The Study Of Rapid Cryopreservation And Evaluation Of Viability Of Thawed Newborn Rats' Ovaries

Objective The aim of this study was to investigate the effect of the rapid freezing to newborn rats, ovaries through observing the survival, growth, endocrine function of ovaries transplanted after cryopreservated and to study the influence on ovarian antigen after cryopreservation.The experiment will provide valuable laboratorial technique and scientific basic for alltransplantation of fetal ovaries after cryopreserved in the clinical. Methods The transplanted ovaries were obtained from female rats with born one day(newborn rat) and adult rats.(1) Cryopreservation of ovaries: A and B solution used as freeing solution contain 1.5M propandial and 1.5M propandial+0.1M sucrose respectively. C solution as thawing contains 0.5M sucrose. Ovaries were rapidly frozen by two-step penetrating balance which ovaries were put in A and B solution in turn under 4℃for 30min respectively and above vapor of liquid nitrogen to cold balance for 5min. Ovaries were plunged directly into liquid nitrogen and stored.(2) The observation of cryopreservative effect: Rapid thaw ovarian tissue cryopreservated after 1-7 days. The effect of cryopreservation was observed by heterotopical allogeneic transplant of ovarian tissue and ultrastructure. Ovarian transplantation: The recipients(adult female rats) were randomly divided into three groups: freezing transplanted group, fresh transplanted group and control group. Ovaries of newborn rats and ovarian tissue of adult rats were transplanted underneath the kidney capsules of ovariectomized female rats. The viability can be judged by under methods.①Observation of oestrous cycle: Cytologic examination of vaginal lavages were performed from 5 days after transplantated to judge both estrum recovery and maintaining of recipient.②Histological observation: The survival grafts were acquired when the oestrous cycle had been resumed for some time and were used HE dying to observe structure of survival ovaries. Enzymohistochemistry was used to study viability and distribution of 3β-HSDH.③Radioimmunological technique was used to detect level of rats' serum E2 in order to quantitily estimate endocrine function of ovaries transplanted.④Immunohistochemistry was used to detect distribution of CD8+ and CD4+ T lymphocyte in transplantative situation. The obvervation of ultrastructure: Ultrastructure of ovaries frozen-thawed was observed through trasmission electron microscopic. Results ( 1 ) The recovery of the estrous cycle: After cryoprevation,transplantation of newborn and adult ovaries to ovarietomized recipients resulted in restoration of estrum within 27.50±6.72 and 28.78±4.30 respectively after transplantation that were obvious longer than the days of themselves fresh transplanted group(14.60±4.84;13.55±2.81)(P<0.01).But there were not significantly different in between freezing transplanting and fresh transplanted group'(P>0.05). Although the recovery rate of oestrus cycle in every freezing transplanted group'(66.67%;75.00%)was lower than that of fresh transplanted group(90.51%;91.67%), they were not significantly different.(2)The observation of histology: When the survival ovarian tissue was obtained from beneath the kidney capsules of rats after transplanted about 40-45 days,the ovarian volume obviously became big in all groups and the ovarian tissue is soft and the surface of them has rich capillary. Under light microscope, there were abundant capillary and similar to those of the normal control group,displaying follicles a lot of growing at all stages of folliculogenesis and interstitial glands and atresic follicles in the survival grafts. Compared with that of 40-45days, the ovarian volume of the fresh adult transplanted group became small and grey. There were seldom capillary on their surface and their texture became rigid about 70 days after transplant. Under light microscope, there were more connective tissue and atresic follicles except less growing follicles. The ovaries' volumes of other transplanted groups did not obviously chang.The survival grafts were observed the differently positive cells of 3β-HSDH showing blue in the enzymohistochemical slices in which the interstitial glands and theca cell showed strongly positive and the stratum granulosum cell were weakly positive. ( 3 ) The level of Serological E2: Serological E2 of normal control group is 34.76±13.53 pmol/L. There were not significantly different among transplanted groups and between each transplanted groups and normal control group. But they all obviously higher than that of ovariectomized group (2.96±1.46pmol/L)(P<0.01). (4) The immunohistochemistry: The numbers of CD8~+ and CD4~+ postive cells of freezing transplanted group of newborn rats were 9.60±6.29 and 10.00±5.16 respectively that obviously lower than that of all fresh transplanted group(P<0.01)and also lower than that of freezing transplanted group of adult rats(28.40±14.52;18.80±9.03)(CD8~+P<0.01,CD4~+P<0.05);There were not significantly different between fresh transplanted group of newborn rat and frozen group of adult rats about the numbers of CD8~+ and CD4~+ positive cells which were obviously lower than that of fresh transplanted group of adult rats(68.00±20.32;40.60±14.39)(P<0.01). The observation of ultrastructure: The ultrastructure of frozen ovarian tissue compared with fresh ovarian tissue was not obviously different. The ultrastructure of frozen ovarian tissue almost was keeped normally. We can obverse clear and integrate of oocyte, the normal conformation of endoplasic reticulum and mitochondrion, obviouse cell junction. Conclusions①The rapid freezing method can effectively cryopreserve newborn rat' ovaries. There is not significantly lagging phenomena compared with time between the working of ovarian function after hetetopic allotransplante of cryopreservated ovaries and the recovery of that after transplant of cryopreservated ovaries.②After heterotopical allogeneic transplant of fresh newborn'ovaries,numbers of T lymphocyte are less than that of fresh adult transplanted group and are further reduced after transplant of cryopreserved ovaries.The result of immunohistochemistry shows that the amount of T lymphocyte cell is less in fresh allotransplanted newborn rats' ovarian tissue than that of adult rats group, that of newborn rat' ovarian tissue is further reduced after cryopreserved. Accordingly, this indirectly shows the immunogenicity of newborn rat' ovarian tissue is relatively low,that of ovarian tissue is further reduced after cryopreserved. The experiment can provide useful methods and scientific basic for clinic use of fetal tissue and organ.