Histological and Immunohistochemical Comparison of Mature Rat Testes with Glycerol, Propanediol and Dimethylsulphoxide as Cryoprotectants

Cryopreservation induced morphological changes of the testicular tissues with different cryoprotectants have been assessed in present study. The testicular tissues of thirty mature male rats (six rats for each group) were used. Group A was control (Fresh sample), samples from group B were cryopreserved using only the freezing media. The others groups were assigned to three freezing protocols using either dimethylsulphoxide (DMSO) group (C), Glycerol (D) or propanediol (PrOH) group (E) as a cryoprotectant. Light and transmission microscopic analysis were carried out on testicular tissue. Immunohistochemical analysis was used for detection of DNA damage. Normal structures were seen in the fresh control group (A), in contrast to the group (B) which showed sever cryoinjury. Different tissue changes in cryoprotectant groups, however, these alterations were vary according to the type of cryoprotectant used. The cryoinjury changes were extreme in propanediol group (E) followed by glycerol group, while the DMSO group showed lighter changes. The damages including irregularities and shrinkage of seminiferous tubules, disruption of interstitial tissue and increase in the intertubular space, abnormal arrangement of testicular cords and desquamated cells in the tubular lumen. The results of DNA fragmentation test revealed that frozen group with DMSO had the significantly lowest rate of tissue damage compared to glycerol and PrOH. These results were confirmed by the ultrastructure observations, that represented by discontinuity of the cell membrane, detachment of the spermatogonia and spermatocytes from the basement membrane and from the supporting cells, swelling of mitochondria and disruption of nuclear membrane and shrinkage of nucleus with dense clumped chromatin. In conclusion, storage of testicular tissue of mature rat using DMSO gave the best results, with little structural alterations of the tissue. Whereas the other cryoprotectant (Glycerol and propanediol) damaged DNA integrity compared to DMSO.