Gonadotropin regulation of ankyrin-repeat and SOCS-box protein 9 (ASB9) in ovarian follicles and identification of binding partners

Abstract

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.

Epigenetics of Skeletal Muscle-Associated Genes in the ASB, LRRC, TMEM, and OSBPL Gene Families

“…The studied ASB proteins, ASB5 and ASB11, display very strong evolutionary conservation of their primary structure with each other and with ASB9 ( Tables S6 and S7 ; [ 38 ]). These proteins have been implicated in regulating the size of the SkM and brain component (ASB5), gastrointestinal expansion and regenerative and embryonic myogenesis (zebrafish asb9/11), and spermatogenesis and ovarian granulosa cell maturation (ASB9) [ 38 , 52 ]. Unlike the evidence for the role of asb9/asb11 in myogenesis in zebrafish, ASB9 and ASB11 genes exhibited negligible expression in human Mb and Mt ( Table S5c ).…”

Interleukins’ expression profile changes in granulosa cells of preovulatory follicles during the postpartum period in dairy cows

“…Nevertheless, the results suggest that these receptors would activate different targets upon cytokines binding and they could play significant roles in signaling pathways controlling either granulosa cells (GC) proliferation and function, ovulation or formation of the CL. We have previously shown that the expression of several genes was dramatically reduced in the ovulatory follicle following the LH surge or hCG injection [30] , [31] . Cytokines whose receptors are induced by LH/hCG, such as the pro-inflammatory cytokine IL15 shown in this study, may be involved in various biological processes such as the ovulation process but could also hinder GC proliferation and follicular growth.…”

Ankyrin‐repeat and SOCS box‐containing protein 9 (ASB9) regulates ovarian granulosa cells function and MAPK signaling

“…Many studies have established that the LH surge positively regulates genes involved in inflammation, cellular movement, tissue remodeling, and angiogenesis, while switching off the expression of genes involved in metabolism and proliferation in multiple species (D. T. Liu et al, 2017; Wissing et al, 2014). The transcription of specific genes that control the growth of a bovine DF is therefore rapidly downregulated or silenced in granulosa cells (GC) as a result of LH‐mediated increases in intracellular signaling while LH/human chorionic gonadotropin (hCG) upregulates or induces the expression of genes involved in ovulation and luteinization as shown in different species including rodents or in the bovine species (Benoit et al, 2019; Christenson et al, 2013; Espey & Richards, 2002; Gilbert et al, 2011; Li et al, 2009; Lussier et al, 2017). We previously identified genes induced in GC of bovine ovulatory follicles following hCG and including Ankyrin‐repeat and SOCS‐box protein 9 (ASB9) (Lussier et al, 2017).…”