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Male Reproductive Tract |
Department of Physiology, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gene regulation, male reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the human and chimpanzee epididymis, the epididymal protein 2/human epididymal protein 2/sperm-associated antigen 11 (EP2/HE2/SPAG11) gene is transcribed into at least nine message variants EP2A–EP2I [12, 14–16]. These message variants encode eight small peptides of 4–11 kDa that have different amino acid sequences [12, 16]. The differences in amino acid sequences result from the alternative use of two promoters (promoters A and B) and eight exons (exons 1–8) and the inclusion or exclusion of exon 5 that shifts the open reading frame of the subsequent exon. Because of this shift, the same 3'-terminal cDNA sequence can result in two different C-terminal peptide sequences.
In the human and chimpanzee, each of the EP2 peptides encoded by the nine EP2 message variants contains a leader sequence characteristic for a secreted protein. There are two leader sequences, one encoded by exon 1 and one encoded by exon 4. After removal of the leader sequence, the peptides consist of combinations of four major peptide modules of 20–40 amino acids and three minor peptide modules of 3–10 amino acids [12, 16]. Among the EP2 peptides, EP2A (HE2
1) consists of modules 1 and 2, EP2B consists of module 2, EP2C consists of modules 1 and 3, EP2D (HE2ß1) consists of modules 1 and 4, and EP2E consists of module 4. Modules 1 and 2 have no recognizable similarity to known proteins or protein fragments. Modules 3 and 4 have a distribution of cysteine residues characteristic for ß-defensins, a family of small peptides with antibacterial activity [17].
An antibacterial function for the EP2 peptides is suggested by the localization of the EP2 gene to human chromosome 8 within a cluster of ß-defensin genes [18–20]. It is hypothesized that the primate EP2 gene originated from the fusion of two ancestral ß-defensin genes [19]. Of the eight known exons of the human EP2 gene, four are derived from the ancestral ß-defensin genes and four were acquired during the gene's evolution. Two of these four acquired exons encode the non-ß-defensin-like peptides EP2A and EP2B. Thus, during its evolution, the EP2 gene evolved new peptides in addition to those of the classical ß-defensins. Moreover, these new peptides are the major message variants observed in human and chimpanzee [12, 16]. The antibacterial function of the EP2 gene has been demonstrated for EP2 peptide message variants EP2A and EP2B [21] and EP2E [22].
EP2 message variants in human and chimpanzee are >98% identical with respect to both mRNA and peptide sequences [16]. Although the EP2 message has been demonstrated in rhesus epididymis [12], it is not known which of the message variants are expressed. Therefore, we analyzed rhesus epididymal RNA by Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) to compare rhesus EP2 messages to those in human and chimpanzee.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epididymides were obtained from three adult male rhesus monkeys (Macaca mulatta) killed at the Yerkes National Primate Research Center of Emory University for reasons unrelated to this study. All experimental procedures were conducted according to the guidelines stated in the United States Public Health Service's Guide for the Care and Use of Laboratory Animals and as approved by Emory University's Institutional Animal Care and Use Committee. Epididymides were subdivided into 1) initial segment, 2) proximal caput, 3) distal caput, 4) proximal corpus, 5) distal corpus, 6) proximal cauda, 7) distal cauda, and 8) vas deferens [23], and the segments were subdivided into 100- to 200-mg pieces. All tissues were frozen immediately in liquid nitrogen and stored at -80°C prior to use.
PCR Analysis
Total RNA was isolated by acid phenol extraction [24] from frozen segments of rhesus monkey epididymis. Aliquots of 1 µg total RNA were reverse transcribed using the SuperScript preamplification system (Life Technologies, Gaithersburg, MD) with oligo-dT as primer. To amplify the EP2 message variant EP2A, PCR was performed using the forward primer EP2PCR3 (5'-AGA CAT GAG GCA ACG ATT GCT CC-3') and the reverse primer EP2PCR4 (5'-GGG ATC AGA GCA AAT GTC ACG C-3'). To amplify the EP2 variant EP2B, PCR was performed using the forward primer EP2PCR5 (5'-GGC AGG GAG GTT CAA CGG AC-3') and the reverse primer EP2PCR4 [16]. These primers were designed to give PCR products that span at least one intron; their location within the genomic sequence is given in Figure 6. The PCR protocol used RedTaq (Sigma, St. Louis, MO) and comprised 30 cycles of 1 min at 90°C, 1 min at 58°C or 60°C and 1 min at 72°C. The PCR amplification products were resolved electrophoretically in a nondenaturing Tris-acetate-EDTA (TAE)-buffered 8% polyacrylamide (40:1 acrylamide:bis) gel and developed with ethidium bromide. They were inserted into pGEM-T Easy (Promega, Madison, WI) and were sequenced by the DNA Sequencing Facility of Emory University.
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Determination of 5'- and 3'-Ends by Rapid Amplification of Complementary DNA
The 5'- and 3'-terminal sequences of the rhesus EP2 message variants recognized by PCR were identified using a rapid amplification of complementary cDNA (RACE) protocol (Marathon cDNA synthesis kit, Clontech, Palo Alto, CA). Total RNA was reverse transcribed with oligo-dT as primer. Using the protocols and reagents provided, single-stranded cDNA was converted to double-stranded cDNA and adapters were ligated onto the double-stranded DNA. These adapters contain a sequence recognized by the adapter primer AP1 (5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3'). PCR using an adapter primer (AP1) as forward primer and EP2PCR4 as reverse primer yielded the 5'-ends, and PCR using EP2PCR3 or EP2PCR5 as forward primer and AP1 as reverse primer yielded the 3'-ends.
Northern Hybridization Analysis
Total RNA was analyzed using the NorthernMax system kit (Ambion, Austin, TX). The RNA (10 µg/lane) was separated electrophoretically in glyoxal-containing 1.5% agarose gels and capillary blotted onto BrightStar-Plus charged nylon membranes (Ambion). The blots were hybridized using 32P-labeled antisense cRNA probes.
Hybridization probes for rhesus EP2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared using the Strip-EZ kit (Ambion) and 32P-CTP. The EP2 probe was transcribed using a plasmid containing a 673-bp PCR product obtained using forward primer EP2PCR3 and reverse primer EP2PCR4. This 673-bp PCR product, which corresponds to the rhesus message variant EP2L, contains exons 1, 2, 3, M2, M3, 5, and 6 and should therefore hybridize to all known EP2 message variants. The GAPDH probe was transcribed using the p-Tri-GAPDH plasmid provided in the Strip-EZ kit (Ambion).
Membranes were preincubated with prehybridization/hybridization solution for 1 h. Hybridization probes were added to the solution, and the membranes were incubated overnight at 63°C. Unbound probe was removed by washing, and the membrane was exposed to a storage phosphor screen (PhosphorImager SI, Molecular Dynamics Inc., Hayward, CA). Before reprobing, membranes were stripped using the solutions and protocol provided in the Strip-EZ kit (Ambion).
Deposition of New EP2 Sequences in the Databases
The newly discovered rhesus EP2 sequences have been deposited in Genbank under accession numbers AF466346 through AF466356.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the segmental expression of EP2 in rhesus epididymis, we analyzed total RNA extracted from eight epididymal segments by Northern hybridization, using a probe expected to hybridize to all known EP2 message variants. As shown in Figure 1, in rhesus epididymis the strongest EP2 message signals appear in distal caput (lane 3) and proximal corpus (lane 4) epididymidis. In proximal corpus (lane 4) and distal corpus (lane 5), the signal consists of at least two bands. The top band that is present in all lanes has an approximate size of 0.9–1.0 kb.
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We analyzed rhesus epididymal total RNA by RT-PCR using primers EP2PCR3 and EP2PCR4 to amplify EP2 variants EP2A and EP2D, which are transcribed off promoter A, and using primers EP2PCR5 and EP2PCR4 to amplify EP2 variants EP2B and EP2E, which are transcribed off promoter B [16]. As shown in Figure 2, amplification of rhesus cDNA using the first primer set produced a pattern of PCR products that was quite different from that of chimpanzee, whereas amplification of rhesus cDNA using the second primer set produced a pattern of PCR products essentially identical to that of chimpanzee [16].
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We also analyzed rhesus epididymal RNA from eight epididymal segments using primers EP2PCR3 and EP2PCR4, and EP2PCR5 and EP2PCR4. As shown in Figure 3, using primers EP2PCR3 and EP2PCR4, the pattern of PCR products varies in different epididymal segments. Distal caput (lane 4) and proximal corpus (lane 5) epididymidis express more of the smaller PCR products than the more proximal (lanes 2 and 3) or more distal (lanes 6–8) segments of the rhesus epididymis. Using primers EP2PCR5 and EP2PCR4, the pattern of PCR products in each of the segments was the same as that shown in Figure 2B, lane 3.
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Rhesus-Specific EP2 Message Variants
By cloning and sequencing the RT-PCR products, we identified 11 EP2 message variants in rhesus epididymis. Among these, we identified rhesus homologs of message variants EP2B and EP2E [16]. Using 3'-RACE experiments to obtain the 5'- and 3'-ends of EP2 message variants, we also found the rhesus homolog of EP2C. As shown in Figure 4, rhesus EP2B peptide is considerably shorter than human EP2B peptide (50 and 29 residues, respectively, including the leader sequence) because of the presence of an earlier stop codon in the rhesus cDNA. Rhesus EP2C and EP2E peptides have the same length as their human equivalents. However, because it uses a later polyadenylation signal, rhesus EP2C message is longer than human EP2C message. Between rhesus and human, EP2B, EP2C, and EP2E peptides together show 87% sequence identity.
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The eight new EP2 message variants in rhesus epididymis, having no known human or chimpanzee counterparts, are designated EP2J–EP2Q (Fig. 5). At their 5'-end, EP2J–EP2Q use exon 1, which encodes the same leader sequence as in EP2C. At their 3'-end, they use exon 6, which encodes the ß-defensin-like peptide EP2E [16]. However, EP2J–EP2Q differ in their use of internal exons 2, 3, M1, M2, M3, and 5. Use of these exons can shift the open reading frame. Therefore, only in EP2Q is the 3'-terminal exon read in the same reading frame as in EP2E. In EP2Q, exon 1 is followed immediately by exon 6. Furthermore, EP2J–EP2P use one of two previously unknown splicing donor sites within exon 3. These splicing donor sites are located 41 and 62 bases upstream of the stop codon used in rhesus EP2C. As a consequence, EP2J–EP2P encode partial ß-defensin-like peptides that contain only the first three of the six ß-defensin-defining cysteines.
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In addition to the previously unknown splicing sites in exon 3, EP2L–EP2O use three previously unknown exons M1–M3. However, sequence comparison shows that exons M1–M3 have highly homologous counterparts in the human EP2 gene. Exons M1–M3 are located within the 9-kb intron located between exons 3 and 4 of the human gene (Fig. 6). Overall, the sequences of the nine exons identified in the rhesus EP2 variants are 92% identical on the DNA level with their corresponding human sequences.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is only moderate overlap among the 11 EP2 message variants detected in rhesus epididymis, the six message variants detected in human epididymis [12], and the five message variants detected in chimpanzee epididymis [16]. Only three of the EP2 message variants, EP2B, EP2C, and EP2E, are found in rhesus, human, and chimpanzee. EP2A, the most abundant variant in human epididymis [12, 14, 16] was not detected in rhesus epididymis.
In human/chimpanzee epididymis, the nine message variants (EP2A–EP2I) use eight exons (exons 1–8), of the EP2 gene. In rhesus epididymis, the 11 message variants (EP2B, EP2C, EP2E, EP2J–EP2Q) use nine exons (1–3, M1-M3, 4–6). In rhesus we detected no variants that use equivalents of exons 7 or 8 of the human EP2 gene [19]. Although our use of reverse PCR primer EP2PCR4 (located in exon 6) would not detect such variants, our 3'-RACE experiments, using EP2PCR3 (located in exon 3) or EP2PCR5 (located in exon 5) as forward primer, could have amplified variants using exon 7 or 8. Using 3'-RACE experiments with EP2PCR3 (located in exon 1) as forward primer did amplify the 3'-end of rhesus EP2C, located in exon 3. Therefore, variant EP2A and variants using exon 7 or 8 homologous to human EP2F-EP2I (HE22, HE2ß2, HE2
1, HE22) [12, 16] do not appear to contribute significantly to rhesus epididymis EP2 splicing variants. Conversely, rhesus epididymis EP2 message variants EP2J–EP2Q were not detected in human/chimpanzee epididymis [12, 16]. We do not exclude the possibility that EP2A is transcribed in rhesus epididymis or that EP2J–EP2Q are transcribed in human/chimpanzee epididymis. However, if they are transcribed, it is at levels so low that they are not readily detected as PCR amplification products.
The features that distinguish rhesus EP2 message variants from human/chimpanzee EP2 message variants are the rhesus-specific use of two 5'-donor splicing sites, located in exon 3, and the use of exons M1–M3 located in the intron between exons E3 and E4. Exon E3 codes for the ß-defensin-like module 3 of EP2C (Fig. 6). Among the eight rhesus message variants that use exon 3, only EP2C codes for a complete ß-defensin-like peptide. Because of the use of earlier splicing sites in exon 3, the other seven message variants code for partial ß-defensin-like peptides (Fig. 6) that have only the proximal three of the six cysteines characteristic of ß-defensins. Depending on which 5'-splicing site is used, the subsequent exons (M1–M3, 5, 6) are read in different open reading frames and produce different C-termini. Exon M1 is read in two reading frames. Exon 5 is read in three reading frames and serves as 3'-untranslated region. Exon 6 is read in two reading frames and serves as 3'-untranslated region. Whether the encoded peptides (EP2J–EP2P) having partial ß-defensin-like sequences exhibit antibacterial activity or play other physiological roles is not known.
Despite the new exons (M1–M3) and the numerous splicing options in the rhesus EP2 gene, its cDNA sequences have readily recognizable counterparts in the human EP2 gene. In the human EP2 gene, sequences analogous to rhesus exons M1–M3 are located in the intron that, in the human EP2 gene, comprises the intergenic region between the two ancestral ß-defensin genes [19]. This suggests that the rhesus and human EP2 genes have a similar, if not identical, structure. Thus, from an evolutionary standpoint, the apparently rhesus-specific exons M1–M3 are not unique to the rhesus monkey but appear to have developed during primate evolution, possibly in parallel with the emergence of the other non-ß-defensin-like exons 2, 5, 7, and 8 of the human EP2 gene [19]. It is unclear, however, why the different species make use of different sets of EP2 variants.
As expected from the close evolutionary relationship, there is significant sequence identity among rhesus, human, and chimpanzee EP2 variants, both on the DNA and amino acid levels. Comparison of DNA sequences of equivalent exons (exons 1–6) shows 92% sequence identity, and comparison of amino acid sequences show 87% sequence identity between rhesus and human. The extent of DNA sequence identity is very similar throughout the different exons, including M1–M3. The sole exceptions are the 3'-terminal region of exon 2 and possibly the 5'-terminal region of exon 3. In this region of exon 3, which codes for amino acids 61–79 of the EP2C peptide (Fig. 4), only 7 of 19 amino acids are identical. However, the physiological significance of this localized sequence divergence is not known.
The rhesus epididymis expresses the ß-defensin-like message variants EP2C and EP2E identified in human/chimpanzee [16]. EP2E has the two-exon structure of the "classical" ß-defensin genes such as DefB1, in which the first exon encodes the leader sequence and the second exon encodes the ß-defensin module [25]. EP2C has a three-exon structure, the two "classical" ß-defensin exons, separated by an intervening exon. As a consequence, the EP2C peptide consists of non-ß-defensin module 1 followed by ß-defensin-like module 3. It is not known whether the entire EP2C peptide is antibacterially active or whether the additional module has to be removed proteolytically to activate the ß-defensin-like module, analogous to the removal of an inhibitory propeptide module in -defensins [26]. Because all the newly identified rhesus EP2 peptide variants contain this additional module, it is possible that they are processed after secretion into the epididymal lumen. The susceptibility of human HE21 (EP2A) and HE2ß1 (EP2D) to cleavage by a furin-like proprotein convertase has recently been demonstrated [21]. In addition, immunocytochemical and immunoblot analyses have demonstrated that EP2-related peptides are present in human epididymal epithelium, epididymal fluid, and ejaculated fluid [12, 15, 21].
Thus far, there is no physiological explanation for the expression of different EP2 message variants among human, chimpanzee, and rhesus. Different EP2 message variants could result from species-specific differences in epididymal splicing enzymes that may be important for reasons unrelated to the EP2 gene or its function. However, as long as a subset of functionally important EP2 message variants is produced, it does not matter how many different variants exist, even if some variants may have no function or physiological importance.
Our experiments showed that the rhesus epididymis produces both ß-defensin-like variants EP2C and EP2E. It is therefore likely that one of the EP2 gene's physiological roles is to help protect the male reproductive tract against microbial invasion. However, in rhesus, as in human/chimpanzee, most of the EP2 message variants code for non-ß-defensin-like peptides or partial-ß-defensin-like peptides. The C-termini of the rhesus EP2 peptides, EP2J-EP2P, have no recognizable similarity to peptides in the databases. Therefore, it is not possible to infer a physiological function by analogy for these peptides.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Otto Froehlich, Department of Physiology, Emory University School of Medicine, 615 Michael Street, 6th Floor, Atlanta GA 30322. FAX: 404 727 2648; froehlich{at}physio.emory.edu
Received: 17 June 2002.
First decision: 6 July 2002.
Accepted: 12 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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