The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.     

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.     

Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter’s karyotype: a FISH analysis

Meiotic segregation of the sex chromosomes was analysed in sperm nuclei from a man with Klinefelter’s karyotype by three-colour FISH. The X- and Y-specific DNA probes were co-hybridized with a probe specific for chromosome 1, thus allowing diploid and hyperhaploid spermatozoa to be distinguished. A total of 2206 sperm nuclei was examined; 958 cells contained an X chromosome, 1077 a Y chromosome. The ratio of X : Y bearing sperm differed significantly from the expected 1 : 1 ratio (χ² = 6.96; 0.001 < P < 0.01). Sex-chromosomal hyperhaploidy was detected in 2.67% of the cells (1.22% XX, 1.36% XY, 0.09% YY) and a diploid constitution in 0.23%. Although the frequency of 24,YY sperm was similar to that detected in fertile males, the frequencies of 24,XX, 24,XY and diploid cells were significantly increased. A sex-chromosomal signal was missing in 4.26% of the spermatozoa. This percentage appeared to be too high to be attributed merely to nullisomy for the sex chromosomes and was considered, at least partially, to be the result of superposition of sex-chromosomal hybridization signals by autosomal signals in a number of sperm nuclei. The results contribute additional evidence that 47,XXY cells are able to complete meiosis and produce mature sperm nuclei. Received: 6 November 1996     

The cleavage rate of digynic triploid mouse embryos during the preimplantation period

Triploidy is a lethal condition in mammals, with most dying at some stage between implantation and term. In humans, however, a very small proportion of triploids are liveborn but display a wide range of congenital abnormalities. In particular, the placentas of human diandric triploid embryos consistently display “partial” hydatidiform molar degeneration, while those of digynic triploids generally do not show these histopathological features. In mice, the postimplantation development of diandric and digynic triploid embryos also differs. While both classes are capable of developing to the forelimb bud stage, no specific degenerative features of their placentas have been reported. Diandric triploid mouse embryos are morphologically normal while digynic triploid mouse embryos consistently display neural tube and occasionally cardiac abnormalities. Previously it was shown that the preimplantation development of micromanipulated diandric triploid mouse embryos was similar to developmentally matched diploid control embryos. In this study, the preimplantation development of micromanipulated digynic triploid mouse embryos is analysed and compared with that of diandric triploid mouse embryos in order to determine whether there is any difference in cleavage rate between these two classes of triploids. Standard micromanipulatory procedures were used to insert a female or a male pronucleus into a recipient diploid 1-cell stage embryo. The karyoplast was fused to the cytoplasm of the embryo by electrofusion. These tripronucleate 1-cell stage embryos were then transferred to pseudopregnant recipients and, at specific times after the HCG injection to induce ovulation, the embryos were recovered and total cell counts made. These results were plotted and regression lines drawn. An additional control group of embryos was subjected to similar micromanipulatory procedures to those used in the experimental study. These embryos had a single pronucleus removed and this was then reinserted into the perivitelline space. Diploidy was immediately restored by electrofusion. These embryos were transferred to recipients and at specific times after the HCG injection the embryos were recovered and total cell counts made. These results were also plotted and regression lines drawn. The results show that the cell doubling time of the digynic triploid embryos was 14.84 h (± 1.19). This was not significantly different from that of the diandric triploid embryos (13.55 h ± 0.86; P > 0.05) or of the manipulated diploid controls (12.12 h ± 0.79; P > 0.05). © 1993 Wiley-Liss, Inc.     

Les formules gonosomiques dites aberrantes chez les Mammifères Euthériens

The chromosome complement of the sloth Choloepus hoffmanni Peters has been investigated in mitosis and also in male meiosis. The karyotype for both males and females is characterized by a diploid number of 49 chromosomes. In the male the Y-material is translocated on an autosome but the meiotic behavior of the gonosomes is normal and therefore the sex determining mechanism may be normal too, despite the translocation. The females have an XO sex-chromosome constitution in somatic cells. An hypothesis, based on a slight deviation of a normal phenomenon is proposed to explain as regular such a formula in normal animals. — Relating to these conclusions, other known deviations of the standard XX/XY sex chromosome constitution in placental mammals are discussed (multiple sexchromosomes, composite gonosomes and XO female formula). The general conclusion is that despite an apparent variability of sex chromosome morphology, all placental mammals seem to retain a truly XX/XY sex constitution.     

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 digynic triploid mouse embryos

LT/Sv strain mice regularly ovulate up to 50% of their eggs as primary oocytes, which are fertilisable and give rise to digynic triploid embryos. A similar number of eggs are ovulated as secondary oocytes and, following fertilisation, give rise to normal diploid embryos. Pregnant LT/Sv females were autopsied at about midday on day 10 of gestation, when normal diploid embryos would be expected to possess between 25 and 30 pairs of somites. While a few of the triploid embryos either consisted of disorganised embryonic masses or were resorbing, most were at readily recognisable embryonic stages. Just over half of the embryos recovered were "unturned," while the remainder had "turned" and possessed between 15 and 25 pairs of somites. The triploids were usually readily recognised, owing to their small size and because they often displayed neural tube and cardiac defects. All of the embryos recovered were analysed cytogenetically by G-banding to establish their ploidy and sex-chromosome constitution. The XY:XX sex ratio of the 105 diploid embryos recovered, all of which had "turned," was 1.06:1, while the overall XXY:XXX sex ratio of the 120 triploids was 1:1. Analysis of only the developmentally most advanced triploid embryos (i.e., the 49 that had "turned") revealed that the XXY:XXX sex ratio in this group was 1.13:1, which was not significantly different from the expected ratio of 1:1. The crown-rump lengths of the XY and XX "turned" embryos were almost identical, as were those of the XXY and XXX "turned" embryos, although the triploids were significantly smaller than the diploids. No obvious effect of sex-chromosome constitution on developmental potential was therefore observed in this study in relation to either the digynic triploid or the control diploid embryos.     

Aneuploidy frequency in sperm of fertile men

Analysis of sperm aneuploidy in 11 healthy men using two-or three-color FISH permitted to determine the average frequency of disomy for chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy for chromosome 18 (0.05%) and to reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We assessed the frequency of meiotic nondisjunction of 13, 21, 18, X, and Y chromosomes and the frequency of XX, XY, and YY diploid spermatozoa. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some forms of chromosomal abnormalities in the off-spring is discussed.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

H-Y antigen expression in a case of XX true hermaphroditism

Summary Cells from an XX true hermaphrodite expressed a reduced amount of H-Y antigen when compared with normal XY cells and with cells from his father, who had an XY/XX chromosomal constitution. His mother had a normal karyotype and was H-Y negative. The four brothers of the patient were clinically and karyotypically normal. An X-Y interchange followed by random inactivation of the X chromosome is proposed to explain the H-Y antigen titer found in the patient.     

Heteromorphic sex chromosome system with an exceptionally large Y chromosome in a catfish Steindachneridion sp. (Pimelodidae)

The chromosomes and banding patterns of Steindachneridion sp., a large catfish (Pimelodidae), endemic to the Igua?u River, Brazil, were analyzed using conventional (C-, G-banding) and restriction enzyme banding methods. The same diploid number (2n = 56) as in other members of the genus and the family was found but the karyotype displayed an XX/XY sex chromosome system. The X chromosome was the smallest submetacentric, while the Y was the largest chromosome in the karyotype. Meiotic analysis showed 27 autosomal bivalents plus one heteromorphic XY bivalent during spermatogenesis. Sex chromosomes had no particular pattern after C-banding but G- and restriction enzyme bandings showed specific banding characteristics. The present finding represents the first report of a well-differentiated and uncommon sex chromosome system in the catfish family Pimelodidae.     

Development of an X-specific marker and identification of YY individuals in spinach

Spinach is a popular vegetable native to central and western Asia. It is dioecious with a pair of nascent sex chromosomes. The difficulties of working with the non-recombining sex determination region of XY individuals have hindered the progress toward sequencing sex chromosomes of most dioecious species. Here we present important advances toward characterizing the non-recombining sex chromosomes in spinach. Of nearly 400 spinach accessions screened, we identified a single accession of spinach in which androdioecious XY individuals segregate YY spinach. The male and female genomes of the spinach cultivar Shami and USDA accession PI 664497 were sequenced at 12–17?× coverage. X-specific sequences were identified by comparing the depth of coverage differences between male and female alignments to a female draft genome. YY individuals were used as a negative control to validate X-specific markers found by depth of coverage analysis. Of 19 possible X chromosome sequences found by depth of coverage analysis, one was verified to be X-specific by a PCR-based marker, SpoX, which amplified genomic DNA from XX and XY, but not YY templates. Androdioecious XY individuals of accession PI 217425 (Cornell #9) were used to develop inbred lines, and at S7 generation, all XY individuals were androdioecious and all YY individuals were pure male. The sex reversal of the XY mutant to hermaphrodite is strong evidence that the sex chromosomes in spinach have a two-gene sex determination system. These results are crucial towards sequencing the X and Y chromosomes to advance sex chromosome research in spinach.     

Sex ratio in early embryonal mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortions sensu stricto with a developed embryo without any significant intrauterine delay of development (n = 100), nondeveloping pregnancies (n = 176), and anembryonic fetuses (n = 66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and 46,XY karyotype. The presence of 46,XY may reflect specific genetic mechanisms of prenatal mortality of embryos with normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for non-developing pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of prevalence of embryos with 46,XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio (1.11) from the control group consisting of medical abortions. Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46,XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical "chances ratio" index also provided evidence that embryos with 46,XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.     

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.     

Fertility effects of the 14;20 Robertsonian translocation in cattle

Superovulation and embryo collection procedures were used to study the effect of the 14;20 Robertsonian translocation on fertility and embryo viability. Karyotypes were successfully completed on cells from 77 of the 279 embryos prepared for such analysis. Embryos from 4 cows heterozygous for the translocation were studied. Two bulls with the same condition were studied by using their semen in artificial insemination of cows with normal karyotypes. The proportions of fertilized ova and transferable embryos were not different between cows with the 14;20 translocation and those with normal karyotypes, indicating that fertilization rates were not affected by the translocation. Twenty-two percent of the embryos which were karyotyped had an unbalanced karyotype and would theoretically not have survived to term. All of the theoretically predicted chromosome complements from such a translocation were observed as were three 58,XX,t karyotypes and a 58,XX karyotype. There was no difference in the percentage of embryos with abnormal karyotypes whether the cow or bull was the carrier. Results therefore indicate that fertility is rather severely impaired in carriers of the 14;20 translocation, as was observed with the 1;29 translocation, with most loss due to embryo mortality rather than a lowered conception rate.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

About the sex ratio in connection with early embryonic mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortionssensu stricto with a developed embryo without any significant intrauterine delay of development (n=100), nondeveloping pregnancies (n=176), and anembryonic fetuses (n=66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and the 46.XY karyotype. The presence of 46.XY may reflect the specific genetic mechanisms of the prenatal mortality of embryos with the normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with the normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for nondeveloping pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of the prevalence of embryos with the 46.XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio from the control group consisting of medical abortions (1,11). Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46.XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical “chances ratio” index also provided evidence that embryos with the 46.XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.     

Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions

Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos. Received: 24 September 1997 / Accepted: 22 October 1997     

Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male

Meiotic segregation of gonosomes from a 46,XY/47,XXY male was analysed by a three-colour fluorescence in situ hybridisation (FISH) procedure. This method allows the identification of hyperhaploid spermatozoa (with 24 chromosomes), diploid spermatozoa (with 46 chromosomes) and their meiotic origin (meiosis I or 11). Alpha satellite DNA probes specific for chromosomes X, Y and 1 were observed on 27,097 sperm nuclei. The proportions of X-and Y -bearing sperm were estimated to 52.78% and 43.88%, respectively. Disomy (24,XX, 24,YY, 24,X or Y,+1) and diploidy (46,XX, 46,YY, 46,XY) frequencies were close to those obtained from control sperm, whereas the frequency of hyperhaploid 24,XY spermatozoa (2.09%) was significantly increased compared with controls (0.36%). These results support the hypothesis that a few 47,XXY germ cells would be able to complete meiosis and to produce mature spermatozoa.     

Isolation and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus L.)

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.