Association of sperm morphology and the sperm deformity index (SDI) with poly (ADP-ribose) polymerase (PARP) cleavage inhibition
Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase (PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.
Association of sperm morphology and the
sperm deformity index (SDI) with poly
(ADP-ribose) polymerase (PARP)
Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase
(PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm
deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.
2011;95:2481–4. 2011 by American Society for Reproductive Medicine.)
Key Words: Sperm morphology, semen analysis, SDI, PARP cleavage, apoptosis, ﬂow cytometry
Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that is
thought to play a role in the changes in chromatin structure and
male germ cell function during postmeiotic differentiation, includ-
ing chromatin condensation, transcriptional inhibition, and the
sequential replacement of histones by transition proteins and
protamines. It also catalyzes the poly(ADP-ribosyl), and the cleaved
(c-PARP) represents the active form of the enzyme and leads to an
immediate DNA-damage dependent post-translational modiﬁcation
of nuclear proteins that contributes to the survival of injured
proliferating cells. Cleavage and inactivation of PARP is a well-
characterized caspase-dependent marker of apoptosis (1, 2).
Recently, we reported the presence of PARP homologs in
ejaculated human spermatozoa and identiﬁed these as PARP-1 (75
kDa), PARP-9 (63 kDa), and PARP-2 (60 kDa)(3) and demonstrated
a positive correlation between PARP and sperm maturity, suggesting
a role for PARP proteins in sperm DNA damage/repair (3).
In contrast to its role as a survival factor in limited DNA dam-
age, PARP-1 promotes cell death under conditions of extensive
DNA damage (4). Therefore, chemical inhibition or genetic abla-
tion of PARP-1 can provide protection against cell death (4–6).We
have demonstrated that mature and immature spermatozoa show
a signiﬁcant decline in the percentage of late apoptotic sperm
after PARP inhibition in chemical and oxidative stress–induced
sperm damage. The response of oxidative stress–induced
damage to PARP inhibition was low compared with that of
chemical damage, suggesting that in addition to DNA damage,
oxidative stress–induced sperm damage may have different
pathway(s). This was evident from the increase in early
apoptotic sperm that was seen in oxidative stress–induced
damage even after PARP inhibition.
Poor spermmorphology correlates with increased formation of re-
active oxygen species(ROS) as well as the frequencies of single- and
double-strand DNA breaks observed in the spermatozoa of infertile
men (7). Increased levels of ROS were observed in samples with
high proportions of sperm abnormalities such as amorphous heads,
damaged acrosomes, midpiece defects, cytoplasmic droplets, tail
defects, and high sperm deformity index (SDI) scores. We demon-
strated a positive correlation between SDI scores and early and
late markers of sperm apoptosis, loss of the integrity of the
mitochondrial membrane potential, and activated caspase-3 (8).
SDI is a novel expression of sperm morphology and a powerful
predictor of the fertility potential of a semen sample both in vivo
and in vitro (9).
The association of c-PARP with sperm morphology and the SDI
scores has not been reported. The aim of our study was to evaluate
the relationship between the SDI scores and different sperm mor-
phological abnormalities with levels of cPARP in the ejaculated
Semen samples were obtained from 10 healthy men at the An-
drology Laboratory of the Cleveland Clinic after 48–72 hours of
sexual abstinence. After liquefaction, semen was loaded onto
a 40% and 80% discontinuous density gradient, 2 mL each (Sage
BioPharma) and centrifuged at 1,600 rpm (400 g) for 20 minutes
at room temperature (10). Immature sperm obtained from the
40%–80% fraction and mature sperm from the 80% fraction
were used for further investigations. A multiple entry scoring tech-
nique was adopted for calculating SDI scores (7). Abnormal sper-
matozoa were classiﬁed more than once if more than one deformity
Nabil Aziz, M.D.
Rakesh K. Sharma, Ph.D.
Reda Mahfouz, M.D.
Rajesh Jha, Ph.D.
Ashok Agarwal, Ph.D.
Liverpool Women’s Hospital, University of Liverpool, United
Center for Reproductive Medicine, Glickman Urological and
Kidney Institute, Cleveland Clinic, Cleveland, Ohio
Received February 1, 2011; revised March 11, 2011; accepted
March 26, 2011; published online April 20, 2011.
N.A. has nothing to disclose. R.K.S. has nothing to disclose. R.M. has
nothing to disclose. R.J. has nothing to disclose. A.A. has nothing to
Reprint requests: Ashok Agarwal, Ph.D., Director, Center for Reproduc-
tive Medicine, 9500 Euclid Avenue, Desk A19.1, Cleveland, Ohio
44195 (E-mail: Agarwaa@ccf.org).
0015-0282/$36.00 Fertility and Sterility
Vol. 95, No. 8, June 30, 2011 2481
doi:10.1016/j.fertnstert.2011.03.095 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
was observed. The SDI was calculated by dividing the total number
of deformities observed by the number of sperm randomly selected
and evaluated, irrespective of their morphological normality.
In vitro sperm DNA damage was induced using  staurosporine
(10 mM), incubation at 37C for 4 hours (10) and  hydrogen per-
, 100 mM) incubation at 37C for 1 hours (10). PARP
inhibition was achieved using 3-aminobenzamide (3-ABA, 1 mM).
Sperm DNA fragmentation was evaluated using the terminal de-
oxynucleotidyl transferase-mediated ﬂuorescein-dUTP nick end
labeling (TUNEL) assay kit (Apo-Direct; Pharmingen) by ﬂow
cytometric analysis (10, 11).
Assessment of caspase-3 activity was performed using the
PE-conjugated monoclonal active caspase-3 antibody apoptosis
kit (BD Bioscience Pharmingen) as established earlier (10).To
perform this assay, the Annexin-V FITC Apoptosis Detection
Kit was used (Pharmingen) (10).
Assessment of cleavage activity was performed using FITC-
conjugated anti-PARP cleavage site-speciﬁc antibody by apoptosis
detection kit (Biosource International Inc.) as established
All ﬂuorescence signals of labeled spermatozoa were analyzed
by the ﬂow cytometer FACScan (Becton Dickinson). A total of
10,000 spermatozoa were examined for each assay at a ﬂow rate
of <100 cells/second. Both the percentage of positive cells and
the mean ﬂuorescence was calculated on a 1,023-channel scale
using the ﬂow cytometer software FlowJo version 6.2.4 (FlowJo,
LLC) (10, 11).
Data are represented as mean SD. Student’s t-test was used to
compare the difference in the percentages of various sperm mor-
phological abnormalities in the mature and immature fractions.
The Pearson correlation test was used to correlate different
covariates. All statistical analysis was done with GraphPad Prism,
version 5.00 for Windows (GraphPad Software). P<.05 was
considered statistically signiﬁcant.
The mature sperm fractions had signiﬁcantly lower mean (SD)
SDI score and signiﬁcantly higher mean percentage sperm with
normal morphology compared with the immature fractions
(Table 1). Positive correlation was seen between the percentage
of sperm with acrosome defects or midpiece defects and c-PARP
when apoptosis was induced with staurosporine in the presence
of PARP inhibitor and when apoptosis was induced by exposure
in the presence or absence of PARP inhibitor. There
was a positive correlation between H
-induced early apoptosis
and the percentage of sperm with amorphous head and sperm
with midpiece deformities (Table 1).
Induced capsase activity was negatively correlated with the per-
centage of sperm with cytoplasmic droplet and sperm with tail
defects, both in the presence and absence of PARP inhibitor. The
proportion of sperm with induced late apoptosis (PI marker) corre-
lated negatively with the percentage sperm with normal morphol-
ogy in the mature and immature fractions and positively with SDI
scores, tail defects, and cytoplasmic droplet in mature fraction and
amorphous head and acrosomal defects in the immature fraction.
The percentage of TUNEL-positive sperm correlated positively
with the percentage of sperm with small heads in the mature frac-
tion before (control) and after induction of oxidative stress. When
the data were considered collectively, a signiﬁcant positive interde-
pendence was seen between the SDI scores and cPARP-positive
sperm in the presence of PARP inhibitor (r ¼0.5; P¼.039). On
the other hand, there was no correlation between the percentage
of sperm with normal morphology and cPARP-positive sperm in
the presence of PARP inhibitor (r ¼0.02, P¼.95).
In this paper, we examined the relationship between sperm
morphology and intracellular levels of cPARP. We correlated dif-
ferent sperm morphological defects with the sperm susceptibility
to apoptotic and oxidative stimuli. We previously reported the im-
pact of apoptotic and oxidative stimuli on mature and immature
sperm populations (10). In the absence of PARP inhibition, expo-
sure to staurosporine resulted in a large increase in the percentage
of early apoptotic sperm. This increase in early apoptotic sperm
with staurosporine declined after PARP inhibition. Furthermore,
we reported a greater than twofold increase in the percentage of
late apoptotic sperm after PARP inhibition in staurosporine-
induced sperm injury. The increase in late apoptotic sperm with
chemical exposure and PARP inhibition was more apparent in im-
mature than in mature sperm fractions, suggesting that mature
spermatozoa are more protected from chemical-induced damage.
In this study, normal morphology correlated negatively with the
late apoptotic marker after oxidative stress both in the presence or
absence of PARP inhibitor. This supports our previous ﬁnding that
oxidative stress–induced sperm damage may have different path-
way(s), such as peroxidation of lipids and proteins, in addition
to DNA damage. The positive correlation of cPARP with percent-
ages of sperm with acrosome defects, midpiece damage, and tail
abnormalities may be attributed to the localization of this enzyme
in the head and midpiece area. When the data were considered
collectively (immature þmature fraction), a signiﬁcant positive
interdependence was seen between SDI scores and cPARP in the
presence of PARP inhibitor.
Previously we reported that immature spermatozoa exhibited
more resistance to oxidative stress–induced damage compared
with mature sperm (10). This ﬁnding is supported by our current
data that the percentage of sperm with small heads in the mature
fraction correlates positively with DNA damage after inducing
oxidative stress. Such a positive relationship was not observed
with any morphological features in the immature fraction. On
the contrary, in the immature fraction, DNA damage (fragmenta-
tion) under similar conditions correlated negatively with different
sperm structural/morphological abnormalities and with SDI
scores. This may be attributed to the fact that the mean percentage
of TUNEL-positive sperm in the immature fraction was reported
to be higher compared with the mature fraction under control
In conclusion, SDI scores are associated with PARP cleavage as
an early marker of apoptosis. Normal sperm morphology corre-
lates negatively with the late apoptotic marker after oxidative
stress both in the presence and absence of PARP inhibitor. The
relationship between acrosomal and midpiece defects appears to
be inﬂuenced by the localization of PARP in these regions in the
2482 Aziz et al. Sperm morphology and cleaved PARP inhibition Vol. 95, No. 8, June 30, 2011
Correlation of morphological features of sperm in the mature and immature fractions and markers of sperm damage using Pearson correlation test.
feature (%) Mature sperm
sperm 95% CI Pvalue
Normal morphology 22.6 11.5 4.4 3.0 11.6 to 24.8 <.0001 hPI 0.646 .044 hPI 0.792(*) .019
haPI 0.786(*) .021
Small head 0.3 0.7 2.6 5.8 5.81 to 1.1 .165 cTUNEL 0.934 <.0001 haAnnexin 0.849(*) .008
hTUNEL 0.683 .021
Amorphous head 50 10.0 66.4 11.0 22.6 to 9.6 <.0001 hAnnexin V 0.801 .005 hPI 0.806(*) .016
haPI 0.816(*) .013
Damaged acrosome 33.5 6.0 46.5 8.3 16 to 9.9 <.0001 hPI 0.732 .016 saPI 0.757 .03
saPARPFacs 0.737 .037
hPARPFACs 0.768 .026
Midpiece defects 12.3 6.3 18.2 13.6 15.6 to 3.7 .2085 hAnnexin 0.642 .045 saPARPFacs 0.73 .04
hPARPFACs 0.78 .023
haPARPFACs 0.75 .033
Cytoplasmic droplet 1.5 2.3 7.6 4.6 8.58 to 3.84 .0001 cPI 0.829 .003 hC3 0.75 .032
hTUNEL 0.67 .034
Tail defects 4 2.8 8 7.9 8.63 to 0.63 .0847 cPI 0.879 .001 sPARPFACs 0.78 .022
sTUNEL 0.660 .038 saC3 0.80 .016
haC3 0.72 .045
Sperm deformity index 1.53 0.12 1.9 0.16 0.45 to 0.3 .0001 sPI 0.691 .027 hTUNEL 0.73 .017
saAnnexin 0.793 .006
hPI 0.764 .01
Note: Values are mean SD. P<.05 was considered signiﬁcant by the two-tailed test. haAnnexin ¼percentage of annexin-positive sperm after treatment with H
and antagonist; hAnnexin ¼percentage of
annexin-positive sperm after treatment with H
; hC3 ¼percentage of active caspase-3-positive sperm after treatment with H
; haC3 ¼percentage of active caspase-3-positive sperm after treatment
and antagonist; saAnnexin ¼percentage of annexin-positive sperm after treatment with staurosporine and antagonist; saC3 ¼percentage of active caspase-3-positive sperm after treatment with
staurosporine and antagonist; cPI ¼percentage of propidium iodide–positive sperm in control aliquots; hPI ¼percentage of propidium iodide–positive sperm after treatment with H
; haPI ¼percentage of
propidium iodide–positive sperm after treatment with H
and antagonist; sPI ¼percentage of propidium iodide–positive sperm after treatment with staurosporine; hPARPFACs ¼percentage of sperm with
PARP fragment after treatment with H
; haPARPFACs ¼percentage of sperm with PARP fragment after treatment with H
and antagonist; sPARPFacs ¼percentage of sperm with PARP fragment after
treatment with staurosporine; saPARPFacs ¼percentage of sperm with PARP fragment after treatment with staurosporine and antagonist; cTUNEL ¼percentage of DNA damage in control sperm aliquots
using TUNEL assay; hTUNEL ¼percentage of DNA damage after exposure to H
using TUNEL assay; sTUNEL ¼percentage of DNA damage after exposure to staurosporine using TUNEL assay.
Aziz. Sperm morphology and cleaved PARP inhibition. Fertil Steril 2011.
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