beta-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells
beta-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells
Authors
Valderrama, Ximena
Ulloa Leal, Cesar
Silva Jiménez, Mauricio
Goicochea, Jose
Apichela, Silvana
Arganaraz, Martin
Sari, Luciana
Paiva, Luis
Ratto, Vicente Francisco
Ratto, Marcelo Hector
Ulloa Leal, Cesar
Silva Jiménez, Mauricio
Goicochea, Jose
Apichela, Silvana
Arganaraz, Martin
Sari, Luciana
Paiva, Luis
Ratto, Vicente Francisco
Ratto, Marcelo Hector
Authors
Date
Datos de publicación:
FRONTIERS IN VETERINARY SCIENCE,Vol.7,,2020
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Abstract
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.