Leydig cells in the adult testis synthesize and secrete testosterone. In the Sprague Dawley rat, cells of the adult Leydig cell (ALC) lineage first become apparent by Day 11 postpartum [1
]. These cells, the progenitor Leydig cells (PLCs), are characterized by their spindle shape and their expression of luteinizing hormone receptor (LHR); aldo-keto reductase family 1, member C14 (AKR1C14; previously 3α-hydroxysteroid dehydrogenase); and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1; previously 3β-hydroxysteroid dehydrogenase) [1
]. The PLCs proliferate and secrete steroids, mainly androsterone [4
]. These cells gradually enlarge, become rounded, and give rise to another intermediate, the immature Leydig cell (ILC). ILCs are most commonly seen in the testis during Days 28–56 postpartum [4
]. They have high lipid content, secrete high levels of 5α-reduced androgens due to high 5α-reductase activity, and have reduced proliferative capacity compared to PLCs. ILCs undergo one or two population doublings before ALC function develops at Postnatal Day 56 [3
]. ALCs have reduced lipid content, and due to reduced levels of testosterone-metabolizing enzymes in comparison to ILCs, they produce high levels of testosterone as their major steroid product [5
The PLCs have been shown to arise from undifferentiated stem Leydig cells (SLCs), the latter being seen 1 wk postpartum as spindle-shaped cells in the testicular interstitium that differ from the PLCs in that they are HSD3B1- and LHR-negative [6
]. The SLCs were shown to be platelet-derived growth factor receptor alpha (PDGFRA)-positive and to contain proteins involved in Leydig cell development, including GATA-binding protein 4, c-kit receptor, and leukemia-inhibitory factor receptor. They were capable of expansion for at least 6 mo without differentiating but became HSD3B1-positive and produced testosterone when treated with media containing thyroid hormone, insulin-like growth factor I, and luteinizing hormone. Moreover, when transplanted into ALC-deficient host rat testes, SLCs colonized the interstitium and subsequently expressed HSD3B1, demonstrating their ability to differentiate in vivo as well as in vitro.
In an early study designed to elucidate the genes that may contribute to the development of ALCs, differential gene expression by PLCs, ILCs, and ALCs was analyzed using Clontech Rat Atlas cDNA microarrays, an approach that evaluated 1176 known genes [7
]. The development of ALCs was characterized by decreased expression of cell-cycle regulators, growth factors, growth factor-related receptors, oncogenes and transcription factors, and by increased expression of genes related to differentiated cell function, including steroidogenic enzymes, neurotransmitter receptors, stress response factors, and protein turnover enzymes. At the time of the previous study, SLCs had not yet been isolated or characterized.
In the present study, Affymetrix Rat Genome RAE230 2.0 arrays were used to conduct DNA microarray analysis of RNA isolated from purified SLCs, PLCs, ILCs, ALCs, and bone marrow stem cells (BSCs). This analysis complements and expands on the previous microarray analysis through its focus on the initial differentiation from SLCs to PLCs, the use of an array that monitors a more complete genome, and the comparison between SLCs and BSCs. Our objectives were to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to gene expression in a well-established stem cell and determine if SLCs share molecular characteristics of other stem cells.