Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.

Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko Gnathopogon caerulescens.

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.

PRDM14 promotes active DNA demethylation through the ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells.

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

Successful vitrification of whole juvenile testis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens).

Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice.

The preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Response to fish specific reproductive hormones and endocrine disrupting chemicals of a Sertoli cell line expressing endogenous receptors from an endemic cyprinid Gnathopogon caerulescens.

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20ß-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions.

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0­2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO, and that recovered PGCs retained ability to differentiate into functional gametes.

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens).

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and ß-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.

Nonylphenol reduced the number of haploids in in vitro spermatogenesis of the endangered cyprinid Gnathopogon caerulescens.

Nonylphenol (NP), an endocrine disrupting chemical, is widely used in industrial and agricultural processes, causing NP influx into aquatic environments. NP induces hormonal imbalance, and male feminization, and reduces germ cell production during spermatogenesis; however, the mechanism by which it affects spermatogenesis remains unknown. Here, we investigated the effect of NP on spermatogenesis in honmoroko (Gnathopogon caerulescens), an endangered fish endemic to Lake Biwa, Japan, using an in vitro differentiation system. We collected spermatogonia from the testes of non-spawning G. caerulescens and subjected them to suspension culture. The spermatogonia differentiated into flagellated spermatozoa in 3 weeks, regardless of the presence of NP. NP concentrations as low as 1 nM caused a decrease in the number of germ cells in a dose-dependent manner, whereas the number of somatic cells decreased only at a high concentration of 1 µM. Flow cytometric analysis revealed that the decrease in germ cell number was attributed to haploids (spermatids and spermatozoa); the number of spermatogonia and spermatocytes was not affected by NP treatment. This result is consistent with the hypothesis that NP might repress the second meiosis or induce apoptosis in haploids. This study demonstrated that the combination of in vitro germ cell differentiation and flow cytometric analysis is useful for evaluating the direct effects of NP on germ cell differentiation in endangered endemic fish.

Global DNA Methylation in Cord Blood as a Biomarker for Prenatal Lead and Antimony Exposures.

DNA methylation is an epigenetic mechanism for gene expression modulation and can be used as a predictor of future disease risks. A prospective birth cohort study was performed to clarify the effects of neurotoxicants on child development, namely, the Tohoku Study of Child Development, in Japan. This study aimed to evaluate the association of prenatal exposure to five toxic metals-arsenic, cadmium, mercury, lead (Pb), antimony (Sb), and polychlorinated biphenyls (PCBs, N = 166)-with global DNA methylation in umbilical cord blood DNA. DNA methylation markers, 5-methyl-2'-deoxycytidine (mC) and 5-hydroxymethyl-2'-deoxycytidine (hmC), were determined using liquid chromatography-tandem mass spectrometry. The mC content in cord blood DNA was positively correlated with Pb and Sb levels (r = 0.435 and 0.288, respectively) but not with cord blood PCBs. We also observed significant positive correlations among Pb levels, maternal age, and hmC content (r = 0.155 and 0.243, respectively). The multiple regression analysis among the potential predictors demonstrated consistent positive associations between Pb and Sb levels and mC and hmC content. Our results suggest that global DNA methylation is a promising biomarker for prenatal exposure to Pb and Sb.

Alteration in peritoneal cells with the chemokine CX3CL1 reverses age-associated impairment of recognition memory.

Cognitive function progressively declines with advancing age. The aging process can be promoted by obesity and attenuated by exercise. Both conditions affect levels of the chemokine CX3CL1 in peripheral tissues; however, its role in cognitive aging is unknown. In the current study, we administered CX3CL1 into the peritoneal cavity of aged mice to investigate its impact on the aging process. In the peritoneal cavity, CX3CL1 not only reversed the age-associated accumulation of cells expressing the senescence marker p16INK4a but also increased peritoneal phagocytic activity, indicating that CX3CL1 affected the phenotypes of peritoneal cells. In the hippocampus of aged mice, intraperitoneal administration of CX3CL1 increased the number of Type-2 neural stem cells and promoted brain-derived neurotrophic factor (BDNF) expression. This treatment, furthermore, improved novel object recognition memory impaired with advancing age. Intraperitoneal transplantation of peritoneal cells from CX3CL1-treated aged mice improved novel object recognition memory in recipient aged mice. It indicates that peritoneal cells have a critical role in the CX3CL1-induced improvement of recognition memory in aged mice. Vagotomy inhibited the CX3CL1-induced increase in BDNF expression, demonstrating that the vagus nerve is involved in the hippocampal BDNF expression induced by intraperitoneal administration of CX3CL1. Thus, our results demonstrate that a novel connection among the peritoneal cells, the vagus nerve, and the hippocampus can reverse the age-associated decline in recognition memory.

Transcriptome analysis revealed bisphenol A and nonylphenol affect reproduction.

Effects of endocrine disrupting chemicals (EDCs) on reproduction have not been fully explained comprehensively. In this study, we tried to validate the common effect of Bisphenol A (BPA) and Nonylphenol (NP) on the differentiation of embryonic stem (ES) cells and found that they modify the expression of germ cell specific genes. To elucidate functional significance on biological process, we performed Gene Ontology (GO)-based microarray analysis comparing with published GeneChip data of primordial germ cell development in vivo. Cluster analysis of gene expression profile revealed that EDC treatment and primordial germ cell (PGC) development shared characteristic cluster consists of GO terms related to "germ cell development" and "reproduction". In the GO term "reproduction", meiosis related genes showed high expression level by EDC exposure. These results suggest that BPA and NP affect not only some of the germ cell specific genes, but functionally interferes germ cell development and reproduction.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis).

In nonhuman primates (NHPs), there have so far been few reports about nuclear transfer (NT), especially using adult somatic cells. The objective of this study was to determine the developmental competence of NT embryos derived from various somatic cells embryonic stem (ES), amniotic epithelial, cumulus, or fetal fibroblast cells] and the nuclear transfer method, such as electro fusion or piezo microinjection, activation with chemical reagent [ionomycine/6-dimethylaminopurine (DMAP), calcium ionophore A23187/DMAP, or cycloheximide (CHX)] and reprogramming time (1, 2, or 4 h; in this study, the duration from injection or fusion to activation was defined as the reprogramming time). Our results showed that a 1-h reprogramming and activation with ionomycin/DMAP are suitable for NT in monkeys. Developing cleaved embryos up to the six-cell stage was similar among all experiments. However, beyond the eight-cell stage, developmental rates were higher in NT embryos reconstructed with fetal fibroblast cells and amniotic epithelial cells, and we were able to produce NT blastocysts from these cells. Interestingly, electro fusion is sufficient for amniotic epithelial cells and piezo microinjection is better suited for fetal fibroblast cells to produce NT blastocysts, thus suggesting that the best method for somatic cell NT may be different between cell types.

In vitro differentiation of fertile sperm from cryopreserved spermatogonia of the endangered endemic cyprinid honmoroko (Gnathopogon caerulescens).

Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

Transient suppression of PPARgamma directed ES cells into an osteoblastic lineage.

Osteoblasts and adipocytes are believed to share a common progenitor. Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the switching of these two cell lineages. Here, we demonstrated the differentiation of ES cells into an osteoblastic lineage using siRNA against PPARgamma without the addition of any osteogenic factors. We found that PPARgamma-siRNA downregulated the expression of aP2 mRNA and lipid accumulation, whereas it upregulated the expression of osteocalcin and calcium deposition. These results suggested that ES cells were directed into an osteoblastic lineage. Therefore, transient suppression using PPARgamma-siRNA may be a novel tool to induce differentiation of ES cells into osteoblasts.

Monkey embryonic stem cells differentiate into adipocytes in vitro.

Production of functional adipocytes is important in adipocyte research and regenerative medicine. In this paper, we describe the differentiation of monkey embryonic stem (ES) cells into insulin-responsive adipocytes. Treatment of embryoid body (EB) outgrowth with adipogenic hormones induced the expression of adipocyte-specific genes, such as PPARgamma, C/EBPalpha, aP2, insulin receptor, and GLUT4. Expression of adipocytokines, leptin and adiponectin, was also detected. Furthermore, translocation of GLUT4 was observed by insulin stimulation in differentiated adipocytes. These results suggested that monkey ES cells can be a useful tool for studying adipogenesis in primate.

Molecular cloning of BNP from heart and its immunohistochemical localization in the hypothalamus of monkey.

Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus.

Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intra-cardially administered microglia in normal mouse brain.

Although the therapeutic use of microglia has received some attention for the treatment of brain diseases, few non-invasive techniques exist for monitoring the cells after administration. Here, we present a technique using magnetic resonance imaging (MRI) to track microglia injected intra-cardially. We labeled microglia expressing enhanced green fluorescent protein with superparamagnetic iron oxide (Resovist) using the hemagglutinating virus of Japan-envelope vector. We injected labeled microglia into the left ventricle of the heart of mice. After monitoring exogenously administered microglia in the mouse brain in vivo using T(2)*-weighted MRI at a magnetic field of 7T, we compared the MR images with histochemical localization of exogenous microglia in vitro. MRI revealed clear signal changes attributable to Resovist-containing microglia in the mouse brain. Histochemistry demonstrated the presence of exogenous microglia in the brain at the same locations shown by MRI. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for brain disease.