A study of X chromosome regulation during oogenesis in the mouse

Mature oocytes of mammals, in contrast to somatic cells, have two active X chromosomes. This situation might arise through either of two possible mechanisms. The germ line might be differentiated from somatic cells prior to X inactivation. Alternatively, an X chromosome in germ cells would be reactivated after prior inactivation. This paper presents data compatible with reactivation of the X in germ cells. X-linked enzymes were compared in oocytes of XX and XO fetal mice. The activity of G6PD is similar in the two classes of cells at early meiotic stages, but an $\text{XX}\text{XO}$ ratio of 2:1 is approached at later times; this suggests reactivation of the G6PD locus. For HPRT, a 2:1 ratio is observed at all meiotic stages. HPRT shows a large increase in enzyme activity during early meiosis, while G6PD does not. Synthesis of this enzyme at early meiotic stages probably accounts for differences between these data and those obtained for G6PD, and places the time of X reactivation at the entry to meiosis.     

Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities.

Oocytes at several stages of growth were isolated by enzymatic digestion of ovaries from infant mice. These oocytes were free of follicle cells and were obtained in sufficient numbers to permit direct biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) activities. Both enzymes increase in total activity as the oocyte grows. However, while the specific activity of G6PD remains constant up to an oocyte diameter of 80–85 μm, the specific activity of LDH increases four-fold. The specific activity of G6PD and especially LDH declines in oocytes over 80–85 μm in diameter, suggesting that the synthesis of these enzymes is almost completed at this stage of growth.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

X linkage of phosphoglycerate kinase in the mouse

The levels of phosphoglycerate kinase (PGK), glucose 6-phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) were measured in one-cell embryos from X/0 and X/X females. Since the level of both PGK and G6PD was dependent on the number of X chromosomes present in the mother, these two enzymes are most likely coded for by X-linked genes. The level of LDH was the same in both types of embryos, indicating autosomal linkage. A search for an electrophoretic variant of PGK was not successful.     

棕色田鼠XO雌体育性研究

通过对棕色田鼠外形特征,卵巢切片,怀胎和生产雌鼠染色体鉴定等方面研究,证实了该鼠ＸＯ雌鼠可孕,并具有生殖能力。染色体鉴定表明,ＸＸ雌体中的两条Ｘ性染以体,一条为Ｍ类型另一条为ＳＭ类型;ＸＯ雌体中的Ｘ性染色体为Ｍ类型。所以ＸＯ雌性的生育能力可能与Ｘ染色体有关,其上可能存在雌性育性基因。     

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.     

Evidence for X-Linkage of Steroid Sulfatase in the Mouse: Steroid Sulfatase Levels in Oocytes of XX and XO Mice

The steroid sulfatase (STS) levels in mature oocytes of XX and XO mice were assayed along with lactate dehydrogenase (LDH), an autosomal marker, and glucose-6-phosphate dehydrogenase (G6PD), a known X-linked gene. LDH levels in XX and XO oocytes were equal, whereas STS and G6PD levels were approximately twice as high in XX oocytes as in XO oocytes. These results indicate that the STS gene is X-linked in the mouse just as it is in humans. Assays of STS in kidney tissue of XX and XO mice indicated dosage compensation for the gene, which is different from that observed in humans.     

Gene dosage effect in human triploid fibroblasts

Summary The activity of 13 cytoplasmic enzymes has been determined in fibroblast extracts from 9 triploid and 13 control lines. The results show a high activity for 2 X-linked enzymes, glucose 6-phosphate dehydrogenase and phosphoglycerate kinase. These data, together with cytogenetic observations, support the contention that 2 X chromosomes were active in the triploid lines.Abbreviations G6PD Glucose 6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - HK hexokinase - PGM phosphoglucomutase - PHI phosphohexoisomerase - PFK phosphofructokinase - ALD aldolase - TPI triosephosphate isomerase - PGK phosphoglycerate kinase - ENOL enolase - AK adenylate kinase - LDH lactic dehydrogenase - HBDH hydroxybutyrate dehydrogenase INSERM U. 129.INSERM U. 73.     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

Human X chromosomes: synchrony of DNA replication in diploid and triploid fibroblasts with multiple active or inactive X chromosomes

We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical stain to measure X-linked glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2) cellulose acetate electrophoresis of G6PD activity in cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of 47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.     

金鱼同工酶的发生遗传学研究——Ⅲ.金鱼同工酶基因座位的加倍与多倍性

以鲫鱼和金鱼为材料,用葡萄糖-6-磷酸脱氢酶(G6PD)、乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)同工酶体系作为基因标志,从检测同工酶的多重组合形式来研究基因的加倍与演化。对彩鲫与金鱼G6PD和LDH同工酶的分析结果表明,它们均具有与四倍体鱼类相应的谱带。因而说明了金鱼的G6PD和LDH同工酶基因座位的加倍与染色体的多倍性有关,为金鱼是四倍体的假说提供了证据。而对MDH同工酶的分析却得到了与二倍体鱼类相同的谱带数。这可能与加倍基因发生突变而不表达有关。     

青海四种雏蝗染色体核型的比较分析

采用常规染色体制片方法对雏蝗属的褐色雏蝗Chorthippusbrunneus(Thunb .) ,异色雏蝗C .big uttulus(Linnaeus) ,小翅雏蝗C .fallax(Zub .) ,青藏雏蝗C .qingzangensis(Ying)的染色体核型进行分析 ,结果 :染色体数目均为 2n(♂ ) =1 7=1 6+XO ;常染色体类型为两类 ,中着丝点染色体 (m ,6条 )和端着丝点染色体 (T ,1 0条 ) ;性染色体类型为端着丝点。褐色雏蝗、异色雏蝗和青藏雏蝗的核型公式和染色体的相对长度组成为K( 2n ,♂ ) =1 7=6m +1 1T =6L +6M +4S +XO ,K( 2n ,♀ ) =1 8=6m +1 2T =6L +6M +4S +XX ;小翅雏蝗的为K( 2n,♂ ) =1 7=6m +1 1T =6L +4M +6S +XO ,K( 2n ,♀ ) =1 8=6m +1 2T =6L +4M +6S+XX。褐色雏蝗性染色体中部有次缢痕。染色体臂数 4种均为NF =2 3(♂ ) ,2 4 (♀ )。