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Centre de Recherche en Biologie de la Reproduction; and Département d’Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Robert Sullivan, Unité d’Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l’Université Laval, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada, G1V 4G2. E-mail: robert.sullivan{at}crchul.ulaval.ca
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Leaving the testis, mammalian spermatozoa transit along the excurrent duct system formed by the vasa efferentia, epididymis, and vas deferens (3). The functions of the epididymis include absorption of seminiferous fluid and the maturation, concentration, transport, and storage of spermatozoa. The epididymis is usually divided into three anatomical regions: caput, corpus, and cauda segments. Sperm membrane modifications occur throughout the male reproductive tract. Caput and corpus epididymidis are involved in the acquisition of sperm fertilizing ability, whereas the cauda segment is specialized in sperm storage. In humans, all portions of the excurrent duct shows high transcriptional activity (4). Many studies have reported that epididymal secretory products are involved in the acquisition of fertilizing ability by mammalian spermatozoa (5, 6). In several species, epididymal proteins have been shown to interact with spermatozoa and to modify their surface properties during epididymal transit (7, 8, 9). Collectively, these modifications are referred to as sperm maturation. These are dependent upon epididymal protein synthesis that is under androgen control (4).
During epididymal maturation, sperm membrane lipids undergo distinct physical and chemical alterations (10, 11). Changes in the distribution of sperm membrane protein occurring during these processes reflect biochemical alterations of both membrane lipids and proteins. The sperm plasma membrane has both membrane-integrated and surface-adsorbed proteins when spermatozoa leave the testis. Some of these surface proteins change their location from one membrane domain to another during sperm maturation (12). Other sperm surface proteins are altered, masked or replaced by new proteins of epididymal origin (13, 14).
Identification of epididymal sperm proteins involved in the acquisition of sperm fertilizing ability has been investigated by many laboratories (4). We have previously described P26h, a 26-kDa protein that is present on the hamster sperm acrosomal surface and in epididymal fluids collected from the caput to the cauda regions (15, 16). The P26h messenger RNA (mRNA) is strongly expressed in the hamster testis, and at a lower level in the corpus epididymidis (16, 17) This protein, which is glycosylphosphatidylinositol anchored to the sperm membrane (18), shows a species-specific affinity for the zona pellucida glycoproteins (17, 19). Based on these observations we have proposed that this sperm protein is involved in fertilization. In fact, polyclonal P26h antiserum inhibits sperm-zona pellucida binding in vitro in a dose dependent manner (20). Using these P26h antibodies, we have identified a 34-kDa human epididymal sperm protein (P34H) that shows structural, antigenic and functional similarities with the P26h. P34H appears on human spermatozoa in the distal caput-proximal corpus epididymidis and its location is restricted to the acrosome (21). Antibodies against this human protein inhibit sperm-zona pellucida binding in vitro, without affecting motility, the ability to acrosome react or sperm-egg plasma membrane fusion (22). P34H appears to be involved in one of the absolute prerequisites of fertilization, i.e. sperm-zona pellucida binding. Therefore, we have proposed that P34H can be used as a marker of epididymal sperm maturation in humans (23). We have also showed that some cases of human infertility are associated with the absence of P34H (23).
In the present study, we report the sequence of the epididymal complementary DNA (cDNA) encoding for the P34H protein. P34H expression pattern along the male reproductive tract is described using Northern blot analysis and in situ hybridization methodologies. Western blot experiments also show that recombinant P34H is antigenically related to the native P34H sperm protein.
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Materials and Methods |
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RNA isolation and Northern blot analysis
Total RNA was isolated after homogenization of human testicular
and epididymal tissues in a guanidium thiocyanate solution (4
M guanidinium thyocyanate, 25 mM sodium
citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-ß-mercaptoethanol)
followed by CsCl fractionation according to Chirgwin et al.
(24). The RNA pellets were resuspended in TES solution (10
mM Tris-HCl, 5 mM EDTA, 1% SDS, pH 7.40), and
extracted once with phenol/chloroform (1:1) and twice with
chloroform/alcohol isoamyl (24:1). RNAs were precipitated with 0.1 vol
of sodium acetate (3 M, pH 5.2) and 2.5 vol of 95%
ethanol. The RNA pellets were resuspended in diethylpyrocarbonate
(DEPC)-treated water, quantitated by spectrophotometry at 260 nm, and
stored at -80 C until used. All solutions were treated with DEPC.
Equivalent amounts of total RNA (20 µg) were denaturated in 50%
formamide at 65 C for 15 min and separated by electrophoresis in 1%
agarose gels containing 2.2 M formaldehyde (25). RNA was
transferred to nylon membranes (Qiagen, Santa Clarita,
CA), UV cross-linked, and prehybridized at 42 C for 4 h in 50%
(vol/vol) formamide, 0.75 M NaCl, 0.05 M
NaH2PO4, 0.005 M EDTA, 2 x
Denhardt’s reagent [0.2% (wt/vol) Ficoll 400, 0.2% (wt/vol)
polyvinylpyrrolidone, 0.2% (wt/vol) BSA, 0.2 mg/ml herring sperm DNA
(Sigma Chemical Co., Mississauga, Ontario, Canada), 10%
dextran sulfate, and 0.1% SDS. Hybridizations were performed in
prehybridization solution supplemented with 1 x 106
cpm/ml of P34H cDNA or actin cDNA at 42 C for 16–20 h. cDNA probes
were random-prime labeled using the T7 Quick-Prime kit (Pharmacia Biotech, Baie D’Urfé, Québec, Canada) with
[
-32P] dCTP. Blots were subsequently washed twice at
room temperature in single-strength SSC, 0.1% SDS for 5 min, and twice
at 65 C in 0.1-strengh SSC for 30 min. Blots were exposed to X-Omat
film (Eastman Kodak Co., Rochester, NY) at -80 C with
intensifying screens for 16–18 h. An RNA ladder (1.6–7.4 kb;
Boehringer Mannheim, Laval, Québec, Canada) was
electrophoresed in parallel and an actin probe was used as a
constitutive internal control. Commercially available nylon membranes
(Invitrogen, San Diego, CA) containing RNA from eight
different human somatic tissues were hybridized in parallel with the
same probes. The P34H mRNA was quantitated by densitometric scanning
and expressed as a ratio of actin transcript.
Cloning and sequencing of P34H
Poly(A)+RNA prepared from human epididymal tissues
were purified from total RNA using a poly(A)+RNA
purification kit (Pharmacia Biotech) according to the
supplier’s instructions. The cDNA library was prepared according to
the instruction provided by the supplier. Briefly, epididymal
poly(A)+RNA was reverse-transcribed and ligated into the
lambda Uni-Zap XR vector (Stratagene, La Jolla, CA). The
lambda library was packaged and amplified using Escherichia
coli XL1-Blue cells and screened with the random-prime labeled
715-bp P26h cDNA cloned in our laboratory (Gaudreault et
al., 1999, submitted for publication). The positive clones
were isolated by plaque purification and screened by PCR. PCR was
conducted in 50 µl total volume containing 5 µl of cDNA (positive
clones), 1 mM of each primer (GGAAACAGCTATGACCATG;
GTAAAACGGCCAGT), 200 mM dNTPs and 1 U of taq DNA polymerase
in the reaction buffer provided by the manufacturer (Pharmacia Biotech). Reaction conditions were 30 cycles at 95 C for 30 sec,
60 C for 30 sec, and 72 C for 1 min. The longest ones were subcloned
into pBluescript KS+. All nucleotide sequences were determined by the
dideoxinucleotide termination method (26) using T7 Sequenase v 2.0 kit
(Amersham Life Science). The labeled reaction products
were analyzed on a DNA sequencer gel. Amino acid sequence was deduced
using Gene Jockey software (Biosoft, Cambridge, UK) and
the corresponding hydropathy plot was drawn according to the Kyte and
Doolittle algorithm (27). Search for amino acid homologies was
performed using "blast search" software.
In situ hybridization
In situ hybridization was performed using digoxigenin (DIG)
(Boehringer Mannheim)-labeled RNA probes as previously
described (28). Testis and epididymis cryosections were fixed with
freshly prepared 4% paraformaldehyde in PBS for 5 min at room
temperature, incubated for 10 min in 95% ethanol/5% acetic acid at
-20 C, and rehydrated by successive baths of decreasing concentrations
of ethanol diluted with DEPC-treated H2O. Target RNA was
unmasked by enzymatic digestion with 10 µg/ml proteinase K
(Boehringer Mannheim) in PBS for 10 min at 37 C, followed
by a 5-min incubation in 0.2% glycine. Sections were postfixed for 5
min with 4% paraformaldehyde in PBS, acetylated with 0.25% acetic
anhydride, 0.1 M triethanolamine, pH 8.0, for 10 min, and
finally washed with PBS.
Tissues were prehybridized for 1 h with a preheated 250 µg/ml salmon sperm DNA in a hybridization solution (0.3 M NaCl, 0.01 M Tris-HCl, pH 7.5, 1 mM EDTA, 1 x Denhardt’s solution, 5% dextran sulfate, 0.02% SDS, and 50% formamide). Sections were then incubated overnight at 42 C, under coverslips, with 25 µl of 2 ng/ml heat-denaturated antisense or sense cRNA probed with DIG according to supplier’s instruction. Sections were washed twice in 2 x SSC at room temperature, followed by two 10-min washes at 42 C in 2 x SSC, 1 x SSC, and 0.2 x SSC, respectively.
Hybridization reactions were detected by immunostaining with alkaline phosphatase-conjugated DIG antibodies. Nonspecific staining was blocked by preincubation for 1 h with 5% (vol/vol) heat-inactivated sheep serum in 0.2 M Tris-HCl, 0.2 M NaCl, and 3% Triton X-100. Sections were then incubated for 2 h at room temperature with the alkaline phosphatase-conjugated anti-DIG antibodies diluted 1:1000 in blocking solution, washed with Tris-HCl/NaCl buffer, and incubated with 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 0.01 M MgCl2. The hybridization signal was visualized after a 10- to 15-min incubation period with the phosphatase substrate nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL, Gaithersburg, MD). Levamisole (2 mM; Sigma Chemical Co.) was added to the reaction mixture to inhibit endogenous alkaline phosphatase. Slides were immersed in 1 mM EDTA, 0.001 M Tris-HCl, pH 7.5, washed 5 min in H2O, counterstained with neutral red, dehydrated through baths of ethanol, cleared in xylene, and mounted with Permount (Fisher Scientific, Nepeau, Ontario, Canada). All sections were processed in parallel to allow comparison.
Antisera against synthetic peptides
The N-terminal 20 amino acid sequence deduced from the human
P34H cDNA (P34pept) and a 10-amino acid sequence deduced
from the hamster P26h cDNA (P26pept) were identified as
potential antigens (see Fig. 2
). P34pept
(MELFLAGRRVLVTGAGKGIG, amino acids 1–20) and P26pept
(CFAKKLKERH, amino acids 196–204) were synthesized on an ABI 433A
Peptide Synthesizer using FastMoc chemistry (Service de séquence
de peptides de l’est du Québec, Ste-Foy, Québec, Canada).
The activation was carried out with HBTU/DIEA. The N-terminal amino
groups were protected by Fmoc, and side chain functional groups were
protected by t-bu (Glu and Thr), Boc (Lys), and Pmc (Arg). The peptides
were cleaved with TFA/thioanisole/water/EDT (90:5:2.5:2.5) for 2–3 h
at room temperature.
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New Zealand female rabbits were immunized sc with 100 µg of keyhole limpet hemocyanin-conjugated P34Hpept or P26hpept emulsified with complete Freund’s adjuvant. Six booster injections were given at 3-week intervals using the same amount of antigen emulsified with incomplete Freund’s adjuvant.
Gamete collection
Sexually mature golden hamsters (Mesocricetus
auratus; Charles River Laboratories, Inc.,
St-Constant, Québec, Canada) were used in these studies. Males
were killed by CO2 inhalation, and the epididymides were
excised, defatted, and dissected. The cauda segments were minced with
fine scissors and gently agitated in D-PBS (Dulbecco’s PBS;
Gibco BRL, Grand Island, NY) to allow for sperm
dispersion. Spermatozoa were washed three times by centrifugation in
D-PBS and subjected to detergent extraction. Sperm membrane proteins
were extracted with 0.4% (vol/vol) Triton X-100 for 10 min at room
temperature. Following centrifugation (800 x g) for 10
min, the supernatant was recovered and the proteins were precipitated
using ice-cold acetone. Proteins were resuspended in SDS-PAGE sample
buffer (50 mM Tris-HCl pH 6.3, 2% (wt/vol) SDS and 5%
(vol/vol) ß-mercaptoethanol).
Human ejaculated spermatozoa from fertile volunteers were washed three times by centrifugation (800 x g) in Dulbecco’s PBS (D-PBS: Gibco BRL) and resuspended in SDS-PAGE sample buffer.
Western blotting
Samples subjected to SDS-PAGE (29) were equilibrated for 10 min
in blotting buffer (10 mM Tris, 96 mM glycine,
10% (vol/vol) ethanol). Proteins were transferred to a nitrocellulose
membrane for 30 min at 4mA/cm2 using a semidry
milliblot-graphite electroblotter system (Millipore Corp., Bedford, MA). The nitrocellulose membrane was
saturated overnight at 4 C with 5% (wt/vol) skim milk in PBS. The
membrane was then incubated for 2 h at room temperature with an
anti-P26h antiserum diluted 1/1000, with an anti-P34Hpept
diluted 1:2000 or with an anti-P26pept diluted 1:2000
supplemented with 2.5% (vol/vol) goat serum in PBS. The anti-P26h used
in this study was previously produced against P26h purified from
detergent-extracted cauda epididymal sperm proteins (19). The antisera
were preabsorbed on a human keratin powder (19). After three washes of
10 min in PBS-Tween (0.2% (vol/vol) Tween-20 in PBS), Western blots
were incubated for 45 min at room temperature with a
peroxidase-conjugated goat antirabbit IgG diluted 1:3000 in PBS
containing 2.5% goat serum. After three more washes in PBS-Tween,
immune complexes were visualized using a chemiluminescent susbtrate of
peroxidase according to the supplier’s instructions (ECL kit,
Amersham Life Science).
Expression of recombinant P34H
The P34H cDNA and the P26h cDNA were subcloned in p-ESP-1
plasmid (Stratagene, La Jolla, CA). This system uses the
yeast Schizosaccharomyces pompe as the expression host and
the glutathione S-transferase (GST) peptide as a protein purification
tag. Unlike the Escherichia coli expression host, S.
pompe allows eukaryotic posttranslational modification of the
expressed proteins. The SP-Q01 S. pompe (leu1–32h)
containing the plasmid were grown on Edinburgh minimal medium
(EMM) agar plates supplemented with thiamine at 30 C for 4 days. The
yeast expression strain was grown to a midlogarithmic phase in yeast
extract supplement (YES) medium, containing sufficient thiamine to
repress the promoter pnmt1. Expression of the fusion gene
was induced by harvesting and growing the cells in EMM broth, which
lacks thiamine, at 30 C for 18 h. Cells were harvested by
centrifugation at 1000 x g for 5 min at 4 C. The cell
pellet was resuspended in 0.5 ml of cold PBS supplemented with 1%
Triton X-100 and protease inhibitors (leupeptin 10 mg/ml; aprotinin 5
mg/ml; AEBSF 0.1 mmol/ml; Pepstatine 5 mg/ml). Acid-washed glass beads
were added to the cell suspension, which was then was vortexed
repeatedly to release the GST fusion protein into the supernatant. The
cell extract was then centrifugated at 12,000 x g for
5 min at 4 C. Fifty ml of supernatant were added to 50 ml of 2 x
SDS-PAGE loading buffer (100 mM Tris-HCl (pH 6.8), 200
mM dithiothreitol, 4% (wt/vol) SDS, 0.2% (wt/vol)
bromophenol blue, 20% (vol/vol) glycerol), boiled for 5 min at 100 C
and subjected to SDS-PAGE.
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). The two
other clones were 600 bp and 1000 bp. The shortest one covered the
300-bp to 900-bp region of the full-length sequence presented in Fig. 1. The 1000-bp clone had two poly A tails. The 912-bp P34H cDNA had a
732-bp open reading frame, starting with an ATG at position 85, a stop
codon at position 817 bp, followed by a poladenylation signal and a
poly A tail (Fig. 1). A 244 amino acid sequence was deduced from the
human epididymal cDNA and had a predicted molecular weight of 26 kDa.
Three N-glycosylation sites were identified in the P34H
sequence, located in the last 100 C-terminal amino acids of the deduced
sequence (Fig. 1).
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). Using these primary
amino acid sequences, the predicted secondary structure of the human
and the hamster proteins were analyzed and compared (Fig. 2B). The
hydropathicity plots of these two proteins appeared to be strikingly
similar. A search for amino acid sequence homologies revealed a 71%
similarity between the epididymal P34H protein and pig lung tetrameric
carbonyl reductase.
Northern blot analysis of P34H mRNA expression
To determine the distribution of the P34H gene expression, P34H
cDNA was used to probe Northern blots of total RNA from different human
tissues. P34H transcript was evident as a single band of 912 bp. As
shown in Fig. 3A, the P34H mRNA was
highly expressed in human epididymis whereas a very weak signal was
detected in the testicular RNA sample. Interestingly, the level of
expression was moderate in the caput, high in the corpus and lowest in
the cauda epididymidis. A faint, but clearly detectable, signal of P34H
transcript was observed in liver, pancreas, muscle, and lung tissues
(Fig. 3B).
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shows a
high signal intensity in the proximal and distal regions of the corpus
epididymidis (Fig. 4, E and F) as expected from Northern blot analysis.
Hybridization signal was detectable in caput (Fig. 4, C and D) and
cauda sections of the epipidymis (Fig. 4, H and I) but at a much lower
intensity than that observed in the distal corpus. No significant P34H
transcript was detected in testis (Fig. 4B). All along the epididymis,
the P34H mRNA staining was restricted to the principal cells of the
epididymal epithelium. The labeling was more intense in the perinuclear
region, especially in the corpus segment (Fig. 4, E and F). No signal
was detectable when negative control hybridization was performed with a
sense strand of P34H cRNA (Fig. 4G).
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). When
similar Western blots were probed with an anti-P34Hpept,
only the P34H was detected in the human protein extract. The hamster
P26h remained undetectable (Fig. 5B). P26h was recognized by the
anti-P26hpept whereas P34H was undetectable on similar
Western blots (Fig. 5C).
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). Western blots of cell extracts from
yeast expressing P26h-GST, revealed a fusion protein of 51 kDa (Fig. 6A). When similar blots were probed with an anti-P34Hpept
serum (Fig. 6B), the same 57 kDa P34H-GST fusion protein was detected.
The P26h-GST protein was undetectable when the
anti-P34Hpept was used (Fig. 6B).
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An intriguing 71% sequence similarity was found between the epididymal
P34H protein and pig lung tetrameric carbonyl reductase (46). Carbonyl
reductase is an NADPH-linked oxidoreductase that catalyzes the
reduction of various carbonyl compounds to corresponding alcohols (47),
and is distributed in tissues of different mammalian species (48).
Carbonyl reductase is known to be a member of the short-chain
dehydrogenase/reductase (SDR) superfamily. Two consensus sequences are
conserved in this family, the NAD(H) or NADP(H) binding domain, a
N-terminal segment (GlyXXXGlyXGly), and the catalytic domain
(TyrXXXLys) (49). P34H deduced amino acid sequence possesses the
catalytic domain in position Tyr149XXXLys153. This sequence represents
the most conserved region of the SDR members (50). Two residues are
strictly conserved in the SDR family: Tyr 152 and Lys 156 (49).
However, they are not present in the P34H deduce amino acid sequence.
Human epididymal sperm P34H protein may even possess an enzymatic
activity, which is not necessarily involved in interaction with the
zona pellucida. This is the case for other sperm proteins, proposed to
mediate binding of spermatozoa to the zona pellucida matrix (45, 51).
These proteins show enzymatic properties, such as proacrosin (52), a
trypsin-like protease (53), a mannosidase (54), a galactosyltransferase
(55), and, P95 (56), a hexokinase (57, 58). The substrate affinity of
these sperm proteins rather than the enzymatic activity is proposed to
mediate the interaction between the sperm and the zona pellucida (59, 60). The similarities of the P34H with the SDR family, particularly
with lung carbonyl reductase, may explain the transcript detected on
Northern blots of human lung tissues (Fig. 3). It has been reported
that carbonyl reductase expression is very high in lung and low in
liver, testis, heart, kidney, fat pad, brain and spleen (61).
The 732-bp open reading frame of the P34H cDNA encodes for a 244 amino
acid protein. A discrepancy exists between the 34-kDa molecular weight
of P34H evaluated by SDS-PAGE (22) and the 26-kDa molecular weight
estimated from the deduced amino acid sequence. P26h and P34H have been
named according to their behavior when submitted to SDS-PAGE.
Considering that the identification of P34H at the protein level was
previously based on cross-reactivity of antibodies raised against the
hamster P26h (22), the discrepancy between P34H molecular weight
deduced from its amino acid sequence and that calculated from its
migration on SDS-PAGE may raise some concern. To ascertain that the
cDNA sequence encodes for the human protein antigenically related to
the hamster P26h, both P26h and P34H cDNAs were expressed in an
in vitro transcription/translation system. When Western
blots of fusion P34H-GST and P26h-GST were probed with an anti-P26h
serum, bands of 57 and 51 kDa, respectively, are detected (Fig. 5).
Considering the GST molecular weight of 27 kDa, the 6-kDa difference
between the two recombinant proteins is close to molecular weight
difference between P34H and P26h revealed by Western blot of sperm
protein extracts (20, 22). When evaluated by SDS-PAGE, the molecular
weight difference between P34H and P26h can partly be explained by
differences in posttranslation modifications of the P34H, especially in
glycosylation patterns. In fact, three N-glycosylation sites
are located in the last 100 amino acids deduced from the P34H cDNA. Of
these sites, two have the same location as those found in the P26h cDNA
sequence (Fig. 1).
P34H and P26h proteins show structural homologies with the SDR
superfamily. Most members of this large family show very high homology
between species (49, 50). P34H and P26h amino acid sequences deduced
from the cloned cDNAs revealed a 65% homology (Fig. 2). If they belong
to the SDR superfamily, a higher level of homology between P26h and
P34H may be expected. P34H distribution and function were described
using a antiserum raised against the hamster P26h purified from
detergent extracted sperm proteins (Fig. 5A) (20). When Western blots
of hamster and human sperm proteins are probed with an antiserum raised
against a synthetic peptide corresponding to the 20 N-terminal P34H
deduced amino acid sequence, the human protein of 34 kDa is detected
(Fig. 5B). Although P34H peptide contains only 7/20 different amino
acids from the P26h, this appears to be sufficient to confer to P34H
antigenic properties different from P26h, at least in this 20
N-terminal amino acid sequence. An antiserum against a peptide sequence
from the P26h sequence amino acids revealed a 26-kDa band only in the
hamster sperm extract (Fig. 5C). These results strongly suggest that
the 35% difference in amino acid sequence between P34H and P26h is not
a consequence of cloning artifacts. The physiology of fertilization
shows great differences from one mammalian species to another (62).
Proteins mediating gamete interaction may have diverged at a higher
degree than other members of the SDR superfamily that catalyze the same
biochemical reaction in different species.
The epididymal segment where spermatozoa acquire their fertilizing ability varies from one species to another (1). Northern blot analysis demonstrated that P34H synthesis occurs principally in the distal corpus epididymidis. The zona-free hamster test suggests that human spermatozoa acquire their fertilizing ability when they reach the corpus epididymal portion of the excurrent ducts (5, 63). The location of P34H synthesis is in agreement with this conclusion. This finding is also supported by previous immunohistological studies showing that P34H starts to accumulate on spermatozoa in this epididymal segment (21, 22). This, however is in contrast with P26h, the hamster analog of P34H, which is highly expressed in the testis, and to a lower level in the corpus epididymidis (Gaudreault, C., C. Légaré, B. Bérubé, and R. Sullivan, submitted for publication). This differential expression may reflect physiological differences between the human and the hamster epididymidis. Other differences in the regionalization of epididymal protein synthesis are known to exist among mammalian species (6).
In situ hybridization studies clearly demonstrate that the P34H transcript is specifically expressed by principal cells of the human epididymis. This cell type represents more than 80% of the cell population of the epididymal epithelium and plays an important role in luminal protein secretion (64). The location of P34H mRNA is thus in agreement with the concept that P34H is an epididymal marker in human. After secretion into the lumen, this epididymal protein gradually accumulates on the sperm surface covering the acrosome, the sperm membrane domain involved in zona pellucida recognition (22).
Epididymal secretory products are essential for mammalian sperm maturation, and current evidence indicates that specific proteins become associated with the spermatozoa during epididymal transit (6). P34H shows functional and structural similarities with the P26h (22), a sperm protein involved in sperm-zona pellucida interaction as shown by the ability of specific antibodies to inhibit both in vitro and in vivo fertilization in the hamster (20). Human P34H protein, like the hamster P26h, is added to spermatozoa during their epididymal transit (18, 21). Both proteins belong to the SDR superfamily, a protein family showed to play a wide range of functions (49). Our results suggest that mediating gamete interactions may be one of these functions.
Recent assisted reproductive technologies have questioned the importance of the human epididymis in sperm maturation (65). Even though the pattern of maturation of spermatozoa in the human epididymis has never been established directly by testing ability to fertilize zona intact eggs, indirect evidence suggests that this ability is acquired in the corpus segment (66). Results following epididymo-vasostomy also support the notion that more efficient spermatozoa are ejaculated if the anastomosis is performed at a distal point of the caput epididymidis (67). These observations are in agreement with our results demonstrating that P34H, a protein essential for sperm function, is largely synthesized by principal cells lining the epididymal lumen. These observations support the importance of the epididymal transit in the acquisition of the fertilizing ability of human spermatozoa. On the other hand, recovery of fertility following anastomosis of the vas deferens to the vas efferent or to testicular tubules challenged the physiological significance of epididymal transit in humans (68). In these pathological situations, the absence of P34H may be overcome by other sperm proteins that normally acts cooperatively at fertilization (69) or by the vas deferens if it has a capacity for P34H secretion (70). More work is needed to clarify the function of the human epididymis and to understand the significance of clinical results obtained in pathological situations affecting the excurrent duct.
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Acknowledgments |
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Footnotes |
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Received October 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase isozyme genes in
epididymis, testis, and liver of the adult rat. J Androl 18:372–377-D-mannosidase of rat sperm plasma membranes:
characterization and potential role in sperm-egg interactions. J
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 G. Frenette, M. Thabet, and R. Sullivan Polyol Pathway in Human Epididymis and Semen J Androl, March 1, 2006; 27(2): 233 - 239. [Abstract] [Full Text] [PDF]
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G. Frenette, C. Legare, F. Saez, and R. Sullivan Macrophage migration inhibitory factor in the human epididymis and semen Mol. Hum. Reprod., August 1, 2005; 11(8): 575 - 582. [Abstract] [Full Text] [PDF]
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C. Legare and R. Sullivan Expression and localization of c-ros oncogene along the human excurrent duct Mol. Hum. Reprod., September 1, 2004; 10(9): 697 - 703. [Abstract] [Full Text] [PDF]
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C. Legare, M. Thabet, and R. Sullivan Expression of heat shock protein 70 in normal and cryptorchid human excurrent duct Mol. Hum. Reprod., March 1, 2004; 10(3): 197 - 202. [Abstract] [Full Text] [PDF]
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C. Legare, N. Verville, and R. Sullivan Vasectomy Influences Expression of HE1 but not HE2 and HE5 Genes in Human Epididymis J Androl, January 1, 2004; 25(1): 30 - 43. [Abstract] [Full Text] [PDF]
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G. Frenette, C. Lessard, E. Madore, M. A. Fortier, and R. Sullivan Aldose Reductase and Macrophage Migration Inhibitory Factor Are Associated with Epididymosomes and Spermatozoa in the Bovine Epididymis Biol Reprod, November 1, 2003; 69(5): 1586 - 1592. [Abstract] [Full Text] [PDF]
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S. A. Joshi, S. A. Ranpura, S. A. Khan, and V. V. Khole Monoclonal Antibodies to Epididymis-specific Proteins Using Mice Rendered Immune Tolerant to Testicular Proteins J Androl, July 1, 2003; 24(4): 524 - 533. [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. Maeda, H. Kaku, M. Shimada, and T. Nishioka Cloning and sequence analysis of D-erythrulose reductase from chicken: its close structural relation to tetrameric carbonyl reductases Protein Eng. Des. Sel., July 1, 2002; 15(7): 611 - 617. [Abstract] [Full Text] [PDF]
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G. Frenette, C. Lessard, and R. Sullivan Selected Proteins of "Prostasome-Like Particles" from Epididymal Cauda Fluid Are Transferred to Epididymal Caput Spermatozoa in Bull Biol Reprod, July 1, 2002; 67(1): 308 - 313. [Abstract] [Full Text] [PDF]
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 J. Nakagawa, S. Ishikura, J. Asami, T. Isaji, N. Usami, A. Hara, T. Sakurai, K. Tsuritani, K. Oda, M. Takahashi, et al. Molecular Characterization of Mammalian Dicarbonyl/L-Xylulose Reductase and Its Localization in Kidney J. Biol. Chem., May 10, 2002; 277(20): 17883 - 17891. [Abstract] [Full Text] [PDF]
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S. Hammami-Hamza, M. Doussau, J. Bernard, E. Rogier, C. Duquenne, Y. Richard, A. Lefevre, and C. Finaz Cloning and sequencing of SOB3, a human gene coding for a sperm protein homologous to an antimicrobial protein and potentially involved in zona pellucida binding Mol. Hum. Reprod., July 1, 2001; 7(7): 625 - 632. [Abstract] [Full Text] [PDF]
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C. Gaudreault, M. El Alfy, C. Legare, and R. Sullivan Expression of the Hamster Sperm Protein P26h During Spermatogenesis Biol Reprod, July 1, 2001; 65(1): 79 - 86. [Abstract] [Full Text] [PDF]
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C. Légaré, M. Thabet, S. Picard, and R. Sullivan Effect of Vasectomy on P34H Messenger Ribonucleic Acid Expression along the Human Excurrent Duct: A Reflection on the Function of the Human Epididymis Biol Reprod, February 1, 2001; 64(2): 720 - 727. [Abstract] [Full Text]
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H.-W. Liu, Y.-C. Lin, C.-F. Chao, S.-Y. Chang, and G.-H. Sun GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation Mol. Hum. Reprod., May 1, 2000; 6(5): 422 - 428. [Abstract] [Full Text] [PDF]
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G.-H. Sun, Y.-C. Lin, Y.-W. Guo, S.-Y. Chang, and H.-W. Liu Purification of GP-83, a glycoprotein secreted by the human epididymis and conjugated to mature spermatozoa Mol. Hum. Reprod., May 1, 2000; 6(5): 429 - 434. [Abstract] [Full Text] [PDF]
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