BIOLOGY OF REPRODUCTION 57, 1502-1508 (1997)
Distribution and Possible Novel Role of Phospholipid Hydroperoxide Glutathione
Peroxidase in Rat Epididymal Spermatozoa'
Cristiana Godeas, 3 Federica Tramer, 3 Fulvio Micali,3 Mariarosa Soranzo, 4 Gabriella Sandri,3
and Enrico Panfili 2 3,
Department of Biochemistry, Biophysics and Macromolecular Chemistry 3 and Institute of General Pathology,4
University of Trieste, 34127 Trieste, Italy
ABSTRACT
The selenoenzyme phospholipid hydroperoxide glutathione
peroxidase (PHGPx, EC 1.11.1.12) is present, in both free and
membrane-bound form, in several mammalian tissues. It utilizes
thiols such as glutathione to specifically scavenge phospholipid
hydroperoxides. The testis exhibits the highest PHGPx-specific
activity so far measured, and interest in the presence and function of the enzyme in this tissue has recently grown. Here we
report the localization of PHGPx in rat epididymal spermatozoa
and its distribution in subfractions obtained by sucrose density
gradient centrifugation. Immunochemical evidence and enzymatic activity revealed for the first time that PHGPx is present
in sperm heads and tail midpiece mitochondria. The binding of
the enzyme to spermatozoa, head, and mitochondria was barely
affected by ionic strength or thiols or detergents, as compared
to the detachment of PHGPx obtained from testis nuclei. Moreover, we demonstrated that pure PHGPx exhibits a higher thioloxidase activity toward isolated epididymal caput protamines
than toward protamines from epididymal cauda. These results
suggest a role for the enzyme in the maturation of spermatozoa
through the metabolism of hydroperoxides and sperm thiol oxidation, in addition to its serving as an antioxidant protector.
INTRODUCTION
Phospholipid hydroperoxide glutathione peroxidase
(PHGPx, E.C. 1.11.1.12) is a 20-kDa selenoenzyme belonging to the group of glutathione peroxidases [1-3]. It
catalyses the oxidation of thiols, such as reduced glutathione (GSH), to specifically reduce certain hydroperoxides
(e.g., phospholipids) that can originate from peroxidative
reactions. From this point of view the enzyme can be considered a bulwark against damage elicited, for example, by
free radicals, especially in the protection of biological
membranes. PHGPx has been identified in several mammalian tissues, where it is both soluble and membrane
bound [4]. It is remarkable that the highest specific activity
of PHGPx so far recorded is exhibited by the rat testisup to two orders of magnitude higher than in the brain or
the liver [5]. In rat testis cells the enzyme is present in
mitochondria, mostly membrane bound [6], and to a lesser
extent in nuclei, where it is associated with chromatin [7].
The crucial importance of selenium (Se) [8] and polyenoic
long-chain fatty acids [9] in the differentiation of testis germinal cells is well established, and the protective role of
PHGPx in these two subcellular compartments is therefore
to be considered of primary importance. It is noteworthy
that the other Se enzyme, glutathione peroxidase, is scarcely present in spermatozoa, where it has been reported only
in the soluble form [10, 11]. Here we present immunologAccepted July 31, 1997.
Received December 17, 1996.
'Financial support by MURST (Rome) and by University of Trieste.
2Correspondence. FAX: 39 40 676 3691; e-mail: [email protected]
ical and enzymatic data concerning the localization of
PHGPx in epididymal spermatozoa of adult rats, demonstrating that the enzyme is present at the level of the sperm
head, mainly in the condensed chromatin, as well as in the
midpiece mitochondria. The specific activities monitored in
these two fractions are similar to those measured in testis
nuclei and mitochondria [6, 7]. Reducing agents (thiols),
ionic strength, or detergents are not capable of quantitatively detaching the enzyme from the spermatozoa and their
subcellular fractions, whereas in the testis the combined
action of thiols and ionic strength [7] is more effective. In
addition, we demonstrate for the first time that PHGPx is
capable of oxidizing reduced sperm protamines, thus suggesting a further role for the enzyme.
MATERIALS AND METHODS
Isolation and Fractionation of Epididymal Spermatozoa
and Protamines
All procedures were carried out at 0-4°C unless otherwise indicated.
Adult (3-mo-old) albino rats were killed and epididymides were rapidly removed and placed in 0.12 M NaCI,
0.01 M Tris-Cl, pH 7.5 (TBS), containing 1 mM PMSF.
Spermatozoa were collected essentially according to Seligman et al. [12] by squeezing two minced epididymides in
2-3 ml of TBS on a glass slide. The resulting sperm suspension was filtered through three layers of cheesecloth and
centrifuged (1200 x g, 10 min). The final sediment was
suspended in a small amount of TBS, and spermatozoa
were counted by using a hemocytometer with Neubauer
ruling (400 squares/mm 2).
Tail midpiece mitochondria were detached and isolated
from epididymal spermatozoa mainly according to the procedure of Pallini [13] for bull sperm. Aliquots of 3 ml of
sperm suspension (3 x 107 cells/ml) were sonicated in an
ice bath for 8 bursts (20 sec each, separated by 40-sec intervals using a Branson sonifier [Branson Ultrasonic Corp.,
Danbury, CT] equipped with a microtip at 2.8 A). The sonicated sperm suspension (about 5 ml) was layered on a
discontinuous sucrose density gradient (9 ml density =
1.29; 9 ml density = 1.2; 9 ml density = 1.11 at 4C), and
mitochondria were obtained by collecting the band between
density of 1.20 and density of 1.11 after a centrifugation at
83 000 x g on a swinging bucket rotor for 4 h.
Sperm heads were separated either by brief sonication
of sperm (15 sec, 107 cells/ml) [14] or by trypsin cleavage
(0. mg/ml trypsin, type IX; Sigma Chemical Co., St. Louis, MO; 108 cells/ml, 5-min incubation at 25°C), followed
by treatment with trypsin inhibitor (type II-S, Sigma) as
described by Millette et al. [15]. Sperm heads were purified
according to San Augustin and Witman [16]. The soluble
fraction (cytosol) was separated as described by Skudlarek
1502
PHGPx IN RAT SPERMATOZOA
et al. [17] after cleavage of whole spermatozoa (107 cells/
ml) with trypsin [15].
Protamines were prepared from spermatozoa isolated
from rat epididymal caput and cauda essentially according
to the method of de Yebra and Oliva [18], by omitting
iodoacetate. The final dry pellet was resuspended in 0.1 M
acetate buffer (pH 4). Acetic acid-urea electrophoresis of
the extracted protamines was performed as reported by Hardison and Chalkley [19].
Fractionation of Testis
Rat testis mitochondria were prepared and subfractionated as previously described [6]. Testis nuclei were isolated
essentially according to Kay and Johnston [20] by means
of centrifugation (in 2.4 M sucrose at 50 000 x g for 1 h)
of the pellet obtained at 700 x g from the testis homogenate.
Assays
PHGPx and succinate cytochrome-c reductase activities
were measured at 30 0C as described by Maiorino et al. [1]
and Sottocasa et al. [21], respectively. The DNA content
was quantified spectrofluorometrically by the method of Labarca and Paigen [22]. Protein evaluation was performed
by the bicinchoninic acid method [23].
PHGPx solubilization assays were carried out by incubating epididymal spermatozoa (about 107 cells/ml, 5-min
incubation with trypsin as described above) and purified
sperm and testis subcellular fractions (0.1-0.2 mg protein/ml for mitochondria; 0.2-0.4 mg protein/ml for nuclei
and sperm heads) for 30 min at 0°C in TBS containing
increasing amounts of KCI, 2-mercaptoethanol (2-ME), and
peroxide- and carbonyl-free Triton X-100. The mixtures
were then spun down (100 000 x g, 20 min) and PHGPx
activity was measured in both the supernatant and the sediment. Supernatant activity of the control (no addition) was
subtracted from supernatant activity of the treated sample,
and the result was divided by the total activity (supernatant
+ sediment); this value represents, in percentage, the soluble PHGPx.
The activity of PHGPx toward extracted protamines was
followed spectrophotometrically in the presence of pure enzyme isolated from testis cytosol (about 0.5-1.0 mol
NADPH/min per milliliter, purified as described by Roveri
et al. [24]) by measuring the disappearance of the titratable
SH (sulfhydryl) groups in the presence of 5,5'-dithio-bis(2nitrobenzoic acid) (DTNB) according to the method of
Ando and Steiner [25]. Pure glutathione peroxidase (GPx;
Calbiochem, La Jolla, CA; about 0.5-1.0 ,umol NADPH/
min per milliliter) was used as a control under the same
conditions as described above.
Light and Electron Microscopy
Immunogold microscopy was carried out on isolated testis tubules and epididymal tissue. Thin slices were fixed
with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1
M phosphate buffer (pH 7.4) for 4 h at 4°C. The slices were
then washed several times with the same phosphate buffer
and then dehydrated in a graded series of ethanol up to
absolute (99%) and embedded in Unicryl (British BioCell
International, Cardiff, UK) as described by Scala et al. [26].
Ultrathin sections (150 nm) were cut and mounted on 300mesh nickel grids. Each grid was floated for 1 h at room
temperature on a 20-uld drop of TBS containing 1% fatty
1503
acid-free BSA (Sigma) and 20% normal goat serum, and
then incubated overnight at 4°C on a drop of anti-PHGPx
rabbit polyclonal antibody diluted 1:50 in TBS containing
0.1% Tween 20 and 5% normal goat serum. The antiserum
raised against pig heart PHGPx [4] was purified by affinity
chromatography against recombinant PHGPxCYs 4 6 [27]. After several washes in TBS containing 1% BSA, the grids
were immediately incubated for 2 h at room temperature
on drops of colloidal gold (10 nm) conjugated to goat antirabbit IgG (BioCell), diluted 1:50 in TBS containing 5%
normal goat serum, 5% fetal calf serum, 0.1% Tween 20,
and 1% BSA. After six washes in TBS containing 1% BSA
and two washes in distilled water, the gold particles were
enhanced by treatment with a silver enhancer kit (BioCell).
Finally, counterstaining was carried out with uranyl acetate
(7 min) and lead citrate (3.5 min). Normal rabbit serum
(diluted 1:50 in TBS) was used as negative control.
Purified mitochondria were negatively stained with 2%
phosphotungstic acid. Electron micrographs were taken using a Philips (Eindhoven, The Netherlands) 201 electron
microscope.
Sperm heads were resuspended in a small amount of
TBS and photographed by a contrast-phase Light Labolux
20 microscope (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany).
RESULTS
Figure A shows the immunochemical localization of
PHGPx in a rat testis tubule section. The majority of the
gold spots are present at the level of elongated spermatids
facing the lumen of the tubule (arrowheads). Figure 1B
shows the negative control obtained with normal rabbit serum.
The subsequent approach for screening the PHGPx distribution outside the testis was accomplished by postembedding immunohistochemical localization on rat spermatozoa in situ in the epididymis. Figure 2 shows typical immunogold results, which clearly indicate the presence of
enzyme within the condensed chromatin on the plasma
membrane of heads (Fig. 2, A and C) and on the mitochondrial sheath (Fig. 2, A and B) that surrounds the fibers
in the midpiece of the tail. Several gold spots are located
at the level of the boundary membranes of the mitochondria
(Fig. 2, A and B, arrowheads), thus confirming the demonstrated location for PHGPx in the testis organelles, where
it is bound at the contact points between inner and outer
mitochondrial membranes [6]. Epididymal cells surrounding the lumen did not exhibit any positive response to immunogold, and no appreciable differences seemed to exist
in the PHGPx distribution between caput and cauda epididymidis sections (data not shown). Figure 2D reports the
negative control carried out with normal rabbit serum. From
these data we can draw the conclusion that the enzyme is
present in spermatozoa outside the testis, where they are
further processed.
To validate the immunochemical data, a biochemical approach was subsequently utilized: the PHGPx-specific activity in the separated sperm subfractions was measured.
Figure 3A shows a pure head population obtained after
sperm sonication [14] and a subsequent density gradient
centrifugation [16]. We extracted the midpiece mitochondria by means of strong sonication, as described by Pallini
[13] for bull spermatozoa, followed by centrifugation in a
sucrose density gradient. This assures excellent purification
of a floating mitochondria population. Figure 3B presents
1504
GODEAS ET AL.
FIG. 1. A) Immunogold localization of rat
testis tubule PHGPx, arrowheads indicating the gold particles at the level of elongated spermatid facing the lumen; B) negative control. Magnification x4200 (reproduced at 68%). Experimental conditions:
see Materials and Methods.
an electron micrograph of the purified mitochondria, showing the characteristic "crescent" shape of the isolated organelle (arrows) and the absence of other contaminating
structures; the arrowhead indicates the typical assemblage
of the helical structure, still maintained for a few mitochondria units.
Table 1 shows the specific activities of PHGPx, of the
mitochondrial marker enzyme succinate cytochrome-c reductase, and the DNA content of the starting spermatozoa,
mitochondria, sperm heads, and soluble material. The highest value for the PHGPx-specific activity was exhibited by
FIG. 2. Immunogold localization of rat
epididymis spermatozoa PHGPx. A) Sperm
head and tail midpiece; B)tail midpiece;
C) sperm head, upper tip section; D) negative control. Magnification x14 000 (A,
D), x13 000 (B), X46 000 (C) (reproduced
at 75%). Arrowheads indicate the boundary membranes of midpiece mitochondria.
Experimental conditions: see Materials and
Methods.
the isolated mitochondria, which also possess the highest
succinate cytochrome-c reductase activity. A lower but significant activity was present in the heads, which remained
uncontaminated by mitochondria and contained the highest
DNA/protein ratio. Soluble PHGPx is also shown; its specific activity was about one order of magnitude lower than
in the other fractions.
Finally, we carried out experiments to characterize the
binding of the enzyme to sperm and purified structures.
Ionic strength, reducing thiols, or detergents were not capable of completely setting free the enzyme on intact sper-
1505
PHGPx IN RAT SPERMATOZOA
FIG. 3. A) Rat epididymal spermatozoa
heads. B) Rat epididymal spermatozoa
midpiece mitochondria (arrows); arrowhead indicates remaining mitochondria helical assemblage. Magnification x850 (A);
x25 000 (B) (reproduced at 68%). Experimental conditions: see Materials and
Methods.
matozoa. Therefore the enzyme seems to be strongly bound
or scarcely accessible to the effectors used. Dose-dependent
behavior for these effectors was also lacking (data not
shown).
We subjected the isolated fractions (testis nuclei and mitochondria, sperm heads and mitochondria) to the same
treatments, which yielded the results reported in Figure 4.
The percentage of solubilization obtained clearly indicates
that for the chromatin-bound PHGPx (Fig. 4A: testis nuclei
and sperm heads), the effect of KCI and 2-ME-both alone
and in combination-and Triton X- 100 was greater in testis
nuclei than in sperm heads. In the case of mitochondria
(Fig. 4B), the solubilizing agents were in general less effective. The detergent had a certain efficacy for testis organelles, whereas it was practically inactive toward sperm
organelles. The effect of ionic strength alone was very low
for testis mitochondria and absent for sperm mitochondria,
and only the combined effect of KCI and 2-ME yielded
some detachment, similar for both mitochondria types.
To collect experimental evidence concerning the possible
role of the enzyme in mature spermatozoa, we monitored
the activity of pure PHGPx toward isolated, reduced rat
protamines extracted from caput and cauda epididymal
spermatozoa. Figure 5 presents the electrophoretic pattern
of the extracted protamines, which were practically free of
other basic proteins. Figure 6 shows the disappearance of
the titratable -SH groups of reduced protamines in the presence of added pure PHGPx and hydroperoxide phosphatidylcholine as oxidized substrate. The graphs clearly indicate that the thiol-oxidizing activity of PHGPx is statistically more pronounced toward the protamines extracted
from caput epididymal spermatozoa (Fig. 6, caput) than toward those coming from the cauda (Fig. 6, cauda). The
activity of the enzyme in the presence of GSH is reported
for comparison, and the results are similar only in the case
of caput protamines (Fig. 6, caput). To evaluate the specificity of PHGPx activity, pure GPx was used under identical
conditions, the only difference being the use of tert-butyl
hydroperoxide as substrate, and no activity toward either
cauda or caput protamines was revealed (data not shown).
DISCUSSION
As far as we have determined, glutathione peroxidase
activity has been reported to be absent or scarce in mammalian sperm [10, 11], whereas it was monitored in mammalian seminal plasma [10, 28]. Recently a new, peculiar
androgen-regulated epididymal secretory glutathione peroxidase subgroup has been described on the basis of comparison of cDNAs [28]. The transcripts do not contain a
selenocysteine codon. Although the rat epididymal glutathione peroxidase belonging to this group and the testis
PHGPx [29] seem to be members of distantly related families (27% sequence identity evaluated by the Intelligenetics
(Mountain View, CA) FAST DB sequence analysis software package), a similar role in sperm protection against
oxidative damage can be postulated for both enzymes. On
the other hand, PHGPx in sperm could assume a specific
role if we consider the importance of Se in spermatogenesis
[8, 30]. Many years ago, Calvin et al. [31] described as the
unique sperm selenoprotein a cysteine-rich protein (15-20
kDa) that was localized in rat mitochondrial capsule but
TABLE 1. PHGPx distribution in rat epididymal spermatozoa.a
Source
Spermatozoa
Mitochondria
Heads
Soluble
PHGPx (nmoles NADPH/min
per mg proteins)
Succinate cytochrome-c reductase
(nmoles c/min per mg proteins)
DNA
(mg/mg proteins)
34.80 ± 14.65
95.37 + 30.63
51.72
15.07*
9.38 + 3.62
38.54 + 10.29
137.40 ± 17.90
7.81 +t 2.78
n.d.
0.32
0.16
0.07 ± 0.01
3.19 ± 0.99
n.d.
Means ± SD of at least 6 different experiments; n.d., not detectable.
*p < 0.05 as compared to mitochondria.
1506
GODEAS ET AL.
U
Lw
w
z
N
+
I-
A
FIG. 5. Acetic acid-urea electrophoresis (8 x 10 cm) [15] of extracted
rat epididymal spermatozoa protamines. Lanes: 1) cauda epididymidis
protamines (9 I.g); 2) caput epididymidis protamines (9 p'g); 3) calf thymus
histones (3 Ig). Coomassie brilliant blue R250 stain.
0Y.
100
1 n
I
- n~~~~~~~~~~~~~~~~~~~~~~~~~~
80
testis MITOCHONDRIA
O sperm MITOCHONDRIA
I
c,
0)
60
X
a
75
-
Pr
-
. .-c
Pr
----
GSH/pc
E
- Pr/PHGPx
a)
0
-
40
50
b
0 20
Un
B
caput
Pr/PHGPx/pc
--- GSH/PHGPx/pc
Q)
0
*
aMns
ai
b
b
I
.
W
LI
W
'4
4-
0
b
I
,
I
I
8
I
I
16
minutes
100
I,
-
-
.
-iiT-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
=
--
Pns
Z
O
I-
I
IfY
FIG. 4. A) Solubilization of PHGPx from isolated testis nuclei and sperm
heads; B) testis mitochondria and sperm mitochondria. KCI (1 M), 2-ME
(5 mM), Triton X-100 (1%). Experimental conditions: see Materials and
Methods. The percentage of the soluble PHGPx activity was calculated
by subtracting from the activity of each sample (mean of 4 different experiments) the activity of the corresponding untreated control (mean of
same 4 experiment) and dividing the result by the total recovered activity.
ap < 0.05, bp < 0.01, and cp < 0.001 on the basis of Student's t-test for
each bar as compared to the corresponding control; ns: not significant.
that had an amino acid composition very different from that
of PHGPx [32]. Recently Behne et al. [33] described, in
addition to the 20-kDa form, another selenoprotein (34
kDa) specific to rat sperm and testis that seems to be transformed in vivo into a 20-kDa protein. At the moment it is
difficult to define how many 20-kDa selenoproteins are
present in rat sperm.
Our results (Fig. 2, Table 1) demonstrate, for the first
time, that PHGPx in the rat reproductive apparatus is present not only in testis [5-7] but also in epididymal spermatozoa, that is, as far as at the end of their maturation
pathway, both in the chromatin domain and in the energy-
0)
75 -
-Pr
-- Pr/pc
(U
--- GSH/pc
E
--
a)
Pr/PHGPx
cauda
-- Pr/PHGPx/pc
-e--GSH/PHGPx/pc
50
0
8
16
minutes
FIG. 6. Thiol-oxidizing activity of pure PHGPx on extracted rat epididymal spermatozoa protamines in the presence of hydroperoxide phosphatidylcholine. The assay contained in 1.5 ml: 0.1 M Tris-CI buffer (pH
7.5), 0.2% Triton X-100, and, when present, 200 Il pure PHGPx, about
150 vIg protamines (Pr) or GSH (both corresponding to about 150 nmol
-SH groups), and 0.014 mM phosphatidylcholine hydroperoxide (pc). The
mixture was incubated at 37°C, and aliquots were withdrawn at intervals
for measurement of the remaining -SH groups at 412 nm in the presence
of DTNB [16]. Caput: protamines from caput epididymal spermatozoa;
cauda: protamines from cauda epididymal spermatozoa. Means t SD of
4 different experiments. *p < 0.001 by Student's t-test, as compared both
to the trace (X) for cauda protamines and to the corresponding blanks
(squares, diamonds, solid triangles, open triangles) monitoring spontaneous or nonspecific -SH oxidation; ns: not significant as compared to the
corresponding blanks (squares, diamonds, solid triangles, open triangles).
PHGPx IN RAT SPERMATOZOA
producing mitochondrial apparatus. The specific activities
of the PHGPx found in spermatozoa subfractions, if compared with those already reported for testis mitochondria
(108.20 + 11.95 nmol/min per milligram protein [6]) and
nuclei (20.66 + 9.18 nmol/min per milligram protein [7]),
are of the same order, being higher in the epididymal sperm
heads than in the testis nuclei. We chose two different protocols in order to prepare heads and mitochondria with a
high degree of purity and integrity. Spermatozoa midpiece
mitochondria are in fact known to be quite peculiar in relation to other types of mammalian mitochondria: they possess an unusual shape and assemblage [13, 15, 31, 34]. A
"capsule" (a keratin-like structure) hardens the outer membrane [34], so that a rather drastic treatment is required for
their solubilization and purification. We avoided high concentrations of thiols [13, 15] because they are responsible
for sensitive structural alterations and for significant solubilization of PHGPx from the structure to which it is bound
[35].
The data on PHGPx solubilization reported in Figure 4
indicate, in addition, a difference in the way the enzyme
binds at the level of mature spermatozoa heads, where it is
more firmly bound than in testis nuclei. The notable solubilization elicited by 2-ME may be linked to the fact that
this agent, maintaining PHGPx in the reduced form [24],
probably removes disulfide interaction in the enzyme itself
or with other groups anchoring it to chromatin structure. In
the case of epididymal spermatozoa, where the cysteinerich protamines in the heads are in the disulfide form [36],
the resulting compactness and resilience of the chromatin
make the enzyme more tightly bound and resistant to the
combined effectors and to KCI alone. The nonionic detergent Triton X-100 is ineffective in solubilizing sperm head
PHGPx, whereas more than 20% is released from testis
nuclei, where about one third of the enzyme was recovered
bound to the nuclear envelope [7]. Both types of mitochondria, in contrast, release PHGPx with difficulty, especially in the case of the sperm organelles, where only the
reduction of thiols exhibited a significant effect. These results could be correlated to the structural diversity of the
subcellular compartments to which the enzyme is bound,
or could suggest more than a single role for PHGPx due to
its differing linking strengths.
The presence in spermatozoa of PHGPx could at first
glance be justified simply by an enzymatic antioxidant role
against hydroperoxides. The results obtained with sperm
protamines (Fig. 6) are, on the other hand, of great interest
in supporting the hypothesis of an additional specific role
of PHGPx in regulating the redox status of -SH groups
other than those of small molecules such as GSH. The catalytic site of the enzyme seems in fact to better recognize
caput than cauda protamines, presumably on the basis of a
different conformation of these proteins, or a different -SH
assemblage and hence accessibility. In the cauda domain,
in fact, protamine thiol oxidation and the subsequent chromatin condensation of the sperm are to be considered complete [36]. Since thiol oxidation is necessary also for mitochondrial capsule stabilization [8], and the absence of free
GSH in rat sperm has been reported [11], we conclude that
the PHGPx described here may be involved in thiol oxidation of specific protein(s) such as protamines, in addition
to fulfilling a protective role. From this point of view, the
hydroperoxides could exert a modulatory function of the
thiol redox status of the protein(s), which has already been
reported [37, 38]. In contrast, this kind of function has to
be ruled out for GPx (which we used as control), very likely
1507
because of the difference in the catalytic site of the enzymes already described [39]. We will therefore further investigate the role of PHGPx in sperm maturation.
ACKNOWLEDGMENTS
The authors acknowledge Dr. N. Kosower (Tel-Aviv University) for
her suggestions and critical reading of the manuscript, Dr. E Ursini (Padova University) for his kind supply of pure PHGPx and PHGPx antibodies, Dr. C. Bruschi (ICGEB, Trieste) for helpful supervision of English
style, and Mr. A. Bianchi for his skillful technical assistance.
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