Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the large SOCS-box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation, identify binding partners in GC of bovine ovulatory follicles, and study its function. GC were obtained from small follicles (SF: 2–4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). Analyses by RT-PCR showed a 104-fold greater expression of ASB9 in GC of OF than in DF. Steady-state levels of ASB9 in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 hours after hCG injection showed a significant induction of ASB9 expression at 12 and 18 hours, reaching a maximum induction of 10.2-fold at 24 hours post-hCG as compared to 0 hour. These results were confirmed in western blot analysis showing strongest ASB9 protein amounts in OF. Yeast two-hybrid screening of OF-cDNAs library resulted in the identification of 10 potential ASB9 binding partners in GC but no interaction was found between ASB9 and creatine kinase B (CKB) in these GC. Functional studies using CRISPR-Cas9 approach revealed that ASB9 inhibition led to increased GC proliferation and modulation of target genes expression. Overall, these results support a physiologically relevant role of ASB9 in the ovulatory follicle by targeting specific proteins likely for degradation, contributing to reduced GC proliferation, and could be involved in the final GC differentiation into luteal cells.

Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

Highlights Interleukin (IL) 4 expression was significantly altered during the postpartum period. IL8 and IL15 were the most significantly induced during the postpartum period. Correlation between increased BHB levels and induction of proinflammatory cytokines. Interleukins analyzed were differentially regulated during follicular development. ILs induced during the post-partum period were also induced 24 h post-hCG injection.

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5’-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2mRNA 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 of bovine preovulatory follicles. This study aimed to further investigate TRIB2mRNA and protein regulation, to identify its binding partners, and study its function in granulosa cells of bovine dominant follicles. Granulosa cells (GC) 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 oestrous), ovulatory follicles 24 hours following injection of an ovulatory dose of hCG (OF), and corpus luteum (CL) at day 5 of the oestrous cycle. In addition to this in vivo model, an in vitro model of cultured GC was used for functional studies using the CRISPR‐Cas9 approach. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF and CL (P < 0.0001). Temporal expression of TRIB2mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 hours after hCG injection. There was a significant reduction of TRIB2 steady‐state mRNA levels in follicular walls, starting at 6 hours through 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 hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression (P < 0.05) while inhibition of TRIB2 using CRISPR‐Cas9 resulted in significantly reduced GC proliferation (P < 0.05). These results provide strong evidence that TRIB2 is a potent regulator of GC proliferation.Support or Funding InformationResearch supported by NSERC of Canada grant #RGPIN‐2018‐04516 to KN.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.