Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS‐PCR). The expression levels of these imprinted genes were also investigated by quantitative real‐time PCR (QRT‐PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Attribute selection and model evaluation for the maternal and paternal imprinted genes in bovine (Bos Taurus) using supervised machine learning algorithms

Imprinted genes display biased expression of paternal and maternal alleles in mammals. They are marked through epigenetic process during gametogenesis. Characterization of imprinted genes has expanded our understanding of the regulation and function of genes. In the current study, 22 experimentally validated imprinted genes in bovine (Bos Taurus) were analysed. Several supervised machine learning algorithms and attribute weighting methods were used to find characteristics of different types of imprinted genes and suggest a classification method for finding maternally and paternally expressed genes in bovine. For assessing the best model and comparing attributes in other organisms, we have also conducted a comparative analysis for human and sheep imprinted genes. According to the results of the present study, GC contents 10 and 100 kb upstream, Gly and Gln amino acids, Ile/ATC codon usage, LINE and SINE in 100kbup and length of first intron were significantly different between the maternal and paternal genes in cattle. Considering all species together, we found that GC content 100 kb up, LINE 100 kb up and the frequency of amino acids like Gly, Gln and Met were the most important attributes for identifying the paternal and maternal imprinted genes. These findings could imply conservation pattern in the attributes among these species.     

Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.     

Genetic variation in monoamine oxidase A and analysis of association with behaviour traits in beef cattle

Husbandry of beef cattle requires animals that do not behave aggressively or timidly. The enzyme monoamine oxidase A and the coding gene (MAOA) play an important role in the complex regulation of behaviour. The complete coding region and a part of the non‐coding sequence of the bovine MAOA gene have been analysed in 20 German Angus and 20 German Simmental bulls and cows with the aim of detecting genetic variability. These two cattle breeds are known to differ regarding their behaviour during handling. Five single nucleotide polymorphisms (SNPs) were identified, three of which were found in the coding region of the gene (exons III and XV). One of the SNPs located in exon XV ( NC_007331.3 :g.80340C>T) was found to be a non‐synonymous mutation. The minor allele frequency of this resulting amino acid substitution was significantly different between 543 German Angus and 417 German Simmental calves (0.39 and 0.49, respectively). The potential functional impact of this polymorphism has been tested by in silico analysis, as well as by association analysis using behaviour scores of the genotyped calves for three behaviour tests that assessed the animals’ temperament during tethering, weighing or social separation. In silico analysis did not deliver consistent results arguing for or against a functional impact of the studied amino acid substitution on the function of the biological protein. No significant association was found between this MAOA polymorphism and the behaviour‐related scores analysed in the study.     

哺乳动物体细胞核移植胚胎发育中表观重编程的研究进展

体细胞核移植（somatic cell nuclear transfer,SCNT）是一种能将已分化的体细胞重编程为全能胚胎的繁殖生物技术,在良种扩繁、濒危物种保护和治疗性克隆等方面有着广泛的应用前景,但极低的克隆效率、克隆动物胎盘异常、出生后胎儿畸形等严重限制了该技术的实际应用。造成克隆效率低和胚胎发育异常的主要原因是供体核表观遗传重编程错误或不完全。1958年,将非洲爪蟾（Xenopus laevis）幼体肠细胞核移入去核卵母细胞,获得了第1例SCNT动物个体;1986年,通过电融合1个卵裂球与去核卵母细胞成功获得了3只存活的羔羊;1997年,将成年母羊的乳腺上皮细胞与去核卵细胞电融合,获得首个SCNT哺乳动物"多利",开启了克隆时代,目前牛、小鼠、山羊、猪、欧洲盘羊、家兔、家猫、马、大鼠、骡子、狗、雪貂、狼、水牛、红鹿、单峰骆驼、食蟹猴等相继成功克隆,其中最引人瞩目的是2018年食蟹猴的成功克隆。作者通过将SCNT胚胎与受精胚胎的发育进行对比,阐述了SCNT过程中DNA甲基化、组蛋白修饰、基因组印迹、染色体状态等的重编程过程和缺陷,并从表观修饰剂、组蛋白去甲基化酶、抑制Xist表达、补充鱼精蛋白和精子RNA方面探讨单独或联合消除表观遗传重编程障碍对克隆效率的影响。随着低样本量测序技术的发展和完善,人们能够在SCNT胚胎中检测到更详细的全基因组表观遗传修饰图谱,进一步揭示SCNT胚胎表观遗传重编程中的缺陷,为提高克隆效率提供了线索。通过上述内容的阐述,希望为后续开发联合消除多种表观遗传障碍而提高克隆效率的策略和思路。     

Manipulation of Domestic Animal Embryos and Implications for Development

Assisted reproductive technologies, as applied to domestic animals, can exert both novel and wide‐ranging influences on the development, viability and welfare of offspring. Some of the changes are evident immediately or soon after the time at which a manipulative procedure is carried out, while other changes may not be evident until later in development or, perhaps, may remain undetected throughout an animal's lifetime. The present review explores some of the consequences – in terms of foetal, placental, neonatal and post‐natal effects – of exposing embryos of cattle, sheep and other species to in vitro culture per se or, during culture, to physically invasive technologies including gene injection and nuclear transfer. The innate sensitivity of oocytes and recently fertilized eggs to their in vitro environment is illustrated by an examination of the later developmental repercussions resulting from apparently innocuous choices related to in vitro culture medium formulations. In contrast, an inherent resilience and paradoxical readiness to resume development following the traumas of nuclear transfer procedures is also in evidence. The extent to which assisted reproductive technologies will succeed, where relevant, in the domestic animal sector will be influenced by our appreciation of embryo requirements, for both short‐ and long‐term developmental fitness, during their earliest developmental stages. Evidence of species‐specific needs is testimony to the challenges ahead. Ultimately, our ability and inclination to resolve the limitations associated with current procedures will probably be greatly enhanced if predictive indicators (genetic, epigenetic or functional markers) of later developmental fitness can be identified.     

Effect of Reproductive Seasonality on Gamete Quality in the North American Bison (Bison bison bison)

The objective was to investigate the effects of reproductive seasonality on gamete quality in plains bison (Bison bison bison). Epididymal sperm (n = 61 per season), collected during the breeding season (July–September), had significantly higher post‐thaw total motility (36.76 ± 14.18 vs 31.24 ± 12.74%), and lower linearity (0.36 ± 0.06 vs 0.39 ± 0.04) and wobbliness (0.49 ± 0.04 vs 0.51 ± 0.03; mean ± SD) compared to non‐breeding season (January–March) samples. Representative samples (n = 4) from each season were used in heterologous IVF trials using cattle oocytes. Cleavage, morulae and blastocyst percentage were higher for breeding vs non‐breeding season sperm samples (81.88 ± 6.8 vs 49.94 ± 6.77; 41.89 ± 13.40 vs 27.08 ± 23.21; and 30.49 ± 17.87 vs 13.72 ± 18.98%, respectively). Plains bison ovaries collected during the breeding (n = 97 pairs) and non‐breeding (n = 100 pairs) seasons were classified as luteal or follicular. Oocytes recovered from these ovaries were classified into five grades based on morphology. There was no significant difference in the number of luteal ovaries or grades of oocytes recovered. Oocytes were matured, fertilized (with frozen sperm from three bison bulls) and cultured in vitro. Cleavage percentage was higher for oocytes collected during breeding vs non‐breeding season (83.72 ± 6.42 vs 73.98 ± 6.43), with no significant difference in subsequent development to blastocysts. In summary, epididymal sperm from non‐breeding season had decreased total motility and resulted in reduced embryo production in vitro. Oocytes collected during non‐breeding season had reduced ability to be matured, fertilized and/or undergo cleavage in vitro. Data suggested that season influenced gamete quality in plains bison.     

Differential promoter methylation of DAZL gene in bulls with varying seminal parameters

In India, cross‐breeding of indigenous cattle with exotic cattle such as Holstein Friesian and Jersey has been going on since last four decades to improve milk production. Although it has led to increased milk yield, the subfertility in male cross‐bred progeny has remained a significant problem. Epigenetic modifications (DNA methylation, histone modifications and chromatin remodelling) are regarded as key players influencing gene expression. DAZL gene plays an important role in germline development and gametogenesis. The methylation and mRNA expression level of this gene have been significantly negatively correlated in the testes of cattle–yak hybrids and their parents. This study analysed the methylation profile of DAZL gene promoter in bull spermatozoa in an attempt to speculate its role in cross‐bred cattle subfertility. Semen samples from Sahiwal, Holstein Friesian and Frieswal bulls (Sahiwal X Holstein Friesian) with varying semen motility parameters were collected, and DNA was isolated. Methylation‐specific primers were used to amplify part of promoter and exon 1 of DAZL gene using bisulphite‐converted DNA. The amplified products were sequenced after cloning in pTZ57R/T vector. Sequence analysis revealed significantly higher DNA methylation of DAZL gene in Frieswal bulls with poor motility (28.26%) as compared to medium (15.21%) and high motility phenotype (6.52%). In pure‐bred counterparts, Sahiwal and Holstein Friesian, epigenetic marks were more in the former (15.21%) than the latter (4.34%), but in both cases, the values were lower as compared to the poor motility Frieswal bulls. This suggests that differential hypermethylation of the CpG islands could possibly influence reproductive parameters in bovines.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co‐culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co‐cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non‐co‐cultured somatic cell nuclear transfer (SCNT‐DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT‐DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation‐related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT‐DO group. Similarly, while the mRNA levels of the deacetylation‐related genes HDAC2 and HDAC3 were significantly higher in the SCNT‐DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co‐culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.     

Identification of sperm motility markers in bovine transition protein genes

Transition proteins (TNPs) are essential in chromatin condensation during spermiogenesis, and hence, they are the candidate genes for identifying sperm motility markers. Coding and in silico predicted promoter regions of these genes were investigated in crossbred and purebred cattle, and also, their mRNA quantification was done to explore its use as a diagnostic tool of infertility. PCR‐SSCP analysis revealed two band patterns in fragment III of TNP1 and fragment II of TNP2 gene. Sequence analysis revealed a deletion of “G” nucleotide in 3′UTR region of TNP1 and C>T SNP in intronic region of TNP2 gene. Least square analysis of variance did not reveal any significant influence of nucleotide deletion on any sperm motility parameters in both crossbred and purebred cattle. However, C>T SNP had a significant effect on initial progressive motility (p < 0.05) in purebred cattle and post‐thaw motility in overall cattle population. RT‐qPCR analysis did not reveal any significant variation in TNP1 and TNP2 gene expression among poorly motile and good quality spermatozoa of Vrindavani bulls.     

WT1基因组蛋白乙酰化修饰对细胞核重编程的影响

利用体细胞移植技术获得克隆动物的成功是几十年来生命科学领域取得的重大突破之一,这项技术引起了社会的广泛关注。然而,由于哺乳动物克隆效率低下,且克隆后代发育异常等问题,已成为目前制约动物克隆技术发展和应用的瓶颈。克隆动物中经常出现后代过大综合征（LOS）,该病导致克隆动物早产、难产和易夭折。LOS类似于人的伯-伟综合征（BWS）,BWS也称为Wlims瘤,表现为巨舌、内脏肿大等症状。研究发现BWS的发病机理与WT1基因（Wilms’tumor 1gene）异常表达有关。本文对体细胞核重编程和表观遗传学调控细胞重编程的研究进展进行综述,并对WT1基因组蛋白乙酰化修饰与体细胞重编程之间的联系进行简要介绍,以期为生命科学领域的进一步探索与研究提供借鉴。