Effect of sucrose on cryopreservation of pig spermatogonial stem cells

摘要

cqvip:Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells(p SSCs) has not been tested. The aim of this work was to study the effect of sucrose during p SSC cryopreservation and to find the most effective concentration in freezing medium. p SSCs were cryopreserved with freezing media containing different concentrations of sucrose(70, 140, 210, and 280 mmol L^(–1)) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue(TB) staining, SYBR-14/propidium iodide(PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^(–1) sucrose. Moreover, the 210 mmol L^(–1) sucrose group yielded the highest survival rate among all the groups(P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR(q RT-PCR) indicated that the m RNA levels of three apoptosis-promoting genes(BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing(P<0.05). Moreover, the mR NA level of one anti-apoptotic gene(XIAP) was significantly lower in thawed cells than in cells before freezing(P<0.05). When comparing the m RNA expression of apoptosis-related genes in thawed cells, the m RNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups(P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^(–1) sucrose group. Both q RT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted p SSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^(–1), which possibly assisted p SSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of p SSCs.