Abstract
BackgroundBesides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.Methodology/Principal FindingsPrimary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-β1, and E2 treatment in turn restored the inhibition of TGF-β1 on GJIC.ConclusionsOur results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.
Highlights
Leydig cells situated in the testicular interstitium are the main sites of testosterone production [1]
Our results indicate, for the first time in adult rat Leydig cells, that Transforming growth factor-b1 (TGF-b1) suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells
Despite the presence of aromatase in germ cells in several species, including the mouse, rat, brown bear, bank vole, rooster and man [7], it is worth noting that Leydig cells in the adult testis have been identified as the major sites of expression of this enzyme [8], which has been shown to be controlled by various factors, such as LH, cyclic cAMP and testosterone, together with other paracrine factors produced by germ cells, such as TNF-a and TGF-b1 [9]
Summary
Leydig cells situated in the testicular interstitium are the main sites of testosterone production [1]. It has become increasingly clear that in the adult testis besides androgens, other steroid hormones commonly synthesized by Leydig cells and the germ cells, estrogens [2], play an important role in the development, growth and differentiation of the male reproductive system and maintenance of spermatogenesis [3]. Aromatase transcription occurs via the alternative use of nine distinct tissuespecific promoters located in the first exon of the Cyp gene [10], and promoter PII is the principal promoter active in rat Leydig cells [11]. This promoter contains several cAMP response element (CRE)-like motifs that mediate the effects of the cAMP transduction pathway that potentiates aromatase gene expression and activity. The objective of the present study is to determine whether TGF-b1 regulates estradiol (E2) synthesis in adult rat Leydig cells and to assess the impact of TGF-b1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells
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