Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes.
Abstract: The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected. Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.
Publication Date: 2017-05-11 PubMed ID: 28518085PubMed Central: PMC5607939DOI: 10.3791/55242Google Scholar: Lookup
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Summary
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The research focuses on methods of collecting immature and mature oocytes from horse ovaries and their subsequent analysis to further understand the reproductive biology of horses and potentially improve assisted reproduction techniques. It identified a higher rate of aneuploidy, differences in spindle morphology, and histone acetylation patterns in oocytes matured in a lab as compared to ones matured in a living organism.
Objective and Approach
- The primary goal of this study is to develop and apply experimental procedures for collecting oocytes (cells that develop into an egg) from horse ovaries.
- Additionally, the research aims to study these harvested oocytes to investigate the occurrence of aneuploidy (abnormal number of chromosomes), characteristics of the meiotic spindle (structure that separates chromosomes during cell division), and patterns of histone acetylation (a gene regulatory process).
- The use of the ovum pick-up (OPU) technique allows for the collection of these oocytes.
- The collected oocytes are then put through a protocol adapted from a previously established method in mice.
Findings
- The research revealed a higher incidence of aneuploidy in oocytes that were matured in a lab (in vitro) as opposed to those matured in a living organism (in vivo).
- Further visualization techniques showed differences in the morphology (shape and structure) of the meiotic spindle in these two groups of oocytes.
- The global pattern of histone acetylation, a process that regulates gene expression, was also found to be different.
- The expression levels of genes coding for histone deacetylases (HDACs) and acetyl-transferases (HATs), enzymes that regulate histone acetylation, were analyzed, but no significant differences were found between in vitro and in vivo matured oocytes.
Implications
- The findings suggest that regulations at the translational level (conversion of RNA to protein) and post-translational level (modifications after protein synthesis) might be affected in oocytes matured in vitro.
- These results can enhance our understanding of the reproductive biology of monovulatory species (species that typically release one egg per cycle) and help in the further development of assisted reproduction techniques.
- The understanding and ability to manipulate these cellular processes are essential because they can improve researchers’ ability to produce embryos from high-performing or high-genetic-value horses without interrupting their career—a critical aspect in the industry of horse breeding and sports.
Cite This Article
APA
Franciosi F, Tessaro I, Dalbies-Tran R, Douet C, Reigner F, Deleuze S, Papillier P, Miclea I, Lodde V, Luciano AM, Goudet G.
(2017).
Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes.
J Vis Exp(123), 55242.
https://doi.org/10.3791/55242 Publication
Researcher Affiliations
- Department of Health, Animal Science and Food Safety, University of Milan; federica.franciosi1@unimi.it.
- Department of Health, Animal Science and Food Safety, University of Milan; IRCCS. Istituto Ortopedico Galeazzi.
- PRC, CNRS, IFCE, Université de Tours, INRA.
- PRC, CNRS, IFCE, Université de Tours, INRA.
- PAO, INRA.
- Clinique des Animaux de Compagnie et des Équidés, Université de Liège.
- PRC, CNRS, IFCE, Université de Tours, INRA.
- University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania.
- Department of Health, Animal Science and Food Safety, University of Milan.
- Department of Health, Animal Science and Food Safety, University of Milan.
- PRC, CNRS, IFCE, Université de Tours, INRA.
MeSH Terms
- Acetylation
- Aneuploidy
- Animals
- Centromere / ultrastructure
- Chromosome Segregation
- Female
- Gene Expression
- Histone Acetyltransferases / biosynthesis
- Histone Acetyltransferases / genetics
- Histone Deacetylases / biosynthesis
- Histone Deacetylases / genetics
- Histones / chemistry
- Histones / metabolism
- Horses / physiology
- In Vitro Oocyte Maturation Techniques
- Metaphase
- Oocytes / metabolism
- Oocytes / physiology
- Ovum
- RNA, Messenger / biosynthesis
- RNA, Messenger / genetics
- Spindle Apparatus / ultrastructure
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Citations
This article has been cited 5 times.- Lodde V, Luciano AM, Musmeci G, Miclea I, Tessaro I, Aru M, Albertini DF, Franciosi F. A Nuclear and Cytoplasmic Characterization of Bovine Oocytes Reveals That Cysteamine Partially Rescues the Embryo Development in a Model of Low Ovarian Reserve. Animals (Basel) 2021 Jun 29;11(7).
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