Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.     

Differential activity of maternally and paternally derived chromosome regions in mice

Although both parental sexes contribute equivalent genetic information to the zygote, in mammals this information is not necessarily functionally equivalent. Diploid parthenotes possessing two maternal genomes are generally inviable, embryos possessing two paternal genomes in man may form hydatidiform moles, and nuclear transplantation experiments in mice have shown that both parental genomes are necessary for complete embryogenesis. Not all of the genome is involved in these parental effects, however, because zygotes with maternal or paternal disomy for chromosomes 1, 4, 5, 9, 13, 14 and 15 of the mouse survive normally. On the other hand, only the maternal X chromosome is active in mouse extraembryonic membranes, maternal disomy 6 is lethal, while non-complementation of maternal duplication/paternal deficiency or its reciprocal for regions of chromosome 2, 8 and 17 has been recognized. We report that animals with maternal duplication/paternal deficiency and its reciprocal for each of two particular chromosome regions show anomalous phenotypes which depart from normal in opposite directions, suggesting a differential functioning of gene loci within these regions. A further example of non-complementation lethality is also reported.     

Embryological and molecular investigations of parental imprinting on mouse chromosome 7

Mouse embryos with duplications of whole maternal (parthenogenetic and gynogenetic) or paternal (androgenetic) genomes show reciprocal phenotypes and do not develop to term. Genetic complementation has identified the distal region of chromosome 7 (Chr 7) as one of the regions for which both a maternal and paternal chromosome copy are essential for normal development, presumably because of the presence of imprinted genes whose expression is dependent on their parental origin. Embryos with the maternal duplication and paternal deficiency of distal Chr 7 are growth retarded and die around day 16 of gestation; the reciprocal paternal duplication embryos die at an unidentified earlier stage. We report here the incorporation of cells with the paternal duplication into chimaeras, resulting in a striking growth enhancement of the embryos. One gene located on mouse distal Chr 7 (ref. 5) is the insulin-like growth factor 2 (Igf2) gene, an embryonic mitogen. In embryos with the maternal duplication of distal Chr 7, the two maternal alleles of the Igf2 gene are repressed. The presence of two paternal alleles of this gene in many cells is probably responsible for the growth enhancement observed in chimaeras. We propose that there are other imprinted genes in this Chr 7 region. We also compare the imprinting of this subgenomic region with phenotypes resulting from the duplication of the whole parental genome in parthenogenones and androgenones.     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.     

Bypassing genomic imprinting allows seed development

In developing progeny of mammals the two parental genomes are differentially expressed according to imprinting marks, and embryos with only a uniparental genetic contribution die. Gene expression that is dependent on the parent of origin has also been observed in the offspring of flowering plants, and mutations in the imprinting machinery lead to embryonic lethality, primarily affecting the development of the endosperm-a structure in the seed that nourishes the embryo, analogous to the function of the mammalian placenta. Here we have generated Arabidopsis thaliana seeds in which the endosperm is of uniparental, that is, maternal, origin. We demonstrate that imprinting in developing seeds can be bypassed and viable albeit smaller seedlings can develop from seeds lacking a paternal contribution to the endosperm. Bypassing is only possible if the mother is mutant for any of the FIS-class genes, which encode Polycomb group chromatin-modifying factors. Thus, these data provide functional evidence that the action of the FIS complex balances the contribution of the paternal genome. As flowering plants have evolved a special reproduction system with a parallel fusion of two female with two male gametes, our findings support the hypothesis that only with the evolution of double fertilization did the action of the FIS genes become a requirement for seed development. Furthermore, our data argue for a gametophytic origin of endosperm in flowering plants, thereby supporting a hypothesis raised in 1900 by Eduard Strasburger.     

Birth of parthenogenetic mice that can develop to adulthood

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.     

Clonal reproduction by males and females in the little fire ant

Sexual reproduction can lead to major conflicts between sexes and within genomes. Here we report an extreme case of such conflicts in the little fire ant Wasmannia auropunctata. We found that sterile workers are produced by normal sexual reproduction, whereas daughter queens are invariably clonally produced. Because males usually develop from unfertilized maternal eggs in ants and other haplodiploid species, they normally achieve direct fitness only through diploid female offspring. Hence, although the clonal production of queens increases the queen's relatedness to reproductive daughters, it potentially reduces male reproductive success to zero. In an apparent response to this conflict between sexes, genetic analyses reveal that males reproduce clonally, most likely by eliminating the maternal half of the genome in diploid eggs. As a result, all sons have nuclear genomes identical to those of their father. The obligate clonal production of males and queens from individuals of the same sex effectively results in a complete separation of the male and female gene pools. These findings show that the haplodiploid sex-determination system provides grounds for the evolution of extraordinary genetic systems and new types of sexual conflict.     

Delayed activation of the paternal genome during seed development

Little is known about the timing of the maternal-to-zygotic transition during seed development in flowering plants. Because plant embryos can develop from somatic cells or microspores, maternal contributions are not considered to be crucial in early embryogensis. Early-acting embryo-lethal mutants in Arabidopsis, including emb30/gnom which affects the first zygotic division, have fuelled the perception that both maternal and paternal genomes are active immediately after fertilization. Here we show that none of the paternally inherited alleles of 20 loci that we tested is expressed during early seed development in Arabidopsis. For genes that are expressed at later stages, the paternally inherited allele becomes active three to four days after fertilization. The genes that we tested are involved in various processes and distributed throughout the genome, indicating that most, if not all, of the paternal genome may be initially silenced. Our findings are corroborated by genetic studies showing that emb30/gnom has a maternal-effect phenotype that is paternally rescuable in addition to its zygotic lethality. Thus, contrary to previous interpretations, early embryo and endosperm development are mainly under maternal control.     

An extrachromosomal factor causing loss of paternal chromosomes

Extrachromosomal inheritance is ubiquitous among plants and animals; however, most extrachromosomal factors are uniparentally inherited through females, but not through males. Examples include chloroplasts, mitochondria and a variety of intracellular symbionts. The only known exception to maternal extrachromosomal inheritance in an animal is a paternally transmitted sex ratio factor (psr) which causes all-male families in the parasitic wasp, Nasonia vitripennis. Normally in this wasp, male offspring are haploid and develop from unfertilized eggs whereas females are diploid and develop from fertilized eggs. The psr factor is either a venereally transmitted infection which prevents egg fertilization (and therefore causes all-male families), or a factor transmitted to eggs by the sperm of males carrying psr, which somehow prevents incorporation of the paternal chromosomes. Here we report that sperm from psr males fertilizes eggs, but that the paternal chromosomes are subsequently condensed into a chromatin mass before the first mitotic division of the egg and do not participate in further divisions. Resulting haploid offspring are male, but have inherited the paternal factor. This extrachromosomal factor promotes its own transmission at the expense of the paternal chromosomes, and therefore can be considered a 'selfish' genetic element.     

腰带长体茧蜂卵和早期胚胎体外发育的初步研究

 通过在体外条件下培养腰带长体茧蜂侧输卵管处解剖得到的脱去滤泡细胞的成熟卵及从寄生早期幼虫血淋巴中获得的初期胚胎,对腰带长体茧蜂卵和胚胎的初期发育进行了初步的研究。结果表明,以TC-100为基础培养基加入一定量寄主幼虫血浆和胎牛血清时,从侧输卵管处解剖得到的卵在合适的体外培养条件下可以进行卵裂,且卵裂产生的初级胚胎可以被释放到培养基中。初级胚胎可以在胚外膜内进行分裂生成二级胚胎细胞,但二级胚胎不能被释放出胚外膜。从寄生初期寄主幼虫体内得到的初级胚胎可以在体外培养条件下长大并持续通过胚外膜凹陷进行增殖,但无法进入个体发育阶段,胚胎最后黑化死亡。     

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.     

Egg investment is influenced by male attractiveness in the mallard

Why females prefer to copulate with particular males is a contentious issue. Attention is currently focused on whether females choose males on the basis of their genetic quality, in order to produce more viable offspring. Support for this hypothesis in birds has come from studies showing that preferred males tend to father offspring of better condition or with increased survivorship. Before attributing greater offspring viability to a male's heritable genetic quality, however, it is important to discount effects arising from confounding sources, including maternal effects. This has generally been addressed by comparing offspring viability from two different breeding attempts by the same female: one when offspring are sired by a preferred male, and one when offspring are sired by a less preferred male. However, here we show that individual female mallard (Anas platyrhynchos) lay larger eggs after copulating with preferred males and smaller eggs after copulating with less preferred males. As a result, females produced offspring of better body condition when paired with preferred males. After controlling for these differences in maternal investment, we found no effect of paternity on offspring condition. This shows that differences between half-sibs cannot always be attributed to paternal or maternal genetic effects.     

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X?chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X?chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X?chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X?chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.     

斑马鱼胚胎发育的功能染色体组

随着人类和其他物种染色体组测序工作的完成 ,人类科学最大的任务就是阐明数以万计的基因的生物功能。人类和动物生命周期都从受精卵开始 ,然后一步一步地发育成具有多元组织和器官的生物体。在胚胎形成过程中 ,伴随着基因生成物的协同运作 ,基因按其固有的程序陆续显现出影响 ,从而决定并实现整个人体程序。如果使用适当的动物做模型的话 ,可以加速胚胎功能染色体组的研究。斑马鱼就是这项研究一个很好的模型。斑马鱼最大的优点就是产卵多、体外胚胎发育、体积小、容易养活 ,除此以外 ,很多的分子、细胞、胚胎和基因操作在斑马鱼身上都很容…     

Seed development: Early paternal gene activity in Arabidopsis.

Both parental genomes are expressed during embryogenesis, although the time of activation of the paternally inherited genes varies between organisms. Results reported by Vielle-Calzada et al. have suggested that delayed activation of the paternal genome seems to be the rule in plant development. We find, however, that during early embryogenesis in Arabidopsis, paternal genes are expressed and are sufficient for normal development. Our findings indicate that there is no overall maternal control of early embryogenesis, and that the contribution of the parental alleles needs to be assessed for each gene individually.     

The Xenopus localized messenger RNA An3 may encode an ATP-dependent RNA helicase

The maternal messenger RNA An3 was originally identified localized to the animal hemisphere of Xenopus laevis oocytes, eggs and early embryos. Xenopus embryos depend on mRNA and protein present in the egg before fertilization (maternal molecules) to provide the information needed for early development. Localization of maternal mRNA gives cells derived from different regions of the egg distinctive capacities for protein synthesis. We show here that An3 mRNA encodes a protein with 74% identity to a protein encoded by the testes-specific mRNA PL10 found in mouse, which is proposed to have RNA helicase activity. Because the gene encoding An3 mRNA is reactivated after gastrulation and remains active throughout embryogenesis, we have examined its distribution in embryonic and adult tissues. Unlike PL10 mRNA, which is primarily restricted to the testes, An3 mRNA is broadly distributed in later development.     

Somatic cell bovine cloning: Effect of donor cell and recipients

Nuclear transfer (NT) is an efficient technique for assessing the developmental potential of a nucleus and for analyzing the interactions between the donor nucleus and the recipient cytoplasm. In amphibians, thought nuclei of adult kerationocytes support development to the juvenile, tadpole stage, no development to the adult stage was re- ported[1], leaving open the question of whether a different- tiated adult nucleus can be fully reprogrammed. The first cloned offspring developed from differ…     

Outbred embryos rescue inbred half-siblings in mixed-paternity broods of live-bearing females

Females commonly mate with more than one male, and polyandry has been shown to increase reproductive success in many species. Insemination by multiple males shifts the arena for sexual selection from the external environment to the female reproductive tract, where sperm competition or female choice of sperm could bias fertilization against sperm from genetically inferior or genetically incompatible males. Evidence that polyandry can be a strategy for avoiding incompatibility comes from studies showing that inbreeding cost is reduced in some egg-laying species by postcopulatory mechanisms that favour fertilization by sperm from unrelated males. In viviparous (live-bearing) species, inbreeding not only reduces offspring genetic quality but might also disrupt feto-maternal interactions that are crucial for normal embryonic development. Here we show that polyandry in viviparous pseudoscorpions reduces inbreeding cost not through paternity-biasing mechanisms favouring outbred offspring, but rather because outbred embryos exert a rescuing effect on inbred half-siblings in mixed-paternity broods. The benefits of polyandry may thus be more complex for live-bearing females than for females that lay eggs.