Effect Of "tonifying Kidney" On Functions Of Auto-transplanted Ovaries After Cryopreservatioii In Mice

Ovarian cryopreservation and transplantation have clinical application value for conservation and utilization of female germ cells. The technique develops rapidly in recent years. However, Chinese Traditional Medicine (TCM) in this field is still in its initial stage. To explore the effect of TCM on growth patterns of follicles and oocytes in ovaries after cryopreservation and auto-transplantation, in the present study we threw and auto-transplanted cryopreserved mice ovaries and then divided them into six groups of Wu Zi Yan Zong Wan (WZYZW), Zuo Gui Wan (ZGW), You Gui Wan (YGW), PMSG,Model and Normol, based on TCM theory that kidney governs reproductive capability. At the same time? the model group was used as control. After7-35days, follicle growth, oocyte development and related gene expressions were analyzed by using methods of histo-morphology, immunohistochemistry (IHC), in vitro maturation (IVM), in vitro fertilization (IVF) and Q-PCR. The results are presented as follows:1) The effect of "TONIFYING KIDNET" on in vivo growth of cryopreserved and auto-transplanted ovaries in miceIn this study,246mice were used and492ovaries were retrieved for cryopreservation and auto-transplantation, in which338ovaries were successfully were retrieved (total retrieve rate is68.70%). There were significant differences in the retrieve rate when compared the control group with the groups of WZYZW1, WZYZW2or PMSG group (all P<0.01or P<0.05). Therefore, it is concluded that WZYZW and PMSG could improve the survival ability of ovaries after cryopreservation and auto-transplantation in mice, and administration of WZYZW before auto-transplantation can improve the survival rate of the auto-transplanted ovaries.On the21st day after auto-transplantation, the original, primary, secondary and antral follicles can be observed. In the groups of WZYZW and YGW, count of antral follicles were more than that in the control group, and luteum was rare. On the35th and42nd days, the group of WZYZW had more antral follicles in ovaries than other groups, and oocytes with cumulus expansion appeared.Immunohistochemical analysis showed that expression of VEGF was increased in the ovarian cortex and the fibrous connective tissue surrounding the ovarian cortex. The VEGF expression in granulosa and theca cells was gradually increased. These results suggested that WZYZW may promote ovarian survival and follicle development via an angiogenetic signal pathway of VEGF.The test of TUNEL in situ detection apoptosis (FITC-labeled) showed that follicles in auto-transplanted ovaries were gradually grown while regions of expression of apoptotic positive cells were gradually reduced with the prolonged time of transplantation. The positive expression of apoptosis in ovarian cortex was also reduced. The ovarian medulla was the main area of expression of apoptotic positive cells.As determined by a specific estradiol ELISA, the highest levels of serum estradiol was found on the21st day after transplantation in the WZYZW group compared with the groups of Control, ZGW and YGW (P<0.05). This suggested that WZYZW had a better effect to improve the follicular development in auto-transplanted ovaries when compared to Zui Gui Wan or You Gui Wan.2) The effect of "TONIFYING KIDNEY" on in vitro oocyte development in ovaries after cryopreservation and auto-transplantationOn the21st day after auto-transplantation, a total of64oocytes were successfully retrieved. And on the35th day,69oocytes were successfully retrieved. However, oocytes at stages of GV and MII with normal morphology could be observed in the WZYZW group. in the Control group compared with the groups of WZYZW, YGW and PMSG (P<0.05). This suggested that WZYZW, YGW and PMSG could improve the production of ooctyes after auto-transplantation of cryopreserved ovaries. During the period of21st-35th day after auto-transplantation, WZYZW and YGW can promote the recovery of ovarian function to further increase the oocyte production,In this study, immature oocytes retrieved from the groups of WZYZW could be maturated to MII stage and pronucleus and second polar body can be observed on the second day after IVM and IVF. These fertilized oocytes grew to2-cell stage and further morula but not blastocysts. Therefore, this indicated that WZYZW was not only able to improve the oocyte production after auto-transplantation of cryopreserved ovaries, but also recover the fertilization ability of oocytes which may be injured during auto-transplantation or cryopreservation.3) The effect of "TONIFYING KIDNEY" on gene expressions related to follicular growth and development in auto-transplanted ovaries after cryopreservation in miceAlthough mRNA of the genes GDF-9, AMH, FSHR, LHR, VEGF, Ang2, Bax, Bcl-2, Fas and Fasl can be detected in both frozen-transplanted and normal ovaries, their expressions in primordial follicles of ovaries after cryopreservation and auto-transplantation were dramatically reduced. In conclusion, WZYZW can improve the survival and growth ability of follicles in cryopreserved ovaries in early time while WZYZW and YGW can improve the survival and growth ability of follicles in cryopreserved ovaries in later time during the7th-35th day after auto-transplantation. The actions may be through inhibition of apoptotic pathway (Bcl-2/Bax) and regulation vascular endothelial growth factor pathway. Moreover, WZYZW and YGW may have more advantages than ZGW in protecting the primordial follicular reserve capacity of ovaries after auto-transplantation and cryopreservation.