?-NGF stimulates steroidogenic enzyme and VEGFA gene expression, and progesterone secretion via ERK 1/2 pathway in primary culture of llama Granulosa cells
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Authors
SILVA JIMENEZ, MAURICIO
Valderrama, Ximena P.
Ulloa-Leal, Cesar
Silva, Mauricio Erciario
Vargas, José Goicochea
Apichela, Silvana A.
Argañaraz, M. E.
Sari, Luciana M.
Paiva, Luis
Ratto, Vicente Francisco
Ratto, M. Hector
Valderrama, Ximena P.
Ulloa-Leal, Cesar
Silva, Mauricio Erciario
Vargas, José Goicochea
Apichela, Silvana A.
Argañaraz, M. E.
Sari, Luciana M.
Paiva, Luis
Ratto, Vicente Francisco
Ratto, M. Hector
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Date
Datos de publicación:
10.3389/fvets.2020.586265
Keywords
Corpus Luteum - Cyp11a1 - Cyp19a1 - Follicle - Hsd3b1 - Mapk - Star - U0126 - 1,4 Diamino 1,4 Bis(2 Aminophenylthio) 2,3 Dicyanobutadiene - 3(or 17)beta Hydroxysteroid Dehydrogenase - Aromatase - Cholesterol Monooxygenase (side Chain Cleaving) - Luteinizing Hormone - Mitogen Activated Protein Kinase 1 - Mitogen Activated Protein Kinase 3 - Progesterone - Vasculotropin A - 1,4 Diamino 1,4 Bis(2 Aminophenylthio) 2,3 Dicyanobutadiene - 3(or 17)beta Hydroxysteroid Dehydrogenase - Aromatase - Cholesterol Monooxygenase (side Chain Cleaving) - Complementary Dna - Luteinizing Hormone - Messenger Rna - Mitogen Activated Protein Kinase 1 - Mitogen Activated Protein Kinase 3 - Nerve Growth Factor Beta Subunit - Nerve Growth Factor Receptor - Progesterone - Protein Tyrosine Kinase A - Vasculotropin A - Adult - Angiogenesis - Animal Cell - Animal Tissue - Article - Cell Culture - Controlled Study - Corpus Luteum - Down Regulation - Enzyme Activation - Enzyme Phosphorylation - Female - Follicular Aspiration - Gene Expression - Granulosa Cell - In Vitro Study - Llama - Luteinizing Hormone Blood Level - Male - Mapk Signaling - Mrna Expression Level - Nonhuman - Ovary Follicle - Ovary Tissue - Ovulation - Primary Culture - Progesterone Release - Progesterone Synthesis - Radioimmunoassay - Real Time Polymerase Chain Reaction - Rna Isolation - Rna Transcription - Semen Analysis - Steroidogenesis - Transvaginal Echography - Western Blotting
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Abstract
The beta-nerve growth factor (?-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 ?-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 ?-NGF,?-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 ?-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?-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. ?-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ?-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ?-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway. © 2021 Elsevier B.V., All rights reserved.
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Keywords
Corpus Luteum , Cyp11a1 , Cyp19a1 , Follicle , Hsd3b1 , Mapk , Star , U0126 , 1,4 Diamino 1,4 Bis(2 Aminophenylthio) 2,3 Dicyanobutadiene , 3(or 17)beta Hydroxysteroid Dehydrogenase , Aromatase , Cholesterol Monooxygenase (side Chain Cleaving) , Luteinizing Hormone , Mitogen Activated Protein Kinase 1 , Mitogen Activated Protein Kinase 3 , Progesterone , Vasculotropin A , 1,4 Diamino 1,4 Bis(2 Aminophenylthio) 2,3 Dicyanobutadiene , 3(or 17)beta Hydroxysteroid Dehydrogenase , Aromatase , Cholesterol Monooxygenase (side Chain Cleaving) , Complementary Dna , Luteinizing Hormone , Messenger Rna , Mitogen Activated Protein Kinase 1 , Mitogen Activated Protein Kinase 3 , Nerve Growth Factor Beta Subunit , Nerve Growth Factor Receptor , Progesterone , Protein Tyrosine Kinase A , Vasculotropin A , Adult , Angiogenesis , Animal Cell , Animal Tissue , Article , Cell Culture , Controlled Study , Corpus Luteum , Down Regulation , Enzyme Activation , Enzyme Phosphorylation , Female , Follicular Aspiration , Gene Expression , Granulosa Cell , In Vitro Study , Llama , Luteinizing Hormone Blood Level , Male , Mapk Signaling , Mrna Expression Level , Nonhuman , Ovary Follicle , Ovary Tissue , Ovulation , Primary Culture , Progesterone Release , Progesterone Synthesis , Radioimmunoassay , Real Time Polymerase Chain Reaction , Rna Isolation , Rna Transcription , Semen Analysis , Steroidogenesis , Transvaginal Echography , Western Blotting
Citation
10.3389/fvets.2020.586265