Culture of preantral follicles in poly(ethylene) glycol-based, three-dimensional hydrogel: a relationship between swelling ratio and follicular developments

Abstract

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)-based hydrogels, creating a three-dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2 week-old mice were cultured in a crosslinked four-arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7-15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60-80%; p = 0.76). Significant (p < 0.04) model effects, however, were detected in the maturation and developmental competence of the follicle-derived oocytes. A swelling ratio of > 21.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel-based, 3D microenvironment.

Keywords: PEG-based hydrogel; mouse; preantral follicle; swelling ratio; three dimensional culture.

Figure 1

Growth of early secondary follicles in a 3D PEG-based hydrogel of different softnesses (as represented by swelling ratio). The follicles were cultured for 9 days in αMEM-based follicle culture medium and their growth was determined by the percentage of the follicles that formed pseudoantrum. No significant (p = 0.76) model effect was detected. Data are indicated as mean ± SE

Figure 2

Morphological change of early secondary follicles growing in 3D PEG-based hydrogel and development of the follicle-derived oocytes after chemical activation. The preantral follicles embedded into the hydrogel and plated on culture dish were cultured for 9 days in αMEM-based follicle culture medium and matured by treatment with human chorionic gonadotrophin and epidermal growth factor. (a–d) Development of preantral follicles in the 3D PEG-based hydrogel: (a) the follicular stage of the follicle at 2 days after culture; (b) the diffuse stage of the follicle at 5 days after culture; (c) the pseudoantral stage of the follicle at 9 days after culture; (d) extrusion of the cumulus–oocyte complexes (arrow) in the hydrogel (*) was notable in the pseudoantral follicle. (e–h) Development of preantral follicles in the conventional, 2D culture system; (e) day 2 after culture; (f) day 5 after culture; (g) the pseudoantral stage of the follicle 9 days after culture; and (h) cumulus-oocyte-complexes (arrow). Overall, granulosa cells of the preantral follicle cultured in the 2D system showed further expansion, compared with those of the follicles cultured in the 3D system. (i) Derivation of the mature oocyte: (j) development to two- to four-cell embryos, 28 h after oocyte activation; and (k) development to the blastocyst stage, 124 h after oocyte activation. Scale bar = 200 µm