Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.     

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC₅₀) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.     

Cultural control of leafhopper-induced maize wallaby ear symptom in forage maize via early planting dates

Maize wallaby ear symptom (MWES) is induced by maize orange leafhopper (Cicadulina bipunctata) feeding and stunts the growth of maize. We examined the relationship between maize developmental stage and MWES occurrence and the effect of planting date on injury from MWES in western Japan. MWES was more pronounced on young maize, and rarely observed on maize older than the 5th or 6th leaf stage, indicating that younger maize is more susceptible to MWES. In a field experiment, early planting (before late July) avoided MWES and resulted in higher yields of forage maize, whereas MWES occurrence and yield loss gradually increased with late planting (from early to middle August) because of a rapid increase in C. bipunctata density during this period. Leaf and stem dry matter of the MWES-susceptible maize variety “3470” decreased together with increase in density of C. bipunctata, whereas leaf and stem dry matter of the MWES-tolerant maize variety “30D44” decreased little. These results indicate that earlier planting together with the use of MWES-tolerant varieties can significantly improve MWES control on forage maize.     

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.     

Analysis of yield-attributing traits for high-yielding wheat lines in southwestern Japan

Development of wheat cultivars that achieve high yields despite the short growing season is essential for increasing wheat production in southwestern Japan. The objectives of this study were to assess the genetic progress in grain yield and to clarify yield-attributing traits of high-yielding wheat lines in southwestern Japan. We conducted field experiments for two growing seasons (2012–2013 and 2013–2014) using three commercial wheat cultivars (Shiroganekomugi, Chikugoizumi, and Iwainodaichi) and four high-yielding wheat lines including Hakei W1380 developed in southwestern Japan. In an ancillary field experiment, we compared a commercial cultivar, Shiroganekomugi, and a high-yielding line, Hakei W1380, in the 2014–2015 season. Across the two seasons, grain yield of high-yielding lines was generally higher than commercial cultivars. Hakei W1380 achieved the highest grain yield across the two seasons, and successfully produced more than 900 g m^?2 in the 2013–2014 season. Correlation analysis showed that recent yield progress of wheat lines in southwestern Japan was derived from enhanced biomass production and grain number m^?2. Larger numbers of grains m^?2 in high-yielding lines than in commercial cultivars were associated with higher crop growth rate at the pre-anthesis stage, and therefore higher spike dry weight m^?2 at anthesis. Genotypic differences in crop growth rate from jointing to anthesis resulted mainly from differences in leaf area index. These results indicate that further improvements in grain yield in southwestern Japan could be achieved by increasing the amount of radiation intercepted at the pre-anthesis stage and grain number m^?2.     

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.     

Reduced intake of dietary lysine promotes accumulation of intramuscular fat in the Longissimus dorsi muscles of finishing gilts

The present study was conducted to elucidate the effect of dietary lysine levels on the intramuscular fat (IMF) content in the Longissimus dorsi (L. dorsi) muscles of finishing gilts. Eleven gilts in total from two litters of pigs aged 110 days were used. The average initial bodyweight of the pigs was 61.7 kg. Six pigs were assigned to the low lysine (LL) diet group (lysine content: 0.43 or 0.40%) and five pigs were assigned to the control group (lysine content: 0.65 or 0.68%). The diets were iso‐energetic and iso‐protein, and contained all essential amino acids (apart from lysine) in the recommended amounts. The pigs were fed these diets until their live weights reached 110 kg. Live weight gain and feed efficiency tended to be lower in the LL group (P = 0.118 and P = 0.052, respectively). Pigs from the LL group took 5 days longer to reach 110 kg (P < 0.01). The IMF content in the L. dorsi of the LL group was twice as high as that of the control group (6.7 vs 3.5%; P < 0.01). The percentage of oleic acid in the L. dorsi of the LL group tended to be higher than that of the control group (P = 0.052), whereas the percentage of linoleic acid and the total percentage of polyunsaturated fatty acids in the L. dorsi were lower (P < 0.05) in the LL group. Free L‐carnitine content in the L. dorsi was lower (P < 0.05) in the LL group. The average abundance of peroxisome proliferator‐activated receptor gamma mRNA in the L. dorsi of the LL group was threefold higher than that of the control group. The leptin mRNA abundance in the L. dorsi of the LL group was 3.3‐fold higher than that of the control group (P < 0.01). These results suggest that a higher activity of adipogenesis may have been involved in the promoted accumulation of IMF in the L. dorsi muscles of pigs, induced by a dietary LL level.