Notch signaling implications in induced luteolysis in pregnant rats

FATIMA HERNANDEZ; GRISELDA IRUSTA; MARTA TESONE

Woods Hole MA

Simposio; Fourteenth Annual Symposium; 2011

Marine biological Laboratory MBL

Notch signaling implications in induced luteolysis in pregnant ratsFatima Hernandez1 Griselda Irusta1, Marta Tesone11 Instituto de Biología y Medicina Experimental (IBYME-CONICET) Buenos Aires, ArgentinaNotch signaling is an evolutionary conserved signaling pathway, which comprises heterodimer transmembrane receptors consisting of an extracellular domain and a noncovalently linked intracellular domain (NICD). In mammals, four Notch receptors (Notch1-4) and five ligands (the Delta-like family, Jagged1 and Jagged2) have been identified. Both the Notch ligands and the receptors are transmembrane proteins; therefore, activation of Notch signaling is based on contact of neighboring cells. Interaction between Notch receptors and these ligands leads to intracellular cleavage of Notch receptors by the gamma secretase complex, the cleaved Notch intracellular domain (NICD) traffics to the nucleus where it interacts with transcriptional factors to regulate cell proliferation, differentiation and apoptosis.The corpus luteum (CL) is a transient endocrine gland whose main function is to secrete the steroid hormone progesterone (P4), essential for implantation of the blastocyst and maintenance of pregnancy in mammals. The CL ceases to produces P4 and regresses (a process called luteolysis) if pregnancy does not occur or if it is no longer required to keep pregnancy.It has been reported that members of Notch signaling are expressed in mammalian ovaries, but the exact function of this pathway in CL regression is still unclear.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 CL collected by microdissection. Total RNA was isolated and protein extraction was performed for real-time PCR and Western Blot assay respectively. Dll4 mRNA levels significantly decreased 4 h 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, 24 h after treatment there were no further changes in the expression of the ligand and receptors. To examine whether Notch signaling influences the function and survival of luteal cells during pregnancy, we administered a γ-secretase inhibitor, DAPT, intrabursally to the ovary of rats on day 19 of pregnancy. We first studied the cleavage of Notch1 in CL of pregnant rats 24 h after DAPT administration to verify if DAPT treatment was able to inhibit the Notch signaling. A significant decrease of NICD in the DAPT group was detected by Western Blot when compared to the Control group. The local injection of DAPT produced a decrease in serum progesterone levels 24 h after the treatment. In addition, Western Blot analysis of CL proteins using an anti-Caspase 3-specific antibody detected a significant increase in the level of the active p17 form of Caspase3 in the DAPT group when compared to the Control group. Injection of DAPT to pregnant rats increased Bax protein expression and decreased the levels of Bcl2 protein, relative to control levels. Consequently, the proapoptotic Bax/Bcl2 ratio was considerably increased in CL obtained from ovaries treated with DAPT.These results support a luteotropic role for Notch signaling to promote both luteal cell viability and steroidogenesis, and suggest that the luteolytic hormone PGF2α may act, in part, reducing the expression of some components of the Notch pathway.