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

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 6 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.

In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function

“…Our previous studies isolated primary Leydig cells using isosmotic continuous Percoll (Gibco Inc.) gradients generated by centrifugation and subsequent BSA gradients ( Browning et al, 1981 ). It was recently demonstrated that Leydig cells could be isolated from adult rats using magnetic-activated cell sorting ( Ji et al, 2022 ), saving a considerable amount of time in experimental procedures. Evidence indicates that the prolactin receptor is a unique Leydig cell surface marker ( Green et al, 2018 ; Guan et al, 2022 ), which was targeted in our MACS protocol.…”

“…Leydig cells were extracted using magnet-activated cell sorting (MACS), as previously described ( Ji et al, 2022 ). Briefly, testes were collected from rats after euthanasia, washed and placed in ice-cold PBS.…”

Impact of vitamin D supplementation as COVID-19 vaccine adjuvant on sperm parameters and sex hormones in men with idiopathic infertility: Two separate pre–post studies

“…Interestingly, they found that a higher level of prolactin correlated with vitamin D deficiency in infertile men [46]. Prolactin receptors, like VDRs, are expressed in the testis and male reproductive system [47]. An increase in the level of prolactin may disrupt spermatogenesis and hormone production through inhibition of VDRs.…”

Hormones and Signaling Pathways Involved in the Stimulation of Leydig Cell Steroidogenesis

“…The membrane receptor for PRL, PRLR, is present in Leydig cells across various species, including humans, mice, rats, and rams [112][113][114][115]. However, in humans, it appears that Leydig cells do not exhibit detectable PRL binding to interstitial cells [116].…”