Antler extracts stimulate chondrocyte proliferation and possess potent anti-oxidative,anti-inflammatory,and immune-modulatory properties

The Sika deer antler is well known for its unique ability to regenerate repeatedly and grow rapidly. Furthermore, it is a precious traditional Chinese medicine and has been widely used for more than 20 centuries. The major bioactive components within the antlers are water-soluble proteins, polypeptides, and free amino acids. Many studies have shown that water-soluble antler extracts play pivotal roles in wound healing, immune system modulation, anti-oxidation, and anti-inflammation. However, the exact effects on chondrocytes are still largely unknown. In this study, we prepared fresh, aqueous extracts from growing deer antlers in a rapid growth stage. We isolated the chondrocytes from neonatal mouse rib cartilage and investigated the effects of antler extracts on chondrocyte viability. We also used the RNA-Seq method to analyze the gene expression pattern under antler extract treatment. We demonstrated that fresh extracts from Sika deer antlers in a rapid growth stage significantly promoted chondrocyte viability and kept chondrocytes proliferating continuously, while blocking maturation and further differentiation. Additionally, our results indicated that antler extracts might serve as a potent anti-oxidant, anti-inflammatory agent, and immune modulator to boost the abilities of chondrocytes against oxidative, inflammatory, and immune stresses. Thus, this study has greatly deepened our current knowledge of the molecular control of antler extracts on chondrocytes. It has also shed light on possible new strategies to further prevent and treat diseases of cartilage and other related diseases.     

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.     

基于转录组学的梅花鹿茸皮组织修复机制研究

为研究不同生长时期梅花鹿东北亚种Cervus nippon hortulorum鹿茸茸皮差异基因表达变化,以期为鹿茸生长机制及组织修复的临床治疗提供理论依据,本研究采用Illumina测序技术和生物信息学方法对快速生长期和骨化期鹿茸生长中心茸皮进行转录组测序和差异基因表达分析。从快速生长期和骨化期茸皮中分别获得45422254条和44530430条clean reads,测序质量值分别为98.50和98.39;通过Trinity组装,分别获得44352条和43194条组装序列,平均长度分别为977nt和1135nt。通过数据库比对和差异基因表达分析筛选出生长因子7种、转录因子25种和胶原分子6种。在快速生长期鉴定出多种参与组织修复的重要生长因子和转录因子,包括Pdgfa、Tgfβ3、Egfl7、Vegfb、Sox4、Sox12、Scx、E4f1、Tcf4和Elf4等。     

Proteome analysis of red deer antlers

Deer antlers are the only mammalian organs capable of repeated regeneration. Although antlers are known to develop from pedicles, which arise from antlerogenic cells of cranial periosteum, their developmental process is not fully elucidated. For example, while endocrine and environmental factors influence the antler development, it is still unclear which signaling pathways are involved in the transduction of such stimuli. To study the developmental process of antlers and identify proteins functioning in their growth, we have established proteome maps of red deer (Cervus elaphus) antlers. With two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry, we analyzed more than 800 protein spots and identified approximately 130 individual proteins derived from the growing tip of antlers. The overall profile of the antler proteome was dissimilar to those of other types of tissue. Also comparison of proteomes derived from proximal bony tissue and the growing tip of antlers revealed substantial differences. Moreover several cell growth or signaling-related proteins are expressed exclusively in the growing tip, suggesting that these proteins function in the growth and differentiation of antlers. Currently, using the antler proteome maps, we are actively searching for the regulatory factor(s) that may control the antler development.     

Nerve growth factor mRNA expression in the regenerating antler tip of red deer (Cervus elaphus)

Deer antlers are the only mammalian organs that can fully regenerate each year. During their growth phase, antlers of red deer extend at a rate of approximately 10 mm/day, a growth rate matched by the antler nerves. It was demonstrated in a previous study that extracts from deer velvet antler can promote neurite outgrowth from neural explants, suggesting a possible role for Nerve Growth Factor (NGF) in antler innervation. Here we showed using the techniques of Northern blot analysis, denervation, immunohistochemistry and in situ hybridization that NGF mRNA was expressed in the regenerating antler, principally in the smooth muscle of the arteries and arterioles of the growing antler tip. Regenerating axons followed the route of the major blood vessels, located at the interface between the dermis and the reserve mesenchyme of the antler. Denervation experiments suggested a causal relationship exists between NGF mRNA expression in arterial smooth muscle and sensory axons in the antler tip. We hypothesize that NGF expressed in the smooth muscle of the arteries and arterioles promotes and maintains antler angiogenesis and this role positions NGF ahead of axons during antler growth. As a result, NGF can serve a second role, attracting sensory axons into the antler, and thus it can provide a guidance cue to define the nerve track. This would explain the phenomenon whereby re-innervation of the regenerating antler follows vascular ingrowth. The annual growth of deer antler presents a unique opportunity to better understand the factors involved in rapid nerve regeneration.     

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。     

Association between melatonin receptor 1A (MTNR1A) gene single-nucleotide polymorphisms and the velvet antler yield of Sika deer

Melatonin, a secretion from pineal gland is ambiguously considered as the key hormone involved in regulation of the antler cycle in Sika deer. To find out more about the roles of melatonin and its receptor gene, we carried out current study to investigate the association between polymorphisms in melatonin receptor 1A (MTNR1A) gene and the antler yield from Sika deer. A total of 251 Sika deer were analyzed in this study, of which consisted of Wusan Sika deer (n = 163) and Dongfeng Sika deer (n = 88). MTNRA gene was amplified by PCR and genotyped by Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). Three polymorphism loci (C518T, C629G and C635T) were detected in exon2 of MTNR1A gene. The restriction site Ecol881 was used for C518T while a C629G polymorphism locus was digested with Mval restriction endonucleases. In Wusan Sika deer the allele frequencies of C and T were 0.637 and 0.363 for C518T, Also C and G alleles in C629G locus were 0.206 and 0.794. Genotypic frequencies of allele CC, CT and TT were 33.7, 59.9 and 6.4 % respectively, It showed 1.8, 37.4 and 60.7 % for frequencies of genotypes CC, CG and GG. In Dongfeng Sika deer the allele frequencies of C and T were 0.518 and 0.482 for C518T, C and G alleles were 0.375 and 0.625 for C629G. Genotypic frequencies were 10.6, 82.4 and 7.1 % for genotypes CC, CT and TT respectively, and they were 1.1, 72.7 and 26.2 % for genotypes CC, CG and GG. Among three SNPs, only C629G showed significant association (P < 0.05) with average antler yield in Wusan Sika deer, while no SNP was significant in Dongfeng Sika deer. These preliminary results implied that the identified SNPs of MTNR1A gene might influence the antler yield in Wusan Sika deer.     

Getting the timing right: antler growth phenology and sexual selection in a wild red deer population

There has been growing interest in the determinants of the annual timing of biological phenomena, or phenology, in wild populations, but research on vertebrate taxa has primarily focused on the phenology of reproduction. We present here analyses of the phenology of the annual growth of a secondary sexual characteristic, antlers in red deer (Cervus elaphus) males. The long-term individual-based data from a wild population of red deer on the Isle of Rum, Scotland allow us to consider ecological factors influencing variation in the phenology of growth of antlers, and the implications of variation in antler growth phenology with respect to the phenotype of antler grown (antler mass) and annual breeding success. The phenology of antler growth was influenced by local environmental conditions: higher population density delayed both the start date (during spring) and the relative end date (in late summer) of antler growth, and warmer temperatures in the September and April prior to growth advanced start and end dates, respectively. Furthermore, there was variation between individuals in this phenotypic plasticity of start date, although not in that of end date of growth. The phenology of antler growth impacted on the morphology of antlers grown, with individuals who started and ended growth earliest having the heaviest antlers. The timing of antler growth phenology was associated with breeding success in the following mating season, independently of the mass of antlers grown: an earlier start of antler growth was associated with siring a higher number of the calves born the following spring. Our results suggest that the phenology of traits that are not directly correlated with offspring survival may also regularly show correlations with fitness.     

Antler mineral composition of Iberian red deer <Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis> is related to mineral profile of diet

Previous studies have suggested that antlers are costly bone structures whose mineral composition may change depending on physiological and other factors. This study examined whether nutrition variation associated with deer management influences antler mineral composition and structural characteristics of whole antler. Mineral distribution and bone structure were examined in antlers from two groups of adult Iberian red deer Cervus elaphus hispanicus Hilzheimer, 1909. They were kept under different feeding regimes at an experimental deer farm and a game estate in southeastern Spain. Protein and mineral contents differed between the diet of captive deer and that of deer in the wild. Significant differences were found for Na, Mg, K and protein. Antler composition seems to reflect the diet, as antlers of deer differed in protein, Na, Mg and K, but not in total mineral content, Ca, Fe or Zn. Thus, management conditions related to nutrition are reflected on antler composition.     

HPLC-MS/MS shotgun proteomic research of deer antlers with multiparallel protein extraction methods

Deer antlers mature rapidly in 60 days, and subsequently shed in 5 days with rapid ossification. During this procedure, the function of deer antlers changes significantly. Therefore, the profiling of antler proteome is helpful to discover important growing and shedding regulation proteins, which might be of great significance for studying development and regeneration. In this study, a parallel protein extraction strategy was developed to extract proteins from antlers of red deer with five different lysis solutions, followed by shotgun proteomic analysis by microflow reversed-phase liquid chromatography/electrospray ionization/tandem mass spectrometry (μRPLC-ESI-MS/MS) with a 30 cm-long serially coupled microcolumn. Our experimental results showed that the identified proteins extracted by five kinds of lysis solution were complementary to each other. In total, 416 unique proteins were identified, with relative molecular masses from 2000 to 600,000, and isoelectric points from 3.84 to 11.57. All these results demonstrate that the combination of parallel protein extraction strategy and μRPLC-ESI-MS/MS analysis with serially coupled long microcolumns might be of great significance for comprehensive proteomic research of deer antler.     

What, if anything, does visual asymmetry in fallow deer antlers reveal?

Fluctuating asymmetry (FA), small directionally random deviations from perfect bilateral symmetry, is thought to reflect individual quality. Furthermore, it has been suggested that FA in secondary sexual characters can be used to assess mate or opponent quality during inter- or intrasexual competition. Studies on fallow deer, Dama dama, have suggested that FA in antlers reflects individual dominance, or the existence of a directional asymmetry (DA) with right antlers being consistently more developed than left antlers. To test these conflicting hypotheses, we analysed relationships between age, dominance and asymmetry in the number of antler points on mature fallow deer males during four rutting seasons in a single population. Age and dominance were only weakly correlated. The number of antler points displayed a pattern of DA (more points on the right than on the left side) that increased with age. Although dominance tended to increase with the total number of antler points, there was no relationship between the level of DA and dominance. These results failed to support the hypothesis that antler asymmetry visually reveals individual quality in fallow deer. Copyright 2000 The Association for the Study of Animal Behaviour.     

梅花鹿重组Galectin-1的表达及多克隆抗体的制备与鉴定

半乳糖凝集素-1（Galectin-1）是最先被报道的哺乳动物半乳糖凝集素,存在于多种组织和细胞内,参与细胞的粘附、增殖、凋亡和炎症反应等多种生理病理过程,并且与免疫系统的调节和肿瘤的发生发展密切相关。鹿茸是哺乳动物中罕见的能够周期性的脱落和再生的附属器官,作为研究哺乳动物器官再生的新模型受到关注。鹿茸再生是一个基于干细胞的过程,定位于角柄骨膜的干细胞是鹿茸再生的基础,Galectin-1在角柄骨膜细胞（pedicle periosteum cell, PPC）中高度表达,提示其在鹿茸再生中发挥着重要的作用。由于尚没有商用的鹿Galectin-1蛋白及其抗体,为进一步研究Galectin-1在鹿茸再生中的生物学功能,需要制备相应的蛋白和抗体,本实验将梅花鹿Galectin-1基因与pET28a连接,并将重组质粒pET28a-Galectin-1转入大肠杆菌（Escherichia coli）BL21(DE3)中诱导表达。用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔子制备多克隆抗体。酶联免疫吸附（enzyme-linked immunosorbent assay, ELISA）法检测抗体效价、Western blot 检测抗体特异性、细胞免疫荧光检测Galectin-1在角柄骨膜细胞中的表达情况。结果表明,本实验成功诱导重组原核表达载体pET28a-Galectin-1在BL21(DE3)中表达,通过Ni纯化获得融合蛋白。ELISA结果显示,抗体效价达到1:64000,Western blot结果表明该抗体特异性良好,细胞免疫荧光显示Galectin-1在PPC全细胞中表达。本实验获得了纯化的鹿Galectin-1蛋白和特异性较好的多克隆抗体,为揭示Galectin-1在鹿茸再生调控中的作用提供了重要的实验材料。     

Does fluctuating asymmetry of antlers in white-tailed deer (Odocoileus virginianus) follow patterns predicted for sexually selected traits?

Secondary sexual characters have been hypothesized to signal male quality and should demonstrate a negative relationship between the size of the trait and degree of fluctuating asymmetry because they are costly to produce. We collected morphometric and antler data from 439 white-tailed deer (Odocoileus virginianus) in Oklahoma, USA, in order to determine whether measures of antler asymmetry follow the patterns predicted for sexually selected characters. Relative fluctuating asymmetry was negatively related to antler size for all deer and within age groups up to five and a half years of age. We did not detect an association between asymmetry and antler size among deer that were six and a half years or older. When categorizing deer by antler size, we found that deer with small antlers (< or = 33rd percentile) had greater levels of relative asymmetry than deer with large antlers (< or = 67th percentile). The relative asymmetry of antlers was negatively related to age and was greatest in deer that were one and a half years old. Relative asymmetry was also negatively related to carcass mass, inside spread, skull length and body length. These data suggest that asymmetry in the antlers of white-tailed deer may be a reliable signal of quality and, as such, may be important in maintaining honesty in intrasexual advertisements during the breeding season.     

Correlates of variation in deer antler stiffness: age, mineral content, intra-antler location, habitat, and phylogeny

To test correlations between the stiffness of deer antler and aspects of cervid ecology, we measured the stiffness (in bending) of antler from white-tailed deer ( Odocoileus virginianus ) and compared our results to previous measurements from antlers of other cervid species. Stiffness of antler specimens did not correlate significantly with mineral content or the location within the antler from which specimens were taken in O. virginianus. However, antler stiffness in white-tailed deer decreased significantly between two- and three-year-old bucks, matching the time in O. virginianus life history when males shift from sparring with mostly larger individuals to sparring with mostly smaller individuals. Stiffer antlers may enable younger, smaller bucks to have a more effective lever through which their smaller muscular forces can be transmitted during sparring with older, larger bucks. The stiffnesses we measured for white-tailed deer antler are similar to values measured previously from other members of the odocoileine lineage, which are lower than those previously measured from the antlers of deer living in tropical habitats. However, confidence limits for maximum likelihood reconstructions of the ancestral stiffness of deer antler span the range of high stiffnesses found among tropical deer; furthermore, parsimony-based reconstructions of ancestral antler stiffness are equivocal. Thus, the high antler stiffnesses of tropical deer may reflect the retention of an ancestral condition, rather than adaptation to year-round antler use.     

Juvenile-to-Adult Antler Development in White-Tailed Deer in South Texas

Abstract: Past studies using penned deer provide conflicting results on the age when reliable predictions about antler growth potential in white-tailed deer (Odocoileus virginianus) can be made. We captured wild whitetail males via aerial net gun on 12 ranches in 5 counties in south Texas, USA, from 1999 to 2007 to determine if a reliable juvenile-to-adult relationship in antler development existed. We individually marked and released captured animals at the trap site after we took antler and body measurements. We recaptured marked animals as possible in subsequent years or until we obtained final measurements after legal harvest. Amount of growth in the first set of antlers in whitetail males was a poor predictor of antler growth at maturity. By 4.5 years of age there were no differences (P > 0.05) in antler measurements regardless of the amount of development of the first set of antlers at 1.5 years. We concluded culling of yearling males based on number of antler points would have little positive effect on overall antler quality in future years.     

Effect of antiandrogen cyproterone acetate on the development of the antler cycle in Southern pudu (Pudu puda)

The antler cycle of pudu is similar to other cervids, but unlike most boreal deer, male Southern pudu (Pudu puda) exhibits two seasonal peaks of LH and testosterone. In that respect, pudu is similar to roe deer. Whereas the antler cycle in some deer species, such as roe deer or white-tailed deer, is very sensitive to variation of testosterone, in other cervids, such as fallow deer or reindeer, a blockade of androgens with cyproterone acetate (CA) has little or no effect on the timing of the antler casting. In order to test the sensitivity of pudu antlers to variations of androgens, CA (administered 2x weekly at 50 mg/buck) was injected intramuscularly for 3 weeks in 5 adult male pudu, starting February 19 (late summer). Four other males of similar age served as controls. The experiment was performed at the University of Concepcion, Chile, latitude 36.6 degreeS. Blood samples were taken once a week between January 19 and April 3. In CA-treated bucks, the antlers were cast approximately 3 weeks after the initiation of CA treatment and a new antler growth began almost immediately. The antlers reached about 5 cm in length, before ceasing to grow at the end of April, when they became mineralized and were subsequently polished. CA had no effect on the already declining levels of LH. Plasma levels of testosterone in controls increased from February 15, whereas in CA-treated bucks remained depresses until March 21. It is concluded that similarly to white-tailed deer, the antler cycle of Southern pudu is very sensitive to manipulation of androgen levels.     

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.