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Abstract
Purpose
The mechanism including changes of proteome within cavernosal tissue after cavernous nerve injury were not evaluated. We performed proteomics and functional analysis to identify proteins of penile corpus cavernosum whose expression was or was not altered by cavernous nerve resection (CNR).
Materials and Methods
Using 8-week-old male WKY rats, sham and CNR operation under microscope were performed. After 8 weeks, penile tissues of sham and CNR group were harvested. We used 2-DE and MALDI-TOF/TOF (AB 4700) to identify of differently expressed penile proteins. 2-DE gels were stained with silver nitrate and the gels were analyzed with PDQuest.
Results
We isolated more than 950 proteins on silver-stained gels of whole protein extracts from normal rat penile corpus cavernous. Of these proteins, 48 prominent proteins were identified using MALDI-TOF/TOF. Protein characterization revealed that the most prominent penile corpus cavernous proteins were those with antioxidant, chaperone, or cytoskeletal structure. Moreover, 11 proteins having levels elevated by CNR were annexin proteins, endoplasmic reticulum protein 29, glutathione s-transferase w-1, and others. In addition, Rho-GDP dissociation inhibitor (RhoGDI), a regulator of Rho proteins, was also increased in CNR rats compared with sham-operated control rats.
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Fig. 1.
Increment in apoptosis of penile corpus cavernosum from CNR rats. Immunohistochemical staining shows higher immunoreactivity in endothelial cells (arrows) and in smooth muscle cells (arrowheads) of CNR group than those of control group. The results are typical examples in 5 independent experiments. 2nd Ab: secondary antibody control. Magnification ×100 or ×400.
Fig. 2.
Images of a silver-stained 2-DE showing whole proteins identified in rat penile corpus cavernosum strips. Proteins were subjected to IEF on IPG strips (pH 3∼10, nonlinear, 17 cm) and then separated by 12% SDS-PAGE. The protein spots were visualized with silver stain. The arrows indicate proteins detected with MALDI-TOF/TOF. The numbers indicated in the gels correspond to the numbers in Tables 1. The results are typical examples in 4 independent experiments.
Fig. 3.
Silver-stained 2-DE gels in whole proteins of penile corpus cavernosum strips from sham (A) and CNR (B) rats. Proteins were subjected to IEF on IPG pH 3∼10 nonlinear (NL) strips and then separated by 12% (w/v) polyacrylamide SDS-PAGE. Arrows show proteins subsequently examined by MALDI-TOF/TOF. The numbers indicated in the gels correspond to the numbers in Table 2. The images are typical of those obtained in gels from 4 independent experiments.
Fig. 4.
Enlargement of a silver-stained 2-DE image showing rat penile corpus cavernosum proteins changed by CN resection. The arrowheads in both the left (sham control) and right (CNR) panels indicate proteins up-regulated compared with controls. The numbers indicated in the gels correspond to the numbers in Fig. 3 and Table 2. Regions containing proteins with statistically significant changed levels following the CNR were cropped and enlarged for this figure (∗p＜0.05). Statistical analysis was performed on gels from 4 independent experiments using PDQuest. White bars, sham control; black bars, CNR.
Fig. 5.
Change of RhoGDI in penile corpus cavernosum strips from CNR rats. (A) Enlargement of a silver-stained 2-DE image showing RhoGDI changed by CNR as described in Fig. 4. (B) Western blot analysis using 1-DE gels. The separated proteins were immunoblotted with anti-RhoGDI or α-tubulin antibodies. Right panels; statistical analysis from left panels. The bands (n=5) of the RhoGDI in the sham control strips were defined as 100%. Denotes significant differences between the sham and CNR rats (∗p＜0.05). The arrow indicates RhoGDI.
Table 1.
Summary of proteins prominently expressed in penile tissue from WKY rats as identified by MALDI–TOF/TO
The identity of each protein is shown. The names of the proteins, peptides representative of the identified sequences and the sequence coverage (SC), theoretical and experimental pI and Mr values, accession numbers in both the SWISS-PROT (SP) and NCBI (NC) databases, and known functions are presented.
Table 2.
Summary of proteins up-regulated in the penile tissue in CNR rats compared with sham-operated WKY rats. Protein of penile tissue were quantified in CNR and sham-operated WKY rats
Increases of at least 1.5-fold in 4experiments were considered to be significant (p＜0.05). The names of the proteins, peptides representative of the identified sequences and the sequence coverage (SC), theoreticaland experimental pI and Mr values, accession numbers in both the SWISS-PROT (SP) and NCBI (NC) databases, and known functions are presented.
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