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.

Advanced mare age impairs the ability of in vitro‐matured oocytes to correctly align chromosomes on the metaphase plate

“…Morphometric analysis revealed that the meiotic spindle in horse oocytes has axial dimensions of approximately 21 9 12 lm. These measurements differ from those previously reported by Franciosi et al [11] (19.89 AE 1.15 lm vs. 17.28 AE 0.81 lm); however, the latter authors measured the spindle axes on the basis of a projected image rather than using confocal microscopic Z-stacks and 3D image reconstruction and analysis, as in the present study. Our results show that the spindle in horse oocytes is larger than that in human oocytes (12 9 9 lm) [15] but smaller than in mice oocytes, where the major axis ranges from 20 to 30 lm [18].…”

“…In particular, advanced maternal age seems to impair the ability of the oocyte to detect and correct the presence of misaligned chromosomes [10]. While in vitro maturation has been shown to compromise the process of chromosome segregation in mares, the effect of maternal age on the ability to correctly form a spindle and align the chromosomes is unknown [11]. The purpose of this study was to determine whether advanced mare age affects spindle morphology and interferes with proper alignment of chromosomes in in vitro-matured MII oocytes.…”

The development of in vitro embryo production in the horse

“… were the first to demonstrate the possibility of producing pregnancies from in vitro matured oocytes transferred to the oviduct for in vivo fertilisation. Elegant studies have subsequently described differences in anatomical and molecular processes between in vivo and in vitro maturation of oocytes which may affect subsequent embryo development . Using only light microscopy, the high lipid content of the equine oocyte makes the cytoplasm dark and difficult to evaluate so other indicators of oocyte quality such as the existence of the perivitelline space, an intact oolemma and segregation of dark and light cytoplasmic zones, have been employed .…”

“…To learn to differentiate among embryo development, fragmentation and parthenogenesis it is important to monitor the development of in vitro produced embryos by using a routine nuclear staining of non‐cleaved and presumptive embryos to document the efficacy of the IVP system in any laboratory . More sophisticated methods of analysis include confocal microscopy and mRNA expression . Useful bioassays of gamete competence include heterologous assays using bovine or porcine oocytes , monitoring the development rate of embryos and, ultimately, the pregnancy success after transfer.…”

“…The excitement in the international equine breeding industry for this technology is an acknowledgement of the incredible progress made by laboratories worldwide, while operating with limited resources. This progress in the equine field of IVP can be applied to the preservation of endangered equids and it can also be a model for human IVP systems . However, the motivation for increasing IVP technology will be driven mainly by the breeders who will want to include sex‐sorted semen , the use of epididymal spermatozoa , genetic testing of embryos and functional IVF culture systems .…”