Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle

Abstract

Vitrified–warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus–oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed using immunofluorescent staining, confocal microscopy and three-dimensional image analysis. Vitrification reduced the ability of oocytes to reach MII and resulted in ultrastructural changes to the meiotic spindle, including shortening of its long axis, and an increased incidence of chromosomes failing to align properly at the metaphase plate. We hypothesise that aberrant chromosome alignment is an important contributor to the reduced developmental competence of vitrified equine oocytes. Contrary to expectation, oocytes from young mares were more severely affected than oocytes from older mares; we propose that the reduced effect of vitrification on oocytes from older mares is related to pre-existing compromise of spindle assembly checkpoint control mechanisms in these mares.

Follicular extracellular vesicles enhance meiotic resumption of domestic cat vitrified oocytes

“…It is likely that ffEVs also contributed to cell structural recovery and to stress response following vitrification. Vitrification was previously described to increase the number of abnormal spindle in MII oocytes from sow, cow, woman, mouse, horse and cat 18,54,[91][92][93] . In our study, no abnormal MII spindles were observed in the non-vitrified controls or in the vitrified oocytes that were incubated with ffEVs.…”

Cryopreservation of equine oocytes: looking into the crystal ball

“…The most extensively studied structures in equine oocytes after cryopreservation are probably the actin cytoskeleton and meiotic spindle (Tharasanit et al 2006a(Tharasanit et al , 2006b(Tharasanit et al , 2009Ducheyne et al 2019), which have been characterised by the disruption of the subcortical actin network, depolymerisation of microtubules, a reduced length and volume of the spindle and an increased metaphase plate thickness. Such changes may impair correct chromosome alignment and cause cytokinesis failure, leading to chromosomal aberrations and subsequent developmental failure.…”

“…Schematic representation of some alterations observed in oocytes after vitrification-warming. (a) Disruption of the actin network, depolymerisation of microtubules and altered length and volume of the spindle, impairing correct chromosome alignment, have been described in vitrified-warmed mature oocytes and in immature equine oocytes that were matured in vitro after warming (Tharasanit et al 2006a(Tharasanit et al , 2006b(Tharasanit et al , 2009Ducheyne et al 2019). (b) Open hemichannels (which are normally closed) have been found in bovine and feline oocytes after vitrification, possibly causing a release of essential ions and allowing the entry of damaging molecules (Snoeck et al 2018;Szymańska et al 2018).…”

“…(c) Fractures and less conspicuous disruption of lipid bilayer membranes affecting the developmental capacity of the oocyte, (d) swelling and changes in the shape and density of organelles such as mitochondria, Golgi apparatus and smooth endoplasmic reticulum and (e) lipid droplets and extruded cortical granules (in vitrified matured oocytes) with debris in the perivitelline space have often been noticed (Hochi et al 1994(Hochi et al , 1996Curcio et al 2014a). 2017; Ducheyne et al 2019). Although repolarisation of the spindle is seen after warming in some human oocytes vitrified at the mature stage (Minasi et al 2012), a similar effect does not seem to occur in vitrified equine oocytes (Tharasanit et al 2006a).…”

“…In domestic animals, live births have been obtained after oocyte cryopreservation in the mouse (Parkening et al 1976), rabbit (Al-Hasani et al 1989), cow (Fuku et al 1992), horse (Maclellan et al 2002;Ortiz-Escribano et al 2017), pig (Somfai et al 2014) and cat (Pope et al 2012). In contrast with the situation in humans, the clinical efficiency in animal species is not satisfactory (Ducheyne et al 2019;Mogas 2019). Oocyte cryopreservation is only of practical use in a particular animal species if the associated assisted reproductive technologies (i.e.…”