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BOR - Papers in Press, published online ahead of print June 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031740
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BIOLOGY OF REPRODUCTION 71, 1453–1460 (2004)
DOI: 10.1095/biolreprod.104.031740
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta)1

Maria Christina W. Avellar2,3, Luciana Honda3, Katherine G. Hamil4, Suresh Yenugu4, Gail Grossman5, Peter Petrusz5, Frank S. French4, and Susan H. Hall4

Section of Experimental Endocrinology,3 Department of Pharmacology, Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, SP 04044-020, Brazil Department of Pediatrics4 Department of Cell and Developmental Biology,5 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7500


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epididymis protein 2 (EP2) gene, the fusion of two ancestral ß-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

antibacterial, epididymis, epididymis protein 2 (EP2) gene, gene expression, human, male reproductive tract, prostate, rhesus monkey, seminal vesicles, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular sperm are unable to swim directionally or to recognize and fertilize an egg. These capabilities are achieved progressively as sperm mature during passage through the epididymis, an androgen-dependent organ, which is also the primary site of sperm storage in mammals [1]. Cell- and region-specific expression, transport, secretion, and absorption of luminal fluid components in the epididymis are able to create sequential changes in the composition of luminal fluid throughout epididymal length, contributing to the development and maintenance of fertile spermatozoa [2]. The epididymis-specific secretory proteins EP2/HE2/SPAG11 (epididymal protein 2/human epididymal 2/sperm-associated antigen 11) are a family of androgen-dependent proteins that bind spermatozoa and are likely involved in sperm maturation [2–4].

The human EP2 gene is located on chromosome 8 (8p23) embedded in a cluster of ß-defensin genes [5, 6]. EP2 spans 19 kilobases (kb), including 3 kb of proximal promoter region, two promoters and 8 exons originally thought to produce a highly human-epididymis-specific single transcript [7, 8]. The EP2 gene is now recognized as the target of regulation at multiple levels. Messenger RNA splicing mechanisms and alternative promoter selection (promoters A and B) generate at least 17 different transcripts reported in human and other primates [9–11]. Subsets of these transcripts are transcribed in different regions of the epididymis [11] and in different species; 9 reported in human and chimpanzee (EP2A-EP2I) [7, 9, 11] and 11 in rhesus monkey (Macaca mulatta) (EP2B, EP2C, EP2E, and EP2J-EP2Q) [11] (Fig. 1). Alternative splicing results in the use of different reading frames, further expanding the diversity of protein coding capacity. Exon E5 encodes portions of the C-termini of EP2A/B and EP2N in two different reading frames and encodes EP2K-EP2M in a third frame. Exon E6, on the other hand, encodes C-terminal regions of EP2A and EP2P in one reading frame, EP2G and EP2D/EP2E/EP2Q in another frame, and EP2J in a third frame. The fusion of two ancestral ß-defensin-like genes is proposed to have produced this complex transcription unit [5]. Although promoters A and B show no sequence similarity, both drive androgen-dependent gene expression [12]. Transcripts include those derived from exons downstream of both promoters, i.e., EP2A and EP2D, as well as independent transcripts containing exons adjacent to only one promoter, i.e., EP2C and EP2E [5, 8–10, 12, 13]. Thus far, exons E7 and E8 are present only in human EP2 transcripts. Whether their homologues are present in the rhesus genome remains to be demonstrated. Conversely, available data suggest that the rhesus variants EP2J-EP2Q are not transcribed in human or chimpanzee [10, 11], although the human genome does contain the additional exons (M1–M3) expressed in rhesus. In rodents, only the E form has been reported [5, 14, 15].



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  		FIG. 1. Genomic organization of the EP2 gene and alignment with alternative transcripts. EP2 alternative splicing variants from human (A) and rhesus monkey (B) are aligned with the EP2 exons present in the human chromosome at 8p23 (GenBank accession number AC134395). Colored boxes indicate the coding region of each variant. The 6 cysteine motif homologous to the active domain of ß-defensins, present in EP2C, EP2D, EP2E, and EP2Q protein variants, is indicated in red (*–*). GenBank accession numbers: human EP2A (AF168616), EP2B (NM-058206.1), EP2C (NM-058203.1), EP2D (AF168617); EP2E (NM-058207.1), EP2F (AF170797), EP2G (AF168618), EP2H (AF168619), EP2I (AF168620); rhesus monkey EP2B (AF466346.1), EP2C (AF466347.1), EP2E (AF466348.1), EP2J (AF466349.1), EP2K (AF466350.1), EP2L (AF466351.1), EP2M (AF466352.1), EP2N (AF466353), EP2O (AF466354.1), EP2P (AF466355.1), and EP2Q (AF466356.1)

Primate EP2 transcripts encode small proteins (4–21 kDa) with an N-terminal leader sequence characteristic of secreted proteins [9, 10]. EP2 transcripts initiated under the control of the A promoter encode a 46-amino acid common region between the signal peptide and the specific C-terminal peptides. The function of the common region is not clear because the whole mature EP2A, EP2D, and EP2G proteins and their C-terminal peptides exhibit similar antibacterial activity [16]. Function is suggested by homology of the 6 cysteine array to the ß-defensin antibacterial motif. But the complete motif is only present in two of the C-terminal peptides (EP2C and EP2D/EP2E/EP2Q). This motif is typically cationic in ß-defensins, but from their calculated isoelectric points (pIs), the EP2C and EP2D/EP2E/ EP2Q peptides are anionic in human and only the EP2E/ EP2Q form is cationic in rhesus monkey and rat. Thus, potent antibacterial properties characterize the EP2A, EP2D/EP2E/EP2Q, and EP2G peptides whether or not the C-terminal peptide is free, the complete 6 cysteine defensin-like motif is present, or the calculated pIs are basic. The common antibacterial property of these isoforms, independent of the structure and charge parameters that are considered to define ß-defensin activities, supports the view that host defense is one of the functions of the entire highly diverse EP2 protein family. The possibility of additional physiological functions is suggested by the presence of at least EP2A and EP2D isoforms on sperm surface [7, 9]. The recently demonstrated release of human DEFB126 from spermatozoa during capacitation suggests the function of this defensin may include blocking a receptor that mediates zona pellucida recognition and binding [17].

Several reports indicated expression of the EP2 gene exclusively in epididymis [7, 9, 14]. However, Jia et al. [6] reported the presence of variants EP2A and EP2D in additional tissues (i.e., gingival keratinocytes and bronchial epithelia), and von Horsten et al. [13] reported EP2A transcripts in testis. Furthermore, other male tissues are sites of abundant antimicrobial peptide expression in mammals [4, 18–20] and invertebrates [21]. In order to understand how broad EP2 expression is in the male reproductive tract, the expression of EP2 variants was profiled in the present study in different tissues from the human and rhesus monkey male reproductive tract. The results indicated that not only do human and rhesus differentially express EP2 transcripts in the epididymis, but these two species express different combinations of these transcripts in different tissues of the male reproductive tract. Analyses revealed the antimicrobial activity of full-length recombinant rhesus EP2C, EP2K, and EP2L, which were not recognized previously as bactericidal, and two novel variants, EP2R and EP2S, were discovered.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Human testis, epididymis, seminal vesicle, and prostate for RNA and immunohistochemical studies were made available by Dr. James L. Mohler, Department of Urology/Surgery, and by the Tissue Procurement Core Facility of the Lineberger Comprehensive Cancer Center, University of North Carolina (Chapel Hill, NC). Human tissues are not accompanied by identifying information and cannot be traced to the donor. Tissues were from patients ranging in age from 58 to 83 yr and were obtained with informed consent. Testis, epididymis, seminal vesicle, and prostate for RNA and immunohistochemical studies were obtained from adult rhesus monkeys 10–12 yr of age, with proven breeding history (Covance Research Products, Inc., Alice, TX).

Reverse Transcriptase-Polymerase Chain Reaction Analyses

Total RNA from human epididymis was isolated by the method of Chirgwin et al. [22]. Total RNA from human and rhesus monkey testis, seminal vesicle, and prostate, as well as rhesus monkey caput epididymis was isolated with Trizol (Gibco-BRL, Gaithersburg, MD) according to manufacturer's instructions. Polyadenylated [poly(A)+] RNA from rhesus monkey seminal vesicle was prepared using Oligotex mRNA midi kit (Qiagen, Valencia, CA) according to manufacture's instructions. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was performed using ThermoScript RT-PCR system for first-strand cDNA synthesis according to the manufacturer's instructions (Gibco-BRL). Oligo(dT)-primed cDNA was synthesized from 5 µg of total RNA from human and rhesus monkey tissues for 1 h, at 55°C, in a reaction volume of 20 µl. The resulting cDNA (2 µl) was amplified by PCR in a final volume of 25 µl containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.4 mM dNTPs, Taq polymerase (Gibco-BRL), and 0.4 µM of each (sense and antisense) specific primer. Primers were designed on the basis of the originally published cDNA sequence for the EP2 gene transcripts (GenBank accession numbers in legend for Fig. 1) in order to amplify PCR products that span at least one intron (Fig. 2). The following forward primers were used: 53F (5'-AGGCAACGATTGC-3'), 53F1 (5'-AATACGGCGCATCCCCTA-3'), and EP2B/EP2E-specific primer 53F2 (5'-GGGAGGTTCAACGGACCTTA-3'). The following reverse primers were used: 53R (5'-CATACGGCAGATGG-3'), 53R1 (5'-CAAGCCACACATTCCTTTAAGC-3'), 57R2 (5'-TTATCCACAGCGTTGTC-3'), and EP2C-specific primers 58R1 (5'-TATCCACATTTACTAGCACAAATTG-3') and 58R2 (5'-TGAACTGTCTGGCACAAGTGGC-3'). PCR protocols generally consisted of an initial cycle of 1 min at 95°C, followed by 35 cycles of 1 min 95°C, 1 min 60°C, and 1 min 72°C, and a final extension of 3 min 72°C. Aliquots of the DNA samples (18 µl) were loaded onto agarose gels (1.8%) containing ethidium bromide (0.5 µl/ml). PCR products were visualized with fluorescent illumination and photographed. Amplicons were subcloned into pCRII (Invitrogen, San Diego, CA) and their identity verified by automated sequencing.



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  		FIG. 2. Positions of EP2-specific forward and reverse primers used in the RT-PCR studies within the genomic organization of the EP2 gene locus

PCR Screening of cDNA Libraries

A directional cDNA library from adult rhesus monkey seminal vesicle [poly(A)+] RNA was constructed in {lambda}ZAPII (Stratagene, Cedar Creek, TX) according to the manufacturer's instructions. The primary library was 98% recombinant and contained 2.6 x 106 unique clones with an average insert size of 1.2 kb. The library was amplified once. Aliquots of a directional cDNA library, constructed from human caput/corpus epididymis in {lambda}ZAPII as described previously [9], were also used. Aliquots of the libraries (5 µl) were screened by PCR analysis, using one of the EP2-specific primers and phage primers T3 (5'-AATTAACCCTCACTAAAGGG-3') or T7 (5'-GTAATACGACTCACTATAGGGC-3') in order to amplify 5' and 3' ends of inserted clones, respectively. Reactions were performed in a final volume of 25 µl, with PCR conditions as described above.

Automated Sequencing

The authenticity of each target gene PCR product was confirmed by direct nucleotide sequencing performed with an ABI PRISM 377 automated sequencer (Applied Biosystems, Foster City, CA) and BigDye Terminator Sequencing kit (Applied Biosystems) at the DNA sequencing facility located at INFAR, Universidade Federal de São Paulo. Insert-contained clones from pCRII were sequenced with vector primers (M13R and M13F).

Immunohistochemical Studies

Tissues for immunohistochemistry were fixed in Bouin solution (75 ml saturated picric acid, 5 ml glacial acetic acid, 25 ml 37% formaldehyde) promptly after excision and embedded in paraffin as previously described [9]. Sections were heated in a microwave oven in 0.01 M citrate, pH 6.0, for antigen retrieval [23]. Sections were stained using antiserum raised in rabbit against the human EP2D C-terminal peptide (CVSNTDEEGKEKPEMDGRSGI) [9]. This peptide is also present in EP2E, EP2Q, and EP2R. For the control staining, antibody was preincubated with this antigen peptide. A Vectastain Standard ABC kit (avidin-biotin-complex horse radish peroxidase) (Vector Laboratories Inc., Burlingame, CA) was employed to demonstrate immunoreactive EP2 using diaminobenzidine as chromogen, resulting in a brown reaction product. Sections were counterstained with toluidine blue. Photographs were taken with a SPOT Cooled Color digital imaging system (Diagnostic Instruments, Inc., Sterling Heights, MI) attached to a Zeiss Photomicroscope III. Photographs were prepared using SPOT image processing software and a Gateway 2000 E-500 computer. Images were arranged using PhotoShop (Adobe Systems Inc., San Jose, CA).

Recombinant Protein Production

Recombinant proteins were prepared as described previously [9] with minor modifications as follows. In brief, Escherichia coli strain OrigamiB-laqIQ (Novagen, Madison, WI) was transformed with modified pQE-80mod vector (Qiagen) containing the following cDNAs: full-length human and rhesus EP2C, full-length rhesus EP2L and EP2K, and grown on Circlegrow media (Qbiogene, Carlsbad, CA) containing 1% glucose, 50 µg/ml carbenicillin, 15 µg/ml kanamycin, 12.5 µg/ml tetracycline, and 34 µg/ml chloramphenicol. Fusion-protein expression was induced with 1 mM isopropyl ß-D-thiogalactoside for 1 h at 37°C. The plasmid pQE-80mod was created by replacing the His tag of pQE-80L with that of the pQE-2 vector. The sequence of the His-tag is MRGSHHHHHHGS. Bacterial lysate, incubated with Ni2+-nitrilotriacetate-agarose (Qiagen) was transferred to a column, washed, and eluted according to the manufacturer's recommendations. Fractions were analyzed on 4–12% gradient NuPAGE gels run in MES buffer (Invitrogen, Carlsbad, CA) and stained with Coomassie Blue G250. Fractions containing purified protein were pooled and dialyzed against 10 mM sodium phosphate buffer (pH 7.4) to remove urea. Recombinant protein yields for EP2C and EP2L/EP2K were 150–200 and 50–100 µg/g cells, respectively.

Antibacterial Assays

The colony forming units (CFU) assay was performed to test the antibacterial activity as described previously [16]. Briefly, overnight cultures of E. coli XL-1 were grown to midlog phase (A600 = 0.4–0.5) and diluted with 10 mM sodium phosphate buffer (pH 7.4). Approximately 2 x 106 CFU/ml of bacteria were incubated at 37°C with 10–100 µg/ml of each recombinant proteins tested. Aliquots of the assay mixture were removed at 2 h after start of incubation, serially diluted with 10 mM sodium phosphate buffer (pH 7.4), and 100 µl of each was spread on a Luria-Bertani agar plate and incubated at 37°C overnight to allow full colony development. The colonies were hand counted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of EP2 Transcripts in Different Tissues from Human and Rhesus Monkey

The distribution of EP2 transcripts in different tissues in rhesus and human revealed that alternative splicing of these mRNAs produces a unique profile in each tissue, implicating tissue- as well as species-specific regulation of EP2 mRNA splicing mechanisms. In the human testis, amplification suggested relatively high abundance of transcripts EP2B, EP2E, EP2A, and EP2D when specific primers were used (Fig. 3). In human epididymis, amplification of transcripts EP2C, EP2B, EP2E, EP2A, EP2D was detected. In human prostate, only EP2C mRNA was amplified, while in human seminal vesicle, low levels of EP2B, EP2E, and EP2A transcripts were detected (Fig. 3). No amplification of EP2C was detected in human testis or seminal vesicle (Fig. 3).



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  		FIG. 3. Analysis of EP2 splicing variants in different male reproductive tissues of adult human and rhesus monkey. Specific pairs of primers used during PCR and specific EP2 DNA products obtained are indicated in each lane. Lane 1: primers 53F1/58R1 and 53F/58R2 amplified EP2C (426 and 295 bp, respectively); lane 2: primers 53F2/53R1 amplified EP2B (411 bp) and EP2E (336 bp); lane 3: primers 53F/53R1 amplified human EP2A (547 bp) and EP2D (469 bp); primers 53F1/53R1 amplified rhesus EP2L (623 bp), EP2K (438 bp), EP2J (360 bp), EP2N (497 bp), EP2R (341 bp), and EP2S (549 bp). Letters indicate most abundant EP2 DNA products. Faint fluorescent EP2J, EP2N, and EP2S DNA products in rhesus epididymis and seminal vesicle visualized during gel analysis were also identified as specific EP2 transcripts, as described in the Results. MW indicates a 100 bp standard DNA ladder. Arrow indicates 600 bp

The analysis of the human EP2 sequences obtained indicated only one nucleotide difference when compared with sequences available in GenBank. This polymorphic site was present in exon E7 (C -> T), resulting in changes in amino acid sequence composition depending on the variant analyzed. In the EP2A variant, this change occurs in nucleotide (nt) 470, causing no change in the predicted amino acid. In variants EP2D (nt 394) and EP2E (nt 120), the predicted amino acid changes from Pro -> Leu, while in variant EP2B (nt 197), the nucleotide is present in the noncoding region. The functional importance of this polymorphism is not known.

In rhesus monkey (Fig. 3), EP2C was the only EP2 transcript amplified when RNA from testis was analyzed. In the corpus epididymis, expression of EP2C, EP2B, and EP2E was detected. When primers 53F1/53R1 were used, major DNA products were visualized and identified by sequencing as EP2L and EP2K variants. Faint DNA products amplified by the same pair of primers were also observed during gel analysis, but are not visible in Figure 3. Cloning and sequencing of these amplicons identified these bands as EP2J and a new EP2 splice variant (named EP2R) containing sequences from exons E2, E3, and E6 (Fig. 4). In the rhesus prostate, EP2C, EP2L, and EP2K were detected. In the rhesus seminal vesicle, strong amplification of EP2C suggesting high transcript abundance and low levels of EP2B transcripts were detected in bands too faint to reproduce in Figure 3. Several products were also amplified in this tissue using primers 53F1/53R1. After subcloning and sequencing, the most highly amplified sequences were identified as the EP2L, EP2K, and EP2R message variants. Less abundant products, observed during gel analysis but not clearly shown in the gel picture, were also identified by cloning and sequencing as EP2N and another new EP2 splice variant (named EP2S), corresponding to sequences contained in exons E2, E3, M2, M3, and E6 (Fig. 4). Variants EP2A and EP2D, the most abundant variants in human epididymis, were never amplified from rhesus monkey tissues. No EP2 splice variants were detected in rhesus tissues when the specific primer to exon E8 (57R2), active in the human EP2 gene, was used (data not shown). Thus, EP2C was the only EP2 splice variant detected in all tissues analyzed from the rhesus monkey. These results are consistent with greater activity at the A promoter than the B promoter in rhesus, as noted previously for human [5]. However, the possibility that undetected isoforms are produced, but at very low levels, cannot be ruled out. Previously reported rhesus isoforms EP2M, EP2O, EP2P, and EP2Q [11] were not detected in this study. The expression levels of these mRNAs were probably lower in the particular rhesus monkey used in this study. These low levels may reflect individual variation in physiologic state. Amplicons unrelated to EP2 appear in these gels similar to those previously reported [11].



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  		FIG. 4. Exon structure of novel EP2 variants R and S. The arrangement of exons observed for the new variants detected in rhesus monkey is aligned with the genomic structure. The GenBank accession numbers for EP2R and EP2S are AY513586 and AY513587, respectively

Few polymorphic nucleotide positions were observed when rhesus monkey EP2 sequences obtained in the present study were compared with sequences available in the GenBank database. Two nucleotide differences were present in exon E2 of variants EP2C, EP2L, EP2K, EP2N, and EP2J (nt 171 A -> G and nt 196 T -> G), leading to changes in amino acid composition (Arg -> Gly and Val -> Gly, respectively). Variant EP2C also presented changes in three more nucleotides in exon E3 corresponding to positions 323 (T -> A), 350 (C -> G), and 437 (A -> G). The changes in positions 323 and 350 were silent, while the nt 437 was present in the 3' noncoding region. In the EP2J variant, nt 774 in exon E6 changed from G -> A, which modifies the last predicted amino acid (Met -> Ile). In the other variants (EP2L, EP2K, EP2N, EP2B, EP2E), the same G -> A change occurred in the noncoding region. The functional impact of these polymorphisms is not known.

PCR screening of the rhesus seminal vesicle cDNA library allowed the amplification of clone inserts corresponding to full-length EP2C, EP2L, EP2K, EP2B, EP2E variants. Sequencing of the amplified DNA products confirmed the same nucleotide changes identified in EP2 DNA sequences during RT-PCR studies. Although PCR screening of this cDNA library with primers 53F1/53R1 was effective to amplify the new EP2 splice variants EP2R and EP2S, predicted 5' ends of these variants were not amplified from the library or from freshly isolated RNA. The amino termini of EP2R and EP2S probably correspond to those of EP2C, EP2K, and EP2L (Fig. 5). In the absence of the 5' mRNA sequences for the R and S variants, the reading frame cannot be assigned with certainty. However, because all known transcripts that contain exon 2 are translated in the same reading frame, the translation indicated in Figure 5 is the most likely one. A comparison of the predicted amino acid sequences for EP2R and EP2S with the different known rhesus EP2 proteins indicated that the C-terminal region of EP2R contains the predicted six cysteine residues similar to the ß-defensins and present also in the EP2Q and EP2E variants (Fig. 5). All the rhesus EP2 sequences obtained in this study were deposited in GenBank under accession numbers AY513586, AY513587, and AY528233– AY528239.



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  		FIG. 5. Alignment of the EP2R and EP2S amino acid sequences with known rhesus EP2 protein sequences. The predicted 6 cysteine residues similar to the ß-defensins are indicated by underlined letters

To determine if EP2 transcripts in the seminal vesicle are translated, tissue sections were immunostained using a polyclonal antibody that recognizes the EP2D, EP2E, EP2Q, and EP2R isoforms. Consistent with the transcript amplification analyses, specific staining in rhesus monkey and human seminal vesicle was located in the epithelial cells and in the lumen of the secretory tissue (Fig. 6). Of the isoforms detectable by the antibody, only EP2E was detected in human and EP2R in monkey seminal vesicle by PCR and cloning (Fig. 3), suggesting that these are the isoforms responsible for the immunostaining.



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  		FIG. 6. Localization of EP2 protein in rhesus monkey (A and B) and in human seminal vesicle (C and D). EP2 was detected within the cytoplasm of the secretory epithelium and in the luminal spaces (A and C). Immunodetection was abolished when the antibody was preincubated with antigen peptide (B and D). Sections were counterstained with toluidine blue. Photographs were taken using a 20x objective

EP2 Antimicrobial Activity

Antibacterial activity of the most widely expressed isoform in this study, EP2C, has not been reported in spite of the presence of the ß-defensin-like 6 cysteine array. A function for EP2K and EP2L could not be inferred from the amino acid sequence as the 6 cysteine array is not present in its entirety and the predicted EP2K and EP2L proteins exhibit no homology with any known proteins. To test these isoforms for antimicrobial activity, a 10-fold concentration range of full-length recombinant rhesus EP2C, rhesus EP2K and EP2L was incubated with E. coli for 2 h (Fig. 7). More than 99.9% of bacteria were killed by 50–100 µg/ ml of each protein. The control protein BSA showed no detectable antibacterial activity when incubated for 2 h at the same concentrations (data not shown).



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  		FIG. 7. Potent antibacterial activity of EP2 isoforms. Survival of E. coli was greatly diminished by a 2-h incubation with recombinant EP2 proteins: rhesus full-length EP2C (black triangle), EP2K (black inverted triangle), and EP2L (black circle)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates species- and tissue-specific EP2 alternative mRNA splicing in epididymis, testis, seminal vesicle, and prostate of human and rhesus macaque. Not only do human and rhesus differentially express EP2 transcripts in the epididymis, but these two species express different combinations of these transcripts in different tissues of the male reproductive tract. Based on the present results and established mechanisms, including species- and tissue-specific transcription, epididymal segment-specific transcription, and alternative splicing as well as alternative promoter selection [7, 9–11, 14], the concept emerges of a finely tuned system that produces different forms of EP2 in different cellular contexts. Two new EP2 splice variants were reported in rhesus tissues, EP2R and EP2S, that bring to 19 the number of different transcripts reported in human and other species [8, 9, 24]. These novel isoforms raise the possibility that still more exonic combinations await discovery. The present report showing bactericidal function for EP2C, EP2K, and EP2L proteins together with reports of the antimicrobial activities of cystatin 11 [25], lactoferrin [26], cathelicidins [27], ß-defensins [28, 29], and epididymis sperm-binding protein ECS42 [30] and EP2/HE2 gene [13, 14, 16] provide strong evidence that numerous secretory proteins of the male reproductive tract contribute to the defense against bacterial infection.

Despite growing interest in how alternative splicing is regulated [31–33], relatively little is known about tissue-specific alternative splicing and its molecular determinants. Although the number of different mechanisms controlling EP2 gene expression is extraordinary, each by itself has been previously reported for different genes in the male reproductive tract and/or elsewhere. Read-through transcription joins the epididymis-specific lipocalin 6 with the adjacent lipocalin-like gene [34] and a eukaryotic initiation factor gene joins the mammalian homologue of Drosophila MASK (multiple ankyrin repeats, single KH domain) [35]. The latter gene fusion is also an example of alternative mRNA splicing and reading frame usage. Splice variants specific to the male system are widely reported [36–39], and underlying mechanisms of this specificity are under investigation [40–42]. Similarly, the mechanisms controlling alternative promoter usage in the EP2 gene are unknown. However, increasing reports describe the combination of alternative promoter selection and mRNA splicing in developing germ cells [43, 44], neuronal communication [45, 46], and cancer [47, 48].

Functional alternative reading frames are not common but have been reported in lymphocytes [35], prostate [38], and neuroendocrine cells [49]. Alternate reading frame usage is sometimes thought to indicate coding for inactive proteins because of the lack of amino acid sequence homology with the original protein [47]. However, whether exon E6 is translated in the EP2A or EP2D/EP2E/EP2Q reading frames, the proteins kill bacteria through a mechanism that involves outer and inner membrane disruption and cell death [16]. Among the rhesus EP2 message variants that contain exon E3 sequence, only EP2R, a new rhesus variant identified in the present study, and EP2C code for a complete ß-defensin-like peptide. Because of the use of earlier splicing sites in exon E3, the other EP2 variants (EP2J-EP2P) code for partial ß-defensin-like peptides that have only the proximal three of the six cysteines characteristic of ß-defensins [11]. Interestingly, the present work demonstrates that EP2C, EP2K, and EP2L have antibacterial activity, confirming that this function is maintained whether the encoded peptide contains a complete or a partial ß-defensin-like sequence. Future studies will address the question whether C- and N-terminal EP2C amino acids are modified or removed posttranslationally or after secretion, as described previously for human EP2A (HE2{alpha}1) and EP2D (HE2ß1) cleavage by a furin-like proprotein convertase [13].

Promoter selection by transcription complexes also exhibits species- and tissue-specific regulation. In chimpanzee epididymis, promoter B of the EP2 gene is equal to or more active than A, whereas in human epididymis, promoter A is more active as judged by the relative abundance of clones in libraries [9]. In human seminal vesicle, variants EP2B and EP2E, originating from promoter B, represent two of three transcript variants and, in human testis, two of four transcript variants. In terms of the total numbers of variant transcripts, far more originate from promoter A than from B. From promoter A, variants EP2F-EP2I have not been detected in chimpanzee and rhesus, while variants EP2A and EP2J-EP2Q have been only detected in human and rhesus monkey, respectively [7, 9, 10, present results]. The mechanisms underlying species- and tissue-specific regulation of EP2 transcript expression are not known. The expression of EP2 splicing variants may be regulated in response to specific cell factors, hormones, and/or extracellular signals. In fact, previous studies have shown that rat and nonhuman primate EP2 gene expression is positively regulated by androgens [9, 14].

Although the level of mRNA in a given tissue does not always correlate with the levels of protein, the location of immunoreactive EP2 observed in the present study in epithelial cells of rhesus and human seminal vesicle confirmed that the synthesis of EP2 protein is not limited to epithelial cells of the epididymis. Immunostaining was performed with a polyclonal anti-EP2 antibody that recognizes EP2D, EP2E, EP2Q, and EP2R isoforms. Taking into consideration the number of EP2 message variants identified by molecular studies in the rhesus and human, selective antibodies will be an important tool to confirm abundance and possible cell populations presenting specific expression of each EP2 isoform in the human and rhesus monkey male reproductive tract.

One question raised by these results is, "What evolutionary forces drove the selection and maintenance of so many variants of EP2?" One possibility is that each variant evolved to control a different subset of microorganisms. The environment in each organ may be conducive to proliferation of different microbial species. In addition to their direct antimicrobial actions, EP2 isoforms may also perform cellular functions needed to combat the consequences of bacterial colonization. The multiple functions of other defensins and the cathelicidins indicate activities involved in integrated mechanisms that go beyond direct pathogen killing to include stimulation of adaptive immunity, wound healing, angiogenesis, and tissue repair as innate host defense responses [50, 51]. The expression of EP2A [3] and EP2D/EP2E [9] isoforms in the male reproductive tract directs attention to the possibility that EP2 isoforms have additional roles in the maturation as well as protection of spermatozoa. Whereas the underlying physiology appears to be comparable in human and rhesus EP2 variants in terms of antimicrobial activity, it will be important to understand if EP2 protein products display other species-specific physiological roles beyond innate immunity and proinflammatory response and, if so, how these mechanisms are involved in sperm maturation, sperm survival, or fertilization mechanisms. EP2 may perform distinct functions in different tissues and, consequently, may have other physiological functions.


   FOOTNOTES
 
1 Supported partially by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 99/1777-1), Brazil, and by the T.W. Fogarty International Center for Training and Research in Population and Health, USA D43TW/HD00627 (subcontract UNIFESP/UNC 5-53284). Support was provided by the CICCR Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR. This work was also supported by grants from the Andrew W. Mellon Foundation and the National Institutes of Health (R37-HD04466, through cooperative agreement U54-HD35041). Back

2 Correspondence: Maria Christina W. Avellar, Section of Experimental Endocrinology, Department of Pharmacology, Universidade Federal de São Paulo—Escola Paulista de Medicina, Rua 03 de maio 100, INFAR, Vila Clementino, São Paulo, SP 04044-020, Brazil. FAX: 55 11 5576 4448; avellar{at}farm.epm.br Back

Received: 6 May 2004.

First decision: 2 June 2004.

Accepted: 16 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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