Ryoichi Hashizume

NER rat strain: a new type of genetic model in epilepsy research.

Summary: Purpose: We characterized and evaluated as an animal model of epilepsy NER, a new epileptic rat strain, which was developed by inbreeding rats with spontaneous tonic‐clonic seizures in a stock of Crj:Wistar.

A 5-bp insertion in Mip causes recessive congenital cataract in KFRS4/Kyo rats.

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.

Asymmetrical allocation of mitochondrial DNA to blastomeres during the first two cleavages in mouse embryos

A recent report showed higher oxygen consumption, adenosine triphosphate (ATP) production and mitochondrial localisation in trophectoderm cells than in the inner cell mass of mouse blastocysts. We hypothesised that this phenomenon was due to the asymmetrical distribution of mitochondria in the blastomeres during the earlier stages. Oocytes, 2-cell embryos and 4-cell embryos were analysed to determine the volume, ATP content and mitochondrial DNA (mtDNA) copy number in the whole egg and individual blastomeres. Significant differences were detected in the volumes of cytoplasm and ATP contents between blastomeres from the 2-cell and 4-cell embryos. Moreover, whilst remaining stable in whole embryos, mtDNA copy number differed between blastomeres, indicating that mitochondria in oocytes are unevenly delivered into the daughter blastomeres during the first two cleavages. Although their volume and ATP content were not correlated, there was a significant correlation between volume and mtDNA copy number in 2- and 4-cell blastomeres. These results indicate that the number of mitochondria delivered to blastomeres during early cleavage is not precisely equal, suggesting that the allocation of mitochondria into daughter blastomeres is affected by uneven cytoplasmic distribution during cytokinesis in the oocyte and mother blastomeres.

The Effects of Cadmium Toxicity on Maturation and Fertilization Process of Mouse Oocytes

ABSTRACT We previously reported that 2-cell embryos obtained after administering cadmium (Cd) to mice immediately before ovulation already demonstrated reduced developmental ability. To determine when this Cd toxicity appears in mouse oocytes before the 2-cell stage, we exposed oocytes to Cd at maturation and during the fertilization period using IVM and IVF techniques. Four experimental groups were defined by the period of Cd exposure: IVM group, exposure at the maturation period; IVF group, exposure at the fertilization period; IVM · IVF group, exposure during both periods; Control group, no exposure. Oocytes obtained from the ovaries were subjected to IVM and IVF, and the fertilization and developmental rates were investigated in all groups. As a result, Cd exposure during the maturation period disturbed fertilization of oocytes, and Cd exposure during the fertilization period disrupted the normal development of embryos after fertilization. This suggested that the manifestation of Cd toxicity in oocytes clearly differed with the exposure period. In addition, it was shown that Cd exposure during the maturation or fertilization period influenced the early development (including the fertilization) at a lower concentration than exposure after the 2-cell stage.

Sister Chromatid Exchange Frequency in Early Embryos and Offspring Derived from IVF or ICSI in Mice

Abstract: To demonstrate chromosomal injuries associated with in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), sister chromatid exchange (SCE), an indicator of DNA damage, was investigated and the cytogenetic influence of embryonic manipulation on chromosomes of early embryos and offspring was investigated. SCE analysis was performed in early embryos and offspring obtained by IVF, ICSI, or in vivo fertilization (control). To label chromosomes of early embryos, the embryos were cultured in BrdU-supplemented medium for 2 cell cycles. For offspring, BrdU solution was repeatedly injected intraperitoneally. Chromosome samples prepared from the embryos and the bone marrow cells of offspring were stained by the Fluorescence plus Giemsa (FPG) method. In the IVF and ICSI groups, the rates of early development, implantation and offspring were lower than those in the control group. The SCE frequency of early embryos was significantly higher in the IVF and ICSI groups than in the control group (P < 0.05). In contrast, the SCE frequency of offspring in the IVF and ICSI groups was not significantly different from that in the control group. These findings suggest that embryos having serious DNA damage due to embryonic manipulation may be eliminated in a relatively early developmental step and may not reach term.

104 Asymmetrical distribution of mitochondrial DNA into blastomeres of two-and four-cell mouse embryos

Mammalian embryos are thought to cleave equally during early development. Therefore, the contents including organelles such as mitochondria (mt) in oocytes are supposed to be delivered symmetrically into each blastomere of the resulting embryo. However, a recent report showed higher oxygen consumption, ATP production, and mt distribution in trophectoderms from mouse blastocysts than that in inner cell masses (Houghton 2006 Differentiation 74, 11–18). We hypothesized that this phenomenon could be preceded by the asymmetrical mt distribution into blastomeres at earlier stages. Oocytes, 2-cell embryos, and 4-cell embryos from BDF1 were measured to determine volumes, ATP contents, and mtDNA copy numbers in the whole egg and individual blastomeres that had been separated in Ca/Mg-free KSOM with added 0.02% EDTA. The volumes of blastomeres were calculated by the sphere volume formula. The ATP contents were determined using a luminometer and commercial kits (BacTiter-Glo, Promega, Madison, WI, USA). The mtDNA copy numbers were quantified absolutely using real-time PCR. The data was analyzed by 1-way ANOVA followed by Fishers least significant difference test. We found about a 10% difference in volume between the largest and the smallest blastomeres from a 4-cell embryo. At the 4-cell stage, the largest blastomeres were significantly larger than the middle-sized and smallest blastomeres, and the next largest blastomeres were significantly larger than the smallest blastomeres (largest: 41 093 µm3, next largest: 39 671 µm3, middle-sized: 37 725 µm3, smallest: 36 518 µm3; n = 25). The procedure for separation of blastomeres did not significantly reduce theATP contents of embryos. We observed the same pattern of significant differences in theATP contents among the 4 blastomeres at the 4-cell stage (largest: 0.141 pmol, next largest: 0.124 pmol, middle-sized: 0.112 pmol, smallest: 0.098 pmol, n = 27). The largest blastomeres from 4-cell embryos contained about 1.4-fold higherATP than the smallest blastomeres. There was no significant difference in the mtDNA copy numbers in oocytes (n = 7), whole 2-cell embryos (n = 6), whole 4-cell embryos (n = 13), the sum of 2 blastomeres from the same 2-cell embryo (n = 6), and the sum of 4 blastomeres from the same 4-cell embryo (n = 6; 245 071 ± 22 696, 267 567 ± 30 989, 262 931 ± 12 952, 239 717 ± 16 813, 247 012 ± 16 166, respectively; ± SEM). However, the larger blastomere of 2-cell embryo had a higher mtDNA copy number than the smaller blastomeres (largest: 137 100 ± 11 493, smallest: 102 617 ± 6205; P < 0.05). The largest blastomeres of 4-cell embryo had about 1.4 times (P < 0.05) higher mtDNA copy numbers than the smallest blastomeres (largest: 72 072 ± 4112, next largest: 66 198 ± 5767, middle-sized: 57 596 ± 3922, smallest: 51 146 ± 3081). We found differences in volumes, ATP contents, and mtDNA copy numbers among blastomeres from the same embryos at the 4-cell stage. These qualitative differences could be related to embryonic metabolism in mouse early development.

Effect of oocyte preincubation and intra-ovarian bursa transfer on the development of oocytes following intracytoplasmic sperm injection in rats

ABSTRACT Rat oocytes are known to be activated in vitro spontaneously. In the present study, we examined the effect of oocyte preincubation on the survival and development of oocytes after intracytoplasmic sperm injection (ICSI) in rats. Some presumptive oocytes produced by ICSI were transferred into the oviduct or ovarian bursa of recipient females to observe the development to term. When ICSI was performed without oocyte preincubation, the rate of oocyte survival was 90.5%, normally fertilized, 82.5%, and cleaved, 58.7%. These rates were reduced by oocyte preincubation for 3 or 5 h and the reduction could be related to the incomplete spontaneous activation of oocytes, which is known to result in no pronucleus formation and no cleavage. Both oviduct and ovarian bursa transfer allowed us to produce live offspring after ICSI (19.5 and 21.7% of transferred oocytes, respectively). Rat offspring could be produced by the ICSI protocol commonly used in mice. In rats, shortening of the period from oocyte recovery to sperm injection might be an important factor in the production of live offspring after ICSI. Intra-ovarian bursa transfer is technically easy and has been successfully applied to the production of rat offspring after various manipulations of oocytes, including ICSI.