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.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: This 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 3ß-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. CONCLUSION: The 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.

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). / 中国幼年林麝（Moschus berezovskii Flerov, 1929ï¼‰é¦™è ºå’Œçš®è‚¤åˆ†æ³Œä¿¡æ¯ç´ çš„é—ä¼ å’Œç»„ç»‡å­¦å ³ç³».

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.

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

BACKGROUND: The 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. METHODS: In 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. RESULTS: We 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.

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.

Anatomical, Histological, and Histochemical Analyses of the Scent Glands of the Scorpion Mud Turtle (Kinosternon scorpioides scorpioides).

Fossil evidence suggests that scent glands are basal features of Testudines. However, we know little about the structure of these glands in the Brazilian Kinosternidae. In this study, we described the macroscopic anatomy, histology, and histochemistry of the scent glands of three males and three females of Kinosternon scorpioides scorpioides from the Marajó mesoregion, Pará State, Brazil. In all of the specimens analyzed, regardless of sex, we found four scent glands, including two axillary and two inguinal glands that were structurally similar to each other. Each gland consisted of a single holocrine secretory lobule, a large lumen surrounded by relatively thin glandular secretory epithelium, an adjacent narrow layer of loose connective tissue, and a thick layer of skeletal striated muscle tissue surrounded by a serous tunic. The secretory epithelium produced a characteristic malodorous yellowish substance that was passed via a single duct through a bone channel in the bridge connecting the carapace to the plastron and excreted through an outer pore in the plate of each respective gland. Histologically, the secretory epithelium presented cells with two types of secretory vacuoles. Type 1 vacuoles stained red were the largest and most frequently found, and stained positively with Periodic acid-Schiff (PAS), suggesting they contained glycoproteic complexes. Type 2 vacuoles were translucent, smaller in size and fewer in number, and negative for PAS staining. Because they are very primitive structures, scent glands must play important roles in the lives of chelonians, but their real function remains unknown. Several hypotheses suggest that they can act as protection against ectoparasites, as a repellent of predators, in addition to attracting mates and eliciting other pheromonal responses. In this study, all animals reacted by exuding malodorous substances when handled, as a form of defense. However, these are just assumptions that need to be clarified with additional studies on animal behavior. Anat Rec, 303:1489-1500, 2020. © 2019 American Association for Anatomy.

Malignant rhabdoid tumor of the musk gland and systemic T-cell lymphoma in a masked palm civet (Paguma larvata).

A 21-year-old male masked palm civet died after 2 months of continuous abdominal distention and poor appetite. Grossly, both musk glands were markedly swelled. Microscopically, round, polygonal and spindle neoplastic cells proliferated diffusely in the right musk gland and a metastatic focus was observed in the lung. The neoplastic cells had abundant cytoplasm with faintly eosinophilic inclusions that ultrastructurally corresponded to whorl aggregates of intermediate filaments. Immunohistochemically, these cells were positive for vimentin, cytokeratins and glial fibrillary acidic protein and negative for desmin. Based on these findings, the tumor was diagnosed as malignant rhabdoid tumor. Papillary adenoma was seen in the opposite musk gland. T-cell lymphoma of the lymph nodes, small intestine and liver was considered as the cause of death.

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.

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.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: This 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 3ß-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. CONCLUSION: The 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.