The Meiosis Of Oocytes Reconstructed By Mouse Pachytene Primary Spermatocyte And GV Oocyte Cytoplasm

The promise for using artificial reproductive technologies such as in vitro fertilization and intracytoplasmic sperm injection is the patients have the ability to produce the normal oocytes or sperms, unless they can get the artificial gametes from their own somatic cells. So, somatic cell haploidization is the the last straw. Theoretically, mitosis and meiosis share some common mechanisms, although many differences are still between them. Meiotic cells especially the huge oocytes are the optimal candidates to produce areificial gametes. Pachytene primary spermatocytes and GV oocytes are both in phrophse of meiosis I. Compared with somatic cells, primary spermatocytes have the superiority in cell cycles to overcome the obstacles that have been found in contructed GV oocytes. We can use the mouse constructed GV oocytes which nuclei are replaced by primary spermatocytes nuclei to study the ability of meiosis, chromosome condensation and segregation, the activity of MPF and MAPK to assess the ability of the GV cytoplasm to haploidize the genetic material of primary spermatocytes even the somatic cells.Mice of the Kunming breed were used throughout the experiments. We injected the parimary spermatocyte nuclei into the enucleated GV oocytes to get the PS+GV constructed oocytes and studied the maturation rate, the size of the first polar bodies, the time needed for extruding the first polar body, the distribution of phosphorylated MAPK (p-MAPK), chromosome condensation and segregation, the activity of spindle assembly checkpoint, the degeneration of the first polar bodies and activation of the maturated constructed oocytes. The results are summarized as follows:1. The constructed oocytes could get through meiosis I, and the maturation rate and time needed for extruding the first bolybody had no difference with GVT oocytes. So the micromanipulation for construction had no impact on maturation.2. The constructed oocytes extruded big polar bodis, but there was no difference in the activity of MPF and MAPK between constructed oocytes with big and small polar bodies. The MPF activity of constructed oocytes was lower than GVT oocytes, but the MAPK activity had no difference with GVT oocytes. 3. The treatment of OA or U0126 which are MAPK activator or inactivator drugs respectively during in vitro maturation had no effect on the size of the first polar bodis of constructed oocytes.4. The difference of the first polar bodis of constructed oocytes whose donor nuclei were injected in the center or peripheral of the GV cytoplasm was not significant.5. The treatment of nocodazole during in vitro maturation for delaying the maturation of constructed oocytes had no effect on the size of the first polar bodies of constructed oocytes.6. The distributions of actin and cortical granules were different between constructed and GVT oocytes, GVT oocytes had the cap shapped actin enriched area and cortical granule-free domain (CGFD) overlied the chromatin of the oocytes maturated in vitro for 6h, but the constructed oocytes did not.7. The chromosome condensation of constructed oocytes was abnormal, there was no well assemblied chromosome in constructed oocytes, but the chromosome condensation of the GVT oocytes was normal.8. The chromosome segregation of the meiosis of constructed oocytes was not equal, but the GVT oocyte was.9. The constructed oocytes had the phenomenon of chromosome bridge during cell division.10. The inhibition of maturation with Nocodazole treatment during in vitro culture, and the acceleration of the extrution of the first polar body with the injection of Anti-Mad2 antibody indicted that the spindle assembly checkpoint was normal during constructed oocyte maturation.11. The degeneration rate of the first polar bodies of constructed oocyties was about 60% and there was no difference between large polar bodies and small polar bodies.12. The treatment of U0126 after maturation decreased the degeneration rate of the first polar bodies of constructed oocyties.13. The p-MAPK segregation was unequal during constructed oocyte division.14. There was no difference in the degree of uneven segregation of p-MAPK between constructed oocytes with big or small polar bodies.15. The parthenogenesis rate of maturated constructed oocytes by SrCl2 after aged 12h was 60%.16. The pronuclear membrane or nucleoli were invisible under light microscopy after activation of matured reconstructed oocytes.17. The cleavage furrow of constructed oocyte was abnormal. Together, the results suggest that the replacement of the GV oocyte nuclei with primary spermatocyte nuclei has no effect on maturation rate, but this replacement is the cause of the big first polar bodies. The abnormal chromosome condensation and segregation is accompanied with the activation of spindle assembly checkpoint. The regulation of MAPK activity during maturation and the delay of the maturation have no effect on the size of the first polar bodies, and there were no actin cap and CGFD in cortical aera near the chromation, these phenomena indict that the first meiotic spindle migration is failure duing the meiosis I of the constructed oocytes. The abnormal cleavage furrow of constructed oocyte affects the segregation of chromatin and p-MAPK during the first meiosis of constructed oocyte. So, the disturbed segregation of p-MAPK during meiosis I of the constructed oocytes caused the abnormal quality of the p-MAPK in matured constructed oocytes and first polar bodies which may be the reason for abnormal first polar bodies degeneration and lower parthenogenesis rate.