Overcoming flat biology: three-dimensional follicle-like structures for domestic cat vitrified oocytes

Follicle-like structures are a combination of living cells and semipermeable 3D matrices, in which physical (3D systems) and chemical (cell-secreted molecules) factors create an in vivolike microenvironment for the in vitro development of female gametes (1, 2). This artificial and viable milieu, successfully applied for fresh oocytes, could be even more useful for stressed gametes, as the low competence vitrified oocytes (VOs). Key players of fertility preservation efforts, VOs poorly achieve full maturation because their inner competence is severely impaired by unavoidable cryodamages also involving their surrounding cumulus cells. The replacement of damaged somatic compartment with species-specific follicle-like structures could restore the developmental competence of VOs. Thus, in this work the creation of a follicle-like structure of granulosa cells (GCs) in 3D barium alginate microcapsules was aimed at improving the nuclear competence of domestic cat VOs. Therefore, a comparison with “flat” GCs culture (monolayer) was performed to test whether 3D spatial organization could be more adequate in preserving GCs nursing ability for stressed gametes. Isolated ovaries (n. 22) from domestic queens were minced to retrieve fresh oocytes that were enzymatically denuded (hyaluronidase 80IU) to collect GCs. After purification (3), GCs were suspended in culture medium [CM=TCM199 supplemented with 0.1% BSA, 5% FBS, 0.5 IU FSH + 0.5 IU LH, 10 µg/mL 22(R)-hydroxycholesterol, 25 ng/ml epidermal growth factor, 0.1% ITS premix (1.0 µg/mL insulin; 0.55 µg/mL transferrin; 0.5 ng/mL sodium selenite), 10- 7 M androstenedione], and the one-step encapsulation technique in barium alginate (2) was performed to obtain the 3D follicle-like structures. Granulosa cells capsules (3D system) or monolayers (2D) were in vitro cultured in CM at controlled atmosphere (38.5°C and 5% CO2 in air) for 2 or 6 days and used as artificial milieu for VOs IVM. Vitrified oocytes were obtained by Cryotop protocol (Kitazato Co., Fujinomiya, Japan) from 127 fresh COCs, and after warming only viable gametes (stained by FDA/PI, n. 112) were in vitro matured (24 h) in 3D and 2D system, either on day 2 or on day 6 of GCs culture. Chromatin configurations were determined by bis-benzimide (Hoechst 33342; Sigma) staining, and data were analyzed by Chi-square test, with the level of significance set at p≤0.05. Very encouraging meiosis resumption rates were obtained in VOs matured in 3D follicle-like structures (45.5%) and monolayer (56.7%) on day 2 of GCs culture. Prolonged culture of GCs for 6 days severely impaired the nursing ability of monolayer (21.7%, p=0.007), but not that of 3D follicle-like structures (26.7%, p=0.07), that supported higher proportions of fullmaturational (TI-MII) stages (17.4%) than GCs cultured for 2 days (0%). Culturing GCs for 2 days allowed lower VOs degeneration rates than 6 days (p=0.001). The ideal culture environment for low competence VOs has to combine physical and chemical factors and 3D follicle-like structures gave promising results for the restoration of VOs fertility potential, but further improvements are needed to promote GCs polarization and self-assembly into tissue-like structures. This research project was funded by EVSSAR Grant 2017. (1) Luyckx V, Dolmans MM, Vanacker J et al. A new step towards the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. Fertil Steril, 2014; 101: 1149–56. (2) Vigo D, Villani S, Faustini M et al. Follicle-like model by granulosa cell encapsulation in a barium alginateprotamine membrane. Tissue Eng, 2005; 11: 709-14. (3) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod, 2015; 12: 931-38.