Analysis of chromosome segregation, histone acetylation, and spindle morphology in horse oocytes
2017
Franciosi, Frederica | Tessaro, Irène | Dalbies-Tran, Rozenn | Douet, Cécile | Reigner, Fabrice | Deleuze, Stefan | Papillier, Pascal | Miclea, Ileana | Lodde, Valentina | Luciano, Alberto M. | Goudet-Guitton, Ghylène | Department of Health, Animal Science and Food Safety ; Università degli Studi di Milano = University of Milan (UNIMI) | Istituto Ortopedico Galeazzi ; Istituto di Ricovero e Cura a Carattere Scientifico | Physiologie de la reproduction et des comportements [Nouzilly] (PRC) ; Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS) | Unité Expérimentale de Physiologie Animale de l‘Orfrasiére (UE PAO) ; Institut National de la Recherche Agronomique (INRA) | Clinique des Animaux de Compagnie et des Équidés ; Université de Liège = University of Liège = Universiteit van Luik = Universität Lüttich (ULiège) | University of Agricultural Sciences and Veterinary Medicine | Regione Sardegna and Regione Lombardia" project "Ex Ovo Omnia" (Grant no. 26096200 to A. M. L.); "L'Oreal Italia per le Donne e la Scienza 2012" fellowship (Contract 2012 to F. F.); FP7-PEOPLE-2011- CIG; Research Executive Agency (REA) "Pro-Ovum" (Grant no. 303640 to V. L.); the Postdoctoral School of Agriculture and Veterinary Medicine, co-financed by the European Social Fund, Sectorial Operational Program for Human Resource Development 2007-2013 (Contract no. POSDRU/89/1.5/S/62371 to I. M.); Institut Français du Cheval et de l'Equitation
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Afficher plus [+] Moins [-]anglais. 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 collectingimmature 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.
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