Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.
Abstract: Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.
Publication Date: 2005-06-11 PubMed ID: 15948165DOI: 10.1002/mrd.20302Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study investigates the occurrence of chromosomal abnormalities in horse embryos both produced in natural conditions (in vivo) and artificial lab conditions (in vitro), using fluorescent in situ hybridization (FISH) probes to mark specific equine chromosomes. Results reveal that although most embryos are exclusively diploid, the chances of encountering chromosomally abnormal cells is slightly higher for embryos developed in a lab.
Objective of the Research
- The main objective of this research is to confirm whether numerical chromosome abnormalities exist in horse embryos produced either naturally (in vivo) or in a lab setting (in vitro).
- This is carried out by validating the use of fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei, the active phase when a cell isn’t dividing.
- The study aims to fill the gap in concrete evidence regarding chromosomal aberrations as a significant cause of early embryonic death in mares.
Methodology
- The study analyzed a total of 42 horse embryos, among which 22 were developed naturally and 20 were developed in vitro.
- FISH probes were used to label specific equine chromosomes in the horse embryos.
- A scoring system was used where the presence of extra FISH signals indicated an increase in ploidy (the number of sets of chromosomes in a cell) meaning the cells were not normal (diploid), but were triploid or mosaic (mixture of different ploidy).
- “Missing” signals were assumed to indicate “false negatives.”
Findings
- The majority of the horse embryos evaluated (30 out of 42) were classified as exclusively diploid, which indicates they have two sets of each chromosome, as is normal.
- However, some embryos developed both in vivo and in vitro showed abnormal ploidy. Four in vivo embryos and seven in vitro embryos were found to have mosaic ploidy, meaning they contained both diploid and polyploid cells.
- One in vitro produced embryo was found to be entirely triploid, carrying three complete sets of chromosomes.
- When examined in more detail, 4.4% of cells in 11 embryos had an uneven number of chromosomes, raising the possibility of other types of aneuploidy (abnormal number of chromosomes) such as trisomy, monosomy, or nullisomy.
- The incidence of apparently mixoploid embryos tended to be higher in in vitro-produced embryos than those developed naturally, suggesting laboratory conditions may increase the likelihood of chromosomal abnormalities.
Conclusion
- The study concludes that chromosomally abnormal cells are present in morphologically normal equine embryos, and in vitro production may increase their likelihood.
- The distinction between different types of ploidy and aneuploidy remains a challenge requiring more advanced techniques such as using more than one probe per chromosome and/or probes for more than two chromosomes.
Cite This Article
APA
Rambags BP, Krijtenburg PJ, Drie HF, Lazzari G, Galli C, Pearson PL, Colenbrander B, Stout TA.
(2005).
Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.
Mol Reprod Dev, 72(1), 77-87.
https://doi.org/10.1002/mrd.20302 Publication
Researcher Affiliations
- Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. B.P.B.Rambags@vet.uu.nl
MeSH Terms
- Animals
- Chromosome Aberrations / embryology
- Embryo Culture Techniques
- Embryo, Mammalian / embryology
- Embryo, Mammalian / pathology
- Female
- Horses
- In Situ Hybridization, Fluorescence
Citations
This article has been cited 11 times.- Jochems R, Canedo-Ribeiro C, Silvestri G, Derks MFL, Hamland H, Zak LJ, Knol EF, Handyside AH, Grindflek E, Griffin DK. Preimplantation Genetic Testing for Aneuploidy (PGT-A) Reveals High Levels of Chromosomal Errors in In Vivo-Derived Pig Embryos, with an Increased Incidence When Produced In Vitro.. Cells 2023 Mar 2;12(5).
- West JD, Everett CA. Preimplantation chromosomal mosaics, chimaeras and confined placental mosaicism.. Reprod Fertil 2022 Apr 1;3(2):R66-R90.
- Antczak DF, Allen WRT. Placentation in Equids.. Adv Anat Embryol Cell Biol 2021;234:91-128.
- Rizzo M, du Preez N, Ducheyne KD, Deelen C, Beitsma MM, Stout TAE, de Ruijter-Villani M. The horse as a natural model to study reproductive aging-induced aneuploidy and weakened centromeric cohesion in oocytes.. Aging (Albany NY) 2020 Nov 2;12(21):22220-22232.
- Shilton CA, Kahler A, Davis BW, Crabtree JR, Crowhurst J, McGladdery AJ, Wathes DC, Raudsepp T, de Mestre AM. Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse.. Sci Rep 2020 Aug 7;10(1):13314.
- Klohonatz KM, Coleman SJ, Cameron AD, Hess AM, Reed KJ, Canovas A, Medrano JF, Islas-Trejo AD, Kalbfleisch T, Bouma GJ, Bruemmer JE. Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy.. Genes (Basel) 2019 Oct 18;10(10).
- Franciosi F, Tessaro I, Dalbies-Tran R, Douet C, Reigner F, Deleuze S, Papillier P, Miclea I, Lodde V, Luciano AM, Goudet G. Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes.. J Vis Exp 2017 May 11;(123).
- Hornak M, Jeseta M, Hanulakova S, Rubes J. A high incidence of chromosome abnormalities in two-cell stage porcine IVP embryos.. J Appl Genet 2015 Nov;56(4):515-523.
- Hornak M, Oracova E, Hulinska P, Urbankova L, Rubes J. Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts.. PLoS One 2012;7(1):e30335.
- Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Vanhaesebrouck E, Galli C, Colleoni S, Vandesompele J, Peelman L. Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts.. BMC Res Notes 2009 Dec 11;2:246.
- Coppola G, Alexander B, Di Berardino D, St John E, Basrur PK, King WA. Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos.. Chromosome Res 2007;15(3):399-408.
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