Different levels of a lack of x-y chromosome pairing in pachytene spermatocytes of red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides)
2011; De Gruyter; Volume: 11; Issue: 1 Linguagem: Inglês
ISSN
2300-8733
AutoresJarosław Sosnowski, Agnieszka Lukaszewicz, L Migalska, Maria Wojnowska, Zbigniew Polański,
Tópico(s)Genetic Mapping and Diversity in Plants and Animals
Resumoresearch was performed on pachytene spermatocytes obtained from 8 male raccoon dogs and 10 male red foxes. cells were immunostained for visualization of synaptonemal complexes and sex chromosome chromatin. fluorescent microscope analysis revealed lack of pairing between the sex chromosomes. additionally, synaptonemal complex structures formed by the B chromosomes and their number in cell were determined. the data showed a large difference in the level of X and Y chromosome pairing failure in the spermatocytes of both species analysed. the described phenomenon was observed in 15.6% of the chinese raccoon dog spermatocytes and only once (0.2%) in the red fox spermatocytes. among the raccoon dog males an interindividual difference in the level of sex chromosome pairing failure was observed. the numbers of B chromosomes observed in analysed cells varied from 2 to 5 in raccoon dogs and from 0 to 6 in red foxes. Different structures of the synaptonemal complexes of B chromosomes were observed such as univalents, bivalents, trivalents and tetravalents. the results of our research showed that with the increase in the number of B chromosomes in spermatocytes of the raccoon dog, the level of sex chromosome pairing failure increases. key words: raccoon dog, red fox, X-Y chromosomes, pachytene spermatocytes, B chromosomes This work was supported by the Polish Ministry of Science and Higher Education, Grant No. 2 P06D 029 29. J. Sosnowski et al. 72 The main aspect of gametogenesis concerns the formation of euploid gametes. Any aberration in the number of chromosomes present in the gametes involved in fertilization will result in aneuploid embryos. The process of chromosome pairing and the formation of bivalents during meiosis is necessary for proper genetic recombination and segregation of chromosomes. The absence of bivalents and the formation of univalents (unpaired chromosomes) at the time of induction of the first anaphase, may lead to random segregation of homologous chromosomes, i.e. aneuploidy. The presence of univalents is mainly associated with the sex chromosomes (Polanski, 2000). The homology of X and Y heterosomes is restricted to a small region called the pseudoautosomal region, where the pairing and recombination occur. The above observations concern A chromosomes. However, there are species that contain B chromosomes in addition to the basic chromosome number. These chromosomes, unlike A chromosomes, do not undergo meiotic division in anaphase and their segregation is random (Świtonski, 1988; Trifonov et al., 2002; Vujośevic and Blagojevic, 2004). As a result, we observe different numbers of B chromosomes in gametes. The occurrence of B chromosomes was described in three species of the Canidae family. They account for the diploid number variability in the red fox (Vupes vulpes) (2n = 34), the Chinese raccoon dog (Nyctereutes procyonoides procyonoides) (2n = 54) and the Japanese raccoon dog (Nyctereutes procyonoides viverrinus) (2n = 38). The B chromosomes of the Chinese raccoon dog and the red fox differ from each other in size, nature and frequency. The B chromosome number in the red fox varies from 0 to 8, (Makinen et al., 1985) but the most common number observed in cells is 2 or 3 (Ellenton and Basrur, 1981), whereas the number described for the Chinese raccoon dog is 0–4. One of the characteristic epigenetic modifications of X and Y bivalent is the phosphorylation of histone H2A.X on serine 139 – γH2A.X. This modification is associated with the inactivation of the sex chromosomes during male meiosis and furthermore, it accompanies the surrounding chromatin of the unsynapsed fragment of autosomes, which is associated with transcriptional silencing by unpaired DNA (Mahadevaiah et al., 2001; Turner et al., 2005). The relationship between this histone modification and the inactivation of the sex chromosomes during spermatogenesis may explain their presence in the “sex body”, i.e. the X and Y chromosome bivalent. The aim of the investigation was to compare the level of sex chromosome pairing failure in two species of Canidae, the karyotypes of which contain B chromosomes, to the number of B chromosomes in pachytene spermatocytes. material and methods Testes of the experimental animals were dissected on the farm during fur collection season (December and January). Level of sex chromosome pairing failure in Canidae 73 Tissues from the testes of 8 adult Chinese raccoon dogs and 10 red foxes (around 20 to 21 months old) were minced into small fragments with small tweezers and left in a hypotonic solution for 30 minutes. Chromosome spreads were prepared by the drying-down method described by Peters et al. (1997). immunocytochemistry Immunocytochemistry was carried out as previously described (Anderson et al., 1999). To avoid non-specific antibody binding, slides were blocked for 30 min. in 3% bovine serum albumin (BSA), 0.1% Tween in PBS in a humid chamber at room temperature. Slides were immunostained overnight at 4°C with primary antibodies against SCP3 (polyclonal rabbit antibody NOVUS, NB 300-232A) to visualize synaptonemal complex lateral elements and against γH2A.X (monoclonal mouse antibody UPSTATE 05-636). Both primary antibodies were used at a 1:1000 dilution in PBS/0.1% Tween20. After incubation with primary antibodies, fluorescenttagged secondary antibodies were used for detection – goat, anti-mouse, Rhodamine conjugated (SANTA CRUZ BIOTECHNOLOGY, sc-2092) and goat, anti-rabbit, FITC conjugated (SANTA CRUZ BIOTECHNOLOGY, sc-2012) diluted at 1:200 in PBS/0.1% Tween20. Chromosomal DNA was stained with DAPI (SIGMA, D-9542) and slides were covered under the Antifade solution (CITIFLUOR Ltd.). microscopy and scoring Slides were analysed under Nikon Eclipse E600 fluorescence microscope. The images were captured with Hamamatsu camera and processed with Lucia software. Slides used for analyses fulfilled the following criteria: all SCs were well visible, without overlapping, countable, and the background staining was low. Because raccoon dog B chromosomes are similar in size to medium acrocentrics and it was not possible to distinguish a fully paired B bivalent from an autosomal bivalent, when more than 26 SC and the sex bivalent were observed, the supernumerary structures were classified as B chromosomes. In all good quality spreads which were subjected to microscope analysis, sex bivalents were closely examined to identify the pairing region – the pseudoautosomal region. The purpose was to identify non-pairing X and Y chromosomes.
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