Isolation of Leydig cells from adult rat testes by magnetic‐activated cell sorting protocol based on prolactin receptor expression
Minpeng Ji, Dan Chen, Xingyi Zhao, Fu Huang, Xiaoju Guan, Xin Wen, Jiexia Wang, Jingjing Shao, Jiajia Xie, Dan Shan, Shuyan Cao, Congde Chen, Haolin Chen
Abstract
BACKGROUND: The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability. METHODS: Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic-activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non-steroidogenic (25-OH-vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24 h. RESULTS: cells/testis), high purity (about 95%) and viability (> 93%). Purified LCs retained high steroidogenic and non-steroidogenic functions in responding to maximal LH stimulations, with more than 10-fold increases in T production in 3 h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24 h. DISCUSSION AND CONCLUSION: We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro.