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.

Meiotic studies of a 38,XY/39,XXY mosaic boar.

Klinefelter's syndrome (KS) is the most common sex chromosome abnormality identified in human males. This syndrome is generally associated with infertility. Men with KS may have a 47,XXY or a 46,XY/47,XXY karyotype. Studies carried out in humans and mice suggest that only XY cells are able to enter and complete meiosis. These cells could originate from the XY cells present in mosaic patients or from XXY cells that have lost one X chromosome. In pig, only 3 cases of pure 39,XXY have been reported until now, and no meiotic analysis was carried out. For the first time in pig species we report the analysis of a 38,XY/39,XXY boar and describe the origin of the supplementary X chromosome and the chromosomal constitutions of the germ and Sertoli cells.

NEMO: a tool for analyzing gene and chromosome territory distributions from 3D-FISH experiments.

UNLABELLED: Three-dimensional fluorescence in situ hybridization (3D-FISH) is used to study the organization and the positioning of chromosomes or specific sequences such as genes or RNA in cell nuclei. Many different programs (commercial or free) allow image analysis for 3D-FISH experiments. One of the more efficient open-source programs for automatically processing 3D-FISH microscopy images is Smart 3D-FISH, an ImageJ plug-in designed to automatically analyze distances between genes. One of the drawbacks of Smart 3D-FISH is that it has a rather basic user interface and produces its results in various text and image files thus making the data post-processing step time consuming. We developed a new Smart 3D-FISH graphical user interface, NEMO, which provides all information in the same place so that results can be checked and validated efficiently. NEMO gives users the ability to drive their experiments analysis in either automatic, semi-automatic or manual detection mode. We also tuned Smart 3D-FISH to better analyze chromosome territories. AVAILABILITY: NEMO is a stand-alone Java application available for Windows and Linux platforms. The program is distributed under the creative commons licence and can be freely downloaded from https://www-lgc.toulouse.inra.fr/nemo

Contribution of radiation hybrids to genome mapping in domestic animals.

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described.

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.

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.

Male meiotic segregation analyses of peri- and paracentric inversions in the pig species.

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 can lead to spermatogenesis impairments and to the production of unbalanced (recombinant) gametes, responsible for early miscarriages, stillbirth, or congenital abnormalities. Sperm samples from boars heterozygote for pericentric inv(2)(p1.1;q1.1), inv(2) (p1.1;q2.1), inv(1)(p2.1;q2.10), or inv(1)(p2.4;q2.9), as well as for paracentric inv(2)(q1.3;q2.5) or inv(1)(q1.2;q2.4) were analyzed using sperm FISH (fluorescent in situ hybridization on decondensed sperm heads) to determine the male meiotic segregation profiles of the rearrangements. Furthermore, the availability of sperm samples for 2 unrelated carriers of inv(2)(p1.1;q1.1) allowed us to check for the occurrence of inter-individual variability of the rates of unbalanced meiotic products for this rearrangement. The estimated proportions of recombinant gametes were very low for all the inversions studied (0.62%, 1.30%, 3.05%, 1.27%, 4.12% and 0.84%, respectively), albeit significantly higher than the control. The rearrangements should therefore have very little impact on the reproductive performance of the carriers. No difference was found between the 2 carriers of inv(2)(p1.1;q1.1), suggesting a lack of inter-individual variability for this rearrangement. Overall, no significant correlation was found between the sizes of the inverted fragments and the proportions of recombinant (unbalanced) gametes for the 6 inversions studied. This is in contradiction with most human results. Further studies (pairing and recombination analysis using immunostaining techniques) should be carried out to elucidate the origin of such an inter-species difference.

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.

Mapping and expression analyses during porcine foetal muscle development of 12 genes involved in histone modifications.

Histone modifications (methylation and demethylation) regulate gene expression and play a role in cell proliferation and differentiation by their actions on chromatin structure. In this context, we studied the temporal expression profiles of genes acting on histone methylation and demethylation during skeletal muscle proliferation and differentiation. Quantitative real-time PCR was used to quantify the mRNA levels of CARM1, JARID1A, JMJD2A, LSD1, PRMT2, PRMT5, SMYD1, SMYD2, SMYD3, SETDB1, Suv39h2 and SUZ12 in foetal skeletal muscle. Our results showed that CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2 were differentially expressed in embryonic muscles of 33 days post-conception (dpc), 65 dpc and 90 dpc. These 12 genes were mapped to porcine chromosomes (SSC) 2q21-24, 5q25, 6q35, 6q12-21, 6p15, 7q21, 3q21-27, 9q26, 10p16, 4q15-16, 10q14-16 and 12p12 respectively. Taking into account the reported QTL mapping results, gene expression analysis and radiation hybrid mapping results, these results suggest that five genes (CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2) could be good candidate genes for growth and backfat thickness traits.

Molecular characterization and expression pattern of the porcine STARS, a striated muscle-specific expressed gene.

STARS (striated muscle activator of Rho signaling) promotes the nuclear localization of MRTFs and mediates SRF transcription, which provides a potential muscle-specific mechanism for linking changes in the actin cytoskeleton structure with muscle gene expression. In this study, the full-length cDNA of the porcine STARS was cloned. The open reading frame of this gene contains 1,155 bp and encodes a protein of 384 amino acids, which is 79, 73, and 77% identical with human, mouse, and rat STARS genes, respectively. RT-PCR revealed that STARS is specifically expressed in heart and skeletal muscles. STARS is also distinctly different in different muscle developmental stages. The result indicates that its expression increased gradually from 33 dpc (days postcoitum) to postnatal muscles, and peaked 28 days postnatal. The porcine STARS was mapped to SSC4p13 using the somatic cell hybrid panel and the radiation hybrid panel IMpRH (LOD score 11.98). The data show that STARS is closely linked to marker SW871. A T/G single nucleotide polymorphism in the coding sequence, detected as Bsh1236I PCR-RFLP, displays allele frequency differences in six pig breeds.

Study of inter- and intra-individual variation of meiotic segregation patterns in t(3;15)(q27;q13) boars.

Constitutional chromosomal rearrangements are relatively frequent genetic anomalies in both man and pigs. Among them, reciprocal translocations, present a specific meiotic segregation pattern. The potential "individual" effect of the t(3;15)(q27,q13) translocation was studied using SpermFISH to analyze the meiotic segregation patterns of three boars carrying this rearrangement. Three samples were taken at different times from one of these boars to analyze a potential "time" effect. No "time" or "individual" effect was found in this study. These results should allow more efficient management of certain reciprocal translocations in pig populations but need to be completed by the study of other kinds of chromosomal rearrangements.

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.

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 gammaH2AX revealed the presence of this modified histone in the chromatin domains of unsynapsed segments (centromeric region of chromosome 14) and spreading of the gammaH2AX 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.

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11.

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility.

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.