从江香猪睾丸生精细胞的分离培养与鉴定

doi:10.16431/j.cnki.1671-7236.2025.05.024

摘要/Abstract

摘要： 【目的】试验旨在建立一种从江香猪睾丸生精细胞的高效体外分离和培养方法，为香猪繁殖特性研究提供材料，并为其他哺乳动物生精细胞分离培养提供技术参考。【方法】从贵州从江粤黔集团香猪原种繁殖场采集60日龄从江香猪睾丸组织，用0.25%胰酶-EDTA溶液和0.1% Ⅳ型胶原酶对其进行消化，再通过差速贴壁的方法对生精细胞进行纯化；利用间接免疫荧光染色对生精细胞进行生殖细胞特异性蛋白4(DEAD-box helicase 4，DDX4)鉴定后，再用PCR方法对3个生精细胞特异表达的标志基因DDX4、泛素羧基末端水解酶同工酶L1(ubiquitin carboxyl-terminal hydrolase isozyme L1，UCHL-1)和Thy-1细胞表面抗原(Thy-1 cell surface antigen，THY1)进行检测，验证生精细胞。采用CCK-8细胞增殖试剂盒检测生精细胞增殖情况，并用HE染色对从江香猪的睾丸组织进行形态学验证分析。【结果】采用0.25% 胰酶-EDTA溶液+0.1% Ⅳ型胶原酶组合酶消化法+差速贴壁法，分别获得大量香猪生精细胞和支持细胞悬液。免疫荧光染色结果显示，生精细胞特异性表达的DDX4和支持细胞特异性表达的GATA结合蛋白4(GATA binding protein 4，GATA-4)的免疫阳性率均超过90%。进一步利用RT-PCR方法验证体外分离的生精细胞，特异表达DDX4、UCHL-1、THY1基因。在此基础上，建立生精细胞-支持细胞2D培养体系，将圆形或椭圆形的生精细胞附着于支持细胞上或悬浮培养基中(含10% 胎牛血清的DMEM/F12，33 ℃，5% CO₂)，可稳定传至第5代。培养24 h内，生精细胞增殖效率较高，进入对数增长期；但经过72～96 h的培养后，生精细胞增殖效率减慢，培养120 h后，细胞活性开始下降，活力变差，原代生精细胞呈S型曲线增长。香猪睾丸组织HE染色验证显示，生精小管里的细胞构成与本研究所分离的生精细胞类型一致。【结论】本试验采用0.25%胰酶-EDTA溶液+0.1% Ⅳ型胶原酶的组合酶消化法对从江香猪生精细胞进行体外分离，采用差速贴壁法进行了纯化，DDX4免疫阳性率达90%以上，生精细胞特异性标志基因DDX4、UCHL-1、THY1均扩增出预期条带。分离的生精细胞采用2D培养体系，在0～24 h达到对数生长期，72～120 h进入平台期，符合生精细胞的常规生长模式和增殖规律。

关键词: 从江香猪; 睾丸生精细胞; 分离培养; 鉴定

Abstract: 【Objective】 The aim of this experiment was to establish an efficient in vitro isolation and culture method for spermatogenic cells from the testes of Congjiang Xiang pigs,providing materials for the study of reproductive characteristics of Xiang pigs and technical references for the isolation and culture of spermatogenic cells from other mammals.【Method】 In this experiment,60-day-old Congjiang Xiang pigs’ testes were collected from Yueqian Group Breeding Farm of Guizhou Congjiang.The tissue was digested using 0.25% trypsin-EDTA solution and 0.1% type Ⅳ collagenase.The spermatogenic cells were purified by differential adhesion.Indirect immunofluorescence staining was used to identify DEAD-box helicase 4 (DDX4).After that,PCR was performed to validate the spermatogenic cells by detecting the expression of three spermatogenic cell-specific marker genes DDX4,ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1),and Thy-1 cell surface antigen (THY1).The proliferation of spermatogenic cells was assessed using the CCK-8 cell proliferation assay.Additionally,histological validation of the testicular tissues from Congjiang Xiang pig was conducted through HE staining to analyze morphological characteristics.【Result】 Using a combination of 0.25% trypsin-EDTA solution,0.1% type Ⅳ collagenase digestion method,and differential adhesion method,a large number of pig spermatogenic cells and Sertoli cell suspensions were obtained.Immunofluorescence staining revealed that DDX4 was specifically expressed in spermatogenic cells,while GATA-4 was expressed in Sertoli cells,with positivity rates exceeding 90%.The expression of DDX4,UCHL-1,and THY-1 genes was confirmed in isolated spermatogenic cells through RT-PCR.A 2D co-culture system of spermatogenic and Sertoli cells was established,in which spermatogenic cells either adhering to the Sertoli cell monolayer or suspended in culture medium (DMEM/F12 with 10% fetal bovine serum,33 ℃,5% CO₂).In this culture system,the Sertoli cell-spermatogenic cells had been successfully subculture to the fifth generation.Within 24 h of culture,high proliferation efficiency was observed in spermatogenic cells,and these cells entered the logarithmic growth period.However,after 72-96 h,the efficiency of spermatogenic cells slowed down,and until 120 h,a decline in cellular activity was noted in this culture system with low viability,and an S-shaped growth curve observed in primary spermatogenic cells.In addition,HE staining of the testicular tissue from Congjiang Xiang pigs confirmed that the cellular composition of the seminiferous tubules was consistent with the types of spermatogenic cells isolated in this study.【Conclusion】 In this study,spermatogenic cells from Congjiang Xiang pigs were isolated in vitro using a combination enzyme digestion method with 0.25% trypsin-EDTA solution and 0.1% type Ⅳ collagenase.Purification was performed via differential adhesion,achieving a DDX4 immunopositively rate of over 90%.The spermatogenic cell-specific marker genes DDX4,UCHL-1,and THY1 were all amplified the expected bands.The isolated spermatogenic cells were cultured in a 2D system,reaching logarithmic growth phase from 0 to 24 h and entering plateau phase from 72 to 120 h,which conformed to the conventional growth pattern and proliferation rules of spermatogenic cells.

Key words: Congjiang Xiang pig; testicular spermatogenic cells; isolation and cultivation; identification

中图分类号:

S811.4

贾雨轩, 李尧江, 曾广湖, 沈祥玉, 龚婷. 从江香猪睾丸生精细胞的分离培养与鉴定[J]. 中国畜牧兽医, 2025, 52(5): 2198-2207.

JIA Yuxuan, LI Yaojiang, ZENG Guanghu, SHEN Xiangyu, GONG Ting. Isolation,Culture and Identification of Testicular Spermatogenic Cells from Congjiang Xiang Pig[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(5): 2198-2207.

参考文献

[1] 高婷.PA1调控小鼠精母细胞减数分裂的机制研究[D].西安：中国人民解放军空军军医大学，2024.GAO T.The study on the mechanism of PA1 regulation of mouse spermatocyte meiosis[D].Xi’an：Air Force Medical University，2024.(in Chinese)
[2] 张鹏飞.水牛睾丸支持细胞和生精细胞的蛋白质组学研究[D].南宁：广西大学，2022.ZHANG P F.Proteomic study of Sertoli cells and spermatogenic cells in buffalo testes[D].Nanning：Guangxi University,2022.(in Chinese)
[3] HE Z,KOKKINAKI M,JIANG J,et al.Isolation,characterization,and culture of human spermatogonia[J]. Biology of Reproduction,2010,82(2):363-372.
[4] 姜禹,赵欣欣,安星兰，等.冷冻保存新生牛睾丸组织中支持细胞的分离纯化[J].中国兽医学报,2019,39(1):158-162.JIANG Y，ZHAO X X，AN X L，et al.Separation and purification of Sertoli cells from cryopreserved testicular tissue of neonatal bulls[J].Journal of Veterinary Science，2019,39(1):158-162.(in Chinese)
[5] 杨璐,黄忍,张焕容，等.新生山羊睾丸支持细胞分离培养方法研究[J].黑龙江畜牧兽医,2021,3:1-4.YANG L，HUANG R，ZHANG H R，et al.Isolation and culture of testis Sertoli cells in newborn goats[J].Heilongjiang Animal Science and Veterinary Medicine，2021,3:1-4.(in Chinese)
[6] 王香祖,席继锋,贾斌，等.小鼠生精细胞的分离和体外培养[J].畜牧与兽医,2017,49(2):15-17.WANG X Z，XI J F，JIA B，et al.Separation and in vitro culture of mouse spermatogenic cells[J].Animal Husbandry & Veterinary Medicine，2017,49(2):15-17.(in Chinese)
[7] 覃兆鲜.水牛生精上皮细胞体外培养的初步研究[D].南宁：广西大学，2008.QIN Z X.Primary studies on buffalo seminiferous epithelium cell in vitro culture[D].Nanning：Guangxi University,2008.(in Chinese)
[8] 邹海军.成年公兔生精细胞和支持细胞的纯化及体外培养[D].扬州：扬州大学，2007.ZOU H J.Purification and in vitro culture spermatogenic cells and Sertoli cells of adult rabbits[D].Yangzhou：Yangzhou University,2007.(in Chinese)
[9] 高艺,黄泳,冯贤辀，等.从江香猪睾丸支持细胞分离培养与鉴定[J].西南农业学报,2022,35(8):1948-1953.GAO Y，HUANG Y，FENG X Z，et al.Isolation,culture and identification of Sertoli cells from the Congjiang Xiang pig testis[J].Southwest China Journal of Agricultural Sciences,2022,35(8):1948-1953.(in Chinese)
[10] 王维勇,龚婷,王婷婷，等.从江香猪生精上皮周期及睾丸发育的形态学分析[J].中国畜牧兽医,2019,46(7):2012-2020.WANG W Y，GONG T，WANG T T，et al.Morphological analysis of spermatogenic epithelial cycle and testicular development in Congjiang Xiang pigs[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2012-2020.(in Chinese)
[11] 龚淼,张雷,赵昱，等.小鼠生精细胞的体外双室无血清培养[J].河北医科大学学报,2016,37(1):1-4.GONG M，ZHANG L，ZHAO Y，et al.Establishment of culture system of mouse spermatogenic cells in vitro without serum[J].Journal of Hebei Medical University，2016,37(1):1-4.(in Chinese)
[12] 刘林洪.大鼠睾丸组织支持细胞/生殖细胞长期共培养的精子发生分析及猪精原干细胞的体外初步研究[D].呼和浩特：内蒙古大学，2012.LIU L H.Long-term culture and spermatogenesis of SGC from rat testis and the primary study on porcine spermatogonia stem cells in vitro[D].Hohhot：Inner Mongolia University,2012.(in Chinese)
[13] 何莹.猪精原干细胞分离纯化及分子标记[D].杨凌：西北农林科技大学，2012.HE Y.Molecular markep，isolation and purification of spermatogonia stem cells in pigs[D].Yangling：Northwest A&F University,2012.(in Chinese)
[14] SEPÚLVEDA B,ARIAS M E,AGUILA L,et al.Gradient sperm selection for reproductive techniques in cattle:Is isolate a suitable replacement for Percoll?[J].Andrologia,2017,50(3):e12921.
[15] 王涵,徐永健,蒙利洁,等.谷氨酸通过T1R1/T1R3-ERK1/2通路调节香猪睾丸间质细胞自噬相关基因表达[J].南方农业学报,2023,54(6):1829-1836.WANG H,XU Y J,MENG L J,et al.Glutamate regulating autophagy-related gene expression in Leydig cells of Xiang pig through T1R1/T1R3-ERK1/2 pathway[J].Journal of Southern Agriculture,2023,54(6):1829-1836.(in Chinese)
[16] 范兴君,谷红梅,杨勇,等.低剂量纳米氧化镍亚慢性暴露对大鼠睾丸生精细胞线粒体功能的影响[J].环境与健康杂志，2025.[2025-01-16].http://kns.cnki.net/kcms/detail/12.1095.r.20241204.1716.002.html.FAN X J,GU H M,YANG Y,et al.Effects of subchronic exposure to low-dose nano-nickel oxide on mitochondrial function of spermatogenic cells in rats[J].Journal of Environment and Health，2025.[2025-01-16].http://kns.cnki.net/kcms/detail/12.1095.r.20241204.1716.002.html.(in Chinese)
[17] 胡凯,杨瑾芸,杨敏,等.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 K,YANG J Y,YANG M,et al.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.(in Chinese)
[18] 海亚楠.BMP4在人睾丸Sertoli细胞的表达、功能、机制及与无精子症和精原细胞瘤的相关性研究[D].上海：上海交通大学，2015.HAI Y N.The expression,role,and mechanism of BMP4 in human Sertoli cells and its association with azoospermia and seminoma[D].Shanghai：Shanghai Jiao Tong University，2015.(in Chinese)
[19] MAYS-HOOPES L L,BOLEN J,RIGGS A D,et al.Preparation of spermatogonia,spermatocytes,and round spermatids for analysis of gene expression using fluorescence-activated cell sorting[J]. Biology of Reproduction,1995,53(5):1003-1011.
[20] LASSALLE B,ZIYYAT A,TESTART J,et al.Flow cytometric method to isolate round spermatids from mouse testis[J].Human Reproduction (Oxford,England),1999,14(2):388-394.
[21] KUBOTA H,AVARBOCK M R,BRINSTER R L.Spermatogonial stem cells share some,but not all,phenotypic and functional characteristics with other stem cells[J].Proceedings of the National Academy of Sciences of the United States of America, 2003,100(11):6487-6492.
[22] HAN S Y,ZHOU L,UPADHYAYA A,et al.TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse[J]. Biology of Reproduction,2001,64(2):507-517.
[23] BRYANT J M,MEYER-FICCA M L,DANG V M,et al.Separation of spermatogenic cell types using STA-PUT velocity sedimentation[J]. Journal of Visualized Experiments，2013，80：50648.
[24] LIU Y,NIU M,YAO C,et al.Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling[J]. Scientific Reports,2015,5:8084.
[25] SUN J,ZHONG L,ZHU Y,et al.Research on the isolation of mouse Leydig cells using differential digestion with a low concentration of collagenase[J]. The Journal of Reproduction and Development,2011,57(3):433-436.
[26] LI B,ZHAO X,JIN T,et al.Efficient isolation and purification of spermatogonia,spermatocytes,and spermatids from mice,piglets,and adult boars using an optimized STA-PUT method[J].Theriogenology,2024,213:97-108.
[27] ZHOU R,WU J,LIU B,et al.The roles and mechanisms of Leydig cells and myoid cells in regulating spermatogenesis[J]. Cellular and Molecular Life Sciences,2019,76(14):2681-2695.
[28] 王雪,王子铭,李晓宇，等.犊牛睾丸支持细胞分离培养与纯度鉴定[J].黑龙江动物繁殖,2020,28(6):1-6.WANG X，WANG Z M，LI X Y，et al.Isolation,culture and purity identification of testis Sertoli cells in newborn calf[J].Heilongjiang Journal of Animal Reproduction,2020,28(6):1-6.(in Chinese)
[29] FRANÇA L R,HESS R A,DUFOUR J M,et al.The Sertoli cell:One hundred fifty years of beauty and plasticity[J]. Andrology,2016,4(2):189-212.
[30] HESS R A,VOGL A W.Sertoli cell anatomy and cytoskeleton[J].Sertoli Cell Biology,2015.Doi:10.1016/B978-0-12-417047-6.00001-6.
[31] SKORUPSKAITE K,GEORGE J T,ANDERSON R A.The kisspeptin-GnRH pathway in human reproductive health and disease[J]. Human Reproduction Update,2014,20(4):485-500.