Research Of Follicle And Oocyte Development After Mice Ovaries Allogeneic Transplantation

In the present study, after castration for a week, the immune-intact male mice at the age of 8-12 week-old were used as recipients for ovarian transplantation. As donors, ten-day-old mice ovaries were transplanted into the back muscle of the castrated male mice which were administrated with immunosuppressants at the same time. Overall, the current study was undertaken to study the ovary survival, follicle growth and oocyte development after ovary allogeneic transplantation; and examine the endocrine function of grafts in the castrated male mice after ovarian transplantation. And the possibility of ovary allografting in immune-intact animals was investigated, which will provide references for ovary allografting in clinical application.In present study, in order to attain the aims, we have conducted our experiments from the following six aspects.Content 1. After ovarian transplantation, the recipients were treated with immunosuppressive therapy. Then we evaluated whether the immunosuppressants could reduce the immunorejection by observing the diameter and recovery rate of the grafts, and the formation of the regenerated blood vessels around the grafts. The results showed that the immunosuppressants might ease the immunorejection reaction during ovarian transplantation.Content 2. Twenty-two days after ovarian transplantation, the development of grafts and the number of follicles in grafts were examined by haematoxylin and eosin staining (HE staining). The results demonstrated that numbers of follicles were lost, while some primordial follicles and antral follicles were found in the grafts. Comparatively speaking, there were few primary and preantral follicles, which were obvious different from 32-day-old mouse ovaries.Content 3. Twenty-two days after ovarian transplantation, the apoptosis of the follicles in grafts was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining (TUNEL staining), and the results showed that many follicles at different developmental stages were apoptotic in the grafts, especially some primordial and primary follicles were tagged with fluorescence. However, on the age-matched ovaries, only some advanced follicles were fluorescently tagged, such as antral follicles.Content 4. In order to observe the potential ability of the follicles and oocytes sequential development in recipients with immuno-therapy, by extending the time of ovarian transplantation to 40 days, and the results demonstrated that the grafts could still survive with recovering cumulus-oocyte complexes (COCs).Content 5. The oocytes were harvested and subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). Embryos were derived from the recovered oocytes after IVM and IVF with 72.4% of cleavage rate and 7.9% of blastocyst rate, and these embryos were then transferred to 14 pseudopregnant recipient mice, and as a consequence, 12 live pups were born from 3 pregnant female mice.Content 6. The hormone fluctuation in host mice was studied by steroid hormone detection, and as a result, the hormone assay showed that the plasma concentration of both estrogen (E₂) and progesterone (P₄) increased obviously after ovarian transplantation.In conclusion, after ovary allogeneic transplantation to immune-intact castrated adult male mice, this is the first time of successfully obtaining live offsprings from the oocytes of grafts by using IVM, IVF and embryo transfer, this result proved completely that the grafted ovaries could survive. Histological observation demonstrated that the follicles and oocytes could further develop in castrated male mice. And we found that the hormone fluctuates in the recipients after ovarian transplantation, which implied that the reproductive axis in castrated male mice might be re-established in future. Overall, these results will not only provide some important references in studying the mechanism of follicles and oocytes in vitro development, but also in clinical study of human ovarian transplantation and the protection of the improved strains in domestic animals.