BIOLOGY OF REPRODUCTION 81, 46–55 (2009)
Published online before print 4 March 2009.
DOI 10.1095/biolreprod.108.075390
Disruption of Poly(ADP-Ribose) Homeostasis Affects Spermiogenesis and Sperm
Chromatin Integrity in Mice1
Mirella L. Meyer-Ficca,3 Julia Lonchar,3 Christine Credidio,3 Motomasa Ihara,3 Yun Li,3 Zhao-Qi Wang,4,5
and Ralph G. Meyer2,3
Department of Animal Biology and Mari Lowe Center for Comparative Oncology,3 University of Pennsylvania School of
Veterinary Medicine, New Bolton Center for Animal Transgenesis and Germ Cell Research,
Kennett Square, Pennsylvania
Genome Stability Group,4 Leibnitz Institute of Age Research-Fritz Lipmann Institute (FLI), Jena, Germany
Faculty of Biology and Pharmacy,5 Friedrich-Schiller-University, Jena, Germany
reduction of the fertilization potential in mature spermatozoa
[1]. Sperm DNA is normally shielded from potentially
compromising environmental effects by extremely tight
packaging into transcriptionally inactive chromatin associated
with protamines linked by covalent disulfite bonds. The
chromatin structure of sperm cells is therefore completely
different from that of somatic cells [2, 3].
The complex chromatin reorganization during spermiogenesis is dramatic, and the steps that lead to packaging of the
paternal genome into the compact transport form are not
entirely understood. Histones are first replaced by the transition
proteins TNP1 and TNP2, which are subsequently replaced by
protamines. Along with the exchange of proteins, the DNA
structure changes from the supercoiled nucleosomal structure
to a relaxed state [4–6]. This requires the occurrence of
controlled transient DNA strand breaks [7–9], particularly
during the exchange of histones by transition proteins in
elongating spermatids such as spermatids of steps 9–12 in mice
[10]. Such DNA strand breaks are apparently mostly afforded
by the activity of enzymes such as topoisomerase II b (TOP2B)
[11, 12] and are therefore presumably mostly protein bound.
However, at a certain frequency, open DNA double-strand
breaks appear to be also formed in this process, posing a threat
to the developing spermatid cell.
Compared with somatic cells, the presence of DNA strand
breaks carries a greater risk for haploid spermatid cells, as they
are not able to undergo homologous recombination, the favored
and most accurate way of repairing DNA double-strand breaks
in diploid cells. Cellular DNA damage repair is facilitated by
two members of the poly(ADP-ribose) polymerase family,
poly(ADP-ribose) polymerase 1 (PARP1) and poly(ADPribose) polymerase 2 (PARP2). DNA strand breaks directly
and immediately recruit the ubiquitous PARP1 and PARP2. On
activation, PARP1 forms a large branched biopolymer
poly(ADP-ribose) (PAR) by cleaving NAD þ into nicotinamide
and ADP-ribose, which becomes polymerized and covalently
attached to acceptor proteins such as PARP1 itself in an
intermolecular automodification reaction. Examples of other
target proteins for this posttranslational modification are
histones, p53, and DNA ligase III. Breakdown of the unique
polymer is facilitated by poly(ADP-ribose) glycohydrolase
(PARG), with PAR turnover being a rapid and dynamic
process mediated by the interplay of PARP1 and PARG [13–
18]. Automodified PARP1 is catalytically inactive and is
released from the DNA strand break. PARP2, another member
of the PARP protein family, has been shown to respond
similarly and as a backup. The ability of PARP1 and PARP2 to
bind to DNA strand breaks and to become activated again is
restored on removal of PAR by PARG, allowing for repeated
ABSTRACT
The major function of sperm is the delivery of the paternal
genome to the metaphase II oocyte, ensuring transmission of the
genetic information to the next generation. For successful
fertilization and healthy offspring, sperm DNA must be protected
from exogenous insults. This is achieved by packaging the sperm
DNA into a condensed protamine-bound form, preceded by the
precisely orchestrated removal of histones and intermittent
insertion and removal of transition proteins. This remodeling
process requires relaxation of supercoiled DNA by transient
formation of physiological strand breaks that spermatids, being
haploid, cannot repair by homologous recombination. In somatic
cells, the presence of DNA strand breaks rapidly induces the
formation of poly(ADP-ribose) by nuclear poly(ADP-ribose)
polymerases, which in turn facilitates DNA strand break
signaling and assembly of DNA repair complexes. We reported
earlier that chromatin remodeling steps during spermiogenesis
trigger poly(ADP-ribose) (PAR) formation. Here, we show that
knockout mice deficient in PARP1, PARG (110-kDa isoform), or
both display morphological and functional sperm abnormalities
that are dependent on the individual genotypes, including
residual DNA strand breaks associated with varying degrees of
subfertility. The data presented highlight the importance of PAR
metabolism, particularly PARG function, as a prerequisite of
proper sperm chromatin quality.
chromatin condensation, chromatin remodeling, condensation,
gametogenesis, PARG, PARP, PARP1, PARP2, poly(ADP-ribose),
poly(ADP-ribose) glycohydrolase, poly(ADP-ribose) polymerase,
residual DNA strand breaks, sonication-resistant spermatid nuclei,
sperm, spermatid, spermatogenesis, spermiogenesis
INTRODUCTION
In humans, incomplete chromatin condensation and the
presence of persistent DNA strand breaks indicate a severe
1
Supported by grants from the National Institutes of Health (NIH R01
HD048837 to R.G.M.) and the Mari Lowe Center for Comparative
Oncology at the University of Pennsylvania. Z.-Q.W. was supported by
Association pour la Recherche sur le Cancer (ARC) France.
2
Correspondence: Ralph G. Meyer, Department of Animal Biology and
Mari Lowe Center for Comparative Oncology, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square,
PA 19348. FAX: 610 925 8121; e-mail: meyerg@vet.upenn.edu
Received: 10 December 2008.
First decision: 13 January 2009.
Accepted: 4 February 2009.
Ó 2009 by the Society for the Study of Reproduction, Inc.
eISSN: 1259-7268 http://www.biolreprod.org
ISSN: 0006-3363
46
POLY(ADP-RIBOSE) METABOLISM AFFECTS SPERM CHROMATIN
cycles of PAR turnover until the DNA strand break is repaired.
Digestion of PAR by PARG is essential to cellular survival,
and a complete knockout of Parg leads to an early embryonic
lethal phenotype [19].
In humans, incomplete chromatin condensation and the
presence of persistent DNA strand breaks in mature spermatozoa indicate a severe reduction of the fertilization potential
[1]. Lack of sperm nuclear integrity often results from
incomplete execution of preceding chromatin remodeling steps
and from faulty DNA strand break management in early steps
of spermatid development. We showed previously that the
nuclear elongation phase of spermatid development involves
formation of PAR concomitantly with the presence of DNA
strand breaks [20].
In the present study, we tested the hypothesis that PAR
metabolism may be required for efficient DNA strand breakmediated chromatin reorganization during postmeiotic germ
cell maturation using Parp1/ and Parg(110)/ mouse
models, as well as a novel double gene-disrupted Parp1//
Parg(110)/ strain. Our studies revealed that mice with a
perturbation in PAR metabolism display sperm nuclear
abnormalities such as alterations in shaping of the nucleus
and in the degree and course of nuclear condensation, as well
as an increase in the amount of residual DNA strand breaks in
epididymal sperm.
47
lg/ml 4 0 ,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich). Fluorescence
microscopy was performed on an inverted fluorescence microscope (Nikon).
Random photographs were taken in the DAPI channel using identical exposure
conditions, and data were collected using automated and manual data collection
modes provided by Image-Pro plus 5.1 software (Media Cybernetics). Minor
and major axes, area, perimeter, and mean density of at least 750 sperm nuclei
per data set per mouse strain were measured using densitometric functions of
this software. First, a polygonic line following the exact perimeter of each
nucleus, including the hooked tip, was drawn automatically based on
standardized background correction and signal threshold parameters that were
kept constant throughout the experiments. Then, minor and major axes were
defined as the longest and shortest possible diagonal lines (Axismin and Axismaj,
respectively) through each marked nucleus. The elongation of nuclei was
computed as the quotient of minor axis / major axis (Axismin / Axismaj), where
Axismin / Axismaj ¼ 1 describes a perfectly round nucleus. Wild-type mouse
sperm were typically measured as having an elongation of ;0.5. To provide an
additional control, roundness of nuclei was also determined by dividing the
expected perimeter (Pexpected) of a nucleus with a given area by the actual
perimeter (Pactual). If Pexpected / Pactual ¼ 1, a nucleus would be perfectly round.
Typical mean values in wild-type mice were ;1.32. The mean density, which
describes the overall brightness of fluorescence of a given nucleus, was also
determined. The result of this measurement reflects the absolute value of optical
extinction over a defined area. The mean density was measured as an additional
estimate of nuclear compaction. Investigations were designed so that samples
from all mouse strains were collected, processed, and evaluated in parallel
using age-matched animals (as in all other investigations presented herein) to
minimize experimental error. Statistical data analyses were calculated using
Excel (Microsoft).
Immunoblot Analyses
MATERIALS AND METHODS
Mouse Strains and Tissue Harvesting
Parp1 gene-disrupted mice (Parp1tm1Zqw [Parp1/] [21]) and Parg genedisrupted mice (Pargtm1Zqw [Parg(110)/] [22]) were maintained according to
the guidelines of the University of Pennsylvania Institutional Animal Care and
Use Committee. Parg(110)/ mice carry a targeted deletion of exons 2 and 3
of the Parg gene, which leads to abrogation of the three large PARG protein
isoforms of 110 kDa, 102 kDa, and 98 kDa, but the two smaller ones of 63 kDa
(ubiquitous) and 58 kDa (mitochondrial) [23, 24] are still expressed. These
animals exhibit increased PAR formation with increased NADþ depletion after
genotoxic insults, but at the same time steady-state levels of the biopolymer are
altered, with an overall depression of cellular PARP1 function [25]. The novel
Parp1//Parg(110)/ double gene-disrupted mice were generated by
conventional crossbreeding of the Parg(110)/ with Parp1/ mice that were
originally in a 129/S1 background (The Jackson Laboratory). Parp1//
Parg(110)/ double-knockout mice were then crossbred with 129/S6/SvEvTac
mice (Taconics) to obtain Parp1/ and Parg(110)/ mice and wild-type
control animals with a comparable background. All animals used in the
investigations were routinely genotyped by PCR using primer sequences for
Parp1 published by Wang et al. [21] and according to The Jackson Laboratory
protocol and using primer sequences for Parg(110) published by Cortes et al.
[22]. Wherever possible, sibling wild-type control animals were used in the
experiments; in all other cases, 129/S6/SvEvTac (Taconics) mice were utilized
as appropriate controls. Experimental procedures were performed by simultaneously analyzing tissue from age-matched animals of all four genotypes.
Computer-Assisted Sperm Analyses
Computer-assisted sperm analyses (CASAs) were performed using an
automated system (Hamilton-Thorne Research Systems). For comparison of
mouse strains, siblings or age-matched (within 2–3 days) sets of 1–5 animals
among 60- to 120-day-old mice were simultaneously used in each experiment.
Cauda epididymes were quickly prepared from euthanized mice, and sperm were
collected by gently swirling sliced epididymes for 5 min in TYH medium (120
mM NaCl, 4.7 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25
mM NaHCO3, 5.5 mM glucose, 0.5 mM sodium pyruvate, and 4 mg/ml bovine
serum albumin [BSA]) (Sigma Aldrich). Measurements were repeated six times
per sample, and all experiments were performed at least in triplicates.
Morphometric Analyses of Sperm Nuclei
Morphometric analyses of cauda epididymal sperm were performed on airdried sperm smears that were fixed in methanol:glacial acetic acid (3:1) and
then embedded in Vectashield mounting medium (Vector Laboratories) with 1
Protein extracts used for SDS-PAGE were obtained by collecting testes
from several mice each per strain, where all mice of all strains used for a given
experiment were of the same age. For SDS protein extraction of whole testes,
decapsulated testes were weighed and then homogenized in an equal volume of
23 Laemmli sample buffer. Protein extracts from sonication-resistant nuclei for
SDS-PAGE were prepared by homogenization of four testes in 5 ml SMTTP
buffer (0.31 M sucrose, 5 mM MgCl2, 10 mM Tris/HCl [pH 7.5], 0.05% Triton
X-100, and 0.5 mM PMSF) in a tight-fitting 15-ml glass homogenizer. After
filtration through a 0.1-mm nylon mesh and centrifugation at 3000 3 g, pellets
were resuspended in 10 ml MTMP buffer (5 mM MgCl2, 10 mM Tris/HCl [pH
7.5], 0.2% beta-mercaptoethanol, and 0.5 mM PMSF) and centrifuged. Nuclei
were resuspended in 8 ml water and were sonicated twice for 1 min at
amplitude 100, with intermittent cooling on ice for 1 min using an ultrasonic
processor (Cole-Parmer). They were centrifuged, resuspended in 2 ml water,
layered over 2 ml SMP (5% sucrose, 5 mM MgCl2, and 10 mM Tris/HCl [pH
6.5]), and centrifuged at 740 3 g for 5 min as previously described [26]. After
resuspending the pellet in 2 ml water, this purification step was repeated, and
then nuclei resistant to ultrasonic shear were diluted and counted using a
hemacytometer. Samples were lysed in an appropriate volume of 23 Laemmli
sample buffer to give a final concentration of 6.7 3 104 nuclei/ll. Cauda
epididymes were obtained by necropsy, and sperm cells were allowed to swim
out of the sliced tissue in 0.75 ml/epididymis prewarmed PBS. Spermatozoa
were washed by centrifugation (700 3 g), resuspended in PBS, and then
pelleted and lysed in 23 SDS Laemmli sample buffer at a final concentration of
2 3 104 cells/ll. An equivalent of 2 3 105 cells/lane was separated by 8% or
10% SDS-PAGE, electrotransferred to polyvinylidene fluoride membranes
(Immobilon-P; Millipore), and subjected to antibody detection. Primary
antibodies used for immunoblot detection were rabbit anti-PARP1 (SA-253,
1:500; Biomol), rabbit anti-PAR (LP96–10, 1:2000; BD Bioscience), rabbit
anti-PARG (C-terminal antigen, 1:500) [23], rabbit anti-PARP2 (Yuc
unpurified, 1:2000; Alexis), mouse anti-phospho-H2AFX (1:500; Upstate),
mouse anti-H2AFX (1:1000; R&D Systems), and mouse anti-beta-actin
(1:5000; Sigma Aldrich). For Western blot detection, horseradish peroxidasecoupled donkey anti-rabbit, donkey anti-mouse, or donkey anti-goat secondary
antibodies (Jackson Immunolabs) were used and were detected in a standard
enhanced chemiluminescence reaction. Signals were quantified using ImageJ
software (Wayne Rasband, National Institutes of Health; http://rsb.info.nih.gov/
ij/). In general, quantification was performed by normalizing the signal
intensities observed for samples of different genotypes to the signal intensity
observed for the respective wild-type sample. In Figure 3, PAR signal
intensities were compared between wild-type and Parg(110)/ animals, as
well as between Parp1/ and Parp1//Parg(110)/ animals. This procedure
adjusts for the different composition in nuclear PARP enzymes in these animals
(being PARP1 and PARP2 in the first data set and only PARP2 in the second
data set) and allows for comparison of PAR accumulation that is dependent on
PARG activity only.
48
MEYER-FICCA ET AL.
TABLE 1. Fecundity of wild-type (Wt), Parp1/, Parg(110)/, and
Parp1//Parg(110)/ mice.
Parameter
Wt 129SVE
Parp1/
No. pups (litters) 572 (101)
419 (78)
Pups/litter 6 SD 5.7 6 2.14 5.4 6 1.95
t-test vs. Wt
NA*
P ¼ 0.34
Parp1//
Parg(110)/ Parg(110)/
287 (68)
4.2 6 2.1
P , 0.0001
328 (62)
5.3 6 2.9
P ¼ 0.35
* NA, not applicable.
FIG. 1. Reduction of sperm motility in Parp1/ mice. Cauda epididymal
spermatozoa from age-matched sets of animals were measured by CASA.
Motile sperm characterized as progressive were each recorded as
percentage of the motile population, and overall motility and progressiveness were expressed relative to the wild type (rel. to wt) in each
experiment. Data represent five independent experiments involving 3–5
animals each. Error bars represent the SEM of three independent
experiments. ** P , 0.001; wt, wild type.
Flow Cytometry
Sperm chromatin integrity assays [27] were used as an estimate of sperm
nuclear integrity in direct comparisons of the investigated mouse genotypes. This
test is based on the metachromatic features of acridine orange (AO), where
fluorescence is green after intercalation into double-stranded DNA chromatin but
is red in a complex with single-stranded DNA or RNA. If DNA integrity is
compromised (e.g., by the presence of strand breaks), sperm nuclei become
sensitive to acid denaturation, causing red fluorescence. A total of 20 000 sperm
cells per sample was analyzed using a FACScan flow cytometer (BD Biosciences)
in combination with Cell Quest Pro software 5.2 for raw data analysis. Results
were expressed as DNA fragmentation index (i.e., the ratio of red:[red þ green]
fluorescence) and as the percentage of cells with intense green fluorescence or
high DNA stainability as an indication of low sperm DNA compaction [27].
RESULTS
Parg(110)/ Mice Have Reduced Fertility
Parg(110)/, but not Parp1/, mice exhibited a mild but
highly significant (P , 0.0001) subfertility phenotype, with
mean 6 SD reduced litter sizes of 4.2 6 2.1 compared with
5.7 6 2.1 in 129SVE wild-type animals of (Table 1). All
animals were routinely removed from the breeding program at
age 6 mo, after which fertility may decline naturally in mice,
and were replaced by 7- to 8-wk-old animals. Unexpectedly, in
Parp1//Parg(110)/ double gene-disrupted mice, reduced
fecundity was not observed in the F1 and F2 generations, and
breeding data averages were statistically no different from
those of the wild type.
Parg(110)/ Animals Exhibit No Reduction
in Sperm Motility
To investigate the reason for reduced fertility of Parg(110)/
mice, physiological sperm parameters of mouse strains were
tested using a CASA system (Fig. 1). While the mean 6 SEM
percentages of motility spermatozoa and progressive motility
spermatozoa isolated from the cauda epididymis were comparable to those of the wild type (with typical motile sperm
fractions being ;55%–60% in individual experimental days) in
Parg(110)/ mice (101.2% 6 6.9% relative to the wild type),
Parp1/ mice had consistently and significantly (P , 0.001)
lower percentages of motile sperm (81.5% 6 3.8% relative to
the wild type) and fewer progressively motile sperm among
those (73.4% 6 11.9% relative to the wild type). Sperm from
Parp1//Parg(110)/ double gene-disrupted animals had
almost normal mean 6 SEM fractions of motile cells (94.2%
6 5.5% relative to the wild type), but progressiveness of motile
sperm was impaired at a level that was statistically comparable
to that of Parp1/ mice (74.8% 6 11.4%). This result is
puzzling and indicates that PARP1 may also have a previously
unrecognized role in sperm cell progressive motility.
Expression of Short PARG Isoforms and Cleavage of PARP2
and PARP1 Are Hallmarks of Late Spermiogenesis
The expression of three major enzymes involved in PAR
metabolism in spermatogenesis (PARP1, PARP2, and PARG)
was compared in lysates of whole testes, lysates of sonicationresistant nuclei (equivalent to the fraction of spermatids in late
steps of spermiogenesis), or lysates of cauda epididymal sperm
(Fig. 2). Immunoblotting of SDS-soluble proteins confirmed
the absence of PARP1 expression in Parp1/ and Parp1//
Parg(110)/ double-knockout mice (Fig. 2, upper panel).
(Note that the upper band is an unspecific signal always
produced by the primary antibody recognizing an unidentified
protein that is lost during spermiogenesis.) It also revealed that
in wild-type control animals PARP1 is still present in the
sonication-resistant nuclei (SRN). The SRN contains testicular
spermatids that should have completed nuclear elongation but
are not yet fully condensed. At this stage, a large portion of the
PARP1 enzyme is present in a cleaved form, reminiscent of the
caspase 3-dependent PARP1 cleavage during apoptosis [28]
(Fig. 2, top middle panel). However, there was no indication of
ongoing apoptosis in spermatid cells as analyzed by TUNEL
assays (data not shown). Apoptotic PARP1 cleavage yields
fragments of 89 kDa and 24 kDa, and only the 89-kDa fragment
is recognized by the antibody used. Epididymal sperm seemed
to be devoid of PARP1 enzyme (Fig. 2, top right panel).
PARP2 was detected at low levels in whole-testis lysates but
seemed to be highly expressed in SRN (Fig. 2, middle panel).
A double band (arrows) indicates that PARP2 is modified or
cleaved in a fashion similar to PARP1. The observed size
differences resulting in a double band could be due to
acetylation, as has been recently described for PARP2 [29].
Acetylation leads to a slight upward shift of the PARP2 protein
band. This indicates that the majority of PARP2 protein in the
whole-testis lysates, as well as the PARP2 comprising the
upper part of the double band in SRN, is modified, while the
lower band represents unmodified protein. Alternatively, the
upper band could consist of unmodified PARP2 protein, while
the lower band represents an as yet undescribed cleavage
product. Again, spermatozoa seem to possess either nondetectable amounts or no PARP2 (Fig. 2, middle right panel).
As expected, expression of at least one large PARG isoform
(110 kDa, 101 kDa, or 98 kDa) was detected in wild-type
POLY(ADP-RIBOSE) METABOLISM AFFECTS SPERM CHROMATIN
FIG. 2. PARP1, PARP2, and PARG expression in testis. The SDS extracts
of whole testis (Wh testis, left column), enriched SRN (middle column),
and sperm (right column) were analyzed in Parp1/, Parg(110)/, and
Parp1//Parg(110)/ double gene-disrupted mice and in wild-type
littermates (Wt 129SVE) using identical starting tissue fresh weight (Wh
testis), identical numbers of isolated nuclei (SRN), or sperm cells. Note
that the 130-kDa band visible in PARP1 (113 kDa) immunoblots is a
background artifact of the primary antibody (dotted arrow, Backg.). The
three PARG bands represent at least five isoforms expressed from
alternatively spliced transcripts of the Parg gene as follows: an unresolved
triple band cluster of full-length PARG(110), PARG(101), and PARG(98)
(upper arrow); PARG(63) (middle arrow); and PARG(58) (lower arrow).
control and Parp1/ whole-testis lysates but not in
Parg(110)/ or Parp1//Parg(110)/ mice (Fig. 2, bottom
panel). While all genotypes are expected to express endogenous 63-kDa and 58-kDa isoforms of PARG, extracts from
Parg(110)/ testes seem to have higher amounts of these
smaller proteins, possibly compensating for the loss of
PARG(110). Most important, no large PARG isoforms were
detectable in SRN (Fig. 2, bottom right panel) of wild-type
control and Parp1/ mice, indicating that the processes
leading to the observed phenotypic effects in Parg(110)
deletion mutants occur before nuclei become sonication
resistant. Arguably, PARG(110) could have been selectively
lost or degraded through the sonication procedure; however,
reprobing of the blot using a PARG N-terminus-specific
polyclonal serum did not show any fragments that would
support this view (data not shown), so that short-enzyme
isoforms that were observed likely originate in alternative
splicing of Parg transcripts. Surprisingly, a single small PARG
isoform remains as an unexpected component of epididymal
sperm (Fig. 2, bottom right panel) in all mouse strains tested.
Poly(ADP-Ribosyl)ation of Testicular Proteins Varies
Among the Parp1 and Parg Mutant Mice
PAR was detected in the immunoblot analyses as a polymer
presumably attached to target proteins, mainly PARP1 and
PARP2 (Fig. 3A). A significant amount of testicular proteins
seemed to be poly(ADP-ribosyl)ated in the wild type, with an
overall ;48% increase in the Parg(110)/ mouse (Fig. 3, A
49
FIG. 3. Parg gene disruption results in increased PAR accumulation. A)
Immunoblot analyses of whole testis (Wh. testis), SRN, and sperm using a
PAR-specific antibody to detect poly(ADP-ribosyl)ated proteins.
Parg(110)/ mice showed increased amounts of PARP1 and PARP2
automodification and other poly(ADP-ribosyl)ated proteins in whole-testis
lysates. Parp1/ and Parp1//Parg(110)/ showed increased PARP2
automodification only. B) Quantification of PAR bands in blotted lysates of
whole testes (such as on the left side of A) showing the mean 6 SD of
three independent experiments. To allow for comparison of PAR
accumulation that is dependent on PARG activity, PAR signal intensities
in genotypes containing the same set of PARP enzymes were quantified
and compared. In the left panel, PAR signals seen in wild-type and
Parg(110)/ animals (both contain PARP1 and PARP2 enzyme) are
compared. In the right panel, PAR signal intensities observed in Parp1/
and Parp1//Parg(110)/ (both are devoid of PARP1 and contain only
PARP2 as a DNA damage-dependent PARP enzyme) are compared.
Overall, Parg(110)/ showed ;1.5-fold increase in PAR formation
compared with the wild type (left), and PARP2-dependent PAR formation
was increased ;3.5-fold in Parp1//Parg(110)/ mice compared with
Parp1/ (right). Units for the data to the left of the gels are kilodaltons.
Error bars represent the SEM of three independent experiments.
and B, left panels). In contrast, in Parp1/ testes very little
PAR was detected, presumably produced by PARP2 (lane 2),
as judged from the molecular weight of the observed band.
This PARP2-dependent PAR formation was increased ;3.5fold in Parp1//Parg(110)/ double-knockout mice (Fig. 3,
A [left panel, upward ‘‘smear’’ in lane 4] and B [right panel]).
This indicates that PARP2 activity is significantly altered in
Parg(110)/ and Parp1//Parg(110)/ double-knockout
mice compared with the wild type. In SRN, immunoblotting
PAR signals mainly seemed to overlap with the PARP1 protein
pattern and therefore point to short polymer lengths. The fact
that PAR formation appeared to be comparable in SRN of the
wild type and the Parg(110)/ mutant supports the notion that
large PARG isoforms may indeed already be absent at this
stage. Faint PAR signals between 55 kDa and 72 kDa indicate
the presence of automodified PARP2 in Parp1/ and Parp1//
Parg(110)/ SRN (Fig. 3A, middle panel, lanes 2 and 4).
Redetection of the blot using PARP1- and PARP2-specific
antibodies (similar to Fig. 2) show that labeled bands (a, b, and
50
MEYER-FICCA ET AL.
nuclei, indicative of interference of the gene deletions with
chromatin condensation. A different method of determining
nuclear elongation based on area and perimeter measurements
(‘‘roundness’’ [Fig. 5D]) consistently supported the results
obtained from elongation measurements. Histogram analyses
further confirmed that the observed highly significant (P ,
0.0001) differences in elongation in Parp1/, Parg(110)/,
and double-knockout mice were not due to the presence of
extremely aberrant subpopulations of nuclei (Fig. 5E).
PAR Metabolism Affects H2AFX Phosphorylation
During Spermiogenesis
FIG. 4. Shaping and condensation of average epididymal sperm are
progressively abnormal from Parp1/ to Parg(110)/ and particularly
Parp1//Parg(110)/ gene-deleted mice with increased sperm nuclear
width (major axes). DAPI-stained nuclei of random images taken from
sperm smears of age-matched animals were analyzed for nuclear area,
exact perimeter (Pm), length (Axismaj), and width (Axismin). Deviations
from normal sperm nuclei shape are indicated by broken lines in the lower
panel sketches.
c) all correspond to PARP1 isoforms absent in the Parp1/
mouse. Band c comprises both PARP1 and PARP2 proteins in
wild-type and Parg(110)/ mice but PARP2 only in Parp1/
and Parp1//Parg(110)/ mice (data not shown). PAR was
not detectable in epididymal sperm samples of either genotype
(Fig. 3A, right panel).
Abnormally Reduced Nuclear Elongation and Chromatin
Condensation in Parp1/, Parg(110)/, and Parp1//
Parg(110)/ Epididymal Spermatozoa
Because motility measurements of sperm cells in the CASA
system initially also indicated an elongation defect in live
Parp1/ and Parg(110)/ sperm heads (data not shown),
further systematic morphometric measurements of fixed sperm
nuclei were performed. These analyses revealed that Parp1/,
Parg(110)/, and Parp1//Parg(110)/ mice all had a
varying degree of abnormal shaping of sperm nuclei (Figs. 4
and 5). While some individual variability of sperm nuclei
within a semen sample can be anticipated, analyses showed a
subtle but highly significant progressive loss of nuclear
elongation (i.e., increasing roundness) in Parp1/,
Parg(110)/, and Parp1//Parg(110)/ sperm (Fig. 4, lower
panel) compared with the wild type. Measurement of the
minimal and maximal axes and the two-dimensionally
projected area of large numbers of nuclei confirmed the notion
that Parp1/, Parg(110)/, and Parp1//Parg(110)/
nuclei were altogether shorter and wider than those in the
wild type (Fig. 5A) with high significance (P , 0.0001).
Compared with the wild type, the mean area was measurably
smaller in Parg(110)/ nuclei but was larger in the Parp1//
Parg(110)/ double gene-disrupted genotype (Fig. 5A). When
calculated as the mean nuclear elongation, sperm nuclei from
all genotypes were indeed found to be rounder than those in the
wild type (Fig. 5B), suggesting a defect in spermatid elongation
during developmental steps 9–12. The mean optical density is
increased in Parp1/ and Parg(110)/ nuclei, which is
consistent with the two-dimensional optical projection of more
spherical objects (Fig. 5C), similar to the area measurements.
In contrast, combined data from measurements of the mean
density (Fig. 5C) and optical area (Fig. 5A) revealed that the
Parp1//Parg(110)/ double gene disruption also leads to the
formation of altogether larger, less condensed, and rounder
Because PARP1 and PARG are intimately involved in DNA
strand break signaling and repair, DNA integrity was evaluated
by studying H2AFX phosphorylation as a marker for the
presence of DNA strand breaks using immunoblot analyses.
H2AFX is a variant histone of the H2A group that is
phosphorylated in the presence of DNA strand breaks and as
part of the heterochromatic XY body during meiosis [30].
Owing to its functions in sex chromatin silencing and
consequently its high abundance during meiosis, no major
differences in H2AFX content or phosphorylation between
genotypes were detectable in whole-testis extracts (Fig. 6, top
two panels). We isolated and extracted SRN, which normally
correspond to testicular spermatids of developmental steps 12
and later. Testicular spermatids isolated in such a way showed
a small increase of c-H2AFX detected in Parg(110)/
compared with the wild type (Fig. 6, middle panels). Overall
H2AFX protein content was reduced to 50%–60% in the
Parg(110)/, suggesting that a comparatively greater portion
of the H2AFX present in SRN of these mice was phosphorylated, which in turn indicates significantly elevated levels of
DNA strand breaks. Comparative immunofluorescence analyses of testes paraffin sections did not show any significant
differences of c-H2AFX signals in stage 12 tubules of the
various mouse genotypes (data not shown). While the amount
of detectable pan H2AFX was not different in sperm of the
different genotypes, Parg(110)/ sperm showed approximately a 2-fold increase in c-H2AFX compared with the wild type;
the mean 6 SEM amounts of c-H2AFX in sperm from animals
with Parp1/ and Parp1//Parg(110)/ genotypes were
reduced to 52% 6 8% and 88% 6 6% (n ¼ 3 for both) of those
in the wild type, respectively. PARP1, PAR, and ATM
physically interact to mediate H2AFX phosphorylation, and it
has been shown that c-H2AFX formation is delayed and
reduced in Parp1/ fibroblasts [31]. This notion is supported
by the results shown in Figure 6, where H2AFX phosphorylation is decreased in Parp1/ and Parp1//Parg(110)/
SRN. Therefore, using H2AFX phosphorylation as an indicator
for the presence of DNA double-strand breaks could produce
false-negative results and underestimate the extent of DNA
strand breaks in Parp1/ and Parp1//Parg(110)/ double
gene-disrupted mice. Therefore, we performed sperm chromatin integrity assays as a sensitive method to test for DNA
integrity in sperm cells.
Abrogation of PARG(110) Expression Results in Reduced
Sperm Chromatin Integrity
To investigate if abnormal H2AFX phosphorylation caused
by the genetic alteration of PAR metabolism indeed correlates
with reduced chromatin integrity, we performed flow cytometry-based sperm chromatin integrity assays. As predicted by
the finding that c-H2AFX is increased in Parg(110)/
spermatozoa, an elevated sensitivity of sperm nuclei toward
POLY(ADP-RIBOSE) METABOLISM AFFECTS SPERM CHROMATIN
51
FIG. 5. Morphometric analyses of increasingly abnormal nuclear shaping in Parp1/,
Parg(110)/, and Parp1//Parg(110)/
sperm. Parp1/ sperm were the least
different from the wild type but had a
significant increase of the nuclear minor axis
(A) and resulting greater mean elongation (B)
(i.e., overall rounder shape). D) Comparisons of area and perimeter (see Materials
and Methods) confirm this notion. C) The
mean optical density is significantly increased in Parp1/ and Parg(110)/
sperm, which is consistent with the twodimensional projection of a three-dimensional more rounded object. However,
Parp1//Parg(110)/ nuclei were the exception, with a decreased mean density
compared with the wild type. Parg(110)/
and Parp1//Parg(110)/ sperm nuclei
had significantly shorter major and longer
minor axes than the wild type (P , 0.0001),
resulting in rounder nuclei. E) Histogram
plotting of the individual elongation data,
pooled from three independent experiments
that each comprised a minimum of 750
nuclei per genotype, demonstrates normal
distributions, indicating that the observed
differences were not due to extremely
abnormal sperm subpopulations. Error bars
represent the SD. * Significantly different
from Wt 129SVE control; ** highly significantly different from WT 129SVE control.
acid denaturation was observed in this strain. The mean 6 SD
increase in DNA fragmentation index was mild but significant
(8.6% 6 0.4%) compared with that in wild-type littermates
(3.9% 6 0.3%) (P , 0.001) (Fig. 7, A and B). The denaturing
acid sensitivity of Parp1//Parg(110)/ sperm nuclei (mean
6 SD, 4.6% 6 0.3%) was intermediate between Parg(110)/
nuclei and the wild-type or Parp1/ nuclei (mean 6 SD, 2.8%
6 0.2%), with the wild-type and Parp1/ nuclei being
statistically indistinguishable from each other (Fig. 7B). This
result indicated that H2AFX phosphorylation (Fig. 6) correlated well with actual DNA strand break frequencies. Therefore,
c-H2AFX formation was likely not selectively impaired in the
Parp1-deleted animals, and Western blot results shown in
Figure 2 suggest that PARP1 may be absent at the spermatid
developmental stage, at which the residual DNA strand breaks
originate. Genotype-specific concentrations of nuclei with high
DNA stainability are similar to the individual DNA fragmentation index values, with Parg(110)/ values being approximately twice as high as those in the wild type (Fig. 7C).
Intense green fluorescence of AO-stained nuclei, as measured
by high DNA stainability, indicates low chromatin compaction
in the absence of DNA strand breaks.
DISCUSSION
An estimated 15% of all couples are affected by a clinical
degree of infertility. In approximately 50% of these couples,
successful conception is affected by the male factor [32].
Crucial prerequisites for successful fertilization in healthy
individuals and in infertility patients are the degree of
chromatin integrity and the frequency of residual DNA strand
breaks in mature sperm cells [33–42]. In this study, we show
that deletion of large PARG isoforms (110-kDa, 101-kDa, and
98-kDa enzymes) leads to increased testicular levels of PAR,
abnormal sperm nuclear shaping that is likely indicative of
interference with the spermatid elongation process, a 2-fold
increase in sperm DNA strand breaks, and finally a mild
subfertility phenotype with significantly reduced litter sizes in
the Parg(110)/ mouse model.
52
MEYER-FICCA ET AL.
FIG. 6. Elevated H2AFX phosphorylation in late developmental steps of
Parg(110)/spermatids and in sperm. a and b) No major differences in
H2AX content or phosphorylation between genotypes were detectable in
whole-testis extracts. In SRN, total H2AFX was significantly (P , 0.001 [n
¼ 3]) decreased in Parg(110)/ (d), indicating that nuclei perhaps become
sonication resistant later in development compared with the wild type, but
c-H2AFX was not reduced (c), indicating that a larger portion of H2AFX
was phosphorylated in this strain in SRN. This was reflected by the
situation in sperm, where H2AFX contents in all genotypes were similar
(f), but the amount of c-H2AFX was ;2-fold in Parg(110)/ (e). f) In
sperm, 56%–68% of the total H2AFX was shifted in size, indicative of
ubiquitination, and only nonubiquitinated H2AFX was in part phosphorylated. Bars represent the SEM of three independent experiments.
ubH2AFX, ubiquitinated H2AFX.
Differentiating spermatids undergo a dramatic change in
DNA topology from bulky histone-bound chromatin to
compact and metabolically inert protamine-bound DNA. This
condensation process is preceded and partially accompanied by
the reshaping of the nucleus from the round early spermatid
nucleus to the elongated final shape. The elongation phase of
differentiating spermatids is marked by a massive occurrence
of physiological DNA strand breaks [12], which likely allow
for the necessary relaxation of the superspiralized DNA during
the DNA topology change. These physiological DNA strand
breaks lead to H2AFX phosphorylation, which has been
observed in elongating and early condensing spermatids [20,
43]. While the nature of the DNA activity that repairs the
occurring DNA strand breaks correctly and to completeness is
not yet well understood, we observed that extensive poly(ADPribosyl)ation takes place concomitantly with these very steps,
indicating that PARsylation participates in the DNA break
management [20].
Results of the present study indicate that Parg(110)/ and
the Parp1//Parg(110)/ mice retain DNA strand breaks in
mature sperm, suggesting that PAR metabolism may be
necessary for proper safeguarding of sperm cell chromatin
integrity during spermiogenesis. Theoretically, the observed
DNA strand breaks can be residual strand breaks remaining
from the physiologically introduced strand breaks during the
extensive DNA remodeling steps during spermiogenesis, or
they can be acquired during the passage of the sperm to the
epididymis and the subsequent epididymal maturation process,
indicating that altered PARsylation leads to more damageprone sperm with inferior chromatin quality.
Sperm nuclear morphology analyses demonstrated that
Parp1/ mice have less elongated nuclei than the wild type,
but this observation was more pronounced in the Parg(110)/
and Parp1//Parg(110)/ double gene-disrupted mice (Figs.
4 and 5). This result suggests that chromatin packaging may be
impaired by a varying degree among the three mutant mouse
strains, with the lowest degree of DNA compaction being
present in the double gene-disrupted mouse. PAR metabolism,
involving interplay of the PARP1, PARP2, and PARG
enzymes, mediates cell recovery from genotoxic stress by a
variety of mechanisms [44–46]. Phosphorylation of H2AFX is
mediated by ATR and ATM and has an important role in DNA
double-strand break signaling involving cofactors such as
BRCA1 [47]. Recent findings revealed an involvement of PAR
formed by PARP1 and PARP2 in DNA double-strand breakdependent phosphorylation of H2AFX [31]. In that study,
downregulation of acute DNA damage-dependent c-H2AFX
formation was demonstrated in Parp1/ mouse embryonic
fibroblasts that could be simulated by treatment with PJ34, a
potent inhibitor of PARP enzymes. Consistent with this
finding, mouse embryonic fibroblasts derived from
Parg(110)/ mice also show such defects [25]. H2AFX
phosphorylation and the percentage of acid-denatured nuclei in
the sperm chromatin integrity assay (Fig. 7, A and B) indicate
that Parg(110)/ mice are most severely affected by the
presence of residual strand breaks. However, it cannot be ruled
out that H2AFX phosphorylation as a response to doublestrand breaks may still be regulated independently from
PARP1-dependent PAR signaling triggered by DNA singlestrand breaks. The morphometry results indicated that, to a
certain degree, all genotypes have mildly atypical sperm nuclei
that are more round shaped (Figs. 4 and 5), indicating that they
may have suboptimal chromatin packaging and quality. Poor
chromatin integrity and residual strand breaks, as measured by
the sperm chromatin integrity assay, are predictive of lower
reproductive performance and specifically of reduced embryonic survival [48, 49], which is consistent with reduced litter
sizes observed in the Parg(110)/ mouse.
H2AFX phosphorylation observed in Parg(110)/ sperm
(Fig. 6e) is another indication of residual DNA strand breaks,
but it cannot be determined based on the data presented
POLY(ADP-RIBOSE) METABOLISM AFFECTS SPERM CHROMATIN
53
FIG. 7. Sperm chromatin integrity assays
indicate poor sperm chromatin quality in
Parg(110)/ mice. A) Acridine orangebased sperm chromatin integrity assays [27]
indicate residual DNA strand breaks (red
fluorescence after acid denaturation) in
Parg(110)/ but not in Parp1/, Parp1//
Parg(110)/, or wild-type 129SVE siblings
(R3 region). B) Data were compared as
DNA fragmentation index (DFI). C) A region
R2 subpopulation with extremely high
green fluorescent stainability (high DNA
stainability [HDS]) was computed from the
raw data and compared. This subpopulation
is characterized by overall low or immature
chromatin condensation, allowing for intercalation of the dye. Representative data
are shown (n ¼ 4). Bars represent the SD of
triplicates in one representative experiment.
** Highly significantly different from Wt
129SVE control.
whether these were newly acquired after the sperm had left the
testis or represent unrepaired DNA lesions that stem from DNA
remodeling during spermatid elongation. These chromatin
reorganization events are accompanied by controlled physiological DNA strand breaks mediated by DNA topoisomerases,
with a probable predominant role of TOP2B [12, 43]. Putative
lesions caused by stalled topoisomerases comprise DNA
double- and single-strand breaks. Most of the DNA singlestrand breaks are repaired using the base excision pathway,
which has been commonly thought to involve PARP1;
however, its exact function there is still under debate [45,
50]. DNA double-strand breaks are repaired by homologous
recombination repair or by nonhomologous end joining in
somatic cells. However, because spermatids, being haploid, are
unable to perform homologous recombination repair, double
strands must be repaired by nonhomologous end joining. The
latter consists of two alternatively used variant pathways, one
involving DNA-dependent protein kinase (default pathway)
and another involving the homolog of the yeast protein RAD18
and PARP1 (backup pathway) [51, 52]. Therefore, it is
conceivable that DNA damage found in mature sperm may
be in the form of residual DNA strand breaks that remained
unrepaired owing to functional PARP1 inhibition caused by
abnormal automodification of the enzyme in the PARG(110)
deletion mutant (Fig. 3A, left panel). The increased levels of
poly(ADP-ribosyl)ated PARP are likely caused by the reduced
PARG activity in Parg(110)/ mice [22] in combination with
altered control of PARG activity [25] due to the loss of the
regulatory A-domain in the mutant. Western blot analyses in
the present study showed that elevated levels of poly(ADPribosyl)ated proteins in whole-testis extracts of Parg(110)/
mice (Fig. 3) were not due to increased apoptosis, as indicated
by in situ TUNEL assays (data not shown) and by the absence
of caspase-dependent PARP1 cleavage. The suggestion that the
observed DNA strand breaks are unrepaired residual lesions
rather than newly acquired lesions is supported by the finding
that SRN and sperm from wild-type mice do not contain
PARG(110). This suggests that the chromatin defects resulting
from abnormal PARP1/PARG protein composition in sperm
from Parg(110)/ and Parp1//Parg(110)/ animals must
arise at earlier time points (e.g., during or before the elongation
phase before chromatin condensation). Because c-H2AFX
formation was only moderately changed in SRN of
Parg(110)/ mice, it seems reasonable to suggest that DNA
strand breaks first exist as unrepaired single-strand lesions that,
depending on their frequency and spacing along the DNA, later
54
MEYER-FICCA ET AL.
develop into double-strand breaks during further condensation
of spermatid nuclei, causing H2AFX phosphorylation in
mature spermatozoa.
Arguably, the deletion of PARP1 may have a similarly
detrimental effect on DNA repair as its inhibition, but recent
evidence shows that inhibition of PARP enzymes (such as by
using a small-molecule inhibitor) results in a delay or defect in
DNA single-strand break repair that is different from, and often
more severe than, the effect caused by genetic ablation of
PARP1 enzyme [50]. In addition, PARP2, which has
substantially overlapping functions with PARP1, was still
expressed in all genetic strains tested in this study, which may
also account for the mild phenotypes observed in the
investigations presented herein. In a similarly mild fashion,
residual DNA strand breaks, compromised chromatin integrity,
and decreased embryonic survival were reported after single
deletion of either transition protein 1 (TNP1) or TNP2 [26, 53],
but if deleted together, the effect was much more dramatic [54].
However, deletion of both Parp1 and Parp2, which are
ubiquitously expressed in the mammalian body, was shown to
be inconsistent with embryonic survival [55].
In summary, the results of this study strongly indicate that
PAR metabolism is involved in proper execution of spermatid
maturation. The study demonstrates for the first time (to our
knowledge) that partial deficiency in PAR metabolism
resulting from deletion of the Parp1 and Parg genes will
negatively affect chromatin integrity in spermatid nuclei, as
measured by abnormal sperm nuclear shaping, altered H2AFX
phosphorylation, and reduced resistance to acid denaturation,
particularly in Parg gene-disrupted mice.
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