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Mice can be cloned from cultured, non-cultured, adult-, fetus-, male-, and female-derived somatic cells at the present day. However, the efficiency of somatic cell cloning is approximately 1–2%, and even in individuals developed to full term, many abnormalities were often shown in cloned mice. Although even the technique itself remains imperfect, embryonic stem (ES) cell lines can be generated from adult somatic cells via nuclear transfer. These cells, which are called ntES cells, exhibit full pluripotency in which they can be differentiated along prescribed pathways in vitro (to produce, for example, dopaminergic neurons) and contribute to the germ line following injection into blastocysts. The rate of establishment of ntES cell lines has been examined for sex and various cell types. This review explains recent advances in mouse cloning to illustrate the strengths and promise of this technique in the study of mammalian biology and biomedicine.
The clinical benefit of Blasotcyst transfer (BT) in IVF-ET is still controversial. Recent study elucidated the benefit of BT to reduce ectopic pregnancy in assisted reproduction. Retrospective analysis of pregnancies with BT showed lower ectopic pregnancy rate compared to the cleaved stage transfer, especially among patients with tubal factor. There could be few chance of embryo migration into the tube after BT because of a limited period from transfer to implantation, and a reduced frequency of uterine contraction at the time of transfer.
In order to evaluate the physiological events in early stage of human embryos after intracytoplasmic sperm injection (ICSI), we have developed new embryo culture system on the stage of inverted microscope as a time-lapse cinematography. Time-lapse recording (Exposure time: 50 ms; Interval: 2 mins) commenced shortly after ICSI and continued up to 40 h. We examined a total of 65 oocytes obtained from 62 patients with ICSI. Fifty five (84.6%) of 65 oocytes showed 2PN/2PB fertilization and all of which cleaved. Out of 55 embryos cleaved, 42 (76.4%) developed into morphologically good quality embryos (GQE), while remaining 13 embryos were classified as poor quality embryos (PQE). There was no significant difference in the time course required for physiological events during early stage of embryos between GQE and PQE. Although no significant difference was observed in the pattern of nucleolar precursor body (NPB) distribution between GQE and PQE, the appearance of cytoplasmic halo during 2PN stage of embryos was significantly related to embryo quality (p<0.05). This study indicates that cytoplasmic halo is a new parameter in the evaluation of good quality embryo. Furthermore, time-lapse cinematography is an excellent tool in elucidating the physiological phenomenon in the early stage of human embryos.
The ultra-rapid cooling vitrification method, which accelerates cooling and warming rates of the specimens by minimizing the volume of the vitrification solution and result in little loss of the cell viability during storage in liquid nitrogen, has been established. The method does not require expensive freezing machineries, and can avoid the time consuming in routine works for its simple protocol. Even matured oocytes, which have been known to their high cryo-sensitivity, can survive through this vitrification procedure. Recently some commercial vitrification containers for ultra-rapid cooling, e.g., Cryotop and Cryotop-animal, have been widely provided. Therefore, they realize general use of ultra-rapid cooling vitrification of oocytes and embryos in human IVF cycles and in animal science. The detail procedure of the ultra-rapid cooling vitrification method is introduced in this article.
Recent advances in culture systems have made it possible to develop IVF embryos into blastocysts (BLs) easily, and BL transfer has proven effective for increasing the pregnancy rate. Therefore, cryopreservation of BLs has become an important technique. However, the cryopreservation of human BLs using both slow freezing method and vitrification with straws resulted in disappointing survival rates. An innovative strategy to circumvent the problem of intracellular ice formation in less permeating embryos is ultra-rapid vitrification, in which embryos are vitrified with a minimal volume of a vitrification solution using minute tools such as cryoloop. Since we started this vitrified BL program using cryoloop, 229 clinical pregnancies (44.1%) and 291 implantations (29.0%) out of 519 BLs transferred cycles were obtained. 147 babies were born in 108 deliveries. Miscarriage rate is 22.7% (52/229). We have applied assisted hatching (AHA) of warmed BLs in order to improve the implantation and pregnancy rates, because cryopreservation including vitrification induces hardening of the Zona pellucida (ZP), which may impair spontaneous hatching after warming. Moreover, we have also applied artificial shrinkage (AS) of the blastoceoleic cavity of expanded or larger stage of BLs in order to increase the survival rate, because the results of our vitrified BL program showed that the efficiency of the vitrification method was dependent on the stage of BL development and was negatively correlated with the size of the blastocoele.
In mammals, both maternal and paternal genomes are required to accomplish normal development to term, because parent-specific epigenetic modification of the genome is imposed during gametogenesis. This leads to inequivalent expression of imprinted genes sorely from the maternal and paternal allele. Production of a viable parthenogenetic mouse individual proved that genomic imprinting governs mammalian ontogeny.