| With the improvement of early diagnosis and treatment of cancer and the progress of radiotherapy and chemotherapy,the survival rate of young women with cancer significantly increased.But the radiotherapy and chemotherapy may cause the female fertility decline or even loss,so fertility preservation before the radiotherapy and chemotherapy has become particularly important.Ovarian tissue cryopreservation and transplantation has been considered an effective way to preserve fertility in cancer patients,but some studies have shown that transplanted cryopreserved ovarian tissue may carry the risk of reintroducing malignant cells.So isolation preantral follicles in vitro culture or transplantation may be safer choice.However,after freezing and thawing the ovarian tissue,it is necessary to determine the effect of cryopreservation on preantral follicular activity before isolating preantral follicles,and to select active preantral follicles for transplantation or in vitro culture.Primordial follicles are the most abundant follicles in ovarian tissue,and only a small part of them are activated through the follicular development pathway,and most of them are blocked.When the number of primordial follicles in the original follicle is less than 1000,menopause will follow,So the primordial follicles are the key to the preservation of female fertility,therefore.Therefore,if the follicles are successfully cultured in vitro,the preservation of female fertility is of great significance.At present,there are a number of studies dedicated to comparing the effect of frozen ovarian tissue between programmed freezing and vitrification,but the results are inconclusive.In addition,there is no research report at home in vitro culture of the best follicular conditions.The first part of this study investigate the influence of two freezing methods on the preantral follicular viability of bovine ovarian tissues,thus laying the foundation for in vitro culture and transplanting follicles.In the second part,alginate gel was used to culture isolated individual primordial follicles after freeze-thawing,and to explore the optimum growth environment in vitro culture primordial follicles.ObjectiveTo investigate the influence of vitrification and programmed freezing on the preantral follicular activity in bovine ovarian tissues,and the optimal concentration of alginate gel in primordial follicle in vitro.Materials and Methods 1、Materials:The fresh bovine ovarian tissue were cut into the tissue block which size were about 0.5cm × 0.8cm × 0.3 cm 2、Methods:(1)The ovarian cortical pieces were randomly divided into three groups: fresh control group,programmed frozen group,vitrified frozen group,the latter two groups were treated with the corresponding method of frozen ovarian tissue and recovery.Ovarian tissues in three groups were randomly selected 3-4 tablets and then fixed with 4% paraformaldehyde for histomorphological analysis.The rest ovarian tissue in three groups were cut into pieces,added Liberase DH enzyme digestion,after termination of digestion,the preantral follicles were randomly selected and counted,then fluorescent staining to identify follicular activity.(2)After ovarian tissue were vitrified freezing and thawing,preantral follicle were isolated and cultured in two-dimensional culture system(control group)and the concentration of 0.5%,1%,2% sodium alginate gel,respectively.Follicle survival and diameter were assessed every second day,and the medium was changed in half volume every second day,The collected culture was stored in a refrigerator at-20℃and the estradiol secretion(E2)level was determined by electrochemiluminescence immunoassay(ECLIA).and cultured for 8 days after fluorescence staining to identify follicular activity.3、Statistical analysis: SPSS21.0 software was used to make statistical analysis,counting data using chi-square test,the other experimental data were expressed as mean±standard deviation(sx ±),t test and one-way analysis of variance were used,differences of P <0.05 were consodered significant.Results1、The results showed that there was no significant difference in the number distribution of different types of preantral follicles both using histological analysis or enzymatic hydrolysis isloated single follicles(P>0.05),and the proportion of primordial follicles was the highest,followed by primary follicles and secondary follicles,no antral follicles.2、After fluorescent staining,the percentage of undamaged preantral follicles(V1)in the program freezing group and vitrification freezing group was lower than the fresh control group,the slightly damaged follicle and moderate damaged follicles(V2+V3)and the severely damaged follicle(V4)were higher than those in the fresh control group,and the differences were statistically significant(P<0.05),there was no significant difference in the number of undamaged follicles(V1)between the two cryopreserved groups(P> 0.05).3 、 There was no significant difference in undamaged primordial follicles between the three groups(P> 0.05).The primary follicles in the programmed frozen group were lower than those in the fresh group,the difference was statistically significant(P<0.05).The primary follicles in the vitrification group were not statistically different from those in the fresh group.Due to the proportion of secondary follicles was low,so there was no statistical analysis,There was no significant difference in undamaged primary follicles between the vitrification and programmed frozen group(P> 0.05).4、After cultured primordial follicles for 8 days in vitro,it was found that the primordial folliclces in two-dimensional culture system was difficult to survive,and the alginate three-dimensional culture system was more suitable for culturing primordial follicles in vitro.5、In the concentration of 0.5%,1%,2% alginate gel,the diameter of follicles have increased to varying degrees,which in 2% sodium alginate gel diameter increased the most,after fluorescent staining,the undamaged follicles in 2% of the sodium alginate gel group were higher than the other groups,compared with 0.5% sodium alginate gel group,the difference was statistically significant(P<0.05),and there was no significant difference compared with 1% sodium alginate gel group(P> 0.05).The levels of E2 in the three alginate gel groups were detectioned,E2 with the extension of the culture time the most secretion,but also in line with the growth trend of follicles in each group,The secretion rate of E2 was the highest in the group of 2% sodium alginate gel group,compared with 0.5% sodium alginate gel group,the difference was statistically significant(P<0.05),And there was no significant difference compared with 1% sodium alginate gel group(P> 0.05).Conclusion1、Frozen-thawn process have some damage for ovarian tissue,part of the primary follicle and secondary follicle were damaged,but it had little effect on the activity of primordial follicles.2、The 2 freezing methods both can better maintain the viability of preantral follicles.3、Alginate three-dimensional culture system is more suitable for the growth of primordial follicle compared with the two-dimensional culture system4、2% sodium alginate gel is more suitable for the growth and development of primordial follicles than lower concentration of alginate gel... |
