Introduction: Microtubule dynamics are essential for the production of a normal meiotic spindle and fertile oocyte. Katanin is an evolutionarily conserved microtubule-severing complex consisting of a p60 severing enzyme and a p80 regulatory subunit. The aim of this study is to investigate the role of Katanin-mediated microtubule severing in oocyte and embryo development.
Methods: We generated oocyte-specific knockouts, using conditional knockout mice (ZP3-Cre), of katanin subunit p80. Breeding trials were conducted to test the effect of p80 on fertility. Meiotic spindles and chromosome organisation were examined in MI and MII stage oocytes using immunofluorescence and hoechst staining. In vitro fertilisation (IVF) and parthenogenetic activation (PA) were performed on control and KO oocytes to determine the role of katanin in oocyte and embryo development.
Results: Breeding experiments show infertility in the p80KO. The first meiotic spindle was normal in oocytes of p80KO mice but defects including, monopolar/multipolar spindles, were observed in the MII eggs (60% vs 0%, p<0.0001). After IVF with wildtype sperm, there was no effect on the rate of 2-cell formation and blastocyst development, although there was a significant decrease in the diameter of the blastocyst (p<0.01) which was reflected in a significant reduction in cell number (49±7 vs 17±2, p<0.0001) as well as other morphological abnormalities. To create homozygous p80 KO embryos, oocytes were parthenogenetically activated. This caused a dramatic failure of preimplantation development with only 16% of the p80KO oocytes reaching blastocyst compared to 68% of controls (P<0.0001). Time-lapse imaging of early embryo development revealed that mitotic failure, asymmetric cell division were prevalent in p80KO parthenogenetic embryos.
Conclusion: This study shows that katanin is essential for fertility and although these microtubule-severing proteins appear to play a minor role in meiosis, they are critical for the fidelity of early embryonic cell division.