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The abnormal activity of PP2A, a dominant member of type 2A serine/threonine protein phosphatase, has been implicated in the development of Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). PP2A is a holoenzyme, and protein methylation of the catalytic subunit, PP2Ac, alters the complex composition. A decrease in PP2Ac methylation levels has been reported in AD and DLB. Aging is the most common risk factor for AD and DLB, but the relationship between aging and PP2A has not been studied in detail. Cynomolgus monkey show increased phosphorylation levels of tau and α-synuclein with aging. In this study, we investigated the alterations in the PP2A activity regulation with aging in monkey brains from 2 to 43 years of age using fractionated proteins. We found that type 2A protein phosphatase activity decreased with aging in cytoplasmic and nuclear-soluble fractions. PP2Ac methylation level was decreased in cytoplasmic and sarkosyl-insoluble fractions. A principal component analysis using PP2Ac, demethylated PP2Ac and PP2A methylesterase PME-1 levels in cytoplasmic and nuclear-soluble fractions as attributes showed that aged monkeys were in the same cluster. Our results show that brain aging in cynomolgus monkeys is closely related to changes in PP2A methylation.

Intrauterine sperm injection for artificial insemination is difficult in cynomolgus macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) due to the complex structure of the cervical canal, which differs from that of humans. Despite the availability of several artificial insemination methods for macaques, pregnancy rates are inconsistent, and details regarding ovulation are unclear, thus warranting more effective methods. Therefore, we developed an effective, ultrasound-guided, transabdominal intrauterine artificial insemination method for cynomolgus macaques that involves timing sperm injection to coincide with the periovulation phase estimated according to rapid hormone measurement. We performed our intrauterine artificial insemination on 6 female macaques; 4 of the 5 animals that were predicted to have ovulated soon after insemination became pregnant, whereas the 1 macaque that was predicted not to have ovulated did not. Furthermore, we saw no evidence of injury, such as a conspicuous needle hole or bleeding on the surface of or inside the uterus, nor did our method result in any abnormalities in the mothers or their offspring. Thus, our ultrasound-guided, transabdominal, intrauterine artificial insemination method is rapid, safe, and effective in cynomolgus macaques.

This study aimed to develop a more suitable ovarian stimulation procedure for cynomolgus macaques (Macaca fascicularis). Macaques were divided into 4 groups, 7AG, 8AG, 7AN, and 8AN, according to the ovarian stimulation procedure administered (i.e., administration of either a gonadotropin-releasing hormone agonist [GnRH-a] or GnRH antagonist [GnRH-ant]) and the number of menstruations (≤ 7 times or ≥ 8 times) in the previous year. In both procedures, oocyte growth and maturation were induced by administration of human follicle-stimulating hormone and human chorionic gonadotropin. The mean numbers of metaphase II mature and metaphase I premature oocytes collected from the 7AG, 8AG, 7AN, and 8AN groups were 12.1 and 10.4, 12.0 and 13.8, 9.1 and 8.3, and 15.5 and 8.8, respectively (P0.05). The fertilization rates of the 7AN and 8AN groups (85.3% and 74.7%) tended to be higher compared with those in the 7AG and 8AG groups (59.1% and 47.3%; P0.05). The 8AN group yielded 19.9 zygotes, which was the largest number per macaque, compared with the other three groups. Furthermore, regarding the decreases in body weight between the start of the procedures and the time of oocyte collection, those of the 7AN and 8AN groups were significantly smaller than those of the 7AG and 8AG groups (P0.05), suggesting that the procedure involving GnRH-ant reduced the burden on the macaques. Thus, controlled ovarian stimulation using a GnRH-ant has some advantages for cynomolgus macaques compared with that using a GnRH-a.

Intrauterine sperm injection for artificial insemination is difficult in cynomolgus macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) due to the complex structure of the cervical canal, which differs from that of humans. Despite the availability of several artificial insemination methods for macaques, pregnancy rates are inconsistent, and details regarding ovulation are unclear, thus warranting more effective methods. Therefore, we developed an effective, ultrasound-guided, transabdominal intrauterine artificial insemination method for cynomolgus macaques that involves timing sperm injection to coincide with the periovulation phase estimated according to rapid hormone measurement. We performed our intrauterine artificial insemination on 6 female macaques; 4 of the 5 animals that were predicted to have ovulated soon after insemination became pregnant, whereas the 1 macaque that was predicted not to have ovulated did not. Furthermore, we saw no evidence of injury, such as a conspicuous needle hole or bleeding on the surface of or inside the uterus, nor did our method result in any abnormalities in the mothers or their offspring. Thus, our ultrasound-guided, transabdominal, intrauterine artificial insemination method is rapid, safe, and effective in cynomolgus macaques.

Aβ metabolism in the brain is mediated by endocytosis, one part of the intracellular membrane trafficking system. We previously showed that aging attenuates the interaction of dynein with dynactin, which disrupts the endosomal/lysosomal trafficking pathway involved in Aβ metabolism, resulting in intracellular accumulation of Aβ. Several studies have shown that in Alzheimer's disease (AD), intraneuronal accumulation of Aβ precedes extracellular Aβ depositions. However, it is unclear what accounts for this transition from intracellular to extracellular depositions. Accumulating evidence suggest that autophagy has an important role in AD pathology, and we observed that autophagy-related protein levels began to decrease before amyloid plaque formation in cynomolgus monkey brains. Surprisingly, experimental induction of autophagosome formation in Neuro2a cells significantly increased intracellular Aβ and decreased extracellular release of Aβ, accompanied by the prominent reduction of extracellular vesicle (EV) secretion. RNAi study confirmed that EV secretion affected intracellular and extracellular Aβ levels, and siRNA-induced downregulation of autophagosome formation enhanced EV secretion to ameliorate intracellular Aβ accumulation induced by dynein knockdown. In aged cynomolgus monkeys, Aβ levels in EV/intraluminal membrane vesicle (ILV)-rich fractions isolated from temporal lobe parenchyma were drastically increased. Moreover, EV/ILV marker proteins overlapped spatially with amyloid plaques. These findings suggest that EV would be an important carrier of Aβ in brain and abnormal accumulation of Aβ in EVs/ILVs may be involved in the transition of age-dependent Aβ pathology.

The endocytic membrane trafficking system is altered in the brains of early-stage Alzheimer disease (AD) patients, and endocytic disturbance affects the metabolism of β-amyloid (Aβ) protein, a key molecule in AD pathogenesis. It is widely accepted that type 2 diabetes mellitus (T2DM) is one of the strongest risk factors for development of AD. Supporting this link, experimentally induced T2DM enhances AD pathology in various animal models. Spontaneous T2DM also enhances Aβ pathology with severe endocytic pathology, even in nonhuman primate brains. However, it remains unclear how T2DM accelerates Aβ pathology. Herein, we demonstrate that cholesterol metabolism–related protein levels are increased and that membrane cholesterol level is elevated in spontaneous T2DM-affected cynomolgus monkey brains. Moreover, in vitro studies that manipulate cellular cholesterol reveal that elevated membrane cholesterol disrupts lysosomal degradation and enhances chemical-induced endocytic disturbance, resulting in great accumulation of Aβ in Neuro2a cells. These findings suggest that an alteration of cerebral cholesterol metabolism may be responsible for augmentation of Aβ pathology in T2DM-affected brains, which, in turn, may increase the risk for developing AD.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide for which 15 disease-associated loci had been discovered. Among them, only 5 loci have been associated with POAG in Asians. We carried out a genome-wide association study and a replication study that included a total of 7378 POAG cases and 36 385 controls from a Japanese population. After combining the genome-wide association study and the two replication sets, we identified 11 POAG-associated loci, including 4 known (CDKN2B-AS1, ABCA1, SIX6 and AFAP1) and 7 novel loci (FNDC3B, ANKRD55-MAP3K1, LMX1B, LHPP, HMGA2, MEIS2 and LOXL1) at a genome-wide significance level (P

It is widely accepted that β-amyloid (Aβ) protein plays a pivotal role in Alzheimer disease pathogenesis, and accumulating evidence suggests that endocytic dysfunction is involved in Aβ pathology. Retromer, a conserved multisubunit complex, mediates the retrograde transport of numerous kinds of cargo from endosomes to the trans-Golgi network. Several studies have found that retromer deficiency enhances Aβ pathology both in vitro and in vivo . Cytoplasmic dynein, a microtubule-based motor protein, mediates minus-end-directed vesicle transport via interactions with dynactin, another microtubule-associated protein that also interacts with retromer. Aging attenuates the dynein-dynactin interaction, and dynein dysfunction reproduces age-dependent endocytic disturbance, resulting in the intracellular accumulation of beta-amyloid precursor protein (APP) and its β-cleavage products, including Aβ. Here, we report that aging itself affects retromer trafficking in cynomolgus monkey brains. In addition, dynein dysfunction reproduces this type of age-dependent retromer deficiency (ie, the endosomal accumulation of retromer-related proteins and APP. Moreover, we found that knockdown of Rab7, Rab9, or Rab11 did not alter endogenous APP metabolism, such as that observed in aged monkey brains and in dynein-depleted cells. These findings suggest that dynein dysfunction can cause retromer deficiency and that concomitant disruption of retrograde trafficking may be the key factor underlying age-dependent Aβ pathology.

The endocytic membrane trafficking system is altered in the brains of early-stage Alzheimer disease (AD) patients, and endocytic disturbance affects the metabolism of β-amyloid (Aβ) protein, a key molecule in AD pathogenesis. It is widely accepted that type 2 diabetes mellitus (T2DM) is one of the strongest risk factors for development of AD. Supporting this link, experimentally induced T2DM enhances AD pathology in various animal models. Spontaneous T2DM also enhances Aβ pathology with severe endocytic pathology, even in nonhuman primate brains. However, it remains unclear how T2DM accelerates Aβ pathology. Herein, we demonstrate that cholesterol metabolism-related protein levels are increased and that membrane cholesterol level is elevated in spontaneous T2DM-affected cynomolgus monkey brains. Moreover, in vitro studies that manipulate cellular cholesterol reveal that elevated membrane cholesterol disrupts lysosomal degradation and enhances chemical-induced endocytic disturbance, resulting in great accumulation of Aβ in Neuro2a cells. These findings suggest that an alteration of cerebral cholesterol metabolism may be responsible for augmentation of Aβ pathology in T2DM-affected brains, which, in turn, may increase the risk for developing AD.

Age-related macular degeneration (AMD) is a leading cause of blindness among elderly people. The appearance of drusen is a clinical manifestation and a harbinger of both exudative and atrophic AMD. Recently, antibody-based medicines have been used to treat the exudative type. However, they do not restore good vision in patients. Moreover, no effective treatment is available for atrophic AMD. We have created small chemicals (Kyoto University Substances; KUSs) that act as ATP regulators inside cells. In the present study, we examined the in vivo efficacy of KUS121 in C-C chemokine receptor type 2-deficient mice, a mouse model of AMD. Systemic administration of KUS121 prevented or reduced drusen-like lesions and endoplasmic reticulum stress, and then substantially mitigated chorioretinal pathologies with significant preservation of visual function. Additionally, we confirmed that long-term oral administration of KUS121 caused no systemic complications in drusen-affected monkeys. ATP regulation by KUSs may represent a novel strategy in the treatment of drusen and prevention of disease progression in AMD.

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.

Concomitant deposition of amyloid -beta protein (Aβ) and neuronal tau as neurofibrillary tangles in the human brain is a hallmark of Alzheimer disease (AD). Because these deposits increase during normal aging, it has been proposed that aging brains may also undergo AD-like changes. To investigate the neuropathological changes that occur in the aging primate brain, we examined 21 brains of cynomolgus monkeys (7–36 years old) for Aβ- and tau-positive lesions. We found, 1) extensive deposition of Aβ in brains of cynomolgus monkeys over 25 years of age, 2) selective deposition of 4-repeat tau as pretangles in neurons, and as coiled body-like structures in oligodendroglia-like cells and astrocytes, 3) preferential distribution of tau in the basal ganglia and neocortex rather than the hippocampus, and 4) age-associated increases in 30–34 kDa AT8- and RD4-positive tau fragments in sarkosyl-insoluble fractions. We further labeled tau-positive structures using diaminobezidine enhanced with nickel, and visualized nickel-labeled structures by energy-dispersive X-ray (EDX) analysis of ultrathin sections. This allowed us to distinguish between nickel-labeled tau and background electron-dense structures, and we found that tau localized to 20–25 nm straight filaments in oligodendroglia-like cells and neurons. Our results indicate that the cytopathology and distribution of tau deposits in aged cynomolgus brains resemble those of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) rather than AD. Thus, even in the presence of Aβ, age-associated deposition of tau in non-human primates likely does not occur through AD-associated mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0385-5) contains supplementary material, which is available to authorized users.

Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; PCD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.

In mice, basic reproductive technologies, such as oocyte/sperm collection, embryo production, micromanipulation, and embryo transfer, have been established. With these technologies, production of transgenic mice has become routine. The cynomolgus monkey, which is one of the laboratory animals closest to human beings, has been used to obtain vaccine approval and for medical research. Therefore, production of transgenic animals in the monkey is a very significant subject. We describe herein the current state of related studies in addition to current findings regarding reproductive technologies.

Although embryonic stem (ES) cell lines derived from mice and primates are used extensively, the development of such lines from other mammals is extremely difficult because of their rapid decline in proliferation potential and pluripotency after several passages. This study describes the establishment of rabbit ES cell lines with indefinite proliferation potential. It was found that the feeder cell density determines the fate of rabbit ES cells, and that maximum proliferation potential was obtained when they were cultured on a feeder cell density of one-sixth of the density at confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Under optimized conditions, rabbit ES cells were passaged 50 times, after which they still possessed high telomerase activity. This culture system enabled efficient gene transduction and clonal expansion from single cells. During culture, rabbit ES cells exhibited flattened monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo respectively. These ES cell lines can be safely cryopreserved for later use. Thus, rabbit ES cells can be added to the list of stable mammalian ES cells, enabling the rabbit to be used as a small animal model for the study of human cell transplantation therapy.

Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (

Aim: To clarify the location of primordial germ cells (PGC) in an embryo of target-age and to examine the culture environment of the PCG. Methods: The days of ovulation and fertilization were estimated by measuring the serum concentration of estrogen. Pregnancy was confirmed by measurement of the serum concentration of the beta subunit of macaque chorionic gonadotropin and by ultrasonography. We also examined the location of PGC in the embryo at the time of retrieval. Results: Results showed that PGC in an embryo were in the hindguts at day 30 postfertilization, arrived at the genital ridges via mesenteries at approximately day 33 postfertilization, and colonized the gonads by day 36 postfertilization. Conclusions: In conclusion, embryos collected on day 33 postfertilization are more suitable for obtaining PGC from cynomolgus monkeys. The PGC collected from cynomolgus monkey fetuses were cultured under conditions for the derivation and culture of human embryonic germ cells; enzymatically dispersed single cells were cultured on a SIM thioguanine-resistant ouabain-resistant cells (STO) feeder layer with recombinant human leukemia inhibitory factor, recombinant human basic fibroblast growth factor and forskolin. The cells from genital ridges and mesenteries at day 33 postfertilization had alkaline phosphatase (ALP) activity in vitro for a maximum of 13 days. In contrast, ALP activity had been held for 2 months under the same culture condition when the cells were derived from the gonads at day 66 postfertilization. Derivation of an embryonic germ cell from a cynomolgus monkey was not achieved from these cultures. (Reprod Med Biol 2007; 6: 203-210).

An embryonic stem (ES) cell differentiation model would facilitate analysis of developmental processes at the cellular level and the effects of embryotoxic and teratogenic factors in vitro. We explored the use of differentiation of embryoid bodies (EBs) from cynomolgus monkey ES cells for embryotoxicity testing. We determined the mRNA expression of various genes using real-time RT-PCR. Oct-3/4 expression was almost completely suppressed on day 14, suggesting that ES cells reached differentiated status in around 14 days. mRNA expression of E-cadherin, connexin 43, caveolin-1, and argininosuccinate synthetase was reproducibly suppressed during EB differentiation in 7-32% of ES cells in three separate experiments. Although these may not be "general stemness marker genes" such as Oct-3/4, they could play a role in readying stem cells for differentiation in response to deletion of signals from feeder cells. Next, we examined the effects of bisphenol A (BPA) on the mRNA expression of several differentiation marker genes for ES cells. That of PAX-6, an ectoderm marker, with 0, 0.1, and 10 microM BPA in 21-day EBs was 3,500%, 6,668%, and 8,394%, respectively, compared with ES cells. The difference between doses of 0 and 10 microM BPA in 21-day EBs was statistically significant (p=0.049). Pax-6 activation in the presence of BPA may interfere with the development of eyes, sensory organs, and certain neural and epidermal tissues usually derived from ectodermal tissues. Differentiation of EBs from cynomolgus monkey ES cells could be a useful model for detecting gene expression changes in response to chemical exposure.

It is widely accepted that β-amyloid (Aβ) protein plays a pivotal role in Alzheimer disease pathogenesis, and accumulating evidence suggests that endocytic dysfunction is involved in Aβ pathology. Retromer, a conserved multisubunit complex, mediates the retrograde transport of numerous kinds of cargo from endosomes to the trans-Golgi network. Several studies have found that retromer deficiency enhances Aβ pathology both in vitro and in vivo. Cytoplasmic dynein, a microtubule-based motor protein, mediates minus-end-directed vesicle transport via interactions with dynactin, another microtubule-associated protein that also interacts with retromer. Aging attenuates the dynein-dynactin interaction, and dynein dysfunction reproduces age-dependent endocytic disturbance, resulting in the intracellular accumulation of beta-amyloid precursor protein (APP) and its β-cleavage products, including Aβ. Here, we report that aging itself affects retromer trafficking in cynomolgus monkey brains. In addition, dynein dysfunction reproduces this type of age-dependent retromer deficiency (ie, the endosomal accumulation of retromer-related proteins and APP. Moreover, we found that knockdown of Rab7, Rab9, or Rab11 did not alter endogenous APP metabolism, such as that observed in aged monkey brains and in dynein-depleted cells. These findings suggest that dynein dysfunction can cause retromer deficiency and that concomitant disruption of retrograde trafficking may be the key factor underlying age-dependent Aβ pathology.

When using animals in experiments, it is important to select the animal that best suits the experiment's purpose. Because non-human primates are closely related to humans, they are invaluable for use in clinical trials. Researchers using non-human primates should understand their characteristics before the start of the study and must execute the experiments efficiently. This article summarizes reproductive manipulation techniques in the study of non-human primates and the current status of this research.

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

Purpose To accelerate the development of new therapies, an inherited retinal degeneration model in a nonhuman primate would be useful to confirm the efficacy in preclinical studies. In this study, we describe the discovery of retinitis pigmentosa in a cynomolgus monkey (Macaca fascicularis) pedigree. Methods First, screening with fundus photography was performed on 1443 monkeys at the Tsukuba Primate Research Center. Ophthalmic examinations, such as indirect ophthalmoscopy, ERGs using RETeval, and optic coherent tomography (OCT) measurement, were then performed to confirm diagnosis. Results Retinal degeneration with cystoid macular edema was observed in both eyes of one 14-year-old female monkey. In her examinations, the full-field ERGs were nonrecordable and the outer layer of the retina in the parafoveal area was not visible on OCT imaging. Moreover, less frequent pigmentary retinal anomalies also were observed in her 3-year-old nephew. His full-field ERGs were almost nonrecordable and the outer layer was not visible in the peripheral retina. His father was her cousin (the son of her mother's older brother) and his mother was her younger half-sibling sister with a different father. Conclusions The hereditary nature is highly probable (autosomal recessive inheritance suspected). However, whole-exome analysis performed identified no pathogenic mutations in these monkeys.

Cloned animals suffer a wide range of severe fetal and placental malformations. Whether these malformations arise from insufficient epigenetic modifications or mutations has not yet been determined. To address this question, we examined siblings from both cloned XO and XY parents. These parents, which exhibited hypertrophic placentas, increased body weights, and open eyelids at birth, were created from the same ES cell sublines. The siblings from all three cloned pairs showed normal body and placenta weights and no open eyelids at birth. The results clearly showed that the phenotypic abnormalities seen in cloned mice were not transmitted to the progeny, a finding that suggests that abnormalities in cloned mice are responsible for insufficient epigenetic modifications/reprogramming. genesis 2:203–207, 2002. © 2002 Wiley-Liss, Inc.

In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.

Induced pluripotent stem (iPS) cells that are potentially similar to embryonic stem (ES) cells can be artificially established by introduction into somatic cells of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4, and c-MYC. In cynomolgus monkeys (Macaca fascicularis), iPS cells generated by using these four allogeneic transgenes should be an important resource for various types of biomedical research because the use of xenogeneic transgenes may cause complications. To establish such iPS cells, cynomolgus monkey somatic cells were infected with amphotropic retroviral vectors, which were derived from Plat-A cells, containing cDNA for the cynomolgus monkey genes POU5F1, SOX2, KLF4, and c-MYC. As a result, iPS cells could be established from somatic cells from fetal liver and newborn skin of cynomolgus monkeys, similarly to the case for mouse and human somatic cells.

Growing evidence suggests that diabetes mellitus (DM) is one of the strongest risk factors for developing Alzheimer's disease (AD). However, it remains unclear why DM accelerates AD pathology. In cynomolgus monkeys older than 25 years, senile plaques (SPs) are spontaneously and consistently observed in their brains, and neurofibrillary tangles are present at 32 years of age and older. In laboratory-housed monkeys, obesity is occasionally observed and frequently leads to development of type 2 DM. In the present study, we performed histopathological and biochemical analyses of brain tissue in cynomolgus monkeys with type 2 DM to clarify the relationship between DM and AD pathology. Here, we provide the evidence that DM accelerates Aβ pathology in vivo in nonhuman primates who had not undergone any genetic manipulation. In DM-affected monkey brains, SPs were observed in frontal and temporal lobe cortices, even in monkeys younger than 20 years. Biochemical analyses of brain revealed that the amount of GM1-ganglioside-bound Aβ (GAβ)--the endogenous seed for Aβ fibril formation in the brain--was clearly elevated in DM-affected monkeys. Furthermore, the level of Rab GTPases was also significantly increased in the brains of adult monkeys with DM, almost to the same levels as in aged monkeys. Intraneuronal accumulation of enlarged endosomes was also observed in DM-affected monkeys, suggesting that exacerbated endocytic disturbance may underlie the acceleration of Aβ pathology due to DM.

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.

Cloning using G(0)-arrested somatic cells has led to the suggestion that this stage of the cell cycle is necessary for the success of cloning. In this study we report that cloned mice can be generated from fetal fibroblasts arrested at metaphase of the cell cycle. The procedure involves fusing a metaphase-arrested fetal fibroblast to an enucleated oocyte. After parthenogenetic activation a polar body and single diploid pronucleus were formed. Some of these were allowed to develop to the blastocyst stage, while others were enucleated and the nucleus was transferred to an enucleated fertilized 1-cell embryo. After the single transfer technique, 2 out of 164 developed to late stages of gestation were dead with gross abnormalities. However, after the serial nuclear transfer, 5 out of 272 embryos were recovered live at Day 19.5, and 2 of these went on to develop into apparently normal adults. All of the cloned embryos showed severe placental hypertrophy and defective differentiation of placental tissues. This study illustrates that reprogramming can occur after nuclear transfer at metaphase of the cell cycle.

Induced pluripotent stem (iPS) cells that are potentially similar to embryonic stem (ES) cells can be artificially established by introduction into somatic cells of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4, and c-MYC. In cynomolgus monkeys (Macaca fascicularis), iPS cells generated by using these four allogeneic transgenes should be an important resource for various types of biomedical research because the use of xenogeneic transgenes may cause complications. To establish such iPS cells, cynomolgus monkey somatic cells were infected with amphotropic retroviral vectors, which were derived from Plat-A cells, containing cDNA for the cynomolgus monkey genes POU5F1, SOX2, KLF4, and c-MYC. As a result, iPS cells could be established from somatic cells from fetal liver and newborn skin of cynomolgus monkeys, similarly to the case for mouse and human somatic cells.

The present study was conducted to clarify changes of intermediate filaments, cytokeratin, vimentin and desmin, in primary, subcultured and cryopreserved bovine oviduct epithelial cells by subculture or cryopreservation and to assess their sustaining ability for the embryo development. Any morphological change of cells isolated from bovine oviducts and cultured in vitro was not observed up to the fifth generation, including cryopreserved cells. Result of indirect immunofluorescent staining study strongly suggested that the primary cells were epithelial cells because immunofluorescence to anti-cytokeratin antibody was detected but not observed anti-vimentin and anti-desmin antibody. From second generation, however, the reaction of vimentin was become to be detected, suggesting that the nature of the epithelial cells changed. Sustaining ability of epithelial cells was assessed by co-culture of bovine parthenogenetic embryos with the oviduct epithelial cells. The results showed that sustaining ability of primary and subcultured-cryopreserved bovine oviductal cells was equivalent in which 29.8% and 28.3% of the parthenogenones in each cases developed to blastocysts. These results suggested that the expression of vimentin in culturing bovine oviduct epithelial cells and their sustaining ability for bovine parthenogenetic embryos had no relation.

We developed a compact culture device that maintains developing embryos in vitro under constant temperature and CO(2) concentration. Using this device, we cultured rabbit embryos from the pronuclear stage to the hatched blastocyst stage and recorded their development digitally for 7 d. Recorded images were converted to a movie, and the developmental movement of individual embryos was analyzed. With this culture system, we can observe embryonic development in a suitable environment continuously for several days; similar long-term observation is not possible in the conventional system. The proportion of embryos that developed from the pronuclear stage to the blastocyst stage was the same in the new system (73.1%; 38 of 52) as in the conventional (control) system (77.6%; 38 of 49). Compaction of embryos occurred from the 8-cell to the morula stage at 32.5 +/- 0.71 h after insemination. The time of blastocyst formation (77.2 +/- 3.2 h after insemination) varied somewhat between embryos. Average hatching time was 98.7 +/- 4.4 h after mating. Therefore, the cleavage, blastomere movement, and hatching processes of blastocysts can be followed clearly and recorded by using this new culture system.

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.

Mice cloned from somatic or ES cells showed signs of phenotypically various abnormalities. These abnormalities are now considered to result from aberrant gene expressions by epigenetic reprogramming errors but it is still unclear when these abnormalities occur and what histological changes occur during the gestation period. To address these issues, we histologically examined the hypertrophic placentas and open eyelids at 12.5, 17.5 and 19.5 days of the gestation period in ES-derived cloned mice that we have previously reported. In the placentas, the histology revealed that the hypertrophy had already occurred at 12.5 dpc and that the main change was the proliferation of trophoblast cells in the labyrinth layer. In the fetuses and placentas at 17.5 and 19.5 dpc, extensive proliferation of spongiotrophoblast and glycogen cells in the spongiotrophoblast layer and enlarged trophoblast giant cells were observed. Open eyelids in cloned mice were observed from 17.5 dpc, whereas the eyelids of the control mice had already been closed. The histology showed the malformation of eyelids where the formation of the stratum corneum and stratum granulosum in the epidermis was insufficient. Based on the histology described here, further comparative studies of the gene expression and histology of abnormalities seen in cloned mice and in gene-targeted and spontaneously mutated mice with similar phenotypic abnormalities could help illuminate these abnormalities and could contribute to the development of somatic cloning technology.

Cloned mice typically display abnormal development, such as overgrowth of fetuses and placentae. Quantitative expression analysis of eight imprinted genes (H19, Igf2, Igf2r, Air, Peg1/Mest, Peg3, Nuronatin (Nnat) and Ndn) and an alternate transcript of Igf2 (P0) in embryonic stem cloned fetuses and placentae at days 9.5, 12.5 and 17.5 after mating was carried out by real time PCR to investigate whether epigenetic modification of imprinted genes is responsible for overgrowth of the fetus and placental hypertrophy. In addition, the methylation pattern through the bisulphite sequencing method in differentially methylated regions of H19 and Igf2r was examined in day 9.5 fetuses and placentae. The results showed clearly that the expression of H19 gene decreased in cloned fetuses at days 12.5 and 17.5 after mating and in placentae at day 17.5 after mating, and Igf2 was also repressed in fetuses at days 9.5 and 12.5 after mating and in placentae at day 17.5 after mating. In contrast, the transcription of P0, which is a placental-specific transcript variant of Igf2, increased at more than four times the control in cloned placenta at day 12.5 after mating. Day 9.5 fetuses that have developed normally revealed only hypermethylated alleles in the H19 differently methylated region (DMR), and both hyper- and hypomethylated alleles in the Igf2r DMR2. These results show that inappropriate reprogramming in some imprinted genes affects the development of cloned embryos, and that aberrant P0 Igf2 transcription in particular may cause the overgrowth of cloned fetuses and placentae.

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.

The transgenic mouse rasH2 line, in which the mouse carries the human c-Ha-ras gene under the control of its own enhancer and promoter, has been proposed as one of the alternative short-term models for carcinogenicity testing. To apply this purpose, we have produced a genetically homogeneous population as C57BL/6JJic-TgN(RASH2) (Tg-rasH2) by continuous backcrossing. In this study, we examined the transgene stability between different generations and the detailed transgene architecture of the integrated human c-Ha-ras gene. Fluorescence in situ hybridization analysis showed that the integrated human c-Ha-ras gene was stably located on chromosome 15E3 in Tg-rasH2 mice at generation number (N) 15 and 20. Southern and Northern blot analysis did not show any differences in the hybridized band pattern in each generation. Southern blot analyses showed that the Tg-rasH2 mouse contained three copies of the human c-Ha-ras gene arrayed in a head-to-tail configuration. We also determined the nucleotide sequence of the transgene in the Tg-rasH2 mouse at N20 and confirmed that the sequence of the coding region was perfectly matched with human c-Ha-ras cDNA. Cloning and sequencing of genome/transgene junctions revealed that integration of the microinjected human c-Ha-ras gene into mouse host genome resulted in a 1820-bp deletion in the rasH2 line. The deleted sequence did not have any sequence homologies with known functional genes. We assumed that either the deletion or the transgene insertion, or both, would not cause insertional mutation. In short-term carcinogenicity testing with a genetically engineered mouse model, confirmation of the transgene or modified gene stability at each generation is one of the important factors that affect the sensitivity to carcinogenic compounds in the same way as the genetic background, age and route of administration.