Effects Of Resveratrol On Dairy Goat Spermatogonial Stem Cells During Cryopreservation

Spermatogonial stem cells(SSCs)are located in the basement membrane of the seminiferous tubules in male animals,and are the only adult stem cells that pass genes to the next generation.SSCs not only self-renew to maintain the stability of the stem cell pool,but also differentiate to participate in spermatogenesis.The research of SSCs is of great significance for understanding the differentiation mechanism of male germ cells,and has clinical application value in the production of transgenic animals and the treatment of male infertility.Cryopreservation maintains the biological characteristics of cells and guarantees their long-term supply.SSC cryopreservation is an important method to preserve the fertility of high-quality immature male dairy goats.However,the freezing-thawing process increases the production of intracellular reactive oxygen species(ROS),causes oxidative stress in SSCs,leads to apoptosis,and reduces cell viability after cryopreservation.So the oxidative damage that results from SSC cryopreservation is a problem that needs to be solved urgently.Resveratrol is a natural plant polyphenol compound with a wide range of biological and pharmacological activities,including antioxidant,anti-aging,anti-tumor,neuroprotection,and cardiovascular disease therapy.At present,there are few studies on the SSC cryopreservation in domestic animals,and there is no report on the effects of resveratrol on the SSC cryopreservation.Therefore,in this study,firstly the SSC expression was determined by section observation and protein detection in the testes of dairy goats at various ages.Then SSCs were separated and purified from dairy goat testes by two-step enzymatic digestion and differential plating.Next,the SSCs were resuspended in freezing media containing different concentrations of resveratrol,and cryopreserved in liquid nitrogen.After thawing,the SSC vitality was tested,and the optimal resveratrol concentration was screened.In addition,kit detection,flow cytometry,Western blot,and transmission electron microscopy were used to explore frozen-thawed cell antioxidant,anti-apoptosis,and AMPK activation,to initially explore the protective mechanism of resveratrol on dairy goat SSCs during cryopreservation.The main results of this study are as follows:1.The testes of dairy goats at different ages(15 days,1 month,3 months,6 months,and10 months old)were observed by hematoxylin-eosin(HE)staining and immunohistochemical staining of PGP9.5.The observation results showed that as the dairy goats grew up,the diameter of the seminiferous tubules increased,and the types of testicular cells increased.The total amount of spermatogonia increased,but the proportion decreased.Western blot was used to detect the protein expression of PGP9.5 and THY1 in the testes of dairy goats at different ages(15,30,45,60,75,90,and 180 days old),and the results showed that the spermatogonia content in the goat testes before 60 days old was significant higher than that after 75 days old(P < 0.05),and suitable for extracting SSCs.2.Testicular cells were separated by mechanical separation and two-step enzymatic digestion,and then purified by differential plating to collect dairy goat SSCs.Immunofluorescence staining was used to identify the cells.The results showed that the purified cells highly expressed markers GFRα1,PLZF,PGP9.5,and THY1,which indicated that SSCs with high purity were obtained by this method.3.Different concentrations of resveratrol(0,0.1,1,10,and 100 μmol/L)were added to the freezing media,and the dairy goat SSCs were cryopreserved by the slow freezing method.After thawing at 37 ℃,the cell viability and antioxidant capacity were determined.CCK-8 and trypan blue staining were used to detect the SSC viability.The results showed that when the concentration of resveratrol was 10 μmol/L,the cell viability was significantly higher than that of the control group and other treatment groups(P < 0.05).Compared with the control group,the addition of resveratrol in the freezing medium significantly increased the frozen-thawed SSC total antioxidant capacity(T-AOC),mitochondrial membrane potential,and antioxidant enzyme activities(SOD,CAT,and GSH-Px)(P < 0.05),while significantly reducing intracellular ROS levels and malondialdehyde(MDA)content(P < 0.05).Among them,the antioxidant effect of SSCs in the 10 μmol/L resveratrol treatment group was significantly better than other treatment groups(P < 0.05),while the ROS levels and MDA content were not significantly different from the 100 μmol/L group(P > 0.05).4.The control group and the 10 μmol/L resveratrol treatment group were selected to further explore the mechanism of resveratrol protecting SSCs during the freezing-thawing process.The results of Annexin V-FITC flow cytometry showed that the proportion of apoptotic SSCs in the resveratrol group was significantly lower than that in the control group(P < 0.05).Resveratrol significantly inhibited the expression of pro-apoptotic protein Bax and promoted the expression of anti-apoptotic protein Bcl-2(P < 0.01),and inhibited the release of Cytochrome C from mitochondria into cytoplasm,thereby reducing the expression of caspase 3 and caspase 9(P < 0.05).In addition,resveratrol activated cellular AMPK phosphorylation,and this effect was partially offset by the AMPK inhibitor Compound C.The cell ultrastructure was observed using transmission electron microscopy,and the results showed that the addition of resveratrol in the freezing medium significantly reduced the proportion of mitochondrial vacuolation,membrane blebbing,and nuclear change in frozen-thawed SSCs(P < 0.05).In conclusion,the addition of resveratrol to the freezing medium effectively improved the quality of frozen-thawed SSCs,and the optimal concentration was 10 μmol/L.Resveratrol protects dairy goat SSCs against damage during the freezing-thawing process by improving the antioxidant capacity,inhibiting mitochondrial apoptosis pathway,and activating AMPK phosphorylation,a key factor in energy metabolism regulation.