β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

Ximena Valderrama,Jose Goicochea,Vicente Francisco Ratto,Marcelo Hector Ratto,Mauricio Erciario Silva,Luciana Sari,Silvana Apichela,Luis Paiva,Martin Argañaraz,Cesar Ulloa-Leal

doi:10.3389/fvets.2020.586265

Abstract

The beta-nerve growth factor (β-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 β-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 β-NGF, β-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 β-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 3β-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. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Highlights

It is well-established that the beta-nerve growth factor present in the seminal plasma of llamas and alpacas is the essential signal inducing the luteinizing hormone (LH) surge and ovulation in these species [1]
Neither β-NGF nor LH affected the mRNA expression of cytochrome P450 aromatase (P450arom encoded by CYP19A1) that is involved in estradiol synthesis (Figures 1A, 2A)
We investigated whether purified llama βNGF exerts luteotropic effects on a primary culture of llama granulosa cells collected from preovulatory follicles

Summary

INTRODUCTION

It is well-established that the beta-nerve growth factor (βNGF) present in the seminal plasma of llamas and alpacas is the essential signal inducing the luteinizing hormone (LH) surge and ovulation in these species [1]. The preovulatory LH surge induced by purified llama β-NGF is more sustained than that the observed after GnRH administration [7, 8], suggesting that the luteotrophic effect of β-NGF could be due to this prolonged LH secretion profile In this sense, systemic administration of β-NGF increased CL vascularization and upregulated the expression of cytochrome P450 side-chain cleavage (P450scc encoded by CYP11A1) and steroidogenic acute regulatory protein (STAR) mRNA transcripts enhancing plasma progesterone concentrations during the early luteal phase in llamas [9]. We investigate the effect of β-NGF on steroidogenic enzymes and VEGFA gene expression as well as progesterone secretion and test whether these effects are mediated via ERK1/2 signaling pathway in vitro using a primary culture of granulosa cells collected from llama preovulatory follicles

MATERIALS AND METHODS

RESULTS

DISCUSSION

ETHICS STATEMENT