Sertoli cell-specific expression of metastasis-associated protein 2 (MTA2) is required for transcriptional regulation of the follicle-stimulating hormone receptor (FSHR) gene during spermatogenesis

Abstract

Background: Desensitization of FSH response by down-regulation of FSHR transcription is critical for FSH action.

Results: Chromatin modifier MTA2 participates in the down-regulation of FSHR transcription.

Conclusion: The FSH/Ar/MTA2 cascade may serve as an indispensable negative feedback mechanism to modulate FSH transduction events in Sertoli cells.

Significance: Our findings provide new insights into mechanisms by which FSH is deregulated in male infertile patients. The effect of follicle-stimulating hormone (FSH) on spermatogenesis is modulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells (SCs). One underlying mechanism is the down-regulation of the expression levels of the FSH receptor (FSHR) gene after exposure to FSH. Here we report that metastasis-associated protein 2 (MTA2), a component of histone deacetylase and nucleosome-remodeling complexes, as a gene product induced directly by testosterone or indirectly by FSH, is exclusively expressed in SCs. Stimulation of SCs with FSH is accompanied by up-regulation of MTA2 expression and enhancement of deacetylase activity. This effect requires the integrity of functional androgen receptor. Furthermore, MTA2 is a potent corepressor of FSHR transcription, because it can recruit histone deacetylase-1 onto the FSHR promoter and participates in the down-regulation of FSHR expression upon FSH treatment. Abolishment of endogenous MTA2 by siRNA treatment disrupted the desensitization of the FSH response and thereafter impaired the FSH-dependent secretory function of SCs. From a clinical standpoint, deregulated expression of MTA2 in SCs of human pathological testes negatively correlates to the deregulated level of serum FSH. Overall, our present results provide the first evidence that the FSH/androgen receptor/MTA2 cascade may serve as an indispensable negative feedback mechanism to modulate the transduction events of SCs in response to FSH. These data also underscore an unexpected reproductive facet of MTA2, which may operate as a novel integrator linking synergistic actions of FSH and androgen signaling in SCs.

FIGURE 1.

MTA2 is exclusively expressed in SCs. A, expression profile of MTA2 was evaluated in different spermatogenic cell lines and in mouse testis at different stages of postnatal development using RT-PCR. Amplification of MTA2 mRNA in mouse blastocyst samples served as a positive control. 18S was used as a loading control. B, immunoblotting analysis demonstrated a single band of MTA2 protein in the testicular lysates, which was absent when samples were incubated with preabsorbed primary antibody or without primary antibody. C, different fusion proteins were constructed for MTA2 as depicted in the left panel and were then subjected to immunoblotting analysis using antibodies against MTA1, MTA2, and MTA3 (right panel). AA, amino acids. D, immunohistochemical analysis in rodent testes revealed a distinct nuclear localization of MTA2 in SCs (arrows). Replacement of the primary antibody with preabsorbed primary antibody abolished the immunostaining, confirming the specificity of the assay. Bar, 25 μm.

FIGURE 2.

Developmental profile of MTA2 expression in murine testis throughout postnatal maturation. A, QRT-PCR analysis of MTA2 transcripts in mouse developing postnatal testis. Top, diagram of the stages of mouse spermatogenesis; P, pachytene spermatocytes; R, round spermatids; ALCs, adult Leydig cells. Bottom, QRT-PCR analysis of MTA2 mRNA level. a, b, c, and d denote groups that are statistically different (p < 0.05; analysis of variance followed by Tukey's test). B, immunohistochemical detection of MTA2 expression in various developmental stages of mouse testes. Bar, 25 μm. Error bars, S.D.

FIGURE 3.

Specific expression of MTA2 in SCs is regulated by androgen signaling. A, serum testosterone (T) level (ng/ml) in rats during androgen manipulation. B, MTA2 mRNA level in rat testis at different time points after administration of the cytotoxic drug EDS was evaluated by QRT-PCR. Data were presented as the mean ± S.D. of at least three determinations (*, p < 0.05; **, p < 0.01, when compared with control). C, SC expression of MTA2 protein (arrows) in rat testis at different time points after administration of the cytotoxic drug EDS was illustrated using immunohistochemical staining. Bar, 25 μm. D, time-dependent up-regulation of MTA2 expression in TM4 cells by testosterone (40 ng/ml) treatment was assessed by Western blotting. E, immunoblot data were densitometrically scanned and compared. Bars, mean ± S.D. (error bars) of n = 3, normalized against actin, wherein the control was arbitrarily set at 1, against which one-way analysis of variance was performed (*, p < 0.05). F, after treatment with testosterone (40 ng/ml) for 6 h, TM4 cells were harvested, and lysates were subjected to a co-IP assay followed by immunoblot analysis to demonstrated the association of endogenous AR with MTA2. G, different GST fusion proteins were constructed for Ar as depicted in the top panel and were then subjected to in vitro pull-down assay along with His-tagged MTA2 protein in the absence or presence of testosterone supplement. H, after Ars were knocked down using siRNA, TM4 cells were transfected with different Ar plasmids and were then incubated with testosterone (100 nm) for 1 h. Immunoblotting analysis were employed to assess the expression level of MTA2. Actin served as loading control. I, primary SCs were pretreated for 2 h with DMSO, PP2 (10 μm), or PD98059 (50 μm) before stimulation for 1 h with EtOH (Vehicle Ctrl) or 100 nm T. Immunoblot analysis of whole-cell extracts was first performed using the antisera against MTA2, pSrc, or pERK followed by reprobing the blots with antisera against total Src or ERK. The figure shown is representative of three experiments. J, up-regulation of MTA2 by the non-classical testosterone signaling pathway in Sertoli cells and the potential enhancement of the interaction between MTA2 and Ar by this process.

FIGURE 4.

FSH regulation of MTA2 expression in SCs. A, RT-PCR analysis of MTA2 expression in freshly isolated and primary cultured SCs. 18S was used as a loading control. B, PCR products from A were then quantified by SYBR Green intercalation as described under “Experimental Procedures.” Quantitative data in terms of MTA2 expression levels were normalized to those of the internal control 18S. Values are the mean ± S.D. (error bars) of at least three determinations. C, RT-PCR analysis of MTA2 expression in SCs was carried out at different time points of oFSH treatment. D, expression of MTA1 and MTA2 in SCs at different time points of oFSH treatment was evaluated at the translational level by Western blotting. E, dose-dependent up-regulation of MTA2 mRNA in SCs upon oFSH treatment. Parallel amplification of 18S mRNA served as internal control.

FIGURE 5.

Up-regulation of MTA2 expression by FSH in SCs is mediated via AR signaling. A, regulation of MTA2 mRNA by FSH in SCs was inhibited by blockage in AR signaling. ActD, actinomycin D. B, after being treated with Ar siRNA or control siRNA for 48 h, TM4 cells were subjected to oFSH treatment for 6 h, followed by QRT-PCR analysis of MTA2 expression levels. Quantitative data in terms of MTA2 expression levels were normalized to those of the internal control 18S. Values are the mean ± S.D. (error bars) of at least three determinations. C, effect of hypophysectomy on mouse testicular morphology was evaluated in H&E-stained transverse testis sections. Bar, 25 μm. D, hypophysectomized mice were treated with oFSH with or without FLUT, oFSH, and testosterone (T), as described under “Experimental Procedures,” and the expression level of MTA2 mRNA was then determined by QRT-PCR. Values are the mean ± S.D. of at least three determinations. #, p < 0.05 when compared with control group; *, p < 0.05 when compared with Hypo group; $, p < 0.05 when compared with the Hypo + oFSH or Hypo + testosterone group.

FIGURE 6.

MTA2 participates in the down-regulation of FSHR expression upon FSH treatment by directly recruiting HDAC1 into FSHR promoter. A, effects of MTA1 siRNA or MTA2 siRNA treatment on HDAC activity in TM4 cells in response to oFSH stimulation. B, expression level of FSHR mRNA in MTA1 siRNA- or MTA2 siRNA-treated TM4 cells upon oFSH stimulation was determined using QRT-PCR. Values are the mean ± S.D. (error bars) of at least three determinations. C, SCs were transfected with FSHR(−100/+123) Luc and expression vectors for His-MTA2 (0.5 μg) and empty vector as indicated. Two days after transfection, the cells were stimulated with vehicle or oFSH (25 ng/ml) for 6 h, after which the cells were harvested, and luciferase activities were normalized for total protein as determined by a Bradford assay. Results represent the mean ± S.D. luciferase activity for four independent experiments using two replicates. D, direct association of MTA2 and HDAC1 in SCs was illustrated by a co-IP assay followed by Western blotting analysis (IB) at different time points after oFSH treatment. E, ChIP analysis showing recruitment of MTA1 and HDAC1 onto the mouse FSHR promoter in SCs upon FSH stimulation. F, double ChIP analysis of MTA2-HDAC1 complex onto the FSHR promoter in SCs at different time points after oFSH treatment. G, after SCs had been deprived of endogenous MTA2 by siRNA treatment, cells were incubated with oFSH for different durations. QRT-PCR analysis was then used to evaluate the MTA2 and ABP mRNA levels at different time points after oFSH treatment. Values are the mean ± S.D. of at least three determinations. *, p < 0.05 when compared with control group. The efficiency of MTA2 deletion was also monitored by immunoblotting analysis (inset).

FIGURE 7.

Deregulated testicular expression of MTA2 negatively correlates with serum FSH level in human infertile patients. A, immunohistochemical analysis of MTA2 expression in human pathological testes. The arrows denote the positive staining of MTA2 in SCs. Bar, 15 μm. B, correlation of relative MTA2 immunoreactive content in human testis with serum FSH level was determined based on Pearson's correlation coefficient with the aid of SPSS 15.0 software.

FIGURE 8.

Summary diagram of the possible mechanisms related to endogenous MTA2 function contributing to the down-regulation of FSHR expression upon FSH treatment in SCs.