Testis Tissue Grafting And Cryopreservation In Newborn Mice

Cryopreservation and transplantation of mouse testis is a significant tool in assisted reproductive technology for preservation of genetic resources and endangered animals. This technique would enable the preservation of the testis cell integrity and the endocrine functions of the testes. The spermatogonial stem cells in the testis of prepubertal males offers clinically relevant options for preservation and restoration of fertility later in life. New approaches based on grafting of testicular tissue can be applied to generate a limited number of sperm cells and could therefore be considered important new alternatives for restoration of fertility in testicular cancer patients by cryopreserving healthy testicular tissue before chemotherapy. The loss of genetic diversity due to premature death of valuable individuals is a significant problem in animal conservation programs, including endangered animals. Since it is impossible to obtain sperm from the testes of these animals, their genetic pool is lost. Testis tissue xenografting provides a tool to obtain spermatozoa from immature animals, promoting spermatogenesis by keeping the microenvironment of the donor testis intact, as evaluated by comparing gene expression in development-matched grafted and donor tissue. Testis tissue xenografting has emerged as a system to obtain spermatozoa from dead immature animals, however protocols to store this tissue before xenografting are still lacking. One purpose of this study is to select the best conditions for testis grafting by comparing the effect of different graft position, different recipient and collection time. The other purpose of this study is to select a better protocol for testis cryopreservation by comparing the effects of different concentration of DMOS and different freezing methods.1. Research on testis tissue graftingTestis from 5 days old C57BL/6J mice are used in present experiment. To select the best conditions for testis grafting, the effect of different graft position, different recipient and collection time are compared. The results were showed as following. (1) There are significant difference of the testicular germ cell differentiation in different transplantation groups. The germ cell differentiation of testicular membrane transplant group are same as control group; the differentiation rate of germ cell was 29.2% in dorsal subcutaneously transplant group; and there was no differentiation in subrenal capsule transplant group. The germ cells differentiation of nude mice recipient 8 weeks group was significantly better than C57BL/6J recipient 8 weeks group (P<0.01). (3)The germ cells differentiation of nude mice recipient 10 weeks group was better than nude mice recipient 8 weeks group (P<0.05). From this study some conclusions can be made as following. (1)The testicular membrane is a favorable position for testicular transplantation, and dorsal subcutaneously transplantation is an alternative choice. Subrenal capsule transplantation are not appropriate for preservation of male reproductive organs in mice. (2) Comparing with same strain recipient, nude mice recipient is better for testicular transplant. (3) Increasing the grafting time from 8 weeks to 10 weeks contribute to the differentiation of germ cells.2. Research on testis tissue cryopreservationTestis from 5 days old C57BL/6J mice are used in present experiment. The proportions of PCNA-positive are used as an indicating meter to evaluate the efficiency of testis cryopreservation, which is effected by the different concentration of DMOS and freezing methods. In addition, testes cryopreserved with optimal conditions are grafted to verify the efficiency of testis cryopreservation. The results were showed as following. (1) Before the cryovials were plunged into liquid nitrogen, three freezing method (Liquid nitrogen vapour freezing method,-20 ℃ standard freezer freezing method and Nalgene freezing container-80℃ freezing method) are used. They show significant difference of PCNA-positive proportion. Nalgene freezing container -80℃ freezing method performs the best result. (2) The effect of different DMSO concentration(5%,10%,15%) on the testicular germ cell differentiation is significant. The best concentration of DMSO is 10%, showing highest proportions of PCNA-positive nuclei. (3) There are significant difference of the testicular germ cell differentiation between freezing group and fresh group. From this study, using Nalgene freezing container-80℃ freezing method, with 10% DMSO, is a favorable protocol, which shows the similar testis grafting efficiency to fresh testis.