INCREASED STRESS-ACTIINCREASED STRESS-ACTI

MEDIATED APOPTOTMEDIATED APOPTOT

PRIMATE RESEARCH LABORATOPRIMATE RESEARCH LABORATO DEVELOPMENTDEVELOPMENT

VIJAY K.VIJAY K. YADAV, YADAV,

VATED PROTEIN KINASEVATED PROTEIN KINASE

IC CELL DEATH IN BUFFIC CELL DEATH IN BUFF

T. NATARAJAN AND R. MEDHA T. NATARAJAN AND R. MEDHA

RY, RY, DEPARTMENT OF MOLECUL DEPARTMENT OF MOLECUL AND GENETICS,AND GENETICS, IISc., BANGALOR IISc., BANGALOR

ACTIVITY DURING PGFACTIVITY DURING PGF22

ALOALO CORPUS LUTEUMCORPUS LUTEUM

AR REPRODUCTION, AR REPRODUCTION, E - 560012E - 560012

MURTHYMURTHY

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 G-

protein 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 F 2–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 :

Time (h) after PGF2injection

Gp#1(n=4)

Gp#2(n=3)

Gp#3(n=3)

Gp#4(n=3)

0 *

*

*

*4

0 18

12

Control

* : CL collection

Note :The experiment was initiated on day 11 of the estrous cycle

PGF2 (750g), i.m., injection.

Serum P4 Concentrations after PGF2 injection in the buffalo: Correlation with StAR protein levels

1

2

3

Time (h) after PGF2 injection

4P

( n

g/m

l )

P4

0 4 12 18

25

50

75

100

125

150

175 StA

R P

rotein

Levels

(% C

han

ge vs tim

e 0)

StAR

30 kD

0 4 12 18

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

Time (h) after PGF2 injection

1240 18

Photomicrographs (40X) of corpora lutea obtained 0,4,12 & 18 h after PGF2injection. 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.

Mid CL 18 h PGF2 SL

Chromatin condensation in the buffalo CL during spontaneous (SL) as well as during PGF2-induced luteolysis

0 h 18 h SL0

10

20

30

40

50

% C

ell D

eath

Corpora lutea obtained from untreated midestrous cycle buffaloes, 18 h after PGF2 injection and during late luteal phase were cryosectioned, stained with DAPI (100g/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 injection0 4 12 180

200

400

600

lo

w M

W D

NA

lab

elin

g

(% C

han

ge

vs t

ime

0)

0 4 12 18 0 4 12 18 0

180

720 540 360

900 1080

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, 30g of DNA was resolved in 2% Agarose gel and stained with EtBr (left). For quantitation, 1g 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

Phospho-BAD

0 4 12 180

25

50

75

100

Time (h) after PGF2 injection

% C

han

ge

vs t

ime

0 BAD

Time (h) 0 4 12 18

Immunoblot analysis of pBAD/BAD; 200g 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

0 0 4 12 12 18 180

50

100

150

200

% C

han

ge

vs t

ime

0

4

Time (h) after PGF2 injection

pp38

p38

Time (h) 0 4 12 18

Immunoblot analysis of phospho-p38/p38; 100g 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

pJNK2

JNK-2

0 0 4 4 12 12 18 180

400

800

1200

1600

2000

Time (h) after PGF2 injection

% C

han

ge

vs t

ime

0

pJNK-1

JNK-1

Time (h) 0 4 12 18 0 4 12 18

0 0 4 4 12 12 18 180

100

200

300

400

500

Immunoblot analysis of phospho-JNK-1/2 and JNK-1/2; 200g 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

4 12 18 0Time (h)

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.

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.

Summary and Conclusion………………………….

These data together suggest a role for the SAP kinases during PGF2

induced apoptotic cell death in the buffalo corpus luteum.