In vitro development of yak (Bos grunniens) embryos generated by interspecies nuclear transfer

Interspecies cloning may be used as an effective method to conserve highly endangered species, but at present it suffers from relatively low levels of efficiency. In order to find a technique that could be used in conservation of the wild yak (Bos grunniens), we designed in six separate experiments to investigate the following factors that might influence the efficiency of interspecies cloning: (1) maturation rates of the recipient bovine oocytes; (2) nuclear donor cell types; (3) age of the yak from which the yak ear skin fibroblast cell line originated; (4) donor cells treated with or without serum starvation; (5) nuclear donor gained from fresh cells or frozen-thawed cells; (6) effect of 0.5 or 1.5 h from fusion to activation. The results of experiment 1 showed that when recipient oocytes in a replicate had a maturation rate of <40% (34+/-3.0%; three replicates) the proportion of nuclear transferred oocytes that developed to blastocyst was 2+/-1.1%, which was significantly lower (P<0.01) than the 25+/-3.2% achieved when the recipient oocyte maturation rate was 71+/-3.7% (three replicates). The efficiency of blastocyst production was increased substantially (P<0.05) when the time from fusion to activation increased from 0.5 h (21+/-2.3%; three replicates) to 1.5 h (35+/-3.5%; five replicates; experiment 6). There was no significant effect of the source of the donor nuclei (ear skin fibroblast or cumulus cells), the age of the animal (3 months or 4 years) from which the donor cells were derived, serum deprivation of the donor cells, or the use of fresh or frozen-thawed donor cells (experiments 2-5). Transfer of three interspecies cloned blastocysts to each of 108 bovine recipients resulted in two pregnancies being established that did not survive to day 120 of gestation.     

Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.     

Development of embryos after in vitro fertilization of bovine oocytes with sperm from either yaks (Bos grunniens) or cattle (Bos taurus)

The objectives of this study were to investigate differences in fertilization and development of embryos after in vitro fertilization of Bos taurus (cow) oocytes with sperm from either yaks (Bos grunniens) or Holstein bulls. Frozen-thawed spermatozoa (Holstein n=5 sires; yak n=5 sires) were evaluated for motility (forward progression) and acrosomal status immediately post-thaw and then 1, 2, 3, and 8h later. In vitro-matured cow oocytes (n=1652) were inseminated with either Holstein bull or yak spermatozoa and after an 18-h co-incubation period, a proportion of the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst production rates. Overall, there were species differences (P<0.05) and an effect of time (P<0.01) in sperm motility and acrosome integrity. An effect (P<0.01) of a species-by-time interaction was detected for motility, but not for acrosome integrity. The percentage of oocytes penetrated and the formation of two pronuclei when cow oocytes were inseminated with yak spermatozoa (97.4% and 81.6%, respectively) were greater (P<0.01) than that achieved with Holstein bull spermatozoa (77.8% and 65.9%, respectively), but the incidence of polyspermy (>2 pronuclei) was similar (P>0.05; 10.8% vs. 15.8%). The yak male symbolxcow combination gave a higher cleavage rate than the Holstein male symbolxcow combination (P<0.05; 76.3% vs. 63.3%), but there was no difference in the blastocyst rate (17.9% vs. 14.5%). It is concluded that yak spermatozoa could successfully fertilize cattle oocytes and their hybrid embryos had normal competence to develop to blastocysts.     

Polymorphism in mitochondrial DNA (mtDNA) of yak (Bos grunniens)

Mitochondrial DNAs (mtDNA) from 21 yaks (Bos grunniens) were assayed for restriction fragment length polymorphisms by using 20 restriction endonucleases, six of which (AvaI, AvaII, BglII, EcoRI, HindIII, and HpaI) detected polymorphism. Four different mtDNA haplotypes were identified. Combining this with previous reports about the mtDNA RFLPs of B. indicus and B. taurus, there are obvious differences in mtDNA polymorphism between the yak and other Bos species. We estimated that the divergence times between the ancestor of B. grunniens and the ancestor of B. taurus or B. indicus were about 1.2–2.2 and 1.01–2.02 million years ago, respectively.     

Skeletal muscle myosins from the yak (Bos grunniens), cattle (Bos taurus) and their hybrids

The skeletal muscle myosins of the yak (Bos grunniens), of cattle (Bos taurus) and of their first and second filial generation hybrids have been studied by ATPase measurements, fluorescence spectroscopy, near-ultraviolet circular dichroism and peptide mapping on polyacrylamide gels. The ATPase activities, the intrinsic tryptophan fluorescence enhancement upon addition of ATP and the circular dichroism spectra of the four myosins were closely comparable. Peptide maps of the myosin heavy chains indicate extensive sequence homologies but do reveal differences between the myosins of the yak and cattle.     

牦牛分子遗传多样性研究进展

遗传多样性研究可有效地揭示牦牛的遗传变异, 是牦牛群体遗传学研究的主要内容之一。自20世纪70年代以来, 人们已对牦牛的体形外貌特征、染色体核型(带型)、生理生化特性和DNA序列变异等进行了较为深入地研究。随着分子遗传学和DNA测序技术的迅猛发展, 近年来的研究主要集中在牦牛的分子遗传多样性。文章对近15年来牦牛mtDNA和核基因组分子标记及侯选基因多样性的研究现状进行了综述, 对前景进行展望, 以期为牦牛群体基因组学等研究提供依据。     

牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化

采集成年母牦牛卵巢,通过光镜和电镜对牦牛卵泡及其卵母细胞不同发育时期的结构变化进行了观察。结果发现当卵母细胞被单层立方卵泡细胞包围时,微绒毛开始出现,而皮质颗粒、透明带则在包被2-4层卵泡细胞时开始出现。随着卵母细胞的继续发育,透明带增厚,微绒毛由粗短变为细长,密度增加;皮质颗粒、线粒体、滑面内质网等细胞器的数目不断增加,并逐渐移行到质膜下;在移行的过程中,皮质颗粒成团存在。在囊状卵泡中,卵母细胞皮质颗粒呈线形分布于质膜下,线粒体、滑面内质网又移向胞质中央。卵母细胞借助微绒毛穿过透明带与卵泡细胞胞质突起相联系。结果证明牦牛卵泡和卵母细胞不同发育时期的结构变化与其它哺乳动物的基本相似。     

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.     

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) was estimated by 16S rDNA homology analysis. Two rumen 16S rDNA libraries were constructed. Of the 194 clones in the library of yak rumen, the sequences were mainly clustered to two phyla, low G+C Gram-positive bacteria (LGCGPB, 54.12% total clones) and Bacteroidetes (30.93%), respectively. While in the 197 clone-library of the cattle rumen, the sequences were mainly related to three phyla, Bacteroidetes (39.59%), gamma-Proteobacteria (26.9%) and LGCGPB (22.34%), respectively. The sequence analysis indicated that more than half of the species harbored in yak rumen belonged to the not-yet-cultured groups at <90% 16S rDNA similarity levels with cultured species, while 36% 16S rDNA sequences amplified from the rumen of Jinnan cattle fell in these catalogues. By comparing the uncultured sequences in yak rumen with those in Jinnan cattle and cow, the former formed distinct clusters loosely related to the later, implying that yak rumen could harbor some special prokaryote phyla. 10.8% sequences retrieved in yak rumen were related to the known rumen fibrolytic bacterial species; however none was related to the known amylolysis species. While 4% and 17.8% sequences retrieved from Jinnan cattle rumen were related to cultured fibrolytic and amylolysis species, respectively. The bacterial structures seemed to be in accordance with the feed of the two kinds of animals. In both rumens, retrieved methanogenic Archaea-related 16S rDNA sequences were at an unreasonable low level; in addition, none sequence was related to Ruminococcus albus, a classical rumen fibrolytic species. The reason can be due to the experimental biases.     

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.     

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and fi0 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of thedonor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES ceils maintainthe capability of sustained growth in an undifferen tiated state, and form embryoid bodies, which, on furtherinduction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that expressmarkers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NTto rabbit eggs retain phenotypes similar to those of conventional human ES ceils, including the ability toundergo multilineage cellular differentiation.     

天祝白牦牛OXGR1基因多态性与体尺性状的关联性分析

为了研究牦牛α-酮戊二酸（盐）受体1（Oxoglutarate receptor1, OXGR1）基因多态性与其体尺性状的相关性,本文以4-8岁天祝雄性（阉）白牦牛的血液DNA（n=192）为实验材料并构建DNA混合池。通过DNA直接测序法检测OXGR1的核苷酸序列潜在的多态位点;利用高分辨率熔解曲线分析技术 (High Resolution Melting,HRM ) 进行分型。采用SHESIS软件对OXGR1基因多态位点进行连锁不平衡分析;运用 PIC-Calc 0.6软件分析多态信息含量;采用卡方检验检测 Hardy- Weinberg平衡;运用SPSS20.0软件对多态位点与体尺性状进行关联分析;运用RNAFOLD、ExPASy和Swiss-model软件对OXGR1基因突变前后的蛋白结构进行预测分析。结果表明：天祝白牦牛OXGR1基因发现有两个多态位点,分别为347(A/G)和678(G/A),每个位点均有3种基因型（AA、AG、和GG）,其优势基因型分别为GG和AA。两个SNPs均达到Hardy-Weinberg 平衡（P＞0.05）,存在强连锁不平衡（D’＞0.75, R²＞0.33）,且均表现为中度多态（0.25< PIC< 0.5）。上述位点不同基因型在体斜长、体高、胸围和管围存在显著差异（P＜0.05）。分子结构预测显示：347 位点处突变为错义突变,其编码的氨基酸由天冬酰胺变为丝氨酸。突变后OXGR1 mRNA二级结构、蛋白质二级结构及三级结构均发生改变。上述结果表明: OXGR1基因可作为牦牛分子育种的候选分子标记,为今后牦牛遗传资源的保护、开发以及新品种的选育提供依据。     

野牦牛（Bos grunniens mutus）mtDNA D\|Loop区的遗传多样性

为从分子水平上评价野牦牛(Bos grunniens mutus)的遗传多样性,对6头野牦公牛线粒体DNA D-loop 区全序列进行了克隆和测定(GenBank登录号为:FJ548840～FJ548845),结合GenBank中已刊登的2条野牦牛的相应序列,使用BioEdit 7 0 9、DnaSP 4.10.1、Arlequin 3.11等生物信息学软件,对全部8条序列开展了比对分析,确定了多态位点与单倍型数目,计算了核苷酸多样度和单倍型多样度.结果表明8条野牦牛线粒体DNA D-loop 区全序列长度在891～894 bp之间,其中A、T、G和C 4种核苷酸的平均含量分别为32.68%、28.65%、13.46%和25.21%,A+T的平均含量为61.33%.排除4处核苷酸的插入或缺失后,共检测到39处转换和颠换位点,占分析序列总长度的4.38%,其中包括4处单一多态位点和35处简约信息位点.依据序列间核苷酸变异共确定了7种单倍型,单倍型多样度(h)为0.9643,核苷酸多样度(π)为0.02144,提示野牦牛群体具有丰富的遗传多样性.     

Production of cloned bovine embryos by somatic cell transfer into enucleated zona-free oocytes

Cloned bovine embryos were produced at the blastocyst stage. Prior to enucleation, oocytes were freed from the zona pellucida. Fibroblasts isolated from the bovine fetus were used as nuclear donors. Pairs of fetal fibroblasts and enucleated oocytes (cytoplasts) were glued in phytohemagglutinin solution under a binocular microscope. The subsequent electrofusion of 39 fetal fibroblast-cytoplast pairs yielded 36 reconstructed one-cell embryos (92.3%). After culturing in synthetic oviduct fluid for 7.5 days, seven cloned embryos developed to the blastocyst stage (19.4%) and six blastocysts were considered fit for transplantation. The applied technique of bovine embryo growth allowed 31.1% zona-free oocytes parthenogenetically activated by to reach the blastocyst stage.     

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens)

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD⁴⁺ T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d _S) and non-synonymous (d _N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d _N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.     

牦牛DBI基因的分子特征及其在乳腺组织中的表达与定位

地西泮结合抑制因子(Diazepam binding inhibitor,DBI)与酰基辅酶A具有高亲和力,在动物组织中广泛存在,与脂肪酸代谢、类固醇激素合成密切相关。为研究DBI基因的分子特征及该基因在乳腺发育中的作用,对牦牛DBI基因编码区进行克隆,进行生物信息学分析;采用实时荧光定量PCR (Quantitative real-time PCR,qPCR)、蛋白免疫印迹技术(Western blotting,WB)和免疫组织化学(Immunohistochemistry,IHC)方法对牦牛泌乳前期、泌乳期和干乳期的乳腺组织中DBI的相对表达量和表达部位进行研究。DBI序列分析显示：牦牛DBI基因编码区序列长264 bp,编码87个氨基酸残基,与牛的同源性高达99.62％;qPCR数据表明：牦牛泌乳前期乳腺组织中DBI基因的相对表达量显著高于泌乳期和干乳期(P< 0.05);WB结果显示：牦牛泌乳前期乳腺组织中DBI蛋白的表达量最高,干乳期次之,泌乳期最低(P< 0.05);IHC结果表明：不同发育时期的牦牛乳腺组织中DBI的表达部位并无明显差异,主要表达于乳腺腺泡上皮细胞、导管上皮细胞及小叶间质细胞。DBI在不同发育时期牦牛乳腺组织中的相对表达量具有明显差异(P< 0.05),揭示DBI可能参与牦牛乳腺发育的过程,这为进一步探究DBI基因在生物体中的作用提供相应的理论参考。