Regulation and Functional Studies of Tribbles homolog 2 (TRIB2) in Ovarian Granulosa Cells

Abstract

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2 mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate TRIB2 mRNA and protein regulation, to identify its binding partners, and study its function in GC of bovine dominant follicles. Granulosa cells were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the estrous), ovulatory follicles (OF) 24 h following injection of an ovulatory dose of hCG, and corpus luteum (CL) at day 5 of the estrous cycle. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF (P < 0.0001). Temporal expression of TRIB2 mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 h after hCG injection. There was a 20‐ and 166‐fold reduction of TRIB2 steady‐state mRNA levels in follicular walls, respectively at 6 and 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by LH/hCG at the protein level. Yeast two‐hybrid screening of a DF‐cDNA library as well as functional studies using the CRISPR‐Cas9 approach are currently being used to identify TRIB2 binding partners and its function in GC. These results support a physiologically relevant role of TRIB2 in follicular development and GC function.Support or Funding InformationResearch supported by internal funds from Université de Montréal to KN and NSERC of Canada grant # 104199 to JGL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.