105CCU/ml组2-细胞胚胎以后的各项胚胎发育指标与对照组比较均有显著性差异（P〈0.05）,终浓度为103CCU/ml组的4-细胞期胚胎形成率、囊胚形成率与对照组比较无显著性差异〉0.05,而D2评分、桑椹期胚胎形成率与对照组比较有显著性差异（P〈0.05）,103CCU/ml组的P值均接近0.05显著性差异临界值。结论 UU能够影响小鼠早期胚胎的发育,8型UU≥104CCU/ml时就可阻碍小鼠早期胚胎的发育。随着UU浓度的增高,对早期胚胎发育的影响也增大。     

脑源性神经营养因子调控热应激条件下小鼠着床前胚胎发育和凋亡

目的 探讨脑源性神经营养因子（BDNF）对正常和热应激条件下小鼠着床前胚胎发育和凋亡的影响。 方法 150只雌性昆明小鼠用于实验,2细胞期胚胎添加或不添加10μg/L BDNF进行体外培养,早期囊胚37 ℃条件下或41 ℃热处理2 h,观察胚胎发育情况,TUNEL法和Hoechst 33342分析囊胚细胞数量和凋亡情况,荧光技术分析Caspase-9的活力。 结果 在37 ℃条件下BDNF能够促进早期囊胚发育,增加囊胚细胞数量,降低凋亡率及Caspase-9的活力,热应激能够降低早期囊胚的发育能力,减少囊胚细胞数量而增加凋亡率,且中等和高Caspase-9活力囊胚的比例热处理后明显增加,当培养基中添加10μg/L BDNF时,能够明显缓解热应激对早期囊胚发育和质量的不利影响。 结论 BDNF对37 ℃或41 ℃热应激条件下小鼠着床前胚胎发育、细胞数量、凋亡和Caspase-9活力起到一定的调控作用。     

血管内皮生长因子受体-2在植入前小鼠胚胎发育中的表达变化

目的：探讨植入前小鼠胚胎血管内皮生长因子受体-2（VEGFR-2/Flk-1）的表达规律及其在小鼠胚胎发育中的作用。方法:对0.5－3.5 d小鼠胚胎行全胚免疫荧光染色及反转录－聚合酶链反应，观察胚胎发育过程中Flk-1表达时相及其变化；取8细胞期胚胎行体外培养，加入5 mg/L Flk-1中和抗体，分析36 h囊胚形成率。结果:在植入前小鼠胚胎中Flk-1 mRNA于4细胞期开始表达，8细胞期达高峰，桑椹胚期和囊胚期无表达；Flk-1蛋白表达定位于胚胎外缘和细胞交界处，于4细胞期和8细胞期表达呈阳性，桑椹胚期、囊胚期表达呈阴性；于体外培养的8细胞期培养液中加入5 mg/L中和抗体后，36 h囊胚形成率降低。结论:在小鼠植入前胚胎发育不同阶段Flk-1表达不尽相同，其作用可能与Flk-1促进植入前胚胎发育有关。     

囊胚培养液对小鼠胚胎的毒性:体外生殖辅助医疗器械安全性评价

背景:囊胚培养液是可以支持胚胎从受精卵发育到囊胚阶段的一种重要培养液,其安全性直接影响囊胚的质量。目的:观察囊胚培养液对小鼠胚胎发育的影响。方法:选用B6D2F1品系小鼠,将雌性小鼠进行超数排卵处理,与同品系雄鼠合笼过夜,次日收集1细胞期受精卵,在M16培养液中培养至4细胞期后,检查胚胎发育情况并转移到受试囊胚培养液中继续培养(实验组),5 d后检查囊胚发育情况并计算囊胚形成率。同时,用含有内毒素的囊胚培养液M16培养小鼠受精卵,作为阳性对照组;用已被证实没有胚胎毒性的囊胚培养液M16培养液培养1细胞期受精卵,作为阴性对照组。结果与结论:阳性对照组囊胚形成率为0,阴性对照组囊胚形成率为87.1%,实验组囊胚形成率为87.3%,实验组囊胚形成率大于80%,说明受试囊胚培养液可以支持小鼠受精卵正常发育到囊胚阶段,对胚胎没有毒性效应。     

四倍体胚胎发育阶段对胚胎干细胞嵌合体小鼠制备的影响

目的 探讨四倍体胚胎发育阶段对胚胎干细胞(ES)嵌合体小鼠制备的影响.方法 通过2-细胞胚胎电融合法制备四倍体胚胎,采用显微注射方法将ES细胞分别注入1-细胞、4-细胞、囊胚3个发育阶段的四倍体胚胎中.所用ES细胞分别为杂交系B6D2F1×129/Sv和近交系C57BL/6J,经胚胎移植和剖腹产以获得ES小鼠.结果 实...     

蛋白激酶C在卵母细胞成熟、受精和早期胚胎发育过程中的作用

小鼠胚胎发育过程中,蛋白激酶C(PKC)家族在调节卵的活化、完成减数分裂和开始有丝分裂、胚胎的致密,以及囊胚形成过程中都有作用,但其多个亚型的功能还知之甚少.研究表明,已知的十种PKC亚型以及PKC锚定蛋白--活化的磷脂酶C受体(RACK)在小鼠胚胎2-细胞期至囊胚期都有表达.本文归纳了近年来对PKC亚型各自不同的动态分布模式和表达水平的研究,这些研究表明它们在早期胚胎发育的各个时期都至关重要,尤其在胚胎4-细胞期的早期,个别亚型在核内的浓度是瞬间升高的,提示我们这些PKC亚型也许控制着早期小鼠胚胎核的组建以及机能调节.本文对核移植胚胎中PKC的可能作用也进行了分析.     

玻璃化冷冻人不同发育时期的胚胎对其发育潜能的影响

目的分析使用玻璃化冷冻法对人D3卵裂期细胞及囊胚进行冷冻,对胚胎发育潜能的影响。方法从2013年3月至2015年12月180周期358枚胚胎行解冻胚胎移植,将胚胎分为卵裂组及囊胚组,统计解冻后存活率,移植后着床率、妊娠率、流产率及多胎妊娠率。结果 D3卵裂组与囊胚组解冻后存活率没有差异,但移植后发育潜能卵裂组明显低于囊胚组。结论冷冻解冻移植囊胚期的胚胎比卵裂期的胚胎更有发育潜能。     

人体外受精-胚胎移植中第3天胚胎发育潜能的评估

目的通过分析第3天(D3)胚胎的优质囊胚形成率,探讨形态学评估在胚胎发育潜能评估中的价值。方法选取接受体外受精-胚胎移植(IVF-ET)治疗的不育症夫妇1 185对,其中女方年龄21~45岁,平均年龄30.88岁。对正常自然受精的8 184个D3胚胎进行囊胚培养,所有的培养都为25μL液滴单培养,对其形成的优质囊胚情况进行比较。结果 D3胚胎中8Ⅰ级胚胎发育形成优质囊胚概率最高(76.49%),明显优于<8细胞的Ⅰ级胚胎(35.10%),与9细胞(68.12%)、>9细胞(70.36%)的Ⅰ级胚胎相比,差异有显著统计学意义(P<0.01);同为8Ⅰ级优质胚胎在高龄人群(≥35岁)优质囊胚形成率明显低于年龄较低人群(<35岁),差异有显著统计学意义(P<0.01);高龄人群优质囊胚形成率8细胞以上Ⅰ级胚胎>8Ⅰ级胚胎>7Ⅰ级胚胎(68.29%vs 68.06%vs 40.82%),差异有显著统计学意义(P<0.01)。结论 8细胞Ⅰ级胚胎仍为卵裂期胚胎移植首选,但传统D3的7、8、9细胞的Ⅰ、Ⅱ级胚胎为优质胚胎的方法值得进一步商榷;高龄人群D3优质胚胎评估标准有待进一步阐明。     

年龄对小鼠IVF早期胚胎发育的影响

目的比较不同年龄小鼠体外受精胚胎发育的结局,探讨以年龄因素为背景的IVF胚胎发育的影响。方法采用相同超数排卵方案,在相同的培养条件和培养时间下,对4-8周龄和44-48周龄小鼠进行体外受精和胚胎培养,观察并记录双原核卵率、≥4细胞率、囊胚率以及孵化囊胚率。结果老龄组在受精率、卵裂率以及孵化囊胚率上与年轻组比较存在极显著性差异（P〈0.01）,但在囊胚形成率上的差异无统计学意义;老龄鼠超数排卵后获卵数明显少于年轻小鼠。结论年龄对卵子数量以及IVF胚胎早期发育有较大影响。     

小鼠非同步胚胎移植技术

目的:比较小鼠非同步胚胎移植技术。方法:将小鼠不同发育阶段的着床前胚胎非同步移植到不同假孕受体小鼠的输卵管或子宫内,观察非同步胚胎移植对小鼠胚胎移植成功率和后代出生重的影响。结果:CD-1小鼠受精卵、2-细胞胚胎、桑椹胚和囊胚都可移植到假孕0.5d受体小鼠输卵管继续发育,妊娠受体在移植后19d出生小鼠,胚胎移植出生率分别为56.3%、62.5%、59.4%和58.3%。桑椹胚和囊胚也可移植到假孕2.5 d受体子宫继续发育,妊娠受体在移植后17 d出生小鼠胚胎移植出生率分别为57.5%和62.5%。囊胚移植假孕0.5 d受体输卵管的出生小鼠的出生重显著大于移植假孕2.5 d受体子宫的出生小鼠。结论:不同发育阶段的胚胎若移植相同的假孕受体,则妊娠受体有相同的妊娠期;相同发育阶段的胚胎若移植不同的假孕受体,则妊娠受体有不同的妊娠期,假孕时间短的受体有较长的妊娠期和显著增加的小鼠出生重。     

Fertilization and Empryology: The effect of epidermal growth factor on growth and differentiation of mouse preimplantation embryos in vitro

The effect of epidermal growth factor (EGF) on embryonic growth,development, attachment and spreading in vitro was studied.EGF was added to 130 embryos at the 4-cell stage; to 128 embryosat the blastocyst stage; and to 147 embryos 24 h following spreading.Development of embryos from the 4-cell to the blastocyst stage,differentiation of the inner cell mass (ICM) and trophectoderm,and the occurrence of attachment and spreading were evaluated.Embryo development was significantly inhibited in cultures supplementedwith 100 ng/ml EGF compared to the controls (P < 0.001).Development of 4-cell embryos to blastocysts occurred in 25%of the EGF group compared to 85% of controls. Spreading occurredin 20% of 4-cell embryos and 30% of blastocysts treated withEGF, compared to 80 and 90% of corresponding controls. In embryosdeveloping from the 4-cell stage, massive growth of the ICMand inhibition of the trophectoderm occurred, whereas both ICMand trophectoderm were inhibited by EGF in embryos developingfrom the blastocyst stage. Following spreading, EGF caused massivegrowth of the ICM and regression of the trophectoderm. Our preliminaryresults show that EGF may be involved in the modulation andcontrol of early embryonic growth and differentiation.     

Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.     

小鼠胚胎毒性试验方法的建立及在评价中药早期胚胎毒性中的应用

目的 建立一种快速、早期的中药胚胎毒性的快速检测技术,用于中药的早期胚胎毒性筛选。方法 ①血清制备:SD大鼠随机分为阳性对照组4只,正常组4只,雄黄组、朱砂组各3只。阳性对照组给予环磷酰胺10 mg/kg,正常组用0.5%CMC-Na灌胃给药,雄黄组和朱砂组分别将雄黄和朱砂（用0.5%CMC-Na稀释）按1.0 ml/100 g灌胃给药。末次给药后1 h,无菌取血并取上清血清;②细胞采集:4~6周雌性C57小鼠,用妊娠孕马血清（PMSG）腹腔注射,48 h后腹腔注射人绒毛膜促性腺激素（hCG）,合笼交配。第2天检查阴栓,用M2培养液将2细胞胚胎从输卵管中冲出,用M16培养液进行培养;③毒性试验:在胚胎2细胞阶段加入正常组、阳性对照组、雄黄组及朱砂组不同稀释比血清,观察囊胚成活率。结果 在1∶4、1∶16、1∶32的稀释比下,正常组的囊胚率为60.00%、73.33%、93.33%,高于阳性对照组的46.67%、60.00%、66.67%;朱砂组血清各个稀释比的囊胚率均未达到80%;除1∶512这个稀释比外,雄黄组血清各个稀释比2细胞胚胎的囊胚率均未达到80%。结论 ①朱砂和雄黄的大鼠含药血清在一定浓度时,对小鼠2细胞胚胎的生长发育有一定的影响,呈现中药的胚胎毒性;②小鼠胚胎2细胞用于中药的早期胚胎毒性筛选方法是可行的。     

The CryoLoop facilitates re-vitrification of embryos at four successive stages of development without impairing embryo growth

BACKGROUND: Vitrification has been shown to be an effective method of cryopreservation, but little is known about re-vitrification of embryos. This study investigated the effect of re-vitrification on mouse embryo preimplantation development and viability post-transfer. METHODS: Mouse embryos at the 1-cell stage were vitrified using the CryoLoop technique. Embryos were warmed and then re-vitrified successively at the 2-, 8-cell and blastocyst stages. The effects of multiple rounds of vitrification on development, differentiation and viability were assessed and compared with non-vitrified embryos. RESULTS: Development to the 8-cell stage on day 3 and blastocyst on day 5 were not affected by re-vitrification. However, better hatching rates were observed in the non-vitrified control group. Total cell number and the number of cells allocated to the inner cell mass (ICM) were not different between treatments. The percentage of ICM development was also not different between treatments. Implantation rate and fetal weights were the same between treatments. However, overall there were fewer fetuses per embryo transferred in the re-vitrified group. CONCLUSION: Re-vitrification of mouse embryos has minimal effect on preimplantation embryo development or implantation potential.     

雌激素受体抑制剂对小鼠囊胚形成的影响

目的观察雌激素受体（estrogen receptor,ER）抑制剂对昆明（Kunming,KM）小鼠体外囊胚形成的影响,为进一步探讨雌激素受体在小鼠囊胚形成中的作用和机制提供实验依据。方法以空白KSOM培养液为对照组,KSOM中添加浓度为1、2、3、4、5p~mol／L的ER仪抑制剂MPP,以及浓度为1、10、50、100μmol／L的ERB抑制剂PHTPP为实验组,收集KM小鼠8．细胞胚进行体外连续培养,计数各组发育至桑葚胚和囊胚两个阶段的数目,比较各组发育到囊胚的比率。结果添加浓度为1~mol／L、2Ixmol／L的MPP实验组囊胚发育率分别为86．36％、86．40％,与对照组（82．26％）相比无明显差别（P〉O．05）,而添加浓度为3μmol／L、4μmol／L、5μmol／L的MPP实验组囊胚发育率分别为46．58％、7．50％、0,发育率显著下降（P〈0．01）。添加浓度为1μmol／L、10μmol／L、50~mol／L的PHTPP实验组囊胚发育率分别为82．61％、87．04％、83．98％,与对照组（82．09％）比较没有明显差异（P〉O．05）,而添加高浓度（100μmol／L）的PHrrPP实验组囊胚发育率（51．39％）较明显下降。结论低浓度（5μmol／L）的ERo的抑制剂MPP可完全抑制KM小鼠8一细胞胚体外发育到囊胚,而50~mol／L的ERB抑制剂PHTPP对KM小鼠囊胚形成无明显影响,高浓度（100μmol／L）的PHTPP才可部分抑制囊胚形成。提示在小鼠囊胚形成中,ERα起着更为主要的作用。     

Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability

In an attempt to enhance embryo development, we have co-cultured1-cell OF1 mouse embryos on bovine kidney epithelial (Madine-Darbybovine kidney; MDBK) cells in a complex medium called complexmouse tubal fluid (cMTF; based on the energy substrate levelsfound in the mouse oviduct, containing non-essential amino acids,glutamine and EDTA). To determine the quality of the blastocystsobtained, we examined several parameters: morphology, totalcell numbers, inner cell mass (ICM): trophectoderm (TE) ratio,glycolytic activity and viability after transfer. A significantlylower number of blastocysts developed on MDBK cells comparedwith cMTF medium. cMTF blastocysts had a significantly higherglycolytic activity and a lower blastocyst cell number thanthose grown in co-culture, while both in-vitro groups had higherICM: TE ratios compared with in vivo. Blastocysts grown on MDBKcells displayed an elevated ICM number compared with those grownin cMTF medium alone. However, the percentage of fetuses aftertransfer remained drastically low in both culture groups comparedwith in-vivo blastocysts. In conclusion, co-culture did notincrease the number of zygotes reaching the blastocyst stage.Although co-culture blastocysts show some similarities to in-vivoembryos in cell number and glycolytic activity, no enhancementin viability was observed.     

The impact of cellular fragmentation induced experimentally at different stages of mouse preimplantation development

It has been demonstrated previously that removal of acellular debris from the preimplantation mouse embryo is beneficial for subsequent development to the hatched blastocyst stage. We have studied the impact of cellular fragmentation induced in the mouse embryo during the late pronuclei and 8-cell stages on the hatching frequency and total cell number at the blastocyst stage. At the late pronuclei stage about one- quarter of the cytoplasm was removed from embryos in the experimental group, in four to six steps, thus creating four to six cytoplasts that were subsequently returned as anucleated fragments under the zona pellucida. Embryos with one-quarter of the cytoplasm removed and with intact cytoplasm after partial zona dissection (PZD) served as controls. At the 8-cell stage, embryos with their nucleoplast removed from two blastomeres served as an experimental group. Groups of embryos with part of the cytoplast removed from two blastomeres (nucleated fragments), embryos with two blastomeres removed and embryos after PZD alone served as controls. After manipulation all embryos were left in culture and analysed at about 100 h after human chorionic gonadotrophin administration. Fragments induced at the late pronuclei stage did not participate in compaction and were often spontaneously expelled from the embryo during hatching. Neither embryo hatching rate nor total cell number was affected when compared with zygotes with reduced cytoplasm. Although both nucleated and anucleated fragments induced at the 8-cell stage participated in recompaction, hatching was not compromised and there was no interference in further development as assessed by the cell number or hatching rate at the blastocyst stage, as compared with embryos with blastomeres removed. We conclude that anucleated cellular fragments formed in an otherwise healthy embryo, both before and after acquisition of the ability for compaction, are benign and that their removal provides no benefit for embryo development, at least to the hatched blastocyst stage.      

Co-culture of 1-cell mouse embryos on different cell supports

The development of 1-cell mouse embryos in explanted oviducts, on mouse and bovine oviduct epithelial cells and on two established cell line supports is compared. The best rates of blastocyst formation were obtained using explanted oviducts; mouse and to a lesser extent, bovine oviduct epithelial cells allow good embryonic development, associated with high viability after transfer of the blastocysts obtained in co-culture. MDBK (from bovine kidney) and Vero (from Green monkey kidney) have been tested. MDBK allows high rates of blastocyst formation (67%) and the blastocysts obtained are viable. Vero does not allow the 2-cell block to be overcome. Maintenance of cell polarity for all the feeder layers did not improve embryo development. A preliminary study on the metabolic modifications induced by the feeder layers showed no modifications at all related to a decrease in glucose, an increase in lactate and early embryonic development. On the other hand, for the free amino acids, cellular supports with high embryotrophic activity seem to mimic tubal secretions, especially with a high level of glycine. Neither a genital tract origin, nor a hormonal contribution are strictly necessary for embryo co-culture, as already demonstrated by co-culture with trophoblastic tissue. Established cell lines, which are easy to handle and control, could be useful tools in embryo biotechnology.     

不同类型的转基因细胞核供体对生产小鼠转基因克隆胚胎的影响

目的 利用体细胞核移植(SCNT)技术生产小鼠转基因克隆胚胎. 方法 利用所构建的含新霉素抗性(Neor)基因和增强型绿色荧光蛋白(EGFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染小鼠胎儿成纤维细胞和小鼠胚胎干细胞,经G418筛选14d后用于核移植.同时以未转基因小鼠卵丘细胞和成纤维细胞为核供体,进行体细胞核移植. 结果 转基因与未转基因胎鼠成纤维细胞的重构胚的囊胚(154/160)发育率为19.23%和22.91%,差异不显著(P＞0.05);转基因胚胎干细胞与胎鼠成纤维细胞的重构胚的囊胚(152/154)发育率为41.54%和19.23%,差异显著(P＜0.05);未转基因卵丘细胞与成纤维细胞的重构胚的囊胚(171/160)发育率为41.17%和22.91%,差异显著(P＜0.05). 结论 利用体细胞核移植技术,小鼠胎儿成纤维细胞和胚胎干细胞可以有效地生产小鼠转基因囊胚.