Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.     

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.     

Expression of amphiphysin I in Sertoli cells and its implication in spermatogenesis

Amphiphysin I is a protein concentrated in nerve terminals and involved in the endocytosis of synaptic vesicle membrane. We show here that amphiphysin I is expressed in the rat testis, localized exclusively in the Sertoli cells. In the postnatal testicular development, expression of amphiphysin I was not evident at birth, but became significant at postnatal day 15 (P15), coinciding with the onset of spermatogenesis. The expression level of amphiphysin I increased 10-fold between P15 and P25 to reach the adult level. In adult testes reversibly damaged by ethane dimethane sulphonate administration, expression of amphiphysin I did not change following the damage, whereas the protein was transiently converted into its phosphorylated form. The increase in levels of phosphorylated amphiphysin I was closely associated with the severe histological damage to germ cells. The present findings suggest that amphiphysin I in Sertoli cells is involved in spermatogenesis, probably through endocytic processes.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.     

Novel (CA)n marker DXYS241 on the nonrecombinant part of the human Y chromosome

The origin of modern humans can be traced by comparing polymorphic sites in either mitochondria or genomic sequences between humans and other primates. The human Y chromosome has both a non-recombining region and X-Y homologous pseudo-autosomal regions. In the nonrecombining region events during evolution can be directly detected. At least a part of homology between Xq21 and Yp11 is a result of rather recent translocations from the X chromosome to the Y chromosome. DNA markers residing in the nonrecombining region of the human Y chromosome are potentially useful in tracing male-specific gene flow in human evolution. However, the number of available markers in the region is limited. Here, we report a novel X-Y homologous (CA)n repeat locus in the nonrecombining region of the Y chromosome. This marker, DXYS241, has several interesting features. Y- and X-chromosome alleles are distinguishable because the Y-chromosome alleles are shorter than the X-chromosome alleles most of the time. We developed 2 primer sets for specific examination of Y- and X-chromosome alleles. The marker should be useful in establishing relationships between populations based on patrilineal gene flow. Sequences homologous to DXYS241 are also found on the X chromosome of primates. Four events during primate evolution that led to the modern human Y chromosome were identified.     

A new species of the genus Hypsibius (Tardigrada: Parachela: Hypsibiidae) from Sakhalin island, far East Russia

A new species of semiterrestrial eutardigrade, Hypsibius stiliferus, is described from Sakhalin Island, Far East Russia. The new species is distinguished from its congeners by having an irregular, polygonal or subtriangular dorsal sculpture, which increases in size posteriorly, two granular macroplacoids, and a cuticular bar near the base of posterior claw 4, and by lacking a microplacoid and septulum. It is currently known from its type locality and several other localities in northern and southern Sakhalin Island. This is the first report concerning tardigrades from Sakhalin Island.     

Oxidative stress and delayed-onset muscle damage after exercise

Reactive oxygen species (ROS) produced during exercise may be involved in delayed-onset muscle damage related to inflammation. To investigate this hypothesis, we studied whether oxidative stress increases nuclear translocation of nuclear factor-kappaB and chemokine expression in skeletal muscle using myotube L6 cells. We also assessed whether prolonged acute exercise could increase these parameters in rats. In L6 cells, H(2)O(2) induced nuclear translocation of p65 and increased the expression of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1), whereas preincubation with alpha-tocopherol limited the increase in these proteins. Sprague Dawley rats were divided into the following groups: rested control, exercised, rested with a high alpha-tocopherol diet, and exercised with a high alpha-tocopherol diet. After 3 weeks of acclimation, both exercise groups ran on a treadmill at 25 m/min for 60 min. Exercise increased nuclear p65, CINC-1, and MCP-1 in gastrocnemius muscle cells, but these changes were ameliorated by the high alpha-tocopherol diet. Increases in myeloperoxidase and thiobarbituric acid-reactive substrates were ameliorated by a high alpha-tocopherol diet, as were the histological changes. Neutrophil activity was not altered by either exercise or a high alpha-tocopherol diet. These results indicate that delayed-onset muscle damage induced by prolonged exercise is partly related to inflammation via phagocyte infiltration caused by ROS and that alpha-tocopherol (an antioxidant) can attenuate such inflammatory changes.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.