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

Abstract

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

CLC Number:

S811.4

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 and Veterinary Medicine, 2025, 52(5): 2198-2207.

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Isolation,Culture and Identification of Testicular Spermatogenic Cells from Congjiang Xiang Pig

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