Dephosphorylated NSSR1 is induced by androgen in mouse epididymis and phosphorylated NSSR1 is increased during sperm maturation

Abstract

NSSR1 (Neural salient serine/arginine rich protein 1, alternatively SRp38) is a newly identified RNA splicing factor and predominantly expressed in neural tissues. Here, by Western blot analysis and immunofluorescent staining, we showed that the expression of dephosphorylated NSSR1 increased significantly during development of the caput epididymis. In adult mice, phosphorylated NSSR1 was mainly expressed in the apical side of epithelial cells, and dephosphorylated NSSR1 in caput epididymis was upregulated in a testosterone dependent manner. In addition, subcellular immunoreactive distribution of NSSR1 varied in different regions of the epididymis. With respect to the sperm, phosphorylated NSSR1 was detected in the mid-piece of the tail as well as the acrosome. Furthermore, NSSR1 was released from the sperm head during the capacitation and acrosome reaction. These findings for the first time provide the evidence for the potential roles of NSSR1 in sperm maturation and fertilization.

Figure 1. Representative images showing the regional expression of NSSR1 in mouse epididymis.

A. Western blot analysis of NSSR1 in three main regions of epididymis: caput, corpus and cauda. The bands detected in epididymis caput were blocked by pre-absorption of the antiserum with specific NSSR1 peptide (Pre-absorbed), but not with non-specific peptide (Contol) (The right panel). B. Representative images showing the immunofluorescent staining of NSSR1 (presented in red) and nuclei (presented in cyan) in different regions of mouse epididymis. Pre-absorption of the antiserum with specific NSSR1 peptide block the immunofluorescent staining (The right panel). Arrows show the principle cells and arrowheads show the apical side of epididymal epithelium. Bar = 40 µm.

Figure 2. NSSR1 expression in mouse caput epdidymis at various developmental stages.

A. Western blot analysis of NSSR1 in mouse caput epididymis at 0-, 1-, 2-, 3-, 4-, 8-week (w). B. Representative immunohistological images showing NSSR1 (red) in the 1-, 2-, 3- and 8-wk caput epididymis with nuclear stain (cyan). Arrows show the principle cells and double arrowheads show the testicular germ cells. Bars = 20 µm.

Figure 3. Representative images showing the hormonal regulation of NSSR1 in mouse caput epididymis.

A. Western blot analysis of hormonal regulation of NSSR1 in caput epididymis (Representative gels and statistical data). S: Sham operation; Isemi: Intact side of unilateral-castrated mice; Csemi: Castrated side of unilateral-castrated mice; C: Bilateral castration; C+Tl: Injection of low dose testosterone (0.125 mg/day) into bilaterally castrated mice; C+Th: Injection of high dose testosterone (0.5 mg/day) into bilaterally castrated mice; C+ Tl +F: Injection of 0.125 mg testosterone together with 1 mg flutamide per day into bilaterally castrated mice; C+E: Injection of 0.2 mg estradiol into bilaterally castrated mice. ** statistically significant different from sham operated group (P<0.05). B. Representative immunohistological images showing NSSR1 proteins (red) in caput epididymis from sham operated (S), bilaterally castrated (C) and testosterone injected bilaterally castrated (C+ Tl) mice, with nuclear staining (cyan). Bar = 40 µm.

Figure 4. Hormonal regulation of NSSR1 in primarily cultured epididymal epithelial cells.

A. Representative IHC images showing the expression of NSSR1 (red) in primarily cultured epididymal epithelium, nuclear were stained with DAPI (presented in cyan). Bars = 40 µm. B. Western blot analysis of hormonal regulation of NSSR1 in primarily cultured epididymal epithelial cells supplemented with 0 nM, 1 nM (10⁻⁹), 1 µM (10⁻⁶) and 0.1 mM (10⁻⁴) testosterone.

Figure 5. NSSR1 expression in mouse sperm.

A. Representative images showing the distribution of NSSR1 proteins (red) in mouse sperms with nuclear stain (cyan). a-d are magnified view of the framed inlet in “Merged” image. Bars = 20 µm. B. Western blot analysis of NSSR1 in sperm lysates. NSSR1 antiserum pre-absorbed with specific NSSR1 peptide was used as the control (Pre-absorbed). C. Western blot analysis of NSSR1 in head and tail of the sperm. β-actin was used as the loading control.

Figure 6. Distribution of NSSR1 in the acrosome of sperms from different regions of mouse epididymis.

A. Representative images showing the distribution of NSSR1 (red) in sperms from different regions of mouse epididymis with nuclear stain (cyan). B. Magnified view of the sperm head in A. In the lowest lane of B, the thin gray curves merged with NSSR1 immunostaining (red) are the outlines of sperm heads. Bars = 10 µm. C. Western blot analysis of NSSR1 in sperms from different regions of mouse epididymis. Proteins from equal number of sperms were uploaded. β-actin was used as the loading control. Int: Initial region; Cap: Caput region; Corp: Corpus region; Caud: Cauda region.

Figure 7. NSSR1 immunostaining in the sperm head disappeared after the acrosome reaction.

A. Spermatozoa were washed from disected mouse epididymis with Whitten's Medium and fixed immediately, or capacitated for 2 h in Whitten's medium and for an additional 30 min with the addition of the calcium ionophore A23187, then fixed. Samples were then immunostained with the NSSR1 antibody, followed by FITC-conjugated secondary antibody (presented in red). Arrows indicate the area of the sperm head where the acrosome is located. Bar = 10 µm. B. Western blot analysis of NSSR1 in pellet lysates before and after acrosome reaction.