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Acta Biochimica et Biophysica Sinica Advance Access originally published online on September 3, 2009
Acta Biochimica et Biophysica Sinica 2009 41(10):809-815; doi:10.1093/abbs/gmp075
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Identification and characterization of an epididymis-specific gene, Ces7

Li Zhang1,2, Zhihong Hu1, Chunfang Zhu1, Qiang Liu1,*, Yuchuan Zhou1,* and Yonglian Zhang1,3

1 Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
2 The Graduate School of Chinese Academy of Sciences, Beijing 100049, China
3 Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China

* Correspondence address. Tel: +86-21-54921267; Fax: +86-21-54921011; E-mail: qliu{at}sibs.ac.cn (Q.L.); Tel: +86-21-54921153; Fax: +86-21-54921011; E-mail: zhouych{at}sibs.ac.cn (Y.Z.)


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
Carboxylesterases (CEs) represent a multigene family of serine-dependent enzymes. Male-dependent CEs are over-expressed in the male reproductive tract of different animal species (bivalve mollusks, fruit-flies, and mammals). Here, a novel rat epididymis-specific gene named Ces7 was cloned and characterized. It was a novel member of CE family, which was mainly expressed and secreted to the lumens of the corpus and cauda epididymis. CES7 protein was highly glycosylated as other mammalian CEs. Furthermore, Ces7 increased with age growth until sex maturation and then maintained at high level. CES7 might be one of the major CEs in male reproductive tract and contribute to the sperm fertilization.

Keywords    epididymis; carboxylesterase; sperm maturation

Received: April 9, 2009; Accepted: May 21, 2009
   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 References
 
Carboxylesterases (CEs) represent a multigene family of serine-dependent enzymes (carboxyl-ester hydrolase; EC 3.1.1.1 [EC] ), which can hydrolyze carboxylester, amide, and thioester bonds in a variety of exogenous and endogenous compounds. Furthermore, the conserved CEs are over-expressed in male reproductive tract [1,2].

In mammalian males, spermatozoa transit along the reproductive tract before their ejaculation. The epididymis is the main part of the male reproductive tract, which assures the sperm maturation and also performs the reservoir function. In this special and continuously changing milieu, spermatozoa gradually acquire their capacity for fertilization, and then they are stored in a quiescent state preventing the sperm activation in the cauda epididymis. During the spermatozoa journey, different biological fluids secreted by accessory sex glands contain a lot of ions, lipid materials, peptides, and proteins for spermatozoa maturation and storage [3,4].

In our current report, a novel gene named Ces7, a member of CE family, was identified in the rat epididymal cDNA library and homologous to the cauxin. With the aid of sensitive and specific polyclonal anti-sera against rat CES7 protein, the characters of rat CES7 were studied. These data suggested that CES7 might be related to the epididymal function.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 References
 
Animals
Healthy male Sprague–Dawley rats and male New Zealand white rabbits were supplied by the Animal Center of the Chinese Academy of Sciences (Shanghai, China). Animals were housed under standard laboratory condition. Experiments were conducted according to a protocol approved by the Institute Animal Care Committee. The protocol conformed to internationally accepted guidelines for the humane care and use of laboratory animals.

Screening of rat epididymal cDNA library for full-length clones
Screening of cDNA library was performed as described previously [5]. The mouse expressed sequence tag (EST) with 213 bp was used to probe the rat epididymal cDNA library. The probe was amplified with mouse epididymal cDNA template by PCR using the forward primer F1: 5'-TTCTGCTTTAGCTGGTGTTTTATGG-3' and the reverse primer R1: 5'-TGACAAGTAATGGGCCAGC-CCTCCT-3' at denaturing, annealing, and extension temperatures of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, respectively, with Ex-TaqTM DNA polymerase (TaKaRa, Dalian, China).

DNA and protein sequence analysis
The mouse ortholog of monkey ESC-615 was obtained by homology, searching the mouse EST database at http://www.ncbi.nlm.nih.gov/BLAST. The signal peptide cleavage sites were predicted at http://www.cbs.dtu.dk/services/SignalP. N-glycosylation sites and phosphorylation sites were predicted at the ProfileScan website http://myhits.isb-sib.ch/cgi-bin/PFSCAN.

RNA isolation and northern blot hybridization
Rat epididymides and other tissues were pulverized in liquid nitrogen and homogenized. Total RNA of each tissue was isolated using the Trizol reagent (Invitrogen, Carlsbad, USA). Northern blot hybridization was performed as described previously [6]. The probe containing 1321 bp was obtained from the full Ces7 clone digested with BamHI at both 291 and 1612 bp sites.

Castration and androgen replacement, developmental pattern
The experiments were performed as described previously [7,8]. In castration and androgen replacement, all animals were divided into nine groups (one control group, four castrated groups, four castrated and androgen-treated groups, each group contained three to seven rats). The castrated groups were killed on days 1, 3, 5, and 7 after castration. Androgen supplementation began on the 7th day after castration, and rats were injected with testosterone propionate (3 mg/kg body weight) every 2 days. Animals in these treated groups were killed on the next day of the injection. The pooled serum samples for each group were subjected for the measurement of testosterone concentration by radioimmunoassay in Shanghai Zhongshan Hospital.

For the developmental pattern analysis, the epididymides of normal male rats were sampled at the age of 15, 30, 45, 60, 90, 120, 270, 360, 450, and 720 days.

The epididymides of each group were excised and mixed for RNA extraction and northern blot analysis.

Antibody preparation
The cDNA fragment encoding the selected peptide (68 amino acids) of Ces7 was cloned into the pQE expression system (Qiagen, Düsseldorf, Germany). The cDNA fragment was amplified by PCR using the following primer pairs: forward primer 5'-GCGGGATCC-ATGAGGGAAGCGCCT-3' with BamHI site and reverse primer 5'-GCGGGTACCTGTAACCAGCCCA-GG-3' with KpnI site. After enzyme digestion, the fragment was inserted to the pQE30 vector (Qiagen). The expression vector was constructed according to the standard protocol provided in the pQE expression manual. The purification of the recombinant protein from the inclusion bodies and the preparation of the rabbit polyclonal anti-sera against the CES7 recombinant peptide were performed as described previously [9].

Protein extract and western blot assay
The experiments were performed as described previously [10]. Total protein extract for each sample was electrophoresed on 8% polyacrylamide gels (SDS–PAGE), electroblotted onto Hybond-P membranes (Amersham Pharmacia Biotech, San Francisco, USA), immunoblotted, and visualized by the ECL Plus system (Amersham Pharmacia Biotech). The anti-CES7 sera (dilution 1:20,000) were used as primary antibody. The secondary antibody (dilution 1:15,000) was the horseradish peroxidase (HRP)-conjugated goat-anti-rabbit immunoglobulin G (Calbiochem, Darmstadt, Germany).

Peptide N-glycosidase F treatment
Peptide N-glycosidase F treatment was performed as described previously [7]. The fresh epididymides were cut into small pieces in PBS buffer. The supernatant of these pieces containing the lumen fluid was collected by centrifuging (16,000–30,000 g). The samples of epididymal lumen fluid were denatured and deglycosylated by PNGase-F (Biolabs, Ipswich, USA). Then N-glycosylation modification of CES7 protein was evaluated by western blot analysis.

Immunohistochemical staining
The tissue section preparation and immunohistochemical staining were performed as described previously [7,8,11] except using the AEC (3-amino-9-ethylcarbazole) staining instead of the DAB (3,3'-diaminobenzidene) staining. Primary and secondary antibodies were diluted in PBS containing 10% (v/v) normal goat serum. The 1:200 diluted anti-CES7 serum was applied overnight at 4°C, and 1:200 diluted HRP-conjugated goat-anti-rabbit IgG was incubated for 1 h at room temperature. As a negative control, serial sections were subjected to the same procedure with pre-immune rabbit sera replacing the primary CES7 anti-sera.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 References
 
Cloning of Ces7 cDNA
Previously, we identified 11 full-length novel genes from monkey epididymis, which included monkey ESC-615 [5]. To clone the rat ortholog of this gene, the mouse EST database was first searched by BLASTN using monkey ESC-615 cDNA sequence because of incomplete rat genomic and EST database at that time. The mouse cDNA sequence of 241 bp (EST No. AV381828) sharing 20% identity with the monkey ESC-615 was acquired from the mouse EST database. The cDNA fragment (213 bp and sharing 30% identity with monkey ESC-615) was amplified by RT–PCR based on this selected mouse sequence and probed to screen the rat epididymal cDNA library. Then the full-length cDNA sequence (2147 bp, named Ces7, GenBank accession No. AF479659 [GenBank] ) was obtained, and the total sequence shared 78% identity with monkey ESC-615 cDNA and, furthermore, the amino acid sequence identity between monkey and rat ESC-615 was 65%.

According to the rat genome database, the gene Ces7 was located on rat chromosome 19. The entire open reading frame was of 1728 bp encoding 575 amino acids [Fig. 1(A), GenBank accession No. AF479659 [GenBank] ]. The N-terminal 28 amino acids probably form a signal peptide as predicted with SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). Based on amino acid sequences of the CE family, the CES7 protein had four conserved catalytic motifs and residues (amino acid sequences with bolded active-site residues, SEDCLY, GGSAG, MQS, ADHTD) among the CE family [Fig. 1(A), black box] [12,13]. Moreover, the C-terminal endoplasmic reticulum (ER) retention tetrapeptide sequence (HXEL, in a single-letter code) can locate mature esterase into the cytoplasm [12]. No ER retention signal in its C-terminal suggested that CES7 should be a secreted protein.


Figure 1
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  		Figure 1 Ces7 sequence with other CE family members (A) Alignment of Ces7 and other esterase. The essential domains of CE B type (in the black boxes) and the antigen peptide used for expression in Escherichia coli (in the red boxes) were highlighted. (B) Phylogenetic tree for Ces7 and other esterases. ratepi615, rat epididymis Ces7 gene; ratkidneyCE4, rat kidney CE4; ratlungCE3, rat lung CE3; ratliverCE1, CE2, and CE6REH, rat liver CE1, CE2, and CE6 retinyl ester hydrolase, respectively.

 
In addition, the multiprotein alignment analysis also showed that CES7 protein was apparently different from typical esterases expressed in other rat tissues [Fig. 1(A,B)]. CES7 protein showed low homology (40–60% identity) with other rat endoplasmic esterases, although all of them had the similar conserved domains of esterases.

The distribution, temporal and androgen regulation of Ces7 mRNA
The Ces7 mRNA of ~2.2 kb was transcribed in the distal and proximal region of the epididymis, but not detected in other tissues tested [Fig. 2(A)]. Moreover, the Ces7 mRNA was more abundant in the corpus and cauda region of the epididymis than that in the caput [Fig. 2(B)].


Figure 2
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  		Figure 2 The tissue distribution pattern of Ces7 (A) The Ces7 mRNA transcription in different tissues. Tissue RNA mixture of each lane loaded is 30 µg. (B) The Ces7 mRNA transcription in different regions of epididymis. Each lane is loaded 10 µg RNA. 18S ribosomal RNA was used for loading control.

 
Since sperm maturation was an androgen-dependent process [14], the androgen effect on the Ces7 expression was analyzed. During this experiment, the testosterone level declined rapidly and was almost undetectable until their supplement [Fig. 3(B)]. Whereas, Ces7 mRNA level did not apparently decrease on the first post-castration surgery day and was slowly down from the 3rd to the 7th day [Fig. 3(A,B)]. Testosterone supplement to the 7th-day animals after castration surgery resulted in a rapid increase in the serum testosterone concentration [Fig. 3(B)]. At the same time, the Ces7 mRNA levels followed the same trend as the serum testosterone out of the physiological level [Fig. 3(A,B)].


Figure 3
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  		Figure 3 Androgen manipulation and developmental pattern for Ces7 (A) Pre-castration control (Epi, 0d); bilaterally castrated for 1, 3, 5, and 7 days (1d–7d); 1, 3, 5, and 7 days (7+1d–7+7d) after the testosterone propionate applied to the 7d castrated rats. RNA mixture of each group was loaded. (B) The relative expression levels of Ces7 mRNA (hybridization density of Ces7 mRNA vs. 18S ribosomal RNA) in the rat epididymides and the serum testosterone level (nM) (>75 nM on 7+1d–7+7d). (C) The epididymis samples at 15d, 30d, 45d, 60d, 90d, 120d, 180d, 360d, 450d, and 720d, respectively, were collected and mixed. RNA samples were pooled from three to seven animals per groups. (D) The relative expression levels of Ces7 mRNA (hybridization density of Ces7 mRNA vs. 18S ribosomal RNA) in the rat epididymides at various development stages.

 
To determine whether Ces7 could be involved in developmental as well as in mature functions, Ces7 mRNAs were analyzed in animals of different ages. Ces7 mRNA could be detected at the age of 30 days or so; after that, they increased gradually and remained at a high level [Fig. 3(C,D)]. Therefore, it indicated that the expression of Ces7 increased in parallel with male development or termed sex maturation.

Expression profiles of CES7 protein
The polyclonal rabbit anti-sera against the recombinant fragment of CES7 protein, amino acid sequence from aa 352 to 419 showed in Fig. 1(A), red box, were raised. The titer of the anti-sera was over 106 as determined by ELISA. Western blotting showed that as low as 2 ng of the purified recombinant antigen could be detected by the anti-CES7 sera (data not shown).

The polyclonal anti-sera against CES7 peptide antigen could specifically recognize CES7 protein in the epididymis. And protein with lower molecular weight (MW) could be also weakly detected in other tissues tested such as testis, prostate, brain, and lung. Moreover, the CES7 proteins were abundantly expressed especially on the corpus and cauda region [Fig. 4(A,B)].


Figure 4
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  		Figure 4 Tissue distribution pattern and deglycosylation of CES7 (A) The CES7 protein expression in different tissues. Protein mixture of each lane loaded is 30 µg. (B) The CES7 protein expression in different regions of epididymis. Lumenal protein extracts loaded are 30 µg. Clusterin was used for loading control. (C) The molecular weight change of CES7 protein before (–) and after (+) deglycosylation treatment by PNGase-F. Primary CES7 anti-sera are diluted 1:20,000. Secondary antibody is diluted 1:15,000.

 
Based on the amino acid sequence of the CES7 protein, its MW of 61 kDa can be calculated (EDITSEQ, DNASTAR software), which is lower compared with the apparent MW of ~70 kDa. The MW of the deglycosylated CES7 was obviously lower than that of untreated CES7 and could be consistent with the calculated MW [Fig. 4(C)]. According to the Prosite database, there were four predicted N-glycosylation sites (amino acid sequences and sites: NVSD at aa 281–284, NEST at 363–366, NESY at aa 515–518, and NIST at aa 524–527) in CES7 protein.

Immunostaining results showed that the CES7 proteins were expressed in the epithelial cells of corpus and cauda epididymis. It is noteworthy that CES7 proteins were almost not localized in caput epididymal lumen, and nevertheless, they were greatly accumulated in the corpus or cauda lumen (Fig. 5). In these regions, the sperms were seemingly covered by CES7. But actually, CES7 protein could not tightly bind the sperm (data not shown). The binding affinity might be weak so that CES7 proteins were easily washed out from sperm surfaces in PBS buffer.


Figure 5
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  		Figure 5 The localization profiles of CES7 in epididymis (A) The expression pattern of CES7 in the whole rat epididymis. Scale bar = 2 mm. (B) The magnified photos of various epididymal regions: (a) initial segment, (b) caput, (c) corpus, and (d) cauda. Scale bars = 100 µm. (e) Magnified corpus. Scale bar = 20 µm. (f) The control: the background level of immuno-reactivity by pre-immune sera at the same condition.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 References
 
In common opinions, CEs should be responsible for the hydrolysis of exogenous or endogenous compounds such as drugs, pro-drugs, and xenobiotic. The functions of over-expressed CEs in male reproductive tract might be related to sperm protection and maturation. CE might hydrolyze glycerides to yield glycerol backbones of glycerol-phospho-choline, which are found in abundance in the epididymal fluids and are believed to serve as organic osmolytes [15,16]. Furthermore, the esterase 6 (EST6) enzyme of Drosophila plays a role in reproductive fitness [17,18]. CES7 as a CE in the epididymis could protect the spermatozoa far away from the chemical damage and also be involved in the similar functions to the above.

In the present study, Ces7 was scarcely sensitive to castration treatment. Nevertheless, with the same RNA sample and northern-blotting membrane, both the RNase9 and HongrES1 found in our lab obviously followed the trend of testosterone levels [7,8]. And the monkey epididymis-specific gene SC-615 homologous to Ces7 also shows testosterone-sensitive [19]. Ces7 transcription was perhaps up-regulated by testosterone and down-regulated by testis factors. On the other hand, inflammatory factors could also be involved in the Ces7 regulation (data not shown).

Recently, cauxin as a urinary CE in cat is also found to present in the epididymal fluid of various animals including cat, ram, boar, and mouse, which is responsible for the major esterase activity of the epididymal fluids in the ram [2]. Comparing the cDNA sequence of cauxin with that of Ces7, it was shown that Ces7 shared 73% identity with cauxin. Both CES7 and cauxin belonged to CE family and showed a relatively low degree of homology (only up to 60%) with other known endoplasmic CEs. Cauxin in the kidney and urine is considered to regulate felinine production, which could be involved in territorial marking [20]. But no functional information of the epididymal cauxin is reported until now.

CE family is considered to be products of ancestral gene duplication events [21]. And the conservation and tissue specificity of CES7 expression in male reproductive tracts of various species suggest its potential functions for sperm fertility. CES7 might have similar esterase activity as other esterases and also contribute to sperm use. In further studies, the Ces7 gene could be inactivated to observe its function in male reproduction.

In summary, Ces7 was an epididymis-specific gene, and CES7 protein was expressed and secreted to the lumens of corpus and cauda epididymis in mature rats. CES7 might be one of the major CEs in male reproductive tract and contribute to the sperm fertilization.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
References
 
This work was supported by grants from the National Basic Science Research and Development Project of China (2006CB504002 and 2006CB944002), the Chinese Academy of Sciences Knowledge Innovation Program (KSCX1-YW-R-54), the National Natural Science Foundation of China (30770473), and the State 863 High Technology R&D Project of China (2004AA221120).


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 

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