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Endocrinology Vol. 141, No. 3 1245-1253
Copyright © 2000 by The Endocrine Society

ARTICLES

HE2ß and HE2{gamma}, New Members of an Epididymis-Specific Family of Androgen-Regulated Proteins in the Human1

Katherine G. Hamil, P. Sivashanmugam, Richard T. Richardson, Gail Grossman, Steven M. Ruben, James L. Mohler, Peter Petrusz, Michael G. O’Rand, Frank S. French and Susan H. Hall

Departments of Pediatrics (K.G.H., F.S.F., S.H.H.), Cell Biology and Anatomy (P.S., R.T.R., G.G., P.P., M.G.O.), and Surgery (J.L.M.) and the Laboratories for Reproductive Biology (K.G.H., P.S., R.T.R., G.G., P.P., M.G.O., F.S.F., S.H.H.), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599; and Human Genome Sciences, Inc. (S.M.R.), Rockville, Maryland 20850

Address all correspondence and requests for reprints to: Susan H. Hall, Laboratories for Reproductive Biology, CB 7500, 375 Medical Sciences Research Building, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500. E-mail: shh{at}med.unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2ß and HE2{gamma} proteins have a 25-amino acid deletion near the C-terminus, and HE2{gamma} isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the ß1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The ß2 form is expressed in principal cells primarily in efferent ducts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOZOA in testicular fluid lack forward motility and the ability to interact with and fertilize eggs (1). As they pass through the efferent ducts, initial segment, caput, corpus, and cauda of the epididymis, spermatozoa undergo further maturation to acquire fertilizing ability. Human efferent ducts branch and anastomose and in the initial segment fuse to form a single convoluted tubule (2) several meters long (3). The epithelial lining of this tubule is characterized by distinct morphological changes along its length and by gradients in gene expression and secretion of specialized proteins (4). Enzymes that alter the sperm surface are secreted from different regions, suggesting sequential surface modification during passage through the epididymis. Sperm surface alterations also involve tight binding of secreted epididymal proteins by integration or adsorption to the plasma membrane, affecting motility and other sperm functions (3). In the cauda, a major storage site, sperm are stabilized by secretions that block their ability to fertilize eggs. In the female tract, sperm capacitation is believed to involve the removal or alteration of these epididymal protective stabilizers from the sperm plasma membrane, leading to sensitization of the membrane and ability to undergo the acrosome reaction (1). Membrane-coating materials known or thought to be removed or altered during this process include caltrin (5), a protease inhibitor (6), several glyco-proteins (7), decapacitation factors (8), and antigens of corpus (9) and cauda (10) epididymal origin.

Principal cells of the epididymal epithelium absorb water and actively transport proteins, small molecules, and ions into and out of the lumen. Testosterone is converted to 5{alpha}-dihydrotestosterone (DHT), which binds the androgen receptor in nuclei and to androgen-binding protein in the lumen, where it reaches a concentration 50–60 times higher than that in caput vasculature (11). High concentrations of DHT and other components of testicular fluid are required to maintain the structure and function of initial segment and proximal caput epithelium (12). If testes are removed, leaving the epididymides intact, spermatozoa in the epididymides are incapable of maturing and rapidly degenerate (13). Androgen-dependent epididymal secretory proteins associate with spermatozoa and promote the ability of sperm to interact with both the zona pellucida (14, 15) and the oolemma (16). However, the mechanisms by which epididymal proteins implement the acquisition of sperm motility and fertilizing ability are largely unknown. Cloning and analysis of human androgen-regulated sperm-binding epididymal proteins are essential to understanding these mechanisms.

Kirchhoff and co-workers isolated six complementary DNAs (cDNAs) that code for proteins (HE1 to HE6) expressed in human epididymis (17). Among these, HE2 localized to the acrosomal and equatorial regions of ejaculated sperm heads (18), suggesting involvement in sperm maturation. HE2 messenger RNA (mRNA; 700 bp) and protein (103 amino acids) sequences revealed no homology with known sequences. The protein contains potential sites for signal peptide cleavage and glycosylation (18). In a study of human epididymal protein targets for male contraception, we discovered multiple size classes of HE2-related sequences and screened a human caput/corpus cDNA library for the corresponding clones. HE2 cDNAs were assigned to three structural subtypes: {alpha}, ß, and {gamma}. Our studies establish the existence of an HE2 family of androgen-regulated epididymis-specific proteins containing identical N-terminal, but different C-terminal, sequences. HE2ß1 is shown to be a sperm-binding protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA isolation and analysis
Total RNA was isolated by the method of Chirgwin et al. (19). Polyadenylated [poly(A)+] RNA was prepared using the Poly(A) Quik mRNA isolation kit (Stratagene, La Jolla, CA) following the manufacturer’s recommendations or by standard oligo-(deoxythymidine) affinity chromatography. Northern hybridizations were performed as previously described (20). Relative expression was determined by scanning exposed Kodak X-Omat AR films (Eastman Kodak Co., Rochester, NY) using an LKB Ultroscan XL laser densitometer (LKB, Rockville, MD).

Library construction
A directional cDNA library from human caput/corpus epididymis was constructed in {lambda} ZAPII (Stratagene) according to the manufacturer’s instructions. The primary library is 98% recombinant and contains 1.2 x 106 unique clones with an average insert size of 1.7 kb. The library was amplified once. RNA used to construct the library was isolated from epididymis obtained from a 58-yr-old Caucasian male and immediately flash-frozen in liquid nitrogen. The patient had received no hormone treatment for prostate cancer.

Library screening
The library was screened at low density [2000 plaque-forming units (pfu)/150-mm dish] using Nitroplus 2000 (Osmonics, Inc., Westborough, MA) membranes according to standard protocols (21). UV cross-linked membranes were prehybridized for 3 h in 5 x SSC (standard saline citrate), 5 x Denhardt’s solution, 1% SDS, and 100 µg/ml sheared, single-stranded salmon sperm DNA at 68 C. cDNAs containing 332-bp HE2{alpha} and the 256-bp HE2ß coding region fragments were 32P-labeled by random priming (Roche Molecular Biochemicals, Indianapolis, IN) and hybridized overnight at 68 C in the same solution in a concentration of less than 1 x 106 cpm/ml. Filters were washed for 15 min in 2 x SSC-0.1% SDS at room temperature and three times for 15 min each time in 0.1 x SSC-0.1% SDS at 50 C. Autoradiographs were exposed for 2 h at -80 C.

Thirty-one positive plaques were picked and stored in 1 ml SM buffer [suspension medium: 100 mM NaCl, 8.0 mM MgSO4, 50 mM Tris (pH 7.5), 0.01% gelatin, and 0.3% chloroform] and rescreened as described above at a plating density of 100 pfu/10-cm dish. Oligonucleotide primers specific for the HE2-coding region were used to amplify the insert of each clone by PCR to verify presence of HE2 sequence.

Amplification of the HE2 coding region
For library screening and Northern blot analysis, the coding region of HE2 (18) was amplified from 108 pfu of the caput/corpus library using the forward primer (53F) GCG GGA TCC AGG CAA CGA TTG C corresponding to nucleotides 171–183 and the reverse primer (53R) GCG GGT ACC CAT ACG GCA GAT GG corresponding to nucleotides 498–485. Reactions contained 1 x QIAGEN TAQ buffer [Tris (pH 8.3), KCl, (NH4)2SO4, and 1.5 mM MgCl2], 50 µM primers, 200 µM deoxynucleotides, 2.5 U TAQ (QIAGEN, Valencia, CA), and 108 pfu of the caput/corpus library in 100-µl reactions. Bands were gel-purified before use for sequencing and as hybridization probes.

Amplification of HE2 5'- and 3'-untranslated regions (UTRs)
UTRs of directionally cloned cDNAs were amplified for sequencing using the following pairs of gene-specific and vector primers. 5'-UTR and adjacent coding region were amplified using the M13R forward vector primer and 53R as reverse. The 3'-UTR and adjacent coding region were amplified using the 53F forward primer and the M13F as the reverse. Plaque-purified clones were amplified using 1000 pfu/reaction. Uncloned PCR products were sequenced.

Amplification of variant HE2 3'-untranslated regions for Northern hybridization
Regions of the 3'-UTR specific to the {alpha}, ß, and {gamma} isoforms were amplified using the M13F primer in the vector as the reverse primer and the following specific forward primers: for {alpha}1 (nucleotides 547–567), CGT GAC ATT TGC TCT GAT CCC; for {alpha}2 (nucleotides +1 to +21), GAA ATG AAA TGA AGG AGG TGG G; and for ß2 (nucleotides +108 to +128), CAG GCC AGC AGC ACA GAC AAC. Plaque-purified {alpha}, ß, and {gamma}1 clones were amplified using 1000 pfu/reaction.

RT-PCR from human and monkey caput RNA
Random hexamers were annealed to 2 µg human or monkey total RNA for 10 min at 25 C. Avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI) was added, and the reaction was continued for 50 min at 37 C in 50 mM Tris (pH 8.3), 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM dithiothreitol, and 40 U RNasin inhibitor (Promega Corp.) for 50 min at 37 C. The enzyme was inactivated at 95 C for 5 min. The resulting first strand cDNAs were used as template for PCR amplification of HE2 coding sequence using 53F and 53R primers and touchdown PCR cycling conditions.

Automated sequencing
DNA was sequenced at the UNC-CH Automated DNA Sequencing Facility using an ABI PRISM model 377 DNA sequencer (PE Applied Biosystems, Foster City, CA) and the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq(R)DNA Polymerase FS. Primers were synthesized on an automated PE Applied Biosystems model 394 DNA synthesizer using standard cyanoethyl phosphoramidite chemistry.

Amplification of specific HE2 coding regions for expression constructs
The cDNA encoding the first 71 amino acids present in all forms of HE2 was amplified using the forward primer 53F and the reverse primer (53TR) GCG GGT ACC TTA TTG GTA AGG TGG GGT G and 106 pfu of plaque-purified {lambda} clone as template. The cDNA encoding amino acids 72–133 of ß1 were amplified using the forward primer 57F GCG GGA TCC GGG GAT GTT CCA C and the reverse primer 57R GCG GGT ACC ACC TTA GAT CCC AGA TCT GCC.

The first 71 amino acids of HE2 were expressed from bacterial vectors pQE-HE2(1–71) and amino acids 72–133 of ß1 from pQE-HE2(72–133). Expression vectors were constructed by cloning the corresponding PCR-amplified DNAs into the BamHI-KpnI sites of the pQE30 vector (QIAGEN) adding 6 N-terminal histidines. Clones free of mutations by double stranded automated sequencing were selected for protein production.

Fusion protein production
Escherichia coli strain M15(pREP4) was transformed with pQE-HE2(1–71) and pQE-HE2(72–133) according to the QIAGEN protocol. A fresh overnight culture of each strain was diluted 1:50 in Luria Bertoni medium supplemented with 25 µg/ml kanamycin and 50 µg/ml carbenicillin and incubated at 37 C. At OD600 0.5, fusion protein expression was induced with 1 mM isopropyl-1-thio-ß-D-galactoside for 4 h at 37 C. Cells were pelleted and frozen at -80 C.

Protein was isolated from 1 g frozen cells resuspended in 5 ml buffer A (6 M guanidine hydrochloride, 0.1 mM NaH2PO4, and 0.01 M Tris-Cl, pH 8.0). After 1 h at room temperature, lysate was centrifuged for 15 min at 10,000x g. Supernatant was incubated for 1 h with nickel-nitilotriacetic acid-agarose. The slurry was transferred to a column and washed with 5 bed vol buffer A, 15 vol buffer B (8 M urea, 0.1 mM NaH2PO4, and 0.01 M Tris-Cl, pH 8.0), and 20 vol buffer C (buffer B at pH 6.3). Recombinant histidine fusion protein was eluted with 5 vol buffer D (buffer B at pH 5.9) and 5 bed vol buffer E (buffer B at pH 4.5). Fractions were analyzed on a 10–20% gradient Tris-Tricine gels and stained with Coomassie blue R250.

Fractions containing purified protein were pooled and dialyzed against PBS to remove urea.

Peptide synthesis
Peptides were synthesized using a Rainin Symphony multiple peptide synthesizer (Rainin Instruments, Woburn, MA) and were purified by HPLC. Peptides were conjugated to keyhole limpet hemocyanin. The amino- terminal lysine was added to the {alpha} and ß2 peptide sequences to facilitate coupling. The peptides correspond to the C-terminal amino acids of the following: {alpha} specific, kVDFLGPRWARGCSTNG; ß1 specific, CVSNTDEEGKEKPEMDGRSGI; and ß2/{gamma}1 specific, kGTGQQHRQRCG. The lowercase k represents lysine added for coupling to keyhole limpet hemocyanin. Antibodies were raised in rabbits by Immunodynamics, Inc. (La Jolla, CA).

Tissue sources
Human epididymides for immunohistochemistry and Northern blot analyses were obtained from prostate cancer patients ranging in age from 58–83 yr. The epididymides were dissected into caput, corpus, and caudal regions before freezing or fixation.

For analysis of androgen regulation, male rhesus monkeys (Macaca mulatta) of similar age, weight, and testicular size underwent subcapsular orchidectomy (22) or sham operation. One orchidectomized monkey was immediately injected im with testosterone enanthate (30 mg/kg BW; 400 mg total); the other was injected with vehicle. Epididymides and remaining testes were removed 6 days later and frozen in liquid nitrogen. All animals used in these studies were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocol follows accepted veterinary medical practice and was approved by the University of North Carolina animal care and use committee. The animals were given analgesics and were monitored closely after surgery.

Rhesus monkeys, 10–12 yr of age with proven breeding history (Covance Research Products, Inc., Alice, TX), provided tissues for Northern analysis and immunostaining. Tissues for immunohistochemistry were fixed in Bouin’s solution (75 ml saturated picric acid, 5 ml glacial acetic acid, and 25 ml 37% formaldehyde) promptly after excision. Surplus human testes and epididymides were made available by Dr. James L. Mohler, Department of Urology/Surgery, University of North Carolina (Chapel Hill, NC). Other human tissues were obtained from the Tissue Procurement Core Facility of the Lineberger Comprehensive Cancer Center, University of North Carolina. Human tissues are not accompanied by identifying information and cannot be traced to the donor.

Immunohistochemical staining
Tissues were fixed in Bouin’s fluid and embedded in paraffin according to standard protocols (23). The double peroxidase-antiperoxidase method was used for immunohistochemical staining (24). Photographs were taken using a Nikon Microphot FXA (Nikon, Melville, NY) and an Apple Power MacIntosh G-3 (Apple Computers, Cupertino, CA) equipped with Scion Image version 1.62C software and a Scion CG7 image capture card (Scion, Frederick, MD). The composite was made using Adobe Photoshop 4.0 and the Fujix Pictrography printer (Fujix, Okayama, Japan).

Surplus swim-up sperm were provided by Dr. Stan Beyler, Assisted Reproductive Technology Clinic, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill. Sperm were washed in PBS and fixed in 4% paraformaldehyde for 30 at Chapel Hill min. They were washed with 50 mM glycine in PBS, smeared on microscope slides, and air-dried. Sperm were permeabilized in methanol for 10 min and blocked in 3% BSA overnight. After two washes in PBS, sperm were incubated overnight at 4 C in primary affinity-purified antibody (1:25) in 3% BSA in PBS. Unbound antibody was removed by two washes with PBS, and biotinylated antirabbit IgG (1:250) was added for 1 h at room temperature. Unbound second antibody was removed by two washes with PBS, and antibody-binding sites were stained with avidin-Texas Red (1:250) for 1 h at room temperature. Unbound stain was removed in two PBS washes. Sperm were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) and observed with a red filter using a Nikon microscope. The image was scanned using a Scan Jet II CX (Hewlett-Packard Co., Palo Alto, CA). Sperm photos were arranged and labeled using Adobe Photoshop 5.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Amplification of the HE2 coding region by PCR using the human caput/corpus epididymal cDNA library as template gave a 256-bp fragment (ß form) in addition to the expected 332-bp ({alpha} form) reaction product (Fig. 1Go, lane 2). Sequencing revealed a deletion of the HE2 coding region in the smaller ß fragment. The same HE2 primers were used for RT-PCR amplification from epididymis total RNA from two humans and one monkey (Fig. 1Go, lanes 3–5). Fragments identical in size to those from the library were obtained from the RNAs, confirming expression of the two isoforms and suggesting that HE2{alpha} is the prototype of a family of epididymal proteins. This was confirmed by screening a human epididymis caput/corpus {lambda} cDNA library using the 32P-labeled PCR products ({alpha} and ß forms). Clones isolated in this screening represented a family of transcripts containing identical N-terminal coding sequence and different C-terminal and 5'- and 3'-UTRs. HE2 transcripts appeared to be a series of precisely defined cassettes variably spliced to generate different linear sequences. Identical sequences and start and stop sites in the corresponding cassettes of different clones suggested that these are alternatively spliced exons encoded by a single gene. Most exons end in the AG of consensus splicing donor sites.



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  		Figure 1. HE2 isoform PCR fragments. Fragments were amplified using PCR with no template (lane 1), PCR using human caput/corpus cDNA library as template (lane 2), RT-PCR using RNA from a 68-yr-old man (lane 3), RT-PCR using RNA from a 58-yr-old man (lane 4), or RT-PCR using RNA from a rhesus monkey (lane 5). The 345- and 270-bp {alpha}- and ß-derived fragments are indicated.

 
Thirty related clones were identified and plaque purified, and each was shown by PCR to contain HE2 sequence. Amplified fragments were of 3 sizes, designated HE2{alpha} for the original HE2 (18); ß, indicating a deleted form; and {gamma}, which was later shown to have a larger deletion. Sequencing revealed 8 HE2{alpha}, 12 HE2ß, and 2 HE2{gamma} clones. From the remaining 8 clones, PCR fragment sizes suggested combinations of exons already seen and were not analyzed further. HE2{alpha} clones contain the entire HE2 coding region for 103 amino acids using the original stop codon (Figs. 2Go and 3Go and Table 1Go). HE2ß and -{gamma} forms encode the first 71 amino acids containing the putative signal peptide and cleavage site (25) at amino acid 25, indicating that they are secreted as demonstrated for the {alpha} form (18). Other potential modifications include glycosylation at asparagine 29, casein kinase 2 phosphorylation at serines 31 and 75, and protein kinase C phosphorylation at serines 75 and 83. {alpha} clones also contain at least 37 nucleotides of the original HE2 5'-UTR and 56 nucleotides of the original 3'-UTR. The truncated 5'-ends of the individual {alpha}2 and {gamma}2 clones resulted from incomplete RT and are indicated in Fig. 2Go by arrows. The {alpha}2 form was unique in this study in encoding variant amino acids at positions 77, 85, and 89, which were also noted by Osterhoff et al. (18). The {alpha}2 clone was isolated from a whole epididymis cDNA library from a different donor from the caput/corpus library, which was the source of all other clones and may account in part for the variant amino acids.



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  		Figure 2. Alignment of HE2 isoform mRNAs. Boxes indicate the coding sequence. Stars indicate stop codons. Line variations represent different nucleotide sequences. Thin V-lines span deletions. The {alpha} clone was isolated by Osterhoff et al. (18 ) from their library. The {alpha}2 clone (Human Genome Sciences, Rockville, MD) was isolated from a whole epididymis cDNA library. The {alpha}1, ß, and {gamma} clones were isolated from our caput/corpus cDNA library. Numbering begins with nucleotide 1 of the longest cDNA clone ({alpha}1), continuing to 742 at the end of the {alpha} and ß1 isoforms. The large and small dotted lines indicate sequences located an unknown distance downstream from 742 and are numbered +1 to +400. Arrows indicate unavailable sequence. Accession numbers: {alpha}, X67697; {alpha}1, AF168616; {alpha}2, AF170797; ß1, AF168617; ß2, AF168618; {gamma}1, AF168619; and {gamma}2, AF168620.

 


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  		Figure 3. HE2 amino acid sequences. Arrows indicate putative signal peptide cleavage site. Three bold underlined amino acids were different in {alpha}/{alpha}2 and {alpha}1. Regions in ß2 identical to those in ß1 or in {gamma}1 are in black boxes. The C-terminal peptides used to immunize the rabbits for immunohistochemistry are in open boxes.

 

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  		Table 1. HE2 protein isoforms

 
The 25-amino acid deletion from nucleotide 382 to nucleotide 457 in every ß form suggests a skipped exon. The deletion results in a frame shift eliminating the HE2{alpha} stop codon at nucleotide 479. In ß1, translation would continue to a stop codon at 645 in the HE2{alpha} 3' UTR. In the resulting ß1 protein of 133 amino acids, the relatively hydrophobic {alpha} form C-terminal region is replaced by a hydrophilic acidic region that contains 6 cysteines and lowers the pI to 8.7 (8.23 without the signal peptide) compared with 11–12 for the other forms (Table 1Go). There are additional consensus casein kinase 2 phosphorylation sites at amino acids 115 and 117.

In ß2, the 3'-UTR of HE2{alpha} stops at nucleotide 535 and skips an exon present in {alpha}2 and {gamma}2 (Fig. 2Go, large dotted line, nucleotides +1 to +103). The remaining 3'-sequence (Fig. 2Go, small dotted line, nucleotides +104 to +400) contains a stop codon after 32 nucleotides. The resulting protein of 108 amino acids contains a potential glycosaminoglycan attachment site at amino acid 102.

The HE2{gamma} forms also contain the HE2{alpha} coding region to nucleotide 381 (amino acid 71) where the frame-shifting ß form deletion begins (Figs. 2Go and 3Go). The {gamma}1 form continues from +104 through sequence identical to the 3'-UTR of {alpha}2 and ß2 and using the same translation stop codon as ß2 at +137. Thus, the C-terminal 11 amino acids of ß2 and {gamma}1 are identical. The {gamma}2 deletion also begins after nucleotide 381, but the coding region is joined to sequence identical to the {alpha}2 3'-UTR beginning at nucleotide +1. A stop codon 11 nucleotides into this segment would terminate translation, resulting in a protein of 74 amino acids.

To determine in which regions of the epididymis HE2 isoforms are expressed, Northern blots of human caput, corpus, and cauda poly(A)+ mRNAs were hybridized to the coding region (which hybridizes to all isoforms) and two 3' probes (Fig. 4Go). All mRNAs were most abundant in caput, where a predominant 0.9-kb mRNA is seen as well as less abundant 1.4-, 2.5-, and 4.4-kb species. The greater intensity of signal in Fig. 4BGo with the +1 to +103 exon probe than the longer +104 to +400 probe in Fig. 4CGo suggests greater abundance of the +1 to +103 sequence, as the hybridization probes were of similar specific activity and G/C content. If more abundant, the +1 to +103 sequence must be present in mRNAs that lack the +104 to +400 region, an arrangement of exons not yet found in the cDNAs in this study.



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  		Figure 4. HE2 mRNAs are most abundant in caput. Northern hybridization to three different exons: A, coding region cDNA (nucleotides 168–381) that binds the entire HE2 family of mRNAs; B, {alpha}2/{gamma}2-specific 3'-UTR probe (nucleotides +1 to +103); and C, {alpha}2/ß2/{gamma}1/{gamma}2 probe (nucleotides +104 to +400). Each lane contains 5 µg poly(A)+ RNA isolated from the indicated regions of human epididymis.

 
We determined whether HE2 isoforms are regulated by testosterone, the key hormone in sperm maturation. RNA was obtained from rhesus monkeys that were sham operated, castrated 6 days, and castrated but given a single injection of 400 mg testosterone enanthate immediately after surgery (Fig. 5Go). HE2 mRNA levels in all regions were at low levels 6 days postcastration. Testosterone replacement maintained higher HE2 mRNA than those in untreated castrated animals in all regions. Serum testosterone levels in sham-operated, castrated, and androgen replaced before surgery animals were 3.0, 3.8, and 1.4 ng/ml, respectively. After surgery, serum testosterone levels were 2.0, 0.15, and 64.9 ng/ml, respectively. Testosterone-replaced animals were given a high dose to mimic the epididymal intraluminal androgen concentrations, which for intact animals are approximately 50 times the normal circulating levels. HE2 mRNA appeared most abundant in the caput of human but in the monkey was most abundant in the corpus, perhaps because of high levels of expression near the border of the two regions and the somewhat different structures in the two species. The {alpha}2/{gamma}2 (+1 to +103) probe hybridized more weakly to the monkey than human mRNAs, but showed the same result as the coding region probe, and the {alpha}2/ß2/{gamma}1/{gamma}2 (+104 to +400) probe failed to hybridize detectably to the monkey mRNAs (data not shown).



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  		Figure 5. Androgen regulation of HE2 mRNA expression. Rhesus monkeys were sham operated, castrated, or castrated and androgen replaced with immediate injection of testosterone enanthate. Epididymides were removed 6 days postcastration. Total RNAs (10 µg/lane) from epididymis regions were analyzed by Northern hybridization to 32P-labeled human HE2 cDNA, nucleotides 168–381.

 
To determine where the HE2ß1and -2 forms are expressed in relation to {alpha}, antisera were raised against {alpha}-, ß1-, and ß2/{gamma}1-specific C-terminal peptides (boxed in Fig. 3Go) and were used to localize the proteins in human epididymis.

Clear, competable {alpha} staining was not observed in this study. Whether the {alpha} form is not sufficiently abundant or the titer too low for detection remains to be determined.

HE2 ß1 expression was strong in the supranuclear region, where the Golgi complex is located in nearly every principal cell in the initial segment (Fig. 6AGo). In some cells, expression extended to the basal lamina. The staining did not develop when antibody preincubated with antigen was used (Fig. 6BGo). Principal cells of the caput and proximal corpus also expressed HE2ß1, but not in every cell (Fig. 6CGo). Heavy brown staining of luminal contents was seen in corpus (Fig. 6CGo). The percentage of cells expressing HE2ß1 decreased gradually through the tubule toward the cauda, where only occasional groups of principal cells were stained (not shown). HE2ß1 expression was variable among morphologically similar cells in the same tubule section, a characteristic of epididymal gene expression (26). The ß1 isoform also localized to the postacrosomal head and neck regions of washed ejaculated human sperm, indicating that this HE2 isoform is a sperm-binding protein (Fig. 7BGo).



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  		Figure 6. Immunohistochemical localization of HE2ß1 and HE2ß2in human epididymis. A-C, HE2ß1 in initial segment (A), initial segment using antibody preabsorbed with peptide antigen (B), and corpus (C). D–F, HE2ß2/{gamma}1 in efferent ducts (D), epithelium III in efferent ducts using antibody preabsorbed with peptide antigen (E), and efferent ducts, epithelium IV (F). Brown color indicates immunostaining. Blue color is the toluidine blue counterstain. Scale bars: A, B, D, and E, 100 µm; C and F, 200 µm.

 


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  		Figure 7. HE2ß1 binds sperm surface. A, Phase contrast of human sperm; B, red immunofluorescent staining of HE2ß1, same sperm as A. Both are x500 magnification.

 
HE2ß2/{gamma}1 was expressed in ciliated goblet cells of the epithelium III efferent ducts, using the efferent duct classification system of Yeung et al. (2) (Fig. 6DGo). The staining was substantially reduced when antiserum was preincubated with antigen (Fig. 6EGo). In the regular cuboidal epithelium IV of the ducts, staining was seen in about a third of the cells (Fig. 6FGo). ß2 was also seen in a few isolated cells of the corpus and in clusters of principal cells in cauda (not shown).

Thus, posttesticular sperm first encounter cells in the efferent ducts that synthesize and probably secrete HE2ß2/{gamma}1 then pass through the lumen of the initial segment, caput, and proximal corpus, where HE2ß1 secretion by the principal cells is abundant, and then into the distal corpus, where both HE2ß1 and HE2ß2/{gamma}1 are expressed in patches of cells in mosaic patterns. Staining of serial sections did not detect individual cells that expressed both ß1 and ß2 (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we demonstrate a family of HE2 proteins generated by alternative splicing of different exons resulting in the attachment of a common 71-amino acid N-terminus to various C-termini. The common N-terminal region of these secreted proteins includes a hydrophobic signal peptide, suggesting that all isoforms are secreted. Androgen regulation and binding of HE2ß1 to sperm suggest that this HE2 isoform has a role in sperm maturation. Interactions of HE2ß1 and other HE2 isoforms with other sperm surface proteins may involve their putative glycosylation and phosphorylation sites. No motifs or homologies to known proteins offer clues to their functions; however, the regionalized epididymal expression of the HE2 isoforms suggests that they exert their effects at different times in the controlled sequence of sperm modifications during epididymal transit. The demonstration of HE2ß1 in the postacrosomal head and neck regions of ejaculated sperm further supports a direct role in fertilization.

We demonstrated expression of HE2ß1 primarily in the initial segment and caput proper. This localization corresponds with that reported previously by Krull et al. (27) using in situ hybridization to a probe recognizing all HE2 isoform mRNAs. Using in situ hybridization, HE2 was not detected in efferent ducts by Krull et al. (27), perhaps due to lower levels of expression. The HE2ß2 expression we observed in efferent ducts by immunostaining was restricted to the distal regions and, in agreement with the in situ result (27), no expression of any isoform was seen in the irregularly shaped epithelium of the proximal efferent ducts. Early posttesticular availability of the HE2 family for interaction with sperm may result in early sperm maturation steps in preparation for possible actions of proteins expressed distally, such as the cauda/vas deferens protein, HE4, a possible protease inhibitor involved in capacitation (28), or a mouse protease inhibitor from seminal vesicle that is removed during capacitation (6).

Weak staining in the postacrosomal head and neck regions of sperm was detected by Osterhoff et al. (18) using their rabbit antibody to recombinant HE2{alpha}. This antigen included amino acids 13–49 also present in HE2ß1. Weak staining could have been obtained if antibodies to this common region were present in lower concentration while their antibody titer to the {alpha}-specific, amino acids 50–81, was higher, accounting for major staining of the acrosomal region. Taken together, our results and those of Osterhoff et al. (18) suggest the {alpha} and ß1 isoforms localize to different regions of the sperm head, which raises the possibility of different functions for the different isoforms.

HE2 expression in testosterone-replaced epididymis was highest in caput instead of corpus, where expression was highest in the intact animal. Similar shifts toward more anterior expression of mRNAs in castrated, testosterone-replaced animals were reported previously (26, 30). Increased expression in the caput could result from loss of testicular factors as suggested previously (30), such as androgen- binding protein. In the presence of a high concentration of circulating testosterone, the loss of androgen-binding protein after castration might result in higher free androgen levels available to bind androgen receptor and regulate gene expression in the epithelial cells.

Reports of sperm achieving fertilizing ability without passing through the entire epididymis led to the speculation that the human epididymis is not essential for the development of fertilizing ability (3). A small number of males with epididymis-vas deferens anastomoses achieved natural mating conceptions; however, additional studies demonstrated that fertility increased in proportion to the length of the epididymis anastomosed to vas deferens. A possible explanation for fertility in some patients with efferent duct-vas deferens anastomoses is that sperm-modifying proteins expressed in the efferent ducts and initial segment initiate posttesticular sperm modification sufficient for the acquisition of low efficiency fertilizing ability.

There is precedent for separate, but related, functions performed by protein isoforms generated by alternative splicing (31). Regulated splicing in different cell types (32) and by different exogenous regulatory factors (33) can control the relative abundance of protein isoforms encoded by single genes. For example, fibronectin secreted from fibroblasts contains exons EIIIA and EIIIB, which encode protein domains that interact with cell surface receptors (34). In contrast, splicing of fibronectin pre-mRNA in hepatocytes skips these two exons and encodes a protein that fails to adhere strongly to cell surfaces and instead circulates in serum. The unique C-termini of HE2 isoforms may confer different functions, as they have different consensus phosphorylation sites and differ in number and arrangement of positively and negatively charged amino acids and number of cysteines available for disulfide bonding. Early studies indicated that disulfide bonds are formed in sperm during epididymal passage (35), possibly involving attachment of luminal proteins.

Maturation of sperm surface composition is critical to fertilization of the egg, and any disruption can impede sperm progress through the cells and carbohydrate matrix of the cumulus oophorus and the glycoproteins of the zona pellucida. Decapacitated sperm and those that have undergone premature acrosome reaction may attach to the surface, but fail to penetrate the cumulus (36). Recent evidence suggests that a major component of the cumulus, polymerized hyaluronic acid, is digested by sperm surface hyaluronidase (37), and that surface acrosin (38), ß-galactosidase, and arylsulfatase (39) also aid cumulus penetration. Zao et al. (40) suggest that the sperm surface adsorbs some of these enzymes during epididymal transit. After successful passage through the cumulus, capacitated spermatozoa bind strongly to the zona pellucida. A 34-kDa sperm antigen acquired during epididymal transit is implicated in this binding (41). Incomplete sperm maturation within the epididymis has been suggested to explain the total failure of sperm binding to the zona in unsuccessful human in vitro fertilizations (42). Zona penetration is facilitated by the lytic action of acrosomal enzymes.

High luminal concentrations of testosterone and DHT are maintained by androgen-binding protein in the initial segment and caput epididymis (43). Androgen-regulated transcription factors, such as the ETS-like factor, PEA3 (44), and the Pem homeobox protein or others, may mediate the regulated production of sperm-modifying proteins. The Pem homeobox transcription factor is transcribed from the proximal promoter only in testis and epididymis and is expressed in epididymis only in the presence of testosterone (45). In addition to HE2, DHT regulates the expression of a number of sperm-binding proteins (26), such as acidic epididymal glycoprotein (46, 47, 48) and epididymal protease inhibitor (49). Based on their possible role in sperm maturation, epididymis-specific, androgen-regulated proteins comprise a source of potential contraceptive targets for the human male.


   Acknowledgments
 
We thank Zang De-Ying, Michelle Cobb, Catharina Weaver, and Raymond Johnson for expert technical assistance.


   Footnotes
 
1 This work was supported by Grant CIG-96–06 from the CONRAD program, the Andrew Mellon Foundation, NIH Grants R37-HD-04466, by NICHHD/NIH through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and Fogarty International Center Training and Research in Population and Health Grant D43TW/HD00627. Back

Received September 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yanagimachi R 1994 Mammalian fertilization. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New York, pp 189–317
  2. Yeung CH, Cooper TG, Bergmann M, Schulze H 1991 Organization of tubules in the human caput epididymidis and the ultra structure of their epithelia. Am J Anat 191:261–279[CrossRef][Medline]
  3. Turner TT 1995 On the epididymis and its role in the development of the fertile ejaculate. J Androl 16:292–298[Free Full Text]
  4. Kirchhoff C 1999 Gene expression in the epididymis. Int Rev Cytol 188:133–202[Medline]
  5. Lardy H, San Augustin J 1989 Caltrin and calcium regulation of sperm activity. In: Schatten H, Schatten G (eds) The Cell Biology of Fertilization. Academic Press, San Diego, pp 29–39
  6. Boettger-Tong H, Aarons D, Biegler B, Lee T, Poirier GR 1992 Competition between zonae pellucidae and a proteinase inhibitor for sperm binding. Biol Reprod 47:716–722[Abstract]
  7. Parry RV, Baker PJ, Jones R 1992 Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma. Mol Reprod Dev 33:108–115[CrossRef][Medline]
  8. Oliphant G, Reynolds AB, Thomas TS 1985 Sperm surface components involved in the control of the acrosome reaction. Am J Anat 174:269–283[CrossRef][Medline]
  9. Okabe M, Takada K, Adachi T, Kohama Y, Mimura T, Aonuma S 1986 Studies on sperm capacitation using monoclonal anti-body—disappearance of an antigen from the anterior part of mouse sperm head. J Pharmacobiodyn 9:55–60[Medline]
  10. Okabe M, Takada K, Adachi T, Kohama Y, Mimura T 1986 Inconsistent reactivity of an anti-sperm monoclonal antibody and its relationship to sperm capacitation. J Reprod Immunol 9:67–70[CrossRef][Medline]
  11. Turner TT, Jones CE, Howards SS, Ewing LL, Zegeye B, Gunsalus GL 1984 On the androgen microenvironment of maturing spermatozoa in the adult rat. Endocrinology 115:1925–1932[Abstract]
  12. Fawcett DW, Hoffer RP 1979 Failure of exogenous androgen to prevent regression of the initial segments of the rat epididymis after efferent duct ligation or orchiectomy. Biol Reprod 20:162–181[Abstract]
  13. Orgebin-Crist M-C, Jahad N 1978 The maturation of rabbit epididymal spermatozoa in organ culture: inhibition by antiandrogens and inhibitors of ribonucleic acid and protein synthesis. Endocrinology 103:46–53[Abstract]
  14. Orgebin-Crist MC, Fournier-Delpech S 1982 Sperm-egg interaction, evidence for maturational changes during epididymal transit. J Androl 3:429–433[Abstract]
  15. Cuasnicú PS, Gonzalez Echeverria F, Piazza A, Blaquier JA 1984 Addition of androgens to cultured hamster epididymis increases zona recognition by immature spermatozoa. J Reprod Fertil 70:541–547[Abstract]
  16. Rochwerger L, Cohen DJ, Cuasnicú PS 1992 Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion. Dev Biol 153:83–90[CrossRef][Medline]
  17. Kirchhoff C, Osterhoff C, Pera I, Schröter S 1998 Function of human epididymal proteins in sperm maturation. Andrologia 30:225–232[Medline]
  18. Osterhoff C, Kirchhoff C, Krull N, Ivell R 1994 Molecular cloning and characterization of a novel human sperm antigen (HE2) specifically expressed in the proximal epididymis. Biol Reprod 50:516–525[Abstract]
  19. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299[CrossRef][Medline]
  20. Hamil KG, Hall SH 1994 Cloning of rat Sertoli cell follicle-stimulating hormone primary response complementary deoxyribonucleic acid: regulation of TSC-22 gene expression. Endocrinology 134:1205–1212[Abstract]
  21. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) 1994 Current protocols in molecular biology. John Wiley and Sons, New York
  22. Zhang X-Z, Donovan MP, Williams BT, Mohler JL 1996 Comparison of subcapsular and total orchiectomy for treatment of metastatic prostate cancer. Urology 47:402–404[CrossRef][Medline]
  23. Preece A 1972 A Manual for Histologic Technicians, ed 3. Little, Brown, Boston
  24. Ordronneau P, Lindstrom PBN, Petrusz P 1981 Four unlabeled antibody bridge techniques: a comparison. J Histochem Cytochem 29:1397–1404[Abstract]
  25. Nielsen H, Engelbrecht J, Brunak S, von Heijne G 1997 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engin 10:1–6[Abstract/Free Full Text]
  26. Orgebin-Crist M-C 1996 Androgens and epididymal function. In: Bhasin S, Gabelnick HL, Spieler JM, Swerdloff RS, Wang C, Kelly C (eds) Pharmacology, Biology, and Clinical Applications of Androgens. Wiley-Liss, New York, pp 27–38
  27. Krull N, Ivell R, Osterhoff C, Kirchhoff C 1993 Region-specific variation of gene expression in the human epididymis as revealed by in situ hybridization with tissue-specific cDNAs. Mol Reprod Dev 34:16–24[CrossRef][Medline]
  28. Osterhoff C, Ivell R, Kirchhoff C 1995 Human epididymal secretory protein HE4 dissociates from spermatozoa during in vitro capacitation. Hum Reprod 10:11 (Abstract 2)[Free Full Text]
  29. Deleted in proof
  30. Winer MA, Wolgemuth DJ 1995 The segment-specific pattern of A-raf expression in the mouse epididymis is regulated by testicular factors. Endocrinology 136:2561–2572[Abstract]
  31. McKeown M 1992 Alternative mRNA splicing. Annu Rev Cell Biol 8:133–155[CrossRef]
  32. Naito Y, Watanabe Y, Yokokura H, Sugita R, Nishio M, Hidaka H 1997 Isoform-specific activation and structural diversity of calmodulin kinase I. J Biol Chem 272:32704–32708[Abstract/Free Full Text]
  33. Scotet E, Houssaint E 1998 Exon III splicing switch of fibroblast growth factor (FGF) receptor-2 and -3 can be induced by FGF-1 or FGF-2. Oncogene 17:67–76[CrossRef][Medline]
  34. Kornblihtt AR, Pesce CG, Alonso CR, Cramer P, Srebrow A, Werbajh S, Muro AF 1996 The fibronectin gene as a model for splicing and transcription studies. FASEB J 10:248–257[Abstract]
  35. Bedford JM 1975 Maturation, transport, and fate of spermatozoa in the epididymis. In: Greep RO, Astwood EB, Hamilton DW, Geiger SR (eds) Handbook of Physiology, sect 7, vol 5. American Physiological Society, Washington DC, pp 303–317
  36. Cummins JM, Yanagimachi R 1986 Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro in relation to the timing of the acrosome reaction. Gamete Res 15:187–212
  37. Joyce C, Jeyendran RS, Zaneveld LJD 1985 Release, extraction, and stability of hyaluronidase associated with human spermatozoa. Comparisons with the rabbit. J Androl 6:152–161[Abstract/Free Full Text]
  38. Tesarik J, Drahorad J, Testart J, Mendoza C 1990 Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction. Development 110:391–400[Abstract/Free Full Text]
  39. Nikolajczyk BS, O’Rand MG 1992 Characterization of rabbit testis ß-galactosidase and arylsulfatase A: purification and localization in spermatozoa during the acrosome reaction. Biol Reprod 46:366–378[Abstract]
  40. Zao PZR, Meizel S, Talbot P 1985 Release of hyaluronidase and ß-N-acetylhexosaminidase during in vitro incubation of hamster sperm. J Exp Zool 234:63–74[CrossRef][Medline]
  41. Boue F, Bérubé B, De Lamirande E, Gagnon C, Sullivan R 1994 Human sperm-zona pellucida interaction is inhibited by an antiserum against a hamster sperm protein. Biol Reprod 51:577–587[Abstract]
  42. Bedford JM, Kim HH 1993 Sperm/egg binding patterns and oocyte cytology in retrospective analysis of fertilization failure in vitro. Hum Reprod 8:453–463[Abstract/Free Full Text]
  43. Hansson V, Ritzén EM, French FS, Nayfeh SN 1975 Androgen transport and receptor mechanisms in testis and epididymis. In: Greep RO, Astwood EB, Hamilton DW, Geiger SR (eds) Endocrinology. American Physiological Society, Washington DC, vol 5:173–201
  44. Xin J-H, Cowie A, Lachance P, Hassell JA 1992 Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev 6:481–496[Abstract/Free Full Text]
  45. Maiti S, Doskow J, Li S, Nhim RP, Lindsey JS 1996 The Pem homeobox gene Androgen-dependent and -independent promoters and tissue-specific alternative RNA splicing. J Biol Chem 271:17536–17546[Abstract/Free Full Text]
  46. Lea OA, Petrusz P, French FS 1978 Purification and localization of acidic epididymal glycoprotein (AEG): a sperm coating protein secreted by the rat epididymis. Int J Androl [Suppl] 2:592–607
  47. Martinez SP, Conesa D, Cuasnicú PS 1995 Potential contraceptive use of epididymal proteins: evidence for the participation of specific antibodies against rat epididymal protein DE in male and female fertility inhibition. J Reprod Immunol 29:31–45[CrossRef][Medline]
  48. Sivashanmugam P, Richardson RT, Hall S, Hamil KG, French FS, O’Rand MG 1999 Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP-1-like protein from the monkey. J Androl 20:384–393[Abstract/Free Full Text]
  49. Sivashanmugam P, Richardson RT, Hall S, Hamil KG, French FS, O’Rand MG Cloning and sequencing of Eppin, a protease inhibitor from human and monkey epididymis and testis. 39th Annual Meeting of the American Society for Cell Biology, Washington DC, 1999 (Abstract 1180)



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