| Objective: (1) In the first part of experiments, this study compared the efficiency of different sucrose concentration in the freezing solution on the survival rate of mouse mature oocyte with slow cryopreservation protocol; After thawing, we investigated whether the culture time was associated with the results of the mouse oocytes intracytoplasmic sperm injection(ICSI). Therefore the inevitably deleterious effect on the oocyte morphology, part of thawed oocytes had to face the fate of fail to fertilization after ICSI. We attempted to remedy these mouse oocytes that fail to fertilization after ICSI by adopting assisted activation with calcium ionophore and puromycin and examined the efficiency. (2) In the second part of experiments, the cryosurvival rates were compared between two groups of human mature oocytes with different sucrose concentation; The oocyte were cultured for different time after thawing and then fixation, immunocytochemical staining and fluorescence microscopy were used to assess the morphology of spindle and chromosome on the mataphase II(MII). Various attempts have to been performed with contrasting rusults to improve success rate of the human cryopreservation oocytes.Method: ICR mouse underwent controlled ovarian hyper-stimulation. Oocytes were obtained to slow cryopreservation. We compared the survival rate of mouse oocytes with 0.2mol/l sucrose and 0.3mol/l sucrose in freeing solution. After thawing, the survival oocytes with normal zona pellucida and cytoskeletal were randomly assigned to five groups to culture different time (group A: 1h, group B: 2h, group C: 3h, group D: 4h, group E:5h) before inseminating. (We compared the percentages of fertilization,embryo cleavage and blastocyst). Furthermore, this study assessed the effect of assisted activation with calcium ionophore and puromycin on mouse oocytes that fail to fertilization after ICSI. Unfertilized mouse oocytes without fragment were randomly assigned to two groups(group I : assisted activation, n=46, group II: without activation, n=22). Another 45 fresh unfertilized oocytes (group III)were activated by the same activated method as group I. (2) Human mature oocytes(n=124) were obtained for slow cryopreservaion and randomly assigned to two groups, the survival rates were compared between two groups with 1.5mol/l 1,2-propanediol + 0.2mol/l sucrose solution and 1.5mol/l 1,2-propanediol + 0.3mol/l sucrose solution. The 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups into HTF culture medium after thawing: group a: culture 1h(n=20), group b: culture 3h(n=22), group c: culture 5h(n=22),the fresh oocytes in control groups(n=18). Fixation and immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome of oocytes in the four groups.Results: A significantly higher survival rate(80.7%) was obtained when the oocytes were cryopreserved in the presence of a sucrose concentration(0.3mol/l) in the freezing solution than the survival rate (60.5%) of oocytes cryopreserved in the freezing solution with lower sucrose concentration(0.2mol/l). For thawed oocytes, ICSI produced higher fertilization rate and blastocyst rate in group C (culture 3h) and group D(culture 4h) with respect to group A(culture 1h) and group B(culture 2h). No significant difference between group C and group D. Part of oocytes in group E(culture 5h) underwent cleavage before ICSI, no significant difference was observed in fertilization rate and blastocyst rate of the remain oocytes in group E compared to other groups. For mouse oocytes that fail to fertilization after ICSI, the activation rate in group I was higher than the consequence of group II(30.4% vs 4.5%, p <0.05). Statistically increase of activation rate and 2PN2PB rate were observed in group III than group I(30.4% vs 71.1%, p <0.05;21.4% vs 62.5%, p <0.05). No significant difference in 1PN2PB rate between group I and group III. (2) Following human mature oocyte cryopreservaion, a significant increase was observed in the survival rate with 0.3mol/l sucrose concentration in freezing solution compared with 0.2mol/l sucrose concentration(80.6% vs 56.5%, p <0.05). The consequence was assessed after culturing and fixation and immunocytochemical staining, a statistically significant decrease rates of normal spindle were observed in three cryoprotectant groups compared to control group(10.0% vs 83.3%,P<0.05; 45.5% vs 83.3%, P<0.05; 40.9% vs 83.3%, P<0.05).The rates of absent spindle in group (a) was significantly higher with respect to control group(45.0% vs 5.6%, p <0.5). Also, the rates of absent spindle in group (a) was higher than group (b) and group (c)(45.5% vs 13.6%, p <0.05; 45.5% vs 13.6%, P<0.05). The rates of normal spindle in group (a) was lower than group (b) and group (c)(10.0% vs 45.5%, p <0.05; 10.0% vs 40.9%, P<0.05);No significant differences were observed in group (b) and group (c). A statistically significant increase rates in abnormal chromosome were observed in group (a) compared to group (b),group (c) and control group(30.0% vs 68.2%,p <0.05; 30.0% vs 63.6%, P<0.05; 30.0% vs 77.8%, p <0.05).No statistically differences in chromosome morphology were observed in group (b),group (c) and control group.CONCLSION: (1)For mouse mature oocyte cryopreservation, our data demonstrated the beneficial effect of increasing the sucrose concentration from 0.2mol/l to 0.3mol/l in freezing medium on improvement of survival rate. After thawing, the 3h～4h incubation could permit restoration of the ultrastructure and improve the normal fertilization rate and embryo developing ability for mouse oocyte. Oocyte assisted activation with calcium ionophore and puromycin may be effective for mouse oocytes that fail to fertilize after ICSI. Because of irreversible injure during cryopreservation, the ability of been assisted activated was significant decrease for mouse thawed oocytes that fail to fertilize after ICSI compared to fresh unfertilized oocyte after ICSI. (2) For human mature oocytes cryopreservation, a Statistically significant increase was observed in the suivival rate in cryopreservation combined with 0.3mol/l sucrose compared to same protocol combined with 0.2mol/l sucrose. With the help of immunofluorescence and confocal laser scanning microscopy(CLSM), we found that the cryoprotectant protocol had a deleterious effect on the organization of the meiotic spindle and chromosome on MII satage and the injured spindle and chromosome rucuperated in a time-dependent process. The 3h～5h post-thawing incubation could permit restoration of the meiotic spindles and chromosome. |
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