Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis

Abstract

Stem Leydig cells (SLCs) play a critical role in the development and maintenance of the adult Leydig cell (ALC) population. SLCs also are present in the adult testis. Their identification, characteristics, and regulation in the adult testis remain uncertain. Using single-cell RNA-seq, we found that the mesenchymal stromal population may be involved in ALC regeneration. Upon ALC elimination, a fraction of stromal cells begins to proliferate while a different fraction begins to differentiate to ALCs. Transcriptomic analysis identified five stromal clusters that can be classified into two major groups representing proliferation and differentiation populations. The proliferating group represents stem cells expressing high levels of CD90, Nes, Lum, Fn and Gap43. The differentiating group represents a progenitor stage that is ready to form ALCs, and specifically expresses Vtn, Rasl11a, Id1 and Egr2. The observation that the actively dividing cells after ALC loss were not those that formed ALCs suggests that stem cell proliferation and differentiation are regulated separately, and that the maintenance of the stromal stem cell pool occurs at the population level. The study also identified specific markers for the major interstitial cell groups and potential paracrine factors involved in the regulation of SLCs. Our data suggest a new theory about SLC identity, proliferation, differentiation, and regulation.

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

“…To reveal the expressions of Prlr mRNAs by adult rodent testicular cells, two datasets of the published single‐cell RNA‐sequence (scRNA‐Seq) of rat and mice testes were reanalysed 31,32 . For rat cells, principal component analysis (PCA) was run on the normalised gene‐barcode matrix by Cell Ranger.…”

Behavior and Functional Roles of CD34+ Mesenchymal Cells in Mammalian Testes

“…Since the co-expressions of Tcf21 and CD34 throughout developmental stages have not been studied, the relationship between Tcf21 + cells and CD34 + cells remains to be clarified. To find and characterize the SLC population, Guan et al [ 73 ] analyzed by sc-RNAseq the mesenchymal cell populations that show proliferative activity (probably including SLCs) after the EDS treatment of adult rats. They identified by transcriptomic analysis five stromal clusters that can be classified into two major groups: proliferation and differentiation populations, the former of which may represent stem cells.…”

“…Photographs showing the localization of CD90 + cells on the surface of freshly isolated seminiferous tubules indicate that the CD90 + cell population density in the interstitium is fairly high [ 68 ], such as that of CD34 + mesenchymal cells. It is, however, unknown how much of the CD90 + cell population overlaps with the CD34 + mesenchymal cell population, because the relationship between the population of mesenchymal CD34 + PDGFRα + cells and that of the other cell types has not been clearly determined [ 73 ]. Sc-RNAseq has shown that a cluster of the mesenchymal cell population expressing Tcf21 isolated from adult mouse testes is heterogeneous, and that three of the five Tcf21 lin clusters express CD34, indicating that the CD34 + cell population is heterogeneous [ 72 ].…”

Basic Leucine Zipper Transcription Factors as Important Regulators of Leydig Cells’ Functions

“…Interestingly, Jun and Fos are highly expressed in PLC, and like Junb , Jund and Fosl2 show a decreased expression upon maturation of PLC to ALC [ 122 ]. In addition, Jun and Fos are highly expressed in rat mesenchymal cells involved in the regeneration of ALC [ 123 ].…”