Seasonal variation in temperate ecosystems influences food availability and the energetic demands associated with thermoregulation and reproduction, shaping physiological traits such as meat composition in wild animals. However, the specific effects of seasonality on meat quality in wild red deer remain insufficiently understood. This study evaluated how seasonality -represented by hunting month (September, January, April, and June)- affects the quality of the Longissimus thoracis et lumborum muscle in 32 adult wild male red deer harvested under minimal stress conditions. We hypothesized that seasonal fluctuations in diet and physiological requirements, particularly those linked to antler growth, would generate measurable differences in carcass traits and meat composition. Deer hunted in September (late summer) showed the highest carcass weight and yield, whereas the lowest values occurred in January (mid-winter) and June (early summer; p < 0.001). Intramuscular fat content was also greatest in September, with significant differences compared to April (p < 0.05). Additional seasonal effects were detected for pH at 72 h post-mortem (pH₇₂), shear force, and fatty acid profile (p < 0.05), indicating that muscle metabolism and textural properties vary across the year. Mineral composition exhibited a distinct pattern, suggesting a stronger association with skeletal mobilization during antler growth than with dietary changes. Mineral composition patterns were consistent with seasonal skeletal mobilization during antler growth: Ca and Mg were higher in April; Fe and Zn were lower in April and June. Overall, these findings demonstrate that both season and antler-growth effort affected meat quality and composition in wild male red deer, providing new insights into nutritional ecology and game meat quality.

Natural extracts tested for potential effects in human health are susceptible to contamination, particularly when samples are often obtained from outdoor environments. Deer velvet antler (DVA) extracts have broad anticancer effects. As sterile samples are required for effective experimentation in cell cultures, we evaluated the most common sterilisation methods to get rid of microbial contamination. We also investigated whether the sterilisation method affected the anti-cancer activity of the DVA extract on human tumour cells. Two antler sections (tip and base) were subjected to water-based extraction. This study compares lyophilised and non-lyophilised DVA extracts. Subsequently, DMEM and LB culture media containing DVA extract were assessed for contamination. The amount of protein was quantified by BCA and visualised by polyacrylamide gel electrophoresis. To analyse the anticancer effect, a cell viability assay was performed on colorectal cancer cell lines. Finally, tumour biomarkers were evaluated by flow cytometry in colorectal cancer cells. Filtration was the sterilisation method that removed the highest microbial load. It reduced cell viability at a concentration of 1 mg/mL of total protein by up to 37% ± 10% (DVA-T) and by up to 69% ± 8% (DVA-B). Furthermore, the protein expression levels of SW480 colorectal cancer cells exhibited a significant increase in response to lyophilised DVA extracts in comparison to non-lyophilised extracts. The data obtained from this study indicate that the selected sterilization approaches allow preservation of protein integrity and in vitro bioactivity of DVA extracts, supporting their standardized preparation for further biological evaluation.

The postnatal development of secondary sexual characteristics is a highly complex process governed by diverse molecular signals and serves as a key marker of sexual maturity. Deer antlers exemplify such traits, distinguished not only by their unique ability to regenerate annually but also by their initiation in postnatal life. It is well established that the antlerogenic periosteum (AP) is the only tissue responsible for postnatal antler formation. Here, we identify a population of RXFP2-positive mesenchymal stem cells within the AP of both male and female deer that are crucial for antler development, primarily through the activation of canonical Wnt signaling. This process also relies on M2 macrophages recruited via IL-34 secretion. Furthermore, these cells exhibit reduced expression of HOX genes, suggesting a high degree of developmental plasticity. Our findings offer new insights into the molecular mechanisms underlying the postnatal development of secondary sexual characteristics, using deer antlers serving as a model system.

Antlers, a male deer secondary sex characteristic, are unique mammalian appendages that fully regenerate annually, under androgen regulation. Stem cells located in the antlerogenic periosteum (AP), a tissue overlaying the frontal crest of both male and female deer, play a crucial role in antlerogenesis. Nonetheless, the underlying molecular mechanisms as to how antlerogenesis is regulated by androgens remain largely unexplored. Here, we show that androgens regulate antler growth via macrophages. Bulk RNA sequencing revealed a significant enrichment of immune-related factors in the androgen-activated antlerogenic periosteum (AAP), and single-cell RNA sequencing identified a cluster of AAP cells overexpressing macrophage chemokine CCL2. Additionally, the presence of a substantial number of monocytes/macrophages was detected in the skin overlying the AAP. Histological examination confirmed macrophage accumulation in the AAP. Removal of macrophages with clodronate effectively inhibited antler generation in male sika deer as well as in nude mice engrafted with the AP. Furthermore, testosterone up-regulated CCL2 expression in the AP cells (APCs), thus enhancing their chemotactic effect on recruitment of macrophages. Remarkably, female sika deer developed antlers following local injection of CCL2, autologous macrophages, or even immune response inducer lipopolysaccharide (LPS). Therefore, macrophages play an essential role in deer antler generation.

Articular cartilage has a low capacity for self-regeneration. Therefore, stem cell therapy has been proposed and is gaining momentum. Antler can self-repair and its cartilaginous tissue can grow at an unprecedented rate (Up to 2 cm/day) and antler regeneration is based on antler stem cells. Therefore, we predicted that antler stem cells or their paracrine factors might be a good source for promoting cartilage repair. In this study, we prepared conditioned medium from antler stem cells (ASC-CM) and evaluated its effect. We implanted the medium into rat cartilage defects and assessed its capacity to promote cartilage repair. The protein composition of ASC-CM was analyzed using via DIA assay. ASC-CM can strongly promote chondrocyte proliferation in vitro; it significantly up-regulates the expression of chondrogenesis-related genes (Aggrecan, Col II, and Sox-9) and promotes glycosaminoglycan formation and type II collagen deposition in cartilaginous tissue; meanwhile up-regulate the apoptosis suppressor gene NAMPT and down-regulate apoptosis gene BAX. In vivo, cartilage defect in rats was significantly repaired using ASC-CM. The compositions of ASC-CM were got and some key proteins such as S100A4 were identified by bioinformatics analysis. The ASC-CM can promote the proliferation of rat-chondrocytes, maintain the chondrocyte phenotype, and inhibit apoptosis of rat-chondrocytes. Therefore, it can promote cartilage repair.

Bone growth and regeneration remain major clinical challenges. Deer antlers, the fastest-growing mammalian bone, regenerate via endochondral ossification and elongate up to 2 cm per day, far surpassing the ~2 cm annual growth of human growth plates. Here, we systematically mapped the cellular landscape of the antler growth center (AGC) using single-nucleus RNA sequencing, chromatin accessibility profiling, and spatial transcriptomics. The AGC harbors a large stem-progenitor pool that drives rapid elongation through vigorous proliferation supported by paracrine signaling. These proliferative cells exhibit a transcriptional program with intrinsically low tumorigenic potential, associated with apoptotic regulation. The AGC also establishes a vascularized niche that supports robust angiogenesis, sustains accelerated cartilage growth, and enables efficient recruitment of osteogenic cells. Notably, antlers employ a hybrid ossification strategy, combining endochondral ossification with direct hypertrophic chondrocyte-to-osteoblast transdifferentiation, likely via PHEX⁺ intermediates. Collectively, these findings refine fundamental concepts of endochondral ossification and offer insights for regenerative bone therapies.

The marked seasonality of temperate habitats in food availability and requirements of energy expenditure in reproduction or temperature homeostasis affects most aspects of wild animals, including meat composition. This study examines for first time the effects of seasonality (assessed as hunting month: September, January, April, and June) on loin muscle quality from 32 adult wild male deer culled stress-free. Deer hunted in September (end of summer) had the highest carcass weight and yield, while the lowest values were observed in January (mid-winter) and June (beginning of summer; p < 0.001). Intramuscular fat content was also higher in September than in the other months, but differences were only significant with April (p = 0.016). Other seasonal effects were found for pH72, shear force and fatty acid composition (p < 0.05). In contrast to these differences, likely caused by food availability, meat mineral composition seems to be caused by the mineral mobilization from the skeleton to grow antlers, as Ca and Mg were higher in April, and Fe and Zn were lower in this month and June. Thus, we can conclude that both, the season and the effort to grow antlers, affecte6d the meat quality and composition in wild male red deer.

Background: Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. Methods: A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. Results: ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco’s modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). Conclusions: ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

Wound healing is a complex process, which involves three stages: inflammation, proliferation, and remodeling. Inflammation is the first step; thus, immune factors play an important regulatory role in wound healing. In this study, we focused on a chemokine, C-C motif chemokine ligand 3 (CCL3), which is often upregulated for expression during wound healing. We compared cutaneous wound healing at the histological, morphological, and molecular levels in the presence and absence of CCL3. The results showed that the wound healing rate in the wild-type and CCL3 -/- + CCL3 mice was faster than that of CCL3 -/- mice ( P < 0.01), and application of CCL3 to wounds increased the healing rate. In the process of wound healing, the degree of reepithelialization and the rate of collagen deposition in the wound of CCL3 -/- mice were significantly lower than those of wild-type mice ( P < 0.01). The number of macrophages and the expression levels of tumor necrosis factor(TNF)-α and transforming growth factor (TGF)-β1 in the wounds of wild-type mice were much higher than those of the CCL3 -/- mice. Removal of macrophages and CCL3 -/- mice share similar phenotypes. Therefore, we infer that the wound healing requires the participation of macrophages, and CCL3 may play an important regulatory role through recruiting macrophages to the wound sites.

Abstract Background Deer antlers are the only known mammalian structure that undergoes full regeneration. In addition, it is peculiar because when growing, it contains vascularized cartilage. The differentiation of antler stem cells (ASCs) into chondrocytes while inducing endochondral extension of blood vessels is necessary to form antler vascularized cartilage. Therefore, antlers provide an unparalleled opportunity to investigate chondrogenesis, angiogenesis, and regenerative medicine. A study found that Galectin-1 (GAL-1), which can be used as a marker in some tumors, is highly expressed in ASCs. This intrigued us to investigate what role GAL-1 could play in antler regeneration. Methods We measured the expression level of GAL-1 in antler tissues and cells by immunohistochemistry, WB and QPCR. We constructed antlerogenic periosteal cells (APCs, one cell type of ASCs) with the GAL-1 gene knocked out (APCGAL-1−/−) using CRISPR-CAS9 gene editing system. The effect of GAL-1 on angiogenesis was determined by stimulating human umbilical vein endothelial cells (HUVECs) using APCGAL-1−/− conditioned medium or adding exogenous deer GAL-1 protein. The effect of APCGAL-1−/− on chondrogenic differentiation was evaluated compared with the APCs under micro-mass culture. The gene expression pattern of APCGAL-1−/− was analyzed by transcriptome sequencing. Results Immunohistochemistry revealed that GAL-1 was widely expressed in the antlerogenic periosteum (AP), pedicle periosteum (PP) and antler growth center. Western blot and qRT-PCR analysis using deer cell lines further supports this result. The proliferation, migration, and tube formation assays of human umbilical vein endothelial cells (HUVECs) showed that the proangiogenic activity of APCGAL-1−/− medium was significantly decreased (P < 0.05) compared with the APCs medium. The proangiogenic activity of deer GAL-1 protein was further confirmed by adding exogenous deer GAL-1 protein (P < 0.05). The chondrogenic differentiation ability of APCGAL-1−/− was impeded under micro-mass culture. The terms of GO and KEGG enrichment of the differentially expressed genes (DEGs) of APCGAL-1−/− showed that down-regulated expression of pathways associated with deer antler angiogenesis, osteogenesis and stem cell pluripotency, such as the PI3K-AKT signaling pathway, signaling pathways regulating pluripotency of stem cells and TGF-β signaling pathway. Conclusions Deer GAL-1, has strong angiogenic activity, is widely and highly expressed in deer antler. The APCs can induce angiogenesis by secreting GAL-1. The knockout of GAL-1 gene of APCs damaged its ability to induce angiogenesis and differentiate into chondrocytes. This ability is crucial to the formation of deer antler vascularized cartilage. Moreover, Deer antlers offer a unique model to explore explore how angiogenesis at high levels of GAL-1 expression can be elegantly regulated without becoming cancerous. Graphical Abstract

Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration. Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum (AP) cells and pedicle periosteum (PP) cells, respectively. As the cells resident in the AP and the PP possess stem cell attributes, both antler generation and regeneration are stem cell-based processes. However, the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized. Here, we took the approach of single-cell RNA sequencing (scRNA-Seq) and identified eight cell types (mainly THY1+ cells, progenitor cells, and osteochondroblasts) and three core subclusters of the THY1+ cells (SC2, SC3, and SC4). Endothelial and mural cells each are heterogeneous at transcriptional level. It was the proliferation of progenitor, mural, and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler. We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis. These may be due to the difference in the degree of stemness of the AP-THY1+ and PP-THY1+ cells. We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration. Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.

Dear Editor, Stem cell therapy holds enormous and revolutionary promise to treat various age-related diseases,such as diabetes,heart failure,and Parkinson's disease.However,low retention and survival rate of delivered stem cells,partially due to immuno-logical rejection,constitute major hurdles for the clinical implementation of stem cell therapy(Lei et al.,2021a).Since mounting evidence showed that several types of stem cells mainly exert their therapeutic effects through the secretion of paracrine effects,exosomes,which are released by stem cells and execute most paracrine functions,have begun to draw attention in the field(Tran and Damaser,2015).

Abstract S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma

目前麝鼠香是麝香最好的天然替代物,麝鼠香来源于麝鼠生殖系统中的香腺,已发现其香腺中分泌细胞和支持细胞是麝鼠泌香的关键.本研究通过组织形态、HE染色、免疫组织化学染色及免疫荧光鉴定方法,初步描述了麝鼠香腺的发育过程.麝鼠香腺的形态结构显示其由小到大再至非泌香期萎缩;HE染色及组化结果显示,香腺在发育初期富含颗粒饱满的腺泡,雄激素分泌处于较低水平,分泌细胞数量较少,随着进一步发育,雄激素水平及分泌细胞数量逐渐升高,在两个月时达到最高,腺泡逐渐变大成熟并开始释放麝鼠香等分泌物;在泌香末期,腺泡逐渐被结缔组织取代.以上结果将有助于麝鼠香腺发育及分泌机制的研究,为提高麝香产量及实现体外泌香建立基础.另外,我们成功分离并鉴定了分泌细胞和支持细胞,以期为后续建立体外泌香体系奠定基础.

Context Immune system has been claimed as the ‘main switch’ of tissue or organ regeneration. Among immune cells, macrophages stand out as important modulators in mutiple regeneration models, such as planarian, axolotl, mammalian hair and liver. As a unique model for mammals, deer antler is considered to ideal for studying complete mammalian organ regeneration. Studies have found that antler regeneration is a stem cell-based process and antler stem cells locate in the pedicle periosteum (PP). Although the regulatory roles of the immune system in other regeneration models have been extensively studied, they remain unstudied in antler regeneration. Aims To explore the possible role of macrophages in the PP cells (PPCs). Methods We treated PPCs with a macrophage-conditioned medium (MCM) and detected effects of MCM on proliferation, migration and apoptosis of the PPCs, and identified differentially expressed genes by using the RNA-seq technique. Key results We found that MCM enhanced proliferation rate and migration rate significantly and stimulated apoptosis of the PPCs. Using the RNA-seq technique, we identified 112 differentially expressed genes in the PPCs (38 downregulated and 74 upregulated) after the MCM treatment. Furthermore, gene-ontology annotation analyses showed that the upregulated genes were mainly involved in cell adhesion, chemotaxis, wound healing, growth factor-stimulated responses, and bone formation, and the downregulated genes were involved in regulation of biosynthesis. Conclusions MCM had a great influence on the antler stem cells, and macrophages might regulate antler regeneration through altering the microenvironment and gene-expression profiles of the PPCs. Implications We believe that the results of the present study would facilitate the discovery of the roles of immune system in antler stem cells and, thus, mammalian organ regeneration in general.

Background: Antler regeneration, a stem cell-based epimorphic process, has potential for applications as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler generation and regeneration is located in the antlerogenic periosteum (AP). However, this antler stem cell pool has not been fully characterized. Finding: We produced a comprehensive transcriptome dataset at the single-cell level for antler stem cells based on the 10x Genomics platform. We generated ~252 million sequence reads representing a large RNA-Seq dataset for 4,731 cells from an individual AP tissue sample. These cells were assigned to four significant clusters. Further screening identified 16 key stem cell markers, of which four mesenchymal (CD29, CD90, vimentin, nucleophosmin) and one embryonic (CD9) stem cell markers showed high expression levels. Our results suggest ASCs are intermediate type between embryonic and mesenchymal stem cells and will help to identify and purify specific ASC types or subtypes. Conclusion: Our results provide the first comprehensive transcriptome dataset at the single-cell level for ASCs, which may hold the key to unveil the secrets about why antlers are the only mammalian organ to fully regenerate.

细胞黏合素C(Tenascin-C)是细胞外基质糖蛋白分子,其在组织损伤时特异性瞬时表达.Te-nascin-C有着许多不同的作用,介导炎症和纤维化过程,参与组织损伤修复,动物发育过程中细胞的分裂、分化和迁移与外周神经系统的再生等.在心脏和动脉损伤、肿瘤血管生成和转移、脊髓再生、骨质疏松以及调节干细胞行为中起重要作用.论文对有关Tenascin-C的生物学功能进行综述,为相关研究提供参考.

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(Esche-richiacoli) BL21 (DE3)中诱导表达.用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔制备多克隆抗体.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.

Antler regeneration, a stem cell–based epimorphic process, has a potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10× Genomics platform (scRNA-seq). A total of 4565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASC resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs, i.e., TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017, and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1, and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.