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Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes

¹ Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 112, 3584 CM Utrecht, The Netherlands² Laboratorio di Tecnologie della Riproduzione, Instituto Sperimentale Italiano Lazzaro Spallanzani, Via Porcellasco 7/F, Cremona 26100, Italy³ Faculty of Veterinary Medicine, University of Bologna, V. Tolara di Sopra 50, Ozzano Emilia, Bologna, Italy⁴ Department of Production Animal Sciences, University of Pretoria, Pretoria 0002, South Africa

Correspondence should be addressed to T A E Stout; Email: t.a.e.stout{at}uu.nl

Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were labelled for connexin-43 or injected with Lucifer yellow dye to determine whether the integrity of the gap junctions between an oocyte and its cumulus was compromised by vitrification. Finally, the effect of denuding immature and mature oocytes on their ability to withstand vitrification was examined. Cryopreserving immature COCs increased the number of dead cumulus cells (13 vs 2.6% for controls; P<0.05). However, an increased proportion of dead cumulus cells did not affect post-warming maturation rates (30% MII) presumably because dead cells were located at the periphery of the cumulus mass and cumulus-oocyte gap junction communication was not disrupted. Moreover, cumulus removal prior to IVM or vitrification indicated that while the cumulus does protect immature oocytes during vitrification it does so by mechanisms other than support during maturation. Cumulus presence was also found to protect mature equine oocytes against vitrification-induced damage since cumulus-enclosed MII oocytes preserved their meiotic spindle quality better during vitrification than denuded oocytes (38.1 vs 3.1% normal spindles; P<0.05).