XX Mice Research Articles

THE GENETICS AND BIOCHEMISTRY OF SEX DETERMINATION

The Y chromosome is a dominant induced of testis development mammals. The identification and cloning of SRY, the testis determining gene, depended on the analysis of the genomes of patients with sex reversal syndromes. By analysis of Y chromosome fragments in the genomes of XX males, it was possible to define a 35kb minimum sex determining region(1); SRY (sex determining region y gene, the equivalent mouse gene is Sry) is located within this region(2). De novo mutations in SRY have been found in 15% of XY females(3,4,5) and XX mice transgenic for SRY are sex reversal males(6). The protein encoded by SKY/Sry contains an HMG box, a protein motif associated with DNA binding activity. Recent experiments have demonstrated DNA-binding activity of SRY protein and associated this biochemical activity with sex determination(7,8). This implies that the genes immediately “downstream” of SRY/Sry in the sex determination pathway will be subject to direct transcriptional regulation. Clues to the identity of these “downstream” genes may come from the analysis of the gnomes of Y-sequence negative XX males. Both “upstream” and “downstream” genes may be affected in XY females with intact SRY genes.

A comparison of "bonus" and "quota" systems utilising the sry gene in beef cattle breeding

The SRY gene controls the presence of testes (i.e. maleness) in mammals. This has been demonstrated by Koopman et al (1991), who bred transgenic XX mice with a copy of the SRY gene on an autosome which were phenotypically male, albeit infertile. A fertile XY male with a copy of the SRY gene on an autosome should, theoretically, produce a greater proportion of offspring which are phenotypically male. This would be advantageous in, for example, breeding Terminal beef sires, where male calves are more valuable than females calves on commercial farms. The possible use of beef cattle transgenic for the SRY gene was investigated by Bishop and Woolliams (1991) who found that there were small genetic advantages in breeding schemes using these bulls, compared to traditional breeding schemes, although there may be practical problems in running such schemes. Their findings pertained to a restricted mating strategy (the "quota" system) and a restricted time horizon (15 years), however. This paper investigates the possible application of the SRY gene considering different mating strategies and a flexible time horizon.

Susceptibility to teratogenicity of hypervitaminosis-A in X-monosomy mice

We previously showed that the incidence of external malformation induced by biotin deficiency did not differ either between XO and XX dams or between XOand XX fetuses. To clarify whether this phenomenon is specific to biotin deficiency or more generally associated with other teratogens, we examined whether XO mice are more susceptible to teratogenic effects of hypervitaminisis-A. Pregnant XO and XX mice were given an excessive vitamin A diets (1.0 to 1.5 × 10 6 IU/kg) from days 0 to 17 of gestation. Maternal hypervitaminosis-A produced a high incidence of external malforamtions (65 to 80%), skeletal anomalies (33 to 47%), and variations (99 to 100%) in the fetuses. However, there is no difference in their incidences between XO and XX dams or between XO and XX fetuses. Together with previous findings, this suggest that developmental stability of the mouse embryo is not affected by missing of one whole X chromosome even with exposure to teratogens.

Effect of growth hormone dosing on ultimate weight and length of XO mice.

Mice with the karyotype 39, XO and XX mice with the same genetic background were injected for a six month period with daily subcutaneous doses of 0.5 IU biosynthetic human growth hormone (hGH) or with placebo. The XO mice dosed with hGH obtained a significantly higher body weight and greater final length than the XO mice dosed with placebo (39.2 g versus 29.0 g and 21.1 cm versus 19.2 cm respectively). The body weight remained constant after cessation of dosing. The XX mice became significantly longer but not heavier as a result of hGH dosing. The placebo dosed XO mice became significantly longer than the placebo XX mice but their body weights were comparable. Dosing with hGH caused a significant increase in growth rate in both XO and XX mice, and the ultimate body weight was reached within a significantly shorter time period. In the XO mice the hGH injections increased significantly the length and thickness of the femoral bones and the length of the tibia bones. Stimulated endogenous plasma growth hormone levels were significantly higher in XX mice compared to XO mice. Plasma IGF-I levels were significantly higher in XO compared to XX mice, but the levels were not affected by hGH dosing. XO mice may be useful in future metabolic and hormonal studies.

38 EFFECT OF GROWTH HORMONE TREATMENT ON ULTIMATE WEIGHT AND LENGTH OF TURNER MICE

Turner mice (xo) and xx mice with the same genetic background were treated for a 6-month period with daily subcutaneous doses of 0.5 IU NorditropinR or placebo, starting at the age of 5 weeks to 5 months. The Turner mice which were treated with NorditropinR reached a significantly higher ultimate body weight (about 38g) and ultimate length (nose to tip of the tail - about 21.5cm) compared to the placebo-treated Turner mice (about 28g and 19cm). The body weight remained constant after cessation of treatment. There was no correlation between age at the start of treatment and ultimate weight or length. Upon sacrifice it was found that the femoral and tibia bones from the NorditropinR-treated Turner mice were significantly longer compared to placebo mice. The ultimate weight and length of the NorditropinR-treated xx mice, as well as the length of their bones, were not significantly different from the comparable parameters in placebo-treated xx mice. Both xo and xx mice reached their ultimate weight significantly faster when they were treated with NorditropinR compared to placebo (means: 22.1 and 19.7 weeks respectively after start of dosing).

Epididymides of sex-reversed XX mice lack the initial segment.

The genetic factor Sxr causes sex reversal of chromosomally female (XX or XO) mice to phenotypic maleness by inducing development of testes that produce androgens. It has been considered that these sex-reversed animals, called pseudomales, confirm the principle originally developed by Jost that adequate androgenization produces normal phenotypic maleness in mammals, irrespective of chromosomal sex. However, we previously discovered that the epididymis of sex-reversed XX mice (pseudomales of genotype XXSxr) lacks EH 9 cells (epididymal head, cell type No. 9, the "principal cell' of the initial segment). The purpose of the present study was to determine whether cell type EH 9 of XXSxr pseudomales is replaced by a principal cell of a different appearance, or whether the initial segment itself is actually absent. We made serial sections of entire epididymal heads and did microdissections to unravel the highly coiled epididymal duct. Using these two approaches, we studied the sequence of epididymal segments, and estimated lengths of the relevant portion of the epididymal duct; we found that the initial segment of XXSxr pseudomales is truly absent. This is the first report of a mutant genotype causing absence of a segment of the epididymis. The XXSxr mutant appears to be an exception to Jost's principle. This finding shows that, even in full androgenization, male phenotype may not always be independent of chromosomal sex.

Retarded development of XO conceptuses during early pregnancy in the mouse.

The growth and development of XO and XX mice were compared from 7 1/4 to 18 1/2 days of gestation. The 7 1/4-day XO egg cylinders were retarded in development (and consequently small) when compared with XX egg cylinders, and this lag in development remained until 10 1/2 days. By 12 1/4 days there was a considerable degree of 'catch-up', but this was not fully maintained. A subgroup of very severely retarded XO fetuses were preferentially located near the cervix. The placentas of XO fetuses were of normal size until 18 1/2 days when they were significantly larger than those of XX controls.

XO monosomy is associated with reduced birthweight and lowered weight gain in the mouse.

The growth of XO mice and their XX sisters was followed from the day of birth up to 15 weeks post partum. XO mice were underweight at birth, and grew more slowly than XX mice in the preweaning period. Some, but not all, of this decrease in growth rate was attributable to an effect of the reduced birth weight.

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.

Male, female and intersex development in mice of identical chromosome constitution.

In the preceding paper1, cytological evidence is presented to show that in ‘sex-reversed’ XX male mice2 the sex-reversing factor, Sxr, is carried on a dark-staining body located on the distal end of the paternal X chromosome. The X-chromosomal location of Sxr raises the possibility that it may be subject to the X chromosome inactivation process3. However, the regular segregation of Sxr and the absence of any indication that intersexes occur with a higher than normal frequency in Sxr stocks4 suggests that either inactivation of Sxr does not occur, or, if it does, that the proportion of cells with the Sxr-bearing X genetically active is sufficient to ensure normal testicular differentiation. Here we present information on the sexual development of chromosomally XX mice carrying Sxr in which the Sxr-bearing X chromosome is held in the inactive condition in most if not all somatic cells by use of the T(X; 16)16H (T16H) X-autosome translocated chromosome5–10 Mice of this genotype were found to develop as normal but sterile males, normal fertile females or intersexes.

Spermatogenesis and sperm-specific phosphoglycerate kinase B and lactate dehydrogenase C4 isoenzymes in sex-reversed mice.

The activities of phosphoglycerate kinase B and lactate dehydrogenase C4 were correlated with the state of spermatogenesis in testes of sex-reversed (Sxr) mice. Both isoenzymes were present in Sxr/+,XO testes, which contained late spermatids. LDH-C4 was detected in testes from 5 of 9 Sxr/+,XX mice, but PGK-B was present in testes of only 1 of the 5 mice. Pachytene spermatocytes were the predominant cell types in those Sxr/+,XX testes that contained spermatogenic cells. These observations confirm that PGK-B is restricted to spermatogenic cells and is not present in somatic testicular cells. Furthermore, they suggest that PGK-B synthesis may be initiated later than pachytene, when synthesis of the LDH-C polypeptide is known to commence.

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.

Enzyme levels in testis and other tissues of genetically sex-reversed mice.

The specific activity of G6PD and PGK were measured in the testes, seminal vesicles, and livers of Sxr/+,XX mice, their Sxr/+,XY littermates and normal mice. While G6PD activity was high in the testes of young normal mice and declined as the testes matured, in the testes of Sxr/+,XX mice activity remained high, suggesting a failure of the Sertoli cells to mature normally. The activity of PGK was low in the testes of young normal mice, and increased as the testes matured. The testes of young Sxr/+,XX mice had high activity of this enzyme which remained high into adulthood. The high activity in young mice suggests an abnormality in the somatic cells. The seminal vesicle and liver measurements of G6PD and PGK confirmed that the Sxr/+,XX mice were phenotypically normal males except with respect to the testis. The developmental patterns of both enzymes in testes lacking germinal cells indicate that the maturation of the somatic cells of the normal testis is influenced by the presence of germinal cells.

Cytotoxic T cells recognize male antigen and H-2 as distinct entities.

XX cells from XX/XY hemopoietic chimeras do not express male determinants in a way to render them either stimulators or targets for male-specific cytotoxic lymphocytes. XX- but not XY-responder T cells from chimeras can be activated to lyse allogeneic male target cells; T cells from normal XX mice depleted of alloreactive T cells, however, cannot be sensitized to lyse allogeneic XY targets. The results imply that T cells recognize the Y-antigen and H-2 as distinct entities, and that in chimeras, they acquire the potential to react against allogeneic XY cells.

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 XX 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.

Cell interactions in the sex organs of sex reversed mice heterozygous for testicular feminization.

The X-linked mutation for testicular feminization (Tfm) described by Lyon and Hawkes (1970) causes androgen insensitivity of the testosterone target cells. The molecular basis of the phenotype is a defect of the androgen receptor protein (Attardi and Ohno, 1974; Gehring and Tomkins, 1974). Due to random X inactivation, XX mice heterozygous for Tfm are mosaics with respect to androgen sensitivity. They can be converted to males by the autosomal dominant mutation for “sex reversal” (Sxr) described by Cattanach, Pollard, and Hawkes (1971). By imitating the Y-chromosome, Sxr induces formation of testes in XX embryos. The testosterone produced by the embryonic testes in turn induces formation of male sex organs. In sex-reversed embryos heterozygous for Tfm, testosterone encounters a mixed population of androgen-insensitive and androgen-sensitive cells. In the androgen-insensitive cells the X chromosome bearing Tfm is active. The Tfm cells express the defective receptor protein and therefore are constitutively bound to form female organs. In the androgen-sensitive cells the wild-type X is active. The wild-type cells therefore express an intact androgen receptor protein. They respond to testosterone and are bound to form male sex organs.

Serological evidence for H-Y antigen in Sxr, XX sex-reversal phenotypic males.

IT is generally considered that the Y chromosome is necessary for the development of the mammalian testis and the consequent male phenotype. Exceptions occasionally occur in man and other animals, where a male phenotype is associated with an apparently normal female karyotype. In some instances it has been possible to show that such “sex reversal” has an autosomal genetic basis. Although this might imply that the Y chromosome is not a prerequisite of maleness, it has not been excluded that a small male-determining region of the Y may have been translocated to an autosome without producing a recognisable karyotypic change1 (reviewed in ref. 2). The best studied case of male development with an apparently female karyotype is provided by the Sxr mutant in the mouse. This autosomal dominant condition causes both XX and XO mice to develop as males, although germ cells are absent in Sxr,XX animals and spermatogenesis is impaired in Sxr,XO mice. Sxr,XY males have a slightly reduced testis size but are usually fertile. Cytological scrutiny of mitotic and meiotic chromosomes (including chromosome banding) has revealed no evidence for a Y–autosome translocation, although a small translocation undetectable by the methods used cannot be ruled out (reviewed in ref. 2). This situation can now be investigated using serological methods for detecting H–Y (histocompatibility–Y) antigen on the surface of mouse sperm3 and male epidermal cells4. These techniques have provided further evidence that expression of this antigen is governed by a gene (or genes) on the Y chromosome even in the absence of the normal male phenotype5. Thus, serological demonstration of H–Y antigen offers a non-karyological criterion for the presence of a translocated portion of the Y in Sxr,XX males, because their cells should express H–Y if Sxr is a translocation involving the H–Y genetic determinant. We report here that Sxr,XX males express H–Y to a degree that may be the same or slightly lower than that of normal males, and that their Sxr,XY siblings seem to have near-normal levels of H–Y. The simplest explanation is that male-determining and H–Y-determining portions of the Y chromosome are present in the animals carrying Sxr.

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice

The meiotic segregation pattern of the X chromosome in XO mice was determined by counting second meiotic metaphase chromosomes. A total of 584 oocytes was collected from superovulated but unmated XO and XX mice. Second meiotic metaphase (MII) counts indicated that the X chromosome in XO mice did not segregate randomly but remained preferentially in the oocyte. Increased maternal age had no effect upon this segregation pattern. Selection against XO and YO preimplantation embryos was evaluated by observing their relative frequencies during first and second cleavage divisions. A combined total of 1650 preimplantation eggs was collected from superovulated and mated XO and XX female mice. Eggs were obtained during metaphase of first (CI) or second cleavage (CII) divisions by injecting mated females with colchicine at 28 or 49 hr, respectively, after human chorionic gonadotrophin (HCG) administration. Prior to analysis, chromosomes were banded by trypsin-Giemsa, which facilitated specific chromosome identification, including the X and Y. The proportion of XX, XY, XO, and YO embryos observed at CI from XO females was not significantly different from expected values based on MII counts. At CII, a significant difference was observed from the expected proportion of embryo classes resulting from a reduction in XO and YO embryos. The results provide cytogenetic evidence emphasizing that, in order for development to continue, at least one functional X chromosome is necessary as early as the two-cell stage. Triploid one- and two-cell embryos were observed more frequently in embryos from XO than XX females. The sex ratio was not significantly different from 1:1 during either cleavage stage regardless of the type of female from which the embryos were derived.

X chromosome activity in oocytes of kangaroo pouch young.

IN female eutherian mammals, inactivation of one of the two X chromosomes occurs randomly from cell to cell in somatic tissues1 but in oocytes (meiotic germ cells) X inactivation does not occur for at least three loci. Oocytes of adult XX mice possess twice as much activity as those of XO mice for the sex-linked enzyme, glucose-6-phosphate dehydrogenase (G6PD)2, hypoxanthine guanine phosphoribosyl transferase (HGPRT)3, and phosphoglycerate kinase (PGK)4. Furthermore, oocytes of adult5 and foetal6 human females, heterozygous for G6PD allelic genes, show activity for both fast and slow electrophoretic forms of the enzyme. In female kangaroos (Marsuipialia: Macropodinae) inactivation of the paternally derived X chromosome occurs in cells of various somatic tissues7 but the question of X chromosome activity in oocytes has not been resolved. We have now examined G6PD expression in ovaries of pouch young known to be heterozygous for the fast and slow forms of the enzyme, and show that only a single X chromosome—the maternally derived one—is active in ovarian cells.

The consequences of X-dosage deficiency in the germ line: Impaired development in vitro of preimplantation embryos from XO mice

Two-cell embryos from XO and XX mice were cultured for 3–4 days. The stage of development reached by the embryos was scored at intervals, using as criteria the number of cells, the degree of compaction, and the presence or absence of a blastocoele. The results show that the embryos from XO mice cleave, compact, and form blastocoeles later than do the embryos from XX mice. Furthermore, many of the XO-derived embryos failed to form blastocysts and became grossly abnormal. It is suggested that this overall impaired development is a consequence of X-dosage deficiency in the maternal germ line through the production of eggs which are deficient in X-linked products. In addition to this overall developmental impairment, a few embryos died at the eight-cell stage. These may have been YO embryos. The breeding characteristics of XO mice, XX↔XY female mouse chimeras, and the sterility of XO (Turner's syndrome) women are discussed in the light of these observations.