Production of Male Cloned Mice from Fresh, Cultured, and Cryopreserved Immature Sertoli Cells1

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.

Comparison of the RNA Polymerase I-, II- and III-Dependent Transcript Levels Between Nuclear Transfer and In Vitro Fertilized Embryos at the Blastocyst Stage

“…Based on the reports that most NT embryos cease development at the pre-and peri-implantation stages [1,41], it is probable that the accumulation of different abnormalities observed in NT embryos at various developmental stages, such as failure to erase some of the features of donor cells [42][43][44], abnormal gene expression [4,8,10,25,[45][46][47], abnormal DNA methylation [4][5][6], abnormal formation of placentae [1,41,[48][49][50], and the abnormalities detected in this study, leads to gradual loss of NT embryos. Abnormalities that have not been detected in previous studies and/or small differences in gene expression may seriously affect embryonic development.…”

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning

“…It has been suggested that the cells in G 0 ͞G 1 of the cell cycle are the most suitable for cloning (16,22,28,32). Although male germ cells at 15.5 dpc are in G 0 mitotic arrest, the success of achieving postimplantation development was much higher than would be predicted when compared with somatic donor cells at a similar cell cycle stage; for example, only 1.7% of Sertoli cell clones (16), 5.3% of cumulus cell clones (this study), and 13.2% of fetal neural cell clones (22) develop to a similar stage.…”

Understanding the X chromosome inactivation cycle in mice

“…The cells were prepared as described previously. 30 Briefly, the collected testicular cells were treated with 0.1 mg/ml collagenase (Sigma-Aldrich) and 0.01 mg/ml deoxyribonuclease (Sigma-Aldrich) for 30 min at 37 °C, followed by 0.2 mg/ml trypsin (Sigma-Aldrich) for 5 min at 37 °C. The testicular cell suspension was washed with Ca 2+ /Mg 2+ -free phosphate-buffered saline (PBS) containing 4 mg/ml bovine serum albumin and were used for injection.…”