Alterations of gene profiles in Leydig-cell-regenerating adult rat testis after ethane dimethane sulfonate-treatment

Abstract

Only occupying about 1%–5% of total testicular cells, the adult Leydig cell (ALC) is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS). In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA) profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.

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

“…Perhaps surprisingly, however, there are quite marked differences in the lists of Leydig cell transcripts identified in the two studies. For example of the 67 known genes identified by Zhang and colleagues (Zhang et al ., 2015) as Leydig cell exclusive only 23 are also identified by O'Shaughnessy and colleagues (O'Shaughnessy et al ., 2014). There are technical differences between the studies (e.g.…”

“…This technique has been used, for example to show that expression and activity of the steroidogenic enzymes, with the exception of HSD17B3, is limited to the Leydig cells (Murphy & O'Shaughnessy, 1992; O'Shaughnessy et al ., 2008b). More recently, arrays have been used in two independent studies to identify a comprehensive set of Leydig cell‐specific transcripts in the EDS rat model (O'Shaughnessy et al ., 2014; Zhang et al ., 2015). In both studies transcripts were identified that showed markedly reduced expression following Leydig cell ablation and in each case this included most of the known Leydig cell‐specific transcripts.…”

“…the arrays used are different) but, perhaps more pertinent, the models are also slightly different. Zhang and colleagues (Zhang et al ., 2015) used a ‘standard’ EDS model (normal adult rats treated once with EDS), whereas O'Shaughnessy and colleagues (O'Shaughnessy et al ., 2014) used a germ cell‐free rat model. The rational for using germ cell‐free animals being that the reduction in androgen levels caused by EDS ablation of Leydig cells will cause germ cell apoptosis (Kerr et al ., 1993) and, therefore, significant loss of germ cell‐specific transcripts.…”

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors

“…Three to six weeks after EDS treatment, the adult Leydig cell population regenerates (Kerr et al 1985, Molenaar et al 1986). This model has allowed investigators to identify factors that regulate Leydig cell differentiation (Molenaar et al 1986, Yan et al 2000, Sriraman et al 2003, Salva et al 2004, O’Shaughnessy et al 2008, Zhang et al 2013, O’Shaughnessy et al 2014, Lobo et al 2015, Zhang et al 2015). Additionally, the EDS model has shed light on stem Leydig cells present in peritubular and perivascular locations within the testicular interstitium (Kilcoyne et al 2014, Chen et al 2017).…”

Platelet-derived growth factor BB stimulates differentiation of rat immature Leydig cells

“…The relative mRNA levels of targeted genes were adjusted to house-keeping gene, ribosomal protein S16 (Rps16), as the internal control. Rsp16 in Leydig cells has been used as the internal control in many studies because it showed consistent expression , Zhang et al 2015. Furthermore, in this microarray, the values for Rsp16 levels in 0, 1 and 10 ng/mL PDGF-BB groups were 40 ± 3, 50 ± 6, and 48 ± 4, respectively, and there were no significant changes between groups.…”