彭波半细毛羊冷冻保存睾丸组织支持细胞分离、纯化及鉴定

doi:10.16431/j.cnki.1671-7236.2025.01.022

Abstract

Abstract: 【Objective】 Testicular Sertoli cells (SCs) are located in the epithelium of seminiferous tubules,which provide physical framework and energy material support for the development of male germ cells at different stages of differentiation,and are very important for spermatogenesis.The purpose of this experiment was to study the technical method of separating SCs from frozen testicular tissue in Pengbo Semi-fine wool sheep under the condition that it was inconvenient to obtain fresh testicular tissue samples in sheep. 【Method】 The cryopreserved testicular tissue of Pengbo Semi-fine wool sheep was resuscitated by cryopreservation solution.The morphology of the recovered testicular tissue was detected by hematoxylin-eosin (HE) staining.The testicular SCs were isolated,purified and cultured by combined enzyme digestion and differential adherence method,the growth pattern was observed,and the growth curve was drawn.The specific genes in testicular SCs were identified by RT-PCR and immunofluorescence staining (IF). 【Result】 The spermatic cord and leydig of frozen-thawed testis were well preserved.Spermatogenic epithelial cell suspension meeting the experimental requirements could be obtained after digestion with combined enzymes.After 2-4 h of separation and culture,the cells adhered to the wall,and the shape was spindle or irregular polygon.After 3-4 d of culture,they converged,and the growth rate was slow after 1-2 d of culture.The proliferation rate was accelerated after 3-4 d of culture,and,the proliferation rate was reduced after 7-8 d of culture.RT-PCR detection showed that GNDF,WT1,ABP and SOX9 genes were specifically expressed in testicular SCs of Pengbo Semi-fine wool sheep,and IF results showed that the specific antibodies GATA4 and Vimentin were immune positive. 【Conclusion】 The primary SCs that met the experimental conditions could be isolated and applied to scientific experimental research after the cryopreservation of testicular tissue pretreated with antifreeze was revived.

Key words: Pengbo Semi-fine wool sheep; cryopreservation; testicular Sertoli cells; separation and identification; cell culture

CLC Number:

S814.8

LIU Haixia, WANG Jian, PINCUO Bandan, ZHU Aiwen, DEQING Zhuoga, WANG Jun, GESANG Jiacuo. Isolation,Purification and Identification of Sertoli Cell in Cryopreserved Testis of Pengbo Semi-fine Wool Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 249-257.

References

[1] HENTRICH A,WOLTER M,SZARDENING K C,et al.Reduced numbers of Sertoli,germ,and spermatogonial stem cells in impaired spermatogenesis[J].Modern Pathology,2011,24(10):1380-1389.
[2] 虎巧燕,翟相钦,李一丹,等.bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响[J].畜牧兽医学报,2024,55(3):1040-1051. HU Q Y,ZHAI X Q,LI Y D,et al.Effects of bta-miR-101 on proliferation,apoptosis and secretion of bovine testicular Sertoli cells[J].Acta Veterinaria et Zootechnica Sinica,2024,55(3):1040-1051.(in Chinese)
[3] 李欣,赵中利,罗晓彤,等.松辽黑猪睾丸支持细胞的制备、体外培养及鉴定[J].黑龙江畜牧兽医,2019,5:55-57. LI X,ZHAO Z L,LUO X T,et al.Preparation culture and identification of testicular Scrtoli cells of Songliao Black pig in vitro[J].Heilongjiang Animal Science and Veterinary Medcine,2019,5:55-57.(in Chinese)
[4] 王菊花,刘丹丹,刘琦,等.山羊睾丸支持细胞分离培养及其氧化应激效果研究[J].中国农业大学学报,2019,29(2):99-106. WANG J H,LIU D D,LIU Q,et al.Isolation,culture and oxidative stress of goat Scrtoli cells[J].Journal of China Agricultural University, 2019,29(2):99-106.(in Chinese)
[5] 张磊,阿果约达,谢拉准,等.牦牛IGF-Ⅱ干扰载体的构建及其转染对睾丸支持细胞的影响[J].畜牧兽医学报,2020,51(9):2138-2146. ZHANG L,AGUO Y D,XEI L Z,et al.Construction of yak IGF-Ⅱ interference vector and its transfection effect on Sertoli cells in testis[J].Acta Veterinaria et Zootechnica Sinica, 2020,51(9):2138-2146.(in Chinese)
[6] 金艳梅,德庆卓嘎,张晓庆,等.西藏彭波半细毛羊育成与养殖新技术[M].北京:中国农业科学技术出版社,2021. JIN Y M,DEQING Z G,ZHANG X Q,et al.New Breeding Technology of Pengbo Semi-fine Wool Sheep in Tibet[M]. Beijing:China Agricultural Science and Technology Press,2021.(in Chinese)
[7] 李讨讨,王霞,马友记,等.藏绵羊BOLL的分子特征及其在睾丸中的表达调控与功能分析[J].中国农业科学,2020,53(20):4297-4312. LI T T,WANG X,MA Y J,et al.BOLL molecular characteristics of Tibetan sheep and its expression regulation and function in the testis of analysis[J].Scientia Agricultura Sinica,2020,53(20):4297-4312.(in Chinese)
[8] LIU X Y,XI H M,HAN S Q,et al.Zearalenone induces oxidative stress and autophagy in goat Sertoli cells[J].Ecotoxicology and Environmental Safety,2023,252:114571.
[9] XU J J,SANG M M,CHENG J M,et al.Knockdown of disheveled-associated activator of morphogenesis 2 disrupts cytoskeletal organization and phagocytosis in rat Sertoli cells[J].Molecular and Cellular Endocrinolog,2023,563:111867.
[10] SEGUNDA M N,DIAZ C,TORRES C G,et al.Comparative analysis of the potential for germ cell (GC) differentiation of bovine peripheral blood derived-mesenchymal stem cells (PB-MSC) and spermatogonial stem cells (SSC) in co-culture system with Sertoli cells (SC)[J].Animals,2023,3(2):318.
[11] WANG Y Y,PAN Y Y,WANG M,et al.Transcriptome sequencing reveals differences between leydig cells and Sertoli cells of yak[J]. Frontiers in Veterinary Science, 2022,9:960250.
[12] ZHENG Y,GAO Q,LI T J,et al.Sertoli cell and spermatogonial development in pigs[J].Journal of Animal Science and Biotechnology,2022,13(1):45-48.
[13] XUE H Y,HUO Y N,HU Y,et al.The role of ALOX15B in heat stress-induced apoptosis of porcine Sertoli cells[J].Theriogenology,2022,185:6-15.
[14] AGRIMSON K S,MINKINA A,SADOWSKI D,et al.Lrh1 can help reprogram sexual cell fate and is required for Sertoli cell development and spermatogenesis in the mouse testis[J].PLoS Genetics,2022,18(2):e1010088.
[15] AN X J,LI T T,CHEN N N,et al.miR-1285-3p targets TPI1 to regulate the glycolysis metabolism signaling pathway of Tibetan sheep Sertoli cells[J].PLoS One,2022,17(9):e0270364.
[16] 张孝忠,徐艳,刘艺丹,等.DON通过TNF信号通路诱导驴睾丸支持细胞凋亡的分子机制[J].青岛农业大学学报(自然科学版),2021,38(3):191-198. ZHANG X Z,XU Y,LIU Y D,et al.Molecular mechanism of DON inducing apoptosis of donkey Sertoli cells through TNF signaling pathway[J].Journal of Qingdao Agricultural University (Natural Science Edition),2021,38(3):191-198.(in Chinese)
[17] 陈雪梅,易川平,罗辉,等.牦牛与犏牛睾丸支持细胞分离培养及生物学特性比较分析[J].畜牧兽医学报,2021,52(6):1603-1615. CHEN X M,YI C P,LUO H,et al Isolation,culture and comparison of biological characteristics of Sertoli cells from yak and cattle-yak[J].Acta Veterinaria et Zootechnica Sinica,2021,52(6):1603-1615.(in Chinese)
[18] 宋连杰,崔迎迎,赵一萍,等.蒙古马睾丸支持细胞的体外分离培养与鉴定[J].中国畜牧兽医,2020,47(9):2751-2758. SONG L J,CUI Y Y,ZHAO Y P,et al.Isolation,culture and identification of testis Sertoli cells in Mongolian horses in vitro[J].China Animal Husbandry & Veterinary Medicine,2020,47(9):2751-2758.(in Chinese)
[19] CHEN L Y,WILLIS W D,EDDY E M,et al.Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(7):1829-1834.
[20] 司蕾,张学明,岳占碰,等.睾丸支持细胞对精原干细胞发育的调节[J].细胞生物学杂志,2009,30(4):479-482. SI L,ZHANG X M,YUE Z P,et al.Regulation of Sertoli cell on the development of spermatogonial stem cell[J].Chinese Journal of Cell Biology,2009,30(4):479-482.(in Chinese)
[21] WONG C H,MRUK D D,LUI W Y,et al.Regulation of blood-testis barrier dynamics:An in vivo study[J].Journal of Cell Science,2004,117(5):783-798.
[22] 梁洺源,朱化彬,陈晓丽,等.支持细胞调控精原干细胞增殖、分化和凋亡的研究进展[J].畜牧兽医学报,2016,47(2):225-231. LIANG M Y,ZHU H B,CHEN X L,et al.The study progress of the proliferation,differentiation and apoptosis of spermatogonial stem cells under the regulation of Sertoli cells[J]. Acta Veterinaria et Zootechnica Sinica, 2016,47(2):225-231.(in Chinese)
[23] ODONNELL L,SMITH L B,REBOURCET D.Sertoli cells as key drivers of testis function[J].Seminars in Cell & Development Biology,2021,121:2-9.
[24] 李丹婷,白利鹏,蔡欣,等.支持细胞功能状态调控精子发生的研究进展[J].中华男科学杂志,2018,24(9):829-833. LI D T,BAI L P,CAI X,et al.Role of Sertoli cells in regulating spermatogenesis:An update[J].National Journal of Andrology, 2018,24(9):829-833.(in Chinese)
[25] 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].Chinese Journal of Veterinary Science,2019,39(1):158-162.(in Chinese)
[26] 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)
[27] OU Y,DORES C,RODRIGUEZ J R,et al.Primary cilia in the developing pigs testis[J].Cell & Tissue Research,2014,358(2):597-605.
[28] SEKIDO R,BAR I,NARVAEZV,et al.SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors[J]. Development Biology,2004,274(2):271-279.
[29] XIAO N W,ZES L,YU R,et al.The Wilmstumor gene,Wt1,is critical for mouse spermatogenesis via regulation of Sertoli cell polarity and is associated with non-obstructive azoospermia in humans[J].Public Library of Science Genetics,2013,9(8):e1003645.
[30] 郑鹏,冯瑞,秦雪,等.新生牛睾丸支持细胞的体外成熟与分化[J].中国兽医学报,2022,42(5):981-985. ZHENG P,FENG R,QIN X,et al.In vitro maturation and differentiation of Sertoli cells from neonatal bovine testis[J]. Chinese Journal of Veterinary Science,2022,42(5):981-985.(in Chinese)
[31] 蔡朦朦,杨习曼,张芳,等.大鼠睾丸支持细胞的分离鉴定及低氧对其增殖的影响[J].山西大学学报(自然科学版),2023,46(3):743-750. CAI M M,YANG X M,ZHANG F,et al.Isolation and identification of Sertoli cells in rat testis and the effect of hypoxia on their proliferation[J].Journal of Shanxi University (Natural Science Edition), 2023,46(3):743-750.(in Chinese)
[32] 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 Ayicultural Sciences, 2022,35(8):1948-1953.(in Chinese)
[33] 王帅,贾琦珍,张玲,等.和田羊睾丸支持细胞的体外培养与分离鉴定[J].西南农业学报,2017,30(8):1918-1922. WANG S,JIA Q Z,ZHANG L,et al.Culture and identification of testis Sertoli cells in Hetian sheep in vitro[J].Southwest China Journal of Agricultural Sciences, 2017,30(8):1918-1922.(in Chinese)

Isolation,Purification and Identification of Sertoli Cell in Cryopreserved Testis of Pengbo Semi-fine Wool Sheep

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