Interaction of orexin/hypocretin-like immunoreactive neurons with melanin-concentrating hormone and α-melanocyte-stimulating hormone neurons in brain of a pleuronectiform fish, barfin flounder

Immunohistochemical localization of orexin/hypocretin in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri was examined as the first step in unraveling the possible function of the hormone in the brain. Orexin-A-like immunoreactive (ir) cell bodies were found to be located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-A-like-ir fibers were detected not only in the hypothalamus but also extensively throughout the brain. The orexin-A-like-ir cell bodies did not project their fibers to the pituitary gland. Since melaninconcentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are suggested to regulate food intake in addition to orexin/hypocretin in the teleost fish, it was examined whether neural connections exist between orexin neurons and the MCH and α-MSH neurons in the barfin flounder brain by using double-staining immunohistochemistry. Some orexin-A-like-ir fibers were in close contact with the MCH-ir and α-MSH-ir cell bodies in the hypothalamus. Moreover, a few MCH-ir and α-MSH-ir fibers were in close contact with the orexin-A-like-ir cell bodies in the hypothalamus. These results suggest that reciprocal connections exist between the orexin and MCH neurons and between the orexin and α-MSH neurons in the brain of the barfin flounder.     

Changes in brain and pituitary GnRH levels during ovarian maturation in wild female Japanese flounder

To elucidate the role of gonadotropin-releasing hormone (GnRH) in gonadal maturation in wild female Japanese flounder Paralichthys olivaceus, we monitored changes in the levels of seabream GnRH (sbGnRH) in the olfactory bulb, telencephalon, hypothalamus, and pituitary during ovarian development together with changes in plasma levels of testosterone (T), estradiol-17β (E₂), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). Fish were caught offshore of the northern mainland of Japan in the Pacific Ocean at 3- to 4-week intervals between April and September by gill net. The netted fish were categorized into six groups based on ovarian stages: previtellogenic (April–early May), early yolk (April–late May), late yolk (late May–June), early spawning (June–August), late spawning (September), and termination (September) stages. The gonadosomatic index significantly increased from the previtellogenic to early spawning stages and decreased thereafter. In the olfactory bulb, no significant differences were observed in sbGnRH levels among the developmental stages. In contrast, sbGnRH levels in the telencephalon and hypothalamus were very high in the previtellogenic stage, lower in the early spawning stage, and relatively high in latter stages. sbGnRH levels in the pituitary were high in the previtellogenic stage and low in the early spawning stage. In addition, the relatively high levels of pituitary sbGnRH were found together with high plasma T, E₂, and DHP levels in fish in the late yolk stage. These results indicate that sbGnRH in the telencephalon, hypothalamus, and pituitary is involved in ovarian maturation and that sbGnRH may play an important role in the initiation of ovarian recrudescence in wild Japanese flounder.     

Effects of Observed Incubation Behavior on Egg Production in Laying Hens of a Commercial Chicken Breed and Detection of Single-Nucleotide Polymorphisms Associated with the Incubation Behavior

Upon contact with laid eggs, avians initiate incubation behavior and stop laying additional eggs. This phenomenon suggests that the productivity of laying hens in free-range facilities may decrease because of frequent contact with laid eggs. Here, we examined whether hens of a commercial breed exhibit incubation behavior in a free-range facility and whether egg productivity subsequently decreases. One-hour observations were performed twice weekly for 3 weeks, during which 9 of 129 hens (7.0%) exhibited incubation behavior (i.e., sitting on eggs) in the free-range facility and were defined as incubating hens. During 4 d of continuous behavioral observation, incubating and non-incubating hens laid the same number of eggs statistically (4.6 and 3.6, on average, respectively); however, incubating hens spent significantly more time on average incubating the eggs (2071.9 min) than did the non-incubating hens (20.9 min; P<0.05), indicating a clear behavioral difference. Subsequently, the incubation behavior and egg productivity of incubating hens and a Silkie Fowl breed hen, which is known to exhibit typical incubation behavior and cessation of laying, were continuously compared for 27 d. The average minutes spent incubating eggs during the observation period increased in both the incubating hens and Silkie Fowl hen and the total time was almost the same (18,088.5 and 23,092 min, respectively). However, the Silkie Fowl hen stopped laying on day 17 after laying 17 eggs, whereas the incubating hens continued laying throughout the observation period. Incubating hens laid an average of 24.5 eggs, indicating that some hens (at least those of the commercial breed used in our study) can continue laying while exhibiting incubation behavior. A single-nucleotide polymorphism associated with incubation behavior was detected on chromosome 4 through genome-wide association analysis.     

Seasonal changes in cellular compartmentation and membrane permeability to sugars in relation to early or high temperature-induced watercore in ‘Orin’ apples

Summary

The intensity of watercore in ‘Orin’ apples (Malus domestica Borkh.) from the outer canopy increased from early-July to mid-August, followed by a decrease through late-October, while no watercore was detected in inner canopy fruit during the same period. Seasonal changes in sorbitol content in each cellular compartment paralleled the development of watercore. Significantly higher sorbitol, fructose and glucose contents in the vacuoles of cells in outer canopy fruit than in inner canopy fruit suggest that active metabolism and uptake of sorbitol might occur in watercored apples in mid-August. The permeability of sorbitol across the tonoplast was significantly higher in outer than in inner canopy fruit after mid-August, whereas no difference was observed between bearing position for the other sugars or for the plasma membrane. These data suggest that elevated sorbitol levels in the intercellular spaces might result from active apoplastic unloading and not from an inhibition of metabolism and/or an increase in leakage from cells.     

Changes of sires in a breeding farm enables maintenance of DNA‐level genetic variation in a produced herd of Hokkaido Native Horses

We investigated whether regular changes of the sire in a breeding farm of Hokkaido Native Horses (HKDs) enables the DNA‐level genetic variation of the produced animals to be maintained. The genotypes of 31 microsatellite markers were identified and analyzed in 207 animals produced in a breeding farm in which the sire was replaced every 3 to 5 years. The mean allele number indicating the degree of genetic variation was 5.97 and was similar to those reported previously. The mean observed heterozygosity was 0.74 and was higher than the expected heterozygosity, 0.69; F_IS was ?0.07, indicating that the analyzed animals reflected frequent outbreeding and had maintained genetic variation. Based on genetic structural analysis, the number of genetic subpopulations of the animals was estimated to be as 6, and the majority (more than 50%) of each subpopulation corresponded to the progeny of one of the sires used in the breeding farm; these observations suggested that genetic variation in the analyzed animals reflected the genetic differences among sires. Pedigree records indicated that the average co‐ancestry coefficient between sires used in the breeding farm was 0.015 corresponding to second cousin. This level of kinship among sires is acceptable for producing HKDs that maintain genetic variation.     

Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1‐positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four‐cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four‐cell stage improves the total number of nuclei and the percentage of POU5F1‐positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four‐cell stage did not exhibit a decrease in the percentage of POU5F1‐positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four‐cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1‐positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers.     

Autophagy defends cells against invading group A Streptococcus

We found that the autophagic machinery could effectively eliminate pathogenic group A Streptococcus (GAS) within nonphagocytic cells. After escaping from endosomes into the cytoplasm, GAS became enveloped by autophagosome-like compartments and were killed upon fusion of these compartments with lysosomes. In autophagy-deficient Atg5-/- cells, GAS survived, multiplied, and were released from the cells. Thus, the autophagic machinery can act as an innate defense system against invading pathogens.     

Development of winter wheat with excellent resistance to pre-harvest sprouting and rain damage

Pre-harvest sprouting and high alpha-amylase activity are the most serious problems for the wheat production in Hokkaido, the northern most part of Japan. It has become more frequent for wheats to be exposed to continuous rain for several days in harvest time. In addition, low temperature below 15 ^°C, combined with rainfall, accelerates the damage of pre-harvest sprouting. Therefore, we started a breeding program in 1998 to develop wheat varieties with excellent resistant to sprouting damage in Hokkaido.Selection and evaluation of pre-harvest sprouting of resistant lines were conducted by two tests. One test was artificial rain treatment of intact spikes and the other was a germination test of hand-threshed grains at 10, 15 and 20 ^°C. The spikes of each line and variety were collected at physiological maturity and at one week after the first sampling date. Falling number was also measured.We have succeeded in breeding a deeply dormant breeding line, Kitakei 1802, from 1,923 doubled haploid lines raised by anther culture from the cross between Kitakei 1616 and Nishikazekomugi. Kitakei 1802 hardly sprouted through 10 days” continuous rainfall treatment at low temperature (15 ^°C) and maintained high falling number (> 300 s) in 2002 and 2003.