Cytogenetic study of some tissues and age-related changes in cell proportions in a goat-sheep chimera.

Different percentages of cells with a female sheep or male goat karyotype were found in kidney (12.0% vs. 88.0%) and lung (42.6% vs. 57.4%) cell cultures from a 10-year-old chimera. Skin biopsies from patches with goat hair or sheep wool showed different, age-related goat-to-sheep fibroblast ratios. Karyotypic analysis of lymphocytes obtained from peripheral blood of the chimera at 6 and 10 yr of age showed no chimerism. Two weeks after birth, however, lymphocytes with both sheep (54,XX) and goat (60,XY) karyotypes were apparent in the blood of this chimera. Twenty percent of the blood cells examined at 2 wk had a caprine karyotype; this proportion declined with time, until it was totally eliminated at age 6.     

Erroneous genetic sex determination of a newborn twin girl due to chimerism caused by foetal blood transfusion. A case report

OBJECTIVE: We present a case of erroneous sex determination in a newborn twin girl (twin A) due to chimerism. CASE REPORT: Amniocentesis and ultrasound examination had pointed towards male sex of both twins. At birth, twin A presented as a phenotypically normal female with 46,XY karyotype, and 46,XY gonadal dysgenesis was suspected. Twin B was a normal male. RESULTS: In our department, further examinations of twin A included undetectable testosterone and inhibin-B and elevated FSH. Ultrasound suspected an infantile uterus, and sequencing of the SRY gene was normal. After gonadectomy, a 46,XX karyotype was demonstrated in both normal infantile ovaries and in the fibroblasts from a skin biopsy. Analysis of X-linked markers in DNA from blood lymphocytes in both twins was identical, consistent with 46,XY karyotypes. CONCLUSION: Twin A is a 46,XX female with a chimeric 46,XY blood cell line due to intrauterine transfusion from her twin brother.     

A successful second delivery outcome using refrozen thawed testicular sperm from an infertile male true hermaphrodite with a 46, XX/46, XY karyotype: case report

We report a successful second delivery of a healthy infant fathered using refrozen thawed testicular sperm from an infertile male chimera. We also examined sex chromosome distribution of the seminiferous tubule. Intracytoplasmic sperm injection (ICSI) was performed using the remaining refrozen testicular sperm, which had been stored during the first treatment. Biopsied testicular cells were examined by fluorescence in situ hybridization (FISH) and the peripheral lymphocyte karyotype was tested using a G-band. Following ICSI, a second pregnancy was established, and a healthy girl was successfully delivered at 40 gestational weeks without complications. Although the husband’s lymphocyte chromosomal analysis revealed a 46, XX [28]/46, XY [2] karyotype, the seminiferous tubule cells on histological examination by FISH were chimeric sex chromosome type XX [18]/XY [82]. In conclusion, this is a very rare case report of a successful subsequent delivery of a healthy infant (46, XX) from an infertile true hermaphrodite (46, XX/46, XY) using refrozen thawed testicular sperm. The seminiferous tubule cells’ karyotype ratio differed from that of the lymphocytes.     

Case report: a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization

We describe a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization. The patient achieved a second pregnancy at 35 years of age. The her blood lymphocyte karyotype was examined by G-band and FISH. Furthermore, cumulus cells and her elbow skin cells were evaluated via FISH. Non-mosaic Turner syndrome was determined by G-banding [100 % (50/50) 45, X]. Lymphocytes were shown as 478/500 (95.6 %) cells of X sex chromosome signal, 15/500 (3.0 %) cells of XXX signal, and 7/500 (1.4 %) cells of XX signal. The cumulus cells were mosaic: 152/260 (58.5 %) were X; 84/260 (32.3 %) were XXX, 20/260 (7.7 %) were XX, and 4/260 (1.5 %) were XY. Moreover, skin cells included a mosaic karyotype [47, XXX(29)/46, XX(1)]. We conclude that the collection of a large number of blood lymphocytes can reveal different mosaic patterns (X, XX and XXX) by FISH in spite of non-mosaic Turner syndrome.     

A case of true hermaphroditism reveals an unusual mechanism of twinning

Traditionally twins are classified as dizygous or fraternal and monozygous or identical (Hall Twinning, 362, 2003 and 735-743). We report a rare case of 46,XX/46,XY twins: Twin A presented with ambiguous genitalia and Twin B was a phenotypically normal male. These twins demonstrate a third, previously unreported mechanism for twinning. The twins underwent initial investigation with 17-hydroxyprogesterone and testosterone levels, pelvic ultrasound and diagnostic laparoscopy. Cytogenetic analysis was performed on peripheral blood cells and skin fibroblasts. Histological examination and Fluorescence in situ hybridization studies on touch imprints were performed on gonadal biopsies. DNA analysis using more than 6,000 DNA markers was performed on skin fibroblast samples from the twins and on peripheral blood samples from both parents. Twin A was determined to be a true hermaphrodite and Twin B an apparently normal male. Both twins had a 46,XX/46,XY chromosome complement in peripheral lymphocytes, skin fibroblasts, and gonadal biopsies. The proportion of XX to XY cells varied between the twins and the tissues evaluated. Most significantly the twins shared 100% of maternal alleles and approximately 50% of paternal alleles in DNA analysis of skin fibroblasts. The twins are chimeric and share a single genetic contribution from their mother but have two genetic contributions from their father thus supporting the existence of a third, previously unreported type of twinning.     

核型异常的两性畸形山羊2例报告

本文报道2例两性畸形的山羊,它们均具雌雄两套生殖器官,染色体组型分析显示它们均为性染色体嵌合体,核型为60,XX/XY,两头畸形山羊的XX细胞系的比例分别为42%和54%,XY细胞系的比例分别为58%和46%。 Abstract:We herein report two cases of goats with sexual deformity.They had two systems of reproductive organs and the karyotype analysis showed mosaicism for their sex chromosomes,i.e.,60,XX / XY.The proportion of XX cell line was 42% and 54%,while XY cell line was 58% and 46%,respectively,in the two abnormal goats.     

家兔早期胚胎细胞发育能力的研究

The developmental potential of rabbit embryonic cells was studied through making chimera by separate introduction of inner cell mass from 96-h-old p. c., 120-h-old p. c., and 144-h-old p. c. of grey rabbits into 96-h-old p. c. blastocysts of New Zealand white rabbits. A total of five overt chimeras were obtained including two fertile males, two fertile females and one sterile male, from the ICM cells of 96-h-old and 120-h-old embryos but none was obtained from 144-h-old cells. Histological examination of the gonad showed that the sterile chimera derived from 120-h-old ICM cells with an ovotestis on both sides. Follicles and seminiferous tubules developed in the cortex and medulla of the gonad, respectively. Neither of them developed into functional germ cells. Analysis of karyotypes of peripheral blood showed that both XX and XY coexisted in lymphocytes. These results indicated that the sterile male chimera was a XX/XY sex chimera derived from ICM cells of donor and recipients with different sex, so as to the chimera with XX and XY genotypic cells. From the results mentioned above we may conclude that the ICM cells at 120-h-old p. c. are still pluripotential, they can not only participate in development into somatic components but also develop into germ cells. The potential of 144-h-old p. c. ICM cells seems to be rather restricted.     

家兔早期胚胎细胞发育能力的研究

本文利用胚泡注射法制作嵌合体对家兔交配后96,120和144小时的ICM细胞的发育能力进行了研究。供体胚胎取自青紫兰灰免,受体胚胎取自新西兰白兔,结果表明96和120小时供胚的ICM细胞与96小时受胚胚泡组合后均能参与发育,形成嵌合兔,144小时者未获得嵌合体。由于120小时的ICM细胞发育的2只表型为雄性的嵌合兔,其中1只不育,其性腺和外周血核型表明不育兔为xx/xy性嵌合,性腺中有处于不同发育程度的卵巢和精细管,外周血含xx和xy两种核型。本实验结果首次证明家兔交配后120小时胚泡的ICM细胞仍具有参与嵌合体发育的能力。它不仅能参与体细胞的分化,并具有形成生殖细胞的能力。交配后144小时胚泡的ICM细胞其发育能力似乎已发生了局限。     

Investigation of genetic markers in a true Hermaphrodite with chi 46,XX/46,XY

Summary We documented a new case of chi 46,XX/46,XY true hermaphroditism substantiated by the evaluation of chromosomal heteromorphism in banded preparations. The patient, a 12-year-old Japanese boy with ambiguous external genitalia, was seen because of abnormal breast development. Surgical exploration showed the right gonad to be an ovotestis and the left gonad to be an ovary. Cytogenetic studies revealed cell admixtures of 46,XX and 46,XY karyotypes in peripheral lymphocytes, skin fibroblasts, and gonadal fibroblasts. From the pedigree studies, the paternal double genetic contributions were evidenced by the differences of sex chromosomes and the blood group types for the ABO and MNSs systems in the two cell lines of the patient. The maternal double genetic contributions were confirmed by the inheritance of Q-fluorescent markers on chromosomes 13 and 22 and by alleles for the Kidd blood group system.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Mosaic variegated trisomy (42,XY/43,XY, + variable) in a male pigtail macaque monkey

We identified an infant male pigtail macaque monkey with a bizarre karyotype which, to the best of our knowledge, has never before been reported in any species. Examination of 107 nuclei from cultured lymphocytes revealed 81 (75.7%) to be trisomic, but with the supernumerary chromosome varying from cell to cell, trisomy 16 being the most common. A small percentage (11.2%) of the nuclei had a normal 42,XY karyotype, and the balance, with the exception of one apparent monosomic (possibly a technical artifact), had multiple chromosome abnormalities. Examination of cultured skin fibroblasts revealed a similar karyotype. We called this karyotype a mosaic variegated trisomy. At birth, the animal had a cleft lip and palate and situs inversus of the heart. He subsequently showed significant developmental delay and apparent mental retardation. There were no clinical symptoms of hematological malignancy, which often have associated acquired chromosome abnormalities such as those described here. The animal survived for 2 yr and 8 mo under intensive care.     

Germ cell chimerism in male marmosets

Marmosets normally produce biovular twins which are connected, via placental vascular anastomoses, as early as pre-somite stages of development. These anastomoses allow the exchange of lymphoid and hematopoietic tissue, as demonstrated by karyotype analysis in heterosexual twins. Because germ cells are also motile during development, i.e., they migrate from the yolk sac endodermal epithelium to the germinal ridges, the possibility exists that germ cells could also be exchanged between heterosexual twins. Testicular squash preparations were examined from 22 adult male marmosets of several species. During the diakinesis stage of meiotic prophase the XY chromosome pair was distinct. Spermatocytes which lacked the conspicuous end-to-end association of the XY pair were considered to have originated from germ cells with an XX sex chromosome constitution. In approximately half the animals examined, all diakinesis figures contained an end-to-end XY pair. In the remaining animals, primary spermatocytes were seen that clearly did not contain an endto-end XY pair. The largest number of such XX primary spermatocytes in any one animal was 21% (9 of 43 cells examined). The effects of germ cell chimerism on the sex ratio of offspring was also investigated.     

Rare case of XX/XY mosaicism and trisomy 13 in early prenatal diagnosis

Coexistence of XX/XY sex mosaicism and autosomal trisomy in prenatal diagnosis is particularly rare. Herein, we report the first, to our knowledge, case of a fetus with cyclopia, ambiguous genitalia and a 47,XX,+13,inv9[47]/47,XY,+13[13] karyotype detected at 13 weeks of gestation after chorionic villus sampling. Molecular analysis after prenatal diagnosis suggests that this is a case of sex mosaicism coexisting with trisomy 13, rather than chimera.     

A cytogenetic survey of 200 unclassifiable mentally retarded children with congenital anomalies and 200 normal controls

Summary A cytogenetic survey was carried out on 200 patients with mental retardation and multiple congenital anomalies, and on 200 normal adult controls. Patients with a known syndrome were excluded from the survey. Chromosome analyses were carried out on blind-coded slides using the ASG banding technique as the routine stain. After the initial analyses (at least 15 cells per person) the slides were decoded, destained and reused for C and Q band polymorphism studies.Five major chromosome abnormalities were detected in the patient group during the survey. They included three patients with de novo, apparently balanced, reciprocal translocations, karyotypes 46,XY,rcp(3;16)(q21;p12); 46,XX,rcp(5;8)(p15;q22); and 46,XX,rcp(5;12)(p11;q24); one with karyotype 47,XX,+mar and one with karyotype 46,XX,der(13),t(13;?)(q34;?). One additional patient whose karyotype in lymphocytes was 46,XX,inv(9)(p11;q13) was found to have a mosaic karyotype 46,XX,inv(9)(p11;q13)/46,XX,inv(9) (p11;q13),der(12),t(12;?)(p13;?) in cultured skin fibroblasts. None of the 200 controls had a major chromosome abnormality.From the combined results of this and previous surveys it is now apparent that about 6.2% of the unclassifiable mentally retarded patients with three or more congenital anomalies and about 0.7% of the controls reveal major chromosome abnormalities.     

The origin of 45,X males.

Maleness in association with the karyotype 45,X is a very rare and hitherto unexplained condition previously described in only four or five patients. This study was carried out to determine whether such males might actually possess Y-chromosomal material. Of the two 45,X males studied, one was found to be a low-grade mosaic with a 46,XY karyotype in less than 3% of fibroblasts; all lymphocytes karyotyped were 45,X. Fibroblast DNA from this individual was found to contain Y-specific repeated sequences in 1%-3% the amount observed in the father, consistent with mosaicism for a 46,XY cell line. No Y-specific repeated sequences were detected in the other patient, in whom all mitoses were 45,X. In neither patient were there detectable amounts of any of the single-copy Y-specific DNA sequences for which we tested. Studies of Xg blood groups and of X-linked restriction fragment length polymorphisms indicated that the single X chromosome was of maternal origin in both 45,X male probands. In contrast to the situation in XX males, we can exclude paternal X-Y interchange as the etiology in the cases described here. Our findings are compatible with mosaicism being the explanation of at least some "45,X" males.     

Gonadal development and growth in 46,XX and 46,XY individuals with P450scc deficiency (congenital lipoid adrenal hyperplasia).

We have investigated gonadal development and growth in 4 individuals (3 with 46,XY and 1 with 46,XX karyotype) with P450scc deficiency. One patient died at 2 months of age from adrenal insufficiency, while the remaining 3 individuals were healthy and developed normally (age at follow-up: 18, 10 and 8 years). In the surviving individuals, the diagnosis was established during the first 2-4 months of life by extensive endocrine studies of blood and urine. In the remaining patient, the diagnosis was made on the basis of karyotype (46,XY), anatomy of internal and external genitalia and adrenal pathology. Gonadectomy was performed in the 2 surviving 46,XY individuals at the age of 7 years, and histological examination showed normal testicular morphology but very few germ cells. Postmortem examination of the testes of the 2-month-old subject showed normal testicular histology, and quantitative analysis revealed a normal number of germ cells. Ultrasound of the 46,XX individual showed normal internal female genitalia including ovaries with follicles. The 3 surviving patients grew along the 75th (46,XY), the 90th (46,XY) and the 50th percentile (46,XX), respectively. The oldest girl experienced normal breast and pubic hair development after oral estrogen replacement and topical testosterone administration. The glucocorticoid and mineralocorticoid replacement was adjusted in accordance with repeated measurements of serum sodium and serum potassium, plasma renin concentration and blood pressure. No attempts were made to normalize serum ACTH. We conclude that prenatal testicular maturation and development of female internal genitalia may take place in the absence of normal steroid hormone production. Normal growth and development may be obtained in P450scc-deficient individuals with adequate hormone replacement.     

Genetic determination of fragile-site expression.

To explore genetic effects on the expression of chromosomal fragile sites in vitro, we studied the expression of common fragile sites (c-fra) in cultured lymphocytes of a human chimera (Chi46,XX/46,XY). Since the two cell lines in the chimera share the same environment in vitro and in vivo on cell culture preceding chromosome analysis, differences in the expression of c-fra must be due to genetic factors. The peripheral lymphocytes were cultured in medium 199 and in medium RPMI 1640 with and without aphidicolin. All lesions were localized after GTG-banding and mapped to the human idiogram. In the cultures with aphidicolin the XX cells showed, at high (0.4 microM) APC levels, a significantly higher expression of c-fra than did the XY cells. This difference between cell lineages was not confined to certain individual c-fra; rather it was seen for practically all of them. Therefore, we conclude that there are genetic factors which influence the propensity of c-fra to be expressed. Whether sex is one of these factors, or perhaps even the most important one, still has to be elucidated.     

Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

Sequential Cross-Species Chromosome Painting among River Buffalo,Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.