ARTICLE IN PRESS
REPRODUCTIVE BIOLOGY
Determination of Poly (ADP-ribose) polymerase
(PARP) homologues in human ejaculated sperm
and its correlation with sperm maturation
Rajesh Jha, Ph.D.,a Ashok Agarwal, Ph.D., HCLD,a Reda Mahfouz, M.D.,a Uwe Paasch, M.D.,b
Sonja Grunewald, M.D.,b Edmund Sabanegh, M.D.,a Satya P. Yadav, Ph.D.,c
and Rakesh Sharma, Ph.D.a
a
Reproductive Research Center, Glickman Urological and Kidney Institute, and c Molecular Biotechnology Laboratory, Lerner
Research Institute, Cleveland Clinic, Cleveland, Ohio; and b Department of Dermatology/Andrology Unit, University of Leipzig,
Leipzig, Germany
Objectives: To investigate the presence of Poly (ADP-ribose) polymerase (PARP) and evaluate its function in
ejaculated spermatozoa.
Design: Experimental study.
Setting: Reproductive Research Center.
Patient(s): Semen specimens from 18 healthy donors and 12 infertile males.
Intervention(s): Ejaculated spermatozoa were subjected to sperm fractionation with a double-layer density gradient, protein extraction, detection, immunoblotting, and mass spectrometry. Semen samples were exposed to PARP
inducer staurosporine, or hydrogen peroxide with or without PARP inhibitor 3-aminobenzimide. Annexin V assay
was examined for apoptosis by flow cytometry.
Result(s): We detected 75 kDa, 63 kDa, and 60 kDa PARP homologues on the immunoblot of mature and
immature sperm fraction isolated from human ejaculate, which were identified as PARP-1 (75 kDa), PARP-9
(63 kDa), and PARP-2 (60 kDa), respectively. Western blot analysis showed a positive correlation between
the amount of PARP protein and sperm maturity. PARP proteins (75 kDa, 63 kDa, and 60 kDa) were evaluated after
inducing apoptosis by hydrogen peroxide and staurosporine exposure.
Conclusion(s): PARP-2 may have a role in prevention of oxygen species/oxidative stress and chemical (staurosporine)-induced sperm cell apoptosis. The presence of the PARP homologue, that is, PARP-1 (75 kDa), suggests
its role in preventing damage of mature sperm. Additional studies are needed to delineate the role of PARP-9 in
sperm physiology. The results from our study indicate an active role for PARPs in sperm cell physiology in preventing apoptosis. (Fertil Steril 2008;-:-–-. 2008 by American Society for Reproductive Medicine.)
Key Words: Sperm, DNA damage, PARP homologues, apoptosis, sperm maturity
The maintenance of genomic integrity is very important to
prevent cancer and other genetic diseases (1). Eukaryotic
organisms protect DNA lesions by numerous mechanisms.
During moderate DNA damage, cell cycle check point proteins such as p53 are activated, and assist in DNA repair
mechanism through a roentgenogram crosscomplementing
factor and other single strand breaks repair/base excision repair factors (DNA polymerase b and DNA ligase III) (2, 3).
However, high doses of DNA damage result in a passive
and unorganized mode of cell death known as necrosis (4, 5).
Received October 26, 2007; revised and accepted December 31, 2007.
R.J. has nothing to disclose. A.A. has nothing to disclose. R.M. has nothing to disclose. U.P. has nothing to disclose. S.G has nothing to disclose. E.S. has nothing to disclose. S.P.Y. has nothing to disclose.
R.S. has nothing to disclose.
Presented at the 63rd Annual meeting of the American Society of Reproductive Medicine, Washington, DC, October 13–17, 2007.
Supported by a Research Grant from the Glickman Urological and Kidney
Institute and Research Programs Committee, Cleveland Clinic, Cleveland, OH.
Reprint requests: Ashok Agarwal, Ph.D., HCLD, Glickman Urological &
Kidney Institute, and Departments of Obstetrics/Gynecology, Cleveland
Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, Ohio 44195 (FAX:
216-636-3118/216-445-6049; E-mail: [email protected]; Web site:
http://www.clevelandclinic.org/reproductiveresearchcenter).
Mono or Poly (ADP-ribosyl)ation is an immediate DNA
damage-dependent posttranslational modification of histones
and other nuclear proteins. This contributes to the survival of
injured proliferating cells [reviewed in (6)]. This modification of Poly (ADP-ribosyl)ation is accomplished by Poly
(ADP-ribose) polymerases (PARP) that help in identifying
region of DNA breaks for recruitment of the DNA base excision repair mechanism. It is also essential for the maintenance of genomic integrity and survival in response to
genotoxic insults (7–10). During excessive DNA damage
and in the presence of sufficient energy supply this enzyme
is cleaved into 24-kDa and 84-kDa domains preventing
0015-0282/08/$34.00
doi:10.1016/j.fertnstert.2007.12.079
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Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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DNA repair, thereby marking apoptosis (11–13). If a damaged
cell lacks energy, PARP is cleaved into 60-kDa fragments
along with ongoing necrosis.
Poly (ADP-ribose) polymerases now constitute a large family
of 18 proteins [reviewed in (6)]. Poly (ADP-ribose) metabolism
is critical in a wide range of biologic structures and processes,
including DNA repair and maintenance of genomic stability,
transcriptional regulation, centromere function and mitotic spindle formation, centrosomal function, structure and function of
vault particles, telomere dynamics, trafficking of endosomal vesicles, apoptosis, and necrosis [reviewed in (6, 14)].
Sperm DNA damage plays an important role in the pathogenesis of male infertility (15–17). Failure of fertilization in
assisted reproduction technology, despite the use of morphologically normal and motile sperm, may be because of such
damage (15–17). Apoptosis or programmed cell death involves a series of cellular, morphologic, and biochemical
alterations, leading to cell suicide without eliciting an inflammatory response. The presence of a key apoptotic protein,
Fas, on mature spermatozoa (18) has been previously detected, but it does not seem to be functional (19, 20). Other
investigators have shown the presence and externalization
of phosphatidylserine residues (21) and caspase activation
(22). PARP cleavage has been reported as an apoptosis or
necrosis marker in other cell types depending on the type
of cleavage (23). This molecule has also been reported in
the testicular germ line maintenance and its development
(24–26). However, its presence on ejaculated sperm is unclear. Although two reports showed its presence (27, 28) another documents its absence (29). In male infertility and
pathophysiology, apoptotic sperm represent poor sperm quality, and this may be attributed to altered functions of normal
apoptosis or oxidative stress (18, 26, 27, 29).
40% to 80% gradient comprising the immature sperm was
carefully aspirated into a tube, washed, and resuspended in
sperm wash medium. This fraction consisted largely of immature, morphologically abnormal sperm with poor motility.
Similarly, the pellet at the bottom of the 80% gradient was
carefully aspirated into another tube, washed, and resuspended in sperm wash medium. This fraction consisted of
morphologically normal, highly motile mature sperm.
Both mature sperm fraction and immature sperm fraction
were washed with Quinn’s sperm wash media (SAGE In vitro
Fertilization Inc.), and final washing was done with phosphate-bufferd saline (PBS; pH 7.4, 10 mM). The experimental
design is shown in Figure 1.
Sperm Protein Extraction
Sperm proteins were extracted according to the method of
Shukla et al. (33). Briefly, postwashed sperm cells pellets
were solubilized in sperm solublization buffer comprising
of 187mM Tris HCl, pH 6.8, 2% SDS, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, and 0.01% Aprotinin.
Sperm suspension was homogenized using a mechanical
homogenizer (Fisher Scientific, Hanover Park, IL) using
three strokes at 10-second intervals. The homogenate was
centrifuged at 12, 000 g for 20 minutes, and the resultant
supernatant was used as a sperm protein extract. All these
procedures were performed at 4 C.
FIGURE 1
Schematic representation of the experimental study
design.
The objectives of our study were to investigate the presence of PARP protein and its homologues in ejaculated
human sperm and to determine its relation to sperm maturity.
We also assessed PARP protein levels through Western blotting after PARP modulation/sperm cell apoptosis induction.
MATERIALS AND METHODS
Sperm Sample Preparation
Following the approval of the institutional review board, semen specimens from 18 healthy donors and 12 infertile males
were collected for this study. Healthy donors were defined as
a group of healthy male volunteers with sperm parameters in
the normal range (normozoospermic) according to the World
Health Organization guidelines (30). These included men
with both proven and unproven fertility. Infertile males
were defined as men who were attending the male infertility
clinic with a history of infertility of at least 1 year.
Sperm count and motility was done before and after double-density centrifugation by a double-density gradient
method (80% and 40% gradient; SAGE In vitro Fertilization
Inc., San Clemente, CA) (31, 32). The interphase between the
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Jha et al.
PARP homologues in human spermatozoa
Jha. PARP homologues in human spermatozoa. Fertil Steril 2008.
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PARP Immunoblotting
Protein concentrations were estimated by BCA Protein Assay
Kit (Pierce Biotechnology, Inc., Rockford, IL). Proteins were
denatured with Laemmli buffer (Bio-Rad Laboratories, Herculus CA) (34) and subsequently heated for 5 minutes at
95 C. Equal concentration of protein (50 mg) was loaded
onto each well and separated on a 12% SDS-PAGE for the protein profile verification. SDS-PAGE resolved proteins were
visualized with 0.1% Coomassie (Brilliant Blue R-250;
Sigma St. Louis, MO) stained in 10% acetic acid and 40%
methanol. Excess Coomassie stain was removed by additional
wash with 10% acetic acid and 40% methanol. Proteins were
resolved on SDS-PAGE then electroblotted onto nitrocellulose membrane (Bio-Rad) in the presence of 25 mM Tris
base, 192 mM glycine, and 20%v/v methanol (34, 35). The
blotted membranes were incubated with 5% nonfat milk followed by anti-PARP-1 (Santa Cruz, Santa Cruz, CA; cat no.
sc-1561) and rabbit antigoat horseradish peroxidase (HRP)
conjugated (sc-2768) (1:1,000) dilutions.
Incubation with primary antibody and blocker (5% nonfat
milk) was done for 2 hours, whereas incubation with secondary
antibody was for 1 hour. Phosphate-buffered saline (10 mM,
pH 7.4 containing 0.1% Tween-20; PBS-T) was used throughout the procedure. Protein bands were visualized by diaminobenzidene tetrahydrochloride reaction (Vector Laboratories,
Burlingame, CA). Secondary antibody specificity was verified
by incubating with secondary antibody only. PARP immunopositive bands were quantified by Totallan 100 software (Nonlinear Dynamics Ltd, Newcastle upon Tyne, UK).
PARP Protein Identification by Peptide Mass
Fingerprinting
Protein extract from mature sperm from rwo donors and one
infertile patient was used for this experiment. Proteins were
resolved on 12% SDS-PAGE; three lanes were used for immunoblotting and other three lanes were stained with coomassie (Brillant blue R-250). The three Western blotted lanes
were probed with PARP-1 antibody to detect the PARP-1-immunopositive band. The corresponding PARP-1-immunopositive bands in the SDS-PAGE were excised and digested by
Trypsin using Trypsin In-Gel Digest Kit (Cat. No. PP 0100,
Sigma). The reaction was performed for 12 hours, following
which the tryptic peptides were detected by MALDI-Tof-Tof
(Applied Biosystem, Foster City, CA) at the Cleveland Clinic
Lerner Research Institute core facility. Bioinformatics analyses were performed through ExPASy Proteomics Server (Protein Prospector; MS-Fit) to identify the corresponding protein
based on the peptide mass fingerprinting.
Immunolocalization of PARP on the Ejaculated Human
Sperm
Immunolocalization of PARP was done on donors and infertile mature and immature sperm fractions according to the
method of Shukla et al. (33). Briefly, spermatozoa were
smeared onto clean glass coverslips precoated with 0.05%
Fertility and Sterility
poly-L-lysine. These smears were allowed to dry at room
temperature. The coverslips were treated with 10% formalin
for 10 minutes to fix the sperm, followed by neutralization in
0.5 M ammonium chloride for another 10 minutes. Sperm
were permeabilized by 0.25% Triton X-100 for 20 minutes,
followed by chilled ethanol for 10 seconds. Blocking was
done with 5% bovine serum albumin, followed by incubation
with 1:300 (vol./vol.) dilution of the anti-PARP-1 (goat antiPARP-1, Santa Cruz; cat no. sc-1561). After washing, coverslips with sperm cells adsorbed were incubated with 1:300
(vol. by vol.) dilution of rabbit antigoat IgG antibody conjugated with fluorescein isothiocyanate (Sigma; Cat no. F
2016) for 1 hour at room temperature. Phosphate-buffered saline with Tween 20 (10 mM, pH 7.4) was used throughout the
procedure. Following each incubation, washing was repeated
thrice with PBS-T at 5-minute intervals. The sperm cells were
counter stained in DAPI (4’, 6-diamidino-2-phenylindole)
(Vector mounting media with DAPI, Vector Laboratories,
Burlingame, CA). The cells were imaged on an upright fluorescence microscope with an exposure time of 1 second (Leica Microsystems Inc, Bannockburn, IL). Negative controls
were prepared by excluding the incubation of the cells in
anti-PARP-1. These experiments were repeated five times,
and representative images were obtained.
PARP Modulation Assay/PARP Induction by Apoptosis
Semen samples were collected from eight donors for this
study. Both mature and immature sperm fractions were separated using the double-density gradient centrifugation
method as described above. PARP activation/apoptosis was
induced by an oxidative stress inducer hydrogen peroxide
(H2O2; 30 mM) and apoptosis by staurosporine (2.5 mM).
PARP activity was inhibited by inhibitor 3-amino-benzamide
(0.3 mM). Each mature and immature sperm fraction was resuspended in HTF media and divided into five aliquots/group,
that is, control/untreated; H2O2 treated; H2O2 þ 3-aminobenzamide; staurosporine alone; and staurosporine þ 3-aminobenzamide. All aliquots were incubated at 37 C for 1 hour.
After incubation, mature and immature sperm aliquots were
processed for gel electrophoresis and Western blotting as
mentioned above. PARP protein bands on immunoblot were
analyzed by Tooltab TL100 (Nonlinear Dynamics).
Sperm Apoptosis Analysis by Annexin V Assay
Annexin V- FITC (BD Biosciences, San Jose, CA; Cat. No.
51-65874X) was used for assessment of sperm apoptosis.
Sperm were washed with cold PBS and then resuspended in
1 annexin V binding buffer (10 mM HEPES/NaOH
(pH7.4), 140 mM NaCl, 2.5 mM CaCl2). To get the concentration 1.0 106 sperm/mL, 2.5 mL Annexin þ 2.5 mL Propidium iodide were added. All test and control tubes were
gently mixed and incubated for 15 minutes at 25 C in the
dark then 400 mL of PBS was added into each tube. After incubation samples were analyzed by flow cytometry (FACScan, BD, San Jose, CA) at a low flow rate using Cellquest
software for about 10,000, and FACs data files were analyzed
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FIGURE 2
Detection of PARP proteins 75 kDa, 63 kDa, and 60 kDa, employing Western blot and their densitometric analysis
for evaluating the protein level in donors and infertile men between (A) immunoblot of mature and immature
sperm fraction; (B) 75 kDa band intensity in mature and immature sperm fraction; (C) 60 and 63 kDa band
intensity in mature and immature sperm fraction; and (D) validation of primary antibody specificity by
immunoblotting with secondary antibody alone.
Jha. PARP homologues in human spermatozoa. Fertil Steril 2008.
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TABLE 1
Analysis of the bands by peptide mass fingerprinting identified these as PARP-1, PARP-9, and PARP-2
cleaved fragment, respectively.
Observed
bands on the
immunoblot
(kDa)
Query
match/percentage
75
31/40 (77%)
human
63
24/43 (55%)
human
60
13/23 (56%)
human
Name of the protein
(PARP homolog)
Gene
Accession #
MW (Da)
Poly [ADP-ribose]
polymerase 1 (PARP-1)
(ADPRT) (NAD(þ) ADPribosyl transferase 1)
Poly [ADP-ribose]
polymerase 9 (PARP-9)
Tankyrase-2 (TANK2)
(TRF1-interacting ankyrinrelated ADP-ribose
polymerase 2) (PARP-2)
P09874
112,954
Q8IXQ6
96,344
Q9H2K2
126,919
Species
Jha. PARP homologues in human spermatozoa. Fertil Steril 2008.
using FlowJo software version 6.4.2 at the flow core facility
of the Lerner Research Institute, Cleveland Clinic.
Statistical Analysis
Results are presented as the mean standard error as well as
median and interquartile ranges for sperm count and motility.
A Student’s t test was used for comparison of two data sets.
Significance was defined as P<.05. This statistical analysis
was performed using Microsoft Excel software.
RESULTS
The median (25th and 75th percentile) for sperm parameters
in unprocessed liquefied seminal ejaculates used in these experiments were: donors, concentration (106/mL) 49 (37.2,
97.7); percent motility, 59.3 (47.8, 70.6); and range of round
cell count was 0.01–2 106/mL. For patients, concentration
(106/mL) 28.1 (17.9, 52.9); percent motility: 57.2 (51.2,
62.8); and range of round cell count was 1.0–5.0 106/mL.
Postwash sperm parameter of mature sperm fractions were:
donors, concentration (106/mL) 56.0 (39.7, 99.2); percent motility: 75.0 (66.0, 84.0); patients, concentration (106/mL): 34.2
(17.0, 58.7); and percent motility: 61.0 (45.7, 72.1). Immature
sperm fraction parameters were: donors, sperm concentration
(106/mL) 27.0 (19.9, 39.7); and percent motility, 33.3 (24.2,
43.9); patients, sperm concentration (106/mL) 14.3 (7.4,
29.7) and percent motility, 43.1 (28.7, 49.7).
Detection of PARP Protein on the Human Ejaculated Sperm
Anti-PARP-1 detected 75-kDa, 63-kDa, and 60-kDa protein bands on the immunoblot in mature and immature sperm
(Fig. 2A). In mature sperm of all investigated samples, the
75-kDa (P<.017) as well as the 63-kDa and 60-kDa
(P<.04) PARP homologues exhibited elevated protein levels
in donors when compared with infertile males (Fig. 2B–C). In
contrast, investigation of the immature sperm fraction did not
Fertility and Sterility
show any significant differences in the 75-kDa, 63-kDa, and
60-kDa protein levels in donors and infertile males (Fig. 2B–
C). Primary antibody specificity was validated by immunoblotting of the secondary antibody alone (negative control) (Fig. 2D).
Sperm PARP-1 Homologue Identification
The above-mentioned PARP-1 immunopositive bands (75
kDa, 63 kDa, and 60 kDa) were subjected to peptide mass finger printing and mass spectroscopy for their identification.
Peptide mass finger printing analysis identified these to be
PARP-1, PARP-9, and PARP-2, respectively (see Table 1).
PARP Immunolocalization on the Sperm
Immunolocalization of PARP on mature sperm from donors
showed intense localization on the head region, particularly
near the acrosomal region (Fig. 3A). In the immature sperm
fraction it was abnormally distributed and present as a distinct
band at the equatorial region of the sperm head (Fig. 3B). The
presence of PARP on the mature and immature sperm fraction of infertile patients exhibited a similar distribution as
seen in mature and immature sperm fraction from donors,
but the intensity of fluorescence was relatively weak (Fig.
3C and D). Specificity of PARP-1 was confirmed by labeling
with secondary antibody alone (Fig. 3E).
PARP Modulation Assay
PARP-1 antibody showed 75-kDa, 63-kDa, and 63-kDa
PARP-immunopositive bands on the blot of donors and infertile mature as well as in immature sperm fractions (Fig. 4A
and B). In mature sperm, incubation with H2O2 alone resulted
in a diminished 75-kDa PARP homologue, which showed further depletion when 3-aminobenzamide was added (P<.045)
(Fig. 4A and C). The 75-kDa PARP of the immature sperm
fraction was not affected by H2O2 at all (Fig. 4B), but the
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FIGURE 3
Immunolocalization study of PARP-1 on (A) mature and (B) immature donor sperm, and (C) mature and (D)
immature sperm from infertile patients. Crossreactivity was tested by incubating with secondary antibody alone
(E).
Jha. PARP homologues in human spermatozoa. Fertil Steril 2008.
60-kDa PARP homologue (PARP-2) was not detectable after
incubation with H2O2 in both the absence and the presence of
3-aminobenzamide in mature and immature sperm (Fig. 4A,
B, and E).
tion with staurosporine and hydrogen peroxide (both with
and without 3-aminobenzamide; Fig. 4E). Interestingly, the
63-kDa PARP homologue exhibited no significant changes
in all the groups studied (Fig. 4D).
In contrast, incubation with staurosporine induced no
changes in the 75-kDa PARP homologue in mature sperm
but led to a reduction of this homologue in immature sperm
(Fig. 4C), particularly when 3-aminobenzamide/PARP inhibitor was added (P<.001). In mature sperm, the 60-kDa PARP
homologue band intensity was slightly reduced by treatment
with staurosporine, whereas 3-aminobenzamide þ staurosporine depleted further the levels of the 60-kDa protein
(P<.004) (Fig. 4E). The immature sperm fraction showed
a complete loss of the 60-kDa PARP homologue after incuba-
Sperm Cell Apoptosis Analysis by Annexin V Assay
Treatment with H2O2 significantly elevated the portion of annexin V positive sperm (P<.003) regardless of the combination with 3-aminobenzamide in mature and immature sperm.
Treatment with staurosporine did not significantly alter the
amount of annexin V positive sperm, but addition of 3aminobenzamide significantly increased annexin V staining
(P<.0005; Fig. 4F). This was true for both mature and immature sperm.
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FIGURE 4
Analysis of PARP proteins by Western blotting after induction of apoptosis by H2O2 and staurosporine in (A)
mature and (B) immature sperm fraction from donors and infertile patients. Quantitative PARP protein level are
represented in graphs (C) 75 kDa, (D) 63 kDa, and (E) 60 kDa. (F) Apoptosis was measured by externalization
of phosphatidylserine/Annexin V staining.
Jha. PARP homologues in human spermatozoa. Fertil Steril 2008.
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DISCUSSION
PARP has been involved in various sperm cell functions such as
gamete differentiation (during spermiogenesis) (36), spermatogenesis (37), and chromatin remodeling (38). PARP has
also been reported in the microsomal–ribosomal fraction along
with its nuclear presence (39). Reports on the presence of PARP
in ejaculated spermatozoa have been controversial. Our present
study puts to rest the controversial debate on the presence or absence of PARP on human ejaculated sperm (27–29). In this
study we have shown the presence of PARP-1 (75 kDa)
and its homologues PARP-9 (63 kDa) and PARP-2 (60
kDa) in both mature and immature sperm. PARP-1 was not detected as expected at 112 kDa; instead, the presence of 75 kDa,
63 kDa, and 60 kDa raised the question of its presence. We
therefore investigated and confirmed PARP-1 immunopositive
proteins by peptide mass fingerprinting analysis. These were
confirmed as PARP-1, PARP-2, and PARP-9.
The presence of low PARP levels in mature sperm of infertile
patients compared with the mature sperm of donors suggests
a role of PARP and its homologues in male infertility. This also
corresponded to the weaker fluorescence intensity seen in sperm
from subfertile males. Furthermore, low levels of PARP-1, -2, and
-9 seen in the immature sperm fractions of both donors and infertile males compared with the mature fractions indicates a potential
role of these homologues during sperm maturation.
PARP homologues have a variety of cellular functions;
among them, DNA damage detection and repair are most
important (6, 36, 40). Particularly, PARP-1 and PARP-2 are
involved in DNA repair mechanisms. DNA fragmentation
is higher in infertility patients as well as in immature sperm
(6, 36, 40). It can be speculated that in such sperm the reduced
levels of PARP homologues might be responsible for the inefficiency to protect/repair posttesticular sperm lesions.
PARP was found to be present on the anterior portion of the
sperm head covering the acrosomal region in the mature fraction of donors and comparatively lower concentration of the
mature fraction of infertile patients. Their localization described abnormal distribution on the immature sperm fraction
of both the fertile and infertile patient. PARP-1 proteins exhibited restricted to the central head region in normal sperm
physiology, but expresses elsewhere than this position. The
presence of high PARP-2 protein levels in the donors shows
its involvement in genomic DNA integrity. Its involvement
in the DNA repair mechanism against genotoxic agents has
also been demonstrated (10, 41), and abnormalities in the
PARP-2 content may affect sperm maturity.
We next examined the effect of oxidative stress and apoptosis by exposing sperm fractions to an external oxidative
stress inducer, H2O2, and an apoptosis inducer, staurosporine.
We did not observe any change in the 63-kDa band, indicating that PARP-9 may not be significantly affected by oxidative stress and sperm apoptosis. In contrast, we observed
a moderate reduction in 75-kDa PARP-1 in mature sperm
in the presence of oxidative stress and after induction of apoptosis in the immature sperm, and this was exaggerated by
8
Jha et al.
PARP homologues in human spermatozoa
addition of the PARP-inhibitor. The 60-kDa PARP-2 was
not detectable under oxidative stress in both the mature and
immature sperm. In the immature fraction, PARP-2 was not
detectable following induction of apoptosis by staurosporine.
The strong modulation of PARP-2 suggests its involvement in
response to oxidative stress and especially to pro-apoptotic
stimuli in immature sperm.
We modulated PARPs function by inducing sperm apoptosis via H2O2 and staurosporine in the physiology of isolated
mature and immotile sperm fraction of the ejaculated sperm.
Evaluation of these protein functions explains the role of
PARP-2 and PARP-1 in providing a protective mechanism
against oxidative stress in normal fertile individuals. This
PARP-dependent mechanism is probably inefficient in infertile men; hence, the sperm are more susceptible to damage.
Earlier reports documented apoptosis via oxidative stress
(42) and chemically induced (43) through caspase-dependent
and -independent pathways. Our results showed prevention
of apoptosis in the presence of the PARP homologue,
whereas PARP inhibition by 3-aminobenzamide increased
apoptosis by twofold. Moreover, PARP-2 levels were also
influenced by apoptotic-inducing agents like hydrogen peroxide (36). Evaluation of the externalization of phosphatidylserine measured by annexin-V staining showed a significant
induction of apoptosis by H2O2. These oxidative stressinduced membrane changes were not preventable by PARP.
However, staurosporine-induced cell apoptosis can be protected by PARP proteins as the PARP inhibitor 3-aminobenzamide escalates phosphatidylserine externalization.
In conclusion, we have demonstrated that human ejaculated sperm contain PARP homologues, PARP-1 (75
kDa), PARP-2 (60 kDa), and PARP-9 (63 kDa). Lower
PARP levels negatively affect sperm maturity, and consequently, may affect male fertility. PARP-1 and PARP-2
may have a role in conferring protection to the ejaculated
sperm, particularly following exposure to oxidative stress
and apoptosis inductors.
Acknowledgements: We acknowledge the support of our Clinical Andrology
laboratory staff in enrolling the donors and patients for the study.
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