| Summary: | The growth of ovarian follicles is well documented in terms of hormonal control, however the fluid dynamics of antral follicle growth is less well understood. Aquaporins (AQP) are transmembrane water channels which facilitate the passive movement of water. In mammals 13 AQPs have been identified in a vast range of tissue types. In terms of ovarian AQPs there is currently a paucity of information. Recent studies in rat, pig and human have revealed the presence of ovarian AQPs, but in doing so have also highlighted a lack of consensus on AQP-type and location. The main aim of this study was to investigate the potential role of AQP in antral follicle growth. The first objective was to identify tissue expression and localisation of AQP proteins in the bovine ovary. This required the characterisation of a panel of polyclonal serum antibodies. Immunohistochemistry (IHC) was then used to identify AQPs and to detect changes in protein expression during follicular growth. Aquaporin 1 was found in most vascular endothelium; it was plentiful in capillaries surrounding antral follicles and increased in abundance as vasculature increased with follicle development. Aquaporin 2 was not found in bovine ovarian tissue and the remaining antibodies were deemed too nonspecific to permit reliable conclusions. The second objective was to investigate, via RT-qPCR, mRNA levels of AQPs in granulosa and theca cells isolated from preantral, through to large preovulatory follicles. Transcripts of AQP 1, -3, -4, -5, -7 and -9 were detected in both the granulosa and theca of antral follicles with expression levels generally higher in theca. The expression of AQP 1, -5, -7 and -9 was initiated in the theca cells of early antral follicles. Finally, swelling assays using bovine and porcine granulosa cells demonstrated the ability of granulosa to swell. This was inhibited by HgCb which is characteristic of AQP function. Porcine granulosa cells incubated with androgen swelled by 27%, this effect was inhibited by hydroxyflutamide. Protein analysis of AQP5 via IHC and Western blotting showed possible up-regulation in porcine follicles. RTqPCR did not reveal AQP5 transcript, the reasons for this currently remain unclear. In conclusion, this study has revealed for the first time the involvement of AQPs in bovine ovarian follicle development, with AQPI, -5, -7 and -9 potentially playing a pivotal role in antrum formation. The AQP system in porcine granulosa cells is androgen sensitive however identification of the AQP/s responsible needs further investigation. The evidence from this investigation suggests a role for AQPs in facilitating follicle growth. The stage-dependent expression of certain AQPs and the androgen sensitive porcine granulosa cells reveals the possibility that AQPs may be modulated by follicle-regulating hormones.
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