Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for “antler” genes that encode α-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.     

Gene expression of axon growth promoting factors in the deer antler

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin.     

利用全基因组重测序分析鹿茸重量相关基因

茸角是鹿科动物特有的器官,具有重要的生物学意义。鹿茸生长是一个复杂的生物代谢过程,其重量与遗传因素有一定关联。本研究对饲养条件基本一致的5个梅花鹿(Cervus nippon)群体进行调查,获得高产和低产梅花鹿个体共100只,利用全基因组重测序分析这些个体与鹿茸重量相关的遗传变异。结果表明,共得到94个与鹿茸重量可能相关的遗传变异,其中有2个变异位点分别定位于OAS2和ALYREF/THOC4基因的外显子区,且ALYREF/THOC4基因在鹿茸中表达量很高。功能富集分析发现,这些遗传变异与鹿茸生长发育密切相关,可作为潜在的鹿茸重量相关遗传变异。本研究首次通过全基因组重测序直接筛选与鹿茸重量相关的遗传变异,并分析关联基因的生物学功能,对揭示鹿茸生长发育和鹿茸重量差异形成的遗传机制具有重要意义。     

Seasonal antler cycle in a herd of pampas deer (Ozotoceros bezoarticus) in Uruguay

To describe the yearling antler cycle of pampas deer bucks (Ozotoceros bezoarticus) 22 bucks and 72 antler cycles have been studied in a semi-captive population. Date of individual antler cast, first day on which the brow and the trez tine were observed, and day of the velvet shedding were registered. Time intervals (days) between first and second antler casts, between first antler cast and the brow tine observation, between brow and trez tines observation and from first antler cast to velvet shedding were calculated. The brow and the trez tine were first observed 22.8±0.6 and 45.9±0.9 days after the first antler cast. Velvet shedding was observed 103.3±2.1 days after the first antler cast. Both antlers casts were observed earlier in adult cycle bucks than in first antler bucks, although the interval between both casts was not different. The interval from the brow tine observation to the trez tine observation was shorter in first antler cycle bucks than in adults. As in other deer species, antler cycle was seasonal, but persistence of cycle-to-cycle antler growth well into adulthood is different from what occurs in most deer species.     

Testosterone and estradiol concentrations in serum, velvet skin, and growing antler bone of male white-tailed deer

The growth and mineralization of antlers correlate with the seasonal variation of serum androgens. Whereas seasonal levels of testosterone (T) in plasma are well established, steroid concentrations have not yet been determined in the tissues of growing antlers. Therefore, RIA was used to determine T and 17beta estradiol (E2) in serum, and three areas (tip, middle, and base) of the antler bone and the antler skin, called velvet. Blood and antler tissues of white-tailed deer (Odocoileus virginianus) were collected from May to August. The difference between levels of T and E2 among the sites was calculated using the square root transformation followed by a mixed model analysis with individual deer and an interaction of individual and year (individual(*)year) as a random factor. Concentrations of T in serum (799+/-82 pg/ml) were higher than T values in the velvet (589+/-58 pg/ml, P<0.01) and in the antler bone (538+/-58 pg/ml, P<0.001). Estradiol concentrations differed among antler tissues and serum (P<0.001) and between years (P<0.01). Estradiol concentrations in serum (25+/-25 pg/ml) were consistently lower than those in antler bone (208+/-11 pg/ml, P<0.001) and velvet (150+/-12 pg/ml, P<0.001). The E2:T ratio in serum was 1:10-60. The same ratio for the antler bone was only 1:2-3 and for the velvet 1:3.5. It is concluded that higher T and lower E2 concentrations found in plasma, as compared to antler bone or antler velvet, may indicate a partial metabolism of systemic androgens into estrogens xin the tissues of growing antlers.     

基于组学技术的鹿茸生物学研究进展

鹿茸是唯一可周期性再生的哺乳动物器官,由软骨、骨、血管、神经及皮肤组织构成。鹿茸再生过程是基于干细胞的增殖和分化,且生长速度极快而不发生癌变。其不仅可作为一种肢体再生的生物医学模型,而且也作为一种研究骨组织生长发育的模型。现代组学技术快速发展,已普遍应用于生物学的各种领域。利用组学技术,在转录和蛋白质水平上,有力地推动了在分子水平上研究鹿茸生物学的进程。本综述拟对组学技术在鹿茸生物学研究中的应用进行总结回顾,并对未来的发展趋势做进一步展望,为鹿茸生物学的深入研究提供参考。     

Identifying novel genes involved in both deer physiological and human pathological osteoporosis

Osteoporosis attacks 10% of the population worldwide. Humans or even the model animals of the disease cannot recover from porous bone. Regeneration in skeletal elements is the unique feature of our newly investigated osteoporosis model, the red deer (Cervus elaphus) stag. Cyclic physiological osteoporosis is a consequence of the annual antler cycle. This phenomenon raises the possibility to identify genes involved in the regulation of bone mineral density on the basis of comparative genomics between deer and human. We compare gene expression activity of osteoporotic and regenerating rib bone samples versus autumn dwell control in red deer by microarray hybridization. Identified genes were tested on human femoral bone tissue from non-osteoporotic controls and patients affected with age-related osteoporosis. Expression data were evaluated by Principal Components Analysis and Canonical Variates Analysis. Separation of patients into a normal and an affected group based on ten formerly known osteoporosis reference genes was significantly improved by expanding the data with newly identified genes. These genes include IGSF4, FABP3, FABP4, FKBP2, TIMP2, TMSB4X, TRIB, and members of the Wnt signaling. This study supports that extensive comparative genomic analyses, here deer and human, provide a novel approach to identify new targets for human diagnostics and therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of PTHrP and the PTH/PTHrP receptor in growing red deer antler

Antler growth is highly co-ordinated, so that trabecular bone and antler skin (velvet) develop together, at a rapid rate and in a manner reminiscent of their development in the fetus. Parathyroid hormone-related peptide (PTHrP) is expressed in both bone and skin, and is therefore a candidate to effect co-ordination between these tissues. The aim of this study was to localize the expression of PTHrP and its principal receptor, the parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrPR), in antler ("spiker") of one-year-old red deer. Using immunohistochemistry and in situ hybridization, intense and overlapping expression of PTHrP and its receptor was seen in developing osseocartilaginous structures and in the underlying layers of velvet epidermis. PTHrP was located on both the cell surface and within the nuclei. Our results strongly suggest that PTHrP, acting via the PTH/PTHrPR and possibly other intracrine mechanisms, plays a central role in the co-ordinated regulation of cell division and differentiation of developing antler bone and skin.     

梅花鹿鹿茸不同产品中氨基酸含量的比较

通过对二杠鹿茸、三杈鹿茸、鹿茸片、鹿茸血、鹿角、鹿角盘等鹿茸产品中氨基酸含量的测定研究,结果表明:鹿茸血中氨基酸含量最高,鹿茸片中的氨基酸含量次之,三杈鹿茸中的氨基酸含量高于二杠鹿茸中氨基酸含量,鹿花盘中的含量高于鹿角中的氨基酸含量,从而为鹿茸这一动物性中药材资源的功能评价、药理作用提供科学理论依据。     

Annulus Fibrosus Cell Characteristics Are a Potential Source of Intervertebral Disc Pathogenesis

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.     

Internal architecture of pampas deer antlers differs in males allocated with and without females

Bone mineralization of antlers and the depth of the antler seal (the basal surface of the cast antlers) are positively related to testosterone concentrations. Pampas deer males that are in permanent contact with females have greater, heavier, and darker antlers than males that are isolated from them. The objectives were to determine if antler compact/spongy bone ratio, antler seal depth, and compact bone darkness are greater in pampas deer males permanently allocated with females than those in males isolated from them. Antlers from males permanently allocated with or without females were cut transversally in seven points and scanned, and the compact/spongy ratio was calculated. The pixel intensity of each image was determined with a software for image analysis. The coronet of the antler was cut longitudinally, and the height of the most prominent protrusion was measured. The compact/spongy ratio was greater in antlers from males that were in contact with females in the second tine (P?=?0.02) and tended to be great in the two other tines (P?=?0.06 and P?=?0.1, respectively). Compact bone pixel color was darker in males in contact with females than that in males isolated from females in two points (P?=?0.02 and P?=?0.05, respectively) and tended to do so in two more (P?=?0.055 and P?=?0.1, respectively). Antler seal convexity was also greater in antlers from males in contact with females (P?=?0.006). We concluded that permanent contact with females stimulated pampas deer males increasing compact bone portion of the antler tines, the seal depth size, and the darkness of the compact bone.     

Effects of public vs. private management on deer antler composition, mechanical and structural variables

Previous studies have shown that antler mineral composition reflects diet. Thus, management involving diet can influence both the mineral composition and mechanical properties of antlers. However, it is not known if reducing population density, which increases availability of food, can compensate mineral deficiencies arising from lack of minerals in plants and, ultimately, in the soil. The present study aimed at assessing if private management often involving a balanced food supplementation produced differences in antler properties compared to both public management and reference antlers from deer farms. We also examined if low population density in a National Park could compensate for mineral deficiencies found in antlers of other public reserves or else, antlers still differed in antler characteristics compared to supplemented deer in private game estates. We used 120 antlers from three public reserves, four private game estates and two deer farms as reference to assess antler composition, mechanical properties, size and structure. Public managed antlers had shorter length compared to private and reference ones, thinner cortex (CT), were less dense, had lower second moment of area (I) and work to fracture (W). In addition, they had content in ash, Ca, Mg, Na, S and Zn lower than antlers from private game estates. In contrast, antlers from public reserves had greater values of Young’s modulus of elasticity (E), Fe, Mn and Si. In most cases, antlers from private management and reference farms showed similar values. When comparing antlers from low population density in a National Park with antlers from private management, differences in antler length, CT, I and ash disappeared, but both differed still in density, E, bending strength, W and content in all minerals mentioned. In conclusion, low density can improve some structural variables, but it cannot compensate for mineral deficiencies whereas food supplementation can.