B. Kociucka

Robertsonian Translocation in a Sex Reversal Dog (XX, SRY negative) May Indicate that the Causative Mutation for This Intersexuality Syndrome Resides on Canine Chromosome 23 (CFA23)

A Bernese mountain dog was subjected for clinical evaluation due to the presence of ambiguous external genitalia (enlarged clitoris). Anatomical and histological studies revealed the presence of one testicle, one ovotestis and a uterus. This dog was classified as a female-to-male sex reversal, with 2 normal X chromosomes and a lack of the Y chromosome-linked genes SRY and ZFY. It is the first case of this syndrome in this breed. Apparently a Robertsonian translocation, rob(5;23), was also identified in this dog and it is again the first case of this type of chromosome abnormality in this breed, as well as the first case of co-occurrence of the sex reversal syndrome along with a centric fusion in the dog. Since on the canine chromosome 23 (CFA23) 3 genes (FOXL2,PISRT1 and CTNNB1) involved in the sex determination process are present, further cytogenetic FISH studies were carried out with the use of BAC probes specific for this chromosome. It was found that a pericentromeric fragment of CFA23 was deleted as a result of the centric fusion. We hypothesize that a cis regulatory sequence for the sex determination genes on CFA23 (e.g. proximally located CTNNB1) is present in the deleted fragment. Thus, a causative mutation responsible for this sex reversal syndrome may reside on CFA23.

Three-dimensional arrangement of genes involved in lipid metabolism in nuclei of porcine adipocytes and fibroblasts in relation to their transcription level.

The 3-dimensional arrangement of chromosomes and genes within a nuclear space is considered to represent the level of transcriptional regulation. Understanding how the nuclear architecture of adipocyte cells contributes to gene expression has become the subject of great interest in the context of obesity research. In this study we investigated nuclear positioning of 3 gene loci involved in lipid metabolism in the pig (Sus scrofa, SSC) which is considered as an important animal model for obesity in humans. We found that the position of the SCD gene in the 3-dimensional space of the cell nucleus is not correlated with transcriptional activity. The gene locus as well as chromosome territory SSC14 occupied the same peripheral location in adipocyte and fibroblast cells, in spite of the fact that their transcription level differs significantly between both cell types. For the 2 other investigated genes, i.e. ACACA and SREBF1 and their chromosome territory (SSC12), slightly different nuclear locations were found. They occupied intermediate nuclear positions in fibroblast nuclei, while in adipocytes they were positioned in the nuclear interior. The more internal location of these genes corresponds to increased transcription levels in fat cells. Our results confirm the non-random position of genes and chromosome territories in nuclei of adult porcine cells and indicate that relationship between transcription activity and gene positioning exists only for some but not all genes.

The relationship between adipocyte size and the transcript levels of SNAP23, BSCL2 and COPA genes in pigs.

Breed-specific differences in fat tissue accumulation in the pig provide an opportunity to study the genetic background of this process. In the present study three pig breeds, differing in fatness, were analyzed in terms of the size of adipocytes derived from three tissues (subcutaneous, visceral and longissimus dorsi muscle) in relation to transcript levels of genes (SNAP23, BSCL2 and COPA), which encode proteins involved in lipid droplet formation. The analysis of adipocyte size revealed significant effects of breed and tissue and confirmed earlier reports that an elevated backfat thickness in some pig breeds is correlated with a larger adipocyte size. Variability in the transcript abundance of the studied genes among breeds and tissues was observed. We found a positive correlation between the abundance of the SNAP23 transcript and adipocyte diameter. The obtained results indicate that SNAP23 may be considered as an interesting candidate gene involved in adipose tissue growth in the pig.

A high incidence of adjacent-1 meiotic segregation pattern, revealed by multicolor sperm FISH, in a carrier boar of a new reciprocal translocation t(6;16)(p13;q23).

Reciprocal translocations pose a serious problem in pig breeding due to the reduced fertility of the carriers. This paper presents a new reciprocal translocation in a phenotypically normal, but hypoprolific (20% reduction) boar. Chromosome banding as well as the FISH technique with the use of BAC and telomeric probes was applied for a detailed characterization of this chromosome rearrangement. The karyotype of the studied boar was described as 38,XY,t(6;16)(p13;q23). The meiotic segregation of the quadrivalent was studied in 1,071 sperms by multicolor FISH. The most frequent segregation patterns were alternate (47.5%) and adjacent 1 (41.9%), while adjacent 2 and 3:1 were less frequent at 1.2 and 9.2%, respectively. Surprisingly, the frequency of the adjacent-1 segregation appeared to be relatively high, when compared with human and pig reciprocal translocations studied by sperm FISH. Our study, along with a review of the literature, shows that a reduction of fertility in the carriers and the incidence of different segregation patterns of the quadrivalent may vary within a broad range, and both aspects seem to be unrelated. A need for obligatory karyotype screening programs of artificial insemination boars is emphasized.

Testicular disorder of sex development in four cats with a male karyotype (38,XY; SRY-positive)

The molecular background of disorders of sex development (DSD) in cats is poorly recognized. In this study we present cytogenetic, molecular and histological analyses of four cats subjected for the analysis due to ambiguous external genitalia. Three cases, with rudimentary penises and an abnormal position of the urethral orifice, represented different types of hypospadias. The fourth case had a normal penis, a blind vulva and spermatogenetically active testes. Histological studies showed structures typical of testes, but spermatogenic activity was observed in two cats only. All the cats had a normal male chromosome complement (38,XY) and the Y-chromosome linked genes (SRY and ZFY) were also detected. Fluorescent in situ hybridization (FISH), with the use of the feline BAC probe harboring the SRY gene, excluded the possibility of chromosome translocation of the Y chromosome fragment carrying the SRY gene onto another chromosome. Sequencing of four candidate genes (SRY--sex determining region Y; AR--androgen receptor; SRD5A2--steroid-5-alfa reductase 2 and MAMLD1--mastermind-like domain containing (1) revealed one SNP in the SRY gene, one common polymorphism in exon 1 of the AR gene (tandem repeat of a tri-nucleotide motif--CAG), six polymorphisms (5 SNPs and 1 indel) in the SRD5A2 gene and one SNP in the MAMLD1 gene. Molecular studies of the candidate genes showed no association with the identified polymorphisms, thus molecular background of the studied DSD phenotypes remains unknown.

Three-Dimensional Positioning of B Chromosomes in Fibroblast Nuclei of the Red Fox and the Chinese Raccoon Dog

Great progress has been achieved over the last years in studies on chromosome arrangement in mammalian cell nuclei. Growing evidence indicates that the genomes spatial organization is of functional relevance. So far, no attention has been paid to the nuclear organization of B chromosomes (Bs). In this study we have examined nuclear positioning of Bs in 2 species from the Canidae family - the red fox and the Chinese raccoon dog. Using 2D and 3D fluorescence in situ hybridization and 2 gene-specific probes (C-KIT and PDGFRA), we analyzed the location of Bs in fibroblast nuclei. We found that small Bs of the red fox occupied mostly the interior of the nucleus, while medium-sized Bs of the Chinese raccoon dog were observed in the peripheral area of the nucleus as well as in intermediate and interior locations. The more uniform distribution of B chromosomes in the Chinese raccoon dog may be the result of differences in their size, since 3 morphological types of Bs are distinguished in this species. Our results indicate that 3D positioning of B chromosomes in fibroblast nuclei of the 2 canid species is in agreement with the chromosome size-dependent theory.

Expression of genes involved in lipid droplet formation (BSCL2, SNAP23 and COPA) during porcine in vitro adipogenesis

Adipogenesis is a complex process of fat cells development driven by the expression of numerous genes. Differentiation of progenitor cells into mature adipocytes is accompanied by changes in cell shape, as a result of lipid accumulation. In the present study, expression of three genes involved in lipid droplet formation (SNAP23, BSCL2 and COPA) was evaluated during porcine adipogenesis. It was found that mRNA levels of BSCL2 and SNAP23, but not COPA, increased during differentiation. Redistribution of SNAP23 protein to different cellular compartments was observed when comparing undifferentiated mesenchymal stem cells and differentiated adipocytes. The BSCL2 protein was found to be highly specific to cells with accumulated lipids, while COPA protein coated the lipid droplets. Obtained results indicated that the studied genes may be considered as candidates for fatness traits in pigs. Moreover, this study has shown that the porcine in vitro adipogenesis system provides a useful tool for the characterisation of novel genes involved in adipose tissue accumulation.

Karyotyping of Chinchilla lanigera Mol. (Rodentia, Chinchillidae)

There is relatively little information about the karyotype structure of the chinchilla (Chinchilla lanigera Mol.), an endemic South American rodent with 2n = 64 chromosomes and “duplicate-type X chromosome”. The species, endangered in nature, is a popular domesticated animal providing one of the most valuable furs in the world. In the present study, detailed karyotype analysis of the domestic chinchilla was performed using selected methods of differential chromosome staining (G-banding, C-banding, C-banding/DAPI, CMA3/DA/DAPI, Ag-NOR staining) as well as fluorescence in situ hybridization (FISH) with rDNA and telomeric (TTAGGG)n repetitive probes. The analysed specimens showed 59 metacentric and five submetacentric chromosomes. C-banding revealed mainly centromeric distribution of heterochromatin on the autosomes, interstitial and centromeric C-positive bands on the X chromosome and almost entirely heterochromatic nature of the Y chromosome. The average amount of heterochromatin in the haploid autosome set (31A) was 11.44%, whereas in the X chromosome 39.84%. Two active nucleolar organizer regions (NORs) and 45S rDNA repeats were located within one pair of big autosomes. In respect of morphology and G-banding patterns, the chinchilla chromosomes were arranged in homologous pairs and a G-banded karyotype was proposed. The obtained results could be the first step towards determination of the standard karyotype for the breeding chinchilla.