Identification and Function of Putative Stem Leydig Cells in the Adult Rat Testis.

Abstract

Stem cells have the capacity to self-renew and to generate differentiated cells. Such cells are found in adult as well as in embryonic tissue. Previous studies have shown that a single injection of adult rats with ethane dimethanesulfonate (EDS) results in the elimination of the adult Leydig cells. Subsequently, a new generation of Leydig cells is restored to the testes, indicating that the elimination of the adult Leydig cells results in the induction of precursor cells to differentiate into functional Leydig cells. We developed an organ culture system with which to test this hypothesis. Interstitial and seminiferous tubule fractions of rat testes were separated from each other and isolated four days after the rats received an EDS injection. The two fractions were cultured in vitro with LH for four weeks. Immediately after their isolation, the tubules did not produce testosterone in response to LH. However, after two weeks in culture, testosterone was detected in the medium of the tubule fraction. Testosterone production by the tubules increased at least through four weeks. Western blots showed that the steroidogenic proteins, P450scc and StAR, which were undetectable immediately after the tubules were isolated, became detectable. Immunhistochemical staining showed that 3β-HSD positive cells were located around the tubules. In contrast to the tubules, the interstitial fraction failed to produce testosterone. The effects of hormones and/or growth factors, including IGF-1, PDGFAA, LIF, T3, TGF-a and PDGFBB, and of Notch inhibitor, on Leydig cell differentiation were tested on cultured tubules. These factors were selected based on previous studies suggesting that they may play roles in Leydig cell differentiation during normal prepubertal development. Among the factors examined, IGF-1, PDGFAA, LIF and Notch inhibitor were found to stimulate testosterone production, while T3, TGF-a and PDGFBB had no effect on Leydig cell differentiation. hese results suggest that Leydig cell differentiation in the adult testis after EDS treatment is similar to Leydig cell differentiation during normal prepubertal development. We suggest that the precursor cells that give rise to the adult cells might be stem Leydig cells. The results support the peritubular localization of these putative stem cells. The organ culture system used for these studies was particularly useful for identifying the factors (hormones and growth factors) involved in the regulation of the differentiation of the putative stem cells. Supported by grants R37 AG021092, RO1 HD050570 and RO1 AG030598. (platform)