Nerve growth factor production in sheep antral follicles

Despite their continuous turnover, sheep antral follicles are always regularly innervated. The local production of neurotrophins is probably involved in the control of ovarian innervation. In this context the present investigation was designed to evaluate the ability of sheep antral follicles to produce neurotrophic factors. In the first part of the paper neurotrophic activity was measured in follicular fluid of sheep antral follicles of different size. Using an in vitro model the effect of gonadotrophins on neurotrophin production was then evaluated. The levels of neurotrophic activity in conditioned medium or follicular fluid and the kind of neurotrophin produced were determined by using the chicken embryo dorsal root ganglia test combined with an immunoneutralization step. Follicular fluid from medium-large follicles (>4 mm) contains high levels of NGF (240-250 ng/ml), whereas the factor is nearly undetectable in small follicles (<3 mm) and in early atretic follicles. Experiments in vitro based on the culture of follicle shells for 12 hr confirmed that medium-large follicles can produce NGF. The production is strictly dependent on gonadotrophin stimulation. When gonadotrophins were not added or were added separately, no detectable levels of neurotrophic activity accumulated in medium. By contrast, in the presence of both LH and FSH the production of NGF became apparent showing a clear dose-response behavior. In addition, this production increased progressively with increasing follicle size from 4 to >5 mm up to values of about 60 ng/follicle, whereas follicles with a diameter of less than 3 mm were insensitive to gonadotrophins stimulation and did not produce significant amount of NGF. The data presented demonstrate that sheep follicles produce relevant amounts of NGF as long as the correct hormonal milieu is provided. Under these conditions the production of the NGF increases with increasing follicle size. This may be responsible for the rapid innervation of the wall of growing follicles and/or take part in other non-neural processes that are generally attributed to gonadotrophin stimulation.