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, New Members of an Epididymis-Specific Family of Androgen-Regulated Proteins in the Human1
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
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proteins have a
25-amino acid deletion near the C-terminus, and HE2 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. |
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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
-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: , ß, and . 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.
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Library construction
A directional cDNA library from human caput/corpus epididymis
was constructed in 
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 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 , ß, and isoforms
were amplified using the M13F primer in the vector as the reverse
primer and the following specific forward primers: for 1
(nucleotides 547–567), CGT GAC ATT TGC TCT GAT CCC; for 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 , ß, and 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 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 and
ß2 peptide sequences to facilitate coupling. The peptides
correspond to the C-terminal amino acids of the following:
specific, kVDFLGPRWARGCSTNG; ß1 specific, CVSNTDEEGKEKPEMDGRSGI; and
ß2/1 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.
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Results |
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form) reaction
product (Fig. 1
, 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. 1, 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 is the prototype of a family of epididymal
proteins. This was confirmed by screening a human epididymis
caput/corpus cDNA library using the
32P-labeled PCR products ( 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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for the original HE2 (18); ß, indicating a
deleted form; and , which was later shown to have a larger deletion.
Sequencing revealed 8 HE2, 12 HE2ß, and 2 HE2 clones. From the
remaining 8 clones, PCR fragment sizes suggested combinations of exons
already seen and were not analyzed further. HE2 clones contain the
entire HE2 coding region for 103 amino acids using the original stop
codon (Figs. 2 and 3 and Table 1).
HE2ß and - 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 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. 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 2
and 2 clones resulted from incomplete RT and are indicated in Fig. 2
by arrows. The 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 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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stop codon at nucleotide 479. In ß1,
translation would continue to a stop codon at 645 in the HE2 3' UTR.
In the resulting ß1 protein of 133 amino acids, the relatively
hydrophobic 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 1
). There are additional consensus casein kinase 2
phosphorylation sites at amino acids 115 and 117.
In ß2, the 3'-UTR of HE2 stops at nucleotide 535 and skips an exon
present in 2 and 2 (Fig. 2, large dotted line,
nucleotides +1 to +103). The remaining 3'-sequence (Fig. 2, 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 forms also contain the HE2 coding region to nucleotide
381 (amino acid 71) where the frame-shifting ß form deletion begins
(Figs. 2 and 3). The 1 form continues from +104 through sequence
identical to the 3'-UTR of 2 and ß2 and using the same translation
stop codon as ß2 at +137. Thus, the C-terminal 11 amino acids of ß2
and 1 are identical. The 2 deletion also begins after nucleotide
381, but the coding region is joined to sequence identical to the 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. 4). 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. 4B with the +1 to +103 exon probe than the longer +104 to +400 probe in
Fig. 4C 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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). 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 2/2 (+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 2/ß2/1/2
(+104 to +400) probe failed to hybridize detectably to the monkey mRNAs
(data not shown).
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, antisera were raised against -, ß1-, and ß2/1-specific
C-terminal peptides (boxed in Fig. 3) and were used to
localize the proteins in human epididymis.
Clear, competable staining was not observed in this study. Whether
the 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. 6A). In some cells,
expression extended to the basal lamina. The staining did not develop
when antibody preincubated with antigen was used (Fig. 6B). Principal
cells of the caput and proximal corpus also expressed HE2ß1, but not
in every cell (Fig. 6C). Heavy brown staining of luminal contents was
seen in corpus (Fig. 6C). 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. 7B).
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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. 6D). The staining was
substantially reduced when antiserum was preincubated with antigen
(Fig. 6E). In the regular cuboidal epithelium IV of the ducts, staining
was seen in about a third of the cells (Fig. 6F). 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 HE2ß2/1 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 HE2ß2/1 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).
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Discussion |
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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. 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 -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 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.
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Acknowledgments |
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Footnotes |
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Received September 9, 1999.
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M. C. W. Avellar, L. Honda, K. G. Hamil, Y. Radhakrishnan, S. Yenugu, G. Grossman, P. Petrusz, F. S. French, and S. H. Hall Novel Aspects of the Sperm-Associated Antigen 11 (SPAG11) Gene Organization and Expression in Cattle (Bos taurus) Biol Reprod, June 1, 2007; 76(6): 1103 - 1116. [Abstract] [Full Text] [PDF]
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 Y. Sang, M. T. Ortega, F. Blecha, O. Prakash, and T. Melgarejo Molecular Cloning and Characterization of Three {beta}-Defensins from Canine Testes Infect. Immun., May 1, 2005; 73(5): 2611 - 2620. [Abstract] [Full Text] [PDF]
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 R. S. McIntosh, J. E. Cade, M. Al-Abed, V. Shanmuganathan, R. Gupta, A. Bhan, P. J. Tighe, and H. S. Dua The Spectrum of Antimicrobial Peptide Expression at the Ocular Surface Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1379 - 1385. [Abstract] [Full Text] [PDF]
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M. C. W. Avellar, L. Honda, K. G. Hamil, S. Yenugu, G. Grossman, P. Petrusz, F. S. French, and S. H. Hall Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta) Biol Reprod, November 1, 2004; 71(5): 1453 - 1460. [Abstract] [Full Text] [PDF]
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S. Yenugu, R. T. Richardson, P. Sivashanmugam, Z. Wang, M. G. O'Rand, F. S. French, and S. H. Hall Antimicrobial Activity of Human EPPIN, an Androgen-Regulated, Sperm-Bound Protein with a Whey Acidic Protein Motif Biol Reprod, November 1, 2004; 71(5): 1484 - 1490. [Abstract] [Full Text] [PDF]
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 S. Yenugu, K. G. Hamil, Y. Radhakrishnan, F. S. French, and S. H. Hall The Androgen-Regulated Epididymal Sperm-Binding Protein, Human {beta}-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial {beta}-Defensin Endocrinology, July 1, 2004; 145(7): 3165 - 3173. [Abstract] [Full Text] [PDF]
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M. I. Nonaka, Y. Hishikawa, N. Moriyama, T. Koji, R. T. Ogata, A. Kudo, H. Kawakami, and M. Nonaka Complement C4b-Binding Protein as a Novel Murine Epididymal Secretory Protein Biol Reprod, December 1, 2003; 69(6): 1931 - 1939. [Abstract] [Full Text] [PDF]
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A. I. Yudin, T. L. Tollner, M.-W. Li, C. A. Treece, J. W. Overstreet, and G. N. Cherr ESP13.2, a Member of the {beta}-Defensin Family, Is a Macaque Sperm Surface-Coating Protein Involved in the Capacitation Process Biol Reprod, October 1, 2003; 69(4): 1118 - 1128. [Abstract] [Full Text] [PDF]
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O. Frohlich, N. M. Ibrahim, and L. G. Young EP2 Splicing Variants in Rhesus Monkey (Macaca mulatta) Epididymis Biol Reprod, July 1, 2003; 69(1): 294 - 300. [Abstract] [Full Text] [PDF]
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K. Doiron, C. Legare, F. Saez, and R. Sullivan Effect of Vasectomy on Gene Expression in the Epididymis of Cynomolgus Monkey Biol Reprod, March 1, 2003; 68(3): 781 - 788. [Abstract] [Full Text] [PDF]
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M.A. Palladino, T.A. Mallonga, and M.S. Mishra Messenger RNA (mRNA) Expression for the Antimicrobial Peptides {beta}-Defensin-1 and {beta}-Defensin-2 in the Male Rat Reproductive Tract: {beta}-Defensin-1 mRNA in Initial Segment and Caput Epididymidis Is Regulated by Androgens and Not Bacterial Lipopolysaccharides Biol Reprod, February 1, 2003; 68(2): 509 - 515. [Abstract] [Full Text] [PDF]
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 S. H. Hall, K. G. Hamil, and F. S. French Host Defense Proteins of the Male Reproductive Tract J Androl, September 1, 2002; 23(5): 585 - 597. [Full Text] [PDF]
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H. H. von Horsten, P. Derr, and C. Kirchhoff Novel Antimicrobial Peptide of Human Epididymal Duct Origin Biol Reprod, September 1, 2002; 67(3): 804 - 813. [Abstract] [Full Text] [PDF]
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 Y. Yamaguchi, T. Nagase, R. Makita, S. Fukuhara, T. Tomita, T. Tominaga, H. Kurihara, and Y. Ouchi Identification of Multiple Novel Epididymis-Specific {beta}-Defensin Isoforms in Humans and Mice J. Immunol., September 1, 2002; 169(5): 2516 - 2523. [Abstract] [Full Text] [PDF]
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K. G. Hamil, Q. Liu, P. Sivashanmugam, S. Yenugu, R. Soundararajan, G. Grossman, R. T. Richardson, Y.-L. Zhang, M. G. O'Rand, P. Petrusz, et al. Cystatin 11: A New Member of the Cystatin Type 2 Family Endocrinology, July 1, 2002; 143(7): 2787 - 2796. [Abstract] [Full Text] [PDF]
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 B. C. Schutte, J. P. Mitros, J. A. Bartlett, J. D. Walters, H. P. Jia, M. J. Welsh, T. L. Casavant, and P. B. McCray Jr. Discovery of five conserved beta -defensin gene clusters using a computational search strategy PNAS, February 19, 2002; 99(4): 2129 - 2133. [Abstract] [Full Text] [PDF]
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Q. Liu, K. G. Hamil, P. Sivashanmugam, G. Grossman, R. Soundararajan, A. J. Rao, R. T. Richardson, Y.-L. Zhang, M. G. O'Rand, P. Petrusz, et al. Primate Epididymis-Specific Proteins: Characterization of ESC42, a Novel Protein Containing a Trefoil-Like Motif in Monkey and Human Endocrinology, October 1, 2001; 142(10): 4529 - 4539. [Abstract] [Full Text] [PDF]
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N. M. Ibrahim, L. G. Young, and O. Frohlich Epididymal Specificity and Androgen Regulation of Rat EP2 Biol Reprod, August 1, 2001; 65(2): 575 - 580. [Abstract] [Full Text] [PDF]
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O. Fröhlich, C. Po, and L. G. Young Organization of the Human Gene Encoding the Epididymis-Specific EP2 Protein Variants and Its Relationship to Defensin Genes Biol Reprod, April 1, 2001; 64(4): 1072 - 1079. [Abstract] [Full Text]
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 P. Li, H. C. Chan, B. He, S. C. So, Y. W. Chung, Q. Shang, Y.-D. Zhang, and Y.-L. Zhang An Antimicrobial Peptide Gene Found in the Male Reproductive System of Rats Science, March 2, 2001; 291(5509): 1783 - 1785. [Abstract] [Full Text]
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 Y. Yamaguchi, S. Fukuhara, T. Nagase, T. Tomita, S. Hitomi, S. Kimura, H. Kurihara, and Y. Ouchi A Novel Mouse beta -Defensin, mBD-6, Predominantly Expressed in Skeletal Muscle J. Biol. Chem., August 17, 2001; 276(34): 31510 - 31514. [Abstract] [Full Text] [PDF]
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