Regulation in vivo and in vitro of G Protein-Coupled Receptor 34 (GPR34) mRNA in Ovarian Granulosa Cells of Cattle and its Role in Steroidogenesis
Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GC) of cystic follicles vs. normal dominant follicles of cattle. The present experiments were designed to determine if: 1) GPR34 expression in GC changes during normal follicular development in estrogen-active (EA) and estrogen-inactive (EI) follicles of cattle, 2) hormones that have been shown to influence steroidogenesis such as IGF-I and FSH regulate expression of GPR34 mRNA, and 3) GPR34 ligands function to regulate GC function. In Exp 1, estrous cycles of non-lactating Holstein cows were synchronized and ovariectomized on either day 3 or 6 after ovulation; a 2 x 2 factorial ANOVA (day 3 vs. 6; EA vs. EI) indicated that GPR34 mRNA abundance in GC increased (P < 0.05) from 6.1 to 14.1 ± 4.3 relative mRNA units between day 3 (n = 5 cows) and 6 (n = 5 cows) post-ovulation but did not differ (P > 0.10) between EA (n = 15) and EI (n = 23) follicles. In Exp 2, ovaries were collected at a local slaughterhouse and GC were isolated and treatments applied in vitro for 24 h; a 2 x 2 factorial ANOVA (+/- IGF-I with +/- tumor necrosis factor (TNF)-α) indicated that IGF-I increased (P < 0.05) GPR34 expression from 3.5 to 7.8 ± 0.3 relative mRNA units and TNFα decreased (P < 0.05) the IGF-I-induced GPR34 mRNA abundance to 6.0 ± 0.4 relative mRNA units in small-follicle (1-5 mm) GC (n = 3 replicates and GC pools/treatment). Also in Exp 2, IGF-I and TNFα decreased (P < 0.05) GPR34 expression from 17.1 to 9.4 and 2.2 relative mRNA units, respectively, in large-follicle (8-22 mm) GC, indicating a change in GPR34 responsiveness occurs during follicle development. Other in vitro experiments (Exp 3-7; n = 3 replicates) revealed that treatment with IL-2, prostaglandin E2 and angiogenin decreased (P < 0.05) GPR34 expression by 62%, 19% and 21%, respectively, whereas FSH, IL-6 and cortisol did not affect (P > 0.10) GPR34 expression in small-follicle GC. In Exp 8, the presumed ligand of GPR34, L-a-lysophosphatidyserine (LPPS), increased GC numbers by 1.74-fold and estradiol production by 5.4-fold (0.19 vs. 1.03 ng/105 cells/24 h) in small-follicle GC (n = 3 replicates). For the first time, we have identified the lysophosphatidylserine receptor GPR34 as a developmentally and hormonally regulated gene in GC, the ligand of which enhances GC proliferation and estradiol production.
Keywords: G protein coupled receptor; granulosa cell; cattle.