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

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.

Development of a serum-free defined system employing growth factors for preantral follicle culture.

This study was conducted to evaluate if mouse preantral follicles can yield developmentally competent oocytes following culture in serum-free, defined medium. Donor follicles were obtained from 14-day-old B6CBAF1 mice, and cultured in α-MEM-Glutamax medium. The replacement of fetal bovine serum with knockout serum replacement (KSR) did not significantly reduce follicle growth or oocyte maturation in vitro, although it significantly reduced the development of oocytes after activation. Regardless of the replacement medium, follicle growth was not influenced by the addition of leukemia inhibitory factor (LIF). The addition of 100 ng/ml stem cell factor (SCF) to the KSR-supplemented serum-free medium significantly stimulated follicle development, which further improved blastocyst formation after oocyte activation. On Day 3 of culture, a significant increase in Bmp7 expression was detected in the SCF-containing medium compared with the serum-containing medium, whereas Gdf9 and Amh were increased in the serum-containing medium. A significant increase in estradiol production was detected under serum-free conditions, but minimal progesterone secretion was detected throughout the culture period. In conclusion, serum-free media can be used to optimize ovarian follicle cultures, and the addition of SCF is beneficial for deriving developmentally competent oocytes through follicle culture.

Generation of viable embryos and embryonic stem cell-like cells from cultured primary follicles in mice.

Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.

Improved viability of freeze-thawed embryonic stem cells after exposure to glutathione.

Adding a potent antioxidant, glutathione (GSH), to a cryoprotective solution consisting of dimethyl sulfoxide and ethylene glycol and/or postthaw culture medium significantly improved the postthaw viability of mouse embryonic stem cells. This effect, which was caused by a decrease in reactive oxygen species, was only induced by exposure of embryonic stem cells during cryopreservation.

Ultrastructural deformity of ovarian follicles induced by different cryopreservation protocols.

Ultrastructural deformities were monitored after cryopreservation of F1 (B6CBAF1) mouse ovaries by either slow freezing or vitrification. Vacuole formation in the ooplasm, zona pellucida, and the cytoplasm of follicular cells, and mitochondrial deformity were detected. These types of cryodamage demonstrated protocol specificity.