title: Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue.
abstract: Propagation of bovine spermatogonial stem cells (SSCs) from the cryopreserved testicular tissue is essential for the application of SSCs-related techniques. To explore the appropriate conditions for in vitro culture of bovine spermatogonia (containing putative SSCs), Sertoli cell monolayer and serum concentration were set as two main control factors. Morphological examination showed that the intactness and structure of adult bovine testicular tissue were well maintained after cryopreservation. The enriched bovine spermatogonia were large round CD9 and promyelocytic leukemia zinc finger protein (PLZF) positive cells, with high nucleocytoplasmic ratios and multiple types including single, paired-, aligned-cells or grape cluster-like colonies in vitro. In Sertoli cell co-culture system, bovine spermatogonia attached quickly and proliferated obviously faster than those in the system without Sertoli cells. Serum-free media was no good for the attachment and proliferation of bovine spermatogonia. When 2.5%, 5% and 10% fetal bovine serum (FBS) was employed in the media, spermatogonia attached easily and divided quickly to form paired-, chained-cells or grape cluster-like colonies with comparable percentages in all groups. However, the contaminated somatic cells proliferated robustly in groups containing 5% and 10% FBS. Together, bovine spermatognia isolated from cryopreserved adult testis tissue express CD9 and PLZF, can survive and proliferate conspicuously in Sertoli cell co-culture system, and low serum provides an optimal condition for the survival and proliferation of bovine spermatogonia because of avoiding the rapid growth of testis somatic cells.
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Huan
authors.initials: H
authors.lastname: Cai
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;; Department of Laboratory Medicines, the 90th Hospital of Jinan, Jinan 250031, Shandong, China.
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Xing-Lan
authors.initials: XL
authors.lastname: An
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Xin-Xin
authors.initials: XX
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Zheng-Zhu
authors.initials: ZZ
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;
authors.forename: Zhan-Peng
authors.initials: ZP
authors.lastname: Yue
authors.affiliation: State & Local Joint Engineering Laboratory for Animal Models of Human Diseases, the First Hospital, Jilin University, Changchun 130062, Jilin, China.
authors.forename: Zi-Yi
authors.initials: ZY
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin, China;. Electronic address: zhangxuem@jlu.edu.cn.
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Zhang
mesh_terms: D000367:Age Factors; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods* / Q000662:veterinary; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods / Q000662:veterinary; D002478:Cells, Cultured; D015925:Cryopreservation; D059247:Fertility Preservation / Q000662:veterinary; D008297:Male; D012741:Sexual Maturation; D013093:Spermatogonia / Q000166:cytology* / Q000502:physiology; D013737:Testis
keywords: Bovine; Cryopreservation; Culture; Enrichment; Spermatogonia; Testis