| Objective:To compare the freezing and thawing effects of different virification medium on MⅡ stage mouse oocytes, the present study used MⅡ stage mouse oocytes for glass micropipette (GMP) vitrification, and utilized in vitro fertilized embryos as control, the dynamic patterns of 5-methyl cytosine (5-mC),5-hydroxymethyl cytosine (5-hmC), 5-formylcytosine (5-fC),5-carboxylcytosine (5-caC) during the development of pre-implantation embryos derived from frozen oocytes were investigated. And the relationship of TET proteins with epigenetic regulation of genome was also studied. The data will provide effective information for studying the molecular mechanism of epigenetic regulation in the developmental embryos derived from frozen oocytes.Methods:The experiment was divided into two groups. The experimental group was in vitro fertilized embryos from freezing thawing mature oocytes from Kunming female mouse and the control embryos were from normal in vitro fertilized embryos. The dynamic patterns of 5-mC,5-hmC,5-fC,5-caC and TET proteins (Tet1,2,3) in different groups were investigated by using immunostaining. The total RNA was extracted from different stages of embryos and reverse transcribed into cDNA. Next, to compare dynamic pattern after freezing, real time PCR was used to quantitatively analyze Tet gene expression.Results:(1) The survival rate of MⅡ stage oocytes and developmental capability of embryos were compared. The results showed that there was no significant difference between commercialized group and self-made group (89.5%±2.2 vs 88.0%±2.6, P> 0.05). And the cleavage rate and blastocyst rate were not significantly different between two groups(68.5%±3.2 vs 66.0%±2.7、51.3%±12.2 vs 49.8%±8.4, P>0.05). But compared to control group, the cleavage rate and blastocyst rate had significant difference (P<0.01), the cleavage rate were 68.5%、66.0% vs 85.9%、51.3%、49.8% vs71.8%. (2) Pre-implantation embryos were co-stained with 5-mC and 5-hmC antibody. There were no significant difference between frozen group and IVF group of 5-mC fluorescence signal (P> 0.05). The strength of the overall signal in frozen group was lower than IVF group, in which both MⅡ stage group and 2-Cell stage group were slightly higher than IVF group. 5-mC signal gradually decreased from pronuclei stage to 2h after fertilization, it reached the lowest at 12h. In the developing embryos, the signal decreased from 2-cell stage and reached to a minimum at EXP-B stage embryo.Fluorescent signal of 5-hmC was no significant difference between pre-implantation embryos from frozen oocyte group and IVF group(P>0.05). The strength of the overall signal in frozen group was lower than IVF group.2-Cell stage group were slightly higher than IVF group.5-hmC signal gradually increased from pronuclei stage to 2h after fertilization, it reached the highest at 12h. In the cleavage embryos, the signal decreased from 2-cell stage and reached to a minimum at 8-cell stage embryo (P<0.05). But the signal gradually increased from 8-cell stage, it reached to a maximum at EXP-B stage. In frozen group,5-mC started to convert into 5-hmC from 4h after fertilization,5-hmC signal reached to maximum and it mainly localized in paternal pronuclei. In IVF group,5-mC started to convert into 5-hmC from 6h after fertilization in paternal pronuclei.(3) There were no significant difference between frozen group and IVF group of 5-mC fluorescence signal (P>0.05). The strength of the overall signal in frozen group was lower than IVF group.5-hmC signal decreased gradually after 2h fertilization and to lowest level at 12h. The signal in IVF group was higher than frozen group at 2h and MⅡ oocytes and had significant difference between them (P<0.05).5-hmC signal started to increase from 2-cell stage and reached to the strongest at 8-cell stage then decreased gradually until to EXP-B stage at which reached the lowest level. The fluorescence of 5-fC had no significant difference in pre-implantation embryos derived from frozen group and IVF group(P>0.05). The strength of the overall signal in frozen group was lower than IVF group. It started to gradually decrease from 2h after fertilization, which reached the lowest at 8h then recovered and increased to the strongest at 12h. The signal of embryos both from frozen group and from IVF group at 6h and 8h stages was dramatically lower than other stage embryos (P<0.05). In cleavage embryos, the signal started to decrease at 2-cell stage and reached to the lowest at 8-cell stage then increased gradually, which reached to the strongest at EXP-B stage. (4) Pre-implantation embryos were co-stained with 5-mC and 5-caC antibody. There were no significant difference between frozen group and IVF group (P>0.05).5-mC fluorescent signal:the signal intensity was lower in frozen group compared to IVF group at pronuclear stage embryos, it started to become weak 2h after fertilization and decreased to minimum at 12h after fertilization. In the cleavage stage embryos, signal strength of frozen group was higher than IVF group, which started to decrease gradually from 2-cell stage, at EXP-B stage embryos to a minimum.5-caC fluorescent signal:at pronuclear stage embryos, the signal intensity was lower in frozen group compared to IVF group, it started to gradually decreased from 2 h after fertilization, and reached to the minimum at 6h and then back to increase to maximamum at 12h, in which the frozen group dramatically lower compared to IVF group. And in cleavage stage embryos, the signal strength of frozen group was higher than IVF group, it started to become weak at 2-cell stage and decreased to minimum at 8-cell stage. In the cleavage stage embryos, signal strength of frozen group was higher than IVF group, which started to decrease gradually from 2-cell stage, at 8-cell stage reached to a minimum then started to get stronger and increase to maximamum at EXP-B stage.Both in frozen group and IVF group,5-mC started to convert into 5-caC from 8h after fertilization. At 12h, fluorencent signal in paternal pronuclei almost disappeared. 5-caC signal mainly and mosty localized in paternal pronuclei. DNA demethylation happened both in frozen group and IVF group,5-caC signal was lowest at 8-cell stage, then began to increase to the highest at expanded blastocysts stage.(5) The fluorencent signal of Tet1, tet2 and Tet3 in pre-implantation embryos derived from frozen oocytes had no significant different compared to IVF group. The strength of the overall signal in frozen group was lower than IVF group. The signal decreased gradually after 2h fertilization and to lowest level at 12h. The signal started to increase from 2-cell stage and reached to the strongest at EXP-B stage. The signal of Tet2 increased strongest at E-B stage.Tet3 signal started to increase gradually from 4h to 10h after fertilization and reached the lowest level at 12h after fertilization. The signal in frozen group was lower than IVF group both at 2h and 4h. In forzen group, signal in 2-cell stage,4-cell stage and 8-cell stage embryos was significant higher than IVF group(P<0.05).(6)Tet gene expression profile in MⅡ oocytes, sperm, pre-implantation embryos was as the followings:the expression of tetl in MⅡ oocytes,sperm, pronuclear embryos and 2-cell stage embryos was very low, but highly expressed both at 4-cell stage and 8-cell stage embryos and which was higher in IVF group than frozen group (P<0.05). The expression of tetl was decreased gradually with the embryonic development and which was higher in frozen group than IVF group at morula stage (P<0.05).tet2 expressed both in MⅡ mouse oocytes of frozen group and pre-implantation embryos of IVF group, which was higher in MⅡ oocytes of IVF group than frozen group (P<0.05). But it was higher in frozen group than IVF group at morula stage (P<0.05). tet3 was highly expressed in MⅡ oocytes and pronuclear (PN) embryos, but it was very low in early cleavage stage embryos. It was higher in frozen group than IVF group at MⅡ oocytes, but it was higher in frozen group than IVF group at pronuclear stage embyros (P<0.05). Conclusions:(1) Using the self-made freezing and thawing medium, we successfully established technology of freezing and thawing MⅡ stage oocytes from kunming mice, and obtained a stable, effective method to get in vitro fertilized early embryos. (2) For the first time, using 5-mC and 5-hmC,5-fC,5-caC antibody co-stain pre-implantation embryos derived from frozen MⅡ oocytes or not, immunofluorescence staining and fluorescence semi-quantitative analysis found that: established the developmental stages of dynamic patterns before implantation. (3) Fluorescent signals of Tetl and Tet2, Tet3 protein in early embryos derived from frozen oocytes or not. The whole signal intensity of frozen group was lower than IVF group, and set up the dynamic changes of the developmental stage before implantation.(4) Tetl was very low in MⅡ oocytes,sperm, pronuclear embryos and 2-cell stage embryos, but highly expressed both at 4-cell stage and 8-cell stage embryos and which was higher in IVF group than frozen group.tet2 expressed both in MⅡ mouse oocytes of frozen group and pre-implantation embryos of IVF group, which was higher in MⅡ oocytes of IVF group than frozen group. But it was higher in frozen group than ⅣF group at morula stage. tet3 was highly expressed in MⅡ oocytes and pronuclear (PN) embryos, but it was very low in early cleavage stage embryos. |
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