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.     

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.     

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.     

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。     

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.     

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.     

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.     

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。     

Antler stiffness in moose (Alces alces): Correlated evolution of bone function and material properties?

The material properties of bone can vary considerably among skeletal elements from different parts of the body that serve different functions. However, functional demands placed on a specific type of skeletal element also can vary at a variety of scales, such as between different parts of the element, among individuals of a species, and across species. Variation in bone material properties might be correlated with differing functional demands at any of these scales. In this study we performed three-point bending tests on bone specimens extracted from antlers of moose (Alces alces) to test for three types of variation in bone material stiffness (Young's modulus): within the antler structure, between populations of moose, and between moose and other deer species. Because superficial portions of the antler are exposed to greater bending stress and strain than deeper portions, and because the antler beam (the basal shaft that attaches to the skull) is subjected to greater bending moments than more distal parts of the antler, we predicted that superficial bone and bone from the beam would be stiffer than bone from other parts of the antler. Instead, we identified no significant differences in these comparisons. There were also no significant differences in antler stiffness between moose from Michigan and the Yukon, even though the rapid growth required of antlers from northern latitudes like the Yukon has the potential to compromise bone material properties. However, moose have significantly stiffer antlers (11.6 +/- 0.45 GPa, mean +/- SE) than any other deer in the odocoileine lineage. Moreover, phylogenetic reconstructions of the evolution of antler stiffness in deer indicate a strong potential that high antler stiffness is a derived feature of moose. The unusual palmate shape of moose antlers likely subjects their antler beams to higher bending moments than found in other odocoileines, a factor that may have contributed to the evolutionary divergence of moose antler stiffness from that of other members of this clade. Although similarities in the mineral composition of bone across species likely limit the overall range of phylogenetic variation in bone material properties, our results demonstrate that evolutionary diversity in bone material properties can show correspondence with phylogenetic differences in mechanical or ecological demands on skeletal elements.     

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.     

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.     

Climate-dependent allocation of resources to secondary sexual traits in red deer

Fitness in highly polygynous male ungulates is related both to body size, weight and antler size. Males must therefore allocate resources both to growth of the body and growth of the antlers, which may lead to tradeoffs whenever resource levels are in limited supply. Several studies have reported how (absolute) growth of antlers and horns are related to environmental conditions, but few have looked for the relative allocation patterns (i.e. relative to body size and weight). We analyzed how the influence of variation in climate (the North Atlantic oscillations, NAO) and population density affected the allocation of resources to antlers, based on data from 2720 red deer stags two years or older harvested during 1965–2002 along the west coast of Norway. Number of antler tines increased up to six years of age, remained stable until 12 years of age, and then decreased significantly (>12 years, n=45). The NAO was positively related to number of antler tines in prime aged males (6–12 years, n=629), also after controlling for both body size and weight. Our study thus suggests that deer have a higher allocation of resources to antlers relative to body weight during favorable environmental conditions.     

The effect of cyproterone acetate on the antler cycle in red deer (Cervus elaphus L.)

The aim of the study was to test the effect of antiandrogen, cyproterone acetate (CA) on the antler cycle in the red deer (Cervus elaphus). CA was administered to three adult red deer stags (Edward, Fuks and Gacek) in weekly intervals. Edward and Fuks were given 600 mg + 600 mg of CA, whereas Gacek was given 600 mg + 300 mg. CA was injected during the hard antler phase: in mid-October (Edward), at the end of November (Fuks) and at the end of January (Gacek). CA caused the antler casting 17 to 22 days after the first injection. In all stags, the casting of antlers was followed by a period of intensive growth of new antlers. Edward was given CA at the end of October. This treatment was responsible for occurrence of the two antler cycles in the year of the experiment. When CA was administered during the middle of the hard antler phase an additional short antler cycle occurs followed by new antler growth. CA treatment in the later part of hard antler phase may cause a prolonged antler cycle.     

The asynchronous timing of antler casting of Reeves’ muntjac (Muntiacus reevesi)

From 1973 until the present time, captive Reeves’ muntjac (Muntiacus reevesi) have been observed in England. The dates of casting of both antlers have been recorded for 25 bucks, totalling 121 antler cycles. All casting dates were between 1 May and 17 July (63.64 % of the cycles occurred in May, 34.71 % in June, and only 1.65 % in July). Only 13.2 % of the antler cycles were synchronous. In 45.71 % of cases, the left antler was cast 1 day or more before the right one, whereas in 54.29 % of cases, the reverse was true. For 57.14 % of cases, the time between castings of the first and second antlers was up to 3 days, and for 32.38 % of the cases, the interval was 4 to 8 days, and in 10.48 %, the difference was 9 to 13 days. All bucks cast their antlers asynchronously at least in some of the antler cycles. No external factor, which would be responsible for this, was detected. Significant variation across season has been shown with no apparent trend. Although records on subsequent seasons were available for most of the bucks, there was a zero repeatability of the process.     

Aspects of breeding biology of chital (Axis axis) and sambar (Rusa unicolor) in the Western Ghats

Antler cycles are convenient external signs indicating internal changes in reproductive status of male deer. Antler phenology of chital (Axis axis) and sambar (Rusa unicolor) were studied in a deciduous habitat of Mudumalai Tiger Reserve, Western Ghats, using vehicle transects for 2 successive years. Apparent breeding seasonality occurred with the majority of adult stags in hard antlers from May to mid October in chital (>87 %), and October to May in sambar (>68 %). Adult hard antler in chital correlated with mean group size, while sambar showed a weak correlation between adult hard antler and group size. Chitals prefer forming large groups at forest edges and open habitats while sambars prefer dense vegetation cover. Adult hard antlers in chital and sambar showed a weak relation to fawning since most adult females were in oestrus during the peak rutting season. Adult hard antlers in chital associated positively with rainfall and day length while sambar responded weakly to rainfall. We conclude that environmental variables determined species-specific mating strategies in the two deer species.     

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.     

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.