Frequency of Aneuploidy Related to Age in Porcine Oocytes

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed

Aneuploidy Detection and mtDNA Quantification in Bovine Embryos with Different Cleavage Onset Using a Next-Generation Sequencing-Based Protocol

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies

Aneuploidy Detection in Pigs Using Comparative Genomic Hybridization: From the Oocytes to Blastocysts

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs

Development and Application of Camelid Molecular Cytogenetic Tools

Cytogenetic chromosome maps offer molecular tools for genome analysis and clinical cytogenetics and are of particular importance for species with difficult karyotypes, such as camelids (2n = 74). Building on the available human-camel zoo-fluorescence in situ hybridization (FISH) data, we developed the first cytogenetic map for the alpaca (Lama pacos, LPA) genome by isolating and identifying 151 alpaca bacterial artificial chromosome (BAC) clones corresponding to 44 specific genes. The genes were mapped by FISH to 31 alpaca autosomes and the sex chromosomes; 11 chromosomes had 2 markers, which were ordered by dual-color FISH. The STS gene mapped to Xpter/Ypter, demarcating the pseudoautosomal region, whereas no markers were assigned to chromosomes 14, 21, 22, 28, and 36. The chromosome-specific markers were applied in clinical cytogenetics to identify LPA20, the major histocompatibility complex (MHC)-carrying chromosome, as a part of an autosomal translocation in a sterile male llama (Lama glama, LGL; 2n = 73,XY). FISH with LPAX BACs and LPA36 paints, as well as comparative genomic hybridization, were also used to investigate the origin of the minute chromosome, an abnormally small LPA36 in infertile female alpacas. This collection of cytogenetically mapped markers represents a new tool for camelid clinical cytogenetics and has applications for the improvement of the alpaca genome map and sequence assembly.Keywords: FISH, BAC library, Translocation, Cytogenetics, Minute chromosome, Alpac

Published frequency of aneuploidy in pigs.

a<p>only chromosomes 1, 10 and Y detected.</p>b<p>only chromosomes 1 and 10 detected.</p>c<p>only hyperhaploidy.</p>d<p>only true polyploidy without mixoploidy.</p>e<p>polyploidy+mixoploidy.</p

The incidence and description of aneuploidies in pig embryos detected by CGH.

a<p>aneuploid embryo was at the late morula stage.</p><p>The table summarizes the numbers of embryos collected and analyzed from individual gilts. Besides that, data on the sex ratio, numbers of abnormal embryos and the description of chromosome abnormalities are provided.</p

The example of WGA-CGH analysis of 2 aneuploid pig embryos.

<p>(A) the male embryo detected with partial loss of chromosome 9q; (B) the female embryo detected with loss of chromosomes 13 and 14. Amplified DNA obtained from the embryo was labelled with red fluorescence and amplified reference male porcine DNA was labeled with green fluorescence. Both DNA samples were mixed and allowed to hybridize to male porcine mitoses. Subsequently, the red and green fluorescence was captured and analyzed using dedicated CGH software. The heterochromatin regions (e.g. centromeres and the q arm of chromosome Y) were excluded from the analysis due to the abundance of repetitive DNA sequences.</p

The incidence and description of ploidy abnormalities in pig embryos detected by FISH.

a<p>embryo contained 98% of triploid cells, therefore it is considered as triploid.</p><p>The frequencies of ploidy abnormalities are grouped with respect to the percentage of abnormal cells within individual embryos (first column). On the right side of the table, the numbers and description of ploidy mosaicism is given; for example, in the group of embryos with ploidy abnormalities 0–5%, 11 embryos contained beside diploid cells only tetraploid cells etc.</p

The occurrence of individual pig chromosomes in aneuploid oocytes, early embryos and blastocysts.

a<p>the results of the CGH analysis of oocytes were published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030335#pone.0030335-Hornak1" target="_blank">[7]</a>.</p>b<p>the results of the CGH analysis of early embryos were published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030335#pone.0030335-Hornak2" target="_blank">[8]</a>.</p>c<p>present study.</p><p>The table shows individual pig chromosomes and their occurrence in aneuploid samples. The aneuploid samples containing >3 chromosome abnormalities per oocyte/embryo (complex aneuploidy with a likely stochastic distribution of chromosome errors) and samples containing segmental chromosome abnormalities were excluded.</p