Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

ATeam technology for detecting early signs of viral cytopathic effect.

Adenosine 5'-triphosphate (ATP), the major energy currency of the cell, is involved in many cellular processes, including the viral life cycle, and can be used as an indicator of early signs of cytopathic effect (CPE). In this study, we demonstrated that CPE can be analyzed using an FRET-based ATP probe named ATP indicator based on Epsilon subunit for Analytical Measurements (ATeam). The results revealed that as early as 3 hr, the virus infected cells showed a significantly different Venus/cyan fluorescent protein (CFP) ratio compared to the mock-infected cells. The ATeam technology is therefore useful to determine the early signs of ATP-based CPE as early as 3 hr without morphology-based CPE by light microscopy, and enables high throughput determination of the presence of microorganisms in neglected samples stored in laboratories.

The complete genomic sequence of Rhinolophus gammaherpesvirus 1 isolated from a greater horseshoe bat.

In a comprehensive research project on bat viruses, we successfully isolated a novel herpesvirus from the spleen of a greater horseshoe bat (Rhinolophus ferrumequinum) in Japan using a cell line established from the kidney of the same bat. This herpesvirus was a novel gammaherpesvirus (Rhinolophus gammaherpesvirus 1; RGHV-1), which belonged to the genus Percavirus. The whole RGHV-1 genome (147,790 bp) showed that 12 of the 84 genes predicted to contain open reading frames did not show any homology to those of other herpesviruses.

A novel method of gene transduction to the murine endometrium using in vivo electroporation.

To investigate the molecular pathways involved in successful embryo implantation in mammals, we developed a novel method for gene transduction into the murine endometrium using in vivo electroporation. Plasmid DNA with an enhanced green fluorescence protein (EGFP) gene was injected into the uterine cavity of non-pregnant female mice, and electrical pulses were subsequently applied to the uterine horn using plate electrodes. EGFP expression was found only in the uterine luminal epithelium (LE), but not in the stroma. EGFP fluorescence in the LE was limited to the site where the positive side of the electrodes was placed during electric stimulation. These results demonstrated that our novel method enabled us to transduce a gene into a desired location of the murine uterus.

The LIF-mediated molecular signature regulating murine embryo implantation.

The establishment of a receptive uterus is the prime requirement for embryo implantation. In mice, the E2-induced cytokine leukemia inhibitory factor (LIF) is essential in switching the uterine luminal epithelium (LE) from a nonreceptive to a receptive state. Here we define the LIF-mediated switch using array analysis and informatics to identify LIF-induced changes in gene expression and annotated signaling pathways specific to the LE. We compare gene expression profiles at 0, 1, 3, and 6 h, following LIF treatment. During the first hour, the JAK-STAT signaling pathway is activated and the expression of 54 genes declines, primarily affecting LE cytoskeletal and chromatin organization as well as a transient reduction in the progesterone, TGFbetaR1, and ACVR1 receptors. Simultaneously 256 genes increase expression, of which 42 are transcription factors, including Sox, Kfl, Hes, Hey, and Hox families. Within 3 h, the expression of 3987 genes belonging to more than 25 biological process pathways was altered. We confirmed the mRNA and protein distribution of key genes from 10 pathways, including the Igf-1, Vegf, Toll-like receptors, actin cytoskeleton, ephrin, integrins, TGFbeta, Wnt, and Notch pathways. These data identify novel LIF-activated pathways in the LE and define the molecular basis between the refractory and receptive uterine phases. More broadly, these findings highlight the staggering capacity of a single cytokine to induce a dynamic and complex network of changes in a simple epithelium essential to mammalian reproduction and provide a basis for identifying new routes to regulating female reproduction.

The contribution of leukemia inhibitory factor (LIF) for embryo implantation differs among strains of mice.

Despite of the claim that maternal leukemia inhibitory factor (LIF) - a member of interleukin 6 (IL6) family of cytokines - plays indispensable roles for murine embryo implantation, these roles remain undefined in humans because the potency of LIF on implantation appears to vary among individuals. Here, we showed that the contribution of LIF for murine implantation was dependent on the strains of mice (ICR, C57BL/6J (B6), ddY, BALB/c, DBA/2Cr and MF1 strains). Inhibition of LIF during the implantation period caused severe disruption of embryo implantation in B6 and MF1 strains. Implantation was partly disrupted in other strains, but some embryos were implanted successfully. We speculated that other IL6 family members compensate for LIF actions on implantation in ICR, ddY, BALB/c, and DBA/2Cr strains. Indeed, the expression level of Ctf1 was upregulated by blockage of LIF function. CT-1 (encoded by Ctf1) treatment induced successful implantation without LIF in delayed implantation mice (ICR and B6) via phosphorylation of the signal transducer and activator of transcription 3 (STAT3) in the uterine luminal epithelium. Simultaneous inhibition of LIF and CT-1 did not block implantation completely in ICR mice, indicating that embryo implantation in this strain was robustly protected by LIF, CT-1 and other potential STAT3 activators. The present study might provide an explanation for the individual variation in the potency of LIF for embryo implantation in humans.

The complete control of murine pregnancy from embryo implantation to parturition.

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17ß-estradiol (E(2)). Continuous exposure to E(2) was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E(2) and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E(2) after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

Kisspeptin neurons mediate reflex ovulation in the musk shrew (Suncus murinus).

The present study investigated whether kisspeptin-G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew (Suncus murinus), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29-induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1-expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1-expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1-positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin-GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Agrin pathway is controlled by leukemia inhibitory factor (LIF) in murine implantation.

Agrin is the heparan sulfate proteoglycan (HSPG) that is well known as the molecule that aggregates acetylcholine receptor (AChR) through muscle specific kinase (MuSK) and rapsyn at neuromuscular junctions. HSPGs are spatiotemporally expressed in embryonic and maternal tissues during implantation. The present study examined the role of agrin in the mouse embryo using leukemia inhibitory factor (LIF)-deficient mice, which show complete sterility. Agrin was detected widely in the cytoplasm of uterine luminal epithelial cells at the third day of pregnancy (Day 3) and Day 4. At Day 5, agrin moved to the apical surface of the luminal epithelium. This migration was not found in LIF-deficient mice. AChR was also found in the apical surface of the uterine epithelium at Day 4 and Day 5 in normal mice. LIF-deficient mice did not show this pattern of expression. Only nAChR b1 subunit mRNA was increased at Day 5 in normal mice. Furthermore, acetylcholinesterase was active in the uterine stroma of normal mice throughout the implantation period and was exclusively active in the uterine epithelium at Day 4. Taken together, agrin signaling was activated in the uterus during embryo implantation in the mice. Here, we suggest that the agrin pathway is involved in closure of the uterine epithelium toward placentation.

Spontaneous endometrial hyperplasia in the uteri of IL-2 receptor beta-chain transgenic mice.

We found frequent and spontaneous proliferation of glandular epithelium and dilated cysts in the uteri of interleukin-2 receptor (IL2R) beta-chain transgenic (Tg2Rbeta) mice. The aim of this study was to examine the involvement of IL2R beta-chain in the pathogenesis of endometrial hyperplasia (EH). Mouse uteri and serum were collected from Tg2Rbeta and normal littermates (NL), which were classified into three groups according to age. The incidence of EH increased in an age-dependent manner in both types of mice. However, in old age, Tg2Rbeta mice showed more serious phenotypes of EH than NL. Immunohistochemical analysis revealed specific localization of IL2R beta-chain in the glandular epithelial cells, with a correlation to the degree of EH, not only in the Tg2Rbeta uteri but also in the NL uteri. Immunoreactions of CD3 and CD25 were detected in the uteri of Tg2Rbeta but were weak in the uteri of NL, and CD25-positive cells were distributed in the endometrial stroma and myometrium in the Tg2Rbeta mice. These findings suggest that the IL2R beta-chain induces growth potential for glandular epithelial cells and an immune-privileged condition mediated by CD25+regulatory-T cells.

Effects of leukemia inhibitory factor on lectin-binding patterns in the uterine stromal vessels of mice.

Lectin histochemistry was performed on mouse uteri to determine what effects leukemia inhibitory factor (LIF) has on carbohydrate epitope expressions at the time of implantation. Twenty-two biotinylated lectins were used in this study. Following injection of LIF, specific binding to the apical surface of the uterine glandular epithelium (GE) was recognized by six lectins. Particularly, binding of the lectin from Griffonia (Bandeiraea) simplicifolia was specific to the glandular epithelium close to the luminal epithelium. Succinylated wheat germ agglutinin (WGA), which has specificity for oligosaccharides recognized by WGA without sialic acid residues, showed weaker binding to the uterine luminal epithelium (LE) and the stroma than WGA, suggesting that terminal residues of glyco-conjugates on these tissues may be modified by sialic acids. Lectin binding to the glandular and luminal epithelium was not influenced by LIF. However, three lectins including a lectin from Dolichos biflorus showed specificity for stromal vessels 6h after LIF injection. Since the lectin from D. biflorus binds to neo-vascular vessels, LIF may play a role in regulating maternal angiogenesis directly and/or indirectly during implantation.

Upregulation of Indian hedgehog gene in the uterine epithelium by leukemia inhibitory factor during mouse implantation.

Leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) are essential for embryo implantation in mice and are regulated by the actions of 17beta-estradiol (E2) and progesterone, respectively. The present study examined the effect of LIF on Ihh and Ihh-related factors in the uterine luminal epithelium during the implantation period using a DNA microarray. Expression of Ihh mRNA reached its peak on the forth day of pregnancy, and progesterone receptor (Pgr) mRNA decreased on the fifth day of pregnancy in wildtype mice. On the other hand, these changes in expression were not seen in LIF-/- mice. Ihh and Pgr mRNA were upregulated by LIF injection in delayed implantation mice. This up-regulation of Pgr was transient and preceded an increase of Ihh mRNA. Ihh mRNA also increased after E2 injection in delayed implantation mice of the LIF-/- genotype. E2 did not affect transcription of Pgr mRNA in the uterine luminal epithelium of delayed implantation LIF-/- mice. Using an antibody against the C-terminal epitope of Ihh, unprocessed Ihh proteins, but not C-terminal peptides, by autoproteolytic cleavage of Ihh were detected by western blot analysis. Unprocessed Ihh did not show quantitative changes between the wildtype and LIF-/- mice during the implantation period. Transcription of hedgehog acyltransferase was not influenced by LIF and E2 injection. In conclusion, LIF, which has a crucial role in E2 action for initiation of implantation, caused transient induction of Pgr mRNA and subsequent upregulation of Ihh mRNA, which mediates progesterone-Pgr actions for successful implantation.

Localization of interleukin-6 receptor mRNA in the pregnant and non-pregnant mouse uterus.

To understand roles of interleukin 6 (IL-6) family cytokines for pregnancy in mice, localization of IL-6 receptor (IL-6R) mRNA was investigated in non- and early pregnant uteri by in situ hybridization. IL-6R mRNA was expressed in all non-pregnant uteri and in pregnant uteri from the third day (Day 3) to the sixth day of pregnancy (Day 6; the day of plug = Day 1). IL-6R mRNA signals were detected in non-pregnant mice in the luminal and glandular epithelium. Signal strength varied according to the sexual cycle. There was no correlation between the signal strength of the IL-6R mRNA and the serum concentrations of progesterone and 17beta-estradiol, which show a monophasic rise in the non-pregnant sexual cycle. In pregnant mice, slight signals were detectable in the luminal and glandular epithelium on Day 3. IL-6R mRNA messages increased with progression towards Day 4, however, localization changed drastically on Day 5. Stromal cells abruptly expressed their mRNA on Day 5, and these cells strongly expressed it on Day 6. The function of IL-6R in the luminal and glandular epithelium might be different from that in the stroma during the implantation period. In addition, few signals were identified in the stromal cells adjacent to the luminal epithelium on Day 6. This suggests that there are two types of stromal cells on Day 6 in mice.

Profiling gene expression in growth-arrested mouse embryos in diapause.

In many mammalian species, embryonic cell proliferation can be reversibly arrested in an embryonic diapause at the time of embryo implantation. A recent report has identified changes in embryonic gene expression that are associated with, and may halt, embryonic cell proliferation.