| Objective: Ovarian tissue cryopreservation plays an important role in female fertility preservation.How to effectively retain the ultrastructure and subsequent function of ovarian tissue during cryopreservation has long been an issue of concern.Vitrification has been considered as an alternative method to slow-freezing of ovarian tissue due to its advantages of avoiding ice crystal formation and less time consumption.However,high concentrations of CPAs in conventional vitrification may exhibit severe osmotic or chemical toxicity to cells.Therefore,it is very important to explore non-toxic and effective CPAs.Late embryogenesis abundant proteins(LEAs)are mainly a family of highly hydrophilic and generally unstructured proteins produced by non-aquatic plants and lower animals;these proteins allow their survival from stressful environments,such as freezing,desiccation and high salinity.Thus LEA protein has the characteristics of CPAs.However,the effect of LEA proteins on ovarian tissue cryopreservation has not been studied.Therefore,the aim of this study was to determine whether LEA proteins supplementation protect mouse ovarian tissue from cryodamage during vitrification,thus optimizing the ovarian tissue cryopreservation protocols for fertility preservation.Methods: The ovarian tissues of 30 female Bal B /C mice at 8-10 weeks weighing about 25 g were randomly divided into three groups: the fresh group,the conventional vitrification group(the control group),the LEA protein vitrification group(the LEA group).After vitrificated for 2 weeks at least,the ovarian tissues were thawing,tissues were fixed for HE staining,used for morphological analysis.Ovarian cell proliferation and apoptosis were tested by immunohistochemistry and the immunofluorescent TUNEL(terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling assay)technique.The ovarian tissues cultured in vitro for 4 days,western blot was used to detect the expression level of apoptosis proteins Bcl-2,BAX and cleaved-Caspase3 to investigate the follicular apoptosis furthermore.The RT-q PCR was performed to test the expression of GAPDH gene for analyzing the difference in m RNA level.Results: 1.After thawing,the proportion of total morphologically normal follicles in LEA protein group was significantly higher than the control group(71.63±2.83% vs.62.72±3.93%)(P < 0.05),and the proportion of morphologically normal follicles at the stage of primordial follicles and primary follicles in LEA protein group was significantly higher than the contral group(76.56±1.96 vs.67.08±3.88),but there was no difference between these two group in the proportion of the morphologically normal secondary and atretic follicles(43.38±11.96% vs.44.15±8.72%)(P > 0.05),indicating that LEA protein could protect the structure of vitrificated ovarian tissues.2.The expression rate of positive Ki67 in LEA protein group was higher than that in control group(65.08±5.53% vs.93.79±7.65%)(P < 0.05),the relative expression rate of positive TUNEL was lower than that of control group(0.83±0.29 vs.1.22±0.23)(P < 0.05),indicating that LEA protein could promote the proliferation and anti-apoptosis of vitrificated ovarian tissues.3.The ratio of Bcl-2/BAX in LEA protein group was higher than that in control group(0.59±0.34 vs.0.44±0.25),and expression of cleaved caspase-3 protein was lower than that in control group(0.78±0.03 vs.1.05±0.04)(P < 0.05),indicating that LEA protein could enhance the anti-apoptosis of vitrificated ovarian tissues.4.At the molecular level,the expression of GAPDH gene in LEA protein group was higher than that in control group(2.33±0.59 vs.1.29±0.20)(P < 0.05),indicating that LEA protein could protect the proliferation of vitrificated ovarian tissues.Conclusions: 1.The LEA protein can better preserve the structure of follicles in mouse ovaries,especially the morphology and structure of thoes follicles near the edge of ovarian tissues.2.The LEA protein could improve r proliferation ability and anti-apoptosis ability of the germ cells in the ovaries. |
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