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

Abstract

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.