key: cord-0021865-mfokdxeb authors: Morishita, Nami; Ochi, Masanori; Horiuchi, Toshitaka title: History and prospects of intracytoplasmic sperm injection (ICSI) and the development of golden hamster ICSI embryos date: 2021-08-26 journal: Reprod Med Biol DOI: 10.1002/rmb2.12410 sha: 9df0662c1858f680b5948528fbaf7c17a2153bda doc_id: 21865 cord_uid: mfokdxeb BACKGROUND: Golden (Syrian) hamsters have many advantages for the study of reproductive biology and developmental biology, including a consistent estrous cycle, a stable superovulation response, and a short gestation period. However, there are serious difficulties in doing in vitro manipulations of hamster embryos, because they are very sensitive to various environmental factors. Thus, biotechnological researches of hamster embryos should be performed with high‐level skills of embryo manipulations. METHODS: The authors summarized the history of hamster intracytoplasmic sperm injection (ICSI) and introduced key points for hamster ICSI, which were found in our previous studies on the production of embryos by ICSI and offspring by embryo transfer. MAIN FINDINGS: The key points for hamster ICSI were in vitro manipulations under the light‐controlled environment, injection of acrosome‐less sperm heads into oocytes as soon as possible before spontaneous oocyte activation occurs, and determination of the optimal culture conditions. CONCLUSION: To our knowledge, there are no available reports on production of offspring from ICSI embryos in hamsters except our reports. Moreover, success rates of hamster ICSI remain very low. For the purpose of spreading hamster ICSI, it is necessary to make further researches to improve manipulation techniques and to resolve experimental problems. The second key to the success of hamster ICSI is to remove the acrosomes from spermatozoa. 8 When hamster oocytes are injected with acrosome-intact spermatozoa, they grow deformed and die. were separated from the tail by applying piezo-electric pulses. In human ICSI, high fertilization and pregnancy rates were achieved by injecting the entire motile spermatozoon, after immobilization. 25 The second culture medium we designed was a modification of HECM-9. Original HECM-9 is a simple composition medium (HECM-3) with 9 amino acids (0.01 mM each of asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, lysine, proline, and serine), 0.5 mM taurine, 3 µM pantothenate and 0.1 mg/ml PVA. 37 With this medium, Yamauchi et al. (2002) 8 showed that ICSI 2-cell embryos could develop into morulae, but not into blastocysts. Therefore, we needed to improve the original HECM-9 to induce ICSI 2-cell embryos to develop into blastocysts. We modified the amino acid content (0.1 mM each of alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, serine), and we substituted the 0.5 mM taurine and PVA with 5 mM taurine and 0.5 mg/ml human serum albumin (HSA) ( Table 2 ). With this modified medium (mHECM-9), in vivo-fertilized 2-cell hamster embryos developed into blastocysts at high rates (>90%). Moreover, in this medium, ICSI 2-cell hamster embryos developed to morulae and blastocysts, and after an embryo transfer, they produced offspring. 10, 11, 17 The time schedule we used for culturing ICSI embryos is shown in Figure 2 . Hamster ICSI is a useful model for improving ICSI technology. Using our in vitro culture system, most of in vivo-fertilized zygotes developed into blastocysts (about 80%). However, in vitrofertilized zygotes (such as with ICSI) could not developed into blastocysts at such a high rate (about 40%). 17 Reduced developmental competence of hamster ICSI-fertilized zygotes into blastocysts is dependent on the fertilization process by ICSI. This result suggested that the ICSI method has some problems that affect embryos before the pronuclear stage, for example, oocyte activation by sperm injection and the following male and female pronuclear formation. Currently, there are two types of human embryo culture media: sequential medium 43 and single medium. 44 Although there is much debate about the superiority of these, they are used differently, depending on laboratory efficiency and patient background. A continuous culture system for hamster embryo production has not been established, because we lack medium with a clear composition that can generate a high rate of development from pronuclear embryos. When a single culture medium for hamster embryos is invented that anyone can reproduce, it will definitely represent a breakthrough. A stress-free hamster embryo culturing method could lead to many new applications. Recently, new genome editing technology has been used to produce knockout (KO) hamsters without in vitro embryo development. The KO hamsters have provided new information, different from that gained previously in mouse models. 45, 46 Many transgenic hamsters can be produced with this technique, without the need for embryo manipulation in vitro. However, in vitro culture systems combined with IVF and ICSI will be needed to investigate issues related to embryo and fetal development. Exactly, there were many restrictions to produce transgenic hamsters due to the developmental block, because it is not easy to culture hamster embryos. In our laboratory, we produced one green fluorescent protein (GFP)-expressing ICSI The golden hamster is a useful animal for research on IVF/ICSI and embryo development, for example, due to evaluate the relationship between fertilization events and the subsequent embryo development. In addition, the use of hamsters in biomedical research might provide new knowledge that is difficult to gain in a mouse model, for example, they have recently attracted attention as a small animal model for SARS-CoV-2 infections and countermeasure development. 50 In the Nami Morishita, Masanori Ochi, and Toshitaka Horiuchi declare no conflict of interest. This review article included no patients, and thus, it did not require approval from an Ethics Committee. 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