Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.     

Gilts and sows produce similar rate of diploid oocytes in vitro whereas the incidence of aneuploidy differs significantly

Oocytes derived from prepubertal gilts show reduced developmental competence when compared to oocytes collected from adult sows. Therefore, the aim of the study was to investigate whether gilts (4-5 months old) and adult sows (average age 3.5 years) of the same breed (Polish Landrace x Polish Large White crossbred) differ with regard to the rate of chromosomally unbalanced oocytes after IVM. COCs derived from individual pairs of slaughterhouse ovaries were matured in vitro and analyzed cytogenetically by conventional staining (Giemsa) and FISH methods (probes corresponding to centromeric regions of pig chromosomes 1 and 10). Altogether, 72 females (31 sows, 41 gilts) and 430 secondary oocytes (194 and 236 oocytes of sows and gilts, respectively) were investigated. Cytogenetic analysis revealed diploid (Giemsa, FISH) and aneuploid (FISH) spreads. The incidence of diploid oocytes was similar for sows (26.0%) and gilts (24.5%) whereas the rate of aneuploid oocytes (nullisomic/disomic) was eight times higher in gilts (10.8%) than in sows (1.3%). Diploid and aneuploid oocytes were observed in 64% of investigated females. Pig chromosome 10 was more frequently disomic/nullisomic compared to chromosome 1 suggesting, that like in human, small porcine chromosomes are often involved in the nondisjunction process. In conclusion, chromosomal imbalance significantly contributes to in vitro embryo production in the pig, since over 60% of females produced diploid or aneuploid gametes. The significantly higher rate of aneuploidy among oocytes derived from gilt ovaries may contribute to the reduced developmental competence of gametes collected from nonmature female pigs.     

Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm.

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

Aneuploidy detection in porcine embryos using fluorescence in situ hybridization

In contrast to human embryos, there are very few studies published on the frequency of chromosomal aneuploidy in farm animals. The objectives of this study were to apply a three-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in porcine embryos using chromosome-specific DNA probes, establish baseline frequencies of aneuploidy in embryos and compare the results with our previous findings of aneuploidy in spermatozoa and oocytes. The embryos were collected from superovulated gilts, which were slaughtered 48 h after insemination. FISH was performed using probes specific for the centromeric regions of porcine chromosomes 1, 10 and Y. Altogether 403 blastomeres from 114 porcine embryos were successfully investigated. Diploidy was observed in 101 (88.6%) embryos, triploidy in 2 (1.8%) embryos, mosaicism/mixoploidy in 9 (7.9%) embryos, and trisomy for chromosomes 1 or 10 in 2 (1.8%) embryos. No blastomere showed aneuploidy for chromosome Y. These findings correspond with the frequencies of aneuploidy we have found previously in porcine germ cells.     

Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients

To further investigate our finding of high levels of spontaneous aneuploidy in somatic cells of Alzheimer's disease (AD) patients (Migliore et al. 1997), we studied the molecular cytogenetics of eight patients with sporadic AD and six healthy controls of similar age. Cytochalasin B-blocked binucleated peripheral blood lymphocytes from the AD patients and unaffected controls were used to measure micronucleus induction or other aneuploidy events, such as the presence of malsegregation in interphase nuclei (representing chromosome loss and gain). Dual-color fluorescence in situ hybridization (FISH) with differential labeled DNA probes was applied. We used a probe specific for the centromeres of chromosomes 13 and 21 combined with a single cosmid for the Down's syndrome region (21q22.2) to obtain information on spontaneous chromosome loss and gain frequencies for both chromosomes (13 and 21). FISH data showed that AD lymphocytes had higher frequencies of chromosome loss (evaluated as fluorescently labeled micronuclei) for both chromosomes, as well as higher frequencies of aneuploid interphase nuclei, again involving both chromosomes, compared to control lymphocytes. However, aneuploidy for chromosome 21 was more frequent than for chromosome 13 in AD patients. This preferential occurrence of chromosome 21 in malsegregation in somatic cells of AD patients raises the hypothesis that mosaicism for trisomy of chromosome 21 could underlie the dementia phenotype in AD patients, as well as in elderly Down's syndrome patients.     

A fluorescent in situ hybridization analysis of X chromosome pairing in early human female meiosis

Fluorescent in situ hybridization (FISH) utilizing an X chromosome whole library probe was used directly to assess the rate of aneuploidy and pairing behavior of the X chromosome in human female meiosis. Over 3000 meiotic cells obtained from fetal ovaries (gestational age 13–22 weeks) were scored for meiotic stage and evaluated for pairing abnormalities. No pairing anomalies were observed in 832 pachytenes. Twenty-two percent (88/398) of cells in zygotene were partially paired, but nonhomologous pairings could not be identified. One aneuploid preleptotene oocyte, presumably from mitotic nondisjunction was detected. To our knowledge, this is the first report of the use of FISH utilizing whole chromosome probes to evaluate the pairing behavior of chromosomes in human female meiosis. The application of this technique to study the relationship between nondisjunction and chromosome pairing behavior in maternal-age-related aneuploidy is discussed.     

Identification of Chromosomal Errors in Human Preimplantation Embryos with Oligonucleotide DNA Microarray

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.     

多色荧光原位杂交检测小鼠精子非整倍体

比多色荧光原位杂交技术评价了２－（４－噻唑）苯丙咪唑（ＴＢ）在雄性小鼠生殖形成过程中的非整倍体诱发效应。根据小鼠精子发育周期,以口饲法处理动物１１天,间隔２２天后取小鼠精子涂片,聚合应用８号,Ｘ及Ｙ染色体特异性ＤＮＡ探针进行多色ＦＩＳＨ,检测精子中出现的二倍体、双体、和缺本频率。结果发现：在２００ｍｇ／ｋｇ剂量组,上述３类异常精子频率均显著高于溶剂对照,其他２个剂量组的非整倍体精子频率与对照无显著     

Frequency of aneuploidy in in vitro-matured MII oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds as determined by dual-color fluorescent in situ hybridization

The current study was undertaken to investigate the aneuploidy rates in in vitro-matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro-matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using “Xcen” and “5” chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was “nullisomic” for chromosome X (1.0%) and one “disomic” for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy + disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1 × 30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the “baseline” level of aneuploidy in the in vitro-matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazards.     

Comparison of aneuploidy frequencies between in vitro matured and unstimulated cycles oocytes by metaphase comparative genomic hybridization (mCGH)

A common observation after in vitro matured oocyte is that they yield poorer embryo quality compared to their in vivo counterparts. This study was designed to assess chromosomal status with metaphase comparative genomic hybridization after in vitro maturation (IVM) in unstimulated cycles and compare the results with those obtained after in vivo maturation. Patients without any obstetrical or gynecological pathology were admitted into the study. IVM oocytes were collected 36 h post hCG and matured in vitro at 37°C in 5% O₂, 6% CO₂, and 89% air for 36 h. All matured (metaphase II) oocytes were subject to polar body 1 (PB-1) biopsy and vitrified individually. PB-1 samples were transferred into 0.25 cc PCR tubes containing 2.5 μl of PBS. PB-1 samples from 12 IVM patients were studied. Twenty-six out of 63 PB-1 samples (41%) were determined as euploid and 37 samples (59%) were aneuploid, whereas these values were 42% euploid and 58% aneuploid in the control group (in vivo matured oocytes). No statistical differences were found between the IVM and the control groups for euploid–aneuploid samples (P = 0.900). More aneuploidy was observed on chromosomes 11, 13, 15, 21, and 22 after IVM. Results show a non-significant rate of abnormal PB-1 formation after IVM compared to in vivo maturation. More aneuploidy was observed in chromosomes 11, 13, 15, 21, and 22 in the IVM group.     

Aneuploidy in pig sperm: multicolor fluorescence in situ hybridization using probes for chromosomes 1, 10, and Y.

The objective of this research was to develop chromosome-specific probes for use in evaluating aneuploidy in boar spermatozoa through the application of fluorescence in situ hybridization (FISH) technology. A multicolor FISH method was developed to detect aneuploidy in the sperm of boars using DNA probes specific for small regions of chromosomes 1, 10, and Y. The average frequencies of sperm with disomy for chromosomes 1, 10, and Y were 0.075%, 0.067%, and 0.094%, respectively. The incidence of disomy did not differ significantly by chromosome. The average frequencies of diploidy were 0.177% for 1-1-10-10 and 0.022% for Y-Y-10-10. Thus, the incidence of overall diploidy (1-1-10-10) was significantly higher than that of disomy for the chromosomes examined (P < 0.01 for disomy of the autosomes and P < 0.05 for disomy of the Y chromosome). No significant age or breed effects on disomy and diploidy rates and no significant interindividual variations in disomy or diploidy were found. The observed level of numerical chromosome aberrations in pig sperm appear to be within the range of the baseline frequencies reported so far in men.     

Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.     

Tyramide signal amplification (TSA)-FISH applied to mapping PCR-labeled probes less than 1 kb in size.

Tyramide signal amplification (TSA)-FISH was used to map one mouse and two human DNA probes of less than 1 kb in size. The two human probes were 319 and 608 bp, and the mouse probe was 855 bp. Probes, made from PCR products, were labeled by incorporating biotin-11-dUTP (human) and biotin-16-dUTP (mouse) during PCR amplification. Signals were readily observed in both interphase and metaphase cells following TSA-FISH for all three genes, whereas conventional FISH experiments produced no signals. The two human ATP-binding cassette (ABC) genes, EST883227 (GenBank Accession No. AA243820) and EST990006 (GenBank Accession No. AA348546), mapped to human chromosomes 7p21 and 17q25. The mouse gene, cmyc (exon 2) mapped to band D2 of mouse chromosome 15. These findings demonstrate the ability of this technique to map small probes (PCR products and expressed sequence tags) of less than 1 kb through highly increased signal amplification.     

Localization of the gene encoding myelin basic protein to mouse chromosome 18E3----4 and rat chromosome 1p11----p12.

The location of a gene encoding myelin basic protein in rat (MBP) and mouse (Mbp) was determined by in situ hybridization using the mouse Mbp cDNA labeled with biotin-11-dUTP as a specific probe. The localization of biotin signals in the mouse was found on Chromosome 18E2----3. The result is consistent with the previous report that the Mbp gene is located on the distal half of Chromosome 18. In the rat, the signals localized on chromosome 1p11----p12, suggesting homology between mouse Chromosome 18 and the short arm of rat chromosome 1.     

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of CRASSOSTREA: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)--the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)--the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of CRASSOSTREA: This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.     

Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes

To increase our understanding about the potential risks of chemically-induced aneuploidy, more information about the various mechanisms of aneuploidy induction is needed, particularly in germ cells. Most chemicals that induce aneuploidy inhibit microtubule polymerization. However, taxol alters microtubule dynamics by enhancing polymerization and stabilizing the polymer fraction. We tested the hypothesis that taxol induces meiotic delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Super-ovulated ICR mice received 0 (control), 2.5, 5.0, and 7.5 mg/kg taxol intraperitoneally immediately after HCG. Females were paired (1:1) with males for 17 h after taxol treatment. Mated females were given colchicine 25 h after taxol and their one-cell zygotes were collected 16 h later. Ovulated oocytes from non-mated females were collected 17 h after taxol. Chromosomes were C-banded for cytogenetic analyses. Oocytes were also collected from another group of similarly treated females for in situ chromatin and microtubule analyses. Taxol significantly (p<0.01) enhanced the proportion of oocytes exhibiting parthenogenetic activation, chromosomes displaced from the meiotic spindle, and sister-chromatid separation. Moreover, 7.5 mg/kg taxol significantly (p<0.01) increased the proportions of metaphase I and diploid oocytes and polyploid zygotes. A significant (p<0.01) dose response for taxol-induced hyperploidy in oocytes and zygotes was found. These results support the hypothesis that taxol-induced meiotic delay and spindle defects contribute to aneuploid mouse oocytes and zygotes.     

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.     

Etude de l'aneuploïdie dans différents naissains d'Ostreidae (Bivalvia)

Study of aneuploidy in spats of Ostreidae (Bivalvia). Chromosomes of cells from gill tissue of juveniles (5–10 mm) from four different populations of Ostrea edulis and five different populations of Crassostrea gigas were examined in order to study aneuploidy and its significance. Mitotic chromosome counts were made for a sample of spats in each population. Cells with the normal diploid complement (2n=20) and with aneuploid complements (2n different from 20) were scored. The total percentage of aneuploid cells in the studied populations of the two species varies from 9% to 34%. Individual patterns of aneuploidy were examined in three populations of Crassostrea gigas. Some animals showed only normal diploid cells, others having both normal and aneuploid cells in variable proportions. The relationship between aneuploidy and growth rate was studied in experimental spats. Mitotic chromosome counts were made on individuals from two groups of animals: the first group was collected at a precise date at a control size of 8 mm, the second group reaching the same size of 8 mm only one month later. The percentage of animals showing aneuploid cells is much greater in the second group. There is a relationship between occurrence of aneuploid cells and growth rate. The karyotypes of 18 aneuploid sets of Ostrea edulis from different spats were analyzed. Chromosome loss was observed only in the submetacentric chromosome pairs. This loss of chromosomes could have an effect on the percentage of homozygotes observed in electrophoregrams. Thus, the percentage of homozygotes would be higher in juveniles showing the greatest number of aneuploid cells. An excess of homozygosity (=heterozygote deficiency) has been reported at a number of enzyme loci in over two dozen bivalve species (including Ostrea and Crassostrea). We suggest the hypothesis that this excess of homozygosity could be related to the occurrence of aneuploid cells. The relationship between aneuploidy and growth rate may prove to be a genetic factor of importance for oyster culturing.     

Multi-locus (ML)-FISH is a reliable tool for nondisjunction studies in human oocytes

In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes.