| Spermatogonial stem cells?SSCs?are in the basement membrane of the seminiferous tubules in the testis of male animals.SSCs can self-renew to maintain a constant number of themselves,and can also differentiate into a class of primitive spermatogonia that produce sperm.It only accounts for a very small proportion in the testicles of male animals,but it plays a vital role in maintaining male fertility.At present,the preservation of male animal fertility is usually achieved by cryopreservation of sperm,but cryopreservation of SSCs is an effective alternative method for males with no sperm,oligozoospermia and pre-sexual maturity.Currently,the liquid nitrogen cryopreservation method?-196??is generally used to cryopreserve cells,but the cells will be stimulated by low temperature,oxidative stress and other external environments during the cryopreservation process,which will cause the cell viability to decrease or even cause apoptosis.Cryopreservation of SSCs can provide an effective way for the preservation of germplasm resources of excellent livestock and poultry.However,the current technology of cryopreservation of SSCs needs to be improved,and there are relatively few studies on how to improve the effect of SSCs cryopreservation.Cryopreservation of SSCs is mostly concentrated on animals such as mice and rats,and there are few reports on the cryopreservation of SSCs in goats and other large domestic animals,which needs further research and improvement.Melatonin has the effects of reducing oxidative stress of cells,reducing apoptosis,resisting inflammation,regulating circadian rhythm and autophagy.It is often used in cell culture in vitro and sperm cryopreservation,which can regulate cell proliferation and exert antioxidant effects.However,the effect of melatonin on the cryopreservation of SSCs in dairy goats is unclear.Therefore,this study screened the formulation of the basic cryopreservation solution,and then added melatonin to the optimal base cryopreservation solution to explore its effects on SSCsl cryopreservation.Aimed to improve the effects of SSCs cryopreservation by improving the formulation of cell cryopreservation solution.In this study,the testes of Guanzhong dairy goats aged 2 to 2.5months were used.The SSCs of dairy goats were obtained by mechanical separation,two-step enzyme digestion and differential adhesion method,and then the obtained SSCs were cryopreserved using liquid nitrogen.Using flow cytometry,Western Blot,transmission electron microscopy to explore the situation of cell oxidation,apoptosis and autophagy,initially revealed the mechanism of melatonin in the cryopreservation of SSCs in dairy goats.This experiment mainly obtained the following research results:1. This experiment used mechanical separation,two-step enzyme digestion combined with differential adherence method to isolate SSCs of dairy goats,and then identified the separated SSCs by immunofluorescence staining.A large number of cells expressed SSCs molecular markers THY1 and PLZF,the results showed that higher purity SSCs can be obtained.2. The experiment compared the cryopreservation effects of three basic cryopreservation solutions on SSCs of dairy goats.It showed that the cell viability of cryopreservation solution II after thawing was significantly higher than that of cryopreservation solution III?P<0.05?.In addition,the cell viability of the cryopreservation fluid II after thawing was higher than that of the base cryopreservation fluid I,but the difference was not significant?P>0.05?.Therefore,the best basic cryopreservation solution is the cryopreservation solution II containing DMSO and FBS,and the cell viability after freezing-thawing reached 55.77%.3. The activity rate of SSCs was detected by trypan blue staining.The results showed that the addition of melatonin at a concentration of 10-6 mol/L in the base cryopreservation fluid II reached 68.99%,which was significantly higher than that of other treatment groups?P<0.05?.Compared with the control group,the melatonin-treated groups can increase the antioxidant level of the cells to different degrees.Compared with other treatment groups,the addition of 10-6 mol/L melatonin to the basic cryopreservation fluid II can significantly reduce the intracellular ROS and MDA content?P<0.05?,and significantly increase the T-AOC and mitochondrial membrane potential?P<0.05?,indicating that the addition of melatonin to the cryopreservation solution can significantly improve the antioxidant capacity of SSCs,and the optimal concentration of melatonin is 10-6 mol/L.4. In order to further explore the molecular mechanism of adding melatonin to improve the activity rate of SSCs after thawing,this experiment was conducted through the following three ways.Firstly,the expression level of antioxidant enzymes was detected by Western Blot.The results showed that the protein expression of catalase?CAT?in the group with 10-6mol/L melatonin was significantly higher than that in the control group?P<0.01?,and the protein expression of dismutase?SOD1,SOD2?and glutathione peroxidase?Gpx5?was significantly higher than that of the control group?P<0.05?.Secondly,this experiment analyzed the signal cascade pathway of apoptosis,and the results showed that the addition of melatonin can significantly reduce the mitochondrial swelling and vacuolation,and inhibit the release of cytochrome C?Cyt c?from mitochondria into the cytoplasm.Furthermore,it significantly inhibited the expression levels of pro-apoptotic proteins Bax,Caspase-3 and Caspase-9?P<0.05?and increased the expression levels of anti-apoptotic proteins?Bcl-2,Bcl-XL??P<0.05?.In addition,the analysis of cell autophagy after thawing by transmission electron microscopy combined with Western Blot technology showed that the addition of melatonin can significantly inhibit the production of cell autophagy bubbles and the expression of autophagy-related proteins?LC3-?,LC3-??,BECN1,ATG7?.These test results show that the addition of melatonin to the cell cryopreservation fluid can improve the viability of cells after thawing by maintaining the antioxidant balance,inhibiting apoptosis and inhibiting the production of autophagic vesicles.The above results indicate that cryopreservation fluid II is the optimal basic cryopreservation fluid.Adding melatonin to cryopreservation fluid II can effectively improve the survival rate of SSCs after freezing-thawing.Among them,adding 10-6 mol/L melatonin had the highest cell viability,reaching 68.99%.Melatonin can increase the antioxidant capacity of SSCs by reducing the production of ROS and increasing the expression of antioxidant enzyme proteins;reducing the release of Cyt c from mitochondria to the cytoplasm,reducing the expression of pro-apoptotic proteins,and increasing the expression of anti-apoptotic proteins to reduce SSCs Apoptosis;in addition,melatonin can reduce the production of autophagic vesicles and reduce the expression of autophagy protein.Melatonin improves the cryopreservation effect of SSCs in dairy goats by improving cell antioxidant,anti-apoptosis and reducing cell autophagy. |
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