Yusuke Atsumi

Phospholipase Cζ mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor

This study was conducted to investigate the role of a sperm‐borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Cζ (PLCζ) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCζ cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca2+ chelator) before SE injection. On the other hand, when the oocytes were injected with PLCζ cRNA at 60 µg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCζ cRNA‐induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCζ cRNA can induce development. In addition, RT‐PCR revealed that PLCζ mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCζ is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis. Mol. Reprod. Dev. 76: 1200–1207, 2009.

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens.

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

Low temperature-induced circulating triiodothyronine accelerates seasonal testicular regression.

In temperate zones, animals restrict breeding to specific seasons to maximize the survival of their offspring. Birds have evolved highly sophisticated mechanisms of seasonal regulation, and their testicular mass can change 100-fold within a few weeks. Recent studies on Japanese quail revealed that seasonal gonadal development is regulated by central thyroid hormone activation within the hypothalamus, depending on the photoperiodic changes. By contrast, the mechanisms underlying seasonal testicular regression remain unclear. Here we show the effects of short day and low temperature on testicular regression in quail. Low temperature stimulus accelerated short day-induced testicular regression by shutting down the hypothalamus-pituitary-gonadal axis and inducing meiotic arrest and germ cell apoptosis. Induction of T3 coincided with the climax of testicular regression. Temporal gene expression analysis over the course of apoptosis revealed the suppression of LH response genes and activation of T3 response genes involved in amphibian metamorphosis within the testis. Daily ip administration of T3 mimicked the effects of low temperature stimulus on germ cell apoptosis and testicular mass. Although type 2 deiodinase, a thyroid hormone-activating enzyme, in the brown adipose tissue generates circulating T3 under low-temperature conditions in mammals, there is no distinct brown adipose tissue in birds. In birds, type 2 deiodinase is induced by low temperature exclusively in the liver, which appears to be caused by increased food consumption. We conclude that birds use low temperature-induced circulating T3 not only for adaptive thermoregulation but also to trigger apoptosis to accelerate seasonal testicular regression.

Lactic acid is a sperm motility inactivation factor in the sperm storage tubules

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). The long-term preservation of sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature.

Novel Method of Gene Transfer in Birds: Intracytoplasmic Sperm Injection for Green Fluorescent Protein Expression in Quail Blastoderms

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Rapid, highly sensitive, and simultaneous detection of staphylococcal enterotoxins in milk by using immuno-pillar devices

Staphylococcal enterotoxins (SEs) have repeatedly caused food poisoning incidents worldwide. Some of the challenges associated with food poisoning outbreaks are that traditional detection methods are expensive and require long processing times and trained technicians. Microchannel devices represent a potential detection method by which these difficulties can be overcome. In this paper, we propose that immuno-pillar devices may represent a rapid, highly sensitive, and low-cost analytical system for the simultaneous detection of staphylococcal enterotoxin types A, B, and D (SEA, SEB, and SED) in milk. To prepare milk samples simulating food contaminated with SEs, commercial milk was spiked with equal amounts of SEA, SEB, and SED. A quantitative analysis of the milk samples was performed within 15 min by using the microchannel device. The analysis required only 0.5 μL of untreated milk sample. The resultant limit of detection was 15.6 pg mL−1 for each SE, and the total assay time and sensitivity were markedly shorter and higher, respectively, than those for commercially available assay kits. The detection range of each enterotoxin using these devices was estimated as 15.6 pg mL−1 to 100 ng mL−1, which completely covers the SE concentrations that can lead to foodborne diseases based on the U.S. Food and Drug Administrations criterion for the infectious SE dose in SE poisoning (1 μg SE). Using our devices, frequent assessment of food potentially contaminated with SE is possible.

Culture System for Bobwhite Quail Embryos from the Blastoderm Stage to Hatching

Quail are divided phylogenetically into two groups, Old World quail and New World quail. Old World quail, such as the Japanese quail (Coturnix japonica), belong to the Phasianidae and distributed in the Palaearctic region (Europe, North Africa, and Asia), whereas New World quail, such as the bobwhite quail (Colinus virginianus), belong to the Odontophoridae and are restricted to North and South America. Both the bobwhite quail and the Japanese quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been limited compared with those on the Japanese quail. We have therefore now developed an ex vivo culture protocol for bobwhite quail embryos from the blastoderm stage through hatching. Of the various culture conditions examined in the present study, a good hatching rate (39%) was obtained when the embryos were cultured ex vivo in a two-step procedure. Unincubated embryos (with egg yolk) were first cultured inside the shell of a Japanese quail egg (11.5 to 13.0 g whole egg weight) together with chicken thin albumen for 63 to 65 h and were then transferred to the shell of a small-sized chicken egg (38 g whole egg weight) until hatching. This ex vivo culture system should provide to be widely applicable to the maintenance and generation of manipulated birds for basic and applied studies on the bobwhite quail.

Different Photoperiodic Responses in Four Lines of Japanese Quail

Organisms measure day length to better adapt to seasonal changes in the environment; this phenomenon is called photoperiodism. The Japanese quail has a highly sophisticated photoperiodic mechanism and is an excellent model for the study of photoperiodism. Various lines of quail have been established during the domestication process. In the present study, we examined the effect of long day (LD) followed by short day (SD) on testicular weight in four lines of quail (L, AMRP, NIES-Br, and WE). When the quail were raised under SD conditions, testicular development was suppressed in all examined lines. The speed of the LD-induced testicular development of NIES-Br line was faster than that of AMRP line, while the speed of the SD-induced testicular regression of L line was significantly faster than that of WE line. These quail lines provide excellent model to uncover the underlying mechanism of seasonal testicular regression.