Effect Of P21 On Bovine Oocytes Meiotic Maturation

Stability of maturation promoting factor (MPF; CDK1/cyclin B1) is needed for completing meiotic cell cycle arrest in mammalian oocytes. P21 is pivotal cyclin-dependent kinase inhibitors (CKIs) which belongs to a member of the Cip/Kip family, and is required for negative regulation of the cell cycle progression. P21 can directly inhibit Thr-161 phosphorylation of CDK1 in the frug oocytes, then inactivation of MPF. This study focues on effect of P21 on mammalian oocytes maturation.The various periods morphological changes of chromosomes during in vitro maturation and the transition from meiosis to mitosis were observed by aceto-orcein staining technique in bovine oocyte. The bovine p21 gene was cloned from cDNA simple of fibroblasts and pVenus-P21 eukaryotic expression vector was constructed. Last, the mRNA of P21 in the bovine oocytes were examined by the Q-PCR, and the protein of P21 were examined by immunofluorescence staining. The effects of P21 on exit from the M-I and M-II were preliminarily researched by over-expression and interference P21. The results were as follows:1. First, The various periods morphological changes of chromosomes during in vitro maturation and the transition from meiosis to mitosis were observed by aceto-orcein staining technique in bovine oocyte. The timetable of oocytes chromosome morphological events were established and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 5.45 h, GVBD to chromatin condensation at 5.45 to 11.4 h, metaphase I at 11.4 to 13.15 h, anaphase I at 13.15 to 17.10, telophase I at 17.10 to 18.35 h, and metaphase II at 18.35 to 24 h. Then the influences of three chemical activation methods(Ionomycin alone, Ionomycin coupled with 6-DMAP, and Ionomycin coupled with Roscovitine)on the second polar body extrusion and pronuclear formation. Results show that there are a large number of second polar body after 2 h of parthenogenetic activation, and pronuclear formation in the 8 h and have the highest number of pronuclear in 16~18 h after parthenogenetic activation by ionomycin alone. But using Ionomycin combination 6-DMAP inhibited the extrusion of the second polar body, and Ionomycin combination Roscovitine also could reached such quantity that the Ionomycin combination 6-DMAP to parthenogenetic activation of bovine oocytes.2. Full-length sequence of p21 gene CDS was cloned and connected to pVenus eukaryotic expression vector for getting vector pVenus-P21. After transfection or microinjection by in vitro transcription cRNA, the fusion protein could be expressed efficiently and localized accurately both in Hela cells and bovine oocytes.3. The content of p21 mRNA were detected in different tissues, and different stage during oocytes maturation and embryo development. The mRNA of p21 in granulosa cells is relatively higher among different tissues; and the content of p21 mRNA at M II stage was higher during oocytes maturation; and the content of p21 mRNA at blastocyst during embryos development. The P21 protein located prather in nuclear than the oocyte cytoplasm. In the process of oocyte maturation, P21 can prevent the oocyte enter the M-I phase, but P21-dependent inactivation of MPF during egg activation and promote its exit from the M-II block.In this study, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined; P21 were expressed in the period of oocyte maturation, P21 was located in the chromosome and the surrounding structure; P21 can prevent oocytes enter into the M-I phase, but it promote oocytes exit from the M-II block.