Human Reproduction, Vol.26, No.1 pp. 47– 58, 2011
Advanced Access publication on November 18, 2010 doi:10.1093/humrep/deq297
ORIGINAL ARTICLE Andrology
Assessment of acrosome and nuclear
abnormalities in human spermatozoa
with large vacuoles
A. Perdrix 1, A. Travers 1, M.H. Chelli 2, D. Escalier 3,4, J.L. Do Rego 5,
J.P. Milazzo 1, N. Mousset-Siméon 1, B. Macé 1, and N. Rives 1,*
1
EA 4308 ‘Spermatogenesis and Male Gamete Quality’, Reproductive Biology Laboratory – CECOS, Rouen University Hospital, Institute for
Biomedical Research, University of Rouen, 76031 Rouen Cedex, France 2Center of Medical Assisted Reproduction, New Parc Clinic, 13 Bis
Hooker Doolitle Street, 1002 Tunis, Tunisia 3Department of Andrologia, Le Kremlin-Bicêtre University Hospital, Le Kremlin-Bicêtre F-94275,
France 4Institut National de la Santé et de la Recherche Médicale (INSERM) U.933, Université Pierre et Marie Curie-Paris 6, Hôpital ArmandTrousseau, 75571 Paris Cedex 12, France 5Institut National de la Santé et de la Recherche Médicale U982, IFRMP 23, University of Rouen,
76821 Mont-Saint-Aignan Cedex, France
*Correspondence address. Tel: +33-2-32-88-82-25; Fax: +33-2-35-98-20-07; E-mail: [email protected]
Submitted on April 19, 2010; resubmitted on September 21, 2010; accepted on September 28, 2010
background: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa.
methods: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a
vacuole occupying .13.0% total head area, isolated under high magnification (×6600), were assessed. Confocal and transmission electron
microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ
hybridization.
results: SLV represented 38.0 + 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in
the anterior and median sperm head (45.7 + 2.90 and 46.1 + 3.00%, respectively). Abnormal acrosomes were increased in SLV compared
with unselected spermatozoa (77.8 + 2.49 versus 70.6 + 2.62%; P ¼ 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P , 0.0001) than SLV, while chromatin condensation was altered in SLV (P , 0.0001). Aneuploidy and diploidy rates were increased in SLV (P , 0.0001).
conclusions: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global
impairment of the spermatogenesis process as a common origin of the morphological alterations.
Key words: acrosome / aneuploidy / chromatin / spermatozoa / vacuoles
Introduction
Basic semen analysis is not sufficient to evaluate male fertility status.
Indeed, 15.0% of infertile males have normal sperm parameters
(Agarwal and Allamaneni, 2005). Consequently, several tests have
been developed over the past decade to improve male infertility
diagnosis (Lewis, 2007). Morphology (using light and electron
microscopy), sperm function (motility, acrosome reaction, spermzona pellucida interaction, oxidative stress) and nucleus evaluation
(chromatin condensation, DNA fragmentation, aneuploidy) have
been the main investigation domains.
Recently, Bartoov et al. (2001) developed a novel method to assess
detailed morphology of motile spermatozoa in real-time at a magnification of up to ×6600: the motile sperm organelle morphology examination (MSOME). Normal spermatozoa have been defined, and new
abnormalities, especially sperm head vacuoles, have been described
(Bartoov et al., 2002). Several authors tried to establish a standardized
sperm MSOME classification (Bartoov et al., 2002; Sermondade et al.,
2007; Saı̈di et al., 2008; Vanderzwalmen et al., 2008; Cassuto et al.,
2009). In our laboratory, Saı̈di et al. (2008) proposed a sperm
MSOME classification based on two main values of vacuole area:
6.4% was considered as the upper limit for a normal vacuole area
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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48
observed in normal sperm, and 13.0% was the vacuole area only
observed in spermatozoa of males presenting severe sperm alterations. The high magnification observation technique has been
adapted to select the best spermatozoa for oocyte injection, introducing a new assisted reproduction technique named intracytoplasmic
morphologically selected sperm injection (IMSI). IMSI has been evaluated and seemed to improve pregnancy rates compared with ICSI
(Bartoov et al., 2003; Hazout et al., 2006; Antinori et al., 2008;
Junca et al., 2009). Sperm morphology (Bartoov et al., 2002; Berkovitz
et al., 2005; Cassuto et al., 2009) and the presence of vacuoles in particular (Berkovitz et al., 2006; Vanderzwalmen et al., 2008; Cassuto
et al., 2009) proved to have a major impact on ICSI outcome.
However, the origin and significance of large vacuoles, as well as
their effect on embryo development, are not clear. The aim of our
study was to evaluate acrosome morphology, chromatin condensation, DNA fragmentation and sperm aneuploidy in spermatozoa
with vacuoles occupying .13.0% of head area. For each patient,
results were compared with those obtained in spermatozoa from
native semen sample. Moreover, confocal and electron microscope
observations were performed to complete our analysis.
Materials and Methods
Patients
A total of 20 patients, aged between 27 and 51 years, consulting for
primary infertility in our Reproductive Biology Laboratory (Rouen University Hospital, France) from January 2009 to April 2009, were included in
this study. All the participants gave their agreement to provide semen
samples for the study. Semen analysis was carried out according to the
World Health Organization (WHO) guidelines (1999), except for sperm
morphology which was explored according to David’s modified classification (Auger et al., 2001). Teratozoospermia defined as ,50% of spermatozoa with typical morphology and confirmed on two semen analyses
spaced by 3 months, was the criterion retained to select our males.
Semen preparation
Semen samples were collected after 3 – 5 days of sexual abstinence and
were liquefied for 20 min at 378C. One semen sample per patient was
used to perform the different analyses, including transmission electron
microscopy (TEM) and confocal microscopy observations for three
patients. From the original sample, two types of preparation were
made: a first fraction of native sperm was washed by centrifugation for
10 min at 540g in phosphate-buffered saline (PBS) (Biomérieux, Marcy
l’Etoile, France), and a second fraction was centrifuged on a gradient
density system (PureSperm 100w, JCD, La Mulatiere, France). The
pellets of native unselected sperm were divided in two equal parts. The
first part was fixed in methanol for 30 min at 2208C, spread onto glass
slides (Polylysine Slidesw, Menzel Gläser, Braunshwein, Germany), airdried and stored at 2208C, for acrosome morphology, DNA fragmentation and aneuploidy analyses. The second part was spread onto glass slides
(SuperFrost Plusw, Menzel Gläser, Braunschwein, Germany) before fixation with 3% glutaraldehyde (Sigma, St Louis, MO, USA), air-dried and
stored at room temperature, for chromatin condensation assessment. Furthermore, a droplet of motile sperm fraction obtained after gradient
density selection was placed into a glass box (Willco Sterilew,
GWST-1000, Biosoft International, Amsterdam, The Netherlands) under
sterile paraffin oil (Ovoilw 100, Vitrolife, Göteborg, Sweden) in order to
perform high magnification observations. For each patient, spermatozoa
Perdrix et al.
morphology was studied at high magnification (MSOME) (×6600) using
an inverted microscope equipped with Nomarski differential interference
contrast optics (Leica DMI 6000B, Leica, Solms, Germany). Twenty-five
spermatozoa were randomly photographed (Leica Application Suite
version 3.4.0, Leica, Solms, Germany) and morphology assessment was
conducted using Leica IM 1000 software (Solms, Germany). Length,
width and surface of head, and number as well as area of vacuoles were
also assessed with this software (Saı̈di et al., 2008). Then, motile spermatozoa containing large vacuoles, occupying .13% of head area (Saı̈di et al.,
2008), were selected at ×6600 magnification using a micropipette injection (ICSI Micropipetsw MIC-50-30, Humagen, Charlottesville, VA, USA)
and a micromanipulator system. Twenty to fifty isolated vacuolated spermatozoa were fixed for each technique: (i) with methanol on two glass
slides (Cytoslidesw, Thermo Fisher Scientific, Runcorn, USA), in order
to perform acrosome evaluation and the terminal deoxynucleotidyl
transferase-mediated dUTP nick end labelling (TUNEL) assay, as well as
fluorescence in situ hybridization (FISH) analysis; (ii) with glutaraldehyde
on one glass slide for aniline blue staining. The number of isolated spermatozoa was limited by the selection duration: a maximum time of 2 h has
been used for this technique in order to avoid possible effects of time
selection on the incidence of vacuolated spermatozoa (Peer et al., 2007)
(Fig. 1).
Electron and confocal microscopy
observations
Ejaculates from three patients (Patients 1, 5 and 8, Table I) presenting 72,
52 and 56% of spermatozoa with large vacuoles respectively were
explored separately using TEM and confocal microscopy analyses.
For TEM analysis, 1 ml from each semen sample was washed twice in
IVF medium (Medicult, Lyon, France) for 10 min at 540 g. The pellets
were fixed in 3% glutaraldehyde. The samples were then washed for
15 min in fresh buffer containing 4% w/v sucrose and embedded in 2%
agar. Post-fixation lasted 1 h in 1% w/v osmic acid in phosphate buffer.
After dehydratation in a graded series of ethanol, small pieces of agar containing spermatozoa were embedded in Epon resin (Polysciences Inc.,
Warrington, PA, USA). Sections were cut on a Reichert OmU2 ultramicrotome (Reichert-Jung AG, Wien, Austria) using a diamond knife. Sections (70 nm) were collected on nickel grids and stained with uranyl
acetate (4% in 70% ethanol, 20 min) and Reynolds lead citrate (10 min),
and examined using a JEOL JEM100 CXII transmission electron microscope (Jeol Ltd, Tokyo, Japan) operated at 80 kV. For each patient, quantitative analyses of sperm head abnormalities were performed on at least
30 longitudinal sections.
Confocal microscopy evaluation was carried out at Regional Platform
for Cell Imaging (PRIMACEN) in Mont-Saint-Aignan (Seine-Maritime,
France). Selected vacuolated spermatozoa were double-stained with
monoclonal antiproacrosin antibody Mab4D4 and 4′ ,6-diamidino-2phenylindole (DAPI) as described below. The preparations were examined
using a filter-free confocal laser scanning microscope (TCS SP2 AOBS,
Leica, Solms, Germany) equipped with a fluorescence DM RXA2UV
optical system, and an argon UV (excitation wavelength 405 nm), an
argon (excitation wavelengths 458, 476, 488 and 514 nm) and three
helium-neon (excitation wavelengths 543, 594 and 633 nm) ion lasers
(Leica, Solms, Germany). The intensity of the immunoreactivity was
measured both in nucleus and vacuoles of the same spermatozoa by
means of the computer-assisted image analysis system of Leica confocal
microscope software LCS. Each spermatozoon was observed on various
focal plans along all its thickness. Each focal plane had a depth of
0.2 mm. Therefore, between 10 and 20 cuts were obtained for each spermatozoon. A total of 15 spermatozoa per patient were scored.
49
Nuclear alterations in large vacuole spermatozoa
Figure 1 Experimental scheme for analysis of spermatozoa from 20 patients. MSOME, motile sperm organelle morphology examination; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling; PBS, phosphate-buffered saline; FISH, fluorescence in situ hybridization.
Proacrosin immunolabelling and
TUNEL assay
beginning, were also scored. The same analysis was performed on vacuolated spermatozoa (Fig. 2A and B).
Immunodetection of proacrosin with monoclonal antibody Mab4D4
allowed characterization of acrosome shape and size (Gallo et al., 1991).
Slides stored at 2208C were pre-incubated in horse serum diluted 1:80
in PBS (Horse Serumw, Sigma) for 30 min at room temperature. Proacrosin immunolabelling was firstly performed with Mab4D4 diluted 1:100 in
PBS (1 h, room temperature). Slides were incubated with a 1:100
diluted biotinylated antimouse antibody (Anti-mouse affinity Isolated
Biotin Conjugatew, Sylenus, Hauthom, Australia) (10 min, 378C). Detection was performed with Avidin Texas Red (Detect B3, StarFishw,
Adgenix, Voisin le Bretonneux, France) for 10 min at 378C. Then, spermatozoa were processed for TUNEL according to the manufacturer’s instructions (In Situ Cell Death Kit Detection PODw, Roche, Mannheim,
Germany) and finally counterstained with a solution of DAPI (Counterstain
1, Adgenix, Voisin le Bretonneux, France) diluted 1:1000 in antifading
mounting medium (Antifadew, MP-QBio-Gene, Illkirch, France).
Spermatozoa were examined with an epifluorescence microscope
(DMRBw, LEICA, Solms, Germany) at a magnification of ×1000 and
images were captured with a digital imaging system (MacProbew version
3.3, Perspective Scientific International LTD, Chester, England). Acrosome
morphology was explored on 200 spermatozoa. Localization, size and
outline regularity were assessed, defining two patterns of acrosomal morphology: normal (i.e. acrosome occupying the anterior half of the sperm
head, with regular outlines) and abnormal (i.e. acrosome absent, small
or irregular), independent of reacted or unreacted acrosome status.
DNA fragmentation was simultaneously characterized on 500 spermatozoa. Spermatozoa were considered with complete fragmented DNA
when the green fluorescence signal occupied .50% of head area. Spermatozoa with green fluorescence points, interpreted as DNA fragmentation
Aniline blue staining
Sperm chromatin condensation was examined on smears fixed with 3%
glutaraldehyde. Slides were stained with 5% aniline blue (Gurrw, BDH Laboratory Supplies, England) at pH 3.5 for 5 min (Terquem and Dadoune,
1983). Five hundred spermatozoa from native semen sample were analysed on each slide under a light microscope (Leitz DMRDw, Leica,
Solms, Germany). Sperm nuclei were considered to be immature with
hypocondensed chromatin when an intense blue staining occupied
.50% of head area. The same analysis was performed on vacuolated
spermatozoa.
FISH
A three-colour FISH was carried out for 15 patients using a-satellite centromeric probes for chromosome X in green (CEP X Spectrum GreenTM ,
Abbott, Rungis, France), chromosome Y in yellow (CEP Y Sat III Spectrum
OrangeTM , CEP Y Sat III Spectrum GreenTM , Abbott, Rungis, France) and
chromosome 18 in red (CEP 18 Spectrum OrangeTM , Abbott, Rungis,
France). The yellow colour was obtained by mixing an equal volume of
CEP Y labelled with Spectrum OrangeTM and CEP Y labelled with Spectrum GreenTM . Spermatozoa DNA fixed with methanol was decondensed
using a solution of 25 mM DTT (dithiothreitol) 1 M Tris– HCl, pH 9.5.
Decondensation was controlled under a phase contrast microscope and
stopped when fixed spermatozoa were decondensed to approximately
twice their original size. After dehydration by incubation in ethanol
series (70, 95 and 100%), spermatozoa DNA and probes were simultaneously denatured at 738C for 5 min on a heated plate (Hybaidw,
Omnigene, Teddington, UK) and hybridized overnight at 378C.
50
Table I Semen characteristics and MSOME assessment of 20 patients enrolled in the study.
Patients
Age
(years)
Semen parameters
.............................................................................
Volume
(ml)
Sperm
count
(106/ml)
Progressive
motility (%)
Normal
morphology
(%)
MSOME
................................................................................................................................
Mean size of the head
(L 3 W) (mm 3 mm)
Mean
vacuole
area (%)
Spz with
large
vacuoles
(%)
Vacuole localization
.......................................................
Ant (%)
Med (%)
Post (%)
..........................................................................................................................................................................................................................................................
1
39
3.0
11
20
21
5.8 × 3.1
18.9
72
34.7
48.0
17.3
2
33
4.9
4
30
26
4.9 × 3.1
9.7
20
28.3
67.4
6.5
3
43
5.0
10
10
33
4.5 × 3.3
8.2
12
33.3
59.0
7.7
4
27
2.3
23
20
20
5.4 × 3.6
10.3
28
36.4
49.1
14.5
5
32
4.3
50
45
31
4.7 × 3.0
13.9
52
62.8
23.3
14.0
6
41
1.2
45
25
48
5.2 × 3.1
10.9
24
48.8
48.8
0.0
7
51
2.0
4
20
12
5.1 × 3.1
12.8
36
44.8
49.3
6.0
8
32
1.9
2
20
10
5.4 × 3.2
16.6
56
43.9
54.4
1.8
9
41
3.5
3
5
15
4.8 × 3.1
10.3
24
56.0
42.0
2.0
10
33
2.5
58
30
26
5.0 × 3.0
8.5
20
51.5
39.4
9.1
11
40
4.3
20
40
21
5.5 × 3.1
10.4
28
26.2
57.1
16.7
12
28
3.3
12
15
17
4.9 × 3.3
19.0
76
60.0
32.5
7.5
13
43
4.5
15
20
16
5.2 × 3.0
6.7
4
48.0
36.0
16.0
14
34
5.5
16
30
42
4.8 × 3.2
8.2
16
58.3
37.5
0.0
15
35
4.5
16
35
23
5.2 × 3.2
17.0
56
55.3
38.3
6.4
16
32
4.5
8
20
15
5.4 × 2.9
12.2
48
54.3
43.5
2.2
17
37
3.5
5
20
18
4.9 × 3.0
15.0
60
46.7
48.9
2.2
18
37
3.0
25
30
42
5.2 × 3.3
9.5
16
14.9
78.7
6.4
19
37
2.4
33
10
10
5.7 × 3.7
16.7
80
61.9
26.2
11.9
20
29
4.9
19
25
16
5.0 × 3.2
11.5
32
48.1
42.6
9.3
Mean + SEM
36.2 + 1.32
3.6 + 0.27
18.9 + 3.60
23.5 + 2.24
23.1 + 2.46
5.1 + 0.08 3 3.2 + 0.04
12.3 + 0.90
38.0 + 5.10
45.7 + 2.90
46.1 + 3.00
7.9 + 1.30
Ant, anterior; L, length; Med, median; MSOME, motile sperm organelle morphology examination; Post, posterior; Spz, spermatozoa; W, width.
Perdrix et al.
51
Nuclear alterations in large vacuole spermatozoa
spermatozoa). Using MSOME observation, the percentage of spermatozoa with large vacuoles ranged from 4 to 80% of motile spermatozoa after gradient density centrifugation, and a significant positive
correlation was found between percentage of spermatozoa with
large vacuole and percentage of spermatozoa with abnormal morphology evaluated on native semen (r ¼ 0.49; P ¼ 0.03). Vacuoles
were mainly located in anterior and median parts of sperm head.
Figure 2 Assessment of acrosome morphology by proacrosin
immunostaining with monoclonal antibody Mab4D4 (red) and detection of DNA fragmentation using TUNEL assay (green) with
4′ ,6-diamidino-2-phenylindole (DAPI) counterstaining (blue) at
×1000 magnification. (A) Human spermatozoon without acrosome
and totally fragmented DNA (left)—normal spermatozoon (right).
(B) Human spermatozoon with abnormally shaped acrosome and
no fragmented DNA (top)—spermatozoon with altered acrosome
and DNA fragmentation initiation (below).
After hybridization, slides were washed once in a solution of standard
saline citrate (SSC) 0.4×/Nonidet-P40 0.3% (Ipegalw, Sigma, Chemical
Co) for 2 min at 738C and once in a solution of SSC 2×/Nonidet-P40
0.1% at room temperature for 1 min. After dehydration, slides were counterstained with a DAPI solution diluted 1:1000 in antifade mounting
medium (Counterstain 1, Adgenix, Voisin le Bretonneux, France).
Spermatozoa were examined with an epifluorescence microscope at a
magnification of ×1000. A triple band-pass filter set [fluorescein isothiocyanate (FITC)/rhodamine/DAPI] was used for the count, and a single
band-pass filter set (DAPI or FITC or rhodamine) to differentiate a spermatozoon from somatic or immature germ cells. Images were captured
with a digital imaging system (MacProbew version 3.3, Perspective Scientific International LTD, Chester, England).
A minimum of 1000 spermatozoa was scored on native semen sample.
Green, red and yellow signals detected chromosomes X, 18 and Y,
respectively. Two fluorescent spots of comparable size and intensity, separated by at least one spot diameter, were considered as two copies of the
corresponding chromosome. Spermatozoa with diffuse fluorescence
signals and overlapping nuclei were classified as ambiguous and were
not included in the count. The same analysis was performed on vacuolated
spermatozoa.
TEM and confocal microscopy analysis
TEM analysis of the spermatozoa from three patients revealed that the
vacuoles were present exclusively in the nucleus, as shown in Fig. 3A
and B. The vacuoles were at various locations in the nucleus depending on the spermatozoon. Large nuclear vacuoles were found in 33, 18
and 40% of spermatozoa from the three patients. Moreover, 56, 33
and 16% of spermatozoa presented a vacuole whose size in section
was moderate. Among them, some could be large vacuoles sectioned
at their margin owing to the TEM ultrathin sections.
For confocal microscopy, the intensity of DAPI immunoreactivity
was measured both in the nucleus and vacuoles of the same spermatozoa for 46 vacuolated spermatozoa from three patients. The mean
DAPI fluorescence intensity was significantly decreased in vacuoles in
comparison with the rest of the nucleus (83.6 + 4.24 versus 211.4 +
3.18; P , 0.0001), independent of the chosen spermatozoon
(P , 0.0001), the chosen patient (P , 0.0001) or the chosen localization of the measure in the nucleus (P , 0.0001). The maximum mean
fluorescence intensity was also higher in the nucleus than in the
vacuole (232.0 + 2.62 versus 111.9 + 5.29; P , 0.0001). Conversely,
Statistical analysis
Statistical analysis was carried out with MedCalcw (MedCalc Software
version 9.3.9.0, Bruxelles, Belgium) in order to compare spermatozoa
from the native semen sample with vacuolated spermatozoa. Student’s
t-test was used for acrosome morphology and chromatin condensation.
Wilcoxon’s rank test was used for DNA fragmentation and x2 test for
aneuploidy frequencies. A value of P , 0.05 was considered to be
significant.
Results
Figure 3 Electron microscopy observation of unselected large
Semen parameters
Semen characteristics are summarized in Table I. Seven patients
presented asthenoteratozoospermia (progressive motility ,50%
spermatozoa; typical forms ,50% spermatozoa) and 13 oligoasthenoteratozoospermia (spermatozoa concentration ,20 × 106/ml;
progressive motility ,50% spermatozoa: typical forms ,50%
vacuole spermatozoa from native human semen samples and confocal
microscope analysis of vacuolated spermatozoa selected under the
MSOME observation. (A and B) TEM observation revealed that vacuoles were located inside the nucleus. (C and D) A lower DAPI signal
intensity was observed in vacuoles under confocal microscopy at
×630 magnification.
52
Perdrix et al.
Figure 4 Evolution of mean DAPI fluorescence intensity between vacuole and nucleus in human spermatozoa with large vacuoles, evaluated with
the confocal microscope along the spermatozoa thickness. AU, Arbitrary Units; M1– M19, cut 1 – 19 along the spermatozoa thickness.
minimum mean fluorescence intensity was lower in the vacuole
than in the nucleus (49.4 + 3.55 versus 161.5 + 5.21; P , 0.0001).
The mean DAPI fluorescence intensity followed the same evolution
in the nucleus and vacuoles but with a significantly decreased fluorescence in vacuoles compared with the rest of the nucleus. Vacuoles
were mainly located in the anterior two-thirds of the sperm head (89%
of spermatozoa) (Fig. 3C and D, Fig. 4).
Acrosome morphology
A total of 200 spermatozoa from the native semen sample and a mean
of 28 spermatozoa with large vacuoles were scored per patient using
proacrosin immunostaining. As shown in Table II, abnormal acrosomes were significantly increased in spermatozoa with large vacuoles
compared with unselected spermatozoa (P ¼ 0.014). The mean percentage of absent acrosome was 23.2 + 3.56% in spermatozoa with
large vacuole and 17.4 + 4.90% in native semen samples.
DNA fragmentation
A mean of 502 unselected spermatozoa (500–530) and 28 (19 –50)
vacuolated spermatozoa were scored with the TUNEL assay. Complete DNA fragmentation was significantly higher in native sperm
than in spermatozoa with large vacuoles (P , 0.0001).
However, the rate of spermatozoa with DNA fragmentation beginning was similar between unselected and vacuolated spermatozoa
(P ¼ 0.68) (Table II).
Chromatin condensation
For each patient, 499 unselected spermatozoa (489–501) and 23
vacuolated spermatozoa (10 –30) were scored. Chromatin condensation assessed by aniline blue was significantly altered in spermatozoa
with large vacuoles in comparison with those from native semen
samples (P , 0.0001) (Table II).
FISH analysis
Hybridization rates were 99% for unselected spermatozoa and 96.9%
for vacuolated spermatozoa. For each patient, an average of 1060
spermatozoa and 27 spermatozoa were scored for spermatozoa
from native sperm and vacuolated spermatozoa respectively. Results
are reported in Table III. Compared with native spermatozoa, in
vacuolated spermatozoa, the frequency of XY hyperhaploid and XX
disomic spermatozoa did not increase significantly (P ¼ 0.11 and
P ¼ 0.07, respectively), mean frequencies of YY and 1818 disomic
spermatozoa were significantly higher (P , 0.0001 and P ¼ 0.0043,
respectively), diploid sperm nuclei were significantly increased
(P , 0.0001) and total aneuploidy frequencies (included hyperhaploid
and disomic spermatozoa) were higher. In conclusion, spermatozoa
with large vacuoles showed a significant increase of chromosome
abnormalities (aneuploidy and diploidy) compared with spermatozoa
from native semen samples (P , 0.0001).
Discussion
Since the first description of sperm head vacuoles using MSOME
(Bartoov et al., 2001), parameters defining a normal vacuole (location,
number and size) have not been clearly established. Large vacuoles
have been defined initially as occupying .4.0% of the total head
area (Bartoov et al., 2002), and considered as abnormal in several publications (Berkovitz et al., 2006; Peer et al., 2007; Sermondade et al.,
2007; Vanderzwalmen et al., 2008). However, more recently,
Franco et al. (2008) considered a vacuole to be large when the area
was .50.0% of total head area. Moreover, Garolla et al. (2008)
Patients
Spermatozoa from native semen sample
.........................................................................................................................
DNA fragmentation
Acrosome morphology
............................................
Complete (%)
Beginning (%)
Abnormal or absent (%)
Spermatozoa with large vacuoles
......................................................................................................
Chromatin
condensation
DNA fragmentation
Abnormal (%)
Total (%)
Acrosome morphology
Chromatin
condensation
Abnormal or absent (%)
Abnormal (%)
.......................................
Beginning (%)
..........................................................................................................................................................................................................................................................
1
9.4
11.6
68.5
53.2
0.0
38.4
61.5
64.2
2
3
11.0
8.0
77.0
21.8
6.4
12.2
48.5
28.1
0.0
0.0
82.8
45.4
0.0
11.5
69.2
4
21.2
11.5
92.5
50.0
35.2
0.0
3.2
71.0
27.7
5
10.0
12.6
57.5
33.4
0.0
22.0
82.0
25.0
6
4.0
7.2
7
5.8
13.2
60.0
9.6
0.0
0.0
75.9
33.3
78.5
18.9
0.0
33.3
80.9
8
10.4
30.0
16.4
71.5
20.0
2.9
2.9
57.1
36.6
9
6.6
9.8
77.0
27.4
5.2
57.8
94.7
46.1
10
10.8
14.0
82.0
38.8
4.5
4.5
86.3
50.0
11
9.4
17.8
68.5
17.8
9.7
16.1
87.1
58.8
12
3.0
6.8
73.0
36.0
5.7
22.9
65.7
72.2
13
5.0
10.6
69.5
19.8
0.0
9.6
80.9
50.0
14
6.0
8.4
64.0
17.4
6.0
12.1
69.7
56.3
15
8.6
18.4
49.0
47.6
0.0
0.0
66.7
64.3
16
9.0
13.2
74.5
30.6
0.0
29.2
91.7
65.2
17
5.8
24.8
74.0
27.0
0.0
12.5
75.0
54.2
18
4.0
4.4
58.5
14.4
0.0
9.5
66.7
59.2
19
20.6
7.4
85.5
15.0
0.0
4.3
91.3
66.6
20
5.4
1.6
83.0
17.2
0.0
0.0
95.0
53.3
Mean + SEM
8.6 + 1.09a
70.6 + 2.62c
26.5 + 2.57d
1.7 + 0.65a
77.6 + 2.54c
50.4 + 3.10d
11.5 + 1.22b
14.5 + 3.45b
Nuclear alterations in large vacuole spermatozoa
Table II Evaluation of DNA fragmentation (TUNEL assay), acrosome morphology (proacrosin immunolabelling using monoclonal antibody Mab4D4) and
chromatin condensation (aniline blue staining) in spermatozoa from native semen samples and spermatozoa with large vacuoles.
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling.
DNA fragmentation was considered completely when green fluorescence occupied .50% of head area, and beginning when only scattered points of fluorescence were observed.
a
P , 0.0001; bP ¼ 0.68; cP ¼ 0.014; dP , 0.0001.
53
54
Table III Aneuploidy and diploidy frequencies for chromosome X, Y and 18 in sperm nuclei from native samples and selected spermatozoa with large vacuoles,
estimated using three-colour FISH.
Patients
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean + SD
..........................................................................................................................................................................................................................................................
Chromosomes
abnormalities (%)
Native semen
samples (%)
Vacuolated
spermatozoa (%)
24 XY
24 YY
24 XX
24, X/Y, +18
Diploidy*
Aneuploidy**
Total chromosome
abnormalities***
24 XY
24 YY
24 XX
24, X/Y, +18
Diploidy*
Aneuploidy**
Total chromosome
abnormalities***
0.49
0
0
0
0
0.49
0.49
0.26
0
0.18
0.09
0.27
0.53
0.80
0.46
0.19
0
0
0.09
0.65
0.74
0.27
0.54
0.18
0.18
0.45
1.17
1.62
0.59
0.49
0.39
0.1
0.3
1.57
1.87
0.38
0.19
0.29
0
2.39
0.86
3.25
0.29
0.29
0.1
0.2
0.59
0.88
1.47
0.57
0.19
0.09
0
0.18
0.85
1.03
0.29
0.1
0
0.1
0.39
0.49
0.88
0.09
0.28
0
0.28
0.09
0.65
0.74
0.45
0.36
0.09
0
0.18
0.9
1.08
0.84
0.19
0.28
0
0.18
1.31
1.49
0.19
0.19
0
0.18
0.27
0.56
0.83
0.57
0.28
0.09
0.09
1.04
1.03
2.07
0
0.38
0.1
0.19
0.57
0.67
1.24
0.38 + 0.06a
0.25 + 0.04b
0.12 + 0.03c
0.1 + 0.02d
0.47 + 0.15e
0.84 + 0.08f
1.3 + 0.18g
0
0
0
0
0
0
0
0
2.56
0
5.12
0
7.68
7.68
0
0
0
0
0
0
0
0
0
0
0
5.88
0
5.88
0
5.88
0
0
0
5.88
5.88
0
0
0
0
29.17
0
29.17
0
0
4.76
0
0
4.76
4.76
0
8.7
4.35
0
4.35
13.05
17.4
0
6.25
0
6.25
0
12.5
12.5
0
3.13
0
0
0
3.13
3.13
7.69
0
0
3.85
0
11.54
11.54
3.45
0
0
0
0
3.45
3.45
3.57
0
0
3.57
0
7.14
7.14
0
0
0
0
0
0
0
0
8.33
0
0
0
8.33
8.33
0.98 + 0.58a
2.32 + 0.86b
0.61 + 0.41c
1.25 + 0.58d
2.63 + 1.96e
5.16 + 1.23f
7.80 + 1.98g
Values are mean + SEM.
*
Sum of frequencies of 1818XX, 1818YY and 1818XY diploid spermatozoa.
**
Sum of frequencies of disomic XX, YY, 1818 and hyperhaploid XY spermatozoa.
***
Sum of frequencies of diploid and aneuploid spermatozoa.
a,c
not significant; b,e,f,gP , 0.0001; dP¼ 0.0043.
Perdrix et al.
Nuclear alterations in large vacuole spermatozoa
analysed ‘large’ vacuoles without specifying their size. In our study, a
vacuole area occupying .13.0% of sperm head was used to describe
abnormal and large vacuoles. This vacuole area value seemed particularly associated with severe sperm alterations (Saı̈di et al., 2008). In
our study, our population was highly selected on teratozoospermia,
and spermatozoa with large vacuoles represented 38.0% of ejaculated
and gradient density selected spermatozoa. This mean value was
lower than published data varying from 30.0 –40.0% (Berkovitz
et al., 2006), to 53.4% (Sermondade et al., 2007) and 73.2%
(Bartoov et al., 2002). The under estimation of large vacuole spermatozoa frequency reported in our study could be explained by our
threshold value of 13%, responsible for a reduced targeted spermatozoa population. Indeed, considering vacuoles occupying .50% of the
sperm nuclear area, the proportion of spermatozoa with a large
nuclear vacuole was 25.2 + 19.2% in an unselected men group
(Oliveira et al., 2010a). The first step of our work was to evaluate
the relationship between nuclear vacuoles and teratozoospermia.
This relationship has previously been studied, however, results are
controversial: Bartoov et al. (2002) did not observe any relationship,
while Oliveira et al. (2009) showed the contrary. In our study, a significant positive correlation was found between the frequency of spermatozoa with large vacuoles and the frequency of spermatozoa abnormal
forms (r ¼ 0.49; P ¼ 0.03). This point can be considered as a supplementary argument for a pathological character of nuclear vacuoles.
The large vacuoles in our study were associated with a global
alteration of spermatozoa morphology.
The origin and consequences of sperm head vacuoles are also
subject to controversy. A large sperm head vacuole may originate
from spermatogenesis impairment, abnormal maturation during male
genital tract transit or acrosome modification during the acrosome
reaction, as suggested by Kacem et al. (2010). In our study, the first
hypothesis was tested, trying to establish a relationship between vacuoles and indicators of spermatogenesis impairment: chromatin condensation, DNA fragmentation and chromosome content. Vacuole
location could be a first element in helping to define their origin.
Indeed, 91.8% of vacuoles were located in the anterior two-thirds
of our sperm heads, as previously reported by Sermondade et al.
(2007). The acrosome and nucleus are the two main structures in
this sperm region.
Acrosome morphology was assessed, in our study, using monoclonal antibody 4D4 immunolabelling (Gallo et al., 1991). Acrosome
alterations detected with this approach are known to be correlated
with IVF failure (Albert et al., 1992). In our males, spermatozoa
with large vacuoles presented more acrosome defects, compared with
spermatozoa from native semen samples. However, in our selected
population with teratozoospermia, acrosome morphology was
deeply altered in native semen, questioning a possible specific association between vacuoles and acrosome defects. In literature, the
association between vacuoles and acrosome abnormalities has previously been reported with TEM. Large head vacuoles may result in
conspicuous deformations of the nucleus and overall head shape,
and are often noted in spermatozoa with small acrosomes
(Zamboni, 1987), and furthermore, sperm head vacuoles and abnormal acrosomes are more frequently observed in infertile males with an
excess of sperm precursors (Mundy et al., 1994): in these two studies,
vacuoles had an exclusive nuclear location and an alteration of spermiogenesis was proposed as a common origin.
55
For the first time, we analysed by TEM samples previously selected
as having a high percentage of large vacuoles by MSOME and found
that these vacuoles were exclusively present in the nucleus. Confocal
microscope observations of large vacuole spermatozoa confirmed that
vacuoles were strictly nuclear. Another study based on an acrosomal
labelling with Pisum sativum agglutinin suggested that the vacuoles
could be of acrosomal origin (Kacem et al., 2010), in contrast to
our observation of proacrosin labelling by means of a specific monoclonal antibody. The possibility that some acrosomes can be vacuolated could not be excluded. However, vacuoles from the acrosome
should be at the margin of the sperm head, contrary to the vacuoles
seen inside the sperm head and found to be in the nucleus, both by
confocal microscopy and TEM.
The exclusive nuclear location of vacuoles supports the previous
observations of their severe impact on sperm quality. This hypothesis
has been supported by the influence of sperm head vacuoles on late
embryo development (Berkovitz et al., 2006; Vanderzwalmen et al.,
2008; Cassuto et al., 2009). Vacuole impact was evident especially
from 3 days after fertilization, time of the onset of paternal DNA
content contribution to embryo development (Tesarik, 2005), underlining the role of paternal genome integrity in blastocyst development.
Besides, recently, three papers have reinforced the concept that an
association between DNA damage and the presence of nuclear vacuoles exists (Franco et al., 2008; Garolla et al., 2008; Oliveira et al.,
2010b). Nevertheless, in our study, DNA fragmentation was not
increased in spermatozoa with large vacuoles. To explain this contradiction with published data, three hypotheses could be proposed.
First of all, the absence of any selection on native sperm before
DNA fragmentation introduced a methodology bias: dead spermatozoa were present in original semen samples, but not in vacuolated
spermatozoa, probably affecting TUNEL results (Barratt et al.,
2010). Second, there was a probable selection bias: Franco et al.
(2008) focused on a very specific spermatozoa population, presenting
a vacuole area .50% of head area; for Garolla et al. (2008) vacuole
area was not specified. Third, fluorescence microscope analysis of
TUNEL slides suffers from subjectivity (Sergerie et al., 2005), and
the positive threshold in our study could be higher in comparison
with the two others studies. Thereby, considering partial labelling
with TUNEL assay, interpreted as DNA fragmentation beginning,
spermatozoa with large vacuoles and spermatozoa from native
sperm presented similar DNA fragmentation rates.
Chromatin condensation appeared as a pertinent criterion for
nucleus quality in large vacuole spermatozoa. Indeed, spermatozoa
with poor morphology are more likely to contain loosely packaged
chromatin, evocating an influence of chromatin condensation during
spermiogenesis on sperm head morphology (Foresta et al., 1992;
Franken et al., 1999). According to Franken et al. (1999), it appears
that abnormal sperm morphology is an indicator of an altered progression of spermatids through a complete spermiogenesis. In our
study, the difference in pretreatment conditions used for aniline blue
staining between unselected spermatozoa and large vacuole spermatozoa seems less important to interpret our results: Franken et al.
(1999) demonstrated an absence of variation between aniline blue
staining performed on spermatozoa from semen samples versus that
after swim-up selection. Moreover, our study population presents
high proportions of altered chromatin compaction in native semen
samples; indeed, chromatin packaging evaluated by aniline blue staining
56
is correlated with sperm morphology (Franken et al., 1999), and our
males were selected on teratozoospermia. The specificity of the
relationship between vacuoles and altered chromatin compaction
can, consequently, be questioned. To argue a possible association
between the vacuole and poor chromatin condensation, we can
refer to the noteworthy increased rate of nuclei with abnormal chromatin in our spermatozoa with vacuoles compared with native sperm:
two times more frequent in large vacuole spermatozoa than in spermatozoa from native semen samples (50.4 + 3.1 versus 26.5 +
2.57%), evocating a particular association between loosely packaged
chromatin and sperm vacuoles. Our results were in agreement with
recent MSOME data focused on large vacuole spermatozoa (Franco
et al., 2008; Garolla et al., 2008) and with historical semen analyses:
Bedford et al. (1973), induced vacuole formation after chromatin
decondensation with sodium dodecyl sulphate (SDS) containing
DTT; Zamboni (1987) suggested that vacuoles, appearing as
non-membrane-bound areas, are the consequence of uneven processes of chromatin condensation, an hypothesis also proposed by
Mundy et al. (1994). Besides, in a recent review, Björndahl and Kvist
(2010) demonstrated that zinc content of seminal plasma plays a
major role in sperm chromatin stability after ejaculation: therefore,
90% of sperm heads underwent chromatin decondensation in SDS,
when exposed to EDTA, a chelator of sperm zinc. As a result, zinc
deficiency induced in spermatozoa by seminal vesicular zinc-chelating
proteins at, or after, ejaculation seems involved in sperm chromatin
instability and would be one mechanism resulting in the appearance
of vacuoles in human ejaculated spermatozoa.
In our study, spermatozoa with large vacuoles presented more
chromosome abnormalities (aneuploidy and diploidy) compared
with native semen spermatozoa. These results, obtained in a small
population of vacuolated spermatozoa, should be interpreted with
caution and confirmed with further studies. Nevertheless, they
seemed in line with previously reported data (Garolla et al., 2008).
In our study, we were confronted with technical difficulties in isolating
vacuolated spermatozoa: each spermatozoon was selected separately;
the duration of manipulation was limited, considering the possible
impact of prolonged incubation on development of vacuoles (Peer
et al., 2007). The number of studied spermatozoa with a large
vacuole was consequently low. To illustrate these difficulties, for five
patients, spermatozoa MSOME selection was stopped before we
obtained a sufficient number of spermatozoa, because the duration
of manipulation was too long: therefore, FISH was performed only
in 15 patients. Recent studies on selected MSOME spermatozoa
were confronted with the same difficulties: Franco et al. (2008),
studied 350 spermatozoa from 30 patients; Garolla et al. (2008) performed analysis on 100 selected spermatozoa from 20 patients. An
uneven distribution of 24,YY and 24,XX incidence in vacuolated
spermatozoa was detected. As can be observed in Table III, the distribution of disomic XX and disomic YY spermatozoa is heterogeneous among the different patients. Our team has previously
published data of FISH evaluation in a large group of infertile patients
(50 males) with distorted semen parameters: heterogeneity of sperm
nuclei disomy frequency between the different chromosomes analysed
and the different subjects was observed, including sex chromosome
aneuploidy (Rives et al., 1999). This discrepancy between disomic
XX and disomic YY spermatozoa has also been reported previously
in patients with severe sperm alterations (Vegetti et al., 2000; Gole
Perdrix et al.
et al., 2001; Härkönen et al., 2001; Nagvenkar et al., 2005). In our
study, we also explored a highly selected spermatozoa population.
We probably cannot apply the same interpretation of meiotic nondisjunctions, as we can do in a normal spermatogenesis process. In
published data, the relationship between sperm aneuploidy rates
and sperm morphology is controversial (Calogero et al., 2001; Gole
et al., 2001; Härhönen et al., 2001; Tempest et al., 2004; Machev
et al., 2005). A correlation between abnormal chromosome content
and altered chromatin condensation has been reported (Morel
et al., 1998, 2001; Ovari et al., 2010). Moreover, several papers
demonstrated the role of chromatin compaction on embryo development (Filatov et al., 1999; Larson et al., 2000; Hammoud et al., 2009).
Finally, sperm chromatin packaging quality seemed to have a determining impact on sperm morphology, sperm chromosome content and
embryo development.
The large vacuoles which we observed in selected spermatozoa
were exclusive of nuclear localization. Vacuolated spermatozoa
seemed to show more frequently an abnormal chromatin condensation and chromosome content. Chromatin organization appeared
of major importance in spermatozoa with large vacuoles. Further
investigations seem necessary to confirm our observations and to
further define the relationship between abnormal chromatin organization and sperm head vacuoles (for example, defects in sperm
nuclear maturation involving protamines and/or the nuclear matrix,
and post-ejaculatory chromatin disorganization owing to abnormal
sequence of ejaculation or particular conditions during semen
liquefaction).
Authors’ roles
A.P. and A.T. performed experiments, collected data, performed statistical analysis and drafted the manuscript. N.R. conceived the study
and revised the manuscript critically M.H.C. performed preliminary
analyses to evaluate the feasibility of this study. D.E. performed
TEM analysis. J.L.D. performed confocal microscope observations.
J.P.M., N.M.-S. and B.M. recruited patients, participated in design of
the study and revised the manuscript critically. All authors have read
and approved the final manuscript.
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