Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes

Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop® vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p = .02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p = .03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro.