Preparation of karyoplasts and cytoplasts from feline oocytes at the germinal vesicle stage

Introduction. The homologous transfer of oocyte nucleus stage I or germinal vesicle (GV-karyoplast) into an enucleated mouse oocyte (cytoplast) at the same developmental stage, resulted in resumption of meiosis (1) and embryo development in vitro (2) of the reconstructed GV-oocytes. The transfer of GVs derived from cryopreserved oocytes also resulted in a normal progression to metaphase II (MII) in vitro of the reconstructed mouse and bovine oocytes (3,4). It has been shown that the GV is potentially more resilient to cooling than the spindle of MII, because chromosomes are decondensed and enclosed within the nuclear membrane. However, thawed immature cat oocytes show a poor developmental competence in vitro (5), probably due to the occurrence of chilling-induced damages to the cytoplasm. This compartment has a pivotal role in the resumption and completion of oocyte maturation, which is essential for the developmental competence of the embryo. Since the integrity of the oocyte nucleus may be better preserved than the cytoplasm after cryopreservation, the transfer of cryopreserved GVs into fresh enucleated oocytes could improve the chance of embryo development in culture. In the feline species there are no reports in the literature concerning GV transfer. Hence, the purpose of this work was to make a preliminary evaluation of the feasibility of enucleating immature oocytes in order to produce GV-karyoplasts and cytoplasts for GV transfer in cat oocytes. Materials and methods. A total of 156 immature (GV) cat oocytes collected from anestrous queens after ovariectomy were mechanically deprived of cumulus cells with a small-bore pipette. The oocytes were centrifuged at 14000 rpm for 16 min to obtain the polarization of the cytoplasm and a better visualization of the GV. The nucleus was measured in order to prepare adequate microtools for manipulation. Prior to enucleation, the oocytes were incubated for 30 min at room temperature in a specific medium containing 7.5 g/mL of cytochalasin B (Sigma Chemical Co., USA) for inducing an increase of the oolemmal elasticity and 50 g/mL of 3-isobutyl-1-methylxanthine (IBMX, Sigma) in order to prevent the GV breakdown (1). Following lancing of the zona pellucida with a sharp-tipped pipette, GV nuclei were extruded using a bevelled glass pipette with a diameter adequate to the size of GV in cat oocytes. The GV was surrounded by a small amount of cytoplasm and encapsulated by a membrane (GV-karyoplast). The enucleated oocytes were considered as cytoplasts. Results. The mean average of GV diameter in oocytes > 120 m of diameter was 35.4 + 5.3 m. A bevelled glass pipette, with inner diameter of 40-45 m, allowed the extrusion of intact and morphologically normal karyoplasts and related cytoplasts in 17.3% (27/156) of micromanipulated oocytes. However, the lancing of the zona pellucida or the extrusion of the karyoplast resulted in rates of 40.4% (63/156) and 42.3% (66/156) of severely damaged oocytes, respectively. Conclusion. These results suggest that is possible to prepare karyoplasts and cytoplasts from feline oocytes, although the efficiency of the technique is low compared to what has been obtained in mouse (around 90%, 1). This is likely due to the thickness and hardness of zona pellucida, and to the larger diameter of the GV of cat oocytes compared to that of mouse (15 m) or bovine (25-30 m) oocytes. Further experiments based on the partial dissection of the zona pellucida with an acidic solution in order to reduce the oocyte damage and to improve the efficiency of GV transfer in feline oocytes, are in progress in our laboratory. References 1) 1) Liu et al., Human Reprod 1999; 14:2357-61. 2) Takeuchi et al., Hum Reprod 2004;19:975–81. 3) Moffa et al., Human Reprod 2002;17:178-83. 4) Luciano et al., Reprod Fertil Dev 2006;18:138. 5) Luvoni and Pellizzari, Theriogenology 2000;53:1529-40.