The effect of seminal plasma β-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

Abstract

Background Nerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. Methods Experiment I: Female llamas ( n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF ( n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas ( n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas ( n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h ( n = 4 replicates) and progesterone secretion at 48 h ( n = 4 replicates) after LH or β-NGF treatment. Results Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro ...

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

“…Primary culture of llama granulosa cells was performed as previously described (10). Chemicals and reagents were purchased from Sigma-Aldrich Co., St. Louis, MO, USA, unless otherwise stated.…”

“…Five biological samples were used in both gene expression and progesterone secretion experiments (n = 15 llamas in each experiment); for each time and treatment, each biological sample was plated in 4 wells (i.e., experimental replicates) and treated as indicated above. For gene expression, cells were treated for 10 or 20 h; this time course was based on a previous report (10), in which significant changes in mRNA expression of STAR and VEGFA in primary culture of granulosa cells treated with β-NGF were detected.…”

“…Primers for PCR were designed using Primer Express Software (PE Biosystems, Foster City, CA, USA). Llama primers for steroidogenic enzymes, VEGFA, and RPLP0 transcripts and their expected size of amplified products and sequences have already been tested and previously described (9,10). The accession numbers of gen data are shown in Supplementary Table 1.…”

“…The presence of NGF and its high-affinity receptor trkA in granulosa, theca, and luteal cells of mammalian species (11)(12)(13) suggests that the luteotrophic effect of β-NGF may be exerted not only by the prolonged LH secretion pattern, but also directly by acting at the ovarian level. In a recent study, the addition of purified llama β-NGF upregulated the expression of STAR and vascular endothelial growth factor (VEGFA) transcripts in primary culture of llama granulosa cells (10).…”

“…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). Also, systemic administration of purified llama β-NGF induced a rapid shift from estradiol to progesterone synthesis in the preovulatory follicle in llamas as evidenced by the in vivo increase of the progesterone/estradiol ratio in the follicular fluid and upregulation of genes related to progesterone production (10).…”