Some characteristics of the XO mouse (Mus musculus L.) I. Vitality: Growth and metabolism

The growth rate of XO mice during the first five weeks of life was shown to be significantly lower (ca. 15%) than the growth rate of normal XX mice. A marker gene Tabby was introduced in order to recognize hemizygous XO females. The presence or absence of this gene had a significant influence on growth rates. XO females could only be compared to XX females in an indirect way. The differences found could not be attributed to maternal influence or to the influence of litter size.Body temperature and thyroid activity were found to be lower in XO mice than in normal females. It is suggested that the lower growth rate characteristic of the XO mice is a consequence of hypothyroidism and a lower basal metabolic rate.The results show that phenotypically XO mice are not entirely normal and at least two normal X's are necessary for complete development.     

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.     

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.     

The development of XO gynogenetic mouse embryos

Diploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.     

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.     

Identification of single-nucleotide polymorphism in the progesterone receptor gene and its association with reproductive traits in rabbits

A total of 598 F₂ does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1–8 were amplified and sequenced. One SNP was found in the promoter region, 2464G>A, three SNPs in the 5′-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes.     

Evidence that the testis determination pathway interacts with a non-dosage compensated, X-linked gene.

In a number of mammals, including mouse and man, it has been shown that at equivalent gestational ages, males are developmentally more advanced than females, even before the gonads form. In mice, although some strains of Y chromosome exert a minor accelerating effect in pre-implantation development, it is a post-implantation effect of the difference in X chromosome constitution that is the major cause of the male/female developmental difference. Thus XX females are retarded in their development by about 1.5 h relative to X(M)O females or XY males; however, they are more advanced than X(P)O females by about 4 h. It has been suggested that this early developmental difference between XX and XY embryos may "weight the dice" in favour of ovarian and testicular development, respectively, although expression of Sry will normally overcome any such bias. Here we test this proposal by comparing the relative frequencies of female, hermaphrodite and male development in X(P)O, XX and X(M)O mice that carry an incompletely penetrant Sry transgene. The results show that testicular tissue develops more frequently in XX,Sry transgenics than in either of the two types of XO transgenics. Thus the incidence of testicular development is affected by X dosage rather than by the developmental hierarchy. This implies there is a non-dosage compensated gene (or genes) on the X chromosome, which interacts with the testis-determining pathway. Since the pseudoautosomal region (PAR) is known to escape X-inactivation, penetrance of the Sry transgene was also assessed in X(M)Y(*X) mice that have two doses of the PAR but have a single dose of all genes proximal to the distal X marker Amel. These mice showed similar levels of testicular development to X(M)O mice with the transgene; thus the non-dosage compensated X gene maps outside the PAR.     

Perinatal oocyte loss in XO mice and its implications for the aetiology of gonadal dysgenesis in XO women

Postnatally, XO mice have approximately half as many oocytes as their XX sisters. A quantitative histological analysis of XO and XX ovaries throughout oogenesis (14 1/2-24 1/2 days post coitum) revealed that this oocyte deficiency in XO mice is due to excess atresia of oocytes at the late pachytene stage (19 1/2 days post coitum). Female mice heterozygous for a large X inversion (In(X)/X mice) were also found to have excess atresia at late pachytene. It was suggested that in XO mice it is the presence of an unpaired X chromosome, and in In(X)/X mice, the incompleteness of X chromosome pairing, which leads to this excess oocyte atresia. A new quantitative histological procedure which was developed for the analysis of perinatal mouse ovaries is also described.     

Somatic damage to the X chromosome of the nematode Caenorhabditis elegans induced by gamma radiation

Summary Wild-type male embryos and young larvae of the nematode Caenorhabditis elegans were more sensitive than wild-type hermaphrodites to inactivation by gamma rays; wild-type males have one X chromosome per cell (XO), whereas wild-type hermaphrodites have two (XX). Furthermore, after transformation into fertile hermaphrodites by a her-1 mutation, XO animals were more radiosensitive than XX her-1 animals; and XX animals transformed into fertile males by a tra-1 mutation did not show increased radiosensitivity. It is concluded that wild-type males are more radiosensitive than wild-type hermaphrodites because they have one X chromosome rather than two, and the predominant mode of inactivation of XO animals involves damage to the single X chromosome. No sex-specific differences in survival were observed after UV irradiation.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

Evidence that postnatal growth retardation in XO mice is due to haploinsufficiency for a non-PAR X gene

XO Turner women, irrespective of the parental source of the X chromosome, are of short stature, and this is now thought to be largely a consequence of haploinsufficiency for the pseudoautosomal region (PAR) gene SHOX. X(p)O mice (with a paternal X) are developmentally retarded in fetal life, are underweight at birth, and show reduced weight gain in the first few weeks after birth. X(m)O mice, on the other hand, are more developmentally advanced than their XX siblings in fetal life; their postnatal growth has not hitherto been assessed. Here we show that X(m)O mice are not underweight at birth, but they nevertheless show reduced weight gain postnatally. The fact that postnatal growth is affected in X(p)O and X(m)O mice, means that this must be due to X dosage deficiency. In order to see if haploinsufficiency for a PAR gene was responsible for this growth deficit (cf SHOX deficiency in Turner women), X(m)Y*(X) females, in which the Y*(X) chromosome provides a second copy of the PAR, were compared with XX females. These X(m)Y*(X) females were also growth-retarded relative to their XX sibs, suggesting that it may be haploinsufficiency for a non-dosage-compensated X gene or genes outside the PAR that is responsible for the postnatal growth deficit in XO mice. The X genes known to escape X inactivation in the mouse have closely similar Y homologues. X(m)YSRY-negative females were therefore compared with XX females to see if the presence of the SRY-negative Y chromosome corrected the growth deficit; this proved to be the case. The postnatal growth deficit of XO mice is therefore probably due to haploinsufficiency for a non-dosage-compensated X gene that has a Y homologue that provides an equivalent function in the somatic tissues of males.     

Teratogen susceptibility of XO mothers and XO embryos in mice

We examined whether the chromosomal imbalance inherent in an XO constitution in mice is more susceptible to teratogenic influence of biotin deficiency using a newly established mouse colony with pure X monosomy. We hypothesized that XO mothers or XO embryos might be more susceptible to certain teratogens. Contrary to our expectation, the incidence of external malformations induced by biotin deficiency did not differ either between XX dams and XO dams or between XX fetuses and XO fetuses.     

单套染色体组在泽蛙雌核单倍体发育中的作用

本文比较了泽蛙(Rana limnocharis Boie)单倍体和二倍体的胚胎发育。结果表明:泽蛙单倍体的生活力低下,器官发生异常,自原肠胚起发育速度减慢。据此,作者讨论了单套染色体组在个体发育中的作用。     

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.     

Post-implantation development and cytogenetic analysis of diandric heterozygous diploid mouse embryos

Diandric heterozygous diploid mouse embryos were produced by standard micromanipulatory techniques using eggs from female mice with a normal chromosome constitution and fertilised by homozygous Rb(1.3)1Bnr males containing a pair of large metacentric marker chromosomes in their karyotype. The constructed diandric eggs were transferred to the oviducts of pseudopregnant recipients and subsequently autopsied midday on the eighth day of gestation. From a total of 85 eggs transferred to females that subsequently became pregnant, 30 implanted. Eighteen implantation sites were found to contain resorptions, and 12 egg cylinder stage embryos were recovered. These were cytogenetically examined. In two cases, no mitoses were observed, and in a third embryo of normal size, only a single paternally-derived marker chromosome was present in its mitoses, indicating that this embryo had a normal chromosome constitution. This presumably resulted from a technical error during the micromanipulatory procedure. The remaining nine morphologically small but normal embryos were diploid, and each had two paternally-derived marker chromosomes, thus establishing their ploidy and confirming their diandric origin. G-banding analysis revealed that all of these embryos had an XY sex chromosome constitution. Since the expected XX:XY:YY ratio of 1:2:1 was not observed, it is clear that the XX class embryos were lost at some stage during the pre- or early post-implantation period, though whether they are represented by the resorption sites is not yet established. The YY class would not be expected to be recovered in any case, as these embryos are believed to be lost during early cleavage. The cytogenetic findings reported here are therefore similar to the results of the chromosomal analyses of the human complete hydatidiform moles of dispermic origin, all of which apparently have an XY karyotype. It is unclear why, both in the human and in the mouse, the XX diandric heterozygous diploid group should develop poorly compared to similar embryos with an XY karyotype.