| The survival rate of cancer patients has dramatically improved as cancer diagnosis and treatment technology has improved,but radiation and chemotherapy for cancer may cause irreversible damage to the ovaries,resulting in premature ovarian failure,thus,the need for female fertility protection is becoming increasingly important.Ovarian tissue cryopreservation is a significant method for female fertility preservation.This approach has the benefit of retaining fertility while maintaining endocrine function,and it is also the ideal choice for teenage female patients and women whose cancer treatment should not be postponed.Vitrification is the application of a highly concentrated cryoprotectant to form a vitrified state of intracellular fluid by rapid cooling,thus minimizing the formation of ice crystals.It has become an important modality for ovarian tissue cryopreservation compared to traditional procedural slow freezing,while having the advantages of simplicity and efficiency.However,due to technological limitations,there is still a lack of uniform application standards for ovarian tissue vitrification cryopreservation,and studies have discovered that oxidative stress injury is one of the major factors affecting the effectiveness of ovarian tissue vitrification freezing.Several antioxidants,such as resveratrol,melatonin,vitamin E,N-acetylcysteine,and others,have been used in the process of ovarian tissue cryopreservation to increase efficiency and later transplanting success.Earlier,our research indicated that at an ideal dose of 0.1 m M,melatonin(MLT)may substantially boost antioxidant capacity and protect follicle cell morphology in vitrified frozen mice ovarian tissues.The phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT) signaling pathway is an important intracellular signaling pathway that regulates a variety of downstream target proteins,including nuclear factor-erythroid 2-related factor 2(Nrf2),the Bcl2 antagonist of cell death(BAD),and the mammalian target of rapamycin(m TOR),in both physiological and pathological situations.Regulation of cell proliferation,oxidative stress,apoptosis,and autophagy.Although sericin,a natural antioxidant,has been utilized for sperm cryopreservation,there have been few studies on the effect of sericin on ovarian tissue freezing.In this study,sericin was added during vitrification freezing and resuscitation of ovarian tissues from KM mice,aiming to improve the efficiency of ovarian tissue freezing by exploiting the antioxidant and anti-apoptotic effects of sericin,and to further investigate whether the protective effects of sericin are related to the PI3K/AKT signaling pathway.The objective of this study was to improve ovarian tissue cryopreservation techniques and offer fresh concepts for maintaining female fertility.Objective:1.Analyze if sericin protects KM mice’s vitrification-freeze-resuscitated ovarian tissues.2.To investigate whether sericin preserves ovarian tissues that have been resuscitated by vitrification freezing via the PI3K/AKT signaling pathway.Methods:1.Mice ovarian tissue collection and experimental grouping: Eighty 8-week-old SPF female KM mice with bilateral ovaries were randomly separated into two groups: fresh(n=24)and vitrified frozen(n=136),with the latter divided into four subgroups: the vitrified control group,the 0.5%(w/v)sericin group,the 1%(w/v)sericin group,and the 0.1m M MLT group.The fresh group was promptly fixed with 4% paraformaldehyde,and the vitrified frozen group was resuscitated after two weeks of vitrification freezing.2.Follicle cell morphological changes were observed: fresh ovarian tissues and frozen resuscitated ovarian tissues were stained with paraffin section hematoxylin-eosin(HE)to observe follicle cell morphology,count the normal rate of follicle morphology at all stages,and measure follicle diameter at all stages.3.Immunohistochemical labeling was used to identify the expression of the apoptosis-related proteins Bcl-2,Bax,and Caspase 3 in each group.4.The levels of cellular lipid peroxidation products MDA and free radical NO,as well as antioxidant substances GSH,antioxidant enzymes GSH-Px,CAT,T-SOD,and total cellular antioxidant capacity T-AOC,were measured using kits.5.Real-time fluorescence quantitative PCR was used to evaluate the m RNA expression of apoptosis-related genes(Bcl-2,Bax),antioxidant-related genes(Nrf2,HO-1,HSP90),and PI3 K,AKT,and m TOR.6.The expression of apoptosis-related proteins(Bcl-2,Bax),antioxidantrelated proteins(Nrf2,HO-1,HSP90),and the protein expression of PI3 K,AKT,p-AKT,and m TOR were determined by Western blotting.7.The kit assessed the amount of LDH,an indication of cytotoxicity.Results:1.Sericin preserved the normal morphology of follicular cells in vitrified frozen ovarian tissue.Follicular cell morphology was seen following HE staining.In the vitrified frozen control group,follicles were distorted,oocytes were cytoplasmic ally wrinkled,and granulosa cells were loosely organized and separated from the basement membrane.2.Sericin improves the morphological normalization rate of follicles at all stages.The morphological integrity of follicles at all stages was increased by adding sericin and MLT during vitrified freezing and thawing(P< 0.05),in which the effect of 1% sericin was better than that of 0.5% sericin.The morphological normalization rates of primordial,primary and secondary follicles in the 1% sericin group were not statistically different from those of MLT group,and the morphological normalization rate of antral follicles was higher than that of MLT group(P<0.05).3.Sericin reduce the follicle diameters at all stages in vitrified frozen ovarian tissues.The follicle diameters at all stages were significantly larger in the vitrified control group in comparison to the fresh group(P<0.05),and were significantly reduced with the addition of sericin and MLT(P<0.05),with the 1% sericin group having a better effect than the 0.5% sericin group,which was not statistically different from the MLT group.4.In vitrified frozen ovarian tissues,sericin reduces free radical levels.The levels of lipid peroxidation products MDA and free radical NO were significantly higher in the vitrified control group(P<0.05)compared to the fresh group,and the addition of sericin and MLT significantly reduced the levels of MDA and NO(P<0.05),with the NO level in the 1% sericin group being lower than that in the 0.5% sericin group(P<0.05),but not statistically different from the MLT group(P>0.05).5.sericin improved the antioxidant capacity of vitrified freezing ovarian tissues.The levels of GSH,GSH-Px,CAT,T-SOD,and T-AOC in the vitrified control group were significantly lower than those in the fresh group(P<0.05),and the addition of sericin and MLT effectively increased the levels of antioxidant substances and the T-AOC of cells,with 1% sericin being more effective than 0.5% sericin(P<0.05).T-SOD and T-AOC levels in the 1%sericin group were not substantially different from those in the MLT group(P>0.05),although CAT levels were greater than in the 1%sericin group(P<0.05).6.Sericin reduces apoptosis in vitrified frozen ovarian tissues.Immunohistochemical staining revealed that Bcl-2,Bax,and Caspase 3were primarily expressed in oocytes.Bcl-2 expression was considerably lower in the vitrified froze control group compared to the fresh group(P<0.05),Bcl-2 expression was significantly higher in the 1% sericin and MLT groups compared to the vitrified control and 0.5%sericin group.In comparison to the vitrified control group,1% sericin and MLT substantially decreased the expression of Bax and Caspase 3(P<0.05).The expression of the three proteins did not change significantly between the 1%sericin and MLT groups(P>0.05).7.Sericin regulates the expression of apoptosis-related genes and proteins in vitrified frozen ovarian tissues.RT-q PCR results showed that Bcl-2 m RNA expression levels were considerably greater in the sericin and MLT groups than in the vitrified control group(P<0.05),and Bcl-2 m RNA expression levels were significantly higher in the 1% sericin group than in the 0.5% sericin and MLT groups(P<0.05).Bax m RNA expression levels were significantly lower in the sericin at different concentrations and MLT groups compared to the vitrified control group,with Bax m RNA expression levels in the 1% sericin group significantly lower than those in the 0.5%sericin group(P<0.05).And no significant difference between the 1% sericin and MLT groups(P>0.05).WB results revealed that the addition of sericin and MLT boosted the expression of Bcl-2 protein compared to the vitrified control group,with the 1%sericin group significantly higher than the 0.5% sericin group and MLT group(P<0.05).The addition of 1% sericin and MLT considerably decreased the expression of Bax protein(P<0.05)compared to the vitrified control group,although there was no statistically difference between the two groups(P>0.05).8.Sericin increased the expression of antioxidant-related genes and proteins in vitrified frozen ovarian tissues.The RT-q PCR findings indicated that the expression levels of Nrf2,HO-1,HSP90 m RNA were significantly higher in the 1% sericin group and MLT group than in the vitrified control group and 0.5% sericin group(P<0.05),with the 1%sericin group being higher than the MLT group(P<0.05).The results of WB experiments showed that the relative expression levels of Nrf2,HO-1,HSP90 proteins in the 1% sericin and MLT groups were significantly higher than those in the vitrified control group and 0.5% sericin group,with Nrf2 protein expression being higher in the 1% sericin group than in the MLT group(P<0.05),while the expression of HO-1 and HSP90 proteins did not change significantly between the 1% sericin and MLT groups(P> 0.05).9.Sericin upregulated PI3 K,AKT,m TOR m RNA and PI3 K,p-AKT,and m TOR proteins levels in vitrified frozen ovarian tissuesThe 1% sericin group and MLT group had substantially greater levels of PI3 K,AKT,and m TOR m RNA expression than the vitrified control group and0.5% sericin groups(P<0.05),according to RT-q PCR data.PI3 K and AKT m RNA expression levels did not differ significantly between the 1% sericin group and MLT groups(P>0.05),however m TOR m RNA expression levels in the 1% sericin group were considerably greater than those in the MLT group(P<0.05).Western blotting experiments revealed that the levels of PI3 K,p-AKT,and m TOR protein expression in the 1% sericin group and MLT group significantly higher than those in the vitrified control group and 0.5% sericin group(P<0.05).The expression levels of PI3 K and p-AKT were not substantially different between the 1% sericin and MLT group(P>0.05),while the expression levels of m TOR protein were considerably greater in the 1% sericin group than those in MLT group(P<0.05).AKT protein expression levels did not change significantly across groups(P>0.05).10.Sericin had no influence on the LDH concentration of vitrified frozen ovarian tissues.The reagent kit was used to detect LDH levels in each group,and the results showed no significant difference in LDH content of ovarian tissues in each experimental group(P>0.05).Conclusion:1.Sericin can maintain the structural morphology of vitrified freezing mice ovarian tissue,increase the antioxidant capacity of vitrified freezing ovarian tissues,and prevent apoptosis,with the optimum effect attained at a concentration of 1%(w/v).2.Sericin may preserve vitrifying frozen ovarian tissue by stimulating the PI3K/AKT signaling pathway,which increases antioxidant protein production and inhibits apoptosis. |
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