Musk Gland Research Articles

Single-nucleus multiomics unravels the genetic mechanisms underlying musk secretion in Chinese forest musk deer (Moschus berezovskii)

Musk secreted by the musk glands in male forest musk deer (FMD; Moschus berezovskii) is highly valued for its pharmaceutical and perfumery applications. However, the regulatory mechanisms underlying musk secretion are not well understood. This study aimed to investigate the genes and transcription factors involved in musk secretion across different periods and ages. We analyzed the musk glands of adult male FMD during the non-secretory and secretory periods, as well as juvenile and adult male FMD during the secretory period, using single-cell multiome ATAC+gene expression technique. Our analysis identified 13 cell types, including acinar cells of Types 1 and 2. Chromatin accessibility analysis and gene expression data confirmed that the genes Map3k2, Hsd17b12, and Jun are critical for musk secretion. Additionally, EHF, NR4A2, and FOXO1 proteins play crucial regulatory roles. Weighted gene co-expression network analysis (WGCNA) highlighted the importance of GnRH signaling pathway in musk secretion. Gene set enrichment analysis (GSEA) showed that the steroid hormone biosynthesis pathway is notably enriched in acinar cells. Furthermore, intercellular communication appears to influence both the initiation and maintenance of musk secretion. These findings provide valuable insights into the molecular pathways of musk secretion in FMD, offering potential avenues for increasing musk production and developing treatment for inflammation and tumors.

Analysis and comparison of blood metabolome of forest musk deer in musk secretion and non-secretion periods

Musk is an important animal product, but the musk secretion mechanism of forest musk deer (Moschus berezovskii) is still unclear. The musk synthesis process in forest musk deer is extremely complex, and many raw materials are directly or indirectly derived from forest musk deer blood. In this study, metabolomics was used to analyze the blood of forest musk deer in secretory and non-secretory phases for the first time, aim at explaining the secretion mechanism from the perspective of blood metabolism. We found that P450-related, choline-related, axonal regeneration and other pathways and related metabolites were significantly enriched during the musk secretion of forest musk deer. These pathways and metabolites related to P450 and choline in blood may have important implications for the mechanism of musk secretion in forest musk deer, because blood components were closely related to musk components and could provide raw materials for musk synthesis in musk gland cells.

Single-nucleus transcriptomics and chromatin accessibility analysis of musk gland development in Chinese forest musk deer (Moschus berezovskii).

Musk secreted by male forest musk deer (Moschus berezovskii) musk glands is an invaluable component of medicine and perfume. Musk secretion depends on musk gland maturation; however, the mechanism of its development remains elusive. Herein, using single cell multiome ATAC + gene expression coupled with several bioinformatic analyses, a dynamic transcriptional cell atlas of musk gland development was revealed, and key genes and transcription factors affecting its development were determined. Twelve cell types, including two different types of acinar cells (Clusters 0 and 10) were identified. Single-nucleus RNA and single-nucleus ATAC sequencing analyses revealed that seven core target genes associated with musk secretion (Hsd17b2, Acacb, Lss, Vapa, Aldh16a1, Aldh7a1, and Sqle) were regulated by 12 core transcription factors (FOXO1, CUX2, RORA, RUNX1, KLF6, MGA, NFIC, FOXO3, ETV5, NR3C1, HSF4, and MITF) during the development of Cluster 0 acinar cells. Kyoto Encyclopedia of Genes and Genomes enrichment showed significant changes in the pathways associated with musk secretion during acinar cell development. Gene set variation analysis also revealed that certain pathways associated with musk secretion were enriched in 6-year-old acinar cells. A gene co-expression network was constructed during acinar cell development to provide a precise understanding of the connections between transcription factors, genes, and pathways. Finally, intercellular communication analysis showed that intercellular communication is involved in musk gland development. This study provides crucial insights into the changes and key factors underlying musk gland development, which serve as valuable resources for studying musk secretion mechanisms and promoting the protection of this endangered species.

Genetic and histological relationship between pheromone-secreting tissues of the musk gland and skin of juvenile Chinese forest musk deer (Moschus berezovskii Flerov, 1929).

BACKGROUND: The musk glands of adult male Chinese forest musk deer (Moschus berezovskii Flerov, 1929) (FMD), which are considered as special skin glands, secrete a mixture of sebum, lipids, and proteins into the musk pod. Together, these components form musk, which plays an important role in attracting females during the breeding season. However, the relationship between the musk glands and skin of Chinese FMD remains undiscovered. Here, the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing (RNA-seq), and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin. METHODS: A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted. Then, based on the anatomical structure of the musk gland, skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD. These tissues were used for RNA-seq, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod, while juvenile FMD did not have any well-developed musk pods. Transcriptomic data revealed that 88.24% of genes were co-expressed in the skin and musk gland tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis found that the genes co-expressed in the abdomen skin, back skin, and musk gland were enriched in biological development, endocrine system, lipid metabolism, and other pathways. Gene Ontology (GO) enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division. Moreover, the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific. qRT-PCR and WB revealed that sex-determining region Y-box protein 9 (Sox9),Caveolin-1 (Cav-1), andandrogen receptor (AR) were expressed in all three tissues, although the expression levels differed among the tissues. According to the IHC results, Sox9 and AR were expressed in the nuclei of sebaceous gland, hair follicle, and musk gland cells, whereas Cav-1 was expressed in the cell membrane. CONCLUSIONS: The musk gland of Chinese FMD may be a derivative of skin tissue, and Sox9, Cav-1, and AR may play significant roles in musk gland development.

Integrated multi-omics analysis reveals insights into Chinese forest musk deer (Moschus berezovskii) genome evolution and musk synthesis.

Among the artiodactyls, male animals belonging to the Family Moschidae have a unique tissue, the musk gland, with the capability of musk synthesis. However, the genetic basis of musk gland formation and musk production are still poorly understood. Here, musk gland tissues from two juvenile and three adult Chinese forest musk deer (Moschus berezovskii) were utilized to analyze genomic evolution events, evaluate mRNA profiles and investigate cell compositions. By performing genome reannotation and comparison with 11 ruminant genomes, three expanded gene families were identified in the Moschus berezovskii genome. Transcriptional analysis further indicated that the musk gland displayed a prostate-like mRNA expression pattern. Single-cell sequencing revealed that the musk gland is composed of seven distinguishable cell types. Among them, sebaceous gland cells and luminal epithelial cells play important roles in musk synthesis, while endothelial cells master the regulation of cell-to-cell communication. In conclusion, our study provides insights into musk gland formation and the musk-synthesizing process.

Sexual dimorphism in Louisiana pine snakes (Pituophis ruthveni).

The Louisiana pine snake, Pituophis ruthveni, is a cryptic, federally threatened snake species with several fragmented populations in Louisiana and Texas, USA. There are currently four captive breeding populations in zoos in the USA; however, little scientific data exists on their life history and anatomy. Accurate sex determination and identification of normal reproductive anatomy are an essential part of a veterinary exam and conservation programs. The authors had encountered various cases of sex misidentification in this species that were attributed to lack of lubrication of the sexing probes and enlarged musk glands. Anecdotal observation led to a hypothesis of sexual dimorphism based on body and tail shape. To test this hypothesis, we measured body length, tail length and width, and body to tail taper angle in 15 P. ruthveni (9 males and 6 females). We also obtained tail radiographs of all animals to document the presence of mineralized hemipenes. Significant dimorphism was identified in relative tail length, width, and taper angle; females consistently exhibited a more acute taper angle. Contrary to previous studies in other Pituophis species, a male-biased sexual size dimorphism was not identified. Mineralized hemipenes were confirmed in all males (a newly described trait in this species), and we found that the lateral view was consistently more reliable for identification of hemipenes compared to the ventrodorsal view. This information contributes to the scientific community's understanding of this species and is of use to biologists and veterinarians working towardconservation of this threatened species.

Forest musk deer (Moschus berezovskii) in China: research and protection

The forest musk deer (Moschus berezovskii) is an endangered artiodactyl species. Males have a musk gland that secretes musk. Musk is the raw material for many medicines and has high economic value. In recent years, because of the impacts of illegal trade and habitat fragmentation, the wild forest musk deer has nearly been driven to extinction, and it has been listed as a key protected animal by many countries. Since 2002, the Chinese government has listed wild forest musk deer populations as first-class nationally protected animals and has conducted many artificial breeding studies. In this article, we review and summarise the biological characteristics and protective measures of wild forest musk deer, the musk synthesis mechanisms and the factors influencing musk yield. We also discuss the problems facing forest musk deer conservation and the development of musk-related medicines.

Single-nucleus RNA and ATAC sequencing uncovers the molecular and cellular characteristics in the musk gland of Chinese forest musk deer (Moschus berezovskii).

The Chinese forest musk deer (FMD; Moschus berezovskii) is an endangered artiodactyl mammal. Musk secreted by the musk gland of male has extremely high economic and medicinal value. However, the molecular and cellular characteristics of the musk gland have not been studied. Here, we investigated the diversity and transcriptional composition of musk gland cell types and the effect of cell type-specific chromatin accessibility on gene expression using single-nucleus RNA sequencing (snRNA-seq) and single-nucleus ATAC sequencing (snATAC-seq) association analysis. Based on uniform manifold approximation and projection (UMAP) analysis, we identified 13 cell types from the musk gland, which included two different acinar cells (cluster 0 and cluster 10). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that many pathways related to musk secretion were enriched in acinar cells. Our analysis also revealed acinar cell core transcription factors and core target genes, and further constructed acinar cell-specific regulatory networks. In cluster 0, 11 core target genes (Nedd4l, Adcy9, Akr1c1, Vapb, Me1, Acsl1, Acss3, Srd5a1, Scnn1a, Acadm, and Nceh1) possibly related to musk secretion were regulated by 24 core transcription factors (SP3, NFIC, NR6A1, EHF, RUNX1, TFAP2A, RREB1, GRHL2, NFIB, ELF1, MAX, KLF5, REL, HES1, POU2F3, TFDP1, NR2C1, ATF7, MEIS1, NR4A2, NFIA, PBX1, ZNF652, and NFKB1). In cluster 10, four core target genes (Akr1c1, Pcca, Atp1b1, and Sgk1) possibly related to musk secretion were regulated by 10 core transcription factors (BARX2, EHF, PBX1, RUNX1, NFIB, FOXP1, KLF3, KLF6, ETV6, and NR3C2). Moreover, the credibility of snRNA-seq and snATAC-seq data was verified by fluorescence in situ hybridization and immunohistochemistry. Finally, cell communication analysis demonstrated that the two types of acinar cells mainly have communications in musk secretion-related processes. In conclusion, we provided important insights and invaluable resources for the molecular and cellular characteristics of the musk gland, which will lay a foundation for the study of musk secretion mechanism in the future.

Differential expression profiles of microRNAs in musk gland of unmated and mated forest musk deer (Moschus berezovskii).

BackgroundThe formation of musk is a complex biophysical and biochemical process that change with the rut of male forest musk deer. We have reported that the mating status of male forest musk deer might result to the variations of chemical composition and microbiota of musk and its yields. Critical roles for microRNAs (miRNAs) of multi-tissues were profiled in our previous study; however, the role for miRNAs of the musk gland remains unclear in this species.MethodsIn this study, we used Illumina deep sequencing technology to sequence the small RNA transcriptome of unmated male (UM) and mated male (UM) of Chinese forest musk deer.ResultsWe identified 1,652 known miRNAs and 45 novel miRNAs, of which there were 174 differentially expressed miRNAs between UM and MM. chi-miR-21-5p, ipu-miR-99b and bta-miR-26a were up-regulated in UM among the 10 most differentially expressed miRNAs. Functional enrichment of the target genes showed that monosaccharide biosynthetic process, protein targeting, cellular protein catabolic process enriched higher in MM. Meanwhile, structural molecule activity, secretion by cell, regulated exocytosis and circulatory system process enriched more in UM, hinting that the formation of musk in UM was mediated by target genes related to exocytosis. The miRNA-mRNA pairs such as miR-21: CHD7, miR143: HSD17B7, miR-141/200a: Noc2 might involve in musk gland development and musk secretion, which need to be verified in future study.

Androgen plays an important role in regulating the synthesis of pheromone in the scent gland of muskrat

The scent (musk) gland is an organ unique to muskrats and other scent-secreting animals, and the pheromones (musk) synthesized and secreted by the scent gland play a role in chemical communication among scent-secreting animals. The musk gland is synchronized with testicular developmental changes; however, little is known regarding androgen secretion from the testis and how this regulates pheromone synthesis and the secretion of scent. To investigate the effect of androgens on the synthesis of pheromones in the musk gland, we established a muskrat castration model by surgical removal of the testis, and analyzed the histomorphology, hormone concentration, gene expression, and changes in the chemical composition of the musk gland in castration and control groups by histomorphological analysis, Enzyme-Linked ImmunoSorbent Assay (ELISA), RNA sequencing (RNA-seq), and gas chromatography–mass spectrometry (GCMS). Histomorphological analysis results showed that after castration, muskrat gland cells underwent significant atrophy (P < 0.05). Hormone measurement results showed that there was a significant decrease in serum testosterone and muskrat musk testosterone (P < 0.05) after muskrat castration. Transcriptome sequencing results showed that 510 differentially expressed transcripts (DETs) were mainly enriched in fatty acid metabolism, terpenoid backbone biosynthesis, fatty acid degradation, PPAR signaling pathway, and fatty acid biosynthesis. GCMS results showed that macrocyclic ketones, steroids, fatty acids, alcohols, and esters in musk were significantly changed (P < 0.05). In conclusion, androgens were found to play an important function in the chemical communication exchange between muskrats through regulating pheromone synthesis in musk cells. This study provides a basis for understanding the mechanism of animal communication influenced by androgens.

Characteristics of steroidogenesis-related factors in the musk gland of Chinese forest musk deer (Moschus berezovskii)

Musk secreted by Chinese forest musk deer (FMD; Moschus berezovskii) is a highly valuable ingredient in the fields of perfumery and medicine, and the main factor affecting the production of musk is the androgen level of male FMD. To clarify whether the musk gland of FMD can synthesize androgen, we compared and analyzed the expression patterns of steroid hormone biosynthesis-related genes in the musk gland and testis of FMD by RNA-seq and RT-qPCR. We obtained 33,308 and 38,602 unigenes from the musk gland and testis, respectively, and 26,780 co-expressed unigenes. Analysis of co-expressed genes revealed that 12,647 genes were annotated to 11,484 Gene Ontology terms and 10,941 genes were annotated to 6120 pathways, including several pathways important in metabolic and synthetic activity. Next, 21 steroid hormone synthesis-related genes were screened from the transcriptome of the musk gland of 4-month-old FMD. The expression levels of three key genes of steroid hormone biosynthesis (CYP11A1, CYP17A1, and HSD3B) in the musk gland differed from their expression levels in the testis based on RT-qPCR. Furthermore, immunohistochemistry indicated that CYP11A1, CYP17A1, and HSD3B were localized in the glandular tubular columnar cells of the musk gland. These results suggested that the musk gland of male FMD has the potential to locally synthesize steroid hormone and thus plays a critically important role in musk secretion.

The transcriptome analysis of males musk gland in Moschus berezovskii (Artiodactyla: Moschidae)

The dwarf musk deer, or Chinese forest musk deer (Moschus berezovskii) is an endangered artiodactyl species native to a narrow region in southern and central China as well as northernmost Vietnam. This species is well known for the habit of male musk deer to secrete musk from the musk gland. Unmated male forest musk deer secrete a greater amount of musk than mated males, which probably allows them to attract a greater number of females. However, transcriptome data for this species are deficient, and the potential transcriptional pathways of musk production remain unknown. In this study, we used RNA-sequencing (RNA-Seq) to perform an in-depth survey of transcriptome data from musk gland and to compare the transcriptome differences between mated- and unmated sexually mature Chinese forest musk deer. There were 13,009 and 12,995 genes were expressed in unmated and mated musk glands tissue. In addition to analyzing these genes, we analyzed the differentially expressed genes in musk gland tissue between mated and unmated Chinese forest musk deer males. One hundred and thirty seven genes were found differentially expressed. We found that some genes in “PPAR signaling pathway”, “PI3K-Akt signaling pathway” and “Thyroid hormone synthesis” pathways may be associated with musk production and the secretion of the musk deer. The annotated genes and differentially expressed genes in this study can be applied in conservation or utilization study of musk deer in the future.

Microbiota Changes in the Musk Gland of Male Forest Musk Deer During Musk Maturation.

The musk gland in an adult male forest musk deer is an organ that synthesizes, stores, and secretes musk, a cream-colored liquid upon initial secretion that gradually transforms into a blackish-brown solid substance upon full maturation. In this study, four healthy adult male forest musk deer were selected and a total of 12 musk samples were collected for analysis. The samples were in three different states depending on the different seasonal collection dates, which were in June, August, and October. High-throughput 16S-rRNA gene sequencing technology was used to detect microbiota changes in the gland. The results indicate that microbial richness gradually declined during the musk maturation process. The microbiota composition between the initial liquid and final solid musk samples was varied significantly (P < 0.05). The dominant bacterial phyla were similar at all three stages included Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. However, the abundances were differences in terms of the dominant bacterial genera. PICRUSt analysis showed the highest represented category was “Amino acid transport and metabolism” (24.8%), followed by “Transcription” (22.04%), and “Carbohydrate transport and metabolism” (20.74%). Our findings indicate that the microbiota in the musk gland plays an important role in the maturation process of musk.

Preparation of Musk-Smelling Macrocyclic Lactones from Biomass: Looking for the Optimal Substrate Combination.

Macrocyclic musk belongs to a well-known and valued class of the fragrance family. Originally, natural musks were obtained from rectal musk glands which often led to the death of the animals. Recently, a lot of effort was invested to obtain such macrocycles in a synthetic way. This research presents a study on the preparation of macrocyclic lactones with the musk scent by ring-closing metathesis (RCM) using biomass-derived starting materials: oleic and 9-decenoic acid. An experimental rule correlating the C-C double bond substitution pattern in the starting diene and the yield for the RCM macrocyclization was proposed.

Isolation, primary culture, and morphological characterization of gland epithelium from forest musk deer (Moschus berezovskii).

Research of epithelial cells in musk gland is lacking. There are no good characterized epithelial cell lines that can provide complementary in vitro models for in vivo research. We successfully cultivated epithelial cells of musk gland for the first time. The protocol described here produces epithelial cell lines from the mature secreting musk gland. Based on morphological observation, epithelial cells of musk gland were isolated and cultured in vitro. After the third passage, the musk gland-derived cells were filled with many lipid droplets and proliferated well. We used gas chromatography and mass spectrometry to explore the chemical composition of lipid droplets in the musk gland-derived cells. The main components of secreted lipid droplet were alkanes, esters, amines, alcohols, ketones, organic acids, and aldehydes. Muscone, which is the main active compound of musk, was not found. This is a new attempt in the field of animal musk to obtain naturally secreted animal musk in vitro by cloning specialized cells. In conclusion, this study provides a reference at the cellular level to further analyze the biology and physiology of the musk gland epithelium and secretion mechanism of musk deer.

Illumina-based de novo transcriptome sequencing and analysis of Chinese forest musk deer.

The Chinese forest musk deer (Moschus berezovskii Flerov) is an endangered artiodactyl mammal. The musk secreted by sexually mature males is highly valued for alleged pharmaceutical properties and perfume manufacturing. However, the genomic and transcriptomic resources of musk deer remain deficiently represented and poorly understood. Next-generation sequencing technique is an efficient method for generating an enormous amount of sequence data that can represent a large number of genes and their expression levels. In the present study, we used Illumina HiSeq technology to perform de novo assembly of heart and musk gland transcriptomes from the Chinese forest musk deer. A total of 239,383 transcripts and 176,450 unigenes were obtained, of which 37,329 unigenes were matched to known sequences in the NCBI nonredundant protein (Nr) database; 31,039 unigenes were assigned to 61 GO terms, and 11,782 to 332 KEGG pathways. Additionally, 592 and 2282 differentially expressed genes were found to be specifically expressed in the heart and musk gland, respectively. The abundant transcriptomic data generated in the present report will provide a comprehensive sequence resource for Chinese forest musk deer as well as lay down a foundation which will help in accelerating genetic and functional genomics research in this species.

Anthropogenic influences on the distribution of a Vulnerable coniferous forest specialist: habitat selection by the Siberian musk deerMoschus moschiferus

AbstractThe Siberian musk deerMoschus moschiferus, categorized as Vulnerable on the IUCN Red List, is a small ungulate associated with coniferous forests of East Asia. In Russia the species is hunted both legally and illegally for the commercially valuable musk gland in males. Steep population declines recorded in recent decades have been generally attributed to intensive illegal hunting, but the decline has coincided with increased logging activity and the concomitant expansion of logging roads. We conducted an occupancy analysis in Primorskii Krai, Russia, to elucidate the relative importance of environmental, ecological and anthropogenic features associated with the presence of musk deer. The top model contained covariates related to the abundance of bearded lichenUsneaspp., the distance to a main road and the distance to logging sites, suggesting that both intensive hunting of musk deer (associated with greater accessibility via roads) and logging of habitat are influencing the occurrence of this species. We propose several management actions to limit the negative influence of logging and logging roads on musk deer in Russia, including encouraging logging companies to set aside high conservation value forests (to retain intact forests) and to close logging roads post-harvest (to reduce access by poachers).

Musk gland seasonal development and musk secretion are regulated by the testis in muskrat (Ondatra zibethicus)

BackgroundThe muskrat is a seasonal breeder. Males secrete musk to attract females during the breeding season. The testosterone binding to the androgen receptor (AR) in musk glands of muskrat may play an important role conducting the musk secretion process.MethodsThe musk gland, testis and blood samples of musk rats are collected in both breeding and non-breeding seasons. Some part of the samples are kept in liquid nitrogen for transcriptome analysis and Western blotting test. Some part of the samples are kept in 70% alcohol for histology experiment, blood samples are kept at −20 °C for the serum testosterone measurement experiment.ResultsThis study demonstrates that the quantity of secreted musk, the volume of the musk glands, the diameter of the gland cells and AR expression are all higher during the breeding season than at other times (p < 0.01). StAR, P450scc and 3β-HSD expression in the Leydig cells of the testis were also higher during this season, as was serum testosterone. AR was also observed in the gland cells of two other musk-secreting animals, the musk deer and small Indian civet, in their musk glands. These results suggest that the testes and musk glands co-develop seasonally.ConclusionThe musk glands’ seasonal development and musk secretion are regulated by the testes, and testosterone plays an important role in the seasonal development of musk glands.

The musk chemical composition and microbiota of Chinese forest musk deer males.

Male musk deer secrete musk from the musk gland located between their naval and genitals. Unmated male forest musk deer generate a greater amount of musk than mated males, potentially allowing them to attract a greater number of females. In this study, we used gas chromatography and mass spectrometry (GC/MS) to explore musk chemical composition of the musk pods of captive mated and unmated sexually mature Chinese forest musk deer and used next-generation sequencing to intensively survey the bacterial communities within them. Analysis of the chemical composition of the musk showed that unmated males have more muscone and cholesterol. Features of the musk16S rRNA gene showed that mated Chinese forest musk deer have both a greater Shannon diversity (p < 0.01) and a greater number of estimated operational taxonomic units than unmated ones; many bacterial genera were overrepresented in unmated Chinese forest musk deer males. Members of these genera might be involved in musk odor fermentation. PICRUSt analysis revealed that metabolic pathways such as aldosterone-regulated sodium reabsorption, metabolism of terpenoids and polyketides, flavone and flavonol biosynthesis, and isoflavonoid biosynthesis were enriched in the musk of unmated Chinese forest musk deer males.

On the nose: genetic and evolutionary aspects of smell.

On the nose: genetic and evolutionary aspects of smell.