| Reproductive technologies are effective in overcoming male and female infertility. Ovarian stimulation combined with IVF or ICSI has become standard treatment in cases of ovarian cycle anomalies, tubal pathology, sperm antibodies, oligoasthenoteratozoospermia and even azoospermia. Only for patients without gametes are there currently no treatment options leading to their own genetic children. Women with ovarian failure or defective oocytes or men with spermatogenic failure are sterile and can only be treated by using donor gametes or embryos or by adoption. Recently, novel technology (somatic cell haploidization)derived from cloning may offer an alternative strategy for treatment of male and female sterility by creating gametes .Somatic cell haploidization is defined as a process whereby a somatic nucleus (2N) undergoes induced meiosis, during which the diploid chromosomes are reduced to haploid (1N). Mammalian oocytes are ideally suited for use as haploidization, because they are easily obtained and manipulation techniques are well established. Despite the success in using germ cells to show that, in principle, haploidization is possible, successful generation and fertilization of artificially hapoidized gametes from somatic cells has not been reported. A careful study of published data revealed similar meiotic abnormalities between the reconstructed oocytes with GV replaced with somatic cells. For examle, reconstructed oocytes frequently produced failure of chromosome segregation, abnormally large first polar bodies, diffused spindles, loosely condensed chromosomes and low activity of protein kinases. Therefore, it is necessary to study some meiotic events of haploization of somatic cells.The meiotic events of mouse oocytes reconstructed with germinal vesicles replaced with primary spermatocytes nuclei were studied by using in vitro culture, drug treatment, confocal microscopy, protein kinase assay in maturation and activation. Besides, we exmined MAP kinase activity in reconstructed oocytes to elucidate the molecular mechanisms regulating meiosis. The results are as follows:1. No difference was found in maturation rates, large polar body formation and chromosome condensation between intact denuded oocytes and germinal vesicle transferred oocytes at the end of the culture period. The results indicatied that the microsurgical procedure had no effect on these oocyts.2. Meiotic maturation could be achieved in reconstructed oocytes matured after primary spermatocyte was transferred into GV ooplasm, but the first polar body was very large and the proportion of oocytes with adequately condensed chromosome was very low. The cytoplasm amount of primary spermatocytes had no influence on the proportion of maturation rates, large polar body formation and adequately condensed chromosome in reconstructed oocytes. OA treatment during maturation reduced the proportion of reconstructed oocytes with large polar body and improved the proportion of reconstructed oocytes with adequately condensed chromosome. However, the proportion of reconstructed oocytes with large polar body was significantly improved by U0126 treatment during maturation, but this treatment had no effected on chromosome condensation.3. After PS was transferred into GV ooplasm, extrusion of the first polar body occurred at 4.5-6.5hr which was approximately 4hr earlier than DO or GV transferred oocytes.4. First polar body did not degenerate after aging for 40 hours in reconstructed oocytes and this was different from DO and GV transferred oocytes. It was OA treatment during maturation not U0126 treatment before activation that significantly improved the degeneration of first polar bodies in reconstructed oocytes with primary spermatocytes and GV cytoplasm. Moreover, when treated with OA during maturation followed by U0126 treatment before activation, the degeneration rates of first polar bodies in reconstructed oocytes was the same as reconstructed oocytes treated only with OA during maturation. The results mentioned above suggested that the events regulating the degeneration of first poalr body in renconstructed oocyte occurred during maturation. Once the first polar body was extrused, no treatment was effective.5. The percentages of activation and pronucleus formation in reconstructed oocytes were significantly lower than that in GV transferred oocytes. Either OA treatment during maturation or U0126 treatment before activation improved the activation and pronucleus formation in reconstructed oocytes and there was no difference between the two treatments. If treated with OA during maturation followed by U0126 treatment before activation, the percentage of activation and pronucleus formation in reconstructed oocytes was significantly higher than that obtained with either treatment only.6. Three different types of immunostaining were established : type I, high staining; type II, intermediate staining; type III, negative staining by using the confocal settings and anti-lamina antibody. DO and GV transferred oocytes showed only type I staining. But reconstructed oocytes with primary spermatocytes showed only type II staining. Either OA treatment during maturation or U0126 treatment before activation significantly improved the proportion of type I staining and the effect of OA treatment during maturation was better than U0126 treatment before activation. If treated with OA during maturation followed by U0126 treatment before activation, the percentage of type I staining in reconstructed oocytes was significantly higher than that obtained with either treatment only.7. MAP kinase and MPF activity were significantly lower in fresh MII reconstructed oocytes than that in fresh GV transferred oocytes at MII. OA treatment during maturation in reconstructed oocytes with primary spermatcytes and GV cytoplasm improved MAP kinase activity significantly and U0126 treatment during maturation had the opposite effect. Howerver, the two treatments during maturation had no influence on MPF activity. From the results mentioned above, we can conclude that large first poalr body formation, long-term persistence of first polar body, inability of bipolar spindle formation and adequately condensed chromosome may be due to the low MAP kinase activity during maturation in reconstructed oocytes.8. MPF activity in GV transferred oocytes from MII stage to the stage before and after activation decreased gradually. But the degree of MPF activity decline in reconstructed oocytes was smaller than that in GV transferred oocytes. OA treatment during maturation and U0126 treatment before activatioh did not affect MPF activity in MII reconstructed oocytes. 9. MAP kinase activity in GV transferred oocytes from MII stage to the stage before and after activation decreased gradually. Howerver, MAP kinase activity in reconstructed oocytes did not change significantly. MAP kinase activity was lower in reconstructed oocytes at MII than that in GV transferred oocytes and OA treatment during maturation improved the MAP kinase activity significantly in reconstructed oocytes at MII, thus MAPK activity in reconstructed oocytes also declined significantly. U0126 treatment before activation significnatly declined MAPK activity in reconstructed oocytes. If treated with OA during maturation followed by U0126 treatment before activation, the degree of MAP kinase activity decline in reconstructed oocytes was higher than that in reconstructed oocytes treated with OA or U0126 only.10. In conclusion, data mentioned above revealed many abnormalities in the reconstructed oocytes. For example, reconstructed oocytes frequently produced abnormally large first polar bodies, loosely condensed chromosomes, first polar body long-term persistence, and failure of pronucleus formation. These meiotic abnormal events were relative to the changes of MAP kianse activity in matured oocytes, oocytes before activation and after activation throughout detection of MAPK activity and drug treatment regulating MAPK activity.
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