The Study Of Mouse Ovarian Tissue Cryopreservation And Preantral Follicles Culture In Vitro

The ovarian tissue cryopreservation has the andvantage that oocytes and embryos cryopreservation can' t replace. It doesn' t need to control the donor's reproductive cycle and pick up oocytes; it can be used to protect the animals in the severe danger or the oocytes of human or animals that are accidental injuried; it can the provide reproductive insurance for people or animals that lose the fertility before sexual maturity and increase the oocytes sourse; it can be used to establish reproductive cells( oocytes) frozen storage; In the same time, there are various phase immature follicles in the ovarian cortex, the primary follicles' sensitivity to cold is lower than mature follicles. So when the low temperature biology technology and reproductive medicine develop, the ovarian cryopreservation has become a most potential choice to protect female's fertility. Despite some articles reported ovarian cryopreservation had been successfully used in clinical practice, the technology of freezing ovarian tissue is not perfect, there are many problems should be resolved. One of the problem is how to establish an optimal freezing protocol, until now people haven't found an optimal freezing protocol. Now the ovarian tissue cryopreservation methods applied to human are autotransplant, heteroctransplant and in vitro maturation (IVM). Now there had been delivered a healthy baby after autotransplant, but IVM is still staying in testing phase, because the maturation system in vitro is not perfect. Objective In this study, we use mouse as an animal model to study the effect of different cryoreserved protect agents on the freezing ovarian tissue, and we study the preantral follicles developed in vitro and the influence of different culture factor, so we want to find a good protocol to freeze the ovarian tissue and culture the follicles in vitro, choose the best CPA and culture system to provide reference to human ovarian storeroom.Materials and methods1. 60 Kunming white female mice at the age of 4 weeks were provided from HenanLaboratory Animal Center. Bright/dark cycle in the environment the mice living in was 10h/14h. The mice drank and ate without time limit.2. We resect the mouse two sides' ovaries, devided them to1×1×1-2 mm pieces using pinhead under microscope. The pieces were put to 1.5 M DMSO, EG, the PROH solution, after one step or three step equilibrium, they were put into the liquid nitrogen directly. When thawing, they were recovered in the 37℃water for 5 rain and CPA were displaced in thawing solution, then incubate 30 min.3. We use defferent method to separate the ovarian follicles under microscope. The mechanical method is seeking out the follicles directly using pinhead after beating upon; the enzyme method is picking out follicles after assimilating 20 min in 0.1% collagen enzyme; the mechanical and enzyme method is assimilating 5 min in 0.1% collagen enzyme first, then separate follicles mechanically.4. There were four kinds of culture medium: the basal culture medium: 10%SPS+10μg/ml insulins+5.5μgs/ml transfermin+ 6.7ng/ml selenium(1% ITS)+100 mIU/ml FSH+a-MEM; the EGF medium: basal culture medium + 0.05mg/ml EGF; GDF-9 medium: basal culture medium+100μg/ mlGDF-9; EGF+ GDF-9 mediums: basal culture medium +0.05 mg/ml EGF+100μg/ml GDF-9+ a- MEM. The normal morphology follicles were put into the drops that include different culture medium,2-4 follicles were put into one drop and cultured for 12 days. The control group is culturing fresh ovarian follicles in basal medium. Refresh the half medium of one drop every 2 days, and see the development of follicles under microscope and measure the diameter of follicles.5. Replace the medium of the drops with maturation medium in the 12 day, after cultured 24 hours, watch the COC release and GVBD, wipe off the granule cells and pick out the mature oocytes.6. Statistical analysis: SPSS10.0 software package was used to do statistical analysis on the experimental data. t-test was used to compare the mean, and thex~2 test was used to compare the rate. The experimental data is expressed asmean tstandard deviations or rate. The confident internal level is chosen at a =0.05, P<0.05 is chosen as statistical significance, P<0.01 is chosen as marked defference.Results1. Effect of different equilibrium protocol on the ovarian tissue morphologyThe rate of normal follicles(43.5%) and preantral follicles(43.7%) in one step equilibrium method is distinctly lower than the control group(82.1%,76.7%) and three step equilibrium method group(74.1%,72.9%), P<0.01. there were no statistical significance between three step equilibrium method group and the control group, P>0.05.2. The component proportions of ovarian various follicles in different groupsThe proportion of preantral follicles is most in every group. The proportion of primary follicles, secondary follicles, preantral follicles and antral follicles in the control group is 5.6%,8.3%,83.3%,2.8%.There was no statistical significance in the proportions of various follicles during all the groups, P>0.05. 3 . Effect of different CPA on the ovarian tissue morphologySome follicles were locked after frozen-thawed, the follicle lose their normal circular structure, the integrity of follicles' membrane was broken; some follicles stay normal shape, but oocytes in them appeared bubble or crimple; some normal follicles separated from frozen-thawed ovaries can be seen the oocytes were dark under the microscope. These were less in the fresh ovaries.The rate of normal oocytes in DMSO group and PROH group was distinctly lower than the control group, P<0.01; the rates of normal follicles and preantral follicles in EG group were distinctly lower than the control group, P<0.01. The rate of normal oocytes in EG group was lower than DMSO group, but had no statistical significance compare to PROH group; there were no statistical significance between DMSO group and PROH group, P>0.05.4. Effect of different CPA on the ovarian follicles development in vitroCompared to the control group, the D4 survival rate and the increase of follicles' diameter were lower than the control group, P<0.05.There was no statistical significance during the three frozen group, P>0.05.5. Effect of different separation method on the follicles' quality and quantityThe follicles number separated in mechanical + collagen separation method was higher than the mechanical separation method, P<0.01; the rate of normal follicles in the collagen enzyme separation method was distinctly lower than the other methods, there was no statistical significance during the left compares, P> 0.05.6. The mouse preantral follicles developed in vitroIn the 2-3 days' culture, the follicles sticked to the wall, fix in the culture vessel; in the 4th day, granule cells increased obviously; in the 5-6 days, the layers of granule cells increased obviously, the granule cells exceed the basal membrane, the oocytes can't be seen clearly, the follicles gradually lose three dimension of the round structure; in the 7-10 days,there appeared antral structure;in the 13th day, the COC released;wipe off the granule cells and saw matured oocytes.In the first few days,the follicles developed very slowly, from the 6th day, follicles development accelerate, and reach the mature folllicle's diameter(>400um).When multi-follicles were cultured together, two or more than two follicles adhere to be conglomeration, so we can't measure the follicles diameter. In the last phase of culture, the follicles that had integrate membrane can form antral structure; the follicle membrane is not integrate, granule cells expand, the follicles still can form antral structure. When cultured in drops in vitro, the preantral follicles can GVBD and the oocytes released.7. Effect of different accretions in the cultured medium on the follicles development in vitro7.1 Effect of different accretions on the follicle survival rate in vitro cultureIn the 12th day, the survival rate of control group,basal culture medium group,EGF group,GDF-9 group and EGF+GDF-9 group is 41.3%, 33.2%,50.0%,49.6%,51.3%. Compare to the control group, the D6, D8, D10, D12 survival rate in basal culture medium group was lower, P<0.05; D10,D12 follicles survival rates of EGF group and GDF-9 group were, P<0.05; the D4,D6 survival rates were higher, P<0.05, the D8, D10,D12 survival rates were distinctly higher, P<0.01. Basal culture medium group,EGF group,GDF-9 group and EGF+GDF-9 group compared with each other, the D6 survival rate in EGF group and GDF-9 group was higher than basal culture medium group, P< 0.05, the D8, D10, D12 survival rate was obviously higher than basal culture medium group, P<0.01;the D4 survival rate in EGF+GDF-9 group was higher than basal culture medium group, P<0.05, the D6, D8, D10, D12 survival rates were obviously higher than basal culture medium group, P<0.01;there were no statistical significance during EGF group,GDF-9 group and EGF+GDF-9 group, P>0.05.7.2 Effect of different culture factors on the follicles and oocytes development in vitro cultureThe follicles in the EGF+GDF-9 group developed faster in vitro, after 12 days culture, most of diameter exceed 400um,the biggest one can be higher than 450um.The follicles and oocytes diameter in DO had no statistical significance during control group, basal culture medium group,EGF group,GDF-9 group and EGF+GDF-9 group, P>0.05.The D12 follicles,oocytes diameter and increased diameter in basal medium group was lower than the control group, P<0.05; the follicles,oocytes diameter and increased diameter in EGF group and GDF-9 group was higher than control group, P<0.05; the follicles,oocytes diameter and increased diameter in EGF+GDF-9 was obviously higher than the control group. Basal culture medium group,EGF group,GDF-9 group and EGF+GDF-9 group compared with each other, the follicles,oocytes diameter and increased diameter in EGF group,GDF-9 group and EGF+GDF-9 group were obviously higher than the basal medium group, P<0.01. there were no statistical significance during EGF group,GDF-9 group and EGF+GDF-9 group, P>0.05.7.3 Effect of different culture factors on the COC,GVBD,MIIThe survival rate,COC rate,GVBD rate,and MII rate of the control group were 41.3%, 63.6%, 65.9%, 13.4%.The survival rate,GVBD rate,and MII rate of the basal medium group were Lower than control, P<0.05;the survival rates in EGF group and GDF-9 group were higher than the control group P<0.05;survival rate,GVBD rate,MII rate in EGF+GDF-9 group were higher than the control group, P<0.05, P<0.01. Basal culture medium group,EGF group,GDF-9 group and EGF+GDF-9 group compared with each other, survival rate,GVBD rate,MII rate in EGF group and GDF-9 group were higher than the basal culture medium group, P<0.05; survival rate,GVBD rate,MII rate in EGF+GDF-9 group were obviously higher than the basal culture medium group, P<0.01. there were no statistical significance during EGF group,GDF-9 group and EGF+GDF-9 group, P>0.05.8. Effect of day of culture on the follicles development after freezing and thawingCompare to the fresh control group, after freezing and thawing, the increased diameter,survival rate,GVBD rate,MII rate in cultured 12 days group were obviously lower than control group, P<0.01; the increased diameter in cultured 14 days group was obviously lower than control group, P<0.01,the MII rate was lower than control group, P<0.05;there was no statistical significance between cultured 16 days group and control group, P>0.05.Cultured 12 days group,cultured 14 days group,cultured 16 days group compared with each other, GVBD rate in cultured 14 days group was higher than cultured 12 days group; the increased diameter,GVBD rate,MII rate in cultured 16 days group were higher than cultured 12 days group, P<0.05, P<0.01. There was no statistical significance between cultured 16 days group and cultured 14 days group, P> 0.05.Conclusion1. The three step equilibrium method can ameliorate the results of ovarian tissue cryopreservation. DMSO is one effective cryopreserved protect agent, decrease the damnification of freezing in ovarian tissue, and can be used to freeze ovarian tissue.2. The mechanical +enzyme method can get more good quantity follicle, is a effective method to separate ovarian follicles.3. EGF and GDF-9 appended into culture medium can accelerate follicles development and maturation in vitro, combined the two factors can more powerfully accelerate follicles development.4. Prolong the culture time can promote follicle diameter increasing,maturation of follicles and oocytes, but compare to the fresh follicles, the development potential of frozen-thawed follicles is decreased.