小鼠休眠胚胎与正常胚胎冻融后体内外发育潜力的比较

目的检验小鼠休眠胚胎冻融后的质量及体内外发育潜力,为胚胎休眠技术的生产应用提供必要的参考。方法采用常规冷冻方法将正常孵化期胚胎和休眠胚胎进行冷冻,之后分别进行体外复苏培养实验和胚胎移植实验。随后利用双重荧光染色的方法分别对冻融前后的小鼠休眠胚胎与正常孵化期胚胎进行细胞计数,观察两种胚胎冻融前后的质量变化。结果休眠胚胎的冷冻解冻回收率、发育率均极显著高于孵化期胚胎(72.1%vs 50.2%,P<0.01;94.2%vs 73.9%,P<0.01)。休眠胚胎的移植妊娠率显著高于孵化期胚胎(40.8%vs 30.1%,P<0.05)。休眠胚胎的内细胞团细胞数显著高于孵化期胚胎(27.83 vs 19.53,P<0.05),滋养层细胞数差异不显著。冻融培养后休眠胚胎的内细胞团数,滋养层细胞数均显著高于孵化期胚胎(25.18 vs 14.68,P<0.05;114.09 vs 73.88,P<0.05)。结论小鼠休眠胚胎冻融后胚胎质量及体内外发育潜力均优于小鼠正常孵化期胚胎。     

利用超数排卵方法高效获取小鼠休眠胚胎

目的为提高小鼠休眠胚胎的制备效率,尝试用超数排卵方法获取小鼠休眠胚胎。方法超排小鼠在注射孕马血清促性腺激素(PMSG)后,按照是否注射抗孕马血清(anti-PMSG serum或A-PMSG)以及注射的不同时间分为5组:在母鼠合笼前注射A-PMSG、在母鼠见栓后注射A-PMSG、在见栓第四天卵巢摘除手术后注射A-PMSG、不注射A-PMSG的超排组和不超排组。结果见栓后注射抗PMSG组的平均出胚数(9.4枚/只)最高,显著高于其他实验组。结论常规超排处理结合注射抗PMSG血清法能有效提高小鼠休眠胚胎的回收率。     

浙贝母鳞片细胞休眠解除前后超微结构的变化

浙贝母休眠解除后,鳞片近轴面表皮附近的几层细胞首先降解。本文观察了这些细胞在休眠解除前后超微结构的变化。与休眠状态时的细胞相比,解除休眠后的细胞中颗粒和丝状物的数量明显增加,一些细胞中出现了复杂的膜结构。线粒体的数目增加,常聚集在细胞核和细胞壁旁。胞间连丝的直径略有增加。休眠解除前后的细胞中,常可见到各类囊泡、多泡体与壁旁体,一些小囊泡正在进入细胞中。表明两种时期的细胞间都发生着物质和信息的交换     

经程序化冷冻的小鼠休眠胚胎的基因表达谱差异分析

目的探讨小鼠休眠胚胎经程序化冷冻后基因表达谱的变化及相关信号通路的改变趋势。方法采用Affymetrix基因芯片检测小鼠正常休眠胚胎和经程序化冷冻后的休眠胚胎的差异表达基因;采用GO分析和Pathway分析等生物信息学方法进一步了解相关信号通路的改变。结果经程序化冷冻后的小鼠休眠胚胎与正常休眠胚胎相比,存在228个差异表达基因,其中50个基因表达上调,178个基因表达下调。Pathway分析显示黏着斑通路、细胞外基质受体相互作用通路、肌动蛋白细胞骨架调节通路、细胞凋亡通路、细胞通讯通路、泛素介导的蛋白质水解通路、甘油磷脂代谢通路、小细胞肺癌通路、TGF-β信号通路、MAPK信号通路等基因差异表达变化趋势明显。结论小鼠休眠胚胎经程序化冷冻后会导致一系列基因调控变化,并可能影响多条信号通路的协同变化。     

改良透射电镜方法观察小鼠ICSI胚胎核仁超微结构

小鼠植入前胚胎的发育过程中,核仁经历从简单到复杂、从致密结构到网状结构的变化。对核仁超微结构的观察有助于揭示早期胚胎发育过程中核仁结构的动态变化及其特定阶段的功能。但由于核仁结构微小,数目较少,并且在胚胎中只处于卵裂球细胞核的内部,难以定位,因而给核仁的超微结构观察带来很大的困难。本实验探索了透射电镜观察小鼠植入前胚胎核仁的方法:先用琼脂对小鼠胚胎进行预包埋,在经过常规的透射电镜样品制备流程后,将整个胚胎先切成半薄切片;经过甲苯胺蓝染色后,选取含核仁结构的切片进行重包埋;最后再对回收来的半薄切片进行超薄切片,醋酸铀染色后上电镜观察;最终成功获得小鼠胚胎植入前发育不同时期核仁清晰的透射电镜图像。     

杜仲休眠枝条中多糖颗粒变化的超微结构研究

在对杜仲（Ｅｕｃｏｍｍｉａ ｕｌｍｏｉｄｅｓ Ｏｌｉｖ．）形成层休眠期枝条的超微结构研究中发现,在筛管分子的筛板附近有一种复合球形体。细胞化学研究证明,这是一种多糖颗粒,但不同于枝条其他薄壁细胞中的多糖颗粒,而与细胞壁中的多糖相似。这种复合球形体只出现于形成层生理休眠期的筛分子中,而在被动休眠期和活动的筛分子中则看不到。     

AFM对小鼠胚胎干细胞的形态学研究

目的：对已建系BALB/c小鼠胚胎干细胞膜表面进行纳米级超微结构的初步形态学研究,从而为从分子水平研究胚胎干细胞的增殖与分化调控机理奠定基础。方法：利用原子力显微镜（AFM）,在空气中对小鼠胚胎干细胞膜表面扫描成像。结果：AFM图像表现BALB/c小鼠胚胎干细胞呈圆盘状,直径约10-15μm,高约2-4μm,胚胎干细胞膜表面比较复杂,随着扫描范围的减小,切向分辨率逐渐增大,可达到纳米分辨,细胞表面有许多紧密堆积的椭球状颗粒。颗粒尺寸（x-y方向）为40-80nm。结果：利用AFM可以得到胚胎干细胞表面高分辨的,可重复的图像。     

包囊游仆虫休眠包囊的超微结构研究

包囊游仆虫休眠包囊中,各类纤毛器的纤毛基体上方的大部分纤毛杆退化,或仅保留毛基体,有时部分额腹棘毛的毛基体也瓦解消失。残留纤毛的纤毛杆周围微管和中央微管仍具有“９＋２”结构特征,也有少数纤毛杆出现２套“９＋２”微管共处于一层纤毛膜内的现象。毛基体中周围三联体微管的中央形成微管形结构聚合体,基体附属结构仅存在基体间连接及纤毛器托架的残余物;非纤毛区皮层表膜下未见微管层。纤毛区皮层含纤毛器腔周围微管层     

大麦根中禾谷多粘菌休眠孢子堆的超微结构

通过扫描电镜和透射电镜观察了禾谷多粘菌Ｐｏｌｙｍｙｘａ ｇｒａｍｉｎｉｓ Ｌｅｄ。休眠孢子堆的超微结构。休眠孢子堆仅分布于寄主根表皮细胞中,休眠孢子堆形状不一,有的呈球状,有的呈棒状,少则由几十个,多则由数百个紧密列的休眠孢子组成。     

小鼠胚胎干细胞与四倍体胚胎的嵌合

The oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos.The embryos were placed twenty at a time between two platinum electrodes laid 1 mm apart in 0.3M mannitol in the electrode chamber.The blastomeres were fused by a short electric pulse(80V for 50μsec) applied by a pulse generator.Fusion of blastomeres was usually completed in 20-60minutes.After 25 hours of culture,most of the tetraploid embryos developed to the four-cell stage.Zonae pellucidae of 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode‘s buffer.The embryos were plated on an ES cell layer,After 40 hours of coculture,248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipinets.Ten recipients were pregnant.but no live fetuses were born.Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and seven abnormal fetuses were obtained.The results demonstrate that ES cells derived from C57BL/6 mice are pluripotential to a certain extent.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Cathepsin L involved in the freezing resistance of murine normal hatching embryos and dormant embryos

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.     

Ultrastructural aspects of mouse cecal epithelium

Summary The ultrastructure of the cecal epithelium of the mouse resembles that of more distal parts of the colon. A brief discussion is presented of the possible significance of apical cytoplasmic granules to the glycocalyx and of the close association of microorganisms with the lining epithelium.This research was supported by N.I.H. grant GM-15289. The author thanks Dr. F. J. Agate for the scanning electron micrograph.     

Successful implantation of in vitro cultured rabbit embryos after uterine transfer: A role for mucin

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 ± 0.2 μm of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 ± 5.5 μm. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20–40 μm thick mucin layer. No live young were obtained from 57 embryos with less than a 20 μm thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos. © 1996 Wiley-Liss, Inc.     

Lower total cell numbers in mouse preimplantation embryos cultured in human assisted reproductive technique (ART) media are not induced by apoptosis

A common feature of assisted reproductive techniques such as IVF or intracytoplasmic sperm injection is the IVC of oocytes or preimplantation embryos in artificial culture media. The IVC conditions are selected to mimic the environment of the female genital tract. We have shown that murine preimplantation embryos respond to different culture media with changes in developmental rates, cellular lineage composition, and gene expression patterns. In this study, we explored whether apoptosis is responsible for cell loss in mouse preimplantation embryos after exposure to different human culture media. We examined total embryonic cell count as well as the pattern of protein expression for caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and the executioner caspase-3 via immunohistochemical staining. Total cell counts decline in embryos cultured either in innovative sequential medium 1 and Blast Assist (Origio) or human tubal fluid and MultiBlast (Irvine Scientific) when compared to KSOM(aa). Few cells were caspase-9 and -3 positive in all experimental groups. Staining for caspase-8 was almost undetectable. We conclude that embryonic cell loss is not associated with higher rates of intrinsic apoptotic cell loss. Our results suggest that the culture medium–dependent decline in total cell count and the developmental restriction in embryos cultured in innovative sequential medium 1/Blast Assist and human tubal fluid/MultiBlast are related to processes affecting cell proliferation.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Role of X-linked inhibitor of apoptosis (XIAP) in frozen and thawed dormant and normal-hatched murine blastocysts

Cryo-injury of mammalian blastocysts occurs during cryopreservation and induces apoptosis in trophoblast cells. This damage affects subsequent embryo development or may even cause death before implantation. X-linked inhibitor of apoptosis (XIAP) is an anti-apoptosis gene that has been widely studied in cancer research. However, only a few studies have investigated the activity of XIAP in cryopreservation. In this study, we investigate the role of XIAP in frozen and thawed murine blastocysts. A total of 1630 blastocysts were divided into fresh and freeze-thaw groups, and XIAP expression was investigated using qPCR, Western blot and confocal analyses. In addition, the effect of the embelin (a XIAP inhibitor) was also evaluated by co-culturing 390 dormant blastocysts. XIAP protein is primarily localized to the mitochondria of trophoblastic cells. Gene and protein expression is significantly down-regulated in blastocysts after cryopreservation, whereas embelin has negative effect on their survivals. These findings further broaden the understanding of mammalian embryonic cryopreservation.