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.

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.

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.

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.