Sex Chromosome Constitution Research Articles

Combined immunophenotyping and FISH with sex chromosome-specific DNA probes for the detection of chimerism in epidermal Langerhans cells after sex-mismatched bone marrow transplantation.

Langerhans cells (LC) of the skin represent bone marrow-derived dendritic antigen-presenting cells and are therefore important in pathophysiological processes such as rejection, graft-versus-host disease, and graft-versus-leukemia-reaction after bone marrow transplantation (BMT). For understanding of these diseases, the evaluation of the chimeric status of LC following BMT is of great interest. To analyze the sex chromosome constitution of LC in the skin, we established a modified and refined technique of combined immunophenotyping and fluorescence in situ hybridization (FISH) and investigated frozen sections of skin biopsies from nine patients after allogeneic sex-mismatched BMT and of two healthy donors for control. LC were specifically labeled using a fluorescent CD1 a antibody and hybridized simultaneously with X and Y chromosome-specific DNA probes. The results of this practical application on nine leukemia patients show the appearance of donor-type LC and the persistence of host-type LC at various times (36 up to 1395 days) after sex-mismatched BMT. Complete chimerism of LC could not be detected in any case. The frequency of recipient-specific LC ranged from 7% to 92% and showed no correlation with time postgrafting. We conclude from our results of 1461 analyzed LC that combined immunophenotyping and interphase cytogenetic analysis by FISH is the method of choice for the assessment of chimerism in a particular cell type after sex-mismatched BMT. Its practical application on other tissues affected by BMT-related pathophysiological processes reveals further knowledge of the time-dependent course of chimeric patterns after BMT.

Neonatal Lupus Erythematosus in an Infant with Turner Syndrome

Neonatal lupus erythematosus (NLE) is characterized by transient, annular cutaneous lesions, congenital heart block, and a variety of systemic or hematologic abnormalities. We describe a white infant girl with onset of skin lesions on the face and scalp at 4 days of age. At age 4 weeks she had generalized, erythematous, scaly, annular skin lesions that underwent spontaneous regression at age 5 months. Her mother had no cutaneous or other lesions, but complement examinations revealed the presence of anti-Ro(SSA) and anti-La(SSB) antibodies, and absence of anti-Sm and anti-RNP antibodies. Karyotyping revealed Turner syndrome (TS) with 45,XO sex chromosome constitution. Ro(SSA) and La(SSB) antibodies were found, and direct immunofluorescence testing on healthy skin was positive. At age 5 months, follow-up immunologic examination of the infant had normal results but the mother still had anti-Ro(SSA) and anti-La(SSB) antibodies. We believe that this is the first reported case of NLE in association with TS.

Analysis of the sex chromosomal equipment in spermatozoa of a 47,XYY male using two-colour fluorescence in-situ hybridization.

The sex chromosomes in spermatozoa of a 47,XYY fertile male were analysed simultaneously by dual fluorescence in-situ hybridization (FISH), with two probes (pHY2.1 and pXBR). Of the 100000 cells analysed, 95179 spermatozoa (95.18%) exhibited one or more hybridization signals. Of the hybridized nuclei, 85.37% showed a normal sex chromosome constitution (37.37% X-bearing cells and 48.00% Y-bearing cells), with an X:Y ratio of 0.78:1. A total of 14.63% of the hybridized nuclei exhibited sex chromosome aneuploidy with a majority of XY- and YY-bearing spermatozoa (9.37 and 4.65% respectively). Even if the majority of spermatozoa have chromosomal haploidy, a large proportion of them exhibits numerical errors for the sex chromosomes. These observations raise questions about the commonly-admitted notions concerning the absence of chromosomal risk for XYY male offspring.

Twin pregnancies with complete hydatidiform mole and coexisting fetus: Use of fluorescent in situ hybridization to evaluate placental X- and Y-chromosomal content

Twin pregnancies with a complete hydatidiform mole (CHM) and a coexisting fetus have an aggressive postevacuation behavior; it is, therefore, important to differentiate these cases from partial hydatidiform moles that rarely require treatment for late sequelae. It has been presumed that twin pregnancies with a CHM and a coexistent fetus are dizygotic gestations, but this has not been confirmed in most cases. The authors investigated the sex chromosomal constitution of paraffin-embedded, formalin-fixed placental tissues in nine pregnancies histopathologically diagnosed as twin gestations with CHM and coexisting fetus, using fluorescent in situ hybridization (FISH) with X- and Y-chromosomal probes. Normal placental tissues showed an even sex distribution—four cases: X signal only, presumably female; four cases: X and Y signals, presumably male. In contrast, all molar tissues of these same pregnancies hybridized with the Xchromosomal probe only. Thus, in four of nine cases, gender differences (ie, different sex chromosome content) in molar villi (X chromosome only, cytogenetic female) versus normal villi (both sex chromosomes, cytogenetic male) confirmed the histopathological diagnosis of dizygotic twinning; a strict relationship between villous morphology (molar vs normal) and chromosomal gender was observed in each instance. This study illustrates that use of FISH on paraffin-embedded tissues can retrospectively establish dizygotic twinning in this unusual type of molar gestation.

Viability, development and incidence of chromosome anomalies of preimplantation embryos from XO mice.

We examined the viability and developmental status of XO embryos at preimplantation stage (day 3 of gestation) by assessing blastocyst formation and counting cell number using our XO mouse colony. We also examined the incidence of chromosome anomalies. Embryos from XO mice (XY, XX and XO) developed more slowly (in cell numbers and blastocoele formation) than those from XX mice (XY and XX). XO embryos also tended to develop more slowly than XX embryos in the XO group. Although litter size at the preimplantation stage of gestation was almost twice as large (12.7) as those at mid-gestation (7.6) and near-term (7.2) in this colony, the adjusted XY:XX:XO ratio (2.8:2.0:1.0) did not differ greatly. This indicates that almost half of the embryos must have been eliminated during the first half of gestation in the XO group, probably regardless of sex chromosome complements. Thus, we consider that maternal XO sex chromosome constitution is disadvantageous for the intrauterine development of the embryo during the early period of gestation. This may be related to precocious aging of XO mice. Further, we confirmed that a high incidence of abnormal karyotypes occurs in embryos from our XO mouse colony.

Recycling the single cell to detect specific chromosomes and to investigate specific gene sequences.

We have developed a new procedure, called cell recycling, which combines the two powerful techniques of polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on the same single cell. A fixed cell is used as the DNA template for PCR, prior to the FISH analysis. Using single blastomeres from mouse embryos as a model system, cell recycling procedures detect the single-copy beta-haemoglobin gene sequence at an efficiency of 70% as well as sex chromosome constitution at an efficiency of 74% in the same single cell. Cell recycling will increase the success rate of pregnancy following preimplantation diagnosis for a specific gene defect by identifying embryos with chromosomal abnormalities and eliminating them from the transfer procedure.

Chromosomal evolution and biogeography of collared lemmings (Dicrostonyx) in the eastern and High Arctic of Canada

Chromosomal variation was examined in 10 populations of Dicrostonyx in the eastern and High Arctic of Canada to determine the extent and nature of chromosomal variation and to examine chromosomal differentiation within and among species and subspecies. Chromosomal data are presented for two species of collared lemmings, D. hudsonius and D. groenlandicus, including D. g. groenlandicus, D. g. clarus, and D. g. lentus. Standard, G-banded, and C-banded karyotypes revealed variation among populations of D. groenlandicus due to Robertsonian fusions and the addition of B chromosomes. G-banding revealed complete homologous banding patterns of autosomal arms between D. groenlandicus and D. hudsonius; however, sex chromosome constitution differed between the two species. Dicrostonyx groenlandicus possesses a neo-XY, formed by a Robertsonian fusion of the sex chromosomes and a pair of autosomes. The neo-XY does not occur in D. hudsonius, and this absence appears to be the primitive state for the genus.

Comparative cephalometric analysis in patients with X-chromosome aneuploidy

The influence of abnormal sex-chromosome constitution on cranial growth, shape and position of the craniofacial structures was analysed. Reduced cranial growth was found in women with Turner's syndrome (45 XO chromosome constitution) and in men with Klinefelter's syndrome (47 XXY chromosome constitution). Lack of or an extra X chromosome produced opposite effects on cranial-base flexion, jaw displacement and maxillary and mandibular inclination to the anterior cranial base. An extra X chromosome affected deviation in sagittal jaw relation while lack of an X chromosome influenced mandibular shape.

Etiopathogenesis, classification, investigations and diagnosis in intersex disorders.

In the last years, advance~ in experimental endocrinology, biochemistry, genetics and molecular biology have all contributed to our understanding of the process of human sex differentiation. In brief, normal sex development consists of three sequential processes. The first is the genetic sex determined at the time of fertilization by the sex chromosome constitution. In the second phase, the genetic information determines that an indifferentiated gonad differentiates either in a testis or an ovary (gonadal sex). Finally, sexphenoO,pe is the result of male differentiation which is an active process resulting of testicular secretions: anti-Miillerian hormone (AMH) produced by the Sertoli cells which inhibits the Miillerian ducts, and testosterone (T) produced by the Leydig cells which is responsible for stabilization of the Wolffian ducts and via its transformation into dihydrotestosterone (DHT) is responsible for virilization of the external genitalia. In the absence of testis, the geni-

Reduced recombination and paternal age effect in Klinefelter syndrome.

The parental origin of the additional sex chromosome was studied in 47 cases with an XXY sex chromosome constitution. In 23 cases (49%), the error occurred during the first paternal meiotic division. Maternal origin of the additional chromosome was found in the remaining 24 cases (51%). Centromeric homo- versus heterozygosity could be determined in 18 out of the 24 maternally derived cases. According to the centromeric status and recombination rate, the nondisjunction was attributable in 9 cases (50%) to an error at the first maternal meiotic division, in 7 cases (39%) to an error at the second maternal meiotic division and in 2 cases (11%) to a nullo-chiasmata nondisjunction at meiosis II or to postzygotic mitotic error. No recombination, and in particular none in the pericentromeric region, was found in any of the 9 cases due to nondisjunction at the first maternal meiotic division. Significantly increased paternal age was found in the paternally derived cases. Maternal age was significantly higher in the maternally derived cases due to a meiotic I error compared with those due to a meiotic II error. There were no significant clinical differences between patients with respect to the origin of the additional X chromosome.

Sex chimaerism, fertility and sex determination in the mouse

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

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.

Chromosome studies in the red howler monkey, <i>Alouatta seniculus stramineus</i> (Platyrrhini, Primates): description of an X<sub>1</sub>X<sub>2</sub>Y<sub>1</sub>Y<sub>2</sub>/X<sub>1</sub>X<sub>1</sub>X<sub>2</sub>X<sub>2</sub>sex-chromosome system and karyological comparisons with other subspecies

In the red howler monkey, Alouatta seniculus stramineus (2n = 47, 48, or 49), variations in diploid chromosome number are due to different numbers of microchromosomes. Males exhibit a Y;autosome translocation involving the short arm of an individual biarmed autosome. Consequently, the sex-chromosome constitution in the male is X1X2Y1Y2, with X1 representing the original X chromosome, X2 the biarmed autosome (No. 7), Y1 the Y;7p translocation product, and Y2 the acrocentric homolog of 7q. In the first meiotic division, a quadrivalent with a chain configuration can be observed in spermatocytes. Females have an X1X1X2X2 sex-chromosome constitution. Chromosome heteromorphisms were observed in pair 13, due to a pericentric inversion, and pair 19, due to the presence of constitutive heterochromatin. Microchromosomes, which varied in number between individuals, were also heterochromatic. NOR-staining was observed at two separate sites on a single chromosome pair (No. 10). A comparison of A.s. stramineus with A.s. macconnelli shows that these two subspecies have identical diploid chromosome numbers (47, 48, or 49), again due to a varying number of microchromosomes, and that they share a similar sex-chromosome constitution. Their karyotypes, however, are not identical, but can be derived from each other by a reciprocal translocation. Further comparisons with other A. seniculus subspecies reported in the literature indicate that this taxon is not karyologically uniform and that substantial chromosome shuffling has occurred between populations that have been considered to be subspecies by taxonomic criteria based on their morphometric attributes.

Determination of sex chromosomal constitution and chromosomal origin of drumsticks, drumstick-like structures, and other nuclear bodies in human blood cells at interphase by fluorescence in situ hybridization

The sex chromosomal constitution has been determined in various types of human leukocytes at interphase by use of fluorescence in situ hybridization with X- and/or Y-specific DNA probes. It is found that during aging and differentiation of myelocytes into polymorphs there is no significant change in the relative frequency of various types of male and female cells with a specific type of sex chromosomal constitution. Non-random variability of the relative proximity between the X chromosomes within the nuclei is also observed in female cells. Moreover, we are the first to determine that sex-specific "drumsticks" and "sessile nodules" in female polymorphs originate from the X chromosomes and that non-sex-specific "drumstick-like" bodies in male polymorphs are of Y chromosomal origin.

Sex chromosome configurations in pachytene spermatocytes of an XYY mouse

Karyotypic investigation of a phenotypically normal but sterile male mouse showed the presence of an XYY sex chromosome constitution. The synaptic behaviour of the three sex chromosomes was examined in 65 pachytene cells. The sex chromosomes formed a variety of synaptic configurations: an XYY trivalent (40%); an XY bivalent and Y univalent (38.5%); an X univalent and YY bivalent (13.8%); or X, Y, Y univalence (7.7%). There was considerable variation in the extent of synapsis and some of the associations clearly involved nonhomologous pairing. These observations have been compared with previously published information on chromosome configurations at metaphase I from other XYY males.

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.

Analysis of the sex chromosome constitution of single bovine spermatozoa by polymerase chain reaction (PCR)

Analysis of the sex chromosome constitution of single bovine spermatozoa by polymerase chain reaction (PCR)

Triploidy and intersexuality in adult commercial layers

The cause of masculinisation of the left ovary and the outgrowth of the vestigial right gonad was investigated in intersexual hens. Tumour-like cell masses, resembling mouse tubular adenomas of the ovary, were observed in the majority of masculinised left gonads. Except for one male and two intersexuals, testosterone concentrations were below detectable levels. For oestrogen, progesterone and the oestrogen : progesterone ratio all differences were significant, except for the difference between the intersex and the male. Histochemically this cell mass showed weak androgen-synthesising activity. These intersexual gonads showed similarities to normal testicular tissue. Karyotyping revealed 3n-autosomes and a ZZW sex chromosome constitution. The early and almost complete absence of cortical follicular structures was most notable and may have been the cause of the sex reversal.

Sex-chromosome constitution of postimplantation tetraploid mouse embryos

Tetraploid mouse embryos were produced at the two-cell stage by blastomere fusion induced by inactivated Sendai virus. The embryos were from chromosomally normal female mice that had been fertilised by homozygous Rb(1.3)1Bnr males carrying a pair of large metacentric marker chromosomes in their karyotype. These "reconstructed" one-cell tetraploid embryos were then transferred to the oviducts of pseudopregnant recipients, which were subsequently autopsied early on the 10th day of gestation. Two-cell stage embryos that did not undergo blastomere fusion after 4-5 h were transferred to a second group of recipients, which were also autopsied early on the 10th day of gestation. From a total of 153 tetraploid embryos transferred to females that subsequently became pregnant, 135 implanted. Sixty-eight implantation sites were found to contain resorptions, whereas 67 contained mostly headfold presomite-stage embryos. Four embryos possessed four to six pairs of somites. All 57 embryos that could be analysed cytogenetically were found to be tetraploid. G-banding analysis revealed that 30 of these embryos had an XXYY and 27 and XXXX sex-chromosome constitution. The presence of two marker chromosomes in all mitotic preparations from each of these tetraploid embryos confirmed that they had all been produced by duplication of their original XY or XX diploid chromosome constitution, respectively. The XXYY:XXXX sex ratio observed was not significantly different from unity. In the control series of transfers, all of the embryos recovered were at the forelimb bud stage and had a diploid chromosome constitution. The results reported here differ from human clinical findings, in which the XXYY:XXXX sex ratio of 120 human tetraploid spontaneous abortions recovered over the last 20 years is 45:75. Possible explanations for these differences are briefly discussed.