Abstract
Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral infection or twin production by blastomere separation, but adverse effects on development have been reported. It would therefore be extremely valuable if the embryo could be covered with a structure similar to that of the ZP. In this study, we sought to determine whether an agarose capsule could serve as a substitute for the ZP. Our results indicate that embryos derived from these agarose capsules were able to develop normally, and could be transplanted to obtain viable offspring, without having to remove the agarose capsule. Furthermore, before compaction, the agarose capsule embryos exhibited good freezing tolerance, and survival rate was extremely high compared to ZF embryos. Thus, agarose capsules represent a valuable tool for utilizing oocytes and embryos that lack a ZP, both in a clinical and livestock setting.
Highlights
Various developmental engineering methods such as in vitro fertilization, intracytoplasmic sperm injection (ICSI), and cryopreservation are applied in the fields of infertility treatment, animal reproduction, and rescue of endangered species
The agarose capsules had the same operability with a micromanipulator as a normal zona pellucida (ZP), and it was possible to cut the capsule with a blade or drill a hole with a piezo drive (Fig. 2A)
This study represents the first demonstration that agarose capsules can be used as a substitute for the ZP in ZF oocytes
Summary
Various developmental engineering methods such as in vitro fertilization, intracytoplasmic sperm injection (ICSI), and cryopreservation are applied in the fields of infertility treatment, animal reproduction, and rescue of endangered species During such processes, oocytes that lack a ZP are often encountered. No zygotes or two-cell-stage embryos that lacked a ZP survived storage in liquid nitrogen[8] It is, possible for embryos to develop normally without a ZP, and the live birth of twins from autologous ZF oocytes was reported in 20169. A similar method has been applied in bovine embryos, where the embryos were covered with a mixture of a sodium alginate solution gelled in a calcium chloride solution, termed a microcapsule This alginate encapsulation of bovine embryos does not affect in vitro development up to the blastocyst stage, but does significantly hinder the hatching process[12]. After placing oocytes into the agarose capsules, we examined embryogenesis after ICSI and assessed the freeze-resistant properties of the resulting embryos
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