Granulosa cells & three-dimensional scaffold: towards a follicle-like structure in the domestic cat

The in vitro development of feline oocytes is still suboptimal, especially in case of low competence gametes, such as cryopreserved or denuded oocytes, valuable resources for fertility preservation purposes. Modifications to media composition alone have not led to truly satisfactory results; therefore, since physical factors can positively influence the chemical conditions (1), a three-dimensional (3D) follicle-like structure could enhance meiosis resumption and further embryo development in vitro of poorly competent oocytes. In the domestic cat, the establishment of granulosa cells (GCs) culture was performed in a bidimensional (2D) system, and steroidogenesis was revealed along culture (2). However, since the follicular environment is three-dimensionally organized, the encapsulation of GCs in a 3D matrix might improve their self-organization, proliferation and endocrine functions. With the final aim of using these follicle-like structures for low competence oocyte IVM, functionality and efficiency of GCs encapsulated in biocompatible 3D matrix were evaluated during culture, and estradiol (E2) and progesterone (P4) concentrations were compared to those obtained from GCs cultured in a 2D monolayer. Cat granulosa cells were collected from isolated ovaries by enzymatic denudation (hyaluronidase 80 IU) of fresh COCs. Cells were processed as in (2) and then re-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]. Granulosa cells were in vitro cultured in a controlled atmosphere for 6 days in 3D follicle-like structures [obtained by one-step encapsulation in barium alginate (3)], or in 2D system (multiwell plates). Aliquots of conditioned CM were collected on days 2 and 6 and stored at -20°C until hormonal (E2 and P4) determination with an Enzyme Linked Fluorescent Assay (ELFA, MiniVIDAS®, Biomerieux, France). Log transformed hormonal concentrations were analyzed by a two-way ANOVA followed by Tukey test; the level of significance was set at p<0.05. Differences in hormonal production among days and systems of GCs culture were observed. Estradiol and P4 concentrations differed significantly during time, resulting higher on day 6 compared to day 2 (p=0.018) and day 0 (p<0.001) of culture. Steroidogenesis was obtained in 3D follicle-like structures as well as in 2D monolayers, although not to the same extent; the systems differed significantly only on day 6 (P4: 2.44 ± 1.28 ng/ml in 3D and 610 ± 607 ng/ml in 2D, p<0.001; E2: 173 ± 145 pg/ml in 3D and 26,709 ± 17,921 pg/ml in 2D, p=0.02). Present results underline that GCs in 3D microcapsules maintained their physiological features, as hormonal secretion. To test whether these 3D conditions could act as proper follicle-like structures, further experiments will be focused on the assessment of their nursing ability for the in vitro maturation of low competence oocytes. This research project was funded by EVSSAR Grant 2017. References (1) Luvoni GC. The never ending search of an ideal culture system for domestic cat oocytes and embryos. Reprod Domest Anim 2018; accepted.(2) Simsek O, Arikan S. Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro. Anim Reprod 2015;12:931-938. (3) 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.