Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

Effects of carprofen and meloxicam with or without butorphanol on the minimum alveolar concentration of sevoflurane in dogs

Sparing effects of carprofen and meloxicam with or without butorphanol on the minimum alveolar concentration (MAC) of sevoflurane were determined in 6 dogs. Anesthesia was induced and maintained with sevoflurane in oxygen, and MAC was determined by use of a tail clamp method. The dogs were administered a subcutaneous injection of carprofen (4 mg/kg) or meloxicam (0.2 mg/kg), or no medication (control) one hour prior to induction of anesthesia. Following the initial determination of MAC, butorphanol (0.3 mg/kg) was administered intramuscularly, and MAC was determined again. The sevoflurane MACs for carprofen alone (2.10 +/- 0.26%) and meloxicam alone (2.06 +/- 0.20%) were significantly less than the control (2.39 +/- 0.26%). The sevoflurane MACs for the combination of carprofen with butorphanol (1.78 +/- 0.20%) and meloxicam with butorphanol (1.66 +/- 0.29%) were also significantly less than the control value after the administration of butorphanol (2.12 +/- 0.28%). The sevoflurane sparing effects of the combinations of carprofen with butorphanol and meloxicam with butorphanol were additive.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Seasonal differences in rumen bacterial flora of wild Hokkaido sika deer and partial characterization of an unknown bacterial group possibly involved in fiber digestion in winter

Rumen digesta was obtained from wild Hokkaido sika deer to compare bacterial flora between summer and winter. Bacterial flora was characterized with molecular‐based approaches and enrichment cultivation. Bacteroidetes was shown as a major phylum followed by Firmicutes, with similar proportions in both seasons. However, two phylogenetically unique groups in Bacteroidetes were found in each season: unknown group A in winter and unknown group B in summer. The ruminal abundance of unknown group A was the highest followed by Ruminococcus flavefaciens in winter. Moreover, the abundance of these two was higher in winter than in summer. In contrast, the abundance of unknown group B was higher in summer than in winter. In addition, this group showed the highest abundance in summer among the bacteria quantified. Unknown group A was successfully enriched by cultivating with oak bark and sterilized rumen fluid, particularly that from deer. Bacteria of this group were distributed in association with the solid rather than the liquid rumen fraction, and were detected as small cocci. Accordingly, unknown group A is assumed to be involved in degradation of fibrous materials. These results suggest that wild Hokkaido sika deer develop a rumen bacterial flora in response to changes in dietary conditions.     

Cytological and genetical studies on male sterility inCryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility. The study was partially supported by Program for Promotion of Basic Research Actives for Innovative Biosciences.     

The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.     

Dispersal ability determines the genetic effects of habitat fragmentation in three species of aquatic insect

• 1. The dispersal ability of species and the geographic scale of habitat fragmentation both may influence the extent of gene flow between fragments, but their interactions have rarely been tested, particularly among co‐occurring species.
• 2. Population genetic structures of three species of aquatic insect were compared in streams fragmented by reservoirs and in unfragmented streams in north‐eastern Japan, using 52, 37, and 58 RAPD markers. The three species studied included a strong disperser Cincticostella elongatula (Ephemeroptera: Ephemerellidae), an intermediate disperser Stenopsyche marmorata (Trichoptera: Stenopsychidae), and a weak disperser Hydropsyche orientalis (Trichoptera: Hydropsychidae).
• 3. The patterns of genetic isolation by distance (IBD) supported a priori hypotheses of dispersal ability. The strong disperser (C. elongatula) exhibited significant IBD only at the largest spatial scale studied (among drainages, r=0.50, P<0.01). The intermediate disperser (S. marmorata) showed IBD both within (r=0.22, P<0.01) and among (r=0.45, P<0.01) drainages. The weak disperser (H. orientalis) did not exhibit significant IBD at any scale.
• 4. Pairwise genetic differentiation (θ) indicated that neither the weak disperser nor the strong disperser were genetically differentiated above and below reservoirs when compared with reference sites. This was in contrast to previous results for S. marmorata, for which subpopulations were genetically fragmented across larger (>4.1 km), but not smaller (<2.9 km) reservoirs.
• 5. We suggest that intermediate dispersers, i.e. those at equilibrium between migration and genetic drift within drainages, are more likely to be affected by fragmentation than either strong or weak dispersers. Intermediate dispersers could therefore be used as indicator species in studies aimed at detecting the effects of distance between habitat fragments (e.g. reservoir size) for conservation planning. Copyright © 2010 John Wiley & Sons, Ltd.

     

Seasonal variation of domoic acid in a bivalve Spondylus versicolor in association with that in plankton samples in Nha Phu Bay,Khanh Hoa,Vietnam

Previously we reported that a significant level of domoic acid, a causative toxin for amnesic shellfish poisoning, was detected in bivalves belonging to a genus Spondylus from various tropical Asian countries. These findings indicate that causative plankton species for domoic acid widely distribute in these areas. In the present study, we monitored seasonal change of domoic acid level of Spondylus versicolor in association with that of plankton samples in Nha Phu Bay, Khanh Hoa, Vietnam from December 2004 to October 2005. The toxicity of S. versicolor showed distinct seasonal variation. During the period when domoic acid level of S. versicolor was increasing, a significant level of domoic acid was detected in the plankton samples, showing the correlation between these two parameters. These findings show that plankton causative for domoic acid occurred in the bay, and S. versicolor accumulate domoic acid during a bloom of the toxic plankton by food web transfer.