Abstract
Oocyte cryopreservation is useful for preserving fertility and storing genetic resources. However, the small number of oocytes acquired using conventional treatment to induce superovulation and the reduction of fertility due to cryopreservation represent significant problems. Herein, we vitrified the oocytes derived through high-yield superovulation using inhibin antiserum and equine chorionic gonadotropin (IAS + eCG: IASe) and examined the yield of cryopreserved oocytes and survival rates relative to those of vitrified–warmed mouse oocytes derived through conventional superovulation using equine chorionic gonadotropin (eCG). Furthermore, we investigated the effects of N-acetyl cysteine on the fertility and developmental potential of vitrified–warmed oocytes derived using IASe. Compared with eCG, IASe increased the yield of cryopreserved oocytes and achieved equivalent survival rates. N-acetyl cysteine (0.5 mM) increased the fertilization rate of vitrified–warmed oocytes derived using IASe. Vitrification decreased thiol levels in the zona pellucida (ZP), while warming followed by N-acetyl cysteine treatment increased free thiol levels in ZP. Moreover, N-acetyl cysteine treatment recovered zona hardening by cleaving disulfide bonds and promoting the expansion of ZP. Two-cell embryos derived via in vitro fertilization using N-acetyl cysteine developed into normal pups through embryo transfer. Therefore, we developed an efficient technique for the production of cryopreserved oocytes using IASe through superovulation and found that N-acetyl cysteine improves the fertility of vitrified–warmed oocytes by cleaving the disulfide bonds and promoting the expansion of ZP.
Highlights
Oocyte cryopreservation is widely used for preserving genetic resources to breed animals for medical research and to support the infertility treatments in human [1]
We found that cysteine analogs such as N-acetyl cysteine (NAC), L-cysteine, Dcysteine, and reduced glutathione enhance the success of in vitro fertilization (IVF) employing freshly harvested oocytes as well as frozen-thawed or cold-stored mouse sperm [11,12,13]
The survival rates of IASe- and equine chorionic gonadotropin (eCG)-derived oocytes were comparable, and the reduced fertilization rate was recovered by NAC treatment
Summary
Oocyte cryopreservation is widely used for preserving genetic resources to breed animals for medical research and to support the infertility treatments in human [1]. Oocytes are collected from donors treated with hormones or drugs to induce superovulation and preserved with cryoprotective agents under liquid nitrogen. Embryos are produced through intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF), and the produced embryos are subsequently preserved or developed. Mouse oocytes can be cryopreserved using vitrification and slow freezing methods [2,3,4,5,6]. We previously developed a simple vitrification method using a highly concentrated vitrification solution [2, 7,8,9], which achieves a relatively high survival rate of mouse oocytes after cryopreservation. The number of embryos and pups derived from vitrified oocytes is insufficient because of the low yield of oocytes induced by conventional superovulation techniques (20–30 oocytes/mouse) and the reduced fertility of vitrified–warmed oocytes used for conventional IVF
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