Effects Of Different Freezing Methods And Autotransplantation On The Expression Of Ang-2 And Its Receptor Tie-2 In Mouse Ovarian Tissue

Objective: Two different freezing methods were used to freeze ovarian tissue of mice and the ovarian tissues were autografted subcutaneously after thawing.The morphological changes of follicles and the expression of Ang-2and Tie-2 were compared between the two methods before freezing,after thawing and after autografting.Methods: Ninty mice were randomly divided into three groups and thirty mice in each group.Because of operational problems during the experiment,74 mice were included in the results(27 in group A,23 in group B,and 24 in group C).Group A was the control group.group B and group C were the experimental groups.In group A,fresh ovarian tissues were directly autografted ubcutaneously.In group B,the ovarian tissues were frozen by programmed freezing method and were autografted ubcutaneously after 21 days.In group C,the ovarian tissues were frozen by vitrifiication method and were autografted ubcutaneously after 21 days.The transplanted ovarian tissues were removed 21 days after autografting in the three groups.The fresh and transplanted ovarian tissues in the three groups,thawed ovarian tissues in group B and group C were dealed with HE staining to observe the changes of follicles and immunohistochemistry to observe the expression of Ang-2 and Tie-2.Results:1.In group A,the normal morphology of primordial follicles,preantral follicles and antral follicles in ovarian tissue after transplantation were significantly lower than before transplantation(P<0.05).In group B,there was no significant difference in the normal morphology of primordial follicles between before freezing and after thawing(P>0.05).the normal morphology ofpreantral follicles and antral follicles of ovarian tissue after thawing were significantly lower than before freezing(P<0.05).In group C,the normal morphology of primordial follicles,preantral follicles and antral follicles of ovarian tissue after thawing were significantly lower than before freezing(P<0.05).After transplantation,the normal morphology of primordial follicles,preantral follicles and antral follicles in ovarian tissue were significantly lower than before freeing and after thawing in group B and group C(P<0.05).There was no significant difference in the normal morphology of primordial follicles,preantral follicles and antral follicles in ovarian tissue before freezing among group A,group B and group C(P>0.05).After thawing,the normal morphology of primordial follicles in group B was higher than in group C(P<0.05).After transplantation,the normal morphology of primordial follicles,preantral follicles and antral follicles in group A were higher than in group B and in group C(P<0.05),there was no significant difference between group B and group C(P>0.05).2.Whether in granule cells or in theca cells,the positive expression of Ang-2 and Tie-2 in the fresh ovarian tissue were significantly higher than after transplantation in group A(P<0.05).In group B and group C,the positive expression of Ang-2 and Tie-2 in the fresh ovarian tissue were significantly higher than after thawing and after transplantation(P<0.05).In the fresh ovarian tissue,there were no significant differences in the positive expression of Ang-2 and Tie-2 among group A,group B and group C(P>0.05).After thawing,the positive expression of Ang-2 and Tie-2 in group B were higher than those in group C(P<0.05).After transplantation,the positive expression of Ang-2 and Tie-2 in group A were higher than those in group B and in group C(P<0.05),there was no significant difference between group B and group C(P>0.05).Conclusions:1.No matter what kind of freezing method was used,compared with preantral follicles and antral follicles,the damage to primordial follicles by freezing was the weakest.The tolerance to cryopreservation of primordialfollicles were the best,programmed freezing method was better than vitrification in the way to protect follicles.2.Freezing could affect the expression of Ang-2 and Tie-2 and had an negative effect on angiogenesis,programmed freezing method was better than vitrification in the way to protect the expression of Ang-2 and Tie-2.3.Programmed freezing and vitrification could damage the follicles and affect the process of blood vessel regeneration.