A. Ducos

Meiotic segregation analysis in cows carrying the t(1;29) Robertsonian translocation.

Heterozygous carriers of Robertsonian translocations generally have a normal phenotype but present reproductive failure. In cattle, the t(1;29) Robertsonian translocation is very common and carriers show a 3–5% decrease in fertility. Some data suggest that female carriers have a higher decrease than male carriers but no direct studies of the chromosome content of oocytes from a t(1;29) carrier cow have been performed so far. Four heterozygous carrier cows underwent hormonal stimulations and follicles punctions and about 800 oocytes were matured in vitro. Six hundred metaphase II preparations were obtained and analysed by fluorescent in situ hybridization with bovine chromosome 1 and 29 painting probes. Proportions of different kinds of oocytes were assessed: 74.11% (292/394) were normal and balanced, 4.06% (16/394) unbalanced and 21.83% (86/394) diploid. For all cows, the number of normal oocytes was not significantly different from the number of translocated oocytes but the diploidy and unbalanced rate were significantly different between them. As found in bulls, the meiotic segregation pattern in cows has shown a preponderance of alternate products. However, the frequency of unbalanced gametes determined in females (4.06%) was significantly higher than the frequency observed in males (2.76%). The divergence in the rate of diploid gametes (0.04% vs. 21.83%) is mainly explained by the difference between males and females.

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics

The aim of this article is not to present an exhaustive review of molecular cytogenetics applications in domestic animal species, but more to illustrate the considerable contribution of these approaches in diagnostics and research in economically important species. A short presentation of the main applications of molecular cytogenetics in humans points out the domains in which it has become an essential tool and underlines the specificities attached to this species in comparison to farm animals. This article is devoted to outlining the current resources available in domestic species and to some examples of fluorescence in situ hybridization applications in the cattle, pig, horse and avian species. From a clinical point of view, these examples illustrate the advantages of FISH for the study of chromosomal abnormalities (identification, characterization and estimation of their effects). Other applications of molecular cytogenetics are also illustrated, particularly ZOO-FISH, an approach which allows the determination of chromosome homologies between species. Finally, a specific emphasis was placed on the usefulness of molecular cytogenetics for the analysis of species such as poultry, which harbour a complex karyotype.

Meiotic studies in an azoospermic boar carrying a Y;14 translocation

A reciprocal translocation between the q arm of the Y chromosome and the q arm of chromosome 14 was identified in a young, phenotypically normal boar presenting azoospermia. Testicular biopsies were analyzed by classical histological and immunolocalization techniques, and by fluorescence in situ hybridization. Meiotic pairing analysis of 85 pachytene spreads showed the presence of an open structure corresponding to a quadrivalent formed by chromosomes 14, X, and the derivative chromosomes 14 and Y in 84.7% of the cases. In the remaining cases (15.3%), a ‘trivalent plus univalent’ configuration was observed. Immunolocalization of γH2AX revealed the presence of this modified histone in the chromatin domains of unsynapsed segments (centromeric region of chromosome 14) and spreading of the γH2AX signal from the XY body throughout chromosome 14 in 7.05% of the cells analyzed. The potential causes of the observed infertility, i.e. activation of meiotic checkpoints and/or silencing of genes necessary for the progression of meiosis, are discussed.

Influence of sex on the meiotic segregation of a t(13;17) Robertsonian translocation: a case study in the pig

BACKGROUND Comparison of male versus female meiotic segregation patterns for Robertsonian translocation (RT) carriers with similar genetic background has rarely been reported in mammalian species. METHODS The aim of this study was to compare the segregation patterns determined for related males and females carrying a 13;17 RT in an animal model (Sus scrofa domestica L.), using dual colour fluorescence in situ hybridization on decondensed sperm nuclei and metaphases II of in vitro-matured oocytes. RESULTS In males, no association between the trivalent and the XY body was observed in any of the 90 pachytene nuclei studied, and the rate of unbalanced spermatozoa ranged between 2.96% and 3.83%. Female meiotic segregation analyses were carried out on 83 metaphase II oocytes. The rate of unbalanced gametes was higher in females than in males (28.91% versus 3.21%, P < 0.001). This difference was due to higher rates of diploid gametes (12.04% versus 0.05%) and unbalanced gametes produced by the adjacent segregation (16.86% versus 3.16%). CONCLUSIONS This study is a new scientific contribution to the comparison of segregation patterns in related males and females carrying an identical chromosomal rearrangement. It allows a better understanding of the meiotic behaviour of RTs. It also clearly illustrates the relevance of swine as an animal model for such meiotic studies.

A new reciprocal translocation in a subfertile bull

Three bulls of the Montbéliarde breed that exhibited fertility rates lower than 30% following more than 400 artificial inseminations were examined. Semen quality (sperm motility and morphology) from these bulls was normal. Fertilizing ability estimated from in vitro embryo production results was studied for two of them. In vitro production rate was very low for one bull (A) and normal for the other (B). Cytogenetic analyses were carried out on the three bulls using chromosome banding techniques. These analyses revealed a reciprocal translocation (12;17)(q22;q14) in bull B. Based on family analyses, the hypothesis of a de novo origin of this rearrangement is proposed.

Studies of male and female meiosis in inv(4)(p1.4;q2.3) pig carriers

Inversions are well-known structural chromosomal rearrangements in humans and pigs. Such rearrangements generally have no effect on the carriers phenotype. However, the presence of an inversion may impair spermatogenesis and lead to the production of recombinant gametes, responsible for early miscarriages, stillbirth, or congenital abnormalities. This is the first report on meiotic segregation and pairing behavior of the inv(4)(p1.4;q2.3) pericentric inversion in pigs. Despite the very large size of the inverted fragment (76% of the chromosome), SpermFISH results showed that only 4.08% of the gametes produced by male heterozygotes were unbalanced. This low proportion could be explained by the particular behavior of normal and inverted SSC4 chromosomes during the initial stages of meiosis. Indeed, immunohistochemistry analyses revealed that heterosynapsis occurred in 92% of the cells, whereas synaptic adjustment was detected in a few spermatocytes only. Unexpectedly, the proportion of unbalanced gametes produced by female heterozygotes, estimated by FISH on metaphase II oocytes, was also very low (3.69%) and comparable to that in males. According to previous results for male and female meiotic processes, different proportions of recombinant gametes in the two genders would have been expected. Complementary studies should be carried out to further document the meiotic behavior of inversions in pigs.

Characterization of Reciprocal Translocations in Pigs using Dual-colour Chromosome Painting and Primed in situ DNA Labelling

We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p arm of chromosome 6, just above the centromere.

Y-autosome translocation interferes with meiotic sex inactivation and expression of autosomal genes: a case study in the pig.

Y-autosome translocations are rare in humans and pigs. In both species, these rearrangements can be responsible for meiotic arrest and subsequent infertility. Chromosome pairing abnormalities on the SSCX, SSCY and SSC1 chromatin domains were identified by analyzing pachytene spermatocytes from a boar carrying a (Y;1) translocation by immunolocalization of specific meiotic protein combined with FISH. Disturbance of the meiotic sex chromosome inactivation (MSCI) was observed by Cot-RNA-FISH and analysis of ZFY gene expression by sequential RNA- and DNA-FISH on spermatocytes. We hypothesized that the meiotic arrest observed in this boar might be due to the silencing of critical autosomal genes and/or the reactivation of some sex chromosome genes.

Analysis using sperm-FISH of a putative interchromosomal effect in boars carrying reciprocal translocations.

The occurrence of interchromosomal effects (ICE) in reciprocal translocation carriers still remains contradictory in the human literature. We used the pig as an animal model to investigate whether the structure of the reciprocal translocations as well as the size and/or type of the chromosomes not involved in the rearrangement may influence the occurrence and the extent of ICE. Analyses of chromosomal sperm content by fluorescence in situ hybridization (FISH) using whole-chromosome painting probes for 7 chromosomes (1, 10, 11, 13, 18, X and Y) were carried out on sperm samples of 2 boars with normal semen parameters carrying different balanced reciprocal translocations: 38, XY, t(3;15)(q27;q13) or 38, XY, t(12;14)(q13;q21). One fertile boar with normal karyotype was also studied as a control. Aneuploidy rates for the 7 chromosomes were estimated by scoring 10,000 to 20,000 spermatozoa for each probe combination. No significant ICE was found except for chromosome 1 in the case of the t(3;15) translocation. Even if statistically significant, this ICE remained very weak and should have very little impact on the reproductive performance of the carrier boar. The size and/or type of chromosomes not involved in the translocation do not seem to have a major influence on the occurrence of ICE. The structure of the translocation could play a role, but complementary studies should be carried out to confirm this assumption.

63,XO/65,XYY mosaicism in a case of equine male pseudohermaphroditism

ing the sex chlr11omosoImes (Long 1988, Power 1990, Crribiu 1 992, Bo3xsling 1 996). The karyotypes observcd in cases of gjonadal intersexualitx are usually balanced XY femllale pseudohermraphroditismII, XX Iale pseudohermiiaphroditisn anid XX/XY freemlartins. Some cases of unbalaniced karvotypes have also beenl reported, for example, 65,XXX (NlorenoMillani and others 1989), 65,XXY (Kulileil anid others 1993), 64,XX/(5,XXY mosaicism (IBoutcrs and otlher-s 1972, 1 975 ), diploid-triploid chimerism 64,XX/96,XXY (Power aind Leadoni 1990) aind 63,XO/65,XYY mosaicistmi (Hohn and oth-