Department of Applied Developmental Biology, Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki, Kanagawa, Japan; Department of Biochemistry, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.
Department of Oncology, Juntendo University Nerima Hospital, Nerima-ku, Tokyo, Japan.
PLoS One. 2014 Apr 21;9(4):e95560. doi: 10.1371/journal.pone.0095560. eCollection 2014.
Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1-2 h, 2-4 h, 4-6 h, 6-8 h, and 8-10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1-2 h was lowest among groups at 2-4 h, 4-6 h, 6-8 h, and 8-10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p>0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets.
胞质内单精子注射（ICSI）是一种治疗男性不育的重要方法，已应用于精子介导的基因转移的转基因技术中。然而，在作为生物医学转化研究模型的小型非人类灵长类动物——普通狨猴中，尚未有研究描述使用 ICSI 成功产生后代。在这项研究中，我们研究了体外成熟的狨猴卵母细胞的囊胚发育和随后的活产后代阶段，并通过 ICSI 进行受精。为了研究进行 ICSI 的最佳时机，校正不成熟的卵母细胞在体外成熟，并在第一极体（PB）挤出后 1-2 小时、2-4 小时、4-6 小时、6-8 小时和 8-10 小时的不同时间点进行 ICSI。然后，成熟的卵母细胞随机分为两组：一组用于体外受精（IVF），另一组用于 ICSI。为了研究 ICSI 后胚胎的体内发育，将 6-8 细胞期胚胎和囊胚非手术转移到受体狨猴中。尽管在第一极体挤出后进行 ICSI 的胚胎受精率在不同时间点之间没有观察到显著差异，但在 2-4 小时、4-6 小时、6-8 小时和 8-10 小时组中，1-2 小时的囊胚率最低。与 IVF 相比，ICSI 的受精率高于 IVF（p>0.05）。ICSI 和 IVF 的囊胚分裂率没有显著差异。在移植 37 个 ICSI 囊胚后，20 名受体中有 4 名怀孕，而在移植 21 个 6-8 细胞期 ICSI 胚胎后，8 名受体中有 3 名怀孕。成功产下了 4 只健康的后代，它们生长正常。这是首例通过 ICSI 产生的狨猴后代，表明 ICSI 是一种有效的狨猴受精方法。
