The Effect on Intracytoplasmic Sperm Injection Outcome of Genotype,Male Germ Cell Stage and Freeze-Thawing in Mice

Background

Intracytoplasmic sperm injection (ICSI) has been widely used to study the mechanisms of mammalian fertilization and to rescue male-factor infertility in humans and animals. However, very few systematic analyses have been conducted to define factors affecting the efficiency of ICSI. In this study, we undertook a large-scale series of ICSI experiments in mice to define the factors that might affect outcomes.

Methodology/Principal Findings

We used a 5×3×2 factorial design with the following factors: mouse genotype (ICR, C57BL/6, DBA/2, C3H/He, and 129/Sv strains), type of male germ cells (epididymal sperm, elongated or round spermatids), and their freeze–thawing treatment. The efficiencies (parameters) of each developmental step were analyzed by three-way ANOVA (significance level P<0.01). The type of male germ cells affected all the four parameters observed: oocyte survival after injection, cleavage of oocytes, implantation, and birth of offspring. Genotype affected the oocyte survival, cleavage and birth rates, whereas freeze–thawing had no effects on any of the parameters. There were significant genotype/cell type interactions for oocyte survival and cleavage, indicating that they were determined by a combination of strain and germ cell maturity. Multiple comparisons revealed that spermatozoa and elongated spermatids gave better implantation and birth rates than did round spermatids, while spermatozoa and elongated spermatozoa were indistinguishable in their ability to support embryonic development. The best overall efficiency (birth rate per oocytes injected) was obtained with frozen–thawed DBA/2 strain elongated spermatids (23.2±4.2%).

Conclusions/Significance

The present study provides the first comprehensive information on ICSI using the mouse as a model and will contribute to the efficient use of materials, time, and efforts in biomedical research and clinics involving ICSI.     

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.     

The FOX hunting system: an alternative gain-of-function gene hunting technique

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.     

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina

Thioredoxin (TRX) plays a variety of redox-related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA-induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA-induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of the MAPKs, p38 kinase and c-Jun N-terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX-treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA-induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.     

Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.     

MIZ1, an essential protein for root hydrotropism, is associated with the cytoplasmic face of the endoplasmic reticulum membrane in Arabidopsis root cells

MIZ1 is encoded by a gene essential for root hydrotropism in Arabidopsis. To characterize the property of MIZ1, we used transgenic plants expressing GFP-tagged MIZ1 (MIZ1-GFP) and mutant MIZ1 (MIZ1(G235E)-GFP) in a miz1-1 mutant. Although both chimeric genes were transcribed, the translational products of MIZ1(G235E)-GFP did not accumulate in roots. Moreover, MIZ1-GFP complemented the mutant phenotype but not MIZ1(G235E)-GFP. The signal corresponding to MIZ1-GFP was detected at high levels in cortical cells and lateral root cap cells and accumulated in compartments in cortical cells. MIZ1-GFP was fractionated into a soluble protein fraction and an endoplasmic reticulum (ER) membrane fraction, where it was bound to the surface of the ER membrane at the cytosolic side.     

Identification and fine mapping of a major quantitative trait locus originating from wild rice, controlling cold tolerance at the seedling stage

Cold tolerance at the seedling stage (CTSS) is an important trait affecting stable rice production in temperate climates and areas of high elevation. In this study, 331 single nucleotide polymorphism (SNP) markers were developed and used along with phenotypic evaluation to identify quantitative trait loci (QTLs) associated with CTSS from a mapping population of 184 F₂ plants derived from a cold tolerant wild rice, W1943 (Oryza rufipogon), and a sensitive indica cultivar, Guang-lu-ai 4 (GLA4). Three QTLs were detected on chromosomes 3, 10 and 11. A major locus, qCtss11 (QTL for cold tolerance at seedling stage), was located on the long arm of chromosome 11 explaining about 40% of the phenotypic variation. Introduction of the W1943 allele of qCtss11 to the GLA4 genetic background increased CTSS. Based on the phenotypic and genotypic assessment of advanced backcross progenies, qCtss11 was dissected as a single Mendelian factor. A high-resolution genetic map was constructed using 23 markers across the qCtss11 locus. As a result, qCtss11 was fine mapped to a 60-kb candidate region defined by marker AK24 and GP0030 on chromosome 11, in which six genes were annotated. Expression and resequence analyses of the six candidates supported the hypothesis that Os11g0615600 and/or Os11g0615900 are causal gene(s) of the CTSS.     

Characterization of Leukocyte Mono-immunoglobulin-like Receptor 7 (LMIR7)/CLM-3 as an Activating Receptor: ITS SIMILARITIES TO AND DIFFERENCES FROM LMIR4/CLM-5*

Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function.     

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD⁺/NADH ratio in cells and results in activation of SIRT1, an NAD⁺-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD⁺ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD⁺ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD⁺ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD⁺, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.     

Chromosome oscillation promotes Aurora A–dependent Hec1 phosphorylation and mitotic fidelity

Most cancer cells show chromosomal instability, a condition where chromosome missegregation occurs frequently. We found that chromosome oscillation, an iterative chromosome motion during metaphase, is attenuated in cancer cell lines. We also found that metaphase phosphorylation of Hec1 at serine 55, which is mainly dependent on Aurora A on the spindle, is reduced in cancer cell lines. The Aurora A–dependent Hec1-S55 phosphorylation level was regulated by the chromosome oscillation amplitude and vice versa: Hec1-S55 and -S69 phosphorylation by Aurora A is required for efficient chromosome oscillation. Furthermore, enhancement of chromosome oscillation reduced the number of erroneous kinetochore–microtubule attachments and chromosome missegregation, whereas inhibition of Aurora A during metaphase increased such errors. We propose that Aurora A–mediated metaphase Hec1-S55 phosphorylation through chromosome oscillation, together with Hec1-S69 phosphorylation, ensures mitotic fidelity by eliminating erroneous kinetochore–microtubule attachments. Attenuated chromosome oscillation and the resulting reduced Hec1-S55 phosphorylation may be a cause of CIN in cancer cell lines.