The frequency of aneuploidy in the secondary spermatocytes of normal and Robertsonian translocation-carrying rams.

A detailed analysis was made of the chromosomes in 1008 M II figures from three different types of heterozygous Robertsonian translocation-carrying rams (53,xy,t1; 53,xy,t3) and 225 M II figures from homozygous Robertsonian translocation-carrying rams (52,xy,t1t1; 52,xy,t3t3) and rams of normal karotype (54,xy). No hypermodal cells were recorded in either the normal or the homozygous rams, but from 4-5% to 9-2% of M II cells from the heterozygous rams were hypermodal. The heterozygous rams also produced a significantly higher level of hypomodal cells suggesting that, in addition to non-disjunction, lagging at anaphase I may have occurred. There were also distinct differences in M II aneuploid spermatocyte frequency between heterozygous versus normal and homozygous rams. Fewer balanced translocation X-carrying M II cells were recorded than expected in three of the four 53,xy,t2 rams. This coincides with mating data which suggest that 26,x,t2 gametes may occur less frequently than expected. Since ewes of normal karotype mated to 53,xy,t rams conceive to first service at a rate equal to or better than normal mating groups, and because no blastocysts with unbalanced karotypes associated with the t1 translocation have been recorded, it is suggested that only euploid spermatozoa are involved in fertilization. In the sheep, aneuploid spermatocytes probably degenerate before sperm maturation.     

Further evidence of normal fertility and the formation of balanced gametes in sheep with one or more different Robertsonian translocations.

Mating experiments are described for sheep with three different Robertsonian translocations in the single heterozygous t1, t2 and t3, homozygous t1t1 and t3t3 and double heterozygous t1t2 and t1t3 state. The experiments were designed to investigate several previously reported unusual chromosome segregation ratios in sheep, to test the fertility of translocation heterozygous ewes mated to rams of normal karyotype and to test both the fertility and segregation patterns of sheep which were double translocation heterozygotes. The fertility of the translocation heterozygous ewes was normal as assessed from conception to first service, numbers of non-conceiving ewes and lambing percentages. Two types of double translocation heterozygous rams mated to ewes of normal karyotype produced regular chromosome segregation patterns in their progeny and the matings were of normal fertility. Double translocation heterozygous ewes were also fertile. Four sheep were bred with 51 chromosomes. Two of these were triple heterozygotes with three different Robertsonian translocations 51,xy,t1t2t3 and 51,xx,t1t2t3 and two were homozygous for one translocation and heterozygous for the others, namely 51,xx,t1t2t3 and 51,xxt1t3t3. All sheep were phenotypically normal. It is concluded that the t1,t2 and t3 Robertsonian translocations of sheep do not affect reproductive performance significantly.     

Additional evidence of the formation of unbalanced embryos in cattle with the 7/21 Robertsonian translocation

To confirm the effect of the 7 21 Robertsonian translocation on fertility in Japanese Black Cattle, cytogenetic studies were performed on embryos collected from the following 3 mating groups: normal bull cross normal cow, translocation carrier bull cross normal cow, and normal bull cross translocation carrier cow. All the analyzable embryos showed normal chromosome complements when the parents had a normal karyotype. In the group sired by the 7 21 translocation heterozygous bulls, a total of 56 embryos had metaphases suitable for chromosome analyses. Out of these embryos, 28 had normal chromosome complements and 25 were embryos with a balanced karyotype. However, 3 (5.4%) were monosomic and trisomic embryos, presumably resulting from the fertilization of normal ova by aneuploid spermatozoa. Unbalanced embryos were also observed in the chromosome analyses of embryos derived from the 7 21 translocation heterozygous cows. These results suggest that the 7 21 translocation in the heterozygous state may be associated with a slight reduction in reproductive efficiency.     

Genetic rescue of lethal genotypes in the mouse

Deletions of gene sequences in chromosome 7 of the mouse are known to interfere with biochemical and cellular development differentiation with lethal effects in homozygotes. The presence of the corresponding wild-type alleles in Cattanach's translocation (chromosomes 7 to X) is able to “rescue” potentially lethal females if they are made heterozygous for the translocation-carrying X chromosome. This holds true for those chromosome 7 deletions with perinatally lethal effects, whereas “rescue” is not readily accomplished with the deletions that cause early embryonic lethality. Females homozygous for the relevant deletion sequences and heterozygous for the translocation-carrying X chromosome are mosaics of two cell types: those in which the wild-type alleles included in the translocated piece complement the depleted sequences, resulting in a normal cellular phenotype, and those with the ordinary X chromosome expressing the lethal phenotype. The developmental interactions between the two cell types and their role in the mechanisms responsible for survival of females homozygous for lethal deletions are discussed. The failure of “rescue” of embryonic lethals reflects as yet unknown temporal and functional aspects of X-inactivation early embryogenesis.     

A first exploration of a Robertsonian translocation heterozygote in the mouse for its usefulness in cytological evaluation of radiation-induced meiotic autosomal non-disjunction.

In this report some data concerning the male meiotic system of mice heterozygous for Rb(11.13)4Bnr are presented and compared with those of a chromosomally normal Swiss random-bred stock. Change of the genetic background from a C3H/Swiss hybrid situation to the fourth backcross generation (to the Swiss random-bred stock), did not alter the average frequency of aneuploid secondary spermatocytes. This was confirmed by studies on post-implantation loss. Spermatogenic characteristics of Rb4/+ mice, such as testis weight, sperm production and the number of diplotene-metaphase-I figures found in stage XII of the seminiferous epithelium, suggest delay and cell death during this period. These data support our working hypothesis that such an aberrant chromosome system may be more prone to radiation effects and therefore is promising in our cytological studies into the causes of spontaneous and in our cytological studies into the causes of spontaneous and induced autosomal non-disjunction during meiosis in the mouse.     

Molecular analysis of nondisjunction in mice heterozygous for a Robertsonian translocation

A Robertsonian translocation results in a metacentric chromosome produced by the fusion of two acrocentric chromosomes. Rb heterozygous mice frequently generate aneuploid gametes and embryos, providing a good model for studying meiotic nondisjunction. We intercrossed mice heterozygous for a (7.18) Robertsonian translocation and performed molecular genotyping of 1812 embryos from 364 litters with known parental origin, strain, and age. Nondisjunction events were scored and factors influencing the frequency of nondisjunction involving chromosomes 7 and 18 were examined. We concluded the following: 1. The frequency of nondisjunction among 1784 embryos (3568 meioses) was 15.9%. 2. Nondisjunction events were distributed nonrandomly among progeny. This was inferred from the distribution of the frequency of trisomics and uniparental disomics (UPDs) among all litters. 3. There was no evidence to show an effect of maternal or paternal age on the frequency of nondisjunction. 4. Strain background did not play an appreciable role in nondisjunction frequency. 5. The frequency of nondisjunction for chromosome 18 was significantly higher than that for chromosome 7 in males. 6. The frequency of nondisjunction for chromosome 7 was significantly higher in females than in males. These results show that molecular genotyping provides a valuable tool for understanding factors influencing meiotic nondisjunction in mammals.     

Analysis of the “Poso” wheat translocation

The two translocation chromosomes in the Poso 5B/7B translocation have been isolated in separate heterozygous aneuploid stocks (19^II+5B+T). The translocation breakpoints are in the long arm of chromosome 7B and the short arm of chromosome 5B. The translocation chromosome bearing the 5B^L pairing inhibitor was obtained as a homozygous aneuploid (19^II+T ₁ ^II ). The heterozygous aneuploid hemizygous for the pairing inhibitor (19^II+5B+T₂) was used to produce intergeneric hybrids. Only a small percentage were of the high-pairing type (17%), the majority having received chromosome 5B through the egg. This indicates a strong selection against eggs containing the translocation chromosome deficient for the pairing inhibitor.     

Mitosis in embryonic trisomy 1 mouse eye

Trisomy 1 embryos consistently show eye defects (e.g., aphakia, microphakia, retention of lens stalk). To determine if the plane of division of mitotic figures is abnormal in the eyes of these animals, trisomic embryos (9.5 through 12 gestational days) were produced from mice doubly heterozygous for Robertsonian translocation chromosomes [Rb(1.3)/Rb(1.10)]. To accommodate for the known delay in trisomic embryo development, animals were grouped according to stages of eye development rather than to gestational age. Serial sections were evaluated without knowledge of karyotype for orientation of mitotic figures (parallel, perpendicular, oblique) in lens, optic cup, and diencephalon. Location of mitotic figures was scored as apical (nearest the lumen), middle, and basal. Numerous anomalies were noted in trisomic eye development. No difference was found between orientation of mitotic figures in the lens and optic cup of trisomy 1 and normal embryos. Location of mitotic figures in trisomy 1 lens was significantly different from that of normal littermates. The data confirm earlier studies that trisomy 1 affects the eye, and they tend to corroborate evidence that this trisomy affects the lens more than it affects the optic cup.     

The influence of the Robertsonian translocation Rb(X.2)2Ad on anaphase I non-disjunction in male laboratory mice

A Robertsonian translocation in the mouse between the X chromosome and chromosome 2 is described. The male and female carriers of the Rb(X.2)2Ad were fertile. A homozygous/hemizygous line was maintained. The influence of the X-autosomal Robertsonian translocation on anaphase I non-disjunction in male mice was studied by chromosome counts in cells at metaphase II of meiosis and by assessment of aneuploid progeny. The results conclusively show that the inclusion of Rb2Ad in the male genome induces non-disjunction at the first meoitic division. In second metaphase cells the frequency of sex-chromosomal aneuploidy was 10.8%, and secondary spermatocytes containing two or no sex chromosome were equally frequent. The Rb2Ad males sired 3.9% sex-chromosome aneuploid progeny. The difference in aneuploidy frequencies in the germ cells and among the progeny suggests that the viability of XO and XXY individuals is reduced. The pairing configurations of chromosomes 2, Rb2Ad and Y were studied during meiotic prophase by light and electron microscopy. Trivalent pairing was seen in all well spread nuclei. Complete pairing of the acrocentric autosome 2 with the corresponding segment of the Rb2Ad chromosome was only seen in 3.2% of the cells analysed in the electron microscope. The pairing between the X and Y chromosome in the Rb2Ad males corresponded to that in males with normal karyotype. Reasons for sex-chromosomal non-disjunction despite the normal pairing pattern between the sex chromosomes may be seen in the terminal chiasma location coupled with the asynchronous separation of the sex chromosomes and the autosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The establishment and application of preimplantation genetic haplotyping in embryo diagnosis for reciprocal and Robertsonian translocation carriers

Background

Preimplantation genetic diagnosis (PGD) is now widely used to select embryos free of chromosomal copy number variations (CNV) from chromosome balanced translocation carriers. However, it remains a difficulty to distinguish in embryos between balanced and structurally normal chromosomes efficiently.

Methods

For this purpose, genome wide preimplantation genetic haplotyping (PGH) analysis was utilized based on single nucleotide polymorphism (SNP) microarray. SNPs that are heterozygous in the carrier and, homozygous in the carrier’s partner and carrier’s family member are defined as informative SNPs. The haplotypes including the breakpoint regions, the whole chromosomes involved in the translocation and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference and embryos. In order to perform this analysis, a reference either a translocation carrier’s family member or one unbalanced embryo is required. The positions of translocation breakpoints are identified by molecular karyotypes of unbalanced embryos. The recombination of breakpoint regions in embryos could be identified.

Results

Eleven translocation families were enrolled. 68 blastocysts were analyzed, in which 42 were unbalanced or aneuploid and the other 26 were balanced or normal chromosomes. Thirteen embryos were transferred back to patients. Prenatal cytogenetic analysis of amniotic fluid cells was performed. The results predicted by PGH and karyotypes were totally consistent.

Conclusions

With the successful clinical application, we demonstrate that PGH was a simple, efficient, and popularized method to distinguish between balanced and structurally normal chromosome embryos.

     

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.     

Fertility of male and female mice heterozygous for the reciprocal translocation T(7;17)3BKM.

The present paper describes the fertility of male and female mice heterozygous for the reciprocal translocation T(7;17)3BKM. This translocation was induced by gamma rays in the spermatozoa of an irradiated parent. It is characterized by "asymmetrical" localization of the breakpoints, distally in Chromosome 7 (7F5) and proximally in Chromosome 17 (17B1). The data presented here relate only those matings in which, for both partners, heterozygosity or normality could be confirmed cytogenetically. The results indicate that both male and female translocation heterozygotes are fertile, their mean litter size being reduced to about 50% of that of normal littermates. This leads to the conclusion that the multivalents mainly undergo either alternate or adjacent-1 2:2 segregation. No viable tertiary trisomics were observed among the progeny of the translocation carriers. Analysis of the frequency of the different types of multivalents in diakinesis-metaphase I spermatocytes showed a significant predominance of chain-type figures (CIV and CIII+I), with chains of four elements (CIV) being more frequent than other configurations. This demonstrates that the small marker chromosome remains attached by one of its segments to the tetravalent.     

Seasonal thyroidal activity and reproductive characteristics of Iranian fat-tailed rams

An experiment was conducted to study seasonal variations of thyroidal activity, serum testosterone concentration and seminal characteristics of two breeds of Iranian fat-tailed sheep. Eight 3 to 4-year-old rams of Ghezel and Mehraban breeds (4 rams/breed) were randomly selected from a flock of fertile rams. Semen was collected by using an artificial vagina, and blood samples were obtained via jugular vein. Seminal characteristics, scrotal length, width, and circumference, and blood parameters were measured at 3-week intervals. Time of the year significantly (P < 0.05) affected the volume of semen, sperm concentration, percent live sperm, percent normal sperm, total sperm number, seminal pH, seminal lactate dehydrogenase (LDH) concentration, scrotal circumference, scrotal width, and serum levels of testosterone, cholesterol, triiodothyronine (T(3)), tetraiodothyronine (T(4)), free T(4) index (FT(4)I), and TSH. Scrotal length was not affected by the time of sampling (P > 0.05). The effect of breed on the serum concentrations of hormones and cholesterol was not significant. Volume of semen, percent normal sperm, and total number of normal sperm were significantly (P < 0.05) higher in Mehraban but scrotal circumference, scrotal width and scrotal length were greater in Ghezel rams. Breedxtime interaction effect was significant for T(4), FT(4)I, sperm concentration, and total sperm number. The largest values for TSH, T(4), FT(4)I, testosterone, total sperm number, percent normal sperm, percent live sperm, sperm concentration, volume of semen and scrotal circumference were found from early summer to winter and the lowest values were recorded at the end of spring and in early summer. In both breeds, high and negative correlations (r = -0.7 to -0.8) were found between LDH levels in semen and percent live sperm, percent normal sperm and total sperm number (P < 0.01). It is unlikely that such variations in seminal quality would affect the fertility under natural mating.     

Morphologic development of the fetal trisomy 19 mouse

Trisomy 19 mice show a characteristic pattern of delayed morphologic development when compared with normal littermates during the second half of gestation. Examinations of the external phenotype and sectioned fetuses, and skeletal maturation at later days, suggest that trisomy 19 has little recognizable effect on morphogenesis prior to 10 days. Trisomic conceptuses collected at subsequent 24-hour intervals show a 1-day lag in weight gain and begin to show a progressive and uniform delay in differentiation so that 14- and 15-day animals resemble 13- and 14-day normal fetuses, respectively. There is no further recognizable increase in this delay through term. There was no striking change in numbers of viable trisomic conceptuses collected throughout the period from 10 days to birth. These observations suggest that most trisomies surviving at 10 days will develop to term, with the major effect of the aneuploid genome being to delay the otherwise normal-appearing growth and development of the affected conceptuses. In all cases, the litters were from crosses between mice doubly heterozygous for the (5.19) and (9.19) Robertsonian translocation chromosomes.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans.

In Caenorhabditis elegans, individuals heterozygous for a reciprocal translocation produce reduced numbers of viable progeny. The proposed explanation is that the segregational pattern generates aneuploid progeny. In this article, we have examined the genotype of arrested embryonic classes. Using appropriate primers in PCR amplifications, we identified one class of arrested embryo, which could be readily recognized by its distinctive spot phenotype. The corresponding aneuploid genotype was expected to be lacking the left portion of chromosome V, from the eT1 breakpoint to the left (unc-60) end. The phenotype of the homozygotes lacking this DNA was a stage 2 embryonic arrest with a dark spot coinciding with the location in wild-type embryos of birefringent gut granules. Unlike induced events, this deletion results from meiotic segregation patterns, eliminating complexity associated with unknown material that may have been added to the end of a broken chromosome. We have used the arrested embryos, lacking chromosome V left sequences, to map a telomere probe. Unique sequences adjacent to the telomeric repeats in the clone cTel3 were missing in the arrested spot embryo. The result was confirmed by examining aneuploid segregants from a second translocation, hT1(I;V). Thus, we concluded that the telomere represented by clone cTel3 maps to the left end of chromosome V. In this analysis, we have shown that reciprocal translocations can be used to generate segregational aneuploids. These aneuploids are deleted for terminal sequences at the noncrossover ends of the C. elegans autosomes.     

Survey for neoplasia in Macoma balthica from the Gulf of Gdansk by flow cytometry

Using flow cytometry, 234 Macoma balthica were examined during a survey to determine frequency of neoplasia in the Gulf of Gdansk (Poland). Clams were collected in 4 locations and DNA content in gill tissue cells was determined by flow cytometry using propidium iodide staining. Cell permeabilization was induced by osmotic shock. Prevalence of neoplasia ranged from 9.6 to 26.7% depending on location. DNA content in aneuploid cells was higher than in normal dividing cells. The fluorescence value for aneuploid cells corresponded to tetraploid/pentaploid cells. Three stages of neoplasia were defined, based on the percentage of aneuploid cells determined by flow cytometry. Histopathological and cytogenetic analyses were also carried out on the same clams for comparative study. Proportions of normal and affected clams detected using flow cytometry were similar to those identified using both methods. In the present study, no clear relationship was demonstrated between prevalence of neoplasia and pollutant detection in the different sampling sites.     

Cytogenetic analysis of sperm from a male heterozygous for a 13;14 Robertsonian translocation

Summary Cytogenetic analysis of 121 sperm from a man heterozygous for a t(13;14) Robertsonian translocation was performed using the technique of in vitro penetration of hamster eggs. The frequency of sperm that were chromosomally unbalanced with respect to the translocation was 27%. The frequency of chromosomally normal (36%) and balanced (38%) complements was approximately equal, as theoretically expected. There was no evidence for an interchromosomal effect since the frequency of numerical chromosomal abnormalities (2.5%) and structural chromosomal abnormalities (10.7%) — both unrelated to the translocation — were within the normal range of control donors. The ratio of X-and Y-chromosome bearing sperm was equal, and there was no evidence for preferential segregation of the X chromosome with the translocation.     

Nondisjunction and transmission ratio distortion ofChromosome 2 in a (2.8) Robertsonian translocation mouse strain

Aneuploidy results from nondisjunction of chromosomes in meiosis and is the leading cause of developmental disabilities and mental retardation in humans. Therefore, understanding aspects of chromosome segregation in a genetic model is of value. Mice heterozygous for a (2.8) Robertsonian translocation were intercrossed with chromosomally normal mice and Chromosome 2 was genotyped for number and parental origin in 836 individuals at 8.5 dpc. The frequency of nondisjunction of this Robertsonian chromosome is 1.58%. Trisomy of Chromosome 2 with two maternally derived chromosomes is the most developmentally successful aneuploid karyotype at 8.5 dpc. Trisomy of Chromosome 2 with two paternally derived chromosomes is developmentally delayed and less frequent than the converse. Individuals with maternal or paternal uniparental disomy of Chromosome 2 were not detected at 8.5 dpc. Nondisjunction events were distributed randomly across litters, i.e., no evidence for clustering was found. Transmission ratio distortion is frequently observed in Robertsonian chromosomes and a bias against the transmission of the (2.8) Chromosome was detected. Interestingly, this was observed for female and male transmitting parents.     

Genetic sexing strains of mediterranean fruit fly (Diptera: Tephritidae): quality in mass-reared temperature-sensitive lethal strains treated at high temperatures

The high-temperature treatment of eggs of mass-reared tsl genetic sexing strains in Mediterranean fruit fly, Ceratitis capitata (Wiedemann), during late embryogenesis (the low-high protocol) conserves more male flies than treatment during early embryogenesis. A tsl strain, AUSTRIA 6-97, was constructed to follow the fate of aneuploid individuals during male-only production. Aneuploid individuals are produced following segregation in the translocation heterozygous males, and they can survive to the pupal stage where they compromise quality because they do not eclose as adults. Hatching, emergence, and male fly production were quantified and the heat-treatment protocol was characterized. The low-high egg treatment conserves the number of euploid-balanced males, and there is a very low survival of aneuploid males. After heat treatment of eggs, at least 95% of the male pupae were euploid compared with only 71% from untreated eggs. The quality of euploid male pupae was diminished with successive daily collections, an effect previously attributed to aneuploid survivors. Reduced yield of euploid males from early heat treatments was the result of an emergence effect, in addition to a maternal effect. A third detrimental effect of heat was found, occurring after hatching and before pupation, that reduces the survivorship of euploid males. The low-high treatment protocol yielded more males, with a higher accuracy than other heat treatments. However, although it avoids both the maternal and emergence effects, the production of euploid males was 30% less than the potential production, implying that the low-high heat protocol for killing female embryos in tsl genetic sexing strains can be fine-tuned.     

Meiotic nondisjunction and translocation segregation in dwarf (dw/dw) and control T(1;13)70H heterozygous mice

The meiotic behavior of translocation heterozygous T70 (1;13)H/+ male mice with a Snell dwarf (dw/dw) genotype was compared with that of nondwarf T70H/+ controls. A four-fold increase in the nondisjunction frequency of the normal bivalents occurred as a consequence of the dwarf genotype. This increase is identical to that seen in karyologically normal dwarf males. No effect of the dwarf condition on the segregation of the translocation multivalent could be noted. Thus, translocation heterozygosity does not enhance the meiotic instability caused by the hypopituitary dwarf condition. From a small sample of oocytes from T70H/+ and chromosomally normal dwarf females it is concluded that nondisjunction in females is not increased by the dwarf condition. In general we conclude that animals with higher spontaneous nondisjunction levels are not necessarily more sensitive to factors increasing nondisjunction.