Germ Cell Nuclear Factor (GCNF/RTR) Regulates Transcription of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in Testicular Germ Cells--The Androgen Connection

Abstract

Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.

Figure 1.

A, Immunohistochemical analysis of the GCNF expression in different stages (I–XII) of spermatogenic cycle of mice testis. Shown below for each stage are IgG negative controls. B, upper panel, Quantitative analysis of immunosignals (DAB staining) in spermatocytes and spermatids at different stages of the spermatogenic cycle. Immunosignal intensities in different cell types were quantified as optical density using ImageJ software. Results are mean ± SE of 3 independent experiments performed. Lower panel, Different stages of spermatogenesis in seminiferous tubules of mice testis (50). The vertically boxed areas present the type of GCs at the different stages corresponding to the expression of GCNF protein shown above. The red lines indicate the GRTH protein expression profile revealed by immunostaining of spermatocytes and spermatids from WT mice testis and nonimmune IgG controls (in yellow) from our previous study (10). P, pachytene spermatocyte; D, diplotene; RS, round spermatid; ES, elongated spermatid; SC, Sertoli cell; SM, spermatocytes in the metaphase of meiosis. Scale bar, 50 μm.

Figure 2.

A, EMSA showing binding of GCNF protein to the putative binding site of the GRTH promoter. Labeled WT GCNF probe was incubated with nuclear extracts prepared from mice testis (lane 2) or in absence of nuclear extracts (lane 1) in a DNA-binding reaction mixture for 30 minutes. Similarly labeled mutant probe was incubated with nuclear extract (lane 4). The DNA-protein complex in lane 2 is indicated by arrow. For competition experiments, a 200-fold molar excess of unlabeled GCNF WT probe (lane 3) was added in DNA-binding reactions. For supershift assay, rabbit polyclonal anti-GCNF was added into the binding reaction (lane 5). B, ChIP assay showing recruitment of GCNF to the GRTH promoter in spermatocytes (SPs) and round spermatids (RSs) of mice testis. The sequence of GRTH 5′-upstream region (-5264/-5273 bp) containing GCNF putative binding site was analyzed by qPCR after immunoprecipitation (IP) with the GCNF antibody. IgG was the negative control.

Figure 3.

Effect of flutamide (Flu) (AR antagonist) treatment on GCNF and GRTH expression in GCs of mice testis. A, Western blotting showing the GCNF and GRTH protein expression bands in spermatocytes and spermatids isolated from WT and Flu-treated mice testis. Actin was used as a loading control. Densitometry analysis of protein bands from 3 independent experiments in each group were quantified and normalized to β-actin. Mean ± SE of 3 independent experiments done in triplicates. *, significant decrease in GCNF (P < .001) and GRTH (P < .05) protein expression in Flu samples compared with WT samples in round spermatids. B, ChIP assay showing the effect of Flu in the recruitment of GCNF to the GRTH promoter in SP and RS of mice. Mean ± SE of 3 independent experiments done in triplicates. *, significant decrease (P < .001) in the recruitment of GCNF to the GRTH promoter in flutamide mice compared with WT mice.

Figure 4.

Effect of flutamide (AR antagonist) on GCNF immunoreactivity in spermatocytes (SPs) and spermatids of mice testis using immunohistochemistry. Upper panel, GCNF immunostaining (dark brown staining as DAB signal) in the nucleus of SPs and round spermatids (RSs) as indicated by arrows in the WT and flutamide mice testicular sections at stage VII of spermatogenesis. Lower panel, Quantitative analysis of GCNF immunosignals in SPs and spermatids different stages of spermatogenic cycle. Immunosignal intensities in different cell types were quantified as optical density using ImageJ software. Results are mean ± SE of 3 independent experiments performed. *, P < .05.

Figure 5.

GCNF regulates GRTH expression in GCs. Top panel (left) region of seminiferous tubule dark zone rich in RS (in red) were used in cultures (right). A, Western blotting showing effect of knock-down of endogenous GCNF protein (upper panel) on GFP and GRTH protein expression (middle panels) in seminiferous tubules (culture prepared from GRTH transgenic mice with GFP as reporter) transfected with 2 different sets of GCNF siRNAs using RNAi technique. Lower panel, β-Actin used as loading control. B, Western blotting showing effect of DHT or flutamide (Flu) or combination of both on the expression of GCNF and GFP in seminiferous tubules. Right side panel, Densitometry analysis of protein bands from 3 independent experiments in each group quantified and normalized to β-actin. Mean ± SE of 3 independent experiments done in triplicates. *, significant difference (P < .05) in GCNF and GFP protein expression levels compared with control.

Figure 6.

Reciprocal regulation of GRTH and GCNF in GCs. A, Real-time PCR analysis of the GCNF message associated with immunoprecipitated total testicular GRTH from WT and GRTH knock out mice. *, significant (P < .001) reduction in association of GCNF mRNA with GRTH protein in GRTH-KO mice compared with WT. B, GCNF mRNA stability assay showing GCNF mRNAs in GCs from WT and GRTH-KO mice using real-time PCR. Seminiferous tubule cultures were incubated with 10-μg/mL actinomycin D (Act D) for 1–10 hours. Data were presented as relative to WT mice at 0 hours (n = 6 mean ± SE). C, Western blotting showing the expression of GCNF protein in spermatocytes and round spermatids of WT and GRTH-KO mice. *, significant increase (P < .01) in GCNF expression in GRTH-KO mice compared with WT mice. D, left, Western blotting showing the effect of depletion of endogenous GRTH protein (above) on the expression of GCNF protein (middle) in seminiferous tubules of WT mice transfected with GRTH siRNAs. β-Actin used as loading control (below). D, right. Densitometry analysis of GCNF protein bands from 3 independent experiments were quantified and normalized to β-actin. *, significant increase (P < .05) in GCNF expression in GRTH siRNA sample compared with scramble siRNA sample. Mean ± SE of 3 independent experiments done in triplicates.

Figure 7.

Schematic diagram showing GCNF regulation of GRTH transcription/expression in testicular GCs linking androgen action and GC gene activation. Androgen (A) synthesized and released from LCs binds to ARs present on Sertoli cells. A/AR complex enters into nucleus, where it activates AR-responsive genes/factors (classical pathway). In addition, the nonclassical androgen pathway should be considered as the mediator of this GRTH activation by TF GCNF (see discussion text). Such Sertoli cells' mediated events/signals is/are in turn passed onto GCs, where activate GCNF, a GC-specific TF, that binds the upstream 5′-region of GRTH gene and promotes its transcription/expression. Association of GRTH with GCNF mRNA and its inhibitory effect on GCNF message stability suggest that this helicase has a role in the regulation of its own transcriptional regulator in GCs.