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Role of the Dll4-Notch system in PGF2alpha (PGF2)-induced luteolysis in the pregnant ratMarta Tesone1, Fatima Hernandez1, Marina C. Peluffo2, Richard L. Stouffer2, Griselda Irusta1, 1 Instituto de Biología y Medicina Experimental (IBYME-CONICET), 2Oregon National Primate Research Center, Oregon Health & Science University (ONPRC-OHSU)The corpus luteum (CL) is a transient endocrine gland in which angiogenesis and vessel regression occur during cyclic ovarian function in adult females. The CL differentiates from the follicle wall after ovulation by tissue remodeling and extensive neo-vascularization. Moreover, the integrity and function of the luteal vasculature decline during regression of the CL near the end of the cycle. The Notch family pathway, particularly Delta-like ligand 4 (Dll4) and its receptors Notch1 and 4 were recently identified as novel factors involved in angiogenesis. This system regulates cell fate decisions, including proliferation and death. The transmembrane receptors are cleaved upon binding of their ligands, leading to the release of the intracellular domain, which translocates to the nucleus where it functions as a transcriptional coactivator of regulatory genes of cellular fate. This processing requires the activity of two proteases, namely tumour necrosis factor α-converting enzyme (TACE) and presenilin/γ-secretase. Given the implication for angiogenesis in the CL lifespan, we investigated the role of the Notch pathway in luteal function and regression of the CL during pregnancy in rats. To determine if the Notch signaling pathway is implicated in CL regression during pregnancy, pregnant rats were injected with a luteolytic dose of PGF2 alpha (400g, ip) or vehicle on day 19 of pregnancy. Ovaries were removed 4 and 24 hours after the treatment and CLs collected by microdissection. Total RNA was isolated and protein extraction was performed for real-time PCR and western blot assay respectively, of Dll4, Notch1 and 4. Dll4 mRNA levels significantly decreased at 4 hours after PGF2α administration, whereas no changes were detected at the protein level. In contrast, both mRNA and protein expression of Notch1 and 4 receptors significantly decreased 4 hours after PGF2α administration. However, by 24 hours after treatment there were no further changes in the expression of the ligand and receptors.To elucidate if the Notch system regulates luteal function during pregnancy, either vehicle (control) or the γ-secretase inhibitor DAPT (10ug/ovary) was injected into the bursa of both ovaries on day 19 of pregnancy. Twenty-four hours after treatment, blood samples and CLs were collected. Luteal function was evaluated by measuring serum progesterone (P4) by RIA and apoptosis was examined by DNA fragmentation in agarose gels. The levels of P4 significantly decreased after DAPT administration. However, apoptotic DNA fragmentation in the CL was not affected by DAPT treatment.These results suggest that the Notch signaling pathway promotes CL function and is acutely suppressed during PGF2-induced CL regression in pregnant rats. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05-06384), NIH-FIRCA RO3-TW007041 and NIH-NCCR RR00163.