Kaoru Takano

Production of Male Cloned Mice from Fresh, Cultured, and Cryopreserved Immature Sertoli Cells

Abstract Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3–10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0–37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.

Birth of mice after nuclear transfer by electrofusion using tail tip cells.

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5–21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult. Mol. Reprod. Dev. 57:55–59, 2000.

Effects of Donor Cell Type and Genotype on the Efficiency of Mouse Somatic Cell Cloning

Abstract Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 × 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 ± 2.1%) was obtained with (B6 × 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 × JF1)F1 and (JF1 × 129)F1 genotypes, whereas no pups were born from cells with the (B6 × JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

Fertilization of Oocytes and Birth of Normal Pups Following Intracytoplasmic Injection with Spermatids in Mastomys (Praomys coucha)

Abstract The mastomys is a small laboratory rodent that is native to Africa. Although it has been used for research concerning reproductive biology, in vitro fertilization (IVF) and intracytoplasmic sperm injection are very difficult in mastomys because of technical problems, such as inadequate sperm capacitation and large sperm heads. The present study was undertaken to examine whether mastomys spermatids could be used to fertilize oocytes in vitro using a microinsemination technique, because spermatids are more easily injected than mature spermatozoa into oocytes. Most mastomys oocytes (80%–90%) survived intracytoplasmic injection with either round or elongated spermatids. Round spermatids had little oocyte-activating capacity, similar to those of mice and rats, and exogenous stimuli were needed for normal fertilization. Treatment with an electric pulse in the presence of 50 μM Ca2+ followed by culture in 10 mM SrCl2 led to successful oocyte activation. After injection of round spermatids into preactivated oocytes, 93% of oocytes were normally fertilized (male and female pronuclei formed), and 100% of cultured oocytes developed to the 2-cell stage. However, none reached term after transfer into recipient females. Elongated spermatids, which correspond to steps 9–11 in rats, activated oocytes on injection without additional activation treatment. After embryo transfer, five offspring (6% per transfer) developed to term. These results indicate that microinsemination with spermatids is a feasible alternative in animal species that are refractory to IVF and sperm injection and that using later-stage spermatids may lead to increased production of viable embryos that can develop into normal offspring.

Microinsemination, nuclear transfer, and cytoplasmic transfer: the application of new reproductive engineering techniques to mouse genetics

Mice are the most commonly used laboratory animals, owing to the availability of large amounts of information on their genetics and biology. As a result, numerous advanced techniques have been developed by using mouse embryos. Studies on murine developmental and reproductive biology are greatly facilitated by short gestation and generation periods of 20 days and approximately 3 months, respectively. Transgenic and knockout mice are of fundamental relevance to mammalian transgenic research. However, in certain areas of reproductive engineering, the murine model is inadequate because of technical difficulties in manipulating unfertilized oocytes. The techniques of microinsemination and nuclear transfer, for example, were successfully implemented in sheep, cows, and rabbits as early as the 1980s (reviewed in First and Prather 1991; Iritani 1991), but their application in mice remained problematic. In 1995, the establishment of a reliable microinjection technique, enabling the transfer of different types of nuclei into mouse oocytes, considerably expanded the field of mouse gamete micromanipulation. Microinsemination also became possible through the use of mature spermatozoa, immature sperm cells (spermatogenic cells) and round spermatids (Ogura et al. 1994; Kimura and Yanagimachi 1995a). Following these early advances, microinsemination and nuclear transfer techniques in the mouse were further developed, to the point that in 1998 normal pups were born after microinsemination with primary spermatocytes, i.e., spermatogenic cells before the first meiotic division (Kimura et al. 1998; Ogura et al. 1998). Similarly, the first murine somatic cell cloning was carried out after nuclear transfer by microinjection with cumulus cells (Wakayama et al. 1998a). Recently, microinseminations with either spermatozoa or spermatogenic cells were used to generate transgenic mice and to propagate infertile mouse strains. Nuclear transfer techniques are expected to increase the efficacy of transgenic/knockout mouse production and enable the conservation of genetically valuable strains of mice. Cytoplasmic transfer, whereby exogenous mitochondrial DNA (mtDNA) is introduced into recipient oocytes/embryos, is another important technique to generate genetic alterations in the mouse. With this technique, heteroplasmic mice with the new phenotype encoded by the introduced mtDNA have been generated to study mitochondrial segregation during development and the mechanisms underlying mitochondrial disease onset (Jenuth et al. 1997; Inoue et al. 2000; Takeda et al. 2000). In this review, we assess the impact of recently established murine reproductive engineering techniques, such as microinsemination, nuclear transfer, and cytoplasmic transfer, on basic research in biology and mouse genetics. Microinsemination

In vitro fertilization and microinsemination with round spermatids for propagation of nephrotic genes in mice

The ICGN is an inbred strain of mice with a hereditary nephrotic syndrome of an unknown cause. The disease progresses to renal failure, resulting in the deterioration of the systemic condition and in a drastic reduction of reproductive capacity. The present study was undertaken to determine if in vitro fertilization (IVF) and microinsemination using round spermatids could ameliorate the reduced fertility of ICGN mice. Mature oocytes (9.4 +/- 1.1 per mouse) were obtained from PMSG/hCG-primed females 2 to 5 mo of age. When spermatozoa from males aged 3 to 4 mo was used for IVF, a high fertilization rate (82.6%) was achieved and a high rate of embryos developed into blastocysts (92.6%). When spermatozoa from males aged 5 to 7 mo was used, the rates of fertilization and development to blastocysts were significantly lower (63.9 and 47.1%, respectively; P < 0.001). However, the production rate of offspring after embryo transfer was satisfactory irrespective of the age of males (59.1%). When males older than 9 mo were used, no fertilization was achieved due to the very poor motility of the spermatozoa. Microinsemination techniques with round spermatids (electrofusion and intracytoplasmic injection) resulted in the production of normal offspring from the older males, including one azoospermic case. These findings indicate that a conventional IVF technique and microinsemination using round spermatids can be used for propagating mutant genes which cause poor reproductive ability in mice.