Proteomic Profiling of Regionalized Proteins in Rat Epididymis Indicates Consistency between Specialized Distribution and Protein Functions Haixin Yuan,†,§ Li Zhang,‡,§ Aihua Liu,‡ Hu Zhou,†,§ Yiguo Wang,†,§ Hong Zhang,†,§ Guoquan Wang,| Rong Zeng,† Yonglian Zhang,*‡ and Zhengjun Chen*,†,⊥ Key Laboratory of Proteomics, State Key Laboratory of Molecular Biology, Graduate School of the Chinese Academy of Sciences, and SHARF Laboratory, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, Peoples Republic of China, and Changhai Hospital, The Second Military Medical University, Shanghai, Peoples Republic of China Received September 25, 2005
The epididymis is a key structure of the male reproductive system; its function is to mature, transport, and store sperm. Most of the research examining the epididymis to date has been limited to the study of the secreted proteins involved in the maturation of spermatozoa. However, it is also very important to understand the protein components, regulation and function of the tissue itself since these are the basis for all of its physiological processes. We investigated the differential expression of proteins among the caput, corpus, and cauda regions of rat epididymis and considered the possible links between the localization of these proteins and the different functions of these epididymal regions. High-resolution 2-D gel electrophoresis followed by mass spectrometry (MS) revealed 28 distinct proteins whose expression levels varied from the caput to the cauda epididymis. Sixteen of them were reported for first time to be expressed in the epididymis. Expression patterns of some proteins were validated by Northern blot or Western blot. Immunohistochemistry revealed that inducible carbonyl reductase (iCR), an important enzyme in the anti-oxidative system, exhibits primary and cell-type specific distribution in the distal cauda region. Moreover, analysis of iCR transcription in castrated animals showed that its expression is androgen-dependent. Together with its known functions, iCR may also be involved in androgen metabolism and maintaining a steady microenvironment in the duct of epididymis. Keywords: rat epididymis • two-dimensional gel electrophoresis • regional distribution • inducible carbonyl reductase • androgen
Introduction The epididymis is a coiled tube attached to the back and upper side of the testis that stores sperm and is connected to the vas deferens. It is a unique organ where the paternal gamete cell, the spermatozoon, acquires motility and fertility in a process called sperm maturation.1 The epididymis also plays an important role in maintaining a stable microenvironment in the ducts by secreting or reabsorbing certain substances in different regions, such as electrolytes, proteins, and steroids. The epididymis is grossly divided into three major regions: * To whom correspondence should be addressed. Dr. Zhengjun Chen, Shanghai Institute of Biochemistry and Cell Biology, 320 Yueyang Road, Shanghai 200031, China. Phone: +86-21-54921081. Fax: +86-21-54921081. E-mail:
[email protected]. Prof. Yonglian Zhang, E-mail:
[email protected]. † Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. ‡ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institues for Biological Sciences, Chinese Academy of Sciences. § Graduate School of the Chinese Academy of Sciences. ⊥ SHARF Laboratory, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. | Changhai Hospital, The Second Military Medical University. 10.1021/pr050324s CCC: $33.50
2006 American Chemical Society
caput, corpus, and cauda (Figure 1). Each region of the epididymis has a distinct physiological function. Briefly, the caput and corpus of the epididymis are the main regions for secretion and absorption, primarily by the epithelial principal cells. The cauda of the epididymis is the main storage depositary for sperm, and helps to expel sperm during ejaculation.1 The epididymis represents a special tissue model that has distinct regional differentiation and carries out sequential functions of a physiological process (sperm maturation). Because proteomics utilizes convenient and powerful techniques to profile total proteins within cells or tissues, it has been applied in several studies examining the mammalian male genital tract. Cartographies of secreted (secretomes) and present proteins (proteomes) in the epididymal fluid of different mammals have been established.2 Umar and colleagues investigated the proteome of rodent epididymis and vas deferens at various development stages.3,4 Some other studies gave insights into the rat spermatogonial proteome5, stallion,6 and boar7 epididymal fluid proteome. Recently, Chaurand and colleagues presented a proteomic study of the mouse epididymis that combined laser capture microdissection (LCM) and Journal of Proteome Research 2006, 5, 299-307
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Materials and Methods
Figure 1. Schematic organization of the rat epididymis. The regions of the epididymis, the caput, corpus and cauda as well as the initial segment, are depicted. Dashed lines indicates the sites where different regions were segmented.
imaging mass spectrometry.8 All of these studies have provided valuable information about the male reproductive system and have provided abundant indications for further research in this field. However, most investigations thus far have focused on the epididymal maturation of spermatozoa and little direct attention has been given to the tissue itself. It is necessary to understand the protein components and tissue changes in detail since they are the determinants of physiological functions. For example, the epididymal duct’s environmental stability maintained by the epithelial cells is critical for the survival and maturation of spermatozoa.9 Thus, mapping of the regional patterns of protein and gene expression along the epididymal tubule is a valuable task for our better understanding the molecular mechanisms of sperm maturation. The epididymis is a classical target tissue for androgens.10,11 It has been demonstrated that androgens can affect several aspects of epididymal functions including mRNA and protein expression.12 In many cases epididymal dysfunction has been shown to be related to abnormal protein expression.13,14 Thus, when identified, such proteins may be targets for diagnosis and treatment of human infertility. On the other hand, they may also be potential targets in the design of specific contraceptive medicines. The present study sought to expand our knowledge of the epididymis by applying a proteomic approach. We employed a method that eliminates sperm and luminal fluid from the duct of epididymis, thereby avoiding contamination to the tissue protein samples. We applied high-resolution 2-D gel electrophoresis to investigate the differential protein patterns of the caput, corpus, and cauda regions of rat epididymis. In total, 28 proteins with regionalized distribution in the epididymis were identified by mass spectrometry (MS) and were classified according to their known or postulated functions. We proceeded to assess how well the putative functions of the individual proteins correlated with their regional and cellular distributions in the epididymis. 300
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Extraction of Protein from Rat Epididymis. Male Wistar rats (aged 100-120 d) were purchased from Shanghai Laboratory Animal Center (Jiu-Ting, Shanghai, China). Rats were anesthetized with ether and the entire epididymis was separated out. Luminal content was eliminated as described previously15 with some modifications. Briefly, the epididymis was divided into caput, corpus and cauda segments. Each segment was cut into small fragments and was incubated in DMEM medium without serum for 3 h at 37 °C to release the content of the ducts. Residual content was then squeezed out with forceps and the fragments were washed twice with 1 × PBS. Each segment was homogenized on ice in 1 mL of cell lysis buffer [50 mM HEPES, pH 7.5, 10% glycerol, 1% TritonX-100, 150 mM NaCl, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 100 mM NaF]. Lysate from homogenization was sonicated and centrifugated at 13 000 rpm for 15 min at 4 °C. Protein in the supernatant was precipitated by 10% trichloroacetic acid (TCA) on ice for 30 min and then centrifugated at 13 000 rpm for 15 min at 4 °C. The pellet was washed by ice-cold acetone and dissolved in lysis solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris base (Amersham Biosciences). Protein concentration was determined by the Bradford assay16 on a Microplate Reader (Bio-Rad, Model 680), using bovine serum albumin (BSA) as the protein standard. The protein samples were stored at -80 °C until used. To exclude individual differences among animals, epididymis samples from 5 rats were used for 2-D gel analysis. 2-DE and Gel Staining. 2-DE was performed according to the methods described previously.17 pH 3-10 nonlinear or pH 4-7 IPG strips (13 cm) were applied in first dimension of isoelectric focusing. For the second dimension, proteins were separated according to their molecular mass using 12% SDSPAGE gels at 20 mA per gel. The electrophoresis gels were stained with Coomassie brilliant blue R-350 (Amersham Biosciences). Analysis of 2-D Gel Spot Patterns. We repeated each 2-D electrophoresis experiment four times to confirm the spot patterns before proceeding with further analysis. The replicates were scanned by a GS-800 calibrated densitometer (Bio-Rad) with standardized parameters and gel images were processed by the software PDQuest 7.0 (Bio-Rad). Gels were normalized using the total volume of all valid spots and spot volumes were determined by modeling the optical density in individual spot segments. Different expression levels of proteins were assessed by comparing the spots volumes, and spots were considered significantly up- (or down-) regulated only if the corresponding volumes showed an increase (or decrease) by a factor of 3. Protein Identification by MALDI-TOF-MS. Protein identification by MALDI-TOF-MS was performed as described previously.18 Peptide mass fingerprinting was performed with the Mascot search engine (http://www.matrixscience.com/, MatrixScience Ltd., UK) against the NCBI nonredundant protein database (http://www.ncbi.nlm.nih.gov) for the species rattus. Errors in peptide mass within 0.05% were allowed. One missed tryptic cleavage site per peptide was allowed during the search. Cysteines were carboamidomethylated and methionines were considered as oxidation events. Proteins that matched at least four peptides and had a Mascot score greater than 63 were considered to be significant (P < 0.05). Protein Identification by 1D-LC-MS/MS. All spots were first analyzed by MALDI-TOF-MS, and those spots that were not
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Profiling of Regionalized Proteins in Rat Epididymis
Figure 2. Panoramic 2-DE images of the protein extracts from the caput, corpus and cauda epididymis. pI ranges (pH 3-10 (A) and pH 4-7 (B)) and molecular weights are indicated. Spots that were identified by MS are indicated by arrows and names in the gels.
identified by MALDI-TOF-MS were further analyzed using a LCQ Deca XP system (Thermo Finnigan, San Jose, CA). Protein identification by 1D-LC-MS/MS was performed as described previously.17 The MS and MS/MS spectra of peptide ions were used by the TurboSEQUEST software (Thermo Finnigan) to search against the NCBI nonredundant database (species: Rattus). The protein identification criteria that we used were based on Delta Cn (g 0.1) and Xcorr (one charge g 1.9, two charges g 2.2 and three charges g 3.75). Total RNA Extraction. The duct contents were removed as described in the protein extraction section, except that the incubation time at 37 °C was decreased to 1 h to prevent RNA degradation. Total RNA from caput, corpus and cauda segments of epididymis was isolated using TRIzol reagent (Invitrogen) as described by Chomczynski and Sacchi.19 RNA was quantified on a GeneQuant Pro spectrophotometer (Amersham Biotech). Northern Blot Analysis. A total of 20 µg of RNA per lane was run on 1.5% agarose gels with 2.2 M formaldehyde. Separated RNA bands were transferred to a nylon membrane and hybridized with an R-32P-labeled probe prepared using a Megaprime DNA labeling kit (Amersham Biosciences) and purified with a QIAquick nucleotide removal kit (QIAGEN) according to the manufacturer’s instructions. The hybridization was performed using Expresshyb Hybridization Solution (BD Biosciences) according to the kit instructions. The hybridized blots were exposed to a phosphorimager and scanned on a Typhoon 9410 imagescanner (Amersham Biosciences). The optical density of each band was determined by the software Quantity One 4.3.0 (Bio-Rad). Western Blot Analysis. Protein was separated by SDS-PAGE or by 2-DE as described above and electro-blotted onto nitrocellulose transfer membrane (Schleicher & Schuell BioScience, Germany). Immunodetection of blots was performed using specific antibodies. Antibodies to galectin-3 and carbonyl
reductase were kindly provided by Dr. Futong Liu (University of California-Davis, Sacramento, USA.) and Dr. Tomoyuki Terada (Osaka University, Osaka, Japan), respectively. Immunohistochemistry. Tissue samples were fixed in Bouin’s fluid and then embedded in paraffin. For immunohistochemical staining of rat epididymis, the fluorescein isothiocyanateconjugated AffiniPure goat anti-rabbit IgG indirect kit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used. Sections were then counterstained with hematoxylin. Photomicrographs of sections were taken by a digital camera (Nikon DXM1200F, Tokyo, Japan) mounted on a light microscope (Nikon Eclipse E600, Tokyo, Japan). Castration and RNA Isolation. Adult male rats (aged 60 d) were castrated surgically. The rats were divided into nine groups depending on their duration of castration (3, 5, or 7 rats per group). Five groups of rats were killed at different times after castration (day 0, 1, 3, 5, or 7). Seven days post-castration the other four groups of rats were injected with testosterone propionate (4 µg/g body weight) every other day (day 7, 9, 11, or 13) and were killed the following day (day 8, 10, 12, or 14). Epididymis samples from each group were pooled for RNA extraction. Testosterone content from pooled serum samples was measured by radioimmunoassay (RIA).
Results General Patterns of Protein Expression. Analysis of the 2-D gels revealed the presence of about 900 spots in pH 3-10 nonlinear2-D gels (Figure 2A) and 700 spots in pH 4-7 2-D gels (Figure 2B). A total of 60 spots were found to be differentially distributed among the caput, corpus and cauda epididymis and were further analyzed by mass spectrometry. Fiftytwo spots, corresponding to 28 different polypeptides, were identified (see Supporting Information Table). This means that as many as 10 proteins were represented by at least two distinct Journal of Proteome Research • Vol. 5, No. 2, 2006 301
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Table 1. Summary of the Proteins Identified by MSa NCBI accession no.
Primary metabolism Lactate dehydrogenase B Triosephosphate isomerase 1 Enolase 1, alpha Creatine kinase, brain Guanidinoacetate methyltransferase Cellular Retinol Binding Protein (Crbp) complexed With All-Trans Retinol Retinol binding protein 1, cellular Isovaleryl coenzyme A dehydrogenase Isocitrate dehydrogenase 3 (NAD+) alpha Similar to ATPase, H+ transporting, V1 subunit A, isoform 1 Amino acid metabolism Gln synthetase Glutamine synthetase (glutamate-ammonia ligase) (GS) Gamma glutamyltransferase D-3-phosphoglycerate dehydrogenase (3-PGDH) Antioxidant system Peroxiredoxin 4 inducible carbonyl reductase Gamma glutamyltransferase Smooth muscle tissue Transgelin (Smooth muscle 22 protein) Tropomyosin 1 alpha chain Others IgE binding protein (galectin-3) Clusterin Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) Epididymal secretory protein 1 Guanine deaminase Chain, Catechol o-methyltransferase Gelsolin Similar to reticulocalbin Similar to ribosomal protein S12; 40S ribosomal protein S12 Immunoglobulin heavy chain
regional abundance
MW
pI
epididymal expression in pub. data
pub. data
our data
NP•036727 NP•075211 NP•036686 NP•036661 NP•036925 1CRB
36613 26849 47116 42984 26675 15864
5.70 6.89 6.16 5.33 5.69 5.10
unknown yes unknown yes yes yes
unknown unknown unknown unknown cap prox. cap
cor/cau cap cap cor/cau cap cap
NP•036865 NP•036724 NP•446090 XP•340988
15835 46436 40044 68268
5.10 8.03 6.47 5.62
yes unknown yes unknown
prox. cap unknown unknown unknown
cap cap cor/cau cap/cor
1717354A P09606
41153 42268
6.38 6.64
yes yes
cap cap
cap cap
A05225 CAA66374
61580 56493
7.21 6.28
yes unknown
cap unknown
cap cap/cor
NP•445964 BAA19007 A05225
31008 30920 61580
6.18 7.64 7.21
unknown unknown yes
unknown unknown cap
cau cau cap
NP•113737 AAK54242
22645 32695
8.87 4.71
unknown yes
unknown unknown
cau cau
AAA41378 NP•036811 NP•058933
27269 51376 24783
9.47 5.47 5.12
unknown yes unknown
unknown cap unknown
cor/cau cap cap
CAD56199 AAF63337 1VID NP•001004080 XP•342482 XP•219903
16364 51439 24960 85492 38113 14449
7.55 5.48 5.11 5.83 4.70 5.93
yes unknown unknown unknown unknown unknown
cau unknown unknown unknown unknown unknown
cau cau cau cau cau cau
AAB21181
51461
8.57
unknown
unknown
cau
protein name
a
Gamma glutamyltransferase is involved in both amino acid metabolism and antioxidant system. For MS parameters please see the supplementary table. Pub: published; cap: caput; cor: corpus; cau: cauda; prox: proximal.
spots on the 2-D gels. As summarized in Table 1, each of these 28 proteins was characterized by its predicated molecular weight, pI, NCBI accession number, and regional abundance. Of these 28 proteins, 8 have been reported to have regionalized behavior within the epididymis, such as Gln synthetase20 and clusterin.21,22 Our results are highly accordant with the reports describing those proteins previously known to be regionalized (Table 1). In addition, our results are the first to reveal the regional distribution of four other previously identified epididymal proteins (Table 1). Furthermore, another 16 proteins were found for the first time to be expressed in the epididymis and these 16 newly identified proteins had notable distinction among the different epididymal regions (Table 1). To facilitate our analysis of these proteins, we have divided them into the following five functional groups: (1) primary metabolism, (2) amino acid metabolism, (3) antioxidant system, (4) smooth muscle tissue, and (5) others or unknown. Expression patterns of some proteins were shown in Figure 3A, B, and C with their normalized relative volume intensity in Figure 3D and E. Analysis on Regional mRNA Levels by Northern Blot. To determine whether the proteins we found were expressed in situ, we compared the mRNA levels of several genes from 302
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different epididymal regions by Northern blot analysis. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) mRNA was detected exclusively in the caput (Figure 4A). Peroxiredoxin 4 and galectin-3 mRNA levels were high in both the corpus and cauda (Figure 4B), whereas mRNA expression of inducible carbonyl reductase (iCR) and transgelin were very high only in the cauda region (Figure 4C). The mRNA data were generally in accordance with the 2-D gel protein findings (Figure 3AC). Modification Analysis of Galectin-3 and iCR Revealed by Western Blot. Two proteins, galectin-3, and iCR, were selected for further analysis on the regional distribution and modification by western blot of 1D- as well as 2D-PAGE. Galectin-3 was highly expressed in the corpus and cauda at a molecular weight of 30 kDa and pI of 9.5. No modified forms of galectin-3 were detected by Western blot analysis (Figure 5A, upper panel and 5B, left lane). iCR was mainly distributed in the cauda, but a weak signal was also detected in the caput and corpus regions. Moreover, diverse forms of iCR were demonstrated in the epididymis by both 1-D and 2-D western blot (Figure 5A, middle panel and 5B, right lane). 2-D-Western blot analysis
Profiling of Regionalized Proteins in Rat Epididymis
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Figure 3. Examples of regionalized proteins in the epididymis. Representative parts of the 2-D gels are amplified to depict some of the identified proteins whose expression levels were predominant in the caput (A), corpus/cauda (B) and cauda (C) regions. (D) & (E) Comparison of the normalized volume intensity of the spots. Test of significant difference from caput (D) or cauda (E) was carried out by unpaired student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 4. Validation of the 2-DE data by Northern blot with R-32P-labeled probes. Genes that were transcribed at high levels in each of the epididymal regions, caput (A), corpus (B), and cauda (C), are shown. 28S rRNA served as a loading control. The numbers under each panel indicate relative intensity of the corresponding bands with the value of caput fixed at 1.0.
demonstrated that iCR consists of 4-6 forms with pI ranging from 7.5 to 9.0. Regional and Cellular Localization of Galectin-3 and iCR. To obtain more information about cellular distribution of some identified proteins in the epididymis, galectin-3 and iCR were selected for immunohistochemistry analysis. As is shown in Figure 6B, galectin-3 was detected in the epithelium of corpus and cauda, but not in the caput. The signal decreased slightly from the corpus to the more distal regions. As is shown in the magnified images (Figure 6B, the lower two panels), galectin-3 was found to be localized primarily in the nucleus of the duct
epithelial cells. No clear signal was detected in the epithelial cells of the caput region. However, weak speckled signals were found in the fibrocytes around the duct of caput epididymis (Figure 6D, right). Immunohistochemistry analysis of iCR revealed that iCR is localized exclusively in the distal cauda epididymis as well as the proximal vas deferens (Figure 6A,C). A boundary could be clearly seen between labeled and unlabeled regions of the cauda epididymis (Figure 6A). iCR was distributed ubiquitously only in the principal cells with a stronger signal in the nucleus as well as on the plasma membrane (Figure 6C, the lower two Journal of Proteome Research • Vol. 5, No. 2, 2006 303
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Figure 5. Western blot analysis for galectin-3 and iCR. Extracts from each region of epididymis were separated by SDS-PAGE (40 µg/gel) (A) or 2-DE (100 µg/gel) (B). Immunoblotting was performed with antibodies to galectin-3 or iCR. β-actin served as a loading control. Note the multiple modification forms of iCR in each region (B).
panels). There was even clear labeling on the microvilli of principal cells, suggesting possible secretion of iCR by these cells. Differential Androgen-Mediated Regulation of iCR and Galectin-3 Transcription. To further reveal the functions of iCR and galectin-3 in the epididymis, we examined the androgen-dependent behavior of these two proteins. A castration animal model was used to determine whether the transcription of the two genes was regulated by androgen exposure. We found that iCR mRNA was sharply decreased 1 day after castration, and nearly absent beyond the second day postcastration (Figure 7A and B). Following an injection of a high concentration of testosterone, its mRNA level was rescued and reached a maximum, even higher than its physiological level, in the third day postinjection. However, this climax could not be maintained and decreased quickly to a normal level. Compared to iCR, galectin-3 mRNA was not down-regulated, after androgen elimination, but a similar change to that of iCR was observed after the testosterone injection (Figure 7C,D).
Discussion We applied high-resolution 2-D gel electrophoresis and mass spectrometry to the investigation of different protein expression patterns of the caput, corpus, and cauda regions of rat epididymis. We identified 28 proteins, whose expression varied among the regions; of these proteins 16 were newly identified in the epididymis. Our analysis revealed that many proteins had diverse modifications and were represented by more than one spot (Figure 2). It has been well documented that protein modifications, such as glycosylation and phosphorylation, are common in the male internal genital tract.23-25 The serial changes of molecular weight and pI of modified proteins clearly displayed in our 2-D maps provide compelling support of such protein modifications. Our methods were designed to exclude tissue contamination by luminal fluid and sperm proteins. Several aspects of our results indicate that this exclusion was effective. For example, although γ-glutamyltransferase is in the luminal fluid throughout the epididymis,26 it was detected only in the caput (Figure 3A), which suggested that γ-glutamyltransferase in the luminal fluid had been removed. In addition, the results from our Northern blot analysis showed that nearly all of the selected proteins were expressed in situ. Chaurand and colleagues carried out a protein profiling and imaging study in the mouse epididymis by an advanced approach that combined LCM and imaging MS.8 The authors identified over 50 proteins that displayed regional selectivity 304
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Figure 6. Regional localization of galectin-3 and iCR revealed by immunohistochemistry. Panoramic image of a section through epididymis is shown (A, left panel, 8×). Note the clear boundary between stained and unstained region of iCR in the cauda epididymis (A, right panel, 40×). Representative examples of the distribution patterns of galectin-3 (B) and iCR (C) within different epididymal regions are shown (200×, upper two rows and 1000×, lower two rows). Note that galectin-3 labeling around the ducts is clearly visible in the caput epididymis (D, indicated by arrows, 200×). NS: nonimmune serum.
of expression within the epididymis. However, although the two species are highly homologous, it was surprising that only the clusterin and epididymal secretory protein 1 findings were consistent between their findings and ours. This difference between the two results may be due to both species differences and method limitations used by the two studies, such as range (pI or molecular weight) and sensitivity limitations for 2-DE
Profiling of Regionalized Proteins in Rat Epididymis
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Figure 7. Androgenic regulation of iCR and galectin-3. Rats were castrated and epididymal mRNA was extracted on the indicated days. After 7-day castration the residual animals were injected with testosterone propionate (4 µg/g body weight) every other day and epididymal mRNA samples were extracted. Northern blot analysis was performed with probes against mRNA of iCR (A) or galectin-3 (C). The relative expression level of mRNA was calculated by mRNA density/18S density and the changes are illustrated graphically for iCR (B) and for galectin-3 (D).
and sample region restriction for LCM. Therefore, it is important to consider the findings of both studies in order to obtain a comprehensive knowledge of regionalized protein patterns within rodent epididymis. The proteins we identified were divided into five functional classes. In general, as we had supposed, localization of most of the proteins was consistent with their putative functions in the tissue. Proteins involved in amino acid metabolism were distributed mainly in the caput epididymis (Table 1), which is the primary region for protein synthesis and secretion.27,28 Smooth muscle proteins, however, were found to be distributed predominantly in the cauda epididymis (Table 1). Since sperm are stored in the cauda region, which helps to drive sperm during ejaculation, the thickness of the periductal smooth muscle gradually increases 2- to 3-fold along the proximal cauda epididymis, and then increases abruptly by the same magnitude where the duct enters the distal cauda epididymis.29 Thus, the abundance of smooth muscle proteins in this region is consistent with its function. Proteins involved in the antioxidant system had no unique expression pattern (Table 1), indicating the universal function of this type of proteins in maintaining the environment all along the epididymis. The epididymis is a rich source of antioxidant enzymes that scavenge excess reactive oxygen metabolites released by spermatozoa during epididymal transit.30,31 Our work demonstrated that peroxiredoxin 4 was expressed in the corpus as well as the cauda, but not in the caput epididymis (Figure 3C). Peroxiredoxin 4 is secreted as a 27-kDa isoform in most tissues, but another unprocessed membrane-bond 31-kDa isoform is found only in testis.32 Our results demonstrated that the 31-kDa isoform is also expressed in the distal region of the epididymis. However, whether the secreted form of peroxiredoxin 4 exists in the epididymis could not be concluded. Our immunohistochemical analysis has provided the first data, to our knowledge, describing the cellular expression and distribution within the epididymis tissue of galectin-3 and iCR.
The nuclear localization of galectin-3 has been observed previously in the proliferating cultures of fibroblasts33 and in the differentiated colonic epithelial cells.34 Galectin-3 has been demonstrated to be a necessary factor in the splicing of nuclear pre-mRNA.35-37 It was interesting that weak speckled signals were detected in the tissues around the duct of caput epididymis where no epithelial cell signal was detected (Figure 6D). However, such weak signals were not detected in the corpus and cauda epididymis. Whether this indicates that galectin-3 may play a distinct role in the proximal epididymis needs further detailed studies. The family of carbonyl reductases in male reproductive system has attracted much attention in previous investigations.38-42 Our results suggested that iCR, a member of the short chain dehydrogenases/reductases (SDR) family, is distributed in a regionally specific manner. It was distributed exclusively in the distal cauda epididymis and had complicated modifications affecting both its molecular weight and pI. Unlike many carbonyl reductases, which are considered to be cytosolic enzymes, iCR in the rat epididymis was found to be concentrated in the nuclei and on the plasma membranes of epithelial cells, whereas the staining in the cytoplasm was relatively weak (Figure 6C). However, some other members of the SDR family, such as aldehyde reductase43 and hamster P26h,44 have been found to be localized primarily in the nucleus of epididymal epithelial cells. Whether iCR is a secreted protein involved in direct interaction with spermatozoa remains to be investigated according to its strong signal on the membrane. Many epididymal genes are regulated by androgens via complicated mechanisms.45,46 For example, the protein level of P26h decreased rapidly after castration and was undetectable by 3 days following castration.47 Interestingly, P26h is a dehydrogenase of the important androgen 5R-dihydrotestosterone in a metabolic process.44 Our work also demonstrated that iCR mRNA levels decreased sharply within one week of castration, yet this deficit was rescued following injection of testosterone (Figure 7A). It is clear that this was a consequence of Journal of Proteome Research • Vol. 5, No. 2, 2006 305
research articles androgen-dependent regulation, rather than an overall decline in RNA synthesis because the mRNA of two other genes, galectin-3 and peroxiredoxin 4, were unaffected (Figure 7B and data not shown). There are also some indications that rat iCR may use androgens as substrates.48,49 Further studies are needed to reveal the precise regulation mechanism between iCR and androgen.
Conclusion The present experiments represent a comprehensive proteomic study of the protein profiles among the different epididymal regions. Twenty-eight proteins were found to be regionally localized within this tissue. Localization of most proteins was closely related to their physiological functions. Our findings made the first demonstration that iCR is expressed highly and distinctly in the distal cauda epididymis and its transcription appears to be regulated by androgens. All these results may provide some useful information for the field of male reproductive biology. Abbreviations. 2-D, two-dimensional; 2-DE, two-dimensional electrophoresis; LCM, laser capture microdissection; IPG, immobilized pH gradient; CHAPS, 3-((3-cholamidopropyl) dimethylamino)-1-propanesulfonate; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; LC, Liquid Chromatography; MS/MS, tandem mass spectrometry; TOF, time-of-flight; DMEM, Dulbecco’s Modified Eagle’s Medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iCR, inducible carbonyl reductase; SDR: short chain dehydrogenases/ reductases.
Acknowledgment. This work was supported by a research grant of Knowledge Creative Program from the Chinese Academy of Sciences (KSCX2-SW-201), and by grants from Ministry of Science and Technology (2001AA233031, 2001CB510205, and 2004CB520802). We thank Feng Zhao for technical assistance, and Dr. Futong Liu (University of CaliforniaDavis, Sacramento, U.S.A.) and Dr. Tomoyuki Terada (Osaka University, Osaka, Japan) for their kindness to offer us excellent antibodies. We also thank Rongxia Li for her helpful suggestions. Supporting Information Available: Fifty-two spots, corresponding to 28 different polypeptides, were identified (see Supporting Information Table). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Robaire, B.; Hermo, L. In The Physiology of Reproduction; Knobil, E., Eds.; Raven Press: New York, 1988; pp 999-1080. (2) Gatti, J. L.; Castella, S.; Dacheux, F.; Ecroyd, H.; Metayer, S.; Thimon, V.; Dacheux, J. L. Post-testicular sperm environment and fertility. Anim. Reprod. Sci. 2004, 82-83, 321-339. (3) Umar, A.; Ooms, M. P.; Luider, T. M.; Grootegoed, J. A.; Brinkmann, A. O. Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development. Endocrinology 2003, 144, 4637-4647. (4) Umar, A.; Luider, T. M.; Berrevoets, C. A.; Grootegoed, J. A.; Brinkmann, A. O. Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line. Endocrinology 2003, 144, 1147-1154. (5) Com, E.; Evrard, B.; Roepstorff, P.; Aubry, F.; Pineau, C. New insights into the rat spermatogonial proteome: identification of 156 additional proteins. Mol. Cell. Proteomics 2003, 2, 248-261. (6) Fouchecourt, S.; Metayer, S.; Locatelli, A.; Dacheux, F.; Dacheux, J. L. Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins. Biol. Reprod. 2000, 62, 1790-1803.
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