骨桥蛋白对小鼠体外受精及早期胚胎发育的影响

目的 探讨骨桥蛋白（OPN）对小鼠体外受精及早期胚胎发育的影响。方法 120只昆明雌鼠和30只雄鼠,采用体外受精、胚胎培养方法,将小鼠精子、卵子、原核期胚胎和2-细胞期胚胎分别用不同浓度的OPN抗体预处理,观察小鼠受精、卵裂以及早期胚胎发育情况。结果 不同浓度的OPN抗体分别预处理精子和卵子后,与对照组比较,受精率显著降低(P＜0.01)。OPN抗体预处理原核期胚胎后,0.01mg/L OPN抗体组的卵裂率较对照组低,但差异无显著性 (P =0.052),1.00mg/L OPN抗体组的卵裂率低于0.01mg/L 组（P ＜0.01）及0.10mg/L组（P ＜0.05）。不同浓度的OPN抗体预处理2-细胞胚胎后可抑制胚胎发育。1.00mg/L OPN抗体组的4-细胞率和8-细胞率与0.10mg/L OPN抗体组相比差异无显著性(P ＞0.05),但前者的囊胚形成率显著低于后者（P ＜0.01）。1.00mg/L OPN抗体组的4-细胞率、8-细胞率以及囊胚形成率均显著低于0.01mg/L OPN抗体组（P ＜0.01）。结论 OPN可促进小鼠的受精和早期胚胎发育。     

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。     

白介素-1β对小鼠胚胎体外发育及胚胎子宫内膜共培养体系分泌Hb—EGF、αυβ3的影响

目的使用不同浓度的白介素-1β(IL-1β)对小鼠胚胎体外发育进行干预并检测与着床相关因子Hb—EGF、αυβ3的分泌,探讨IL-1β在胚胎着床中的作用。方法将IL-1β稀释成不同浓度,研究其对小鼠2-细胞期胚胎囊胚形成率及囊胚孵出率的影响,并用酶联免疫吸附法检测不同浓度的IL-1β对胚胎子宫内膜共培养体系Hb—EGF、αυβ3表达的影响。结果 2-细胞胚胎体外培养,经含不同浓度的IL-1β培养液干预,培养72h的囊胚率明显高于对照组,有显著性差异(P<0.01)。1ng/ml和10ng/ml组培养96h的胚胎孵出率明显高于对照组,有显著性差异(P<0.05)。100ng/ml组的孵出率与对照组相比,无显著性差异(P>0.05)。胚胎-子宫内膜共培养体系经1ng/ml、10ng/mlIL-1β干预后Hb—EGF、αυβ3蛋白表达上调,与对照组相比,有显著性差异(P<0.05);共培养体系经100ng/ml IL-1β干预后Hb—EGF、αυβ3蛋白表达与对照组相比,无显著性差异(P>0.05)。结论合适的IL-1β可以促进体外培养小鼠胚胎发育及胚胎着床。     

IGF-Ⅱ对小鼠2-细胞胚胎体外发育的影响

目的研究胰岛素生长因子-Ⅱ对小鼠2-细胞胚胎体外发育的影响。方法在mKSOM培养液中添加不同浓度的IGF—Ⅱ（insulin—like growth factor—Ⅱ）对小鼠2-细胞胚胎进行体外培养,观察囊胚发育率、孵化率和囊胚细胞数的变化。结果（1）培养到72h,实验组囊胚率显著高于对照组。（2）培养到96h,0．1、1、10ng／ml IGF-Ⅱ添加组的孵化率显著高于对照组和100ng／ml IGF—Ⅱ添加组。（3）进行囊胚细胞计数,实验组与对照组比较,差异无显著性;四个实验组之间比较,差异亦无显著性。结论IGF-Ⅱ浓度为0．1、1、10ng／ml时,能促进小鼠2-细胞胚胎的体外发育。     

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。     

内毒素对去卵透明带2-细胞小鼠胚胎体外发育的影响

目的 观察不同剂量浓度内毒素对去卵透明带2—细胞小鼠胚胎体外发育的影响。方法 获取小鼠2—细胞胚胎，用胰蛋白酶法去除卵透明带后，分别置于含有0，1pg/ml，5pg/ml和10pg/ml内毒素的CZB液滴中培养，此外，用蛋白酶法去除卵透明带，在不含内毒素的CZB液滴中培养，观察胚胎体外发育情况。结果 用胰蛋白酶法去卵透明带的2—细胞小鼠胚胎，其囊胚率随着培养液中内毒素剂量的增加而降低，且5pg/ml和10pg/ml时与对照组(不含内毒素)相比差异显著(P＜0．001)。在有内毒素存在时，停滞在2—细胞或4细胞期的胚胎彼此分离排成列，不再呈球形。部分卵裂球出现空泡或碎裂。另外，用蛋白酶法去除透明带的2—细胞鼠胚的囊胚率为61．7％，显著低于胰蛋白酶法的73．8％(P＜0．001)。结论 微量内毒素即明显抑制去卵透明带2—细胞小鼠胚胎的体外发育，并表现出剂量效应；去卵透明带2—细胞小鼠胚胎试验可应用于培养液的微量内毒素检测；胰酶法去除卵透明带后胚胎的囊胚率优于蛋白酶法。     

两种小鼠胚胎体外发育系统对内毒素敏感性的比较研究

目的 :探讨小鼠 1细胞和 2细胞胚胎体外发育系统对内毒素的敏感性。方法 :获取小鼠 1细胞胚胎和 2细胞胚胎 ,分别与 3个内毒素剂量 (0 .7,7,70EU/ml)组共培养 ,计算各阶段胚胎的发育率。结果 :小鼠 1细胞和 2细胞胚胎的内毒素0 .7EU/ml组 ,从 2细胞至囊胚各期发育率与对照组比较没有显著性差异 (P >0 .0 5 )。内毒素在 7EU/ml水平显著降低小鼠 1细胞胚胎的体外发育率 (P <0 .0 1) ,但对 2细胞胚胎未见明显影响。内毒素在 70EU/ml水平则完全抑制 1细胞和 2细胞胚胎的体外发育 (P <0 .0 1)。培养液中含有内毒素引起碎裂胚胎增多 ,卵裂球退化和轮廓不清。结论 :内毒素对体外培养的胚胎有明显毒性 ,小鼠 1细胞胚胎体外发育试验对内毒素较 2细胞胚胎试验敏感     

胚胎的形态学指标与囊胚形成的相关性分析

目的研究胚胎的形态学指标与囊胚形成的关系。方法收集321对行辅助生殖技术助孕的夫妇D3移植和冷冻后剩余胚胎共1208枚,放入培养液中继续培养至囊胚期,观察囊胚形成率和优质囊胚形成率。结果共形成囊胚210枚,囊胚形成率17.4%,优质囊胚62枚,优质囊胚形成率为5.1%。D3卵裂球数目为6-10细胞组囊胚形成率和优质囊胚形成率极显著高于其它组(P0.01)。不同胚胎碎片组比较差异不显著(P0.05)。胚胎发育迟缓组囊胚形成率和优质囊胚形成率比正常发育组低,差异极显著(P0.01),其中延时受精组、发育阻滞组的囊胚形成率极显著低于发育迟缓组(P0.01)。结论 D3剩余胚胎仍有继续发育成囊胚的潜能,D3细胞数和胚胎发育速度与囊胚形成密切相关。     

输卵管积水对小鼠胚胎体外发育能力的影响

目的探讨输卵管积水对小鼠胚胎体外发育能力的影响。方法收集人输卵管积水，小鼠促超排卵，收集2细胞胚胎和囊胚，随机分配到含有不同浓度输卵管积水的培养液中，观察胚胎的发育，计算成囊率，囊胚孵出率。结果输卵管积水组的胚胎成囊率和囊胚孵出率低于无积水组，并呈剂量依赖性。结论输卵管积水能显著影响胚胎的早期发育潜能。     

Total ablation of paternal accessory sex glands curtails developmental potential in preimplantation embryos in the golden hamster

The effects of total removal of paternal accessory sex glands (TX) on preimplantation embryonic development was studied in the golden hamster model. Cell numbers of the two groups of embryos did not differ up to 60 h p.c., but at 66 and 70 h p.c., each TX embryo has 2 and 3 cells less respectively (P<0.05, TX vs SH). At 70 h p.c., 46.6±4.4 of the TX embryos blastomeres were labelled with the terminal deoxynucleotide transferase – mediated dUTP-nickend-labelling technique, compared with 31.5±2.1 in the SH group (P<0.01, TX vs SH). No difference was found in the SDS-PAGE profiles of two-cell embryos from the two groups. An extra band corresponding to 136.5 kDa was consistently found in the four-cell TX embryos. The nascent proteins profiles of four-cell embryos from the two groups were similar. As the embryos progressed from two to four cells, the protein content decreased by 16% in the SH embryos (P<0.05) and 7% in the TX embryos. These observations suggest that total ablation of paternal accessory sex glands could result in developmental aberrations from the two-cell to morula stages and a higher incidence of apoptosis at 70 h p.c. Accepted: 28 March 2001     

p38MAPK在小鼠早胚中的表达及其对胚泡发育的作用

目的观察p38丝裂原活化蛋白激酶(p38MAPK)在小鼠早胚中的表达及其作用,探讨p38MAPK与胚泡植入的相关关系。方法取小鼠胚胎的2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。用体外胚胎培养技术:将2细胞期小鼠早胚随机分组,接种到添加不同浓度p38MAPK特异性抑制剂SB220025的培养液内进行培养,台盼蓝染色观察p38MAPK特异性抑制剂对小鼠早胚发育的影响。结果 p38MAPK在小鼠胚胎发育各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。体外实验显示:抑制剂组早胚不能发育到胚泡阶段,抑制剂恢复实验发现抑制剂组胚卵仍具存活能力。结论 p38MAPK在小鼠胚胎发育过程中起作用,p38MAPK特异性抑制剂可抑制小鼠早胚的发育。     

Globulin-Enriched Protein Supplements Shorten the Pre-Compaction Mitotic Interval and Promote Hatching of Murine Embryos

PROBLEM: To determine whether Synthetic Serum Substitute (SSS), which contains human globulins in addition to Human Serum Albumin (HSA), is superior to HSA alone as a protein supplement for embryo culture. METHOD: Development of mouse zygotes to eight-cell/compacting morulae and to hatching/hatched blastocysts was assessed in Human Tubal Fluid (HTF) medium containing either SSS or HSA. RESULTS: Although there was no difference in the overall blastocyst rate at 120 h in HTF+SSS versus HTF+HSA, significantly more embryos at 54 h were at the eight-cell/compacting morula stage in HTF+SSS. At 120 h, there were more hatching/hatched blastocysts in HTF+SSS, and hatching correlated with SSS concentration. Addition of isolated globulins to HSA significantly stimulated the number of hatching/hatched blastocysts. Hatching could be “rescued” by transfer of embryos grown in HTF+HSA to globulin-containing media and prevented by removal of globulins as late as the compacted morula stage (54 h). CONCLUSIONS: SSS is superior to HSA alone for embryo culture. The stimulatory effects on mitosis and hatching may be mediated directly by globulins or by other components in the globulin-enriched fraction.     

The effect of injected cycloheximide on early amphibian development

Cycloheximide is a known inhibitor of cytoribosomal translation. Previously published results indicated that embryos cultured in cyclo-heximide developed only to the midblastula stage. The current study involved injecting one μg of cycloheximide into each developing embryo of Rana pipiens. The three stages selected for the injection were the two-cell, late four-cell, and morula. The embryos stopped developing at the four-cell, sixteen-cell and midblastula stages, respectively. Control embryos injected at the same stages with Holtfreter's solution developed normally. The results would indicate that de novo protein synthesis is necessary, not just at the midblastula stage, but throughout the entire cleavage period. The results are discussed in light of previous investigations.     

Facilitated glucose transporters play a crucial role throughout mouse preimplantation embryo development

The role of glucose fluctuates during preimplantation mouse embryo development, indicating that a specific interplay exists between glucose metabolism and uptake. In this study, attempts were made to characterize the role of the Na(+)-coupled active and the facilitated glucose transporters (GLUT) during preimplantation development by using specific glucose analogues and transport inhibitors and by examining the expression of GLUT1. One-cell outbred mouse embryos were cultured in medium M16 (5.5 mmol/l glucose), M16 without glucose (M16-G), M16-G + 2-deoxyglucose, M16-G + 3-O-methylglucose, M16 + phlorizin and M16 + phloretin and development to the blastocyst stage assessed. The absence of glucose, or the presence of 3-O-methylglucose, which is taken up but not metabolized, did not inhibit blastocyst development. 2-Deoxyglucose, which is phosphorylated but not metabolized, inhibited blastocyst development. Culture in M16 supplemented with phlorizin, an inhibitor of Na(+)-coupled active glucose transport did not inhibit blastocyst formation. Phloretin had no effect on the cleavage of two-cell embryos to the four-cell stage, but inhibited the morula/blastocyst transition. Both phloretin and phlorizin inhibited glucose uptake in two-cell embryos. Finally, GLUT1 expression was 10-fold less in blastocysts cultured in M16 compared to in-vivo blastocysts and those cultured in M16-G. The results show that both types of glucose transporters influence preimplantation embryo development and that the embryo has an innate ability to control the uptake of glucose by regulating the expression of GLUT1.     

Lamins A and C are present in the nuclei of early porcine embryos,with lamin A being distributed in large intranuclear foci

Gametogenesis and embryogenesis are dynamic developmental stages marked by extensive modifications in the organization of the genome and nuclear architecture. In the literature it is conveyed that only B-type lamins are required in these early stages of development and that A-type lamins are not present or required until differentiation of specific cell types associated with specialized tissue is initiated. To assess the presence of nuclear structures that are putatively involved in genome regulation, we investigated the distribution of lamin proteins throughout the early stages of porcine embryonic development, using testes tissue sections, oocytes and in-vitro fertilized (IVF) porcine embryos and employing anti-lamin antibodies. We have shown that anti-lamin A staining is present at the one-cell, two-cell, four-cell, and six- to eight-cell stages of early porcine embryo development, but diminishes at the morulae and blastocyst stages. Large intranuclear anti-lamin A foci are prominent in the early preimplantation stages. Both anti-lamin A/C and anti-lamin B staining were clearly present in all embryonic stages. Immature porcine oocytes revealed lamin rings using the monoclonal anti-lamin A/C antibody and many immature oocytes exhibited a pale rim staining pattern with anti-lamin A antibody. A-type lamins were not observed in sperm precursor cells. Thus, we have shown that A-type lamins and B-type lamins are present at the nuclear envelope in very early porcine embryos and that lamin A is also found in large intranuclear aggregates in two-cell to eight-cell embryos but is lacking from later embryonic stages. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Induction of sister chromatid exchange in preimplantation mouse embryos in vitro by 3H-thymidine or ultraviolet light in combination with caffeine

Preimplantation mouse embryos were exposed in vitro to 3H-thymidine (25, 100, or 250 Bq/ml) or ultraviolet (UV) light (1.35 or 4.05 J/m2), either alone or in combination with caffeine (1 mM with 3H-thymidine and 0.5 mM with UV light). Exposure to 3H-thymidine lasted for 2 days, from the two-cell stage to the late morula/early blastocyst stage, and UV radiation was applied acutely at the late morula/early blastocyst stage. The effects were quantified by the sister chromatid exchange (SCE) assay. All three agents induced SCEs when used singly. 3H-thymidine was effective in inducing SCEs only at 250 Bq/ml, whereas UV light was effective at both fluences. Although caffeine did not induce SCEs when it was added before exposure to bromodeoxyuridine (BrdUrd), which is used to visualize SCEs, it did induce SCEs when present during the entire culture period (3H-thymidine experiments) or during incubation in BrdUrd (UV experiments). Caffeine markedly enhanced the SCE-inducing effect of UV light but did not influence the effect of 3H-thymidine.