The Effects Of Cryoprotectants On The Iconic Enzymatic Activities And Key Genes Expression Of Calf Testicular Tissue

The cryopreservation technology of testicular tissue especially of young animals,couldnot only offer a new way of saving the germ cell of fine livestock and endanger species,butalso provide a theoretical basis for fertility restoration of juvenile male patients with cancerand tissue cryopreservation, promoting the biological reproduction technology of animals. Inorder to research feasible cryopreservation system of calf testicular tissue, in this study,6kinds of cryoprotectants that used Glycerin, DMSO, Glycol, Trehalose, BSA and TesticularLiquid as main ingredients were designed. First, the experiment measures the activity oficonic enzymes as AKP, ACP, LDH andβ-D-Glucosidase in frozen calf testicular tissue.Second, the relative expression of CREM, Stra8, HSP70-2, Oct4, SCF and C-KIT gene intesticular tissue were measured by RT-PCR technology. The thawed testicular tissue wasmade into single-cell suspension and cell viability was determined, then select testis cell withhighest motility rate proceed SSCs purification culture, identify the achieved cells apply themethod that alkaline phosphatase staining and reverse transcription PCR. The main results asfollows:1. After cryopreservation of calf testicular tissue for7d with15mg/ml bovine serumalbumin (BSA), the activities of AKP, ACP, LDH and β-D-Glucosidase of calf testiculartissue are314.04,106.05,1721.39and462.97U/gprot, significantly higher than other groups(P<0.05). After cryopreservation for7d with40mg/ml trehalose, the activities of AKP, ACP,LDH and β-D-Glucosidase of calf testicular tissue are454.88,114.63,1862.79and457.8U/gprot，less than15mg/ml BSA group but higher than the other groups, significantly(P<0.05). After cryopreservation for7d with10%DMSO, the activities of AKP, ACP, LDHand β-D-Glucosidase of calf testicular tissue are314.04,106.05,1721.39and462.97U/gprot, less than40mg/ml trehalose group but higher than the other groups, significantly(P<0.05). After cryopreservation for7d with testicular liquid and ethylene glycol,4kinds oficonic enzyme activities were less than10%DMSO group as a whole, not significantly（p>0.05）. The effect of glycerin group was worst.2. After cryopreservation for7d with15mg/ml BSA, testicular tissue with good spermproduction activity and the activity of SSCs. The expression quantities of CREM, Stra8,HSP70-2, Oct4, SCF, C-KIT gene were all significantly higher than other groups (P <0.05).After cryopreservation for7d with40mg/ml trehalose, testicular tissue was also with good sperm production activity and the activity of SSCs. The expression quantities of the6keygenes were slightly lower than BSA group But significantly higher than other groups (P <0.05). The expression quantities of the6key genes of testicular liquid group were all lower.Compared with glycerol and ethylene glycol, DMSO had good freeze protection of testiculartissue, after cryopreserved by10%DMSO, the expression quantities of the6key genes werebelow trehalose group but higher than ethylene glycol and glycerin group, not significantly (P>0.05). Freeze protection of glycerin was the worst.3. By the two-step enzyme digestion method with1mg/ml Type IV collagenase and0.25%trypsin，testicular single cell suspension can be got. After cryopreservation for7d with15mg/ml BSA, the live rate of testicular tissue cells was78.93%, significantly higher thanother groups (P <0.05). After cryopreservation for7d with40mg/ml trehalose, the live rate oftesticular tissue cells was74.17%, which was below BSA group but significantly higher thanother groups (P <0.05). After cryopreservation for7d with10%DMSO and20%testicularliquid, the live rates of testicular tissue cells respectively were61.59%and68.14%, that werebelow the trehalose group but higher than glycerine and ethylene glycol group, notsignificantly (P>0.05). After cryopreserved by glycerin and ethylene glycol, the live rates ofcells were all lower than60%, freeze protections were poorer. Testicular cells that werecryopreserved by15mg/ml BSA were cultured in vitro. The improved differential adherencemethod can separate SSCs from sertoli cells and got feeder layer of sertoli cells. SSCs stainedfor alkaline phosphatise were positive and dyed brown or brown. With Oct4, SCF and C-KITas molecular markers, the identification of these3marker genes were used to identify SSCs.Combined two kinds of identification result, the cells of cultivation were SSCs.