Identification and Cryopreservation of Type a Spermatogonia

发布时间：2021-03-23 05:19

　　1.中国杂交奶水牛A型精原细胞的分离、鉴定和纯化从睾丸细胞中分离A型精原细胞部分是本实验的必要组成部分,因此本实验有相当一部分是用来研究水牛犊牛（3-6月龄）A型精原细胞的分离、鉴定和纯化。组织学和DBA免疫组化研究结果表明5月龄时A型精原细胞数量达到最大值。一种改进的睾丸组织分离细胞方法是剪碎之后研磨,然后再用胶原酶、透明质酸酶和脱氧核糖核酸酶消化两次。利用层粘连蛋白、多聚赖氨酸和明胶微分度可以显著影响A型精原细胞的纯度（P<0.05）。在这些细胞外基质分子（ECMs）中,层粘连蛋白和明胶效果很好,纯度分别达到39.38±1.21%and32.15±1.60%。另外,利用层粘连蛋白和明胶混合后细胞分离液离心效果最好,可以达到>90%的纯度。另外,细胞的活力并没有因为组分不同而受到影响（P>0.05）。2.A型精原细胞的低温储藏A型精原细胞可以持续产生雄性配子。低温储藏是保存精原细胞的重要方法,但是改善低温保存过程以免影响活力和减少的活性氧（ROS）的损害是一个迫切解决的问题。本研究的目的是利用牛磺酸（2-aminoethanesulfonic acid）作为抗氧化剂... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：75 页

【学位级别】：博士

【文章目录】：
ABSTRACT
摘要
1. Review of Literature
    1.1 Preamble
    1.2 Testis
    1.3 Spermatogenesis
    1.4 Stages of Spermatogenesis
        1.4.1 Mitosis
        1.4.2 Meiosis
        1.4.3 Spermiogenesis
    1.5 In vivo spermatogenesis
    1.6 In vitro spermatogenesis
    1.7 Comparison of spermatogenesis in rodents and bovine
    1.8 Strategies for isolation of spermatogonial stem cells
        1.8.1 Mechanical Isolation
        1.8.2 Enzymatic Isolation
    1.9 Purification/Enrichment of spermatogonial stem cells
        1.9.1 Laminin Selection
        1.9.2 Flow Cytometry
        1.9.3 Percoll density gradient separation
        1.9.4 Morphology based enrichment
    1.10 Identification of Spermatogonia
        1.10.1 Cluster Designation 34
        1.10.2 Podocalyxin
        1.10.3 Caudal type homebox 2
        1.10.4 Zinc finger protein 42
2. Isolation,identification and enrichment of type A spermatogonia from the testis of Chinese crossbred buffaloes（Swamp×River）
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Testes collection and tissue preparation
        2.2.2 Testicular Histology
        2.2.3 Cell viability and yield
        2.2.4 Immunohistochemistry and Immunocytochemistry
        2.2.5 Experimental Design
        2.2.6 Statistical Analysis
    2.3 Results
        2.3.1 Testicular histology
        2.3.2 Identification of spermatogonia in the testis and after isolation
        2.3.3 Application of erythrocyte lysis buffer solution
        2.3.4 Selection of isolation strategy
        2.3.5 Application of ECM molecules for the enrichment of type A spermatogonia
    2.4 Discussion
3. Cryopreservation of Type A spermatogonia in Chinese crossbred buffaloes （Swamp ×River）
    3.1 Introduction
    3.2 Material and methods
        3.2.1 Testes collection and tissue preparation
        3.2.2 Histology and immunohistochemistry
        3.2.3 Isolation of Type A spermatogonia
        3.2.4 Cell viability
        3.2.5 Cryopreservation and thawing
        3.2.6 Analysis of oxidative stress parameters
        3.2.7 Statistical Analysis
    3.3 Results
        3.3.1 Testicular histology and immunohistochemistry
        3.3.2 Effect of taurine on viability after thawing
        3.3.3 Analysis of oxidative stress parameters
    3.4 Discussion
4. Expression analysis of spermatogonia specific markers in Chinese crossbred buffalo（Swamp x River）
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Testes collection and tissue preparation
        4.2.2 Isolation of spermatogonial stem cells（SSCs）
        4.2.3 Immunohistochemistry of testis tissues
        4.2.4 RNA extraction and cDNA synthesis
        4.2.5 Polymerase Chain Reaction,cloning and sequencing of CD34,PODXL,CDX2 and REX1
        4.2.6 Bioinformatic Analyses
        4.2.7 Quantitative real-time PCR
        4.2.8 Western-Blot Analysis
        4.2.9 Statistical analysis
    4.3 Results
        4.3.1 CD34,PODXL,REX1 and CDX2 mRNA is expressed in testis of prepubertal and adult buffalo bulls
        4.3.2 Spermatogonia express CD34,PODXL,REX1 and CDX2
        4.3.3 Quantitative real-time PCR
        4.3.4 Western blotting
    4.4 Discussion
5. Conclusions and Future Directions
References
Curriculum Vitea
ACKNOWLEDGEMENTS



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