Developmental competence of interspecies cloned embryos produced using cells from large Japanese field mice (Apodemus speciosus) and oocytes from laboratory mice (Mus musculus domesticus).

The large Japanese field mouse (Apodemus speciosus) is endemic to Japan and may be used as an animal model for studies related to environmental pollution, medical science, and basic biology. However, the large Japanese field mouse has low reproductive ability due to the small number of oocytes ovulated per female. To produce experimental models, we investigated the in vitro developmental potential of interspecies somatic cell nuclear transfer (iSCNT) embryos produced by fusing tail tip cells from the large Japanese field mouse with enucleated oocytes from laboratory mice (Mus musculus domesticus). Only a small number of iSCNT embryos developed to the 4-cell (0-4%) and blastocysts (0-1%) stages under sequential treatment using trichostatin A (TSA) and vitamin C (VC) supplemented with deionized bovine serum albumin (d-BSA). This sequential treatment led to the reduction in H3K9 trimethylation and did not affect H3K4 trimethylation in at least the 2-cell stage of the iSCNT embryos. Moreover, iSCNT embryos that received tail tip cells with exposure treatment to ooplasm from cell fusion to oocyte activation or VC treatment prior to cell fusion did not exhibit significant in vitro development improvement compared to that of each control group. This suggests that large Japanese field mice/laboratory mice iSCNT embryos that received sequential treatment using TSA and VC with d-BSA may have slightly better developmental potential beyond the 4-cell stage. Our results provide insights into the reprogramming barriers impeding the wider implementation of iSCNT technology.

Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Development of feline embryos produced by Piezo-actuated intracytoplasmic sperm injection of elongated spermatids.

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules.

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.

RecQ5 protein translocation into the nucleus by a nuclear localization signal.

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.