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In vitro differentiation of spermatogonial stem cell with testicular cells from Guangxi Bama mini-pig
Huimin Zhao1,2, Junyu Nie1, Xiangxing Zhu1, Yangqing Lu1, Xingwei Liang1, Huiyan Xu1, Xiaogan Yang1, Yunkai Zhang2, Kehuan Lu1, Shengsheng Lu1,*
1State key laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi High Education Key Laboratory for Animal reproduction and Biotechnology, Nanning, 530005, China
2College of Life Science and Technology,Guangxi University, Nanning, 530005, China
Correspondence to: Tel: +86-07713235707; E-mail: shengshenglu@sina.com
Received: August 1, 2017; Revised: April 28, 2018; Accepted: May 9, 2018; Published online: June 21, 2018.
Abstract
In this study, we attempted to establish a culture system for in vitro spermatogenesis from spermatogonial stem cells (SSCs) of Bama Mini-pig. The dissociated testicular cells from one-month pig were co-cultured to mimic in vivo spermatogenesis. The testicular cells were seeded in Minimum Essential Media alpha (α-MEM) supplemented with KnockoutTM Serum Replacement (KSR). Three-dimensional (3D) colonies were formed after 10 days culture. The colonies showed positive staining for SSC-associated markers such as UCHL1, PLZF, THY1, OCT4, Dolichos biflorus agglutinin (DBA) and alkaline phosphatase (AP). Induction of SSCs was performed in α-MEM and KSR supplemented with retinoic acid (RA), bone morphogenetic protein 4 (BMP4), activin A, follicle stimulating hormone (FSH) and testosterone (T). The results showed that STRA8, DMC1, PRM1, and TNP1 were upregulated significantly compared to testis from one-month pig, while expression levels of the genes were significant low compared to pubertal testis. However, the upregulation of ACROSIN was not significant. Replacement of α-MEM and KSR with Iscove’s Modified Dulbecco Medium (IMDM) and fetal bovine serum (FBS), did not upregulate expression of these genes significantly. These data indicated that SSCs of Bama mini-pig could undergo differentiation and develop to post-meiotic stage in α-MEM supplemented KSR and the induction factors with testicular cells
Keywords: Bama Mini-pig; Spermatogonial stem cells; Proliferation; Differentiation


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