Biomedical subjects

H Nakasaka

Publications and source records attributed to H Nakasaka.

4 recordsLinked to original sources

PubMed2001

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus.

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 microM A23187 for 5 min were treated with 10 microg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Adenine↗

PubMed2000

Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes.

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 microg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

Animals↗

PubMed2000

Effects of preservation of mouse spermatozoa in electrolyte-free solution at 4 degrees C on the outcome of mouse in vitro fertilization.

PURPOSE: The aim was to assess the fertilizing capacity of spermatozoa cool-preserved in electrolyte-free (EF) solution. METHODS: Mouse spermatozoa were cool-preserved in EF solution and the acrosomal status of the spermatozoa was compared before and after preservation using chlortetracycline stain. Mouse oocytes were inseminated by spermatozoa cool-preserved in EF solution for 2, 4, or 7 days and fertilization and blastocyst rates were evaluated. RESULTS: Acrosomal status of spermatozoa cool-preserved in EF solution was not different from spermatozoa before preservation, but the capacitated and acrosome-reacted spermatozoa significantly increased after reinitiation. Cool-preservation in EF solution for up to 4 days did not affect fertilization rate. Blastocyst rate of embryos derived from spermatozoa cool-preserved for 4 or 7 days in EF solution was significantly lower than that of embryos derived from fresh spermatozoa. CONCLUSIONS: Mouse spermatozoa cool-preserved in EF solution possesses as much fertilizing capacity as fresh spermatozoa. However, prolonged preservation affects the embryonic development.

Acrosome Reaction↗

PubMed2000

Fertilization failure and oocyte activation.

Recent reports have suggested that when unfertilized oocytes with a spermatozoon after intracytoplasmic sperm injection (ICSI) are properly activated, the activated oocytes develop normally similar to fertilized oocytes. However, human oocytes do not easily respond to universal activators of mammalian oocytes like ethanol or calcium ionophore A23187, which induce a calcium transient in ooplasm. Puromycin activates human oocytes at a rate of 90%, but more than two thirds of the parthenogenesis possess 2 haploid pronuclei or 1 diploid pronucleus without extrusion of the second polar body. Therefore, the activation method which produces one pronucleus with extrusion of the second polar body in oocytes without a spermatozoon is necessary for producing embryos with normal karyotypes. Recently, we found the oocyte activation method which produced parthenogenesis displaying one pronucleus with extrusion of the second polar body. Using our method (a combination of calcium ionophore A23187 and puromycin), the activation rate was approximately 90% and the proportion of parthenogenesis displaying one pronucleus with extrusion of the second polar body was approximately 80% in human aged and mouse young oocytes. When human unfertilized oocytes following ICSI were activated by this method, two pronuclei were formed with extrusion of the second polar body in 30% of the oocytes. Four cleaved parthenogenesis (or embryos) showed normal karyotypes. However, the cytotoxic, teratogenetic and mutagenetic activity of Ca ionophore and puromycin should be approved prior to the clinical adaptation of the method.

Animals↗