Sequence diversity of the MHC Ⅱ DRB gene in Chinese tree shrews (Tupaia belangeri chinensis)

The major histocompatibility complex (MHC) is a cluster of genes involved in vertebrate immune response regulation. MHC class I and II cell surface proteins are crucial for discrimination of self versus non-self by the adaptive immune system. Due to their special phylogenetic position within the Euarchontoglires and as a relative of primates, tree shrews have been proposed as an alternative experimental animal model for biomedical studies. However, information about the genetic structure of the tree shrew populations is largely unknown. In this study, we characterized diversity in exon 2 of the MHC II DRB gene isolated from Chinese tree shrews (Tupaia belangeri chinensis). We identified 12 different DRB exon 2 alleles from 15 Chinese tree shrews, 1 to 4 alleles were observed per individual with high levels of sequence divergence between alleles. There were more non-synonymous than synonymous substitutions in the functionally important antigen-binding site (dN/dS = 2.7952, P < 0.01), indicating that the DRB exon 2 in Chinese tree shrews has been influenced by positive selection.     

Positively selected genes of the Chinese tree shrew (Tupaia belangeri chinensis) locomotion system

While the recent release of the Chinese tree shrew(Tupaia belangeri chinensis) genome has made the tree shrew an increasingly viable experimental animal model for biomedical research, further study of the genome may facilitate new insights into the applicability of this model. For example, though the tree shrew has a rapid rate of speed and strong jumping ability, there are limited studies on its locomotion ability. In this study we used the available Chinese tree shrew genome information and compared the evolutionary pattern of 407 locomotion system related orthologs among five mammals(human, rhesus monkey, mouse, rat and dog) and the Chinese tree shrew. Our analyses identified 29 genes with significantly high ω(Ka/Ks ratio) values and 48 amino acid sites in 14 genes showed significant evidence of positive selection in the Chinese tree shrew. Some of these positively selected genes, e.g. HOXA6(homeobox A6) and AVP(arginine vasopressin), play important roles in muscle contraction or skeletal morphogenesis. These results provide important clues in understanding the genetic bases of locomotor adaptation in the Chinese tree shrew.     

Rhombomere-specific patterns of apoptosis in the tree shrew Tupaia belangeri

Whether rhombomere-specific patterns of apoptosis exist in the developing hindbrain of vertebrates is under debate. We have investigated the sequence of apoptotic events in three-dimensionally reconstructed hindbrains of Tupaia belangeri (8- to 19-somite embryos). Apoptotic cells were identified by structural criteria and by applying an in situ tailing technique to visualize DNA fragmentation. Seven rhombomeres originated from three pro-rhombomeres. Among pre-migratory neural crest cells in the dorsal thirds of the neural folds, the earliest apoptotic concentrations appeared in the developing third rhombomere (r3). Dorsal apoptotic maxima then persisted in r3, extended from r3 to r2, and also arose in r5. Transverse apoptotic bands increased the total amount of apoptotic cells in odd-numbered rhombomeres first in r3 and, with a delay, also in r5. This sequence of apoptotic events was paralleled by an approximate rostrocaudal sequence of neural crest cell delamination from the even-numbered rhombomeres. Thus, large-scale apoptosis in r3 and r5 helped to establish crest-free zones that segregated streams of migrating neural crest cells adjacent to r2, r4, and r6. The sequence of apoptotic events observed in the dorsal thirds of rhombomeres matches that reported for the chick embryo. Other shared features are apoptotic peaks in the position of a circumscribed ventricular protrusion of fusing parts of the neural folds in r1 and r2, and Y-shaped apoptotic patterns composed of apoptotic maxima in the dorsal and lateral thirds of r1, r2, and r3. These rhombomere-specific patterns of apoptosis may therefore represent a conserved character, at least in amniotes.This work was supported by the Deutsche Forschungsgemeinschaft (KN 525/1–1, KN 525/1–2, BR 1185/4–1, and former Sonderforschungsbereich 89: Cardiology)     

Recognition memory in tree shrew (Tupaia belangeri) after repeated familiarization sessions

Recognition memories are formed during perceptual experience and allow subsequent recognition of previously encountered objects as well as their distinction from novel objects. As a consequence, novel objects are generally explored longer than familiar objects by many species. This novelty preference has been documented in rodents using the novel object recognition (NOR) test, as well is in primates including humans using preferential looking time paradigms. Here, we examine novelty preference using the NOR task in tree shrew, a small animal species that is considered to be an intermediary between rodents and primates. Our paradigm consisted of three phases: arena familiarization, object familiarization sessions with two identical objects in the arena and finally a test session following a 24-h retention period with a familiar and a novel object in the arena. We employed two different object familiarization durations: one and three sessions on consecutive days. After three object familiarization sessions, tree shrews exhibited robust preference for novel objects on the test day. This was accompanied by significant reduction in familiar object exploration time, occurring largely between the first and second day of object familiarization. By contrast, tree shrews did not show a significant preference for the novel object after a one-session object familiarization. Nonetheless, they spent significantly less time exploring the familiar object on the test day compared to the object familiarization day, indicating that they did maintain a memory trace for the familiar object. Our study revealed different time courses for familiar object habituation and emergence of novelty preference, suggesting that novelty preference is dependent on well-consolidated memory of the competing familiar object. Taken together, our results demonstrate robust novelty preference of tree shrews, in general similarity to previous findings in rodents and primates.     

冷暴露对中缅树鼩适应性产热特征的影响

在冷暴露(5 ± 1℃)1 d,7 d,14 d 和21d 对中缅树鼩的肝脏、心脏、膈肌、腓肠肌和褐色脂肪组织(BAT)的总蛋白和线粒体蛋白含量、线粒体的状态Ⅳ呼吸能力、细胞色素C 氧化酶活力及血清中甲状腺激素的水平等指标进行了测定。结果表明:冷暴露过程中肝脏线粒体蛋白含量对低温的反应比总蛋白的反应剧烈,心脏和BAT 组织的线粒体蛋白含量也随着冷驯化时间的延长而显著增加,但腓肠肌的反应较为温和;各组织线粒体的状态Ⅳ呼吸能力均显著增强,除腓肠肌外其它各组织细胞色素C 氧化酶活性也随冷暴露时间的延长而急剧增加。说明在低温条件下,肝脏、腓肠肌、心脏和BAT 等组织都参与了体温调节的过程,在自然生境中,低温是重要的刺激产热的调节因子。     

Horizontal cells invest retinal capillaries in the tree shrew Tupaia belangeri

Fibroblast growth factor (FGF) receptors constitute a family of four membrane-spanning tyrosine kinases (FGFR1-4) which serve as high-affinity receptors for 17 growth factors (FGF1-17). To study functions of FGF/ FGFR signals in development, mice that carry mutations in each receptor have been created by gene targeting. Analysis of these mutant mice revealed essential functions of FGF receptors in multiple biological processes, including mesoderm induction and patterning, cell growth and migration, organ formation and bone growth. In this review we discuss recent work with FGF receptors to illustrate mechanisms, through which the FGF/FGFR signals specify vertebrate limb initiation, outgrowth and patterning.     

Evaluation of hypothalamo-pituitary-adrenal activity in the tree shrew (Tupaia belangeri) via salivary cortisol measurement

Saliva sampling is frequently used in humans for adrenal glucocorticoid hormone analysis because of advantages such as non-invasiveness, the ease of collection, and storing of the samples. To transfer this advantageous method to laboratory mammals, potentially confounding factors such as stressful handling procedures have to be excluded. In the present study we established a method for collecting saliva for cortisol measurement in freely moving adult male tree shrews (Tupaia belangeri). The practicability of the procedure was demonstrated (i) by stress-induced changes in cortisol levels revealing a significant increase during the stress phase (control = 0.91 nmol/l vs stress = 1.71 nmol/l), and (ii) by reporting no significant differences in salivary cortisol levels before and after performance of a learning task. The present study emphasizes the use of salivary cortisol analysis especially for monitoring acute changes in the hypothalamo-pituitary-adrenal axis activity in male tree shrews.     

中缅树鼩精子形态特征及冷冻损伤

树鼩作为一种新型的、接近灵长类的实验动物,在医学生物学上的应用受到越来越多的重视。精子的结构特性研究及冷冻后结构的完整性分析是精子生物学的主要内容,也有助于树鼩的实验室快速繁殖。该研究采用人工饲养的中缅树鼩(Tupaia belangeri chinensis),结果显示其睾丸占总体重的(1.05±0.07)%,总体积为(1.12±0.10)mL。附睾尾及输精管精子总量估计在2.2×107～8.8×107,其运动度和顶体完整率分别为(68.8±3.9)%和(90.0±2.1)%。利用扫描电子显微镜和透射电子显微镜对树鼩附睾精子的超微结构进行的观察和分析显示精子头部呈圆形或卵圆形;头部长度、宽度平均分别为6.65和5.82μm;精子尾部中段、主段、尾段和精子总长度平均分别为13.39、52.35、65.74和73.05μm;尾部中段的线粒体螺旋数量为48个,其轴丝结构为典型的"9+9+2"结构。冷冻解冻后的精子主要表现在顶体与质膜不完整、精子断裂、尾部扭曲和膨大。上述结果提示树鼩精子与其他哺乳动物精子的结构特征相似,但是精子大小和线粒体螺旋数目有明显的差别,且超微结构改变仍是冷冻精子运动和受精能力下降的主要原因。     

Body temperature rhythm of the tree shrew, Tupaia belangeri.

The circadian rhythm of body temperature of the tree shrew Tupaia belangeri was studied by telemetry. The amplitude of the daily (or circadian) variation was found to be much larger than that of most endotherms (amplitude approximately 5 degrees C) and the bimodal shape of the rhythm differed from the cosine waveform that characterizes the temperature rhythms of most other species. In free-running conditions, as well as in the entrained state, the temperature rhythm remained synchronized to the rhythm of locomotor activity.     

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew (Tupaia belangeri chinensis)

The Chinese tree shrew (Tupaia belangeri chinensis) is a small experimental animal with a close affinity to primates. This species has long been proposed to be an alternative experimental animal to primates in biomedical research. Despite decades of study, there is no pure breed for this animal, and the overall genetic diversity of wild tree shrews remains largely unknown. In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations, we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew. An analysis of a wild population of 117 individuals collected from the suburb of Kunming, China, showed that these loci exhibited a highly expected heterozygosity (0.616). These 12 microsatellites were sufficient for individual identification and parentage analysis. The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.     

Establishment of basal cell carcinoma animal model in Chinese tree shrew (Tupaia belangeri chinensis)

Basal cell carcinoma (BCC) is the most common skin cancer worldwide,with incidence rates continuing to increase.Ultraviolet radiation is the major environmental risk factor and dysregulation of the Hedgehog (Hh) signaling pathway has been identified in most BCCs.The treatment of locally advancedand metastatic BBCs is still a challenge and requires a better animal model than the widely used rodents for drug development and testing.Chinese tree shrews (Tupaia belangeri chinensis) are closely related to primates,bearing many physiological and biochemical advantages over rodents for characterizing human diseases.Here,we successfully established a Chinese tree shrew BCC model by infecting tail skins with lentiviral SmoA1,an active form of Smoothened (Smo) used to constitutively activate the Hh signaling pathway.The pathological characteristics were verified by immunohistochemical analysis.Interestingly,BCC progress was greatly enhanced by the combined usage of lentiviral SmoA1 and shRNA targeting Chinese tree shrew p53.This work provides a useful animal model for further BCC studies and future drug discoveries.     

Spontaneous malignomas in Tupaia (tree shrew)

Nine spontaneous malignomas of the tree shrew were detected and analysed during an observation period of nine years. The tumours were histopathologically examined and classified. All malignomas developed in imported Tupaia only. From the tumour cells of two different animals new Tupaia herpesviruses were isolated. This is the first. report on spontaneous malignomas of Tupaia in captivity.     

Growth and reproductive development in the male tree shrew (Tupaia belangeri) from birth to sexual maturity

The growth and reproductive development of the male tree shrew were studied from birth to sexual maturity. An infantile phase from birth to Day 30 was characterized by the rapid involution of the testis and adrenal gland from a fetal condition followed by a nadir in testosterone levels and slow growth and differentiation of the testis and accessory sex organs. The initiation of puberty occurred collaterally with the emergence of the young from the nest and was marked by a sharp rise in testosterone levels from Days 30 to 35 to maximum levels at Days 40-55. Peak testosterone levels were temporally correlated with the onset of maximum growth and differentiation of the testis and accessory sex organs, descent of the testis, development of the scrotum, and a pronounced peak in the weight-velocity curve. The rapid growth of males at puberty contributed to a moderate degree of sexual dimorphism in this species. Puberty was attained at about Day 90 with the completion of spermatogenesis and the functional differentiation of the accessory sex organs. The postnatal development of the tree shrew conforms with the general primate pattern. The precise endocrine correlates established during puberty make Tupaia belangeri a useful small animal model for the study of puberty in primates.     

Histopathological changes in the liver of tree shrew (Tupaia belangeri chinensis) persistently infected with hepatitis B virus

Herpes simplex virus type-1(HSV-1) and HSV-2 are important human pathogens that cause significant ocular and urogenital complications, respectively. We have previously shown that HSV-1 virions lacking glycoprotein K (gK) are unable to enter into neurons via synaptic axonal membranes and be transported in either retrograde or anterograde manner. Here, we tested the ability of HSV-1 (F) gK-null to protect against lethal challenge with either highly virulent ocular HSV-1 (McKrae strain), or genital HSV-2 (G strain). The gK-null virus vaccine efficiently protected mice against lethal vaginal infection with either HSV-1(McKrae) or HSV-2 (G). Female mice were immunized via a single intramuscular injection with 106 PFU of the gK-null virus. Immunized mice were treated with Depo-Provera fourteen days after vaccination and were challenged via the vaginal route one week later. Ninety percent of mice vaccinated with the gK-null virus survived HSV-1 (McKrae) challenge, while 70% of these mice survived after HSV-2 (G) challenge. Moreover, all vaccinated mice exhibited substantially reduced disease symptoms irrespective of HSV-1 or HSV-2 challenge as compared to the mock vaccinated challenge group. T-cell memory immune responses to specific glycoprotein B (gB) and glycoprotein D (gD) peptide epitopes were detectable at 7 months post vaccination. These results suggest that the highly attenuated, non-neurotropic gK-null virus may be used as an effective vaccine to protect against both virulent HSV-1 and HSV-2 genital infections and induce lasting immune responses.     

HS-SPME-GC/MS法分析中缅树鼩尿液的化学成分

正尿液是动物新陈代谢的产物,在动物的化学通讯中发挥着重要作用,动物依靠它识别个体,选择高质量的配偶,判断亲缘关系,吸引异性等,以增加个体的适合度(Macdonald and Brown,1985)。各种动物尿液的化学成分复杂,含有挥发性和非挥发性两类化学信号物质。尿液的化学组成和其功能在许多动物中已进行报道,如大熊猫(Ailuropoda melanoleuca)(刘玉良等,2012)、水獭(Arvicola