The effect of preovulatory administration of b-NGF purified from llama seminal plasma on folicular 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-NGF will up-regulate steroidogenic enzymes and VEGF gene expression resulting in a change in the progesterone/estradiol ratio in the follicular fluid during the preovulatory stage in llamas. Methods: 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 in both time points. Results: 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 CYP19A1/P450 aromatase 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 (20h) significantly up regulated the mRNA expression of HSD3B whereas the expression of STAR and CYP11A1/P450scc where significantly up regulated only by mating (20h), or β-NGF at 10 or 20 h after treatment. VGEF was up regulated only in those llamas submitted to mating (10h) or treated with purified β- NGF (10 and 20h). Conclusions: Systemic administration of purified β-NGF from llama seminal fluid, induced a rapid shift from estradiol to progesterone production in the preovulatory follicle in llamas as a result of a change in steroidogenic gene expression. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β- NGF-treated llamas suggest local effects of seminal components, which could be direct or associated with LH increase. Finally, the angiogenic factor VEGF was significant up regulated after β-NGF treatment at all-time points.