Role of Dll4-Notch system on PGF2alpha (PGF2a)-induced luteolysis in the pregnant rat

TESONE, M; HERNANDEZ, F; PELUFFO, MC; STOUFFER, RL; IRUSTA, G

Milwaukee, Wisconsin

Congreso; 43rd SSR Annual Meeting; 2010

SSR (Society for the Study of Reproduction)

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. Notch family pathway, particularly Delta-like ligand (Dll4) and its receptors Notch1 and 4 were recently identified as novel factors involved in angiogenesis regulation. This system regulates cell fate decisions, proliferations, and death. The transmembrane receptors are cleaved on 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 á-converting enzyme (TACE) and presenilin/ã-secretase. Given the implication of the 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 the CL regression during pregnancy, pregnant rats were injected IP with a luteolytic dose of PGF2 alpha (400mg) or vehicle on day 19 of pregnancy. Ovaries were collected 4 and 24 hs after the treatment and CL’s were dissected by microdisection. Total RNA was isolated and protein extraction was performed for real time PCR and western blot techniques respectively for Dll4, Notch1 and 4. Dll4 mRNA expression significantly decreased at 4 hs after PGF2á administration. In contrast no changes were detected at protein level. The mRNA and protein expression of the Notch1 and 4 receptors significantly decreased 4 hours after PGF2á administration. However, 24 hours after the treatment there were no significant changes in the expression of the ligand and receptors analyzed. To elucidate if the Notch system has a per se effect in luteal function during pregnancy; pregnant rats were injected on day 19 of pregnancy with either g-secretase inhibitor DAPT (10mg/ovary) into the bursa of both ovaries or vehicle (control). Twenty four hours after the treatment blood samples and CLs were collected. Luteal function was evaluated by measuring serum progesterone (P4) by RIA and apoptosis was examined by fragmentation of DNA in agarose gels. The levels of P4 significantly decreased after ã-secretase administration. However, in our experimental model CL apoptotic DNA fragmentation was not affected by DAPT treatment. These results suggest that the Notch signaling pathway is involved in CL function and it is associated to PGF2a-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.