基于催乳素受体表达的磁激活细胞分选方案从成年大鼠睾丸中分离莱迪希细胞。

Isolation of Leydig cells from adult rat testes by magnetic-activated cell sorting protocol based on prolactin receptor expression.

机构信息

Zhejiang Provincial Key Laboratory of Anesthesiology, Department of Anesthesiology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.

Department of Gynecology and Obstetrics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.

出版信息

Andrology. 2022 Sep;10(6):1197-1207. doi: 10.1111/andr.13211. Epub 2022 Jul 11.

DOI:10.1111/andr.13211

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

Reanalysis of scRNA-seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 10 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.

摘要

背景

睾丸间质细胞（LCs）的主要功能是产生睾酮（T）。细胞的体外培养是研究雄激素产生及其调节的非常重要的方法。已经开发出各种从睾丸中富集细胞的方法。然而，获得大量高纯度和高活力的细胞仍然具有挑战性。在这里，我们描述了一种从大鼠睾丸中大量分离高纯度和高活力 LCs 的新方法。

方法

用催乳素受体（PRLR）抗体标记成年大鼠睾丸酶消化的细胞。阳性细胞通过磁激活细胞分选（MACS）方案分离。在黄体生成素（LH）存在的情况下，体外检测纯化的 LCs 的类固醇生成（T 产生）和非类固醇生成（25-羟维生素 D 产生和 Insl3 和 Cyp2r1 表达）功能，最多可达 24 小时。

结果

对 scRNA-seq 数据的重新分析表明，Prlr 表达在成年大鼠睾丸 LCs 中高度特异。基于 PRLR 表达的 MACS 程序能够以非常高的产量（每个睾丸约 10 个细胞）、高纯度（约 95%）和活力（>93%）分离 LCs。纯化的 LCs 在对最大 LH 刺激的反应中保持高类固醇生成和非类固醇生成功能，在 3 小时内 T 产生增加 10 倍以上，在 24 小时内 Insl3 和 Cyp2r1 表达增加 42%和 103%。