Localization and androgen regulation of metastasis-associated protein 1 in mouse epididymis

Abstract

Background: Metastasis-associated protein 1 (MTA1), the founding member of the MTA family of genes, can modulate transcription by influencing the status of chromatin remodeling. Despite its strong correlation with the metastatic potential of cancer cells, MTA1 can also regulate crucial cellular pathways by modifying the acetylation status. We have previously reported the presence of MTA1/MTA1 in human and mouse testes, providing the evidence for its involvement in the regulation of testicular function during murine spermatogenesis. The objective of present study was to further assess the localization of MTA1 in mouse epididymis on both transcriptional and translational level, and then to explore whether MTA1 expression is regulated by androgens and postnatal epididymal development.

Methodology/principal findings: Mice were deprived of circulating androgen by bilaterally castration and were then supplemented with exogenous testosterone propionate for one week. MTA1 was immunolocalized in the epithelium of the entire epididymis with the maximal expression in the nuclei of principal cells and of clear cells in proximal region. Its expression decreased gradually after castration, whereas testosterone treatment could restore the expression, indicating that the expression of this gene is dependent on androgen. During postnatal development, the protein expression in the epididymis began to appear from day 7 to day 14, increased dramatically from postnatal day 28, and peaked at adulthood onwards, coinciding with both the well differentiated status of epididymis and the mature levels of circulating androgens. This region- and cell-specific pattern was also conservative in normal human epididymis.

Conclusions: Our data suggest that the expression of MTA1 protein could be regulated by androgen pathway and its expression level is closely associated with the postnatal development of the epididymis, giving rise to the possibility that this gene plays a potential role in sperm maturation and fertility.

Figure 1. Expression level of MTA1 in different segments of adult mouse epididymis.

(A) The products of a representative semi-quantitative RT-PCR were subjected to electrophoresis on a 1.5% agarose gel. GAPDH was used as an internal control for each PCR amplification. (B) Relative expression levels in RT-PCR were obtained in each sample by normalization of absolute optical densities (ODs) of the specific target to that of the GAPDH signal. (C) Western analysis of MTA1 protein in total tissue protein extracts from caput, corpus and cauda of the epididymis and testis. The blot was reprobed with a β-actin (42 kDa) monoclonal antibody to confirm equal loading of proteins in all lanes. (D) Semi-quantitative values are normalized to those of loading controls (β-actin) to express arbitrary units of relative expression. Comparison of the relative densities between groups in both assays was performed by ANOVA. (* p<0.05 and ** p<0.01).

Figure 2. The localization of MTA1 protein in the adult mouse epididymis.

(A) The immunohistochemical staining showed the region-specific expression pattern of MTA1 protein in the whole epididymis even at a lower magnification. Gradually decreased expression was observed from initial segments to the distal cauda. Original magnification ×4 (B) Higher view of IHC staining revealed a detailed expression pattern. MTA1 was mainly localized in the nuclei of principal cells (black arrows) and of clear cells (black arrow heads) with the maximal expression level along initial segment, caput and proximal corpus. The narrow cells (Empty arrow heads), the luminal contents and the intertubular space were all completely negative for MTA1 staining. Staining of testicular section was served as positive control. All sections were slightly counter-stained by hematoxylin. Abbreviations: Ser, Sertoli cell; Ley, Leydig cell; pachy, pachytene spermatocyte; rsd, round spermatid. Bar = 10 µm.

Figure 3. Cellular localization of mouse MTA1 in different cell types of the adult mouse epididymis.

(A) The subcellular localization of MTA1 protein was determined by using cell-specific antibodies. The immunofluorenscence of MTA1 (FITC-labeled, green), ATP6E (Rhodamine labeled for clear cells, red), CLU (Rhodamine labeled for principal cells, red) and their colocalization in the distal caput were shown by confocal microscope. DAPI was used to stain nuclei of epithelium. Bar = 20 µm (B) RT-PCR analysis of prm-2 (601 bp) and MTA1 (482 bp) expression was conducted in isolated caudal sperm and luminal fluid, with testis and caput as positive controls. Left penal depicted representative isolated caudal sperms.

Figure 4. Characterization of MTA1 expression in normal human excurrent duct system.

(A) Lysates from human efferent ductules, caput epididymis, corpus epididymis and cauda epididymis were separated over 10% SDS-PAGE and analyzed by immunoblotting with anti-MTA1 polyclonal antibody, respectively. For control purposes, loading of tissue extract for SDS-PAGE was corrected for levels of β-actin. (B) Comparative analysis of the expression of MTA1 in different segments of excurrent duct system by immunohistochemistry. The positive cells were identified as ciliated principal cells of epididymis (arrows) and basal cells (empty arrows). Columnar ciliated cells of efferent ductule (arrow heads) were barely stained. (IS, interstitial space) Negative control was performed using a nonimmune serum instead of primary antibody as demonstrated in NC. Bar  = 20 µm.

Figure 5. Expression of MTA1 in mouse epididymis during postnatal development.

(A) RT-PCR analysis of MTA1 expression at different time points during postnatal development. GAPDH was served as internal control. (B) Western blot analysis of MTA1 expression at different time points during postnatal development. β-actin was served as internal control. (C) Quantitative analysis of MTA1 expression in mouse epididymis during postnatal development by RT-PCR and western blot was carried out using band densitometry by Image J software. (* p<0.05, ** p<0.01 when comparing D70 with D21) (D) Immunohistochemistry localization of MTA1 protein in mouse proximal caput epididymis on different postnatal days 7, 14, 21, 28, 35, 49, 70, and 90 (a–h). Negative control was performed using a nonimmune serum instead of primary antibody as demonstrated in penals a'–h'. Red arrows labeled for clear cells; white arrows labeled for basal cells; black arrows labeled for principal cells, and small black arrows labeled for narrow cells. Bar = 2 µm.

Figure 6. Expression level of MTA1 in mouse epididymis following castration and subsequent testosterone manipulation.

(A) RT-PCR assay of adult mouse epididymis RNA from precastration (C0); bilaterally castrated for 1, 3, 5, and 7 d (C1, C3, C5, C7) and 1, 3, 5 and 7d after a single injection of testosterone propionate applied to the 7-d castrated mice (C7+1, C7+3, C7+5 and C7+7). The total RNAs were pooled from six animals at each time-point. (B) Western blot analysis of MTA1 and β-actin proteins from adult mouse epididymis at different time points as described above. (C) The relative expression levels of MTA1 mRNA (expression density of MTA1 mRNA/GAPDH mRNA) and MTA1 proten (expression density of MTA1/β-actin) in the mouse epididymis were compared with the serum testosterone level (expressed in nanomoles per liter) during androgen manipulation (n = 6).

Figure 7. Immunohistochemical localization of MTA1 protein in the castrated mouse epididymis.

Sections of the epididymal segments from various groups of mice including sham group (A) were immunolocalized with a MTA1 antibody and nonimmune goat serum (negative control) (D) as described under “Materials and Methods” and counterstained with hematoxylin. The data depicted represents staining for the MTA1 protein in epididymis of day 7 postcastration mice (B) with testosterone propionate supplementation (day 7) (C). Bar = 20 µm.