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Dibutyryl Adenosine Cyclic Monophosphate Control the Maturation of Porcine Ooctytes Cultured in Vitro

作　者: 赵明辉
导　师: 尹熙俊
学　校: 延边大学
专　业: 动物遗传育种与繁殖
关键词: dbcAMP pig IVM labour-cost oocytes
分类号: S828
类　型: 硕士论文
年　份: 2011年
下　载: 1次
引　用: 0次
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内容摘要

The main object of this study was to establish the basic porcine COCs IVM system fitting for our Lab., and evaluate the effect of N,2’-O-Dibutyryladenosine 3’:5’-Cyclic Monophosphate (dbcAMP) on porcine oocytes maturation and development in vitro in order to control the in vitro maturation of pig oocytes.Pig oocytes were matured for different maturation durations, with or without exogenous hormone supplements after the first 20h of in vitro maturation, treated with CB or 6-DMAP after electronic activation and were fused with somatic cell used different voltage. Rates of reached to MⅡstage, development to blastocyst and the rate of fusion and lysis were checked.Use the basic IVM system established above, effect of dbcAMP on pig oocytes at GV stage and development ability of pig PA and SCNT embryo were investigated.Oocytes reached to MⅡstage cultured for 33h significant lower than 41,49 and 57h(52.54% vs.79.19%,93.25% and 93.75%, respectively p<0.05), there is no significant difference among 41,49 and 57h, while oocytes matured for 41h reached to normal 2-4 cells significant higher than them at 57h group(92.26% vs.56.17%, p<0.05) and the blastocyst rate was also higher than 33h,49h and 57h(41.16% vs.11.96,21.20 and 26.92%, respectively p<0.05). Hormone free after the 1st 20h of IVM culture didn’t improve the cleavage(hormone free vs. hormone not free:86.71% vs.81.77%, p>0.05) and blastocyst formation(hormone free vs. hormone not free:30.06% vs.26.60%, p>0.05). Treat pig PA embryos with CB of 6-DMAP after electric activation, there were no significant difference in cleavage(CB vs.6-DMAP:70.44% vs.79.01%) and blastocyst(CB vs.6-DMAP:28.57% vs. 34.89%).Effect of different fusion voltages on rate of fusion and lysis in somatic cell oo-plasm complex after electronic fusion were also been investigated. Results show that when somatic cell oo-plasm complex fused with voltage at 150V/mm, the fused rate significantly lower than that used 180V/mm (66.38% vs.86.88%, p<0.05), while the lysis rate of 150V/mm group(4.26%) was also significantly lower than 180V/mm(19.24%) group but higher than control group(2.49%).Oocytes treated with 1mM dbcAMP for 22,34,37,39, and 72h respectively. Results show that in each group, most oocytes were synchronized at GVII stage. At the check point of 39h, we found that there were 23.50% oocytes reached to GVIV stage, significantly higher than others. And at 72h, about 35.47% oocytes reached to MⅡstage, and just 29.62% oocytes still arrested at GVII stage. COCs were matured in IVM-Ⅲat the first 0,22,24,26,28h of the culture and then released the dbcAMP and hormone, after time released the dbcAMP and hormone, rate of oocyte reached MⅡwere recorded and compared at 11-18h after dbcAMP released. Results shows that at 13h after dbcAMP and hormone free, oocytes in 26 and 28h (28.97% and 31.11%) reached MⅡstage were higher than that which at 22h matured (0.00%, p<0.05). While there were no significant difference among each group at 11,15,16,17,18h, respectively.At 22h,24h and 26h groups(46.93,61.90 and 57.09%), oocytes reached MⅡstage at 15h were significant higher than that at 13h (0.00%,10.98%,28.97%, respectively p< 0.05). At 26h group, oocytes at 13h after dbcAMP release reached MⅡstage were significant higher than 11h(28.97% vs.1.59% p<0.05). While there were no significant difference between two adjacent check point in 28h group.In conclusion oocytes being treated with dbcAMP for 22,24,26, and 28h were used in SCNT and PA. Our results suggested that there were no significant difference at rates of fusion, cleavage, lysis, fragment and blastocyst in SCNT group, and in parthenogenetic activation group, there were also no any statistic difference between 22 and 28h group in rates of cleavage, lysis, fragment and blastocyst.

全文目录

Abstract  6-12
Chapter 1 Literature review  12-25
  1.1 Introduction  12-14
  1.2 Maturation of pig oocytes in vitro  14-22
    1.2.1 Oocyte meiotic resumption  14-15
    1.2.2 Cytoplasmic maturation  15-16
    1.2.3 MPF and MAPK  16-17
      1.2.3.1 MPF  16-17
      1.2.3.2 MAPK  17
    1.2.4 Factors affecting the development and quality of pig oocytes matured in vitro  17-22
      1.2.4.1 Donor age & Follicular Factors  17-20
      1.2.4.2 Anti-oxygen  20
      1.2.4.3 Growth facotrs  20
      1.2.4.4 Hormone  20-21
      1.2.4.5 Other  21-22
  1.3 cAMP and PKA pathway  22-24
  1.4 Aim of this study  24-25
Chapter 2 Methods and materials  25-34
  2.1 Chemicals and media  25-26
    2.1.1 Basic meida  25-26
    2.1.2 IVM media  26
    2.1.3 SCNT media  26
  2.2 Main equipment  26-27
  2.3 Methods  27-32
    2.3.1 Collection and transportation of ovaries  27-28
    2.3.2 Oocytes collection and IVM  28
    2.3.3 Cumulus cell removal and polar body searching  28
    2.3.4 Establishment of porcine fibroblast cell lines  28-29
    2.3.5 Preparation of donor cells  29
    2.3.6 Nuclear transfer  29-30
    2.3.7 In vitro culture of embryos  30
    2.3.8 Staining  30-32
  2.4 Experiments Design  32-33
  2.5 Statistical analysis  33-34
Chapter 3 Results and analysis  34-40
  3.1 Effects of duration of maturation,hormone free and chemical treatment on development of pig embryo cultured in vitro  34-36
    3.1.1 Effect of difference in vitro maturation durations on development of porcine parthenogenetic activation embryo  34-35
    3.1.2 Effects of the duration of exposure to hormone supplements on development of pig oocytes cultured in vitro  35
    3.1.3 Effect of CB and 6-DMAP treatment on development of pig parthenogenetic embryos cultured in vitro  35-36
  3.2 Effect of fusion voltage on rate of fusion and lysis of oo-plasm  36
  3.3 Effect of dbcAMP on oocytes were at GV stage  36-40
    3.3.1 Ability of dbcAMP inhibit GVBD of pig oocytes matured in vitro  36-38
    3.3.2 Effect of different dbcAMP treatment durations on maturation of pig oocytes matured in vitro  38-39
    3.3.3 Effect of different dbcAMP treatment durations on development of SCNT and PA embryo cultured in vitro  39-40
Chapter 4 Discussion  40-45
  4.1 Effects of duration of maturation,hormone free and chemical treatment on development of pig embryo cultured in vitro  40-42
    4.1.1 Effect of different maturation durations on maturation of pig oocytes cultured in vitro  40-41
    4.1.2 Effect of the duration of exposure to hormone supplements on development of pig oocytes cultured in vitro  41
    4.1.3 Effect of CB and 6-DMAP treatment on development of pig parthenogenetic embryo cultured in vitro  41-42
  4.2 Effect of fusion voltage on rate of fusion and lysis of oo-plasm  42
  4.3 Effect of dbcAMP on pig oocytes and embryo cultured in vitro  42-45
    4.3.1 Ability of dbcAMP inhibited GVBD of pig oocytes matured in vitro  42-43
    4.3.2 Effect of different dbcAMP treatment durations on maturation of pig oocyte matured in vitro  43
    4.3.3 Effect of different dbcAMP treatment durations on development of SCNT and PA embryo cultured in vitro  43-45
Chapter 5 Conclusion  45-47
Reference  47-53
Appendix Ⅰ Components of basic medie  53-54
Acknowledgement  54

Dibutyryl Adenosine Cyclic Monophosphate Control the Maturation of Porcine Ooctytes Cultured in Vitro

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