Production of mouse offspring from inactivated spermatozoa using horse PLCζ mRNA

Yamamoto, Y; Hirose, Naoki; Kamimura, Satoshi; Wakayama, Sayaka; Ito, Junya; Ooga, Masatoshi; Wakayama, Teruhiko

doi:10.1262/jrd.2019-043

Cited by 12 publications

(14 citation statements)

References 35 publications

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“…The washed zygotes were then cultured in CZB at 37°C with 5% CO 2 until use. ICSI was performed as described previously [ 13 , 17 , 18 ]. To disperse the cumulus cells, the COCs were treated with 0.1% bovine testicular hyaluronidase for 3 min.…”

Section: Methodsmentioning

confidence: 99%

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

et al. 2022

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Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80 °C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80 °C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80 °C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.

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Section: Methodsmentioning

confidence: 99%

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

et al. 2022

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“…We also confirmed whether we might have accidentally injected FD elongated spermatids rather than round spermatids. Spermatozoa and elongated spermatids already contain sperm factors, such as PLC ζ , that activate the oocytes after fertilization/injection 30 , 31 . Because FD treatment does not cause loss of sperm factors 4 , the oocytes should be activated by sperm factors if FD elongated spermatids are injected instead of round spermatids.…”

Section: Resultsmentioning

confidence: 99%

Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

Wakayama

Ito

Ooga

et al. 2022

Sci Rep

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Mouse cloning by nuclear transfer using freeze-drying (FD) somatic cells is now possible, but the success rate is significantly lower than that of FD spermatozoa. Because spermatozoa, unlike somatic cells, are haploid cells with hardened nuclei due to protamine, the factors responsible for their tolerance to FD treatment remain unclear. In this study, we attempt to produce offspring from FD spermatid, a haploid sperm progenitor cell whose nuclei, like somatic cells, have not yet been replaced by protamine. We developed a method for collecting FD spermatids from testicular suspension. Despite the significantly lower success rate than that of FD spermatozoa, healthy offspring were obtained when FD spermatids were injected into oocytes. Offspring were also obtained from FD spermatids derived from immature male mice that had not yet produced spermatozoa. These results suggest that nuclear protaminization, rather than haploid nuclei, is one of the key processes responsible for tolerance to FD treatment.

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“…ICSI was performed as previously described [ 12 , 13 ]. Briefly, for the microinjection of spermatozoa, 1–2 µl of the sperm culture solution were directly moved to the injection chamber.…”

Section: Methodsmentioning

confidence: 99%

Improvement of a twice collection method of mouse oocytes by surgical operation

Inoue

Harada

Wakayama

et al. 2020

Journal of Reproduction and Development

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Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

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Production of mouse offspring from inactivated spermatozoa using horse PLCζ mRNA

Cited by 12 publications

References 35 publications

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Mouse <i>in vivo</i>-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

Improvement of a twice collection method of mouse oocytes by surgical operation

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