Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

Abstract

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3b-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17b-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif ), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place. Reproduction (2008) 135 851-858

Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes

“…Cells on the tubule surfaces first underwent division, and then differentiated and produced T (17). These results suggested that in addition to reported perivascular locations in the interstitial compartment (18), stem cells also were located on the surfaces of the seminiferous tubules (16,19,20).…”

“…----+ ----+ ----+ We now know that stem Leydig cells are located both on the tubule surfaces (9,16,19,20) and associated with blood vessels (18,24). In the event of loss of the adult Leydig cells (14,15), stem cells in both locations are capable of regenerating new Leydig cells (16,(20)(21)(22); Fig.…”

Cell‐specific ablation in the testis: what have we learned?

“…By following the progressive nature of germ cell loss after busulfan treatment, it is clear that the Sertoli cells are most influenced by post‐meiotic germ cells, with significant changes in Sertoli cell gene transcript levels apparent only when these germ cells are lost from the seminiferous epithelium, ~15–30 days post busulfan injection. Whilst there are also gene expression changes at earlier time points, these are not as widespread suggesting spermatogonia and spermatocytes exert less influence over Sertoli cell gene expression (O'Shaughnessy et al ., 2008b). …”

“…The identity of the adult Leydig cell precursor population has been contentious for a number of years with pericytes, peritubular cells, fibroblasts, and endothelial cells all suggested (Jackson et al ., 1986; Kerr et al ., 1987; Teerds et al ., 1990; Davidoff et al ., 2004; O'Shaughnessy et al ., 2008b; Stanley et al ., 2012). Recent data indicates, however, that the major source of adult Leydig cells after EDS treatment are peritubular cells (O'Shaughnessy et al ., 2008b; Stanley et al ., 2012) although a contribution from other sources cannot be ruled out. The role of gonadotrophins in Leydig cell regeneration was established in early EDS studies using hypophysectomised rats or testosterone implants.…”

“…Subsequent studies have shown that similar control mechanisms regulate normal adult Leydig cell development at puberty (Clark et al ., 2000; Gnessi et al ., 2000; Baker & O'Shaughnessy, 2001; Zhang et al ., 2001, 2004; Baker et al ., 2003). Other factors such as DHH, PDGF and anti‐Müllerian hormone (AMH) have been implicated in the development of Leydig cells both during development and re‐generation (Salva et al ., 2004; Josso et al ., 2006; O'Shaughnessy et al ., 2008b) and further studies on EDS regeneration have also suggested that nerve growth factor (NGF) and Sertoli cells are involved in Leydig cell proliferation, differentiation and survival (Yan et al ., 2000; Zhang et al ., 2013). …”