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

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.

Differential regulation of Janus kinase 3 (JAK3) in bovine preovulatory follicles and identification of JAK3 interacting proteins in granulosa cells.

BACKGROUND: Janus kinase 3 (JAK3) is a member of the membrane-associated non-receptor tyrosine kinase protein family and is considered predominantly expressed in hematopoietic cells. We previously identified JAK3 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. The present study aimed to further investigate JAK3 regulation, to identify protein binding partners and better understand its mode of action in bovine reproductive cells. RESULTS: GC were obtained from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). RT-PCR analyses showed greatest expression of JAK3 in GC of DF, while JAK3 expression was downregulated in OF (P < 0.0001). In addition, there was a 5- and 20-fold reduction of JAK3 steady-state mRNA levels in follicular walls, respectively at 12 and 24 hours post-hCG as compared to 0 h (P < 0.05). Similarly, JAK3 expression was downregulated by the endogenous LH surge. These results were confirmed in western blot analysis showing weakest JAK3 protein amounts in OF as compared to DF. Yeast two-hybrid screening of a DF-cDNA library resulted in the identification of JAK3 partners in GC that were confirmed by co-immunoprecipitation and included leptin receptor overlapping transcript-like 1 (LEPROTL1), inhibin beta A (INHBA) and cyclin-dependent kinase inhibitor 1B (CDKN1B). In functional studies using bovine endometrial cells, JAK3 increased phosphorylation of STAT3 and cell viability, while the addition of JANEX-1 inhibited JAK3 actions. CONCLUSION: These results support a physiologically relevant role of JAK3 in follicular development and provide insights into the mode of action and function of JAK3 in reproductive tissues.