Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a negative regulator of luteinizing/chorionic gonadotropin hormone-induced steroidogenesis in Leydig cells: central role of steroidogenic acute regulatory protein (StAR)

Abstract

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.

FIGURE 1.

Steroidogenic response to gonadotropin stimulation in WT and GRTH KO mice. A, testosterone response (serum and Leydig cell) to an in vivo single dose of hCG (0.5 μg, sc, 24 h) in WT and GRTH KO mice. B, testosterone response to in vitro hCG (100 ng, 3 h) stimulation in purified Leydig cells prepared from WT and KO mice. Data are presented as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05.

FIGURE 2.

Abnormal mitochondrial morphology in Leydig cells of GRTH KO mice associated with high cholesterol content in the inner mitochondrial membrane. A, EM analysis of Leydig cells in WT and GRTH KO mice. Small arrows, mitochondria with normal morphology; large arrows, swollen mitochondria without normal central cristae. N, nuclear. Scale bars = 1 μm. B, cholesterol content in the inner mitochondrial membrane (Mito. Mem.) of purified Leydig cells from WT and KO mice. Animals were treated with a single dose of hCG (0.5 μg, sc, 24 h). Data are presented as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05.

FIGURE 3.

Expression of mRNAs (basal and hCG treatment) of genes involved in cholesterol synthesis/transfer in Leydig cells from WT and GRTH KO mice. A, mRNA levels were assessed in purified Leydig cells prepared from adult mice (WT and GRTH KO) 24 h after in vivo injection of hCG (+; 5 units, subcutaneous) or vehicle in the control group (−). Gene expression was analyzed by real-time RT-PCR and normalized to β-actin. Results are presented as -fold change relative to the WT control (−; dotted line). Data are presented as the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.05 compared with the WT control; #, p < 0.05 compared with the KO control. ND, not detectable. B, general scheme highlighting genes involved in the classical SREBP2-regulated pathway for cholesterol biosynthesis and transfer to mitochondria, followed by testosterone production.

FIGURE 4.

Effect of hCG on the expression of proteins involved in cholesterol synthesis/transfer in Leydig cells from WT and GRTH KO mice. A, Western blotting was used to analyze protein levels in purified Leydig cells prepared from adult mice (WT and GRTH KO) 24 h after in vivo injection of hCG (+; 5 units, sc) or vehicle in the control group (−). β-Actin was used for normalization. Data are representative of three independent experiments. B, diagram presentation of -fold change relative to the WT control (dotted line). Signals were quantitated and normalized to β-actin. Values are means ± S.E. *, p < 0.05. ND, not detectable.

FIGURE 5.

Colocalization of GRTH and StAR in Leydig cells (A) and StAR mRNA association with GRTH protein (B). A, purified mouse Leydig cells were immunostained with rabbit anti-GRTH (rGRTH) and goat anti-StAR (gStAR) polyclonal antibodies, followed by Alexa Fluor 647 donkey anti-rabbit and Alexa Fluor 568 donkey anti-goat secondary antibodies. IgG were used as the background control. DAPI indicates nuclear staining. The merged images show DAPI-stained StAR and GRTH. B, real-time PCR analysis of the StAR message associated with immunoprecipitated (IP) total testicular GRTH complexes from WT and KO mice (left) or at the testicular cytoplasmic site of WT animals treated with in vivo hCG (+; 5 units, sc, 24 h) or vehicle in the control group (−) (right). Data are expressed as a relative ratio of messages from immunoprecipitated GRTH (GRTH-IP) to the immunoprecipitated IgG negative control group (IgG-IP). Data are representative of three independent experiments. *, p < 0.05.

FIGURE 6.

GRTH as a negative regulator of StAR mRNA stability. A, real-time PCR analysis of StAR and SREBP2 mRNAs in Leydig cells from WT and GRTH KO mice. Cells were incubated with 10 μg/ml actinomycin D (Act D) for 1–10 h. Data are presented as relative to WT mice at 0 h. Values are means ± S.E. of three independent experiments performed in triplicate. B, isolated nuclei of Leydig cells from WT and KO mice were used for nuclear run-on analysis of the nascent StAR RNA transcript. Data were normalized to β-actin as the mean ± S.E.

FIGURE 7.

Postulated model for GRTH negative autocrine molecular mechanism of regulation of androgen synthesis in Leydig cells. Steps 1–12 indicate the sequence of gonadotropic hormone stimulation of androgen synthesis through PKA-regulated StAR action and GRTH negative regulation (see below). Dotted arrows, multiple steps of cholesterol and steroidogenesis; AcCoA, acetyl-CoA; ?, not determined; bent arrow, transcriptional initiation. After gonadotropin binds to the luteinizing hormone receptor (LHR) at the cell surface of Leydig cells, cAMP-activated PKA type II (step 1) facilitates StAR mRNA tethering, protein synthesis (step 2a), and activation (phosphorylation; step 2b) in the vicinity of mitochondria (10). In turn, StAR enhances the transfer of cholesterol to the inner mitochondrial membrane (step 3) to initiate pregnenolone synthesis (step 4) as the rate-limiting step for the consequent androgen production (step 5). Androgen transcriptionally activates GRTH gene expression through binding to the androgen receptor (AR; step 6) (1, 5). GRTH transport its own RNA message from the nucleus (step 7) to the cytosol to be translated at polysome sites (step 8) (6). GRTH further associates with StAR mRNA as a GRTH-StAR mRNA complex (step 9) to enhance StAR message degradation presumably through the small RNA pathway system (step 10). This negative regulatory action of GRTH on the fate of StAR mRNA (step 11) controls androgen homeostasis in the Leydig cell. In the absence of GRTH, increased HMGCR protein levels through a yet-to-be-identified mechanism increase de novo cholesterol synthesis (step 12) as a compensatory reaction to restore the intracellular cholesterol pool. The prolonged StAR mRNA half-life and enhanced protein levels facilitate cholesterol accumulation at the inner mitochondria and the increased androgen production observed in GRTH null mice upon gonadotropin stimulation.