105 P75 Neuronal Cells and Fibers in the Bovine Ovary

Abstract

Neurotrophins are molecules involved in the development and survival of neurons and its cellular projections. Results of recent studies have implicated the local role of the high affinity neurotropin receptor, trkA, in bovine ovarian follicle selection and early luteogenesis (Carrasco et al. 2016 Reprod. Biol. Endocrinol. 14, 47), but innervation and neuropeptide control remains an unexplored aspect of ovarian function. P75 is the low-affinity receptor for all neurotrophins and is expressed in ovarian tissue. The objective of this study was to explore the distribution of P75 neurons and fibres within the ovary and to examine the relationship of these components with follicular development. The ovaries of cows (n = 5) were collected at the time of slaughter, 36 h after induced luteolysis (i.e. proestrus). The ovaries were fixed in 4% paraformaldehyde for 48 h, and samples from the ovarian hilus, medulla, and cortex (3 blocks per ovary) were cryo-sectioned (20–50 µm). Tissue sections were incubated for 48 h with a rabbit antibody against rat P75 or a mouse monoclonal antibody against neurofilament. Immunodetection was visualised by an amplification procedure with horseradish peroxidase using nickel DAB as a chromogen. Sections were counterstained with nuclear fast red for follicle identification. Immunoreactive cell bodies were counted in 10 to 20 fields (40×) per section, and data were expressed based on ovarian areas (cortex, medulla, or hilus) as an average count per 40× field per animal. Data among ovarian regions were compared by ANOVA; differences were considered significant when P < 0.05. Antral follicles ≤5 mm displayed strong immunoreactivity in the theca layer, without reaction in the granulosa cells. In contrast, preovulatory follicles were devoid of P75 immuno-reactivity in the theca layer. Oval P75 immunoreactive neuron-like cells were present in all ovarian areas studied. The neuronal nature of the P75 immunoreactive cells was confirmed by the presence of a similar pattern when adjacent sections were stained for neurofilaments, a protein characteristic of neurons. In the stroma of the ovarian cortex and medulla, neurons were present individually (scattered) rather than grouped; however, a dense network of neurons and fibres was detected immediately beneath the ovarian surface epithelium. No differences between the cortex, medulla, and hilus were found in the mean number of immunoreactive cells (10.6 ± 2.8, 14.4 ± 3.6 and 13.9 ± 2.0 cells/40× field, respectively). Immunoreactive neuron-like cells and fibres were in close proximity to blood vessels in the ovarian medulla. Corpora lutea were devoid of P75 immunoreactivity. In conclusion, results document the existence of a neuronal network in the bovine ovary, displaying an association with follicles at different stages of development. The abundance of neuronal components (i.e. neuron cell bodies and axons) in the ovarian stromal and surface epithelium implies a role of innervation (either extrinsic or intrinsic) in the control of ovarian follicular development and function.

Research was supported by the Natural Sciences and Engineering Research Council of Canada.