beta-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells
- Valderrama, Ximena - Ulloa Leal, Cesar - Silva Jiménez, Mauricio - Goicochea, Jose - Apichela, Silvana - Arganaraz, Martin - Sari, Luciana - Paiva, Luis - Ratto, Vicente Francisco - Ratto, Marcelo Hector
- Datos de publicación:
- FRONTIERS IN VETERINARY SCIENCE,Vol.7,,2020
- MAPK - U0126 - corpus luteum - follicle - STAR
- Migración Web of Science 
- The beta-nerve growth factor (beta-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama beta-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with beta-NGF, beta-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of beta-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3 beta-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. beta-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that beta-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that beta-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.