Highly successful production of viable mice derived from vitrified germinal vesicle oocytes.

The vitrification of immature germinal vesicle (GV) oocytes is an important way to preserve genetic resources and female fertility. However, it is well known that cryopreserved GV oocytes have very poor developmental ability and that further improvement in this technique is needed. We previously reported the successful vitrification of matured mouse oocytes with enclosed cumulus cells using the calcium-free vitrification solution supplemented with ethylene glycol (EG) by the minimal volume cooling (MVC) method. In this study, we investigated whether our method is applicable to the vitrification of mouse oocytes at the GV stage (GV oocytes). Following maturation and fertilization in vitro, vitrified GV oocytes showed high survival (94.3 ± 2.0%) and maturation (94.3 ± 2.1%) rates. Although the fertilization and blastocyst rates of vitrified oocytes (fertilization: 46.6 ± 4.9% and blastocyst: 46.6 ± 3.0%) were significantly lower than those of fresh oocytes (fertilization: 73.0 ± 7.1% and blastocyst: 71.6 ± 8.0%) (P < 0.01), there were no differences in the ability to develop to term between fresh oocytes (50.0 ± 8.4%) and vitrified oocytes (37.5 ± 4.6%) (P > 0.05). In conclusion, we here show, for the first time, the efficient production of live mice derived from vitrified GV oocytes.

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine.

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

Ethylene glycol-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.