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From * Biologie de la Reproduction, CECOS, and
U.F. Enzymologie CHU, Clermont-Ferrand,
France; and UMR CNRS 6547 Equipe
épididyme et maturation du gamète male, Aubière
Cedex.
| Correspondence to: André Force, Service de Biologie du Développement et de la Reproduction, CHU Hôtel-Dieu, Boulevard Léon Malfreyt, 63003 Clermont-Ferrand, France (e-mail: andre.force{at}wanadoo.fr). |
| Received for publication November 14, 2003; accepted for publication April 21, 2004. |
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Abstract |
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Key words: Human spermatozoa maturation, enolase isoforms, epididymal sperm, testicular sperm
(ENO-), a ubiquitous form, distributed
in most adult cell types, and enolase S (ENO-S), a sperm-specific isoform
(Edwards and Grootegoed, 1983;
Force et al, 2002). Both
enolase isoforms seem to reflect opposite aspects of sperm cell quality:
ENO- is associated with abnormal and/or immature spermatozoa,
and ENO-S is associated with normal spermatozoa. In the present study, sperm
ENO-S was characterized in testicular, epididymal, and ejaculated spermatozoa
to determine whether epididymal maturation provoked any change in the
electrophoretic character of this enzyme. |
Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Patients
Testicular sperm extraction or microsurgical epididymal sperm aspiration
was performed during the diagnostic work-up of the azoospermic patients, and a
cryoconservation of the retrieved spermatozoa was generally performed for
eventual future in vitro fertilization using the intracytoplasmic sperm
injection (ICSI) technique. Our protocol received the patients' approval, and
the enolase evaluation was conducted only when sufficient spermatozoa were
retrieved without consequence for ICSI success.
Epididymal sperm was retrieved from 11 obstructive azoospermic patients, 6 of whom presented with congenital bilateral absence of the vas deferens (CBAVD), 1 with a failed reversal of vasectomy, 1 with an epididymal cyst, and 3 with no defined etiology.
Testicular spermatozoa were obtained from 8 cases of obstructive azoospermia (3 with CBAVD, 3 with infectious syndromes, and 2 with no clear etiology). From 4 patients, both testicular and epididymal spermatozoa were retrieved.
Extracts were also performed from testicular biopsies obtained from 2 patients with secretory azoospermia and elevated plasma follicle-stimulating hormone levels (the absence of spermatozoa observed by histologic examination corresponds to a spermatogenetic arrest at the level of the spermatocyte, and no spermatids were detectable; data not shown).
The ejaculated sperm used for comparison were provided from normospermic fertile patients.
Testicular Sperm Extraction
The surgical technique for testicular biopsy retrieval was previously
described (Silber et al,
1995). Briefly, testicular tissue was excised and placed directly
in a petri dish with 2 mL of Earle medium. For sperm extraction, the
seminiferous tubules were rinsed 2–3 times in Earle medium and gently
dissected and minced with a lancet. The preparation was incubated in 5%
CO2 at 37°C for 3 hours. The supernatant was then collected and
centrifuged at 500 x g for 10 minutes. The pellet was suspended
in Earle medium, the sperm concentration was estimated, and the cells were
finally either cryopreserved according to Grizard et al
(1999) or processed
immediately (5 cases) for enolase assay.
Epididymal Sperm Extraction
Epididymal tubule dissection and aspiration were performed using an
operating microscope under magnifications of between 15x and 20x.
Individual epididymal tubules were entered with microscissors, and fluid with
spermatozoa was aspirated into small syringes and subsequently distributed
into sterile 15-mL test tubes (Becton Dickinson, Franklin Lakes, NJ)
containing Earle medium. After centrifugation, the sperm pellet was either
cryopreserved according to Grizard et al
(1999) (4 cases) or processed
immediately (7 cases) for enolase assay.
Spermatozoa Purification
All sperm samples (of ejaculated, epididymal, or testicular origin) were
layered on top of a 2-step discontinuous gradient obtained with 1 mL of both
47.5% and 95% Percoll (Nidacon). For epididymal and ejaculated samples, the
P95 fraction was collected and processed for protein extraction (see next
section). For testicular samples, because the sperm quantity obtained in the
95% Percoll fraction was lower than 100 000 spermatozoa, the enolase analysis
was performed using spermatozoa from the 47.5%–95% Percoll interface.
When no spermatozoa were observed after histologic evaluation, the cell
mixture was layered on a 4-step Percoll gradient (30%, 35%, 40%, and 45%) for
separation of the different germ cells according to Gandini et al
(1999).
Enolase Extraction and Electrophoretic Determination of Enolase Isoforms
To extract ENO-S isoforms correctly, the
octyl-ß-D-glucopyranoside (OGP; Sigma) detergent was
necessary. This nonionic detergent allows a solubilization without the
denaturation of hydrophobic proteins interacting with membrane structures or
other proteins.
The sperm samples obtained after Percoll selection (epididymal, testicular, or ejaculated) were washed extensively with 20 mM Tris, 150 mM NaCl, and 5 mM MgCl2, pH 7.4, and centrifuged at 1200 x g for 10 minutes at 4°C. To extract proteins, the pellet was then vortexed for 1 hour at 4°C in 500 µL of 20 mM Tris and 5 mM MgCl2 devoid of NaCl and supplemented with 0.5% OGP. Finally, each extract was stored at -80°C until use. Before analysis, each sample was thawed at room temperature and then centrifuged at 10 000 x g for 10 minutes at 4°C. Total enolase activity was assessed on the supernatant according to a method described by Viallard et al (1985). A minimum of 300 000 spermatozoa were used to perform this assay. The contamination by immature germ cells was about 10% and 40% in the epididymal and testicular preparations, respectively. Finally, for the samples showing secretory azoospermia (absence of spermatozoa in the preparation), the enolase assay was realized from the cells collected in the 40% Percoll fraction. A minimum of 200 000 immature cells were necessary to perform the assay.
Enolase isoforms were separated by electrophoresis on cellulose acetate
plate "Titan III Iso-Flur" 3905 from Helena (Beaumont, Tex). To
compare different extracts, an identical total enolase activity was used in
each electrophoretic well. The separated enolase isoforms were detected by
overlaying the plate with a specific enolase substrate (2-phosphoglycerate),
which, via a multistep reaction, produces NADPH (the reactions involve
pyruvate kinase, hexokinase, and glucose-6-phosphate dehydrogenase; for
details, see Viallard et al,
1986; Force et al,
2002). The bands corresponding to the enolase isoforms were
detected by measuring the NADPH-related fluorescence (
= 340 nm) with
a scanning fluorometer, "Cliniscan II Astron Densitometer," from
Helena. In the absence of a specific enolase substrate in the revelation
medium, no fluorescent bands were observed.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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was found in all of the samples (testicular, epididymal,
and ejaculated) with the same electrophoretic characteristics
(Figure 1).
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ENO-S was found in all of the samples containing spermatozoa but with different electrophoretic characteristics, depending on the origin of the spermatozoa (Figure 1).
Percoll-selected ejaculated sperm used as reference from a normozoospermic patient presented a unique S band after electrophoresis (ENO-S2). The electrophoretic profile determined on testicular and epididymal spermatozoa clearly demonstrated 2 different patterns of ENO-S (Figure 1a and b). The electrophoretic profile of testicular spermatozoa showed 2 bands, S1 and S3. Such a profile was observed in the 8 analyzed samples. Whatever the epididymal sperm sample, a prominent S2 band appeared, with intermediate electrophoretic mobility, between S1 and S3, whereas the S1 and S3 bands were very slightly detected (Figure 1). This profile is similar to that of ejaculated spermatozoa.
A comparison of the ENO-S electrophoretic profile between the testicular and epididymal sperm of the same patient was made for 4 different patients (Figure 2). These profiles were similar to those obtained from either epididymal or testicular samples evaluated from distinct patients. The study performed on the testicular and epididymal spermatozoa from the same patient clearly demonstrated that the differences in ENO-S characteristics were strictly related to the origin of the spermatozoa and did not come from interindividual variations.
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Finally, enolase isoform profiles of testicular immature germ cells from an
azoospermic patient (nonobstructive azoospermia) were determined
(Figure 3). No ENO-S isoform
was found in these extracts, whereas ENO- was still present.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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isoenzyme and of an unusual isoform (ENO-S) characterized by
its particular electrophoretic migration and sperm specificity. Our results
using electrophoresis on acetate cellulose confirmed those obtained by Edwards
and Grootegoed. Recently, we demonstrated quantitative and qualitative
differences in enolase activity between spermatozoa of abnormospermic and
normospermic men (Force et al,
2002). We suggested that ENO- characterizes abnormal
and immature spermatozoa, whereas ENO-S characterizes normally developed
spermatozoa.
In the present study, the electrophoretic pattern of enolase isoforms in
spermatozoa retrieved from different regions of the male genital tract was
evaluated. As shown previously, the ENO- and ENO-S isoforms were
pre sent in ejaculated spermatozoa. Furthermore, 3 variants of the S isoforms,
named S1, S2, and S3 according to their pronounced acidic migration
characteristics, were identified in epididymal and testicular spermatozoa. In
testicular sperm, only 2 bands, S1 and S3, were present, whereas in epididymal
sperm, these bands were reduced, and a prominent S2 band appeared. This latter
electrophoretic pattern was similar to the ENO-S pattern of ejaculated
spermatozoa. Thus, the comparison of electrophoretic ENO-S isoform profiles
between testicular and ejaculated sperm revealed changes that seemed to take
place in the epididymis. The importance of sperm epididymal transport in men
is a long-standing question. It is well accepted that spermatozoa are not
fully mature when they leave the testis and that they develop the capacity to
be motile along with biochemical transformations during their epididymal
transit (Cooper, 1995). The
present study shows that, during sperm maturation in the epididymis, S1 and
perhaps S3 isoforms are modified in favor of the S2 isoform. In addition, the
fact that S2 was the prominent isoform in ejaculated sperm provided evidence
that its determination in these spermatozoa was an interesting marker of
epididymal sperm maturation. Moreover the presence of ENO-S (S2) in normally
developed spermatozoa was demonstrated previously
(Force et al, 2002). That we
found a majority of S2 isoforms in ejaculated sperm suggests an association of
this latter isoform with matured spermatozoa. Most of our results were
obtained on mature ejaculated sperm; however, studies of immature ejaculated
sperm have shown a heterogeneity in S isoforms with the presence of S1 and S3
bands (unpublished data).
Different explanations can be proposed concerning the heterogeneity of the
S isoforms and/or the remodeling of the ENO-S. Because the S3 isoform is the
most neutral isoform, S1 or S2 could derive from this one by a sialylation.
Sialyltransferases involved in the glycosylation process are present in the
epididymis (Singer et al,
1988; Tulsiani et al,
1993). In the rat, a similar variant of
-L-fucosidase has been found during epididymal maturation
(Abascal et al, 1998), and the
differentially sialylated isoforms of -L-fucosidase
indicated a significant trend of increased thermostability with increasing
sialylation (Alhadeff and Andrews-Smith,
1980). Compared to ENO-, the increased
thermostability of ENO-S (Edwards and
Grootegoed, 1983) suggests that this enzyme contributes to the
highly specialized performance of mature spermatozoa.
The variation in S isoforms could also be due to phosphorylation. Phosphorylated variants of ßß enolase were observed in muscle (Asaga and Konno, 1975; Nettelblad and Engstrom, 1987). The phosphorylation of glycolytic enzyme in human spermatozoa improves enzyme activity and glycolytic flux (Harrison et al, 1991; Knull and Minton, 1996; Ovadi and Srere, 1996; Leclerc and Goupil, 2002).
The presence of the isoform in immature testicular cells
suggests an early expression in spermatogenesis. That the S isoform was found
only in mature testicular sperm and was absent in the testicular extract from
one patient showing a spermatogenesis arrest (confirmed by histologic
examination) suggest that the S isoform was synthesized in the final stages of
spermatogenesis, during spermiogenesis. In mouse sperm, the S isoform is
present only in elongating spermatids or in washed spermatozoa and is absent
in middle and late pachytene spermatocytes or in round spermatids
(Edwards and Grootegoed, 1983).
Two hypotheses can be proposed concerning ENO-S expression: 1) ENO-S is the
product of a gene locus distinct from those determining the somatic tissue
enolases and is expressed in the late stage of spermatogenesis in the haploid
genome, or 2) the gene locus is the same as that for the
isoenzyme, and a stable messenger RNA (mRNA) is transcribed premeiotically and
stored untranslated until the late spermatid stage of development
(Erickson et al, 1980). Enolase
studies in different tissues are in favor of 2 genes. Indeed, in brain and
muscular tissue, the specific enolase isoforms (
and ß enolase
genes) were encoded by a gene distinct from the ubiquitous gene
(Sakimura et al, 1985,
1990).
Another hypothesis concerning the origin of ENO-S variants can be set forth. According to the studies carried out on rat spermatozoa, enolase would be localized to the tail of mature spermatozoa, and an association with microtubules, dependent on the concentration of the medium in ATP, cytosine triphosphate, and guanosine triphosphate, was demonstrated (Gitlits et al, 2000). These results confirm several reports of glycolytic enzyme associated with microtubules of the flagellum (Storey and Kayne, 1975; Durrieu et al, 1987; Knull and Walsh, 1992). In addition, Westhoff and Kamp (1997) demonstrated the existence of a multienzyme complex on the tail of human spermatozoa. The glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath and is associated with other glycolytic enzymes, namely triose isomerase and phosphoglycerate kinase. Together with our results, these reports showing the presence of the ENO-S isoforms probably during spermiogenesis, which correspond to the tail formation of the spermatozoa, are in favor of a flagellar localization of ENO-S isoforms.
Moreover, we have indirect evidence of the subcellular localization of
enolase isoforms provided by the extraction protocol used. A complete
extraction of ENO- is obtained by a mechanical treatment
(high-speed vortex, 1 hour at 4°C) in the presence of a Tris buffer
without NaCl. By using identical conditions, the lactate dehydrogenase and the
creatine kinase, both of which are enzymes with cytosolic localization, are
also completely extracted (data not shown). These experimental elements added
to the positive correlation found between the ENO- activity and
the spermatozoa carrying residual cytoplasmic material
(Force et al, 2002) and suggest
the cytosolic localization of ENO-. Contrary to
ENO-, no relation was found between ENO-S activity and the
percentage of spermatozoa carrying residual cytoplasmic material
(Force et al, 2002). Moreover,
to extract ENO-S isoforms, a detergent (OGP) was necessary to obtain a
solubilization without the denaturation of hydrophobic proteins in interaction
with membrane structures or other proteins. Thus, it is possible that ENO-S
electrophoretic variants are generated from ENO-, either by
aggregation of the molecules or connection to other glycolytic enzymes or
sperm cell protein structures.
In conclusion, we have shown that a heterogeneity in the S isoform existed in testicular sperm with the S1 and S3 variants. This heterogeneity disappeared during epididymal maturation with the appearance of a prominent S2 isoform, which was equal in amount to the major S isoform in normally developed ejaculated spermatozoa. These results show that the ENO-S isoforms are interesting markers of sperm maturation and clarify the role of the epididymis in human sperm maturation. In the future, the clinical use of ENO-S profiles could be proposed for cases of obstructive azoospermia (infectious syndrome) to help choose the better surgical sperm source for ICSI.
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Acknowledgments |
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