59 BOVINE OOCYTE RECONSTRUCTION FOLLOWING CRYOPRESERVATION AND GERMINAL VESICLE TRANSPLANTATION

Abstract

The aim of the present study was to assess the maturational competence and the cytoskeletal pattern of bovine oocytes reconstructed by a germinal vesicle transplantation (GVT) technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. Cumulus-oocyte complexes were isolated from slaughtered ovaries and denuded oocytes were cryopreserved by vitrification in open pulled straws (Vajta et al. 1998 Mol. Reprod. Dev. 51(1), 53-58) after cumulus cell removal (Modina et al. 2004 Eur. J. Histochem. 48(4), 337-346). Denuded oocytes were maintained in meiotic arrest with 0.5 mM of 3-isobutyl-1-methylxanthine, centrifuged for 20 min in for visualization of the germinal vesicle and then treated with 7.5 µg/mL of cytochalasin B for 20 min before enucleation. By means of micromanipulation and electrofusion procedures, female gametes were reconstructed from karyoplasts and cytoplasts obtained from either cryopreserved or freshly isolated oocytes, and then in vitro-matured for 24 h (Luciano et al. 2005 Mol. Reprod. Dev. 71(3), 389-397). After in vitro maturation (IVM), the nuclear stage was assessed by Hoechst 33342 staining and oocytes were fixed for analysis of the organization of microtubules and microfilaments, which provide the framework for chromosomal alignment and meiotic division. A total of 179 oocytes were reconstructed by the GVT procedure. All data were analyzed by chi-square test. No differences were observed in the reconstruction efficiency after fusion between fresh and cryopreserved oocytes (77.6% and 81.2%, respectively). The maturational competence of artificial gametes originated from cryopreserved oocytes was not different from that of gametes reconstructed with fresh oocytes (21.3% and 26.7%, respectively) and cytoskeleton organization analysis of artificial oocytes demonstrated that the correct distribution of microtubules and microfilaments was associated with a proper progression through metaphase II (MII) of meiotic division. However, the maturation rate of reconstructed oocytes was significantly lower than that of the control group, represented by untreated oocytes (86.7%; P < 0.05), and pre-enucleation procedures significantly affected the capability of oocytes to reach the MII stage of meiotic division (73.3%; P < 0.05). We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature bovine oocytes are suitable for the GVT procedure, and that they generate oocytes able to progress to MII in vitro. The possibility of using cryopreserved immature oocytes as a source of nuclei and cytoplasm could help in applying the GV transfer procedure, both in research and clinical settings and in fertility preservation programs.

This work was supported by a PRIN 2003 MIUR Grant.