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

Gabriel Benoit,Kalidou Ndiaye,Aly Warma,Jacques G Lussier,Peter J Hansen

doi:10.1371/journal.pone.0212571

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.

Highlights

It is well documented that the cyclic ovarian activity results in profound modifications that require spatio-temporal coordination of proliferation, apoptosis and differentiation of various cell types within the ovarian follicle leading to changes in gene expression [1,2,3,4]
In order to investigate ASB9 mRNA expression and regulation in bovine follicles, total RNA extracts of bovine granulosa cells (GC) from small follicles (SF; 2–4 mm in diameter), dominant follicles obtained at day 5 of the estrous cycle (DF), ovulatory follicles isolated 24 hours post-hCG (OF), and corpora lutea obtained at day 5 of the estrous cycle (CL) were analyzed using RTqPCR
Because ASB9 transcript was expressed to high amounts in ovulatory follicles, the gonadotropin-dependent regulation of ASB9 mRNA during the periovulatory period was further investigated in ovulatory follicles using total RNA obtained from follicular walls of preovulatory follicles collected at 0, 6, 12, 18 and 24 hours post-hCG injection

Summary

Introduction

It is well documented that the cyclic ovarian activity results in profound modifications that require spatio-temporal coordination of proliferation, apoptosis and differentiation of various cell types within the ovarian follicle leading to changes in gene expression [1,2,3,4]. The transcription of specific genes that control the growth of a bovine dominant follicle is rapidly downregulated or silenced in GC as a result of LH-mediated increases in intracellular signaling [3, 6, 7] while LH upregulates or induces the expression of genes involved in ovulation and luteinization [8]. These observations demonstrate the importance of gene functional studies during the final stages of follicular development and ovulation to better coordinate the ovarian activity. In light of these results, we further investigated the regulation and function of ASB9 in GC of ovarian follicles

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