两种激活处理促进小鼠孤雌胚胎原核形成

不同人工处理方法激活哺乳动物卵母细胞的机理相似,但其激活效率存在差异。本研究以昆明(KM)、129/Sv×KM F1和C3H×KM F1雌鼠来源的卵母细胞为对象,利用氯化锶(SrCl2,Sr2+)联合细胞松弛素B(cytochalasin B,CB)(Sr2++CB)和离子霉素(ionomycin,Ion)联合6-二甲胺基嘌呤(6-dimethylaminopurine,6-DMAP)(Ion+6-DMAP)两种激活方法处理下对比分析不同品系小鼠卵母细胞的激活效率,并以卵母细胞原核形成率、原核数量和孤雌胚胎体外发育来评价两种激活剂的激活效率。研究结果表明,Ion+6-DMAP激活卵的1原核比率显著高于2原核(p0.05),Sr2++CB激活卵的2原核比率显著高于1原核(p0.05);KM、129/Sv×KM F1和C3H×KM F1各组孤雌胚胎卵裂率和激活率没有显著差异(P0.05),但129/Sv×KM F1和C3H×KM F1囊胚发育率显著高于KM组(p0.05)。3种小鼠品系的卵母细胞用Sr2++CB处理的孤雌胚胎发育率显著高于Ion+6-DMAP。结果证明,Sr2++CB处理小鼠卵母细胞的激活效率明显优于Ion+6-DMAP;129/Sv×KM F1和C3H×KM F1的孤雌胚胎体外发育率显著高于KM小鼠,为研究小鼠遗传背景影响孤雌胚胎发育的机理提供参考。     

小鼠单个植入前胚胎cDNA文库的构建及应用

哺乳动物合子基因组激活和植入前胚胎阶段特异性表达基因的研究一直是发育生物学研究的重点。发现和克隆植入前胚胎阶段特异性表达的基因需要有效的方法,阶段特异性cDNA文库的构建和筛选是一种较好的方法。用小鼠单个成熟卵细胞、受精卵、2细胞、4细胞和8细胞胚胎分别构建了cDNA文库。滴度分别为:62×105、83×105、104×106、151×106和162×106。随机挑选了29个克隆并进行了序列分析,结果表明,和已知表达序列标签(ESTs)同源的序列为6552%(1929),未知序列为1379%(429),并发现了2个重复序列。用特异性引物,分别对小鼠持家基因(βactin)和发育特异基因(OCT4)进行PCR扩增,结果表明,所建立的cDNA文库可以反映小鼠胚胎在不同发育阶段整个基因群体的转录活性,为阶段特异性基因的筛选和小鼠早期阶段基因表达模式的研究提供了一种有价值的资源库 。     

C3d3通过促进脾脏B细胞高表达CXCR4增强hCGβ DNA免疫体液免疫效应

本文旨在探讨分子佐剂C3d3与hCGβ融合在基因免疫中增强抗hCGβ体液免疫效应的机制。分别用质粒pCMV4-hCGB-C3d3、pCMV4-hCGβ和pCMV4免疫BALB/c小鼠,间接ELISA法检测免疫小鼠外周血IgG/IgA类抗hCGβ抗体水平;ELISPOT分析免疫鼠脾脏组织IgG/IgA类抗体分泌细胞水平(ASC);RT-PCR分析免疫鼠脾脏B细胞趋化因子受体表达,RT-PCR和FCM分析CXCR4表达水平;RT-PCR和ELISA检测脾脏组织CXCL12表达水平。结果显示,pCMV4- hCGβ-C3d3免疫组外周血IgG类抗hCGβ抗体水平明显高于pCMV4-hCGβ免疫组;而IgA类抗hCGβ抗体水平在两组间无明显差异。pCMV4-hCGβ-C3d3免疫组脾脏组织IgG类ASCs水平明显高于pCMV4-hCGβ组;两组间IgA类ASCs水平无明显差异。经pCMV4-hCGB、pCMV4-hCGβ- C3d3免疫鼠脾脏B细胞CXCR4表达明显高于对照组;且pCMV4-hCGβ-C3d3组明显高于pCMV4-hCGβ免疫组。CXCR4~ 细胞与ASCs呈正相关,r=0.966,(P<0.05)。pCMV4-hCGβ-C3d3和pCMV4-hCGβ组脾脏组织CXC L12表达均显著高于对照组。结果表明,分子佐剂C3d3与hCGβ基因融合,在基因免疫小鼠后能够显著升调节脾脏ASCs CXCR4表达,从而可能增强抗hcGβ基因疫苗的体液免疫效应。     

昆明小鼠体内发育早期胚胎葡萄糖代谢关键酶转录产物的分析

首次报道了昆明小鼠体内发育的早期胚胎1-细胞至桑椹期阶段葡萄糖代谢的3种关键酶-6-磷酸葡萄糖脱氢酶（G6PDH）、6-磷酸果糖激酶（PFK）和磷酸葡萄糖变位酶（PGM）的基因转录情况,其分别体现了磷酸戊糖、糖酵解、糖原的合成和分解等途径,根据G6PDH、PFK、PGM的cDNA序列分别设计和合成3套共6对内、外引物,采用巢式RT-PCR方法对其进行检测。结果表明：早期胚胎1-8细胞阶段均有G6PDH基因的转录,叠椹期胚胎不存在该基因的转录,说明早期胚胎1-8细胞阶段可能存在磷酸戊糖,而桑椹期则不存在;1-细胞至桑椹期均存在PFK基因的转录,说明该阶段的胚胎可能存在糖酵解代谢途径;1-细胞至桑椹期均不存在PGM基因的转录,说明该阶段的胚胎可能不存在糖原的合成与分解代谢途径。     

小鼠2-细胞胚胎ATP合成酶6基因特异表达分析及鉴定

合子基因组活化是小鼠胚胎早期发育由细胞质调控向核调控转变的关键 .小鼠合子基因组活化发生在 2 细胞胚胎阶段 ,通过对 2 细胞胚胎阶段特异性表达基因的分析 ,可以从分子水平上揭示早期小鼠胚胎的发育机理 .用DD RTPCR技术 ,从单个小鼠 2 细胞胚胎与成熟卵母细胞 (MII细胞 )中分离了 2个差异片段 ,片段 2同小鼠睾丸中表达的一个未知片段具有高度同源性 .经过cDNA文库构建、筛选 ,分离到其全长cDNA .序列分析结果表明 ,该基因为小鼠ATP合成酶亚单位 6基因 .ATP合成酶亚单位 6基因由线粒体DNA编码 ,与细胞内ATP的合成相关 .小鼠 2 细胞胚胎特异表达的ATP合成酶亚单位 6基因可能与胚胎正常发育相关     

小鼠体外受精、胚胎培养及胚胎快速冷冻的研究

目的　为扩大胚胎来源并获取特定胚龄胚胎 ,建立小鼠冷冻胚胎库。方法　运用超数排卵、体外受精与胚胎培养及胚胎冷冻技术系统研究了小鼠受精卵的体内发育与运行规律。卵母细胞的体外成熟与受精、单细胞胚胎培养及胚胎快速冷冻。结果　 (1)注射hCG后 12～ 2 0h受精卵发育至原核期 ,4 2～ 4 8h为 2 细胞期 ,4 8～ 6 0h为 4 细胞期 ,6 0～ 6 8h为 8 细胞期 ,以上各期受精卵均处于输卵管中 ;75～ 78h为桑椹胚 ,78～ 80h为致密桑椹胚 ,90～ 92h为早期囊胚 ,92～ 96h为囊胚 ,以上各期均处于子宫角中。 (2 )培养液中添加促性腺激素 (FSH与hCG) ,能显著提高卵母细胞的体外受精率 ,添加FCS和激素组的体外受精率又显著高于单独添加激素组 ,FCS还能显著提高胚胎发育。 (3)在培养液中添加EDTA ,能有效克服小鼠胚胎的 2 细胞阻断 ,其 2 细胞胚的发育率达 10 0 % ,8 细胞胚发育率达 5 5 %以上 ;牛、羊上皮细胞培养液上清也能有效克服 2 细胞阻断。添加乳酸钠和丙酮酸钠可使 2细胞与 8细胞期胚的发育率显著提高。 (4)以D PBS +甘油 +蔗糖为冷冻液 ,以D PBS +蔗糖为稀释液 ,对小鼠胚胎进行快速冷冻 ,桑椹胚的存活率为 6 9 3% ,早期囊胚的存活率为 6 0 4 %。结论　研究为将生物技术应用于小鼠 ,扩大卵子和胚胎来源     

Ghrelin在绵羊体内卵母细胞和早期胚胎的表达

为了明确ghrelin是否参与了卵母细胞成熟及胚胎早期发育进程,本研究利用免疫荧光技术和实时定量RT-PCR技术检测了绵羊卵母细胞和体内早期胚胎中ghrelin蛋白的表达定位和ghrelin mRNA水平相对表达变化规律。免疫荧光染色结果表明,ghrelin蛋白主要分布于卵母细胞胞质内;实时定量RT-PCR结果揭示绵羊卵母细胞和早期胚胎ghrelin mRNA的相对表达量依据发育阶段的不同而呈现一定变化规律,即在成熟卵母细胞,2细胞胚胎期和8细胞胚胎期显著高于未成熟卵母细胞和4细胞胚胎期(P<0.05),囊胚期表达量最高。卵母细胞和早期胚胎中ghrelin蛋白的表达及ghrelin mRNA特定的表达模式,揭示这一新型分子在绵羊卵母细胞成熟以及胚胎早期发育过程中具有潜在的调控作用。     

胸腺肽-β4在小鼠卯母细胞和早期胚胎中的表达与分布规律

胸腺肽-β4(thymosin-β4,Tb4)是一种重要的G-actin遮蔽因子(G-actin sequestening factor),在细胞微丝活动中有着调节G-actin活性的作用.以往报道证实了Tb4在细胞中具有广泛的生理作用,但其在哺乳动物卵母细胞成熟和早期胚胎发育等方面的作用,迄今还没有系统的研究报道.以昆明白小鼠卵巢、卵母细胞和早期胚胎作为实验材料,以免疫(荧光)组织化学和RT-PCR技术为主要研究方法,对Tb4的表达与分布进行了研究.结果显示,Tb4在相关发育过程中,存在差异性的表达和定位变化.结果表明,在小鼠卵母细胞成熟和早期胚胎的发育过程,Tb4能够通过表达与分布的变化对细胞微丝活动和细胞增殖活动进行调控,对小鼠卵母细胞成熟、早期胚胎发育以及胚胎着床过程有重要的作用.     

人类21号染色体转录调节相关基因作为早期分子诊断标记物的可行性研究

目的分析与转录调节相关的人类21号染色体(HC21)基因在小鼠植入前胚胎发育过程中的表达模式,初步阐明这些基因与早期胚胎发育的关系,发现这些基因作为分子诊断标记物的可行性。方法应用植入前胚胎的GlobalRT-PCR方法,对BACH1、RUNX1、SIM-2、ERG、KIAA0136、GCFC、SON、PKNOX1、HSF2BP和NRIP1小鼠同源基因在植入前发育阶段的表达模式进行研究。结果RUNX1、ERG和SON在整个植入前发育过程中都未见表达。其他基因的表达呈现出阶段特异性表达的特点:BACH1几乎在整个发育过程中都有表达,但是存在着胚胎个体之间的差异,不适于作为分子标记物;SIM-2只在1和2细胞期表达;Kiaa0136在2、4细胞期以外的各个阶段均表达;除了1-和2-细胞期,GCFC在其它阶段普遍表达;PKNOX1只在1、8细胞以及桑椹期表达;而HSF2BP和NRIP1的表达仅见于成熟的卵母细胞和1细胞期胚胎。结论与转录相关的小鼠HC21同源基因大部分参与了早期胚胎发育的转录调节,这些基因的阶段特异性表达说明他们参与了早期胚胎发育的不同转录调节环节,对这些基因的深入研究是认识唐氏综合症发生的分子机制和发现早期分子诊断标记的基础。     

斑马鱼母源因子在胚胎发育中的作用

动物受精时,精子主要是将雄原核释放到卵子中,形成的合子中雌、雄原核融合为合子核,但受精卵基因组在前几次有丝分裂过程中不转录,合理的逻辑性推测是其早期发育完全依赖于卵质中储存的RNA和蛋白质,即母源因子.上世纪80年代对无脊椎动物的正向遗传研究发现,母源因子在卵子和胚胎极性的决定、早期胚胎的图式形成等方面发挥了决定性作用.过去10多年来,通过对斑马鱼和小鼠突变体的研究,也证明母源因子在脊椎动物胚胎早期发育中起着重要作用.本文主要综述斑马鱼母源因子在卵母细胞的极性、卵子的激活、早期细胞分裂、母源mRNA的清除、合子基因转录激活以及胚层的形成和分化、体轴的建立等方面的作用,相关知识对于研究人类生育障碍和先天性疾病的发生机制和诊治有借鉴意义.     

The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus—including deer mice—are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.     

Identification of male-specific amh duplication,sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus)

Background

The probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development.

Results

Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY ‘supermales’ resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20β-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10⁻⁹). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20β-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-β domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20β-hsd, down-regulated in males.

Conclusions

This study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-β domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-774) contains supplementary material, which is available to authorized users.     

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

Background and Aims

Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.

Methods

Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.

Key Results

In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.

Conclusions

The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.