Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.     

A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.     

Improving ovulation in gilts using anti-inhibin serum treatment combined with fixed-time artificial insemination

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6–8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-β (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG.     

Effect of shading on zinc deficiency symptoms in rice plant

Under Zn deficiency, some major deficiency symptoms were observed on rice plants, i.e., reduction of young leaf elongation and development of necrosis on the expanded leaves. To clarify the former phenomena, the physiological role of Zn was studied from the standpoint of protein synthesis (Kitagishi and Obata 1986; Obata et al. 1994, 1996) and metabolism of auxin (Takaki and Arita 1986; Domingo et al. 1992). In contrast, the direct cause of the latter phenomenon has not yet been studied.     

Histological observation of the reproductive system in a viviparous teleost Xenotoca eiseni Rutter 1896 (Cyprinodontiformes: Goodeidae)

Xenotoca eiseni is a viviparous teleost belonging to the family Goodeidae. Here, we report histological observations of the reproductive organs in an adult male, an adult female, a pregnant female with intraovarian embryo and an extracted embryo of X. eiseni. High-resolution images of haematoxylin–eosin-stained sagittal sections revealed the detailed structure of gonads, gametes and reproductive components of the mother–embryo relationship. In the male, mature spermatozoa in the epididymis formed sperm packages. In the female, oogenesis proceeded asynchronously in the ovarian wall, and various stages of oocytes were observed in single ovary. In both sexes, genital openings were located between the anus and anal fin. Developing embryos were observed in an ovary of the pregnant female. Fine structures of components of the mother-to-embryo nutrient supply, ovarian septum and trophotaenia were observed in the pregnant ovary. An immature gonad prior to gamete formation was identified in the extracted embryo. With the aim of supporting the development and extension of studies on this viviparous teleost, we have shared our histological images as raw data in an open online archive, the 'NAGOYA repository ( http://hdl.handle.net/2237/00032456 )'. Our goal is a comprehensive understanding of the viviparous system in fish using both histological observation and molecular biology methods including genomics and proteomics.     

3-Methylthiopropionic acid ethyl ester, isolated from Katsura-uri (Japanese pickling melon, Cucumis melo var. conomon), enhanced differentiation in human colon cancer cells

The fully ripened fruit of Katsura-uri Japanese pickling melon ( Cucumis melo var. conomon) has rarely been used for food because the midripened fruit is utilized for making pickles, but the fully ripened fruit is no longer valuable for pickles due to the fruit body being too soft. We have considered the utilization of the fully ripened Katsura-uri fruit that may be used for nonpickling products, particularly if the fully ripened fruit demonstrated health benefits such as anticarcinogenic properties. The phytochemical extract from the fully ripened fruit of Katsura-uri Japanese pickling melon was purified via a bioassay-guided fractionation scheme, which was based on the induction of differentiation in a RCM-1 human colon cancer cell line. On the criteria of two differentiation markers (duct formation and alkaline phosphatase activity), the most potent fraction contained a compound identified as 3-methylthiopropionic acid ethyl ester, based on GC retention time, EI-MS, (1)H NMR, and (13)C NMR spectra. Previously, the role of 3-methylthiopropionic acid ethyl ester was considered as an odor producing compound in many fruits, but this study indicates potential medical benefits of this compound.     

The developmental ability of vitrified oocytes from different mouse strains assessed by parthenogenetic activation and intracytoplasmic sperm injection

Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 x DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr(2+) treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.     

Succession and Phylogenetic Profile of Eubacterial Communities in Rice Straw Incorporated into a Rice Field: Estimation by PCR-DGGE Analysis

PCR-DGGE analysis followed by sequencing was conducted to estimate the succession and the phylogenetic profile of the eubacterial communities responsible for the decomposition of rice straw (RS) that was incorporated into a rice field. Leaf sheath and leaf blade parts were separately put in nylon mesh bags, and were placed in the rice field soil under drained conditions during the off-cropping season and under flooded conditions after transplantation of rice. In addition, RS samples that had been placed under drained conditions in the off-cropping season were placed again in flooded rice field soil after transplantation of rice. DGGE patterns of the bacterial communities in the RS samples were classified into two groups, namely leaf sheaths and leaf blades. Principal component analysis of the DGGE patterns revealed the succession along with the duration of placement. These results indicated that the RS part (sheath or blade) mainly determined the structure of the bacterial communities responsible for the RS decomposition, followed by the duration of placement. Sequence analysis of the characteristic DGGE bands indicated that most of the closest relatives associated with the bands belonged to α-, β-, γ-, and δ-Proteobacteria, CFB group, and Spirochaetes. Some bands were closely related to Acidobacteria and Verrucomicrobia. CFB members and α-Proteobacteria predominated commonly in both RS parts, while γ- and δ-Proteobacteria, and Spirochaetes and β-Proteobacteria specifically colonized sheath and blade parts, respectively. In addition, Proteobacteria and CFB members characterized the differences in the bacterial communities under flooded or drained conditions. These results suggest that Proteobacteria, CFB group, and Spirochaetes were responsible for RS decomposition in rice field soil under both flooded and drained conditions.     

Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain

The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases.