INCREASED STRESS-ACTI MEDIATED APOPTOT

1. INCREASED STRESS-ACTI MEDIATED APOPTOT VIJAY K.YADAV, PRIMATE RESEARCH LABORATO DEVELOPMENT

2. VATED PROTEIN KINASE IC CELL DEATH IN BUFF T. NATARAJAN AND R. MEDHA RY, DEPARTMENT OF MOLECUL AND GENETICS, IISc., BANGALOR

3. ACTIVITY DURING PGF2 ALOCORPUS LUTEUM MURTHY AR REPRODUCTION, E - 560012

4. 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 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.

5. 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 ? * Control Gp#1(n=4) 0 * Gp#2(n=3) • 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 ? 4 * Gp#3(n=3) 12 • What is the role played by MAP kinases in the CL death process ? * Gp#4(n=3) 18 0 Time (h) after PGF2injection * : CL collection Experimental Design : PGF2 (750g), i.m., injection. Note :The experiment was initiated on day 11 of the estrous cycle

6. 30 kD ( ng/ml ) 4 P 0 4 12 18 0 4 12 18 Serum P4 Concentrations after PGF2 injection in the buffalo: Correlation with StAR protein levels 3 175 P4 150 StAR 125 2 (% Change vs time 0) StAR Protein Levels 100 75 1 50 25 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.

7. 18 0 4 12 Morphological characteristics of luteal cells during PGF2- induced Luteolysis in the buffalo corpus luteum 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 arrowsindicates morphologically deformed cells and green arrow indicates condensed nuclei.

8. 18 h PGF2 SL Mid CL Chromatin condensation in the buffalo CL during spontaneous (SL) as well as during PGF2-induced luteolysis 50 40 30 % Cell Death 20 10 0 18 h SL 0 h 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.

9. 1080 900 720 540 360 180 Qualitative and Quantitative Biochemical analysis of DNA Integrity during PGF2-induced luteolysis in the buffalo CL Time (h) AfterPGF2 Injection 0 4 12 18 0 4 12 18 0 600 400 low MW DNA labeling (% Change vs time 0) 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.

10. Time (h) 0 4 12 18 Phospho-BAD BAD 100 75 % Change vs time 0 50 25 0 0 4 12 18 Time (h) after PGF2 injection Increased BAD activation duringPGF2-induced apoptosis in the buffalo CL 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.

11. p38 MAPK: Phosphorylation dependent and independent levels duringPGF2-induced apoptosis in the buffalo CL Time (h) 0 4 12 18 pp38 200 p38 150 100 % Change vs time 0 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.

12. JNK-1 and 2: Phosphorylation dependent and independent levels duringPGF2-induced apoptosis in the buffalo CL Time (h) 0 4 12 18 0 4 12 18 pJNK-1 pJNK2 2000 500 JNK-2 JNK-1 1600 400 1200 % Change vs time 0 300 800 200 400 100 0 0 0 0 4 4 12 12 18 18 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.

13. Phospho-JNK: Localization duringPGF2-induced apoptosis in the buffalo CL 0 4 12 18 Time (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.

14. 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.