PARENT SESSION

(W621) EXPRESSION AND ACTIVITY OF CASPASE-2, -3, -8, AND -9 IN THE RAT CORPUS LUTEUM AT DIFFERENT STAGES DURING THE NATURAL ESTROUS CYCLE.

Peluffo, Marina¹, Bussmann, Leonardo¹, Stouffer, Richard², Tesone, Marta¹, ¹ IBYME (Instituto de Biologia y Medicina Experimental), Buenos Aires, Argentina² ONPRC-OHSU (Oregon National Primate Research Center- Oregon Health and Science University), Beaverton, Oregon

ABSTRACT- Apoptosis is associated with regression of the corpus luteum (CL) in many species. Since caspase enzymes play a central role in apoptosis, we studied the expression of several initiators (-2, -8 and -9) and the main effector (-3) caspase in the CL during the natural estrous cycle of the rat. CLs were dissected from ovaries obtained at estrus (E) and diestrus II (DII)) under a stereoscopic microscope. Old and new CLs were identified and weighed to confirm differences between groups. CL proteins were isolated for assessment of fluorometric caspase (-2, -3, -8, -9) activity. Ovaries (n = 6-7) were fixed in formalin and used for immunohistochemistry (IHC) of caspase-2, -3, -8, -9, and for hematoxylin-eosin analyses. Weight of the old and new CLs at both E and DII revealed significant differences (Old vs New CL at E: 11.3 ± 0.7 vs 3.9 ± 0.4 mg; DII: 5.2 ± 0.4 vs 12.2 ± 0.6 mg; p<0.05). No significant differences were found at DII between the old and new CL for any of the caspase activities. However, at E, caspase-2, -3 and -9 activity was significantly (p< 0.05) greater in old CL (7.6, 1.7 and 1.4 fold increase, respectively) as compared to new CL. Only caspase-8 activity did not change in the CLs at E. IHC analyses showed a dynamic expression of the caspases during the CL lifespan. Specific staining was detected in the small and large luteal cells, as well as in the endothelial cells, but not in stroma cells. Immunostaining for caspase-2 appeared to increase as the luteal phase progress, peaking at DII in the old CLs. However, caspase-3 protein staining appeared to peak at DII in the new CLs. IHC results for caspase-8 and -9 showed little change in expression with a slight increase at E in the old CLs. Hematoxylin-eosin analyses revealed an increase (p<0.05) in the number of apoptotic cells at DII of the old CL. In summary, dynamic expression of the caspases was observed during the formation and regression of the rat CL. Caspase-2, -3 and -9 activity peaked in the old CL at E, when regression is just beginning, whereas the number of apoptotic cells increased as luteal regression progresses, peaking at DII in the old CL. These results suggest an important role for this protease family during the early events of the luteolysis in the rat during the natural estrous cycle. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05-06384), and RR00163.

KEY WORDS: CASPASE, CORPUS LUTEUM, LUTEOLYSIS, APOPTOSIS