INCREASED STRESS-ACTI
MEDIATED APOPTOT
VIJAY K. YADAV,
PRIMATE RESEARCH LABORATO
DEVELOPMENT
VATED PROTEIN KINASE
IC CELL DEATH IN BUFF
T. NATARAJAN AND R. MEDHA
RY, DEPARTMENT OF MOLECUL
AND GENETICS, IISc., BANGALOR
ACTIVITY DURING PGF2
ALO CORPUS LUTEUM
MURTHY
AR REPRODUCTION,
E - 560012
Introduction
Corpus Luteum (CL) is an ephemeral endocrine structure that develops from a preovulatory follicle /
Graffian follicle after ovulation. It plays a pivotal role in the control of reproduction in mammals.
Regression of CL in the absence of conception is obligatory for initiation of a new reproductive cycle to
allow for reovulation and another chance for conception to occur. On the other hand, during a
reproductive cycle in which conception occurs and implantation ensues the prolongation of luteal
function beyond its life span is obligatory for maintenance of pregnancy. In a number of farm animals
including the buffalo, prostaglandin F2 (PGF2) is recognized as the physiological luteolysin that is
responsible for regression (luteolysis) of CL at the end of a non-fertile cycle. Despite the central role of
PGF2 in luteolysis, actual mechanisms during spontaneous luteolysis at the end of non-fertile cycle or
luteolysis that occurs following exogenously administered PGF2 are poorly defined. However, it is
now well established that apoptosis or programmed cell death plays a central role in the regression of
CL that occurs during PGF2-induced or spontaneous luteolysis in CL of several species including the
cow. Prostaglandin F2 in the CL acts by binding to specific receptors belonging to the family of Gprotein coupled receptors localized mainly to large luteal cells, but are also present on small luteal and
endothelial cells of the CL. Upon binding to the receptor, PGF2 has been reported to induce activation
of membrane-bound phospholipase-C that catalyzes the hydrolysis of phosphotidyl inositol 4,5
bisphosphate to inositol trisphosphate (IP3) and diacyl glycerol (DAG). Prostaglandin F2–increased
IP3 levels have been reported to stimulate mobilization of intracellular Ca2+ dependent protein kinase
C (PKC). Although many of the antisteroidogenic actions of PGF2 in large luteal cells appear to be
mediated by PKC, stimulation of PKC in CL by pharmacological agents does not induce cell death even
though they bring about decreased steroidogenesis, indicating that PGF2 has additional effects and the
signaling pathways for steroidogenesis and cell death in luteal cells may be different.
Questions :
 What is the time course of decrease in functionality (P4 is an index of CL function)
of Buffalo CL in response to exogenous PGF2 injection ?
 Does Buffalo corpus luteum, like the cow CL, die by apoptosis in response to
exogenous PGF2 injection, if so, what is the time course of onset of apoptosis ?
 What is the role played by MAP kinases in the CL death process ?
Experimental Design :
Gp#1(n=4)
0 * Control
*
Gp#2(n=3)
4
*
Gp#3(n=3)
12
*
Gp#4(n=3)
0
* :
18
Time (h) after PGF2injection
CL collection
PGF2 (750g), i.m., injection.
Note :The experiment was initiated on day 11 of the estrous cycle
Serum P4 Concentrations after PGF2 injection in the buffalo:
Correlation with StAR protein levels
30 kD
175
150
StAR
125
2
100
75
1
50
StAR Protein Levels
P4
(% Change vs time 0)
P 4 ( ng/ml )
3
25
0
0
4
4
12
12
18
18
Time (h) after PGF2 injection
Each bar ( ) represents M ± SEM values and each ( ) bar represents quantitative
analysis of the western blots which were probed using StAR specific antibody and data
are presented as % change vs time 0. A qualitative western blot for StAR is also shown.
Morphological characteristics of luteal cells during PGF2- induced
Luteolysis in the buffalo corpus luteum
0
4
12
18
Time (h) after PGF2 injection
Photomicrographs (40X) of corpora lutea obtained 0,4,12 & 18 h after PGF2injection. Sections
(3-5 m) were stained with hematoxylin and eosin. Black arrows indicate normal cells, blue
arrows indicates morphologically deformed cells and green arrow indicates condensed nuclei.
Chromatin condensation in the buffalo CL during spontaneous (SL) as well as
during PGF2-induced luteolysis
Mid CL
18 h PGF2
SL
% Cell Death
50
40
30
20
10
0
0h
18 h
SL
Corpora lutea obtained from untreated midestrous cycle buffaloes, 18 h after PGF2 injection and during
late luteal phase were cryosectioned, stained with DAPI (100g/ml) for 10 min and observed by confocal
microscope. DAPI stained nuclei were counted as normal or condensed nuclei. Percentage of death was
determined by percentage of condensed nuclei vs total number of nuclei. Blue arrows indicate normal
nuclei and green arrows indicate condensed nuclei.
Qualitative and Quantitative Biochemical analysis of DNA Integrity during
PGF2-induced luteolysis in the buffalo CL
Time (h) After PGF2 Injection
0
4
12 18
0 4 12 18 0
1080
900
720
540
360
180
low MW DNA labeling
(% Change vs time 0)
600
400
200
0
0
4
12
18
Time (h) after PGF2 injection
Genomic DNA isolated from corpora lutea obtained 0,4,12 and 18 h after PGF2 injection to buffaloes was
analyzed for oligonucleosomal DNA fragmentation, considered as hallmark of apoptosis. For qualitative
analysis, 30g of DNA was resolved in 2% Agarose gel and stained with EtBr (left). For quantitation, 1g
of DNA was end labeled with 32P-deoxy ATP using tdt enzyme, resolved in 2% gel, transferred to nylon
membrane and analyzed in the Phosphor-imager (right). Bar diagram shows the quantiatation of low MW
DNA fragments.
Increased BAD activation during PGF2-induced apoptosis in the buffalo CL
Time (h)
0
4
12
18
Phospho-BAD
% Change vs time 0
BAD
100
75
50
25
0
0
4
12
18
Time (h) after PGF2 injection
Immunoblot analysis of pBAD/BAD; 200g of total cell lysates from corpora lutea obtained 0,4,12 and 18 h
after PGF2 injection were resolved on SDS-PAGE, transferred to PVDF membrane and probed with pBAD
stripped and reprobed with tBAD antibody.The blot shown is from one of three independent experiments.
Quantitative analysis of western blots were done using amersham gel-doc software. The results presented
are from three independent experiments, with error bars representing SEM.
p38 MAPK: Phosphorylation dependent and independent levels during PGF2induced apoptosis in the buffalo CL
Time (h)
0
4
12
18
pp38
200
% Change vs time 0
p38
150
100
50
0
0
0
4
4
12
12
18
18
Time (h) after PGF2 injection
Immunoblot analysis of phospho-p38/p38; 100g of total cell lysates from corpora lutea obtained 0,4,12
and 18 h after PGF2 injection were resolved on SDS-PAGE, transferred to PVDF membrane and probed
with pp38, stripped and reprobed with p38 antibody.The blots shown are from one of three independent
experiments. Quantitative analysis of western blots were done using amersham gel-doc software. The
results presented are from three independent experiments, with error bars representing SEM.
JNK-1 and 2: Phosphorylation dependent and independent levels
during PGF2-induced apoptosis in the buffalo CL
Time (h)
0
4
12
0
4
12
18
pJNK-1
2000
% Change vs time 0
18
JNK-1
1600
pJNK2
500
JNK-2
400
1200
300
800
200
400
0
100
0
0
4
4
12 12 18 18
0
0
0
4
4 12 12 18 18
Time (h) after PGF2 injection
Immunoblot analysis of phospho-JNK-1/2 and JNK-1/2; 200g of total cell lysates from corpora lutea
obtained 0.4,12 and 18 h after PGF2 injection were resolved on SDS-PAGE, transferred to PVDF
membrane and probed with pJNK-1/2, stripped and reprobed with JNK-1/2 antibody respectively.The
blots shown are from one of three independent experiments. Quantitative analysis of western blots were
done using amersham gel-doc software. The results presented are from three independent experiments,
with error bars representing SEM.
Phospho-JNK: Localization during PGF2-induced apoptosis in the
buffalo CL
Time (h)
0
4
12
18
Immunohistochemical staining for phospho-JNK in the buffalo corpus luteum collected 0,4,12 and
18 h after PGF2 injection to buffaloes. pJNK was detected using phospho-JNK antibody with FITC
conjugated goat anti-rabbit antibody.
Summary and Conclusion………………………….
PGF2 injection into the buffaloes during mid-estrous cycle caused
decreased P4 concentrations within 4 h and the levels were decreased to
25% of the pre-treatment by 18 h.
Morphological analysis of the CL tissue obtained 18 h after PGF2 injection,
revealed increased nuclear condensation, a characteristic feature of
apoptotic cells. Apoptosis was further confirmed biochemically by analysis
of cellular DNA which revealed increased low MW DNA fragments.
Western blot analysis of tissues revealed increased Stress Activated Protein
Kinases (SAP Kinase) activity 18 h post PGF2 injection, which was further
confirmed by immunofluorescence analysis of pJNK.
These data together suggest a role for the SAP kinases during PGF2
induced apoptotic cell death in the buffalo corpus luteum.