Two embryonic stem (ES) cell lines (OKB6-I and -II) derived from C57BL/6 strain mice were established by the whole blastocyst culture method, and the net isolation ratio was 3.9% (2/51). They had pluripotency and normal karyotype, and were positive for alkaline phosphatase activity. It was indicated that OKB6-I was a female cell line, and OKB6-II was a male, judged by the size and morphology of their chromosomes. By coculture with ICR embryos, a chimeric mouse and a hermaphrodite were generated from OKB6-II, but no chimera was generated from OKB6-I. OKB6-II⇔ICR chimera generated 3 albino type viable offspring, when mated with ICR strain mouse. OKB6-II⇔ICR hermaphrodite was sterile, and had a contralateral ovary and testis-like structure.

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The sex of human preimplantation embryos was determined by fluorescent in-situ hybridization (FISH), and the reliability of this method was evaluated. Preimplantation sex determination by FISH was performed with blastomeres dissociated from pre- implantation embryos with 2 pronuclei in the 2-8 cell stage that were obtained by intracytoplasmic sperm injection (ICSI). Dual color FISH analysis was performed with DXZ1 and DYZ1, X and Y chromosome-specific probes, respectively. The detection sensitivity of DXZ1 and DYZ1 was evaluated with interphase nuclei of human lymphocytes obtained from normal males and females. The signal detection rate in these nuclei was 92.0% (460/500) in the males and 89.4% (447/500) in the females. The fixation rate of single blastomeres determined by FISH was 88.7% (86/97). The sex determined by FISH was the same in all blastomeres within the embryo in 20 (80%) of 25 embryos; 11 embryos were determined to be males and 9 to be females. The other 5 embryos (20%) showed sex chromosomal mosaicism. When the results in 2 blastomeres are normal and consistent, the probability that the remaining part of the embryo was mosaic was 12% (3/25). When the sex of the entire embryo is determined by biopsy of 2 blastomeres instead of 1 blastomere, mosaicism can therefore be excluded with a reliability of 88%, and more accurate diagnosis is possible.

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The incorporation and oxidation of glucose and pyruvate were compared when embryos were cultured in M16 medium and M16 plus EDTA. In both culture conditions the incorporation and oxidation of glucose increased a little but then significantly decreased by 40 and 45 h after hCG injection. On the other hand, the incorporation and oxidation of pyruvate increased with the development of embryos, was reduced at the blastocyst stage, but had not significantly decreased by 40 and 45 h after hCG injection. After mouse embryos were cultured for 6 h in M16 medium, the incorporation and oxidation of glucose and pyruvate were significantly decreased compared with culture in M16 plus EDTA at 40, 45 and 50 h after hCG injection. These results suggest that EDTA had the ability to maintain a large degree of viability of mouse embryos for further development.

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Spermatozoa of mutated mice whose appearance quite resembles the rhino mouse were frozen and thawed, then submitted to in vitro fertilization and embryo transfer (IVF-ET). The cryopreservation solution was an 18% raffinose and 3% skim milk mixture, and the spermatozoa were stored in liquid nitrogen at -196°C. Two and half months later, the samples were thawed and fertilized with ICR mouse oocytes in vitro. The motility rate after thawing was approximately 20% and the fertilization rate was 14% (13/95), but 92% (12/13) of the fertilized eggs developed into the blastocyst stage. These twelve embryos were transferred into the uterine horns of a pseudopregnant recipient, and 75% (9/12) were born. The heterozygous F1 offspring did not show signs of the rhino-like phenotype, such as wrinkled skin without hair, but among a total of 39 offspring (F2) derived from brother-sister mating of F1 siblings, 11 (5 males and 6 females) pups showed signs of the rhino-like phenotype, e.g. at two- to three-weeks of age hair fell out, and at 6 months their skin became thick and wrinkled. Histopathological observations showed pilary canal cysts in the cortex of the skin, and dermal cysts were observed in the middle and inner area. These results demonstrate that the gene resources of rhino-like mutated mice can be preserved by the cryopreservation of spermatozoa and re-expressed via IVF-ET.

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A total of 1,923 follicular human ova were collected for use in intracytoplasmic sperm injection (ICSI). Timed ovulation was induced by means of a standard ovarian stimulation protocol involving set dosages of follicle stimulating hormone (FSH), human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (hCG). Prior to performing ICSI, all of the ova were subjected to thorough examination which led to the discovery of only two parthenogenetic oocytes, also referred to as parthenogens, out of the total 1,923. These oocytes were retrieved from different patients. Although genetic analysis revealed that one of the parthenogens had a haploid number of chromosomes, it was impossible to analyze the second because it had not yet reached the metaphase stage. The 0.1 percent incidence of spontaneous parthenogenesis in this study clearly indicates that this phenomenon is extremely rare.

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Effect of some reducing agents, their combination with other agents, and their concentration on changes in sperm nuclei and pronuclear formation was investigated. First, while treated with 50 mM DTT, 80% of the sperm had partly decondensed their nuclei after 0.5 h of treatment, and complete decondensation was observed in all the sperm after 10 min incubation in 5 mM DTT and 1% SDS. None of the sperm was seen dispersed 2 h after treatment with 50 mM GSH and 5% SDS. Second, the decondensation rate was dependent on the NaCl concentration when DTT was used as a reducing agent. Finally microinjection of sperm pretreated with 5 mM DTT resulted in a significantly higher male pronuclear formation rate 6 h postinjection (49%) than that (5.3%) of the heparin pretreated control. These results suggest that pretreatment of bovine sperm with DTT may affect the fertilization rate after microinjection.

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In the dog, copulation can occur 2-3 days before ovulation and oocytes are ovulated at the stage of the germinal vesicle (GV). It is therefore probable that GV oocytes can be penetrated by spermatozoa and developed normally. In the present study, we examined the penetration rate and male and female nuclear changes in canine oocytes which were inseminated just after collection from preovulatory follicles and cultured for up to 72 h. The penetration rate reached its maximum (64.4%) at 24 h after insemination, although only 6.9% of penetrated oocytes were mature at that time, indicating that GV oocytes were penetrated by spermatozoa. Thereafter, the maturation rate increased to 17.9% and 60.0% of penetrated oocytes at 48 and 72 h after insemination, respectively. Both male and female pronuclei were developed in 26.7% of matured-fertilized oocytes at 72 h after insemination. These results suggest the possibility of the participation of GV oocytes in the normal fertilization processes in the dog.

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We examined whether we can detect multiple gene expressiom in single early bovine embryos by using antisense RNA (aRNA) and RT-PCR (aRNA-RT-PCR). Embryos, from the 2-cell stage to the blastocyst stage, were produced by the IVM-IVF method, cryopreserved and used as material for aRNA synthesis. Antisense RNA solution was yielded after reverse-transcription (RT) with a synthetic primer containing the T7 RNA polymerase binding site, double strand cDNA synthesis and transcription by the T7 RNA polymerase. For the reverse transcription of aRNA (aRNA-RT), we used a 5 μl of total 100 μl aRNA solution (1/20) and either sense primer or an antisense one encoding bovine beta-actin (bActin). After the aRNA-RT and PCR, the presence of the expected products was confirmed by electrophoresis. We confirmed the bActin products when the sense primer was used in aRNA-RT, but we did not confirm the products with the antisense one. These results indicate that we could detect multiple gene expression in a single early bovine embryo by means of aRNA-RT-PCR.

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