Study On The Cryopreservation, In Vitro Culture And Transplantation Of Rabbit Ovarian Tissue

PartⅠ:Effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary folliclesObjective:To observe the effects of four kinds of different frozen-thawed methods on the morphology of rabbit ovarian tissue and the PCNA expression of the oocytes in the primordial and the primary follicles to investigate the optimal protocol of cryopreserving rabbit ovarian tissue.Methods:12 healthy female rabbits were randomly divided into four groups. Rabbit ovarian tissues were cryopreserved by procedure slow-freezing protocols including PROH and DMSO method (PROH group, DMSO group) and vitrification protocols including DMSO+PROH and DMSO+EG methord(vitrification group 1, vitrification group 2). Thawed after cryopreserved by liquid nitrogen for one month, HE staining and immunohistochemistry staining were used to observe the changes of the structure of frozen-thawed ovarian tissues and the morphology of different stages of follicles and PCNA expression of the oocytes in the primordial and the primary follicles.Results:1. The rates of the healthy primordial follicles and primary follicles of rabbit ovarian tissue declined to 80.08% and 55.00%,70.53% and 52.94%,67.55%and47.06%,69.65% and 55.56% in four frozen-thawed groups respectively,while that of fresh control groups were 90.14%and 86.05%,91.45% and 89.29%,91.76%and 84.62%,92.15% and 89.47% respectively. The differences between the fresh control groups and the freezing groups were significant (P<0.05). In all freezing groups, the rate of the healthy primordial follicles in PROH group was the highest. Compared the rate of the healthy primordial follicles in PROH group with that of DMSO group(70.53%),vitrification group1(67.55%) and vitrification group2 (69.65%), the difference was significant (P<0.05).After merging the freezing groups, the rate of the healthy primordial follicles was 72.7%.The rate of the healthy primary follicles was 52.77%. The difference was significant (P<0.05).In the four kinds of freezing groups, the ovarian tissue structures were damaged. Some follicles were separated with the stromal cells. The connections among stromal cells were losse, disordered, and the gaps had formed by microscope.2. The results of immunohistochemistry staining: the PCNA positive rate of the oocytes in the primordial and the primary follicles in PROH group was the highest (76.98%), the second was vitrification group2 (72.44%). Compared with the fresh control group (78.04%), the difference was not significant(P>0.05), while the PCNA positive rate in DMSO group(58.97%) and vitrification group1(48.00%) declined significantly compared with that of the fresh control group(P<0.05).Conclusions:1. Four kinds of frozen-thawed protocols damaged the structure of rabbit ovarian tissue in a certain extent. The rates of the healthy follicles at all stages declined significantly and the damagement appeared in the srtomal cells.2. PROH procedure slow- freezing method is the best among DMSO and two vitrification methods for cryoperserving primordial follicles of ovarian tissue.3. Cryoperserving methods have more damagement to the primary follicles than to the primordial follicles.4. PROH procedure slow-freezing method and DMSO+EG vitrification protocol are the optimal protocols for keeping the proliferative activity of the oocytes in the primordial follicles and the primary follicles. PartⅡEffects of procedure slow-freezing protocol and vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the folliclesObjective:To investigate the effecst of PROH procedure slow-freezing protocol and DMSO+EG vitrification protocol on ultra microstructure, ER and MHC-Ⅱantigen expression of rabbit ovarian tissues and proliferative activity of the follicles.Methods:1. Nine healthy female rabbits were randomly divided into three groups, one group was the fresh control group and the other groups were the freezing groups. The freezing groups were cryopreserved by PROH procedure slow-freezing method and DMSO+EG vitrification method.2. After thawing, the effects of two freezing methods on ultra microstructure of rabbit ovarian tissues were observed by transmission electron microscope.3. The effects of two freezing methods on ER and MHC-Ⅱantigen expressions of rabbit ovarian tissues were observed by SP immunohistochemistry method.4. Follicles were mechanically dissected from ovarian tissue. Twenty small OGCs(diameter100-150μm, wrapped up by 2~5 layers of granulosa cells)and twenty big OGCs(diameter200-300μm, wrapped up many layers of granulosa cells)with normal appearance from every groups were tested with 3H-TdR uptake method respectively.Results:1. In PROH group, partial chondriosomes of the oocyte from primordial follicle swelled which appeared vacuole, and some chondriosomes were normal. The nucleus appearance was normal, and nuclear membrane was integrity and clear. The nuclear membranes of granular cells were integrity; in which chondriosomes were normal and gap junctions among cells were intact. In DMSO+EG groups, the most chondriosomes in the oocyte from primordial follicle swelled and its organelles were disaggregated, but cell appearance was nomal.The nuclear membrane was integrity and clear. In surrounding granular cells chondriosomes were acceptable, and chondriosomes of stromal cells were obviously swollen and appeared vacuole.2. The result of immunohistochemistry suggested that there was no difference on ER expression of rabbit tissues in two frozen-thawed groups and the fresh group (P>0.05). MHC-Ⅱantigen expression in DMSO+EG group was significantly decreased compared with the fresh group (P<0.05), but there was no significant difference in PROH group(P>0.05).3. The proliferative activity of big OGCs in two frozen-thawed groups significantly decreased compared with the fresh group (P<0.05), but there were no significant difference in small OGCs (P>0.05).Conclusions:1. The effect of PROH procedure slow-freezing protocol on ultra microstructure of rabbit ovarian tissues was less than DMSO+EG vitrification protocol.2. There was no significant effect on ER expression of rabbit ovarian tissues by two frozen-thawed methods.3. The DMSO+EG vitrification method obviously degraded the immunogenicity of ovarian tissues.4. Two frozen-thawed methods did not have significant effect on the proliferative activity of small OGCs with intact appearance and structure, which may easily develop and maturate in vitro. But big OGCs packed by multilayer granular cells appearing normal morphology, its proliferative activity were significantly suppressed. PartⅢ:Research on in vitro culture of cryopreserved rabbit ovarian tissueObjective:To investigate the suitable method of in vitro culture of ovarian tissue.Methods:1. Took fresh rabbit ovarian tissues and thawed the frozen rabbit ovarian tissues preserved by PROH procedure slow-freezing method, then used partial-isolated culture method and tissue mass culture method to continually culture them in vitro for 14d as fresh partial-isolated culture group, fresh tissue mass culture group, frozen partial-isolated culture group and frozen tissue mass culture group.2. The results of culture were observed by inverted microscope every two days. The tissue culture medium was collected for 500μl to detect the content of E2, and an identical amount of fresh culture medium was added. The secretion production of E2 was measured in each group by ELISA method.The ovarian tissues were separated mechanically on D14th of culture. The cpm of small secondary follicles (small OGCs ,diameter 100-150μm, wrapped up by 2~5 layers of granulosa cells) and the cpm of big secondary follicles (big OGCs ,diameter 200-300μm , wrapped up many layers of granulosa cells) were measured by 3H-TdR uptake method.Results:1. Small follicles wrapped by monolayer granulosa cells in fresh and frozen partial-isolated culture group were found failed to increase in diameter and become dark and necrotic in appearance through inverted microscope after continual culture for 14 days. 2. There were antral follicles forming in the fresh and the frozen partial-isolated culture group after cultured for 14 days. The follicles of GV phase but not MⅡwere found after further separation in two partial-isolated culture groups .3. There were no antral follicles forming in two tissue mass culture groups after cultured for 14 days.4. There was highest secretion production of E2 on 6th day and then it began to decrease on 8th day in the fresh tissue mass group, while the secretion production of E2 decreased continually in the frozen tissue mass group, and kept increasing in two partial -isolated culture groups.5. There were no significant differences of cpm in small OGCs among fresh and frozen partial -isolated culture groups and fresh tissue mass culture group.,but the cpm of small OGCs in frozen tissue mass culture group decreased significantly.6. There were no big OGCs with normal morphology in the frozen mass culture group. There were no significant differences in the cpm of big OGCs between the fresh and frozen partial -isolated culture groups(P>0.05),while there was significant decrease of the cpm in big OGCs in the fresh tissue mass group compared with the above two groups(P<0.05).Conclusions:1. The partial -isolated culture method of in vitro culture of rabbit ovarian tissue was better than tissue mass cultural method. The secretion production of E2 increased and there were antral follicles forming in the former groups. The secretion production of E2 decreased and there were no antral follicles forming in the latter groups.2. There were good proliferative competences of the big and small OGCs cultured from the fresh and frozen partial-isolated culture groups, which suggested PROH procedure slow-freezing method can maintain the development potentiality of survival follicles.3. The culture system is not suitable for primordial and primary follicles in vitro development. 4. Tissue mass culture method is not suitable for the culture frozen ovarian tissue.PartⅣ:Research on transplantation of the rabbit ovarian tissueObjective:To investigate the suitable transplantation position of ovarian tissue and observe the physiological function of transplanted survival.Methods:1. 20 healthy rabbits were chosen, and randomly divided into control group (5 rabbits), castration group (5 rabbits) and transplantation group(10 rabbits).2. Broad ligament beside the uterus was chosen as the position of transplantation in the transplantation group and both ovaries were cut ,then medullary substance of two ovaries were removed, 1×1×1mm3 tissue were cut and implanted into the subserosa of the left broad ligament. After the operation,HMG 10IU /d were injected i.m every other day for half month. Both ovaries were cut in the castration group.3. The change of E2 level was assessed by vaginal cytology from the second day to one and a half month after operation.4. Venous blood of the ear was taken and the serum E2 level was assessed by ELISA in each rabbit three times every 5 day from one month after the operation.5. One and a half month after operation, the endometrium and ovaries of the control group, the endometrium of the castration group and the endometrium and survival ovarian tissue of the transplantation group were taken and then HE staining was made to observe the morphology.6. The expression of PCNA and VEGF were measured by immunohistochemistry in the ovarian tissue of the transplanted survival and the control group. Results:1. There were signs of low estrogen level in the vaginal cast-off cells in the castration group 4~6 days after operation. A rabbit died after the operation and 7 rabbits showed sighs of low estrogen level in the vaginal cast-off cells and recovered 10~20 days after operation in the transplantation group, but the other 2 rabbits didn't recover.2. The serum E2 level returned normal one month after operation in the 7 rabbits of the transplantation group, which had no significant difference compared with the control group(P>0.05). The serum E2 level didn't return the normal level in the other 2 rabbits. The serum E2 level decreased significantly in the castration group compared with the control group(P<0.05).3. The epithelial cells of endometrium were composed of monolayer columnar cells, and the glands were few and shrinked, the stromal cells arranged compactly in the castration group.In the transplantation group, the surface layer cells of the endometrium showed jagged and composed of pseudoambilayer columnar cells and there were large amounts of glands which arranged tightly and shaped well-stacked, the stromal cell arranged rarefactly in 7 rabbits similar to the control group. The morphology of endometrium in the other 2 rabbits with low E2 level was similar to the castration group.4. The survival ovarian tissue of 6 rabbits was found in the broad ligament in the transplantation group. The transplanted ovarian tissues were wrapped by fibrous tissues and fatty tissues. And follicles of each phase with eumorphism and large amount of new vessels were found in the survival transplanted ovarian tissue .5. The positive rate of PCNA expression in the oocytes of the primordial and the primary follicle in the transplantation group was 78.67%,which had no significant difference compared with the control group(77.55%)(P>0.05).There were no significant difference in the expression of VEGF between the two groups(P>0.05). Conclusions:1. There is abundant blood supply in broad ligament beside the uterus, and its internal environment is similar to the physiological condition of ovary. The operation is convenient and suitable for transplantation of ovarian tissue. The method of transplantation combining with using Gn after operation harvested a high survival rate.2. There was smooth endocrinological function in the survival ovarian tissue in broad ligament beside the uterus and E2 with physiological dose could be secreted, which played an important role to the epithelium of vaginal and endometrium.3. The oocytes in the survival ovarian tissues had smooth proliferative activity and had the ability to secrete VEGF, which played an important role in maintaining the production and structure of blood vessels.