Purification and Transcriptomic Analysis of Mouse Fetal Leydig Cells Reveals Candidate Genes for Specification of Gonadal Steroidogenic Cells1

Abstract

Male sex determination hinges on the development of testes in the embryo, beginning with the differentiation of Sertoli cells under the influence of the Y-linked gene SRY. Sertoli cells then orchestrate fetal testis formation including the specification of fetal Leydig cells (FLCs) that produce steroid hormones to direct virilization of the XY embryo. As the majority of XY disorders of sex development (DSDs) remain unexplained at the molecular genetic level, we reasoned that genes involved in FLC development might represent an unappreciated source of candidate XY DSD genes. To identify these genes, and to gain a more detailed understanding of the regulatory networks underpinning the specification and differentiation of the FLC population, we developed methods for isolating fetal Sertoli, Leydig, and interstitial cell-enriched subpopulations using an Sf1-eGFP transgenic mouse line. RNA sequencing followed by rigorous bioinformatic filtering identified 84 genes upregulated in FLCs, 704 genes upregulated in nonsteroidogenic interstitial cells, and 1217 genes upregulated in the Sertoli cells at 12.5 days postcoitum. The analysis revealed a trend for expression of components of neuroactive ligand interactions in FLCs and Sertoli cells and identified factors potentially involved in signaling between the Sertoli cells, FLCs, and interstitial cells. We identified 61 genes that were not known previously to be involved in specification or differentiation of FLCs. This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development. In addition, it provides a basis for targeted studies designed to identify causes of idiopathic XY DSD.

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

“…The existence of such non-steroidogenic interstitial progenitor cells was also observed in cell-sorting studies in which interstitial cells with high Sf1 expression represent fetal Leydig cells, whereas low Sf1-expressing interstitial cells are potential non-steroidogenic cell progenitors (Inoue et al, 2015;McClelland et al, 2015). These nonsteroidogenic interstitial progenitor cells appear to be enriched for Ptch1, suggesting that these cells respond to Hedgehog signaling (Inoue et al, 2015;McClelland et al, 2015). Consistent with these observations, genetic manipulation of Hedgehog signaling in the fetal testis significantly influences the adult Leydig cell population (Barsoum et al, 2013), supporting the presence of a Hedgehogresponsive GLI1 + progenitor population for adult Leydig cells.…”

Alterations in Fetal Leydig Cell Gene Expression during Fetal and Adult Development

“…Several studies using microarray or deep-sequencing to investigate gene expression of cells in fetal testes have been published [Jameson et al, 2012;McDowell et al, 2012;McClelland et al, 2015]. In these studies, Sertoli cells, germ cells, FLCs, and interstitial cells from transgenic mice carrying Sox9-EGFP , Mafb-EGFP , Oct4-mCherry , or SF1-BAC-EGFP as the transgenes were fractionated by FACS.…”

Leveraging Online Resources to Prioritize Candidate Genes for Functional Analyses: Using the Fetal Testis as a Test Case

“…While scientists have gathered a number of missing puzzle pieces, unexplained cases of disorders of sexual development (DSD) and infertility clearly indicate that many key pathways have not yet been placed in the picture [Ono, and Harley, 2013]. In order to fill in the missing pieces, the field has used mouse models to generate numerous transcriptomic studies that reveal variations in gene expression between sexes, cell types, developmental stages and different genotypes [Jameson et al, 2012; McClelland et al, 2015; Beverdam, and Koopman, 2006; Nef et al, 2005; Bouma et al, 2010; Bouma et al, 2007; Albrecht, and Eicher, 2001; Rolland et al, 2011; Coveney et al, 2008; Munger et al, 2013]. Each of these studies had the goal to identify new genes that control key processes in sex determination of the gonads.…”

“…Subsequently, to move beyond male and female comparisons and to gain more cell type specific information, scientists generated transcriptomic data from enriched cell populations isolated from cell type-specific reporter mice. This strategy was used to isolate somatic cells (Sertoli, Leydig, and others) and germ cells to identify key differences between lineages in addition to sexes [Nef et al, 2005; Beverdam, and Koopman, 2006; McClelland et al, 2015; Rolland et al, 2011; Inoue et al, 2015; Bouma et al, 2007]. This approach was used in the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) Consortium-backed microarray of four key cell types (supporting, germ, interstitial, and endothelial cells), aiming at differences in cell population gene expression over a developmental time-course [Jameson et al, 2012].…”

“…Efforts by other groups continued to fill in the puzzle with the advent of RNA-seq and whole exome sequencing. RNA-seq allowed gene expression to be assayed at a greater sensitivity in a more unbiased way than microarrays on whole gonads and sorted cells [McClelland et al, 2015; Inoue et al, 2015; Lindeman et al, 2015]. In tandem, the decreasing cost for next-gen sequencing has made whole exome sequencing of DSD patients economically feasible [for review see Ostrer, 2014; Ono, and Harley, 2013].…”