Girdin蛋白在小鼠受精卵微丝聚集调控作用的初步研究

目的:探讨Girdin蛋白在调控小鼠受精卵早期发育微丝聚集中的作用。方法:采用免疫荧光染色观察Girdin蛋白和F-actin在小鼠受精卵中的共定位情况。激光共聚焦显微镜观察Girdin表达敲低后小鼠受精卵分裂形态及分裂率。结果:在小鼠受精卵中存在Girdin蛋白并且Girdin蛋白与F-actin存在共定位。敲低Girdin蛋白的表达,小鼠受精卵出现不规则分裂,10μmol/L Girdin si RNA处理组小鼠受精卵有28.93%到达2-细胞期,而注射20μmol/L Girdin si RNA组小鼠受精卵只有15.1%到达2-细胞期。并且Girdin表达敲低的受精卵微丝不能正确聚集。结论:Girdin蛋白在调控小鼠受精卵早期分裂微丝聚集中发挥作用。     

RNA结合蛋白Rbpms 2在小鼠卵母细胞及早期胚胎中的表达及意义

目的:检测分析多重剪接RNA结合蛋白2(RNA binding protein with multiple splicing 2,Rbpms2)在小鼠卵母细胞及早期胚胎中的表达及分布情况。方法:运用生物信息学资源分析Rbpms2基因表达的组织分布情况以及Rbpms2蛋白的功能结构域;通过免疫印迹法、免疫组织化学及免疫荧光检测,分析Rbpms2在小鼠卵母细胞及早期胚胎中的表达和分布。结果:生物信息学检索显示Rbpms2 mRNA在卵细胞及受精卵中高表达,其蛋白具有一个RNA识别结构域。免疫印记法证实Rbpms2蛋白在小鼠卵母细胞中高表达,同时免疫组织化学显示阳性信号主要集中在卵母细胞胞质中,免疫荧光显示在早期胚胎中,Rbpms2的信号集中在细胞核中。结论:Rbpms2在小鼠卵母细胞及早期胚胎中呈高水平表达,提示其可能在卵母细胞和早期胚胎的发育过程中发挥重要作用。     

14-3-3蛋白在小鼠受精卵及2-细胞期胚胎中的表达与定位

目的:研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法:采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达,利用间接免疫荧光技术观察其在细胞中的定位。结果:在小鼠受精卵和2-细胞期胚胎中,全部表达14-3-3蛋白,并且为同一亚型;14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论:14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中,其定位可能影响胚胎的早期发育。     

14-3-3蛋白在小鼠受精卯及2-细胞期胚胎中的表达与定位

目的：研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法：采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达，利用间接免疫荧光技术观察其在细胞中的定位。结果：在小鼠受精卵和2-细胞期胚胎中，全部表达14-3-3蛋白，并且为同一亚型：14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论：14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中，其定位可能影响胚胎的早期发育。     

着床丝氨酸蛋白酶-2(ISP2)蛋白在小鼠卵子和早胚组织中的表达及其定位

目的:了解ISP2蛋白在小鼠卵细胞和早胚组织中的表达情况。方法:收集小鼠未成熟卵、成熟卵、受精卵和着床前胚泡,利用特异性抗ISP2抗体,采用免疫荧光和激光共聚焦显微镜技术对ISP2在卵细胞和早期胚胎组织中的表达模式以及亚细胞定位进行检测分析。结果:在小鼠未受精卵、受精卵和着床前胚泡中均能检测到特异的ISP2蛋白信号;通过激光共聚焦显微镜观察到ISP2在细胞膜上的信号强于其在细胞质。结论:ISP2蛋白在小鼠卵子和早胚组织中均有表达,并主要分布在细胞膜上。     

水通道蛋白3在小鼠早期胚胎发育过程中的表达

目的 了解水通道蛋白3(aquaporin3,AQP3)在小鼠早期胚胎发育中的表达分布情况.方法 建立昆明小鼠控制性超排卵模型,收集小鼠四细胞期、八细胞期、桑葚期胚胎及早期囊胚,采用免疫荧光显微镜和激光共聚焦显微镜对AQP3在小鼠早期胚胎中的表达模式及亚细胞定位进行检测分析.结果 在小鼠早期胚胎的4个时期中均能检测到特异性AQP3荧光信号,激光共聚焦显微镜观察到AQP3的分布部位在各时期不完全相同,在四细胞期胚胎及八细胞期胚胎主要定位于卵裂球核膜,桑葚期胚胎主要定位于细胞膜,而早期囊胚主要定位于滋养层细胞膜及细胞质.结论 AQP3对小鼠早期胚胎的发育可能具有潜在的调控作用.     

Aurora激酶A、B在小鼠受精卵第一次有丝分裂进程中的表达与定位

目的:研究Aurora激酶A(AURKA)、Aurora激酶B(AURKB)在小鼠受精卵第一次有丝分裂进程中的表达与亚细胞定位。方法:分别利用实时荧光定量PCR和Western blotting法从mRNA和蛋白水平上检测AURKA、AURKB在小鼠受精卵第一次有丝分裂进程中的表达规律,并用间接免疫荧光法观察AURKA/B蛋白在各时相的细胞定位。结果:AURKA、AURKB在受精卵的G2、M期呈高表达,G1、S期呈低表达,AURKA/B蛋白主要定位于胚胎的细胞质和细胞核中,但细节上各有特点。结论:AURKA、AURKB在小鼠受精卵第一次有丝分裂进程各期呈时空特异性表达,提示其对小鼠早期胚胎发育过程发挥重要的调控作用。     

蛋白激酶B(AKT)抑制剂对小鼠1-细胞期胚胎分裂的影响

目的:研究蛋白激酶B(AKT)在小鼠1-细胞期受精卵中的作用。方法:激酶活性分析检测2种AKT的抑制剂在小鼠1-细胞期胚胎中对MPF活性的调节;G2/M期的细胞计数检测AKT抑制剂对小鼠1-细胞期胚胎细胞分裂的影响;Western blotting检测AKT抑制剂对小鼠1-细胞期胚胎中pCDC2Tyr15的去磷酸化状态的改变。结果:AKT抑制剂抑制了MPF活性、G2/M期的转换,pCDC2Tyr15的去磷酸化状态。结论:AKT存在于小鼠1-细胞期胚胎中并通过调节MPF活性和pCDC2Tyr15的去磷酸化状态调节细胞分裂的进程。     

乳酸对着床前小鼠胚胎丙酮酸摄入和代谢的调节

分析胚胎营养摄入特点发现,最早期胚胎摄入丙酮酸,而8细胞期以后丙酮酸摄入下降,到囊胚期则以葡萄糖为主要营养物质。尽管乳酸并不能单独支持受精卵的首次分裂,但乳酸可以被受精卵和二细胞胚胎氧化。乳酸可以导致二细胞胚胎丙酮酸的氧化下降。为明确乳酸对着床前胚胎丙酮酸摄入和代谢的调节,观察不同浓度乳酸对丙酮酸摄入和     

人类卵母细胞及植入前胚胎中母性效应基因Mater mRNA的表达

目的:了解母性效应基因Mater表达与人类卵母细胞及早期胚胎发育不同阶段的关系。方法:用巢式逆转录多聚酶链式反应(single cell nested RT-PCR)方法检测人类卵子和植入前各阶段胚胎的母性效应基因Mater mRNA的表达。结果:Mater在人类卵母细胞GV、MⅠ、MⅡ都有表达,植入前胚胎1、2、4、6细胞期表达量逐渐下降,到8细胞期、囊胚、孵出囊胚期均未检测到Mater mRNA表达。结论:人类卵母细胞和植入前胚胎Mater基因的表达量随发育时间的改变而变化,对卵子生长和胚胎发育有重要意义,与母性效应基因在小鼠等其他哺乳动物中的表达模式相似。     

Downregulation of gene expression and activity of GRIM-19 affects mouse oocyte viability,maturation, embryo development and implantation

PurposeTo investigate the expression of GRIM-19 (Gene associated with retinoid-interferon-induced mortality 19) in mouse oocytes and preimplantation embryos, and to study the effect of GRIM-19 on the developmental competence of mouse oocytes and embryos.MethodsGRIM-19 was evaluated at both mRNA and protein levels. The expression of GRIM-19 gene was downregulated in mouse oocytes cultured in vitro by specific small interfering RNA (siRNA) injection, while the activity of GRIM-19 was decreased by microinjection of a GRIM-19 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocytes matured in vitro were then fertilized by intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate, cleavage rate, blastocyst formation rate and implantation rate.ResultsGRIM-19 is expressed throughout oocyte maturation and preimplantation embryo development stages. GRIM-19 was localized primarily in the cytoplasm of all cells examined. Downregulation of gene expression and activity of GRIM-19 resulted in decreased oocyte viability, potency of oocyte maturation, embryo development and implantation.ConclusionsGRIM-19 may play important roles in mouse oogenesis and early embryonic development and implantation.     

应用荧光原位杂交技术进行植入前胚胎染色体诊断的价值

目的 初步探讨应用荧光原位杂交(FISH)技术进行植入前胚胎染色体诊断的价值。方法 对10对不孕夫妇进行植入前遗传学诊断(PGD)周期的超促排卵和卵母细胞浆内单精子注射,于受精后第3天进行胚胎活检及FISH分析,第4天选择染色体组成正常或平衡的胚胎进行移植。结果 10个PGD周期共获卵158个,对其中54个胚胎进行活检,51个胚胎获得明确诊断,诊断率为94％(51／54)。对染色体组成正常或平衡的24个胚胎进行官腔内移植,共4例获得妊娠,其中3例已足月分娩健康婴儿,1例为异位妊娠。结论 应用FISH技术进行植人前胚胎染色体诊断,是预防流产和染色体异常患儿出生的有效手段。     

HLA-G蛋白在人类着床前胚胎的表达

目的:探讨HLA-G在人类胚胎早期发育和免疫耐受中的作用和意义。方法:以体外受精-胚胎移植技术助孕过程中剩余的、自愿捐赠的人类早期胚胎作为研究对象,采用免疫荧光染色技术,检测人类着床前胚胎HLA-G蛋白的表达。结果:采用MEM-G/9单抗进行了HLA-G免疫荧光染色,20个2-8-细胞期的人类早期胚胎中15个胚胎HLA-G蛋白检测阳性;3个桑椹胚和5个囊胚均为阳性。结论:着床前人类早期胚胎表达HLA-G蛋白。     

Passive immunization with antisperm monoclonal antibody ZAP-7 increases preimplantation embryo mortality in the mouse

Objective: To evaluate the effect of VIC-1 and ZAP-7 antihuman sperm monoclonal antibodies on in vivo fertility in the mouse.

Design: A randomized blinded study using a mouse model.

Setting: University-based laboratory.

Animals: B6CBAF1 mice (n = 6 per experimental group).

Intervention(s): Antisperm antibodies were administered intravaginally to female mice before mating. Control mice received no treatment, saline, or nonspecific antibodies. Number and viability of preimplantation embryos were determined by microscopic observation. Mouse sperm, oocytes, and normal preimplantation embryos were used in indirect immunofluorescence assays with antisperm antibodies. The effect of antibody treatment on sperm motility and vitality was evaluated.

Main Outcome Measure(s): Antigen expression, sperm motility and vitality, number and viability of embryos.

Result(s): ZAP-7 antibody recognizes a sperm antigen expressed in zygotes and early preimplantation embryos. Passive immunization with ZAP-7 increases embryo mortality significantly (more than 40% above controls). Passive immunization with VIC-1 has no deleterious effect.

Conclusion(s): ZAP-7 monoclonal antibody disrupts fertilization and embryogenesis in the mouse.     

Serum anti-Müllerian hormone levels are not associated with aneuploidy rates in human blastocysts

Research questionAnti-Müllerian hormone (AMH) is the most established biomarker for estimating ovarian reserve. No reliable marker of oocyte quality, however, is available. Is there an association between the rates of aneuploidy and the different ranges of serum AMH levels?DesignRetrospective, single-centre study of 1718 patients undergoing intracytoplasmic sperm injection and preimplantation genetic testing with aneuploidy at the blastocyst stage between January 2015 and December 2019. Patients were stratified into six different categories of AMH (ng/ml) according to percentile distribution.ResultsAlthough a higher number of biopsied embryos were found for higher AMH levels (P = 0.017), a lower rate of biopsied blastocysts per metaphase II (P = 0.019) and per fertilized oocyte (0.023) was observed in this group of high AMH. A higher number of euploid embryos was found for higher AMH values (P = 0.031); however, the rate of aneuploid embryos per metaphase II or per fertilized oocyte was not significantly different across the six groups. No differences were observed in the implantation, pregnancy and ongoing pregnancy rate, or in the miscarriage and biochemical loss rate. Regression analysis did not show any significant correlation between AMH and aneuploid embryos.ConclusionsIn this large series of patients, AMH was not related to embryo aneuploidy.     

Exposure of Preimplantation Embryos to Platelet-Activating Factor Increases Birth Rate

PROBLEM: Platelet-activating factor (PAF) plays a significant role in fertility. Preimplantation stage embryos produce PAF (ePAF) which is required for development. PAF's mechanism of action is receptor-mediated and its presence has been reported in the developing mouse and human embryo. Exposure of preimplantation stage mouse embryos results in higher implantation rates. However, the effect of such treatment on live-birth rates and birth weights has not been reported. Therefore, the objective the study was to determine the effect of exposing preimplantation mouse embryos to PAF on subsequent birth rate and weight. DESIGN: Two-cell stage preimplantation stage mouse embryos exposed to PAF (10(-7) M) for 15 min prior to intraoviductal transfer. METHODS: Preimplantation stage embryos were recovered from eCG/hCG primed BDF1 female mice. Embryos were exposed to synthetic PAF (10(-7) M) for 15 min. PAF-treated embryos were transferred to the oviducts of pseudopregnant female CD-1 female mice. Superovulated and cultured BDF1 embryos not treated with PAF served as in vitro controls and naturally ovulated embryos with no collection/culture served as in vivo controls. Embryos were permitted to develop to term (18-21 days). The number of pups born per litter and litter weights subsequently were recorded. RESULTS: A total of 160 BDF1 mouse embryos were collected, treated, and transferred (20 per CD-1 recipient) as described. There was a significant (P < 0.05) increase in the number of pups born to the PAF treatment group (56/80; 70%) as compared to the control group (44/80; 55%). There was also a significant difference (P < 0.05) in litter birth weights between the PAF (1.31 g/litter) and controls groups (1.25 g/litter). There was a significant difference (P < 0.05) in birth weights between the PAF treatment group and the in vivo group (1.51 g/litter). There was a significant difference in birth weights between the in vitro-control and in vivo groups (1.51 g/litter). There were no observational malformaties to pups born in any group. CONCLUSIONS: Brief exposure of preimplantation stage embryos to PAF will result in a significant increase of delivery rates (pups/litter) as well as birth weights. However, the increase of birth weight was significantly below that found naturally. Additional studies are warranted to elucidate the mechanism of PAF's action in the preimplantation stage embryo and subsequent uterine development.