Vitamin C treatment of embryos,but not donor cells,improves the cloned embryonic development in sheep

Vitamin C is not only an antioxidant but also a regulator of epigenetic modifications that can enhance the activity of the ten-eleven translocation (TET) family dioxygenases and promote the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we investigated the effects of vitamin C in regulating DNA methylation in sheep somatic cells or embryos in an effort to improve the cloned embryo development. Vitamin C treatment of sheep foetal fibroblast cells significantly increased the 5hmC levels but did not affect the 5mC levels in cells. After nuclear transfer, vitamin C-treated donor cells could not support a higher blastocyst development rate than non-treated cells. Although combination of serum starvation and vitamin C treatment could induce significant 5mC decrease in donor cells, it failed to promote the development of resultant cloned embryos. When cloned embryos were directly treated with vitamin C, the pre-implantation development of embryos and the 5hmC levels in blastocysts were significantly improved. This beneficial role of vitamin C on embryo development was also observed in fertilized embryos. Our results suggest that vitamin C treatment of the embryos, but not the donor cells, can improve the development of cloned sheep embryos.     

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5‐methylcytosine‐5mC and 5‐hydroxymethylcytosine‐5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT‐♀ and SCNT‐♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT‐♀ embryos was greater than that of SCNT‐♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT‐♀ embryos, whereas 5hmC was majorly expressed in SCNT‐♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT‐♂ embryos were higher than those of SCNT‐♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight‐stage of the IVF, SCNT‐♀ and SCNT‐♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT‐♂ embryos at the eight‐cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two‐cell, eight‐cell and blastocysts of SCNT‐♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT‐♀ embryos than that of SCNT‐♂ embryos.     

5-甲基胞嘧啶到5-羟甲基胞嘧啶的转变过程参与小鼠雄原核的DNA主动去甲基化

利用5-甲基胞嘧啶（5-methylcytosine，5mC）和5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5-hrnC）特异性抗体对小鼠原核时期胚胎进行免疫荧光染色。同时使用荧光定量PCR方法检测Tet（Ten eleven translocation）基因在小鼠早期胚胎中的表达。免疫荧光染色结果显示早期原核阶段（PN1到PN3），雄原核中5mC的含量逐渐减少，而5hmC的含量逐渐增加。但是雌原核中5mC和5hmC的含量基本不变。在原核后期阶段（PN4到PN5）5hmC主要存在于雄原核中。荧光定量结果显示在小鼠MⅡ卵母细胞和植入前胚胎中Tet 1和Tet 2基因表达量较低，但是Tet 3在卵母细胞和原核时期表达量较高，随着胚胎的发育其表达量逐渐降低。结果表明，在小鼠原核时期阶段5mC到5hmC的转变过程主要是由TET3蛋白催化的，并且此过程参与小鼠雄原核的DNA主动去甲基化。     

TET介导的DNA主动去甲基化的机制与功能研究进展

TET蛋白是一种α-酮戊二酸/Fe²⁺依赖的双加氧酶家族，可以氧化5-甲基胞嘧啶（5mC）产生5-羟基甲基胞嘧啶（5hmC）、5-甲酰基胞嘧啶（5fC）和5-羧基胞嘧啶（5caC）。TET蛋白在DNA去甲基化过程中发挥关键作用，并参与哺乳动物早期发育过程。现在被广泛认可的一种途径是TET蛋白氧化5mC，接着由胸腺嘧啶糖苷酶（thymine DNA glycosylase，TDG）氧化5fC、5caC，且TDG更易切割5caC，最后经过碱基切除修复得到未被修饰的胞嘧啶，达到去甲基化的目的。去甲基化过程中调控方式主要包括调节TET蛋白水平和调节代谢产物及辅助因子。作者主要对胚胎发育前后去甲基化的作用进行了阐述。     

DNA甲基化酶抑制剂5-Aza-CdR对多能基因及甲基化相关基因在徒手克隆胚胎中表达模式的影响

本试验通过实时荧光定量PCR方法对多能基因Oct4和Nanog及DNA甲基化相关基因Dnmt1和Tets在徒手克隆（handmade cloning,HMC）胚胎中的表达模式进行初步研究,并探讨5-Aza-CdR处理重构胚对这些基因表达模式的影响。结果显示,Oct4、Nanog和Tet3的表达在2细胞时期达到顶峰,Dnmt1和Tet2基因的表达随HMC胚胎发育而下降,而Tet1基因随HMC胚胎发育表达上升。使用5-Aza-CdR处理重构胚没有改变Oct4、Tet1和Tet3基因的表达模式,使Nanog基因在胚胎发育初期表达增加,Dnmt1和Tet2基因在胚胎发育初期表达降低。研究初步确立了Oct4、Nanog、Dnmt1和Tets基因在HMC胚胎的表达模式,5-Aza-CdR对重构胚的处理可对HMC胚胎的甲基化模式产生影响。     

Effect of DNA Methylation Inhibitor 5-Aza-CdR on Patterns of Pluripotent-related Genes and Methylation Genes in Handmade Cloning Embryo

The expression pattern of pluripotent gene Oct4 and Nanog,and methylation related genes Dnmt1 and Tets of handmade cloning (HMC) embryos were studied by Real-time quantitative PCR assay.The effect of genes expression pattern by 5-Aza-CdR on HMC reconstructed embryos was also explored. The results showed that the expression of Oct4,Nanog and Tet3 genes reached peak on 2-cell stage,the expression of Dnmt1 and Tet2 genes declined with the embryo development,while the expression of Tet1 gene increased.The use of 5-Aza-CdR didn't change the expression pattern of Oct4,Tet1 and Tet3 genes,but increased the expression of Nanog gene at the beginning of embryo development,while decreased the expression of Dnmt1 and Tet2 genes.The expression pattern of Oct4,Nanog,Dnmt1 and Tets genes in the development of HMC embryo was established,the use of 5-Aza-CdR could influence the methylation process of HMC embryo.     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

产蛋前期和产蛋高峰期鸡全基因组甲基化差异及其对肝脏转录组影响的研究

旨在通过对产蛋前期和产蛋高峰期鸡肝全基因组甲基化差异进行分析,解析基因组甲基化对不同发育阶段肝中基因表达差异的影响。本研究采用全基因组重亚硫酸盐测序（WGBS）技术对产蛋前期（20周龄）和产蛋高峰期（30周龄,各3只DNA混池）卢氏绿壳蛋鸡肝全基因组的甲基化水平进行检测,并与已有的肝mRNA转录组数据进行整合分析,探讨基因组甲基化对不同生理阶段基因表达差异的影响。结果表明,全基因组范围约有4%的胞嘧啶（C）发生了甲基化（mC）;两个生理阶段的总体甲基化水平基本一致。共检测到670个差异甲基化区域（DMRs）和356个差异甲基化基因（DMGs）。基因本体（GO）和相关信号通路（KEGG）分析发现,超甲基化DMGs显著富集在发育的正向调控、细胞形态改变的调控、VEGF信号通路、肌动蛋白细胞骨架的调控、粘着斑及间隙连接等相关过程,低甲基化DMGs显著富集在胚胎消化道形态的发生、间充质细胞增殖的正向调控、淀粉和蔗糖代谢及Wnt信号通路等相关过程。基因不同功能区域甲基化水平与基因表达水平有关,启动子（promoter）及基因体（gene body）区域甲基化水平与基因表达水平呈显著负相关,其他区域（内含子、3'UTR）的甲基化水平与基因的表达水平无明显关系。其中,与肝脂质代谢相关的候选基因RASD1、HAO1、UBE2O、MSRB3受甲基化调控。本研究绘制了不同生理时期卢氏绿壳蛋鸡全基因组甲基化图谱,结合mRNA转录组数据阐述了DNA甲基化在基因表达方面的调控作用,并鉴定出了不同生理时期受甲基化调控的基因,为深入研究表观遗传调控在不同生理时期蛋鸡肝代谢中的作用机制提供参考。     

Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi

C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990bp, containing a 71bp 5'-untranslated region (UTR), an open reading frame (ORF) of 723bp, and a 196bp long 3'-UTR. mC1qL2 cDNA was 1193bp, containing a 100bp 5'-UTR, followed by an ORF of 756bp and a 3'-UTR of 337bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed (p<0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection.     

Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor

B-cell activating factor (BAFF), belonging to the TNF family, is critical for B cell survival and maturation. cDNA of goose BAFF (gBAFF) was amplified from goose spleen by RT-PCR. The open reading frame (ORF) of gBAFF encodes a protein of 288-amino acid. The gBAFF shows 98, 92, 44 and 55% amino acid sequence identity with duck (dBAFF), chicken (cBAFF), mouse (mBAFF) and human BAFF (hBAFF), respectively. RT-PCR results showed that gBAFF mRNA is expressed in thymus and more highly expressed in the bursa of Fabricius and spleen. Recombinant soluble gBAFF (gsBAFF) expressed in Escherichia coli has molecular weight of approximately 19kDa. In vitro, purified gsBAFF was able to promote bursa B cells survival/proliferation in goose, duck and chicken. Furthermore, recombinant dsBAFF and csBAFF have a positive effect on goose, duck and chicken bursa B cells survival/proliferation. These findings indicate that gBAFF plays an important role in the survival/proliferation of goose B cells and, owing to its high evolutionary conservation, functional cross-reactivity exists between chicken, duck and goose BAFF.     

Functional restoration of the bursa of Fabricius following in ovo infectious bursal disease vaccination

The primary role of the avian bursa of Fabricius is to provide an essential microenvironment for B-lymphocytes to diversify their immunoglobulin genes by gene hyperconversion. Infectious bursal disease (IBD) vaccination using intermediate plus vaccine strains can temporarily deplete the bursal follicles and interrupt the normal B-cell development, which is generally followed by B-cell repopulation and histological regeneration. To find evidence that functional restoration of the bursa of Fabricius occurs in addition to the histological regeneration, we have analysed the chB1 gene expression, which indicates active bursal B-lymphocytes, and also the surface expression of a carbohydrate structure Lewis(x), a marker which identifies those bursal B-lymphocytes that are undergoing gene hyperconversion. In ovo vaccination with an immune complex vaccine (IBDV-BDA) caused transient bursal destruction in both the SPF and the maternally protected broiler groups with differences evident in the starting time, the severity and the duration of the effect. After the depletion phase, signs of histological regeneration appeared together with chB1- and Lewis(x) expression indicating that B-lymphocytes were functionally active and the bursa of Fabricius was serving again as an efficient primary lymphoid organ providing an appropriate microenvironment for B-cell development.     

Avian B cell development: lessons from transgenic models

The bursa of Fabricius is critical for the development of B lymphocytes in avian species. Despite considerable advances in our understanding of the molecular mechanisms by which avian antibody diversity is generated, many stages of B-cell development in the bursa and the means by which they are regulated remain unclear. Here we discuss the use of productive chicken retroviral vectors which allow gene transfer in vitro or in vivo as tools to probe the requirements for bursal B-cell development. Expression of a truncated form of bursal cell surface IgM, lacking variable region encoded determinants, is sufficient to promote the initial colonization and clonal expansion of B-cells within the bursa. Expression of this truncated IgM does not, however, protect developing bursal cells against the apoptosis that occurs within the bursa after hatch. Conversely, over-expression of the proto-oncogene bcl-2, following retroviral gene transfer, protects cells against apoptotic cell death but is not sufficient to allow B lineage progression in the absence of sIgM expression. Finally we discuss the use of regulated promoters within the retroviral gene transfer system to show that while bursal cells are susceptible to transformation by the v-rel oncogene in vitro, this oncogene preferentially targets mature peripheral cells in vivo.     

New Insight into the Structure,Development, Functions and Popular Disorders of Bursa Fabricii

Humoral immune responses in birds, contrary to mammals, depend on the normal functioning of bursa Fabricii. Recent studies have delivered new information about the structure, development and origin of cells that compose the bursa environment. Several viral infections affect bursa, causing lymphocyte depletion or excessive proliferation. This review summarizes data on the development and histology of healthy bursa and introduces some common disorders that affect this organ.     

Short-term exposure to subacute doses of aflatoxin-induced depressed mitogen responses in young mallard ducks

Mallard ducklings were fed diets containing corn naturally contaminated with mixed aflatoxins, purified T-2 toxin, or no detectable mycotoxin in two trials. The aflatoxin level used was 12 ppb in the first trial and 33 ppb in the second. T-2 was added at 2 ppm in both trials. No pathology was associated with the aflatoxin used in this study, and T-2--induced lesions were described in a previous publication. The weights of primary (thymus and bursa of Fabricius) and secondary (spleen) lymphoid organs were significantly reduced in the T-2--treated birds. The total number of viable cells recovered from the thymus was significantly reduced in aflatoxin-treated birds. The numbers of viable cells recovered from thymus, bursa of Fabricius, and spleen were all significantly reduced after treatment with T-2. In each trial, significantly lower mitogenic responses were seen to pokeweed mitogen and concanavalin A in birds fed aflatoxin or T-2, representing reduction in both B-cell and T-cell mitogenesis. Birds fed aflatoxin also had significantly reduced Escherichia coli O55 lipopolysaccharide-induced mitogenic responses. These studies indicate that subacute oral exposure to aflatoxin caused a loss of normal lymphocyte reactivity in mallard ducklings. This finding supports the hypothesis that waterfowl that ingest even small quantities of mycotoxin-contaminated waste grain are likely to be more susceptible to bacterial or viral infections.     

Review of studies on the immunological capacity in the bursectomized chick

This paper summarizes data on the immunological capacity in the bursectomized chick. A series of experiments described by Glick and Sadler represented the functional importance of the bursa of Fabricius for the humoral immunity in chicken. Later studies of immune responses in bursaless chickens reported by Lerner et al. contributed to our knowledge of bursa-independent humoral immunity and demonstrated an extra-bursal site for B-cell differentiation. Bursectomy at an early stage of chicken development changes the immune responses after hatching. Here I present my current understanding of embryonic B-cell populations (bursa-dependent and independent) following in ovo bursectomy which may influence B-cell differentiation with reference to our experiments on J chain production.     

Atrophy of the primary lymphoid organs of meat pigeons in Italy associated with circoviruslike particles in the bursa of Fabricius

During a survey effected in a meat pigeon slaughterhouse of central Italy, atrophy of primary lymphoid organs (bursa of Fabricius and thymus) and hypoplasia of bone marrow were observed. Histologic, ultrastructural, and hematologic examinations were performed on a total of 80 randomly selected 30-day-old meat pigeons. By histologic studies, lymphocytic depletion and necrosis with cyst formations in the bursa of Fabricius were detected in all subjects that showed thymus and bursa atrophy at necropsy. Basophilic intranuclear inclusions were also observed in bursal cells. After ultrastructural studies, these inclusions were proved to be viral particles resembling circoviruslike particles in morphology and size. Severe lymphocytic depletion of the bursa was plausibly associated with the presence of the viral particles.