Two maternally derived X chromosomes contribute to parthenogenetic inviability

In certain extraembryonic tissues of normal female mouse conceptuses, X-chromosome-dosage compensation is achieved by preferential inactivation of the paternally derived X. Diploid parthenogenones have two maternally derived X chromosomes, hence this mechanism cannot operate. To examine whether this contributes to the inviability of parthenogenones, XO and XX parthenogenetic eggs were constructed by pronuclear transplantation and their development assessed after transfer to pseudopregnant recipients. In one series of experiments, the frequency of postimplantation development of XO parthenogenones was much higher than that of their XX counterparts. This result is consistent with the possibility that two maternally derived X chromosomes can contribute to parthenogenetic inviability at or very soon after implantation. However, both XO and XX parthenogenones showed similar developmental abnormalities at the postimplantation stage, demonstrating that parthenogenetic inviability is ultimately determined by the possession of two sets of maternally derived autosomes.     

Effects of sex chromosome dosage on placental size in mice

Mice of the XO genotype with a paternally derived X chromosome (XpO) have placental hyperplasia in late pregnancy, although in early pregnancy the ectoplacental cone, a placental precursor, is smaller in XpO mice than in their XX sibs. This early size deficiency of the ectoplacental cone is apparently a consequence of Xp imprinting, because XmO embryos (with a maternally derived X chromosome) are unaffected. In the present study we sought to establish whether XpO placental hyperplasia in late pregnancy is also a consequence of Xp imprinting. Placental weight data were first collected from litters that included XpO or XmO fetuses and XX controls. Comparison of XO placentae with XX placentae showed that XpO and XmO placentae are hyperplastic. This finding suggested that the hyperplasia might be an X dosage effect, and this hypothesis was supported by the finding that XY male fetuses from the same crosses also had larger placentae than their XX sibs. Further analysis of a range of sex-chromosome variant genotypes, including XmYSry-negative females and XXSry transgenic males, showed that mouse fetuses with one X chromosome consistently had larger placentae than littermates with two X chromosomes, independent of their gonadal/androgen status.     

Timing of mitotic chromosome loss caused by the ncd mutation of Drosophila melanogaster.

We studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin-like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX-XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin-like proteins in Drosophila.     

An extra maternally derived X chromosome is deleterious to early mouse development

An extra copy of the X chromosome, unlike autosomes, exerts only minor effects on development in mammals including man and mice, because all X chromosomes except one are genetically inactivated. Contrary to this contention, we found that an additional maternally derived X (XM) chromosome, but probably not a paternally derived one (XP), consistently contributes to early death of 41,XXY and 41,XXX embryos in mice. Because of imprinted resistance to inactivation, two doses of XM remain active in the trophectoderm, and seem to be responsible for the failure in the development of the ectoplacental cone and extraembryonic ectoderm, and hence, from early embryonic death. Discordant observations in man indicating viability of XMXMXP and XMXMY individuals suggest that imprinting on the human X chromosome is either weak, unstable or erased before the initiation of X-inactivation in progenitors of extraembryonic membranes.     

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).     

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.     

Studying Early Lethality of 45,XO (Turner's Syndrome) Embryos Using Human Embryonic Stem Cells

Turner''s syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos.     

Genomic imprinting: male mice with uniparentally derived sex chromosomes

Although it has been known that there is an X-chromosome imprinting effect during early embryogenesis in female mammals, it remains unknown if parental origin of the X chromosome has an effect in males. Furthermore, it has not been possible to produce animals with normal sex chromosomes of uniparental origin to further evaluate such imprinting effects. We have devised a breeding scheme to produce male mice, designated XPYP males, in which both the X and Y chromosomes are paternally inherited. To our knowledge, these are the first mammals produced that have a normal sex chromosome constitution but with both sex chromosomes derived from one parent. Development and reproduction in these XPYP males and the sex ratio and chromosome constitution of their offspring appeared normal; thus there is no apparent effect in males of having both sex chromosomes derive from one parent or of having the X chromosome derived from an inappropriate parent. Although we have detected no X-chromosome imprinting effect in these males, evidence from other sources suggest that the X chromosome is parentally imprinted. Thus detection and definition of an imprint can depend on the assay used.     

Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results

Summary Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.     

Expression of X-linked genes in androgenetic,gynogenetic, and normal mouse preimplantation embryos

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplan-tation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androge-netic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blas-tocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both and rogenones and gynogenones at the blas-tocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, where as the developmental defects of gyno-genones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those far-thest away from the X inactivation center. © 1995 Wiley-Liss, Inc.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

Abstract. In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara . It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara . Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.     

The Expression of X-Linked Phosphoglycerate Kinase in the Early Mouse Embryo

Abstract. During early mouse embryogenesis, the activity of X-chromosomally linked maternal and paternal phosphoglycerate kinase (PGK-1) alleles was determined using electrophoretic separation of their gene products and a sensitive fluorometric enzyme assay. In the embryos collected from females homozygous for PGK-1^b mated with PGK-1^a males and vice versa, the paternally derived allozyme was first detected after implantation on day 6. Expression of the maternally inherited allele was studied in embryos from females heterozygous for PGK-1^b and PGK-1^a. From day 1 to day 4, the embryos maintained a constant ratio of enzyme activity of PGK-1B to PGK-1A. Prior to implantation of the embryos between day 4 and day 5, the activity ratio of the two PGK-1 allelic variants changed significantly due to the first appearance of newly synthesized PGK derived from the maternally inherited allele.
Our data demonstrate a temporal difference in the onset of PGK synthesis depending on whether this particular gene product is of maternal or paternal origin. Therefore, we conclude that the maternal PGK-1 locus is already activated during late preimplantation development whereas the paternally inherited gene locus remains silent at the preimplantation stage but is subsequently expressed at approximately the time of X-chromosomal inactivation.     

The relationship between DNA methylation and chromosome imprinting in the coccid Planococcus citri

The phenomenon of chromosome, or genomic, imprinting indicates the relevance of parental origin in determining functional differences between alleles, homologous chromosomes, or haploid sets. In mealybug males (Homoptera, Coccoidea), the haploid set of paternal origin undergoes heterochromatization at midcleavage and remains so in most of the tissues. This different behavior of the two haploid sets, which depends on their parental origin, represents one of the most striking examples of chromosome imprinting. In mammals, DNA methylation has been postulated as a possible molecular mechanism to differentially imprint DNA sequences during spermatogenesis or oogenesis. In the present article we addressed the role of DNA methylation in the imprinting of whole haploid sets as it occurs in Coccids. We investigated the DNA methylation patterns at both the molecular and chromosomal level in the mealybug Planococcus citri. We found that in both males and females the paternally derived haploid set is hypomethylated with respect to the maternally derived one. Therefore, in males, it is the paternally derived hypomethylated haploid set that is heterochromatized. Our data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.