Immunolocalization of Orexin A and its receptors in the different structures of the porcine ovary

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within “health”, proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock.