| In recent years, with the advances of oncological treatments, cure rates of childhood cancers are as high as 80%. However, unfortunately these treatments often cause permanent sterility as a result of the loss of spermatogonial stem cells. Hence, the preservation of fertility in pre-pubertal boys is becoming an urgent need in clinical. Sperm freezing is the optimal way for fertility protection in the male who already has spermatozoa in his semen.Since the first success of artificial fertilization using frozen semen, Cryopreservation of semen and spermatozoa became a well-developed technique at present, and routinely used in clinical practice. But the shortage is that it is only applicable for adults rather than pre-pubertal boys. For young boys who do not yet produce spermatozoa, cryopreservation of testicular tissue or testicular cell suspension might be an optimal choice to preserve their fertility. However, both of these methods have advantages and disadvantages. Testicular tissue which containing impaired cells or cancer cells will affect the survival rate of germ cells and may also cause infection of cancer cells during implantation. In contrast, cryopreservation of testicular cell suspension provided a better option.Nevertheless, this method will increase the required amount of sample; aggressive enzymatic treatment may influence the viability of cells. Extensive handling may lead to direct mechanical damage on the cell-cell adhesion, increased cell hypoxia, which further damage the cells. The negative influence of these treatments can be avoided when testicular tissue is cryopreserved. For pre-pubertal boys, when full spermatogenesis is not yet ongoing, it is essential to store the undamaged spermatogonia (SG) and the neighbouring cells as integrated tissue. Because preservation of Sertoli cells (SCs) and cell-to-cell contacts in testicular tissue has been proved as an important role in subsequent maturation of SG.So,it is more significant to study the cryopreservation of immature testicular tissue. Jahnukainen suggested that the type of cryoprotective agent employed seems to be much more critical in freezing testicular tissue for graft survival. There was a clear difference freezing effect between difference cryoprotective agents. The concentration of cryoprotective agent in cryopreservation media also had a significant effect on the result of cryopreservation. In present study, we used an identical un-controlled freezing program and tested three different cryoprotectants. The effect of cryopreservation was evaluated by light microscopy and transmission electron microscopy (TEM). Our aim was to find the optimal cryoprotectant for cryopreservation of immature testicular tissue.Methods: 10-day-old Kunming mouse suckling mouse, degermed with75% alcohol and sacrificed, obtained the two sides testicular tissue. Testes were decap- sulated and fat pad under the anatomical lens.The obtained testicular tissue were randomly divided into fresh control, glycerol group, Dimethylsulphoxide (DMSO) group and DMSO with sucrose group. The fresh control didn't receive any treatment, fixed immediately.The rest three group were transferred to cryovials containing a final volume of 1.5 ml of homologous cryoprotectant. The cryovials were first equilibrated on ice for 40 min and thereafter placed in-20℃standard freezer for 60 min. The cryovials were then plunged into liquid nitrogen and stored for one month.The cryovials were thawed in 37℃water bath and the testes were washed in fresh ice cold medium. Then the testes were fixed by 4% paraformaldehyde, embedded in paraffin and sliced, observed with light microscope and immuno- histochemistry. For TEM the tissue samples were fixed in 2.5% gluta- raldehyde, postfixed in osmium tetroxide,embedded in epoxy resin, sliced ultrathin section and then examined with TEM.Results:1 Light microscope:⑴HE staining :The morphological structure changes in testicular tissue was studied in four groups. In the DMSO with sucrose group, the percentages of the seminiferous tubules (STs) with an intact undamaged structure , signs of central necrosis and those totally damaged were identical to those in the fresh control(P>0.05). The numbers of ST with intact structures were significantly decreased in DMSO group when compared with fresh control and DMSO with sucrose group.The proportions of ST with signs of central necrosis and total necrotic changes were significantly increased in DMSO group(P<0.05). When compared with fresh control ,DMSO with sucrose group and DMSO group, the percentages of ST with intact structures were significantly decreased and the proportions of ST with signs of central necrosis and total necrotic changes were significantly increased in glycerol group(P<0.05).⑵Immunohistochemistry: The percentage of SG with normal undamaged morphology in DMSO with sucrose group was87.693±2.408%, in fresh control was 91.971±3.209%,there was no significant difference between the two groups(P>0.05).In DMSO group was 71.707±3.512%, when compared with fresh control and DMSO with sucrose group it was significantly decreased (P<0.05). Whereas in glycerol group was 57.895±6.083%, compared with DMSO group it was significantly decreased(P<0.05). The percentage of detached SG from neighbouring SCs and degenerated SG was no statistical difference between DMSO with sucrose group(10.769±3.049% and 1.538±0.212%, respectively)and fresh control(7.299±4.690% and 0.730±0.093% ,respectively)(P>0.05). The percentage in DMSO group( 22.439±5.603% and 5.854±2.307% ,respectively ) was significantly increased(P<0.05). This percentage in glycerol group(31.579±3.847%and 10.526±2.143% ,respectively ) was significantly higher than in DMSO group(P<0.05).2 Transmission electron microscopy: In the DMSO with sucrose group the spermatogonia were more mildly influenced and showed a morphology similar to that in the control material, with well-preserved and well-attached cells. At higher magnification, increased vacuolization were observed.However, these changes were very slight. In DMSO group, the well attachment of the spermatogonia to the basement membrane were observed.However, the number of vacuoles in the cytoplasm was significantly increased. The nuclei of the spermatogonia were dense. In glycerol group,detachment of spermatogonia from the basement membrane was observed. The nuclei of the cells were dense, pyknotic and dark. More vacuoles were observed in the cytoplasm and the sizes of the vacuoles were larger.Mitochondria were swollen.Conclusion: When the immature testicular tissue was freezing, DMSO as a cryoprotectant proved to maintain the structure of spermatogonia after cryopreservation better than glycerol. Whereas DMSO with sucrose as a cryoprotectant proved to maintain the structure of spermatogonia after cryopreservation better than DMSO.This indicates that the optimal cryoprotectant for cryopreservation of immature testicular tissue was the combination of penetrating cryoprotectant and nonpenetrating cryoprotectant.
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