Effects of post-ovulatory aging on centromeric cohesin protection in murine MII oocytes.

Purpose: Post-ovulatory aging causes a high frequency of aneuploidy during meiosis II in mouse oocytes, irrespective of maternal age. In this study, we evaluated the effects of post-ovulatory oocyte aging on the protection of chromosomal cohesion involved in aneuploidy and verified the relationship between PP2A or SGO2 expression and the phosphorylation level of REC8 in oocytes. Methods: Murine ovulated oocytes were incubated for 6 or 12 h in vitro after collection and denoted as the aged group. The oocytes examined immediately after collection were used as the control group. Immunofluorescent staining was used to detect the localization of PP2A, SGO2, BUB1, AURORA B, and MAD2 in the chromosomal centromere. Immunoblotting was used to quantify the expression of proteins describe above and REC8 in the oocytes. Results: PP2A expression involved in the de-phosphorylation of REC8 decreased over time in oocytes, suggesting a deficiency in PP2A in centromeres. This indicated an increase in the level of phosphorylated REC8, which destabilizes centromeric cohesion in oocytes. In contrast, SGO2 expression was significantly high in aged oocytes. Conclusions: The findings show that post-ovulatory aging destabilizes the centromeric cohesin protection in oocytes and can cause aneuploidy, which is often observed in aged oocytes during meiosis II.

Mitochondrial DNA variations in Japanese farmed emu populations.

The emu (Dromaius novaehollandiae) is a new poultry. In this study, we investigated the haplotype composition of mitochondrial DNA among emu populations farmed in Japan. We sequenced the D-loop region in 109 individuals, and detected four substitution sites and three haplotypes (Hap-a, -b, and -c). Hap-a was the most frequently observed haplotype in the Japanese populations. Although Hap-c was a rare haplotype in not only Japanese but also Australian populations, it was detected with high frequency in the Japanese farmed population. The AMOVA indicated that 9% of total variance was "among population". The FST value was 0.087 and genetic differentiation was significant (P<0.01). These results may contribute to conserving the genetic resources available for the Japanese emu industry.

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations.

The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan are unknown and the number of microsatellite markers for genetic analysis of the emu is insufficient. In this study, we isolated 16 microsatellites from the emu genome and developed ten new microsatellite markers. These microsatellite markers were used to characterize three farm emu populations in Japan. The number of alleles ranged from 3 to 13 and the expected (HE) and observed heterozygosity (HO) of these microsatellite loci was 0.187-0.802 and 0.179-0.647, respectively. The polymorphic information content ranged from 0.176 to 0.786. Positive inbreeding coefficient (FIS) values were detected in all tested populations, and they ranged from 0.027 to 0.540. These results suggest that farm populations of the emu in Japan resulted from inbreeding. The fixation index (FST) values ranged from 0.026 to 0.061, and phylogenetic trees and population structure analysis confirmed no definitive genetic differentiation among the three populations. Therefore, these populations are at a relatively low level of genetic differentiation at present. The microsatellite markers developed in our study can be utilized for genetic analysis and preservation of genetic resources in the emu.

Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes.

Mammalian oocyte quality degrades over time after ovulation in vitro, which can cause fatal defects such as chromosomal aneuploidy. As various oocyte manipulations employed in assisted reproductive technology are time consuming, post-ovulatory aging is a serious problem to overcome in reproductive medicine or ova research. In this study, we investigated the effects of postovulatory aging on the incidence of chromosome aneuploidy during meiosis II, with a focus on the expression of functional proteins from the spindle assembly checkpoint (SAC). Chromosome analysis was used to assess the rate of aneuploidy in in vitro aged oocytes, or in early embryos derived from aged oocytes. Immunofluorescent staining was used to detect the localization of MAD2, which is a SAC signal that monitors the correct segregation of sister chromatids. Immunoblotting was used to quantify cohesin subunits, which are adhesion factors connecting sister chromatids at the metaphase II (MII) centromere. It was shown that post-ovulatory oocyte aging inhibits MAD2 localization to the sister kinetochore. Furthermore, oocyte aging prevented cohesin subunits from being maintained or degraded at the appropriate time. These data suggest that the destabilization of SAC signaling causes sister chromatid segregation errors in MII oocytes, and consequently increases the incidence of aneuploidy in early embryos. Our findings have provided distinct evidence that the post-ovulatory aging of oocytes might also be a risk factor for aneuploidy, irrespective of maternal age.

A 1-bp deletion in Fgf5 causes male-dominant long hair in the Syrian hamster.

Hair length in mammals is generally regulated by the hair cycle, and its disruption leads to abnormal hair morphogenesis in several species. FGF5, one of the hair cycle regulators, has a role in inducing catagen, and that mutation causes abnormal hair length in both sexes in humans, mice, dogs, and cats. Male-dominant long-haired coat (MALC) is an inbred strain of Syrian hamster exhibiting spontaneous long hair in males. After castration, MALC exhibited significantly shorter hair than the control individuals, but testosterone administration to castrated MALC showed reversion to the original phenotype. Moreover, flutamide administration led to MALC phenotype repression. Histological analysis revealed that hair follicle regression was shown in the wild-type 4 weeks after depilation, but that of MALC remained in the anagen phase. We detected a c.546delG of Fgf5 in MALC (Fgf5malc) that might lead to truncation resulting from a frame shift in FGF5 (p.Arg184GlyfsX6). Additionally, homozygous Fgf5malc was only detected in long-haired (Slc:Syrian×MALC)F2 and (J-2-Nn×MALC)F2 progenies, and all homozygous wild and heterozygous Fgf5malc individuals showed normal hair length. Thus, Fgf5malc leads to male-dominant long hair via a prolonged anagen phase which is affected by testosterone in hamsters. To our knowledge, this report is the first to present the sexual dimorphism of hair length caused by the Fgf5 mutation.

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.