University of Montreal Saskatoon, Saskatchewan, Canada
Soma Nosrat Pour (University of Montreal)| Kalidou Ndiaye (University of Montreal)
Ankyrin-repeat and SOCS-box Protein 9 (ASB9) was previously identified as a differentially expressed gene in ovulatory follicles post-luteinizing hormone (LH) surge or hCG injection in ovarian granulosa cells (GC). ASB9 interactions in GC were also identified and included PAR1, TNFAIP6/TSG6, and TAOK1. However, ASB9 mechanisms of action in GC is still undefined. We investigated the role of ASB9 in GC through specific binding partners, its effects on MAPK signaling and on GC proliferation. An in vivo model of GC from follicles at different developmental stages, small follicles (SF), dominant follicles (DF), and ovulatory follicles (OF) and an in vitro model of cultured GC along with the CRISPR/Cas9 approach to inhibit ASB9 were used. Inhibition of ASB9 in GC resulted in significant increase in PAR1, PCNA, CCND2, and CCNE2 and significant decrease in TAOK1, TNFAIP6, and CASP3 expression. From in vivo samples, PAR1 was differentially expressed in DF as compared to OF while TSG6 and TAOK1 were induced in OF. Further analyses showed an increase in GC number and a decrease in apoptosis and caspase3/7 activity following ASB9 inhibition. Western blot analyses demonstrated that ASB9 induction in OF by hCG from 12h post-hCG through 24h was concomitant with a significant decrease in MAPK3/1 phosphorylation levels while pMAPK3/1 increased following ASB9 inhibition. These results provide strong evidence that ASB9 is a regulator of GC activity and function by modulating MAPK signaling pathway likely through specific binding partners such as PAR1, therefore controlling GC proliferation and contributing to GC differentiation into luteal cells.