Investigating the Antioxidant Effects of Quercetin in Freezing-Thawing Process of Mouse Spermatogonial Stem Cells

Background and Aim: Current cancer therapy approaches can be harmfulto stem cells, in particular for spermatogonial stem cells (SSCs) which canlead to infertility problems. SSCs preservation techniques can be helpfuland applied to pre-pubertal boys before starting their treatment. In otherhand, cryopreservation protocols are associated with increased reactiveoxygen species (ROS) production which subsequently leads to cellulardamages.Methods: In this study, we treated mouse SSCs by Quercetin beforecryopreservation procedure and then evaluated its antioxidant effects oncell viability, ROS contents and apoptosis in SSCs after thawing. SSCs wereisolated from 3-6-day-old neonate mice and were cultivated in a culturemedium containing 40μM Quercetin for 48 hours and then frozen for2 weeks. Cell viability was evaluated by methylthiazoltetrazolium (MTT)test and ROS content was determined using the dichlorofluoresceindiacetate (DCFDA) assay. Apoptosis was analyzed by detection ofPhosphatidylserine externalization using flow-cytometry and also bygene expression evaluation of Bax and Bcl-2 using Real-time PCR.Results: Our result indicated that pre-treatment of SSCs by Quercetinsignificantly decreased apoptotic cell numbers (P<0.001) and ROScontent (p<0.05) after the freezing-thawing process in comparing tothe control group. MTT assay confirmed that Quercetin decreased themortality of SSCs from damage induced by vitrification. The resultsshowed that the survival rates of the frozen-thawed SSCs in the Quercetinpretreatedgroup were significantly increased compared with thecontrol group (OD: 0.5396 ± 0.006638 vs 0.486 ± 0.01047, P<0.001).Phosphatidylserine (PS) externalization was detected using AnnexinV. A significant difference between the mean percentage of vital SSCs(AnV-/PI-) in the Quercetin-pretreated group and the mean percentage ofthese cells in control group was observed (61.9 ± 1.0 vs 28.6 ± 5.4, P<0.01). The mean percentage of apoptotic SSCs (AnV+/PI-) significantlydecreased in the Quercetin-pretreated group (9.345 ± 0.75 vs 29.65 ±1.65, P<0.001). Also, the mean percentage of necrotic SSCs (AnV+/PI+)in Quercetin-pretreated group decreased in comparison with the controlgroup (19.29 ± 1.11 vs 35.3 ± 4.3, P<0.001). Gene expression level ofBcl2 was increased (P<0.05) and Bax expression was decreased (P<0.01)by Quercetin. Conclusion: Our results suggest that Quercetin can increase antioxidantpotential of the mSSCs for high production of oxidative species duringcryopreservation, and so, our approach can be a promising strategy toimprove the fertility preservation techniques.

کلیدواژه ها:

Spermatogonial stem cell ، Cryopreservation ، Oxidative stress ، Quercetin