Journal of Andrology, Vol. 27, No. 2, March/April 2006
Copyright q American Society of Andrology
Polyol Pathway in Human Epididymis and Semen
GILLES FRENETTE,* MICHEL THABET,† AND ROBERT SULLIVAN*
From the *Centre de Recherche en Biologie de la Reproduction and the Département d’Obstétrique-Gynécologie et
†chirurgie, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada.
ABSTRACT: Two enzymes are involved in the polyol pathway: an
aldose reductase that reduces glucose in sorbitol followed by its
oxidation in fructose by sorbitol dehydrogenase. It has been previously shown that both enzymes are presented in the bovine epididymis, where they are associated with membranous vesicles called
epididymosomes. Based on the distribution of these enzymes, it has
been hypothesized that the polyol pathway can modulate sperm motility during the epididymal transit. In the present study, polyol pathway was investigated in semen and along the epididymis in humans
in order to determine if sperm maturation can be associated with this
sugar pathway. Western blot analysis shows that both aldose reductase and sorbitol dehydrogenase are associated with ejaculated
spermatozoa and prostasomes in humans. These enzymes are also
associated with epididymosomes collected during surgical vasectomy reversal. Western blot, Northern blot, and reverse transcription–
polymerase chain reaction analysis show that aldose reductase and
sorbitol dehydrogenase are expressed at the transcriptional and
translational levels along the human epididymis. Unlike what occurs
in the bovine model, distribution of these enzymes is rather uniform
along the human excurrent duct. Immunohistological studies together with Western blot analysis performed on epididymosomes preparations indicate that the polyol pathway enzymes are secreted by
the epididymal epithelium. These results indicate that the polyol
pathway plays a role in human sperm physiology.
Key words: Spermatozoa, sperm maturation, sperm motility.
J Androl 2006;27:233–239
eaving the testis, the mammalian spermatozoon has
to transit along the epididymis. This long convoluted
tubule is involved in the remodeling of the sperm surface
and in modifications of macromolecular constituents, culminating in the storage of fully functional male gametes.
This process, called sperm maturation, includes the acquisition of flagellar forward motility and the ability to
efficiently encounter the oocytes and their investments
(Robaire and Hinton, 2002; Ecroyd et al, 2004).
We have previously shown that the epididymal epithelium secretes in an apocrine mode small membranous
vesicles that interact with the spermatozoa during epididymal transit (Sullivan et al, 2001, 2003; Saez et al, 2003).
Complex patterns of proteins are associated with these
vesicles, called epididymosomes (Légaré et al, 1999a;
Frenette and Sullivan, 2001). Prostasomes are similar vesicles secreted by the prostate and present in semen. Within the epididymal intraluminal compartment, selected proteins of epididymosomes are transferred to define sperm
surface domains (Frenette et al, 2002). Using a proteomic
approach, we showed in bovine that aldose reductase is
one of the major proteins associated with epididymosomes (Frenette et al, 2003). Aldose reductase uses
NADPH as an electron donor to reduce aldoses and ketoses. This enzyme is involved in the reduction of glucose
in sorbitol in the polyol pathway. The second step of this
sugar pathway involved sorbitol dehydrogenase, which
oxidizes sorbitol using NAD1 as an electron acceptor to
produce fructose (Oates, 2002) (Figure 1). Using the bovine as a model, we showed that both enzymes of the
polyol pathway are synthesized by the epididymal epithelium and that aldose reductase is highly expressed in the
proximal portion in the epididymis, while sorbitol dehydrogenase has a maximum activity in the distal portion
(Frenette et al, 2004). We thus proposed that sorbitol is
accumulating along the bovine epididymis and is oxidized
in the distal portion of the epididymis to produce fructose.
Based on this concept, we hypothesized that the polyol
pathway can modulate the flagellar motility of the transiting epididymal spermatozoa.
Epididymal physiology is poorly understood in humans
and shows characteristics when compared to usual animal
models (Légaré and Sullivan, 2004). In this context, factors modulating the metabolism and flagellar motility of
epididymal spermatozoa have to be revisited in humans.
The objective of the present work was to investigate the
existence of the polyol pathway along the epididymis of
normal men.
L
Supported by a Canadian Institutes for Health Research grant to R.S.
Correspondence to: Robert Sullivan, Unité d’Ontogénie-Reproduction,
Centre de Recherche, Centre Hospitalier de l’Université Laval, 2705 Blvd
Laurier, Ste-Foy, PQ, Canada, G1V 4G2 (e-mail: robert.sullivan@
crchul.ulaval.ca).
Received for publication June 2, 2005; accepted for publication August
30, 2005.
DOI: 10.2164/jandrol.05108
233
234
Journal of Andrology · March/April 2006
Figure 1. Polyol pathway. AR indicates aldose reductase; SODH, sorbitol dehydrogenase; NAD1, nicotinamide adenine dinucleotide; and NADPH,
nicotinamide adenine dinucleotide phosphate. Note that sorbitol is a linear sugar.
Materials and Methods
Biological Material
Human epididymides were obtained with the collaboration of our
local transplantation program after we obtained informed consent from relatives. Donors ranged in age from 18 to 55 years
and had no previous history that would affect the reproductive
function. Donors had experienced accidental death, and tissues
were dissected while artificial circulation was maintained to preserve those tissues destined for transplantation. Epididymides
were dissected on ice in caput, corpus, and cauda segments (Légaré et al, 1999b). Some tissue pieces were fixed in 4% paraformaldehyde for immunohistochemistry or were snap-frozen in
liquid nitrogen for protein and RNA extraction. Vas deferens
intraluminal fluid was obtained during surgical vasectomy reversal, and semen samples were obtained from the andrology
laboratory of our hospital. All procedures were approved by the
ethics committee at our institution.
Epididymosomes/Prostasomes
Epididymosomes were prepared from fluids collected from the
vas deferens during vasectomy reversal surgery. Prostasomes
were prepared from semen samples obtained by masturbation.
Only normospermic samples, according to World Health Organization criteria, were used in this study (World Health Organization, 1999). Vas deferens fluids and semen samples were
diluted with 2 volumes of Tris (30 mM) NaCl (130 mM) (TN;
pH 7.5) and centrifuged twice at 3000 3 g for 10 minutes. Supernatants of vas deferens were centrifuged at 120 000 3 g for
2 hours; pellets were resuspended in TN and ultracentrifuged a
second time. Pellets from vas deferens fluid containing epididymosomes were frozen until used. Prostasomes from semen
samples were prepared using the same protocol, with the exception that pellets obtained after the first centrifugation were chromatographed on a Sephacryl S-500 HR (Pharmacia, Baie d’Urfé,
Québec, Canada). The void volume containing prostasomes was
than ultracentrifuged a second time at 120 000 3 g for 2 hours
(Ronquist and Brody, 1985). The quantity of epididymosomes
present in seminal fluid is negligible when compared to the
quantity of prostasomes.
Western Blots
Epididymal tissues were homogenized in water containing 1%
sodium dodecyl sulfate (SDS) and centrifuged at 16 000 3 g for
20 minutes. Supernatants were precipitated with MeOH/CHCl 3,
and proteins were resuspended in sample buffer and submitted
to SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli,
1970). Protein concentrations were determined by amido black
staining of dot blots (Chapdelaine et al, 2001).
Semen samples were diluted with 2 volumes of TN, and spermatozoa were peletted at 1200 3 g for 10 minutes. After 2
washings by centrifugation, sperm proteins were extracted with
1% SDS and submitted to SDS-PAGE. Pellets of epididymosomes and prostasomes were resuspended in sample buffer for
Western blot analysis.
Electrophoretic protein patterns were transferred on nitrocellulose and probed with antibodies against enzymes of the polyol
pathway. The anti-aldose reductase antiserum was raised in rabbit immunized against a recombinant human aldose reductase
(AKR1B1) encoded by a cDNA cloned from a human endometrium biopsies cDNA library (generous gift of Dr MA Fortier
from Univ Laval, Québec, Canada). The anti-sorbitol dehydrogenase rabbit antiserum was produced against a peptide derived
from the sorbitol dehydrogenase–deduced amino acid sequence
(Ng et al, 1998) (gift from SK Chung, Hong Kong University).
Blots were incubated with the anti-aldose reductase antiserum
diluted 1:5000 or with 3.5 mg/mL of rabbit immunoglobulin G
(IgG) anti-sorbitol dehydrogenase. Enzymes were detected with
a goat anti-rabbit IgG coupled to peroxidase and revealed using
enhanced chemiluminescence (ECL) substrate (Amersham,
Buckinghamshire, United Kingdom).
Aldose Reductase and Sorbitol Dehydrogenase mRNAs
Epididymal tissues were homogenized in 4 M guanidine thyocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M mercaptoethanol; extracted with phenol-chloroform; and RNAs were
precipitated with ethanol (Chomczynski and Sacchi, 1987).
RNAs were separated on agarose gels and transferred on a nylon
membrane. A probe corresponding to the first 690 nucleotides
of the aldose reductase cDNA was random primed with Ready
Prime II kit (Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom) and used to probe the nylon membrane. Prehybridization was performed in ‘‘Express hyb’’ (Clontech, Calif)
and hybridization was done in the same solution containing 3–
4 3 106 dpm/mL labeled probe. The ubiquitous expressed gene
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used
to probe the same membrane.
Northern blot analysis was not sensitive enough to detect sor-
Frenette et al · Polyol Pathway in Epididymis
Figure 2. Western blot of protein from human epididymosomes (E),
prostasomes (P), and ejaculated spermatozoa (SP) probed with an antialdose reductase (Panel A) or an anti-sorbitol dehydrogenase (Panel B).
The quantities of protein or the number of spermatozoa used to perform
the Western blots are indicated under each lane. Aldose reductase and
sorbitol dehydrogenase are indicated by arrows and molecular weight
standards (3103) are indicated on the left.
bitol dehydrogenase mRNA in epididymal tissue homogenates.
Reverse-transcribed cDNAs from different epididymal segments
were amplified using 59-ATC-TTC-TTC-TGT-GCC-ACG-CC39
and 59-GGC-CAC-GTG-TTG-CAG-TAT-CG-39 probes. Reaction conditions were as follows: initial denaturation for 5 minutes at 958C followed by 32 cycles of denaturation at 948C for
1 minute, annealing for 1 minute at 558C, and elongation at 728C
for 1 minute. A 566-bp cDNA fragment was amplified from
RNA extracted from each epididymal segment and directly sequenced by the core facilities at our institution.
Immunohistological Localization of Aldose Reductase
and Sorbitol Dehydrogenase in the Epididymal Tissues
Paraformaldehyde epididymal tissues were embedded in optimal
cutting temperature (OCT) immediately after dissection and kept
at 2808C until use. Cryosections were washed in phosphatebuffered saline (PBS) and treated for 30 minutes in 3% H2O2 in
methanol to inhibit endogenous peroxidases. Histological sections were saturated in 10% goat serum in PBS and incubated
with the rabbit anti-aldose reductase antiserum diluted 1:1000 in
PBS supplemented with 0.5% goat serum or with 5 mg/mL of
rabbit anti-sorbitol dehydrogenase IgG prepared in the same solution. Following incubation with a biotinylated goat anti-rabbit
IgG antiserum, the immune complexes were revealed with avidin-peroxidase preformed complex (Vectastain ABC reagent,
Vector, Burlingame, Calif). Negative controls were performed
with normal rabbit serum diluted 1:1000 in PBS. Histological
sections were counterstained with Harris hematoxylin. All tissue
sections were processed in parallel to allow for comparison.
Results
Polyol Pathway in Semen
Presence of the polyol pathway enzymes in epididymosomes and prostasomes as well as in proteins extracted
235
Figure 3. Western blot of protein from caput (Ca), corpus (Co), and
cauda (Cd) human epididymidis probed with an anti-aldose reductase
(Panel A) or an anti-sorbitol dehydrogenase (Panel B). Aldose reductase
and sorbitol dehydrogenase are indicated by arrows and molecular
weight standards (3103) are indicated on the left.
from ejaculated spermatozoa was investigated (n 5 7).
Aldose reductase was detectable in protein extracted from
epididymosomes, but at a much lower level than the signal associated with the same amount of protein extracted
from prostasomes (Figure 2A). A high signal was obtained when proteins extracted from prostasomes were
probed with an anti-sorbitol dehydrogenase. This enzyme
was almost undetectable on Western blots of the same
amount of protein extracted from epididymosomes (Figure 2B). Both aldose reductase and sorbitol dehydrogenase were associated with ejaculated spermatozoa and
showed similar levels of detection on a given semen sample. Both enzymes, however, showed some variability
from one semen sample to the other (Figure 2).
Polyol Pathway in the Epididymis
Western blots performed 4 times on human epididymal
protein extracts revealed that both aldose reductase and
sorbitol dehydrogenase are immunodetectable (Figure 3).
Aldose reductase was detectable at a similar level of expression in the caput and corpus epididymidis, whereas a
slightly more intense signal was revealed at the cauda
epididymal segment (Figure 3A). Sorbitol dehydrogenase
was immunodetectable as a band corresponding to a molecular weight of 39–40 kd in the 3 epididymal segments.
A very faint signal was detectable in the corpus when
compared to the caput and cauda epididymidis (Figure
3B). Additional bands were associated with high background due to poor antibody titer.
At the immunohistological level, both aldose reductase
and sorbitol dehydrogenase were detected in the 3 segments of the human epididymis. The signal was less intense in the caput epididymidis, especially when it was
probed with aldose reductase antiserum. The signal was
mainly detected in the epididymal epithelium, with a
Journal of Andrology · March/April 2006
236
Figure 4. Immunohistological localization of aldose reductase (AR) (Panel A) and sorbitol dehydrogenase (SODH) (Panel B) in human caput (Ca),
corpus (Co), and cauda (Cd) epididymidis. Presence of antigen is revealed by red staining. Tissue sections were counterstained in blue. Small insertions
(Co9) are negative controls using preimmune serum.
higher labeling at the apical portion of the epithelial cells
(Figure 4). Small intraluminal structures are labeled and
are thought to be the result of fixation artifact.
At the transcriptional level, both aldose reductase and
sorbitol dehydrogenase mRNAs were detectable in human
epididymal tissues. Aldose reductase transcript was detected as a single band of the expected size of 1.5 kb on
Northern blots of epididymal RNA (Figure 5). Sorbitol
dehydrogenase transcript was undetectable using standard
Northern blot protocol but was amplified at the expected
size by reverse transcription–polymerase chain reaction
nonquantitative assay (Figure 6). Thus, sorbitol dehydrogenase gene was expressed at a much lower level compared to aldose reductase. For both mRNAs, the amount
detected was comparable from one epididymal segment
to the other (Figures 5 and 6).
Discussion
Both aldose reductase and sorbitol dehydrogenase are immunodetectable on protein extracts of ejaculated human
spermatozoa. The cellular compartment distribution of
these enzymes in spermatozoa, however, remains to be
determined, thus limiting the understanding of the role of
the polyol pathway in sperm physiology. In human semen, aldose reductase and sorbitol dehydrogenase are not
restricted to spermatozoa; they are detectable as components of prostasomes and epididymosomes. Prostasomes
are abundant in human seminal plasma and have been
shown to play many functions in sperm physiology and
modulation of the female reproductive tract (Saez et al,
2003; Ronquist and Nilsson, 2004). The 2 polyol pathway
Frenette et al · Polyol Pathway in Epididymis
237
Figure 6. Detection of sorbitol dehydrogenase transcripts by reverse
transcription–polymerase chain reaction (RT-PCR) on human caput (Ca),
corpus (Co), and cauda (Cd) epididymidis.
Figure 5. (Panel A) Northern blot analysis of aldose reductase (AR)
mRNA in the human caput (Ca), corpus (Co), and cauda (Cd) epididymidis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as
a housekeeping gene. (Panel B) Densitometric determination of the relative amounts of AR mRNA expressed as ratios of GAPDH.
enzymes have also been identified in bovine (Frenette et
al, 2004) epididymosomes. Although it is well demonstrated that exosomes from the epididymis intraluminal
compartment transfer selected proteins to the sperm surface, the involvement of exosomes from the epididymis
or the prostates in the acquisition by spermatozoa of the
polyol pathway enzymes remains to be determined. Both
sorbitol and fructose are constituents of seminal plasma,
and all accessory glands of the rat male reproductive
tracts can synthesize these carbohydrates (Kobayashi et
al, 2002). In humans, both exosomes and spermatozoa
have the enzymes necessary to produce both fructose and
sorbitol. Both of these sugars can be used as energy
sources by ram (O’Shea and Wales, 1965) and human
(Murdoch and White, 1968) spermatozoa. In human semen, involvement of exosomes vs sperm-associated polyol pathways remains to be determined.
The polyol pathway is believed to be involved in the
etiology of diabetic complications. It has thus been extensively studied in kidney, retina, and peripheral neurons, the main tissues affected by diabetes (Yabe-Nishimura, 1998; Oates, 2002). Whereas aldose reductase is
present in all tissues examined (Markus et al, 1983), sorbitol dehydrogenase tissue distribution shows great interspecies variability (Vaca et al, 1984). As shown in bovine
(Frenette et al, 2004) and rat (Kobayashi et al, 2002) mod-
els, both aldose reductase and sorbitol dehydrogenase are
expressed at the transcriptional and translational levels in
human epididymal tissues. Localization of both enzymes
in the epididymal epithelium together with their association with human epididymosomes indicate that these enzymes are secreted in the intraluminal compartment. In
fact, epididymosomes present in the intraluminal compartment of the excurrent duct are secretory products of
the epididymal epithelium that follow an apocrine mode
of secretion that has been described in different mammalian species, such as rat (Hermo and Jacks, 2002),
mouse (Rejraji et al, 2002), ram (Ecroyd et al, 2004; Gatti
et al, 2004), bovine (Frenette and Sullivan, 2001; Frenette
et al, 2002), and hamster (Yanagimachi et al, 1985; Légaré et al, 1999a). Association of aldose reductase and
sorbitol dehydrogenase with epididymosomes has been
shown in bovine (Frenette et al, 2003, 2004) and ram
(Ecroyd et al, 2004; Gatti et al, 2004).
In bovine, aldose reductase protein and enzymatic activity is high all along the excurrent duct, except in the
distal portion of the cauda epididymidis and vas deferens.
Considering that aldose reductase is the limiting enzyme
of the polyol pathway (Hers, 1956) and that reduction of
glucose in sorbitol is optimum at a slightly acidic pH
corresponding the epididymal fluid pH, production of sorbitol is expected to be favored in the epididymis. Sorbitol
is a linear sugar and is thus poorly permeable through
biological membrane. Thus, sorbitol in the epididymal
fluid is not accessible to sperm glycolysis. The high activity of aldose reductase along the epididymis can thus
contribute to maintaining sperm motility in a repressive
state. A decrease in the aldose reductase activity in the
distal cauda epididymidis and vas deferens together with
an increase in sorbitol dehydrogenase will favor formation of fructose, a product of sorbitol oxidation that will
be more accessible for sperm glycolysis and production
of the energy necessary for sperm motility. In bovine, the
polyol pathway may thus modulate sperm motility during
the epididymal transit (Frenette et al, 2004). The situation
appears different in the human excurrent duct. Even
though both enzymes of the polyol pathway are expressed
at both the transcriptional and translational levels, their
Journal of Andrology · March/April 2006
238
distribution along the epididymis seems quite homogeneous. We can thus predict that there is no modulation of
sorbitol along the excurrent duct in humans. For obvious
reasons, intraluminal concentrations of polyols along the
human excurrent duct are, however, unknown.
The need for maintaining sperm motility in a quiescent
status within the epididymis may be different in human
compared to other mammalian species. Compared to domestic animals and usual laboratory species, morphology
of the human epididymis shows many peculiarities. The
human caput epididymidis is mainly formed by multiple
branched efferent ducts (Yeung et al, 1991), and there is
no differentiated initial segment. The cauda epididymidis
in human is poorly developed, indicating a poor sperm
storage capacity. Whereas the bovine cauda epididymis
stores a number of sperm equivalent to 10 to 12 ejaculates
(Curtis and Amann, 1981), in humans the epididymis
storage capacity is less than 3 ejaculates (Johnson and
Varner, 1988; Bedford, 1994). These morphological characteristics of human epididymis reflect the poor development of this single convoluted tubule, which reaches 6
m in length when uncoiled in rat, more than 50 m in large
domestic animals, and a modest 5 to 6 m in humans (Bedford, 1994). The sperm transit along the human epididymis is rapid, 2 to 4 days (Bedford, 1994), when compared
to the 10- to 12-day journey in other mammalian species
(Robaire and Hermo, 1988). In light of these observations, it is obvious that the mechanisms maintaining spermatozoa in a quiescent state during the epididymal transit
will not be as critical in humans as they are in animals
with more developed excurrent ducts. While the polyol
pathway is present in human, it is not differentially expressed along the epididymis as it is in bovine (Frenette
et al, 2004) and may thus perform functions other than
modulating epididymal sperm motility. Obviously, in humans as in other mammals, mechanisms other than the
polyol pathway are involved in the regulation of epididymal sperm motility and metabolism. In many animal
models, many genes have been shown to be expressed in
specific segments of the epididymis. This appears to not
be the case for the polyol pathway enzymes, as has been
shown for other gene products (Légaré and Sullivan,
2004). Extrapolation of results from animal models to human epididymal physiology should be handled with caution.
Acknowledgments
We wish to thank Drs MA Fortier and E Madore (Université Laval) for
providing us with recombinant aldose reductase and antibodies as well
as Dr SK Chung (Hong Kong) for the generous gift of anti-sorbitol dehydrogenase. Mrs C Légaré is acknowledged for technical help and stimulating discussion.
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