Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.     

Detection of Thiophanate-methyl-resistant Strains in Diplocarpon mail, Causal Fungus of Apple Blotch

Isolates of Diplocarpon mali, causal fungus of apple blotch, collected from four prefectures in Japan in 1997–1998 were tested for sensitivity to thiophanate-methyl. Results from mycelial growth tests showed that MIC values of the fungicide were 0.19 μg/ml against all isolates from Akita, Nagano and Saga prefectures but 100 or 200 μg/ml against all isolates from the Tokusa area in Yamaguchi prefecture. Detached apple leaves sprayed with the fungicide developed severe symptoms when inoculated with the isolate from Tokusa, but developed no symptoms with the isolate from Nagano. These results are the first confirmation of thiophanate-methyl-resistant strains in D. mali. Received 1 September 1999/ Accepted in revised form 15 October 1999     

Age-related changes of reproductive hormones in young Meishan boars

Meishan pigs are known for their early sexual maturity. On the other hand, they grow slowly. There is no information currently available about the combination of these two characteristics in Meishan pigs. To study the developmental characteristics of Meishan pigs, the plasma concentrations of LH, FSH, inhibin, testosterone, estradiol-17beta, progesterone and insulin-like growth factor-I (IGF-I) in young Meishan boars were determined using RIA and ELISA. Inhibin decreased with age in weeks, while testosterone and estradiol-17 beta increased. Testosterone increased gradually, and an increase in estradiol-17beta occurred after sexual maturity. IGF-I increased before puberty and subsequently decreased just after puberty like a pubertal IGF-1 surge. FSH, LH and progesterone did not change with age. There was no significant correlation among the hormones. During the experimental period, the Meishan boars showed large individual differences. These differences may depend on the fact that Meishan boar reach maturity at 12 weeks of age and continue to grow thereafter.     

Development of DNA markers that discriminate between white- and blue-flowers in Japanese gentian plants

We developed molecular markers for discrimination of white and blue flower color in Japanese gentian plants. White-flowered gentians can be classified into two types, based on genetic and physiological features. One type includes four allelic variations (gtmyb3-1, gtmyb3-2, gtmyb3-3, and gtmyb3-4) of an anthocyanin biosynthetic regulator gene (GtMYB3), distinguished by three PCR-based molecular markers. The other type contains a newly identified inactive allele (ans1) of the anthocyanidin synthase (ANS) gene with a premature stop codon generated from a 4-bp deletion in the second exon. The ans1 allele was distinguished from the active ANS allele by a cleaved amplified polymorphism sequence (CAPS) marker. The genotypes of 12 white-flowered gentian cultivars/lines could be identified and classified as either ans1 or gtmyb3 using these four molecular markers. No white-flowered gentians contained ans1 and gtmyb3 alleles simultaneously. The mutated ANS gene co-segregated with white flower color in an F₂ population, demonstrating that the CAPS marker is useful to discriminate between white and blue flowers in gentian. Markers to discriminate flower color in Japanese gentian will be useful for early selection of progeny and for breeding management.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.