(P-336) Role of Ankyrin-repeat and SOCS-Box Protein 9 (ASB9) in PAR1 Inhibition and MAPK Signaling Pathway in the Bovine Ovary

Abstract Authors: Naranjo Gonzalez Daniela 1; Ndiaye Kalidou 1

1. Centre de Recherche en Reproduction et Fertilité, Faculté de médecine vétérinaire, Département de biomédecine

Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, QC, Canada

Abstract Text: The Ankyrin-repeat and SOCS-box protein (ASB) family, part of the E3 ubiquitin ligases, regulates proliferation, differentiation, carcinogenesis, and cell cycle in granulosa cells. We previously identified ASB9 as an hCG-induced gene in granulosa cells (GC) of bovine ovulatory follicles. Although we reported ASB9 interactions with target binding partners including protease-activated receptor 1 (PAR1), the exact roles of ASB9 in GC activity and in various signaling pathways need to be fully investigated. In this project, we aimed to study the mechanisms of action of ASB9 in GC and determine whether ASB9 induction by LH/hCG is necessary for the regulation of target genes such as PAR1, and signaling pathways involved in GC function and activity. Cultured GC were treated with different doses of FSH, LH and Thrombin. RT-qPCR analyzes showed that thrombin increased PAR1 expression in GC (P < 0.05) while FSH had no effect on PAR1. Treatment with LH reduced significantly PAR1 (P < 0.05), even in the presence of thrombin, demonstrating downregulation of PAR1 by LH, possibly via induction of ASB9. Western blot results show that ASB9 expression in GC in vitro is increased 12 hours after treatment with LH (P < 0.05). Furthermore, the phosphorylation profile of MAPK3/1 (ERK1/2) in thrombin-treated GC suggests a PAR1-mediated control while the presence of ASB9 in GC appears to have a negative effect on the MAPK pathway. Finally, thrombin could block the negative effect of ASB9 on PAR1 very early (within 1 hour treatment) while this protease does not seem to affect ASB9 at 6 and 12 hours of treatment. Proliferation assays showed that ASB9 negatively regulates GC number as well as proliferation marker CCND2 while apoptosis marker BAX2 was increased with LH treatment. Together, these data show functional effects of ASB9 on GC activity through PAR1 and potential consequences on the ovulatory process in bovine species.