318 INHIBITION OF FIRST POLAR BODY EXTRUSION BY CYTOCHALASIN B DURING IN VITRO MATURATION OF PORCINE OOCYTES LEADS TO RE-ARRANGEMENT OF THE SEGREGATED CHROMOSOMES AND IMPROVEMENT IN THE QUALITY OF THE PARTHENOGENETIC BLASTOCYSTS

Abstract

Diploid parthenotes are usually obtained by the inhibition of second polar body (PB2) extrusion after activation of metaphase II (MII) oocytes. However, diploid embryos can be generated by the inhibition of the first polar body (PB1) extrusion as well, using cytochalasin B (CB) during in vitro maturation prior the activation procedure. A higher percentage of mouse embryos generated by the activation of MII oocytes and the inhibition of PB2 extrusion were proven to be homozygous than for parthenotes obtained by the latter method (Kubiak et al. 1991 Development 111, 763-769). The aim of the present study was to examine if such difference has any effect on the development of parthenogenetic embryos in vitro. Nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to CB during in vitro maturation (IVM) was investigated in the present study. The tendency of nuclear maturation was similar in oocytes matured in the presence of 1 ¼g/mL CB (IVM-CB group) and control oocytes matured without CB after 37 h of IVM; at this time the frequency of oocytes that had reached/or passed through anaphase-I stage did not differ significantly (P < 0.05) between the IVM-CB and the control groups (61.3% and 69.9%, respectively), however, no polar body extrusion was observed in the IVM-CB group and the two lumps of homologue chromosomes remained in the oocyte and turned into two irregular sets of condensed chromosomes. By 41 h of IVM, the double sets of chromosomes re-united in 89.5% of IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached metaphase-II stage (MII) by this time. When IVM-CB oocytes were electrically (1.5 kV/cm for 100 ¼s) activated and subsequently cultured without CB, 39% of the oocytes extruded a polar body (PB) and 82.9% of them had a female pronucleus. When those oocytes with PB were cultured, the blastocyst rate of the cleaved embryos did not differ (P < 0.05) from those of the control that were stimulated at MII and subsequently treated with CB (43.3% and 48.2%, respectively). The number of blastomeres in Day 6 blastocysts was significantly higher (P < 0.05) in the IVM-CB derived embryos than in those in the control group (47.8 and 40.7, respectively); moreover, the ratio of dead blastomeres (dead cells : live cells) was higher (P < 0.05) in the control than in the IVM-CB blastocysts (0.047 and 0.031, respectively). A possible explanation for this result might be a lower frequency of homozygous genes in IVM-CB parthenotes, in which segregation of sister chromatids were promoted instead of segregation of homologous chromosomes to obtain diploid embryos. In such embryos the expression of recessive lethal, sublethal and subvital genes might have a lower probability.

This work was supported by the Japanese-Hungarian bilateral scientific and technological cooperation (TET JAP-11/02).