Mutations induced in male germ cells after treatment of transgenic mice with ethylnitrosourea.

The present study was undertaken to clarify whether the transgenic mouse mutagenesis assay system can be used instead of dominant lethals or specific locus test after treatment of male germ cells in mouse with ethylnitrosourea (ENU). Male Big Blue transgenic mice (BB) carrying a lacI target gene were given a single intraperitoneal injection of 150 mg/kg ENU. Vasa deferential sperm, caudal epididymal sperm or whole testes were assayed for mutation at 3, 14, 22 and 93 days after treatment with ENU. The average of background lacI- mutant frequencies was 2.05 x 10(-5). The MF observed in post spermatogonial stage after treatment with ENU were slightly increased over background. On the other hand, ENU induced high MF in the spermatogonial stage. MF detected after treatment of BB male germ cells with ENU were lower than those detected in the mouse visible specific-locus mutations in previous reports. Nevertheless, it is clear that this assay is a practical alternative to the specific locus test for detecting mutations induced in spermatogonial stage.

Typing of X chromosomes bearing Tabby allele in mouse preimplantation embryos by detection of a microsatellite marker.

Tabby (Ta) is a semidominat allele of the locus on the mouse X chromosome, which causes a characteristic coat pattern and developmental defects in endocrinic glands. To establish a method for identifying the X chromosome bearing the Ta allele in early preimplantation embryos, we performed PCR amplification of an X chromosomal microsatellite marker locus from preimplantation embryos obtained from mating between XO female and XY male mice. The microsatellite marker locus was shown to be polymorphic between X chromosomes bearing the Ta and wild-type alleles. The amplification of the marker locus from early preimplantation embryos demonstrated that the Ta locus can be correctly typed by this method from embryos as little as 2 cells.

Sex-linked differences in developmental potential of single blastomeres from in vitro-fertilized 2-cell stage mouse embryos.

Single blastomeres were isolated from 2-cell mouse embryos and analyzed for their sex using the partially deleted Y chromosome as a marker. Sex identification of 83% of the embryos was achieved with conventionally Giemsa-stained chromosome preparations. The other half-embryos were cultured individually and were transferred to pseudopregnant recipient females. The implantation rate was significantly higher in the male half-embryos. Although 6% of the half-embryos were male. The male half-embryos contained more blastomeres in the blastocyst stage than female ones. These facts suggests that the potential for the development of male half-embryos into fetuses is different from that of female half-embryos.

PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro.

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos.