| ObjectiveThe first part:The protective role of Antifreeze protein Ⅲ on mouse embryo in vitrificationAntifreeze protein Ⅲ was applied into vitrification of two-cell stage embryos to observe the freeze protection of cytoskeleton microfilament and their potentiality development after recovery.The second part:The protective role of Antifreeze Protein Ⅲ on the structure and function of mature mouse oocyte in vitrificationTo investigate the damage in vitrification of mice MⅡ phase (metaphase Ⅱ) oocytes, and to observe whether AFP Ⅲ plays a protective role in the process of vitrification on their cellular structure and function.MethodsThe first part:The protective role of Antifreeze protein Ⅲ on mouse embryo in vitrification1. Retriveal of two-cell stage embryos:Eight to twelve weeks old female kunming mice weighing30-35g were treated with i.p. injections of10IU pregnant male serum gonadotropin, after48hrs, they were treated with i.p. injections of10IU human chorionic gonadotropin, after that they were closed to cage with sexually mature male kunming mice for the night according to1:1, the female vaginal suppositories were observed next morning and female and male mice were fed separately if there were vaginal suppositories in female mice, then mice were killed by cervical dislocation46-48hrs later, both ovaries and part of uterus were excised then the fallopian tubes were separated under the stereoscope, two-cell stage embryos were collected under stereo microscope by puncturing fallopian tube at the ampulla,2. Groups:1200two-cell stage embryos were collected and three groups were divided randomly, fresh control group, vitrification group and vitrification group with additional AFPⅢ repectively. AFPⅢ was added in vitrification liquid to make the concentration of500ng/mL,400two-cell stage embryos were in each group,200two-cell stage embryos of each group were used for blastocyst culture after recovery and others were used for immunofluorescent staining of microfilament.3. Vitrification and warming of embryos:After vitrification, the surviving two-cell stage embryos were used for developing to observe the blastocyst formation to compare the recovery rate of frozen groups and following blastocyst formation rate of three groups.4. The observation of embryo microfilament:After fixed, penetrated and blocked, two-cell staged embryos were incubated with Phalloidin-FITC diluted in PBS at a concentration of0.1ug/mL for60minutes in dark for immuno fluorescent staining, the rates of intact microfilament in three groups were used for statistical analysis.5. Statistical Analysis:Data was analyzed with SPSS17.0. The means were compared using unpaired Student’s t-tests. Ratios were compared using Chi-squared tests. The inspection level was0.05. P-values<0.05was considered statistically significant.The second part:The protective role of Antifreeze Protein III on the structure and function of mature mouse oocyte in vitrification1. Retrieval of mature oocyte:Eight to twelve weeks old female kunming mice weighing30-35g were treated with i.p. injections of10IU pregnant male serum gonadotropin, after48hrs, they were treated with i.p. injections of10IU human chorionic gonadotropin, then mice were killed by cervical dislocation18-20hrs later, both ovaries and part of uterus were excised then the fallopian tubes were separated under the stereoscope, oocytes were collected under stereo microscope by puncturing fallopian tube at the ampulla.2. Groups:1200oocytes were collected and four groups were divided randomly, fresh control group, fresh control group with additional cryoprotectant, vitrification group and vitrification group with additional AFPIII repectively. AFPIII was added in vitrification liquid to make the concentration of500ng/mL,300oocytes were in each group,100oocytes of each group were used for in vitro fertilization and blastocyst rates were observed,100oocytes were used for immunofluorescent staining of microtubule and microfilament,100oocytes were used for Real-time fluorescent quantitative PCR.3. Vitrification and warming of oocytes:The surviving oocytes of vitrification groups were used for in vitro fertilization.4. Retrieval of sperm:Sperm suspension was collected from the cauda epididymidises of male kunming mice and processed with gradient centrifugation, then was incubated at37℃with6%CO2in humidified air, after half an hour, the hierarchical dynamic was used to make the fast forward movement sperm draw in preheated G-IVF medium and the sperm density was adjusted to3-6×106/mL under inverted microscope.5. In vitro fertilization:The mature oocytes were placed in G-IVF medium and were fertilized with epididymal sperm, then they were incubated at37℃with6%CO2in humidified air. The formation of male and female pronuclei was observed after5to9hours, after fertilization,2,4,8and blastocyst development were observed in days2,3,4,5respectively. The fertilization rate and blastocyst formation rate were calculated.6. The observation of microtubule in oocytes:After fixed, penetrated and blocked, the oocytes were incubated with anti-alpha tubulin antibody diluted in PBS at a concentration of1:250at4℃overnight, then they were incubated with fluorescein-conjugated Goat Anti-Rabbit IgG diluted in PBS at a concentration of1:100for120minutes in dark for immunofluorescence staining.7. The observation of microfilament in oocytes:After fixed, penetrated and blocked, the oocytes, two-cell, four-cell, eight-cell staged embryos and blastocyst were incubated with Phalloidin-FITC diluted in PBS at a concentration of O.lug/mL for60minutes in dark for immunofluorescence staining.8. The observation of chromosome in oocytes:After staining of the microtubules and microfilament, the oocytes and embryos were incubated in DAPI for3minutes in dark conditions for DNA maker.9. The determination of the expression of genes by Real-time fluorescent quantitative PCR: The oocytes were lysed, separated, sedimented, eluted, redissolved, the purity and concentration of mRNA was measured by Ultraviolet Spectrometry Photometer, reverse transcription reaction, Real Time PCR was used to measure the concentration of gene of Mad2、Eg5、Cenp-e and Cirbp (10μL system).10. Statistical Analysis:Data was analyzed with SPSS17.0, The means were compared using unpaired Student’s t-tests. The ratios were compared using Chi-squared tests. Gene expression in four groups was compared with a Mann-Whitney U test using GraphPad Prism5(GraphPad Software, Inc, USA), while mRNA expression experiments were repeated at least three times. The inspection level was0.05. P-values<0.05was considered statistically significant.ResultsThe first part:The protective role of Antifreeze protein on mouse embryo in vitrification.1. The recovery rate in vitrification group with additional AFPIII was higher than the vitrification group, they showed statistical significance (P<0.05).2. The ratio of blastocyst formation rate and intact actin filaments were higher in fresh control group and vitrification group with additional AFPIII compared to the vitrification group, they all showed statistical significance (P<0.05); while they showed no statistical significance in fresh control group and vitrification group with additional AFPIII (P>0.05).The second part:The protective role of Antifreeze Protein3on the structure and function of mature mouse oocytes in vitrification1. The ratio of microtubules, actin filaments, chromosomal integrity was more intact in vitrification group with additional AFPIII compared to the vitrification group, they showed statistical significance (P<0.05), while they showed no statistical significance in fresh control group and vitrification group with additional AFPIII (P>0.05). The ratio of intact organelles was more higher in fresh control group and fresh group with additional cryoprotectant containing ethyleneglycol (EG)、dimethyl sulfoxide (Me2SO) and Sucrose than the other two vitrification groups, these results showed statistical significance(P<0.05).2. Real-time PCR analysis revealed that the relative quantification of Mad2which stands for mitotic arrest deficient2and Cenp-e which stands for centromere protein E were significantly higher in vitrification group with additional AFPIII, fresh control group and fresh group with additional cryoprotectant than in vitrification group, and showed statistical significance (P<0.05). By contrast, the expression of Cirbp which stands for cold inducible RNA binding protein and Eg5which stands for kinesin-5motor protein were up-regulated in vitrification group compared to others and showed statistical significance(P<0.05). They showed no statistical significance in fresh control group, fresh group with additional cryoprotectant containing ethyleneglycol (EG)、dimethyl sulfoxide (Me2S0) and Sucrose, and vitrification group with additional AFPIII (P>0.05).3. The fertilization rate in fresh control group and fresh group with additional cryoprotectant was higher than the other two vitrification groups and showed statistical significance (P<0.05). Furthermore the recovery rate and the fertilization rate in vitrification group with additional AFPIII were higher than vitrification group and showed statistical significance (P<0.05). But the blastocyst formation rate in four groups showed no statistical significance (P>0.05).ConclusionsThe first part:The protective role of Antifreeze protein on mouse embryo in vitrification.1. The recovery rate in vitrification group with additional AFPIII was higher than the vitrification group.2. The ratio of blastocyst formation rate and intact actin filaments were higher in fresh control group and vitrification group with additional AFPIII compared to the vitrification group.3. They showed no statistical significance in fresh control group and vitrification group with additional AFPIII.The second part:The protective role of Antifreeze Protein3on the structure and function of mature mouse oocytes in vitrification1. The ratio of microtubules, actin filaments, chromosomal integrity was more intact in vitrification group with additional AFPIII compared to the vitrification group. They showed no statistical significance in fresh control group and vitrification group with additional AFPIII. The ratio of intact organelles was more higher in fresh control group and fresh group with additional cryoprotectant containing ethyleneglycol (EG)、dimethyl sulfoxide (Me2SO) and Sucrose than the other two vitrification groups.2. The expression of Mad2, CENP-E in vitrification group with additional AFPIII, fresh control group and fresh group with additional cryoprotectant was higher than in vitrification group. The expression of Cirbp and Eg5was up-regulated in vitrification group compared to others and showed no statistical significance in other three groups.3. The fertilization rate and the recovery rate in fresh control group and fresh group with additional cryoprotectant were higher than the other two vitrification groups. The recovery rate and the fertilization rate in vitrification group with additional AFPIII were higher than vitrification group. The blastocyst formation rate in four groups showed no statistical significance. |
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