Reprod Dom Anim 39, 39–42 (2004)
2004 Blackwell Verlag, Berlin
ISSN 0936-6768
Poly(ADP-Ribose) Glycohydrolase in Bovine Retained and Not Retained Placenta
M Kankofer1, L Guz2 and J Wierciński3
1
Department of Biochemistry, Faculty of Veterinary Medicine, Agricultural University, Lublin, Poland; 2Department of Fish Diseases and Biology,
Faculty of Veterinary Medicine, Agricultural University, Lublin, Poland; 3Subdepartment of Instrumental Foodstuff Analysis, Faculty of Agriculture,
Agricultural University, Lublin, Poland
Contents
Poly(ADP-ribose) glycohydrolase (PARG) is the enzyme
which degrades poly(ADP-ribose) polymers synthesized by
poly(ADP-ribose) polymerase (PARP). Both enzymes are
activated in response to different stimuli like oxidative stress
and are involved in DNA repair processes. The retention of
bovine foetal membranes (RFM) is supposed to be connected
with oxidative stress conditions. The aim of the study was to
detect the presence of PARG protein in bovine placenta in
order to find the relationship between the process of releasing/
retaining placenta and DNA repair. Placentomes, collected
after spontaneous delivery or caesarian section were divided
into maternal as well as foetal part of placenta, homogenized
and subjected to electrophoresis. Animals were divided into six
groups as follows: A – caesarian section before term with
RFM; B – caesarian section before term without RFM; C –
spontaneous delivery at term with RFM; D – spontaneous
delivery at term without RFM; E – caesarian section at term
with RFM; F – caesarian section at term without RFM.
PARG protein was detected in nitrocellulose membranes using
commercially available bovine anti-PARG antibody and
Western blotting technique. Single bands referred to bovine
PARG standard were observed in all examined tissues as well
as in human placenta used as the control of procedure. In
addition, the intensity of staining was stronger in retained than
properly released term placenta and in foetal than in maternal
part of the placenta. These results may suggest the differences
in enzyme protein content and careful conclusions can be
drawn that the activities of PARG may be altered between
compared groups of animals. It may confirm the presence of
oxidative stress conditions and their consequences on metabolic pathways, the content of biologically active substances
and processes of proper releasing placenta. Further experiments on PARG activity in bovine foetal membranes with
respect to proper and improper placental release are necessary.
Introduction
Although the activity of poly(ADP-ribose) glycohydrolase (PARG) was described in the early 1970s (Ueda
et al. 1972), its characteristics still require investigation.
PARG, together with poly(ADP-ribose) polymerase
(PARP), is involved in poly(ADP-ribose) metabolism.
Upon binding to DNA breaks, activated PARP cleaves
NAD+ into nicotinamide and ADP-ribose and polymerizes the latter onto nuclear acceptor proteins including histones, transcription factors, and PARP itself
(Virag and Szabo 2002). Poly(ADP-ribose) polymers
synthesized by PARP in response to different stimuli are
degraded later on by PARG. The activity of both
enzymes is supposed to be associated with DNA repair
processes (Satoh et al. 1993; Davidovic et al. 2001),
programmed cell death (Lazebnik et al. 1994) and
ischemic cell damage (Eliasson et al. 1997). Oxidative
U.S. Copyright Clearance Centre Code Statement:
stress-induced overactivation of PARP consumes NAD+
and consequently ATP, culminating in cell dysfunction
or necrosis (Virag and Szabo 2002).
The retention of bovine foetal membranes, which is
one of the most important post-partum disorders,
warrants the investigation of its biochemical background
as well. It is supposed to be connected with oxidative
stress conditions. These conditions not only induce the
imbalance between production and neutralization of
reactive oxygen species (ROS), but also increase the
peroxidation processes of proteins and lipids (Kankofer
2001a,b). Oxidative damage to DNA molecules may be
a consequence (Kankofer and Schmerold 2002) and may
lead to stimulation of DNA repair processes that require
PARP and PARG activity (Pieper et al. 1999; Shall and
de Murcia 2000). ROS-induced alterations in protein,
lipid and DNA pathways result in changes in hormone
level which may disturb the proper separation of foetal
membranes.
To check this hypothesis, the study on PARG activity
in bovine placenta was undertaken. The aim of this
study was to detect the presence of PARG protein in
bovine retained and properly released placenta by means
of the Western blotting technique and bovine antiPARG antibody.
Materials and Methods
All pregnant cows included into this study were clinically healthy and of Holstein-Friesian breed, 2–6 years of
age. They were fed a standard diet, i.e. sugar beet pulp,
barley straw and grass silage. Days of gestation were
calculated using dates of insemination. Preterm Caesarean sections were indicated for teaching purposes, term
sections because of fetus size.
Placentomes were collected from pregnant horn (one
per cow) immediately after normal delivery of a calf at
term (282–288 days of pregnancy), after extraction of a
calf during Caesarean section before term (272–277 days
of pregnancy), and during Caesarean section at term.
The remainder of foetal membranes were left in situ until
they were released spontaneously within 8 h after
parturition or removed by a veterinarian after 8 h and
defined as retained placenta (Grunert 1983). Cows were
divided into six groups according to the time of
expulsion and the mode of delivery as follows: A –
Caesarean section before term with retained placenta;
B – Caesarean section before term without retained
placenta; C – normal delivery at term with retained
placenta; D – spontaneous delivery at term without
retained placenta; E – Caesarean section at term with
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40
M Kankofer, L Guz and J Wierciński
retained placenta; F – Caesarean section at term without
retained placenta. Three randomly chosen animals per
each group were analysed in duplicate by Western
blotting technique. Placental tissues were divided into
maternal and foetal part of placenta, washed in cold
0.9% NaCl, and stored frozen until analysis.
Placental maternal and foetal tissues were homogenized in phosphate buffer (0.05 mol/l, pH 7.0) using an
Ultra Turrax T 25 (Ikawerk Janke and Kunkel Inc.,
Staufen, Germany) for 5 min and centrifuged for 20 min
at 3000 · g. The whole procedure was performed at 4C.
The protein content of supernatants was determined
using Lowry’s method and bovine serum albumin as
standard (Lowry et al. 1951). The supernatants were
subjected to sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE).
Human placental sample, collected after normal
(uncomplicated) pregnancy and parturition, was included into analysis as the control of experimental procedure. Antibody used in the experiment recognized
human PARG also.
0.05% Tween 20 for 5 min, then 10 min in PBS
containing 0.5% Tween 20 (PBST). Before hybridization with the second antibody, the membranes were
washed 4· for 20 min with PBSTM.
The second antibody anti-IgG (anti-rabbit conjugated
to alkaline phosphatase, DAKO A/S, Glostrup, Denmark) was diluted 1 : 500 in PBSTM and the membranes were incubated for 2 h. The blot was washed in
PBST 3· for 10 min and in substrate buffer (0.1 M/l Tris
pH 9.5; 0.1 M/l NaCl; 50 mM/l MgCl2) for 10 min.
Finally, the blot was developed in a substrate solution
which was freshly prepared by adding 44 ll of nitroblue
tetrazolium chloride solution [NBT; 75 mg/ml in 70%
(v/v) dimethylformamide] and 33 ll of 5-bromo4-chloro-3-indolyl phosphate p-toluidine salt [BCIP;
50 mg/ml in 100% (v/v) dimethylformamide, Life
Technologies GmbH, Karlsruhe, Germany] to 10 ml
of substrate buffer. Positive reactions revealed purple
bands. The staining was stopped by washing the
membrane in distilled water. The specificity of the
primary antibody had previously been verified by
comparing the reactivity of first and second antibody
to sample preparations on Western blotting. Bovine
PARG standard was purchased from Alexis Biochemicals.
Densitometric analysis was performed by use of Gel
Doc 1000 Gel Documentation System (Bio-Rad, Hercules, CA, USA) and computer program Sigma Gel
(Sigma, St Louis, MO, USA). Peak areas were expressed
in arbitrary units.
Electrophoresis and Western blotting
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was carried out in 12% polyacrylamide gels
using the method of Laemmli (1970) at 120 V. Samples
were resuspended in a reducing loading buffer (62.5 mM/l
Tris, pH 6.8; 6 M/l urea; 10% glycerol; 2% SDS; 0.003%
bromophenol blue; 5% 2-mercaptoethanol) and before
application, were incubated for 15 min at 65C. The
same amount of protein was loaded per each well
(20 lg).
After electrophoresis, proteins were transferred onto
Immun-Blot Polivinylidene Difluoride Membranes (PVDF)
membrane (BioRad, Richmond, CA, USA). The transfer
buffer consisted of 25 mM/l Tris, 192 mM/l glycine, 20%
methanol, pH 8.3. Electrotransfer was performed at
150 V for 1 h.
The PVDF membranes were washed with PBSTM
solution (PBS 1x, pH 7.4; 3% non-fat powdered milk;
0.1% Tween 20) for 2 h. Anti-PARG polyclonal antibody (cat. no 210–773-R050; Alexis Biochemicals,
Nottingham, UK) was diluted in PBSTM solution
(1 : 5000) before incubation with the membranes for
18 h at 4C. After incubation, PVDF membranes were
washed in PBS containing 2 M/l deionized urea and
1
2
3
4
5
6
7
Results
The presence of PARG protein is showed in Fig 1.
The enzyme was defined with respect to PARG
bovine standard. Bovine placental samples, as well as
human placenta, showed one band of molecular weight
compared with enzyme standard (110 kDa).
Western blotting technique allows to confirm the
presence of examined protein by the use of a specific
antibody. To visualize the results, by colour, enzymatic
reaction was implemented. Obtained intensity of staining only suggests the differences in amount of enzyme
protein. The intensity of staining differed between
examined samples what might, however, only semiquantitatively and indirectly, also suggest the alterations in
enzyme activity.
8
9
10 11
12
13 14
kDa
- 110
Fig. 1. Western blotting of PARG from bovine and human placenta. Lanes: 1 – human placenta; 2, 3 – maternal and foetal part, respectively;
from Caesarean section before term with RFM group; 4, 5 – maternal and foetal part, respectively; from Caesarean section before term without
RFM group; 6, 7 – maternal and foetal part, respectively, from spontaneous delivery at term with RFM group; 8, 9 – maternal and foetal part,
respectively, from spontaneous delivery at term without RFM group; 10, 11 – maternal and foetal part, respectively, from Caesarean section at
term with RFM group; 12, 13 – maternal and foetal part, respectively, from Caesarean section at term without RFM group; 14 – enzyme standard
Poly(ADP-Ribose) Glycohydrolase in Bovine Placenta
In addition, densitometric analysis showed stronger
staining in foetal than in maternal part of the placenta.
In placental samples collected at term, a stronger
intensity of staining was observed in retained than properly released foetal membranes as well as in Caesarean
section than normal delivery groups. In preterm samples
the intensity tended to be stronger in control cows than in
RFM animals. For example the following peak areas were
determined in membrane presented in Fig. 1: lane 2 – 111;
lane 3 – 448; lane 4 – 176; lane 5 – 556; lane 6 – 253; lane 7 –
199; lane 8 – 1; lane 9 – 86; lane 10 – 441; lane 11 – 452; lane
12 – 55; lane 13 – 57.
Such densitometric analysis is valuable only together
with the results of direct determinations of enzyme
activity. In the present study this is the only suggestion
for further experiments.
Discussion
Well evidenced changes in the levels of steroid hormones
(Grunert 1983) and prostaglandins (Leidl et al. 1980;
Slama et al. 1993), which are observed during the
retention of foetal membranes in cows, are connected
with the disturbances in their pathways. These disturbances reflect the alterations and/or inhibition of activities of enzymes which are involved in hormone
metabolism. Enzyme activities may be changed because
of ROS excess, causing protein peroxidation and proteolysis. Disturbed NAD/NADH ratio may also influence the proper activity of dehydrogenases responsible
for steroid metabolism (Takayanagi et al. 1986).
Local interaction of ROS with prostaglandin F2a
(PGF2a) on progesterone release in ewes was described.
The results demonstrated that PGF2a may induce
ROS increase during ovine luteolysis (Hayashi et al.
2003).
The evidence for ROS imbalance during RFM is from
experiments describing not only the alterations of antioxidative enzymes activity, but also the changes in the
level of non-enzymatic antioxidants (Kankofer 2001c,d).
The concentration of protein (Kankofer 2001a) and lipid
peroxidation products (Kankofer 2001b) increase in
RFM- affected cows in comparison with control animals.
The determinations of 8-OH-dG, which is a marker of
oxidative DNA damage, suggest the presence of this
damage during improper placental release (Kankofer
and Schmerold 2002). These determinations facilitated
finding the relationship between RFM, ROS imbalance,
and PARG activity.
PARG was purified from different tissues and partly
characterized (Tanuma and Endo 1990; Maruta et al.
1991; Braun et al. 1994). Bovine cDNA encoding this
enzyme was also isolated recently (Lin et al. 1997). It is a
cytoplasmic and nuclear 110 kDa protein responsible
for poly(ADP-ribose) polymers catabolism (Desnoyers
et al. 1995; Winstall et al. 1999a). The known inhibitors
of human placental PARG are three classes of tannins
(gallotannins, ellagitannins, condensed tannins) (Aoki
et al. 1993) as well as ADP dihydroxypyrrolidine (Slama
et al. 1992). There are suggestions that PARG shows
different affinity for long and short poly(ADP-ribose)
molecules resulting in the hypothesis that polymer size
and numbers are rate-determining factors in poly
41
(ADP-ribose) metabolism (Hatakeyama et al. 1986;
Winstall et al. 1999b). There is evidence for a 59-kDa
protein with PARG activity which is supposed to be the
product of proteolysis or the result of purification
procedure (Winstall et al. 1999a,b).
PARG acts through endo- and exoglycosidic mode to
remove poly(ADP-ribose) polymers (Braun et al. 1994).
The synthesis of poly(ADP-ribose) polymers, catalysed
by PARP, appears in response to different stimuli such
as oxidant injury. This is the reason why the activity of
both enzymes is closely connected. The presence of
PARP protein was previously detected in bovine
placenta by use of Western blotting technique (Kankofer and Guz 2003). The intensity of staining of enzyme
bands differed between examined groups of animals with
and without retained placenta.
Western blotting technique and anti-bovine PARG
antibodies allowed to describe also PARG protein
presence in bovine released and retained placenta.
Although it is only a semiquantitative method, it was
possible to see the differences in intensity of staining of
obtained bands. It might indirectly suggest the differences in enzyme activity between cows affected with
RFM and control animals. It may also provide
evidence for oxidative/antioxidative imbalance present
in examined animals. Human placental tissue which
was used as the control showed the same electrophoretic pattern and also only one band of enzyme
protein. The intensity of staining, however, was weaker
as in bovine tissue.
There are reports indicating that poly(ADP-ribose)
catabolism may be involved in local increase of ATP,
which could serve as an energy store for DNA repair
enzymes as well as for the ligation step (Maruta et al.
1997; Oei and Ziegler 2000).
PARG, like other proteins, is cleaved during apoptosis. Responsible for this cleavage is caspase-3 which
releases two enzymatically active fragments of 85 and
74 kDa in human cells (Affar et al. 2001). Partial
examination of bovine placental proteins by means of
SDS-PAGE and zymography of metalloproteinases
showed differences in the number of fractions as well
as differences in metalloproteinases activities in cases of
retained and released placenta (Maj and Kankofer 1997,
1998). This may suggest the alterations in proteolytic
processes in bovine placenta during improper placental
release (Gross et al. 1985).
The present study brings additional evidence that
bovine placenta is sensitive to ROS imbalance, leading
to DNA damage. This damage is repaired by PARP and
PARG action which are both present in bovine released
and retained placenta. However, the control and
efficiency of repair processes in bovine retained placenta
requires further elucidation.
References
Affar EB, Germain M, Winstall E, Vodenicharov M, Shah
RG, Salvesen GS, Poirier GG, 2001: Caspase-3 mediated
processing of poly(ADP-ribose) glycohydrolase during
apoptosis. J Biol Chem 276, 2935–2942.
Aoki K, Nishimura K, Abe H, Maruta H, Sakagami H,
Hatano T, Okuda T, Yoshida T, Tsai YJ, Uchiumi F, 1993:
42
Novel inhibitors of poly(ADP-ribose) glycohydrolase. Biochim Biophys Acta 1158, 251–256.
Braun SA, Panzeter PL, Collinge MA, Althaus FR, 1994:
Endoglycosidic cleavage of branched polymers by poly
(ADP-ribose) glycohydrolase. Eur J Biochem 220, 369–375.
Davidovic L, Vodenicharov M, Affar EB, Poirier GG, 2001:
Importance of poly(ADP-ribose) glycohydrolase in the
control of poly(ADP-ribose) metabolism. Exp Cell Res
268, 7–13.
Desnoyers S, Shah GM, Brochu G, Hoflack JC, Verreault A,
Poirier GG, 1995: Biochemical properties and function of
poly(ADP-ribose) glycohydrolase. Biochimie 77, 433–438.
Eliasson MJL, Sampei K, Mandir A, Hurn P, Traystman RJ,
Pieper A, Wang ZQ, Dawson TE, Snyder SH, Dawson VL,
1997: Poly(ADP-ribose) polymerase gene disruption renders
mice resistant to cerebral ischemia. Nat Med 3, 1089–1095.
Gross TS, Williams WF, Manspeaker JE, Lewis GS, Russek E,
1985: In vitro proteolytic activity of the late pregnant and
periparturient bovine placenta. J Anim Sci 61(suppl. 1), 391–
392.
Grunert E, 1983: Ätiologie, Pathogenese und Therapie der
Nachgeburtsverhaltung beim Rind. Wien Tierärztl Mschr
70, 230–235.
Hatakeyama K, Nemoto Y, Ueda K, Hayaishi O, 1986:
Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small
poly(ADP-ribose). J Biol Chem 262, 14902–14911.
Hayashi K, Miyamoto A, Konari A, Ohtani M, Fukui Y,
2003: Effect of local interaction of reactive oxygen species
with prostaglandin F2a on the release of progesterone in
ovine corpora lutea in vivo. Theriogenology 59, 1335–1344.
Kankofer M, 2001a: Protein peroxidation processes in bovine
retained and not retained placenta. J Vet Med A 48, 207–
212.
Kankofer M, 2001b: The levels of lipid peroxidation products
in bovine retained and not retained placenta. Prost Leukotr
Essent Fatty Acids 64, 33–36.
Kankofer M, 2001c: Antioxidative defence mechanisms
against reactive oxygen species in bovine retained and notretained placenta. Activity of glutathione peroxidase, glutathione transferase, catalase and superoxide dismutase.
Placenta 22, 466–472.
Kankofer M, 2001d: Non-enzymatic antioxidative defence
mechanisms against reactive oxygen species in bovine
retained and not-retained placenta. Vitamin C and glutathione. Reprod Dom Anim 36, 203–206.
Kankofer M, Guz L, 2003: Poly(ADP-Ribose) polymerase in
bovine retained and not retained placenta. Reprod Dom
Anim 38, 390–393.
Kankofer M, Schmerold I, 2002: Spontaneous oxidative DNA
damage in bovine retained and non-retained placental
membranes. Theriogenology 57, 1929–1938.
Laemmli UK, 1970: Clevage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–
685.
Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG,
Earnshaw WC, 1994: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371,
346–347.
Leidl W, Hegner D, Rockel P, 1980: Investigations on the
PGF2a concentration in the maternal and fetal cotylodons of
cows with and without retained fetal membranes. J Vet Med
A 27, 691–696.
Lin W, Ame JC, Aboul-Ela N, Jacobson EL, Jacobson MK,
1997: Isolation and characterization of the cDNA encoding
bovine poly(ADP-ribose) glycohydrolase. J Biol Chem 272,
11895–11901.
M Kankofer, L Guz and J Wierciński
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ, 1951:
Protein measurement with the Folin phenol reagent. J Biol
Chem 193, 265–275.
Maj JG, Kankofer M, 1997: Activity of 72 kDa and 92 kDa
matrix metalloproteinases in placental tissues of cows with
and without retained fetal membranes. Placenta 18, 683–
687.
Maj JG, Kankofer M, 1998: Placental proteins from cows with
and without retained fetal membranes. The SDS-PAGE
analysis. Rev Med Vet 149, 75–80.
Maruta H, Inageda K, Aoki T, Nishina H, Tanuma S, 1991:
Characterization of two forms of poly(ADP-ribose) glycohydrolase in guinea pig liver. Biochemistry 30, 5907–5912.
Maruta H, Matsumura N, Tanuma S, 1997: Role of (ADPribose)n catabolism in DNA repair. Biochem Biophys
Commun 236, 265–269.
Oei SL and Ziegler M, 2000: ATP for the DNA ligation step in
base excision repair is generated from poly(ADP-ribose).
J Biol Chem 275, 23234–23239.
Pieper AA, Verma A, Zhang J, Snyder SH, 1999: Poly (ADPribose) polymerase, nitric oxide and cell death. Trends
Pharmacol Sci 20, 171–181.
Satoh MS, Poirier GG, Lindahl T, 1993: NAD(+)-dependent
repair of damaged DNA by human cell extracts. J Biol
Chem 268, 5480–5487.
Shall S, de Murcia G, 2000: Poly(ADP-ribose) polymerase-1:
what have we learned from the deficient mouse model?
Mutat Res 460, 1–15.
Slama JT, Simmons AM, Harson ME, Aboul-Ela N, Jacobson
MK, 1992: A new transition state inhibitor specific for
poly(ADP-ribose) glycohydrolase. In: Poirier GG, Moreau
P (eds), ADP-ribosylation Reactions. Springer Verlag, New
York, pp. 400–410.
Slama H, Vaillancourt D, Goff AK, 1993: Metabolism of
arachidonic acid by caruncular and allantochorionic tissues
in cows with retained fetal membranes (RFM). Prostaglandins 45, 57–75.
Takayanagi R, Kato KJ, Ibayashi H, 1986: Relative inactivation of steroidogenic enzyme activities of in vitro vitamin Edepleted human adrenal microsomes by lipid peroxidation.
Endocrinology 119, 464–471.
Tanuma S, Endo H, 1990: Purification and characterization of
an (ADP-ribose)n glycohydrolase from human erythrocytes.
Eur J Biochem 191, 57–63.
Ueda K, Oka J, Narumiya S, Miyakawa N, Hayaishi O, 1972:
Poly(ADP-ribose) glycohydrolase from rat liver nuclei, a
novel enzyme degrading the polymer. Biochem Biophys Res
Commun 46, 516–523.
Virag L, Szabo C, 2002: The therapeutic potential of
poly(ADP-ribose) polymerase inhibitors. Pharmaceutical
Rev 54, 375–429.
Winstall E, Affar EB, Shah R, Bourassa S, Scovassi AJ, Poirier
GG, 1999a: Poly(ADP-ribose) glycohydrolase is present and
active in mammalian cells as a 110-kDa protein. Exp Cell
Res 246, 395–398.
Winstall E, Affar EB, Shah R, Bourassa S, Scovassi IA, Poirier
GG, 1999b: Preferential perinuclear localization of poly
(ADP-ribose) glycohydrolase. Exp Cell Res 251, 372–378.
Submitted: 06.08.2003
Author’s address (for correspondence): Marta Kankofer, Department
of Biochemistry, Faculty of Veterinary Medicine, Agricultural University, 20-123 Lublin, ul. Lubartowska 58 a, Poland. Tel.: (0048 81)
747 40 23; fax: (0048 81) 445 60 06; E-mail: kankofer@agros.ar.
lublin.pl