不同月龄波尔山羊睾丸中精原干细胞活性及增殖特征研究

doi:10.12160/j.issn.1672-5190.2024.04.004

摘要/Abstract

摘要： [目的]探究不同月龄波尔山羊睾丸中精原干细胞（spermatogonial stem cells,SSCs）在体外培养过程中的活性及增殖特征。[方法]分别采集1、3、5、58月龄的波尔山羊睾丸,通过组织学方法分别观察不同月龄波尔山羊睾丸的发育情况;分别收集不同月龄波尔山羊睾丸组织,利用三步酶消化法分离成单细胞,通过台盼蓝染色观察睾丸消化细胞总数和死亡细胞数;采用0.2%明胶富集差速贴壁法纯化SSCs并培养10 d,通过流式细胞术鉴定细胞悬液纯化前、纯化后及培养10 d后的Thy-1阳性（Thy-1⁺）细胞数;将纯化的SSCs培养10 d后,采用碱性磷酸酶染色鉴定SSCs克隆,分别计算不同月龄波尔山羊睾丸SSCs形成的克隆数和克隆面积。[结果]睾丸组织学观察发现1月龄波尔山羊曲细精管管腔生殖细胞数量最少,随着波尔山羊月龄的增加,逐渐发育出各级生殖细胞,但58月龄波尔山羊睾丸生精上皮明显退化。不同月龄波尔山羊睾丸组织经过三步酶消化,在纯化前及纯化后获得的睾丸细胞总数以5月龄波尔山羊最高,显著（P<0.05）高于1月龄波尔山羊;不同月龄波尔山羊纯化前及纯化后的睾丸细胞存活率之间均无显著（P>0.05）差异。在纯化前、纯化后的睾丸组织细胞以及培养10 d的纯化SSCs中,Thy-1⁺细胞数占睾丸细胞总数的比例均以1月龄波尔山羊最高,显著（P<0.05）高于其他月龄波尔山羊。培养10 d后,不同月龄波尔山羊睾丸来源的SSCs克隆均表现为碱性磷酸酶阳性,1月龄波尔山羊睾丸SSCs形成的平均克隆数显著（P<0.05）高于其他月龄波尔山羊,不同月龄波尔山羊的SSCs平均克隆面积无显著（P>0.05）差异。[结论]5月龄波尔山羊睾丸中细胞总数最多,1月龄波尔山羊睾丸中细胞总数最少,但SSCs活性和增殖效率最高。

关键词: 波尔山羊, 睾丸, 精原干细胞, 活性, 增殖, 流式细胞术

Abstract: [Objective] The aim of the present study was to characterize the activity and proliferation of spermatogonial stem cells （SSCs） from testes of Boer goats at different months of age during in vitro culture. [Method] Testes of Boer goats at 1, 3, 5 and 58 months of age were sampled, and the development of them was observed by histological method. The testicular tissues from goats at different months of age were separated to single cell by three-step enzyme digestion method. The total number of digestive cells and the number of dead cells was observed by trypan blue staining. The SSCs were purified with differential adhesion method and cultured for 10 days. The number of Thy-1⁺ cells in suspension before purification, after purification and cultured for 10 days was determined by flow cytometry. After cultured for 10 days, the SSCs clones were identified by alkaline phosphatase staining, and the number and area of clones formed by SSCs from testes of goats at different months of age were statistically measured. [Result] Testicular histological observation demonstrated that goats aged 1 month had the least number of germ cells in lumen of contorted seminiferous tubule, germ cells of all levels gradually developed with the increase of months of age, while seminiferous epithelium of goats aged 58 months was evidently degraded. After three-step enzyme digestion, the highest total number of testicular cells obtained before and after purification was observed in goats aged 5 months, which was significantly （P<0.05） higher than that in goats aged 1 month. There was no significant （P>0.05） difference in survival rate of testicular cells before and after purification for goats at different months of age. Among pre- and post-purification testicular cells and purified SSCs cultured for 10 days, the highest proportion of Thy-1⁺ cells in the total number of testicular cells was found in goats aged 1 month, which was significantly （P<0.05） higher than that in goats aged the other months. After 10 days of culture, the SSCs clones from testes of goats at different months of age were positive for alkaline phosphatase. The average number of SSCs formed clones from testes of goats aged 1 month was significantly （P<0.05） higher than that of goats aged the other months, while no significant （P>0.05） difference in average area of SSCs formed clones among goats at different months of age was observed. [Conclusion] Boer goats aged 5 months had the highest total number of cells in testes. Those aged 1 month had the lowest total number of cells in testes, while had the highest activity and proliferation efficiency of SSCs.

Key words: Boer goat, testis, spermatogonial stem cell, activity, proliferation, flow cytometry

中图分类号:

S827.891

胡凯, 杨瑾芸, 杨敏, 张金龙, 范彩云, 朱冰玉, 程建波, 王菊花. 不同月龄波尔山羊睾丸中精原干细胞活性及增殖特征研究[J]. 畜牧与饲料科学, 2024, 45(4): 24-33.

HU Kai, YANG Jinyun, YANG Min, ZHANG Jinlong, FAN Caiyun, ZHU Bingyu, CHENG Jianbo, WANG Juhua. Characterization of Activity and Proliferation of Spermatogonial Stem Cells from Testes of Boer Goats at Different Months of Age[J]. Animal Husbandry and Feed Science, 2024, 45(4): 24-33.

参考文献

[1] 朱立发,孙嘉辰,吴鑫.精原干细胞命运调控的研究进展[J].中国细胞生物学学报,2024,46(4):580-595.
[2] MCLEAN D J,FRIEL P J,JOHNSTON D S,et al.Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice[J].Biology of Reproduction,2003,69(6):2085-2091.
[3] CREEMERS L B,DEN OUDEN K,VAN PELT A M M,et al. Maintenance of adult mouse type A spermatogonia in vitro:Influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture[J].Reproduction,2002,124(6):791-799.
[4] DIRAMI G,RAVINDRANATH N,PURSEL V,et al.Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM[J].Biology of Reproduction,1999,61(1):225-230.
[5] IZADYAR F,SPIERENBERG G T,CREEMERS L B,et al.Isolation and purification of type A spermatogonia from the bovine testis[J].Reproduction,2002,124(1):85-94.
[6] IZADYAR F,DEN OUDEN K,CREEMERS L B,et al.Proliferation and differentiation of bovine type A spermatogonia during long-term culture[J].Biology of Reproduction,2003,68(1):272-281.
[7] 张茂,赵鑫,蔡更元,等.猪精原干细胞体外培养体系的建立[J].浙江农业学报,2021,33(12):2254-2263.
[8] 闫慧敏. 牛精原干细胞的分离培养与标记基因PLZF报告载体的构建[D].呼和浩特:内蒙古大学,2023.
[9] 代贵超,任发,李倩,等.枸杞多糖对山羊精原细胞体外增殖的影响[J].家畜生态学报,2022,43(3):40-46.
[10] BAHADORANI M,HOSSEINI S M,ABEDI P,et al.Short-term in-vitro culture of goat enriched spermato gonial stem cells using different serum concentrations[J].Journal of Assisted Reproduction and Genetics,2012,29(1):39-46.
[11] RODRIGUEZ-SOSA J R,DOBSON H,HAHNEL A. Isolation and transplantation of spermatogonia in sheep[J].Theriogenology,2006,66(9):2091-2103.
[12] HAMRA F K,CHAPMAN K M,WU Z R,et al.Isolating highly pure rat spermatogonial stem cells in culture[J].Methods in Molecular Biology,2008,450:163-179.
[13] ZHU H J,LIU C,LI M Z,et al.Optimization of the conditions of isolation and culture of dairy goat male germline stem cells (mGSC)[J].Animal Reproduction Science,2013,137(1/2):45-52.
[14] CHRISTIN-MAITRE S,YOUNG J.Androgens and spermatogenesis[J].Annales D′Endocrinologie,2022,83(3):155-158.
[15] XIE T,SPRADLING A C.A niche maintaining germ line stem cells in the Drosophila ovary[J].Science,2000,290(5490):328-330.
[16] LUI W Y,LEE W M,CHENG C Y.Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway[J].Biology of Reproduction,2003,68(6):2189-2206.
[17] KANATSU-SHINOHARA M,OGONUKI N,INOUE K,et al.Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells[J].Human Reproduction,2003,18(12):2660-2667.
[18] KANATSU-SHINOHARA M,MIKI H,INOUE K,et al.Germline niche transplantation restores fertility in infertile mice[J].Human Reproduction,2005,20(9):2376-2382.
[19] ABU ELHIJA M,LUNENFELD E,SCHLATT S,et al.Differentiation of murine male germ cells to spermatozoa in a soft agar culture system[J].Asian Journal of Andrology,2012,14(2):285-293.
[20] APONTE P M,VAN BRAGT M P A,DE ROOIJ D G,et al. Spermatogonial stem cells:Characteristics and experimental possibilities[J].APMIS,2005,113(11/12):727-742.
[21] FARD A H H,KOUCHESFEHANI H M,JALALI H. Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats[J].Andrologia,2020,52(9):e13660.
[22] RYU B Y,ORWIG K E,KUBOTA H,et al.Phenotypic and functional characteristics of spermatogonial stem cells in rats[J].Developmental Biology,2004,274(1):158-170.
[23] HERMANN B P,SUKHWANI M,SIMORANGKIR D R,et al.Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques[J].Human Reproduction,2009,24(7):1704-1716.
[24] KIM Y H,KANG H G,KIM B J,et al.Enrichment and in vitro culture of spermatogonial stem cells from pre-pubertal monkey testes[J].Tissue Engineering and Regenerative Medicine,2017,14(5):557-566.
[25] REDING S C,STEPNOSKI A L,CLONINGER E W,et al.THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis[J].Reproduction,2010,139(5):893-903.
[26] ABBASI H,TAHMOORESPUR M,HOSSEINI S M,et al.THY1 as a reliable marker for enrichment of undifferentiated spermatogonia in the goat[J].Theriogenology,2013,80(8):923-932.
[27] WU J,SONG W C,ZHU H J,et al.Enrichment and characterization of Thy1-positive male germline stem cells (mGSCs) from dairy goat (Capra hircus) testis using magnetic microbeads[J].Theriogenology,2013,80(9):1052-1060.