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

Abstract

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.

Keywords: Forest musk deer; Musk gland; Pheromones; Sebaceous gland; Skin tissues; Transcriptome.

Fig. 1. Unigene length distribution in forest musk deer (FMD). The x-axis indicates the length of sequenced unigenes and the y-axis indicates the number of sequenced unigenes.

Fig. 2. Venn diagram indicating gene expression (transcriptome datasets) in the back skin, abdominal skin, and musk gland tissue of forest musk deer (FMD). The overlapping sections indicate the genes co-expressed in different tissues.

Fig. 3. Gene Metascape enrichment in forest musk deer (FMD) musk gland tissues. The Metascape software was used to analyze gene specificity in tissue and cells.

Fig. 4. Gene Ontology (GO) functional classification of co-expressed genes. The GO database allows for the annotation of genes and gene products across three categories: biological process, molecular function, and cellular component.

Fig. 5. Histogram illustrating the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway classification of genes co-expressed in the abdominal skin, back skin, and musk gland tissues. The x-axis indicates the percentage of genes annotated to a pathway. The y-axis indicates the KEGG pathways, with different colors corresponding to the six categories in the top layer.

Fig. 6. Hematoxylin-eosin (HE) staining of the musk gland and skin tissues of 4-month-old forest musk deer (FMD). (a) General structure of the musk gland (4× objective lens). Green arrow, loose connective tissue (LCT); blue arrow, musk gland part (MGP); white arrow, acinar cavity (AC). (b) Musk gland skin junction (40× objective lens). The white arrow points to the hair follicle (HF), and the black arrow points to sebaceous gland (SG). (c) Part of musk gland (40× objective lens). The blank space represents the AC. (d) HE-stained sections of FMD skin tissue (10× objective lens): the figure shows the epidermal layer (EL), subcutaneous tissue (ST), adipose tissue (AT), and muscle tissue (MT). (e) The section of HE-stained FMD abdomen skin tissue. The black arrows indicate SG, and the white arrow points to hair shaft (HS) tissues in three different developmental stages. (f) Structure of the HS structure in FMD. The image shows the medulla (Med), cortex (Cor), and cuticle (Cut), which are pointed by black arrows. (g) The section of HE-stained FMD skin tissue. The rectangular box denotes a structural unit consisting of an SG and HF (SG-HF unit), including an SG (black arrow), HF (white arrow), and hair shaft (HS, blue arrow). (h) Microstructure of the HF (white arrow) and SG (black arrows). (i) The complete structure of the HS, including the Med, Cor, and Cut.

Fig. 7. Immunohistochemical results of sex-determining region Y box protein 9 (Sox9), Caveolin-1 (Cav-1), and androgen receptor (AR) in the abdominal skin, back skin, and musk gland tissues of 4-month-old male forest musk deer (FMD). (a‒c) Negative control; (d‒l) Expression and localization of sox9 (d‒f), Cav-1 (g‒i), and AR (j‒l) proteins in the abdominal skin (d, g, j), back skin (e, h, k), and musk glands (f, i, l). Brown color indicates a positive signal for the corresponding protein (40× objective lens). The arrows indicate positive signals.

Fig. 8. mRNA expression of Sox9 (a), Cav-1 (b), and AR (c) in the abdominal skin, back skin, and musk gland detected by qRT-PCR. The mRNA expression levels of Sox9, Cav-1, and AR were normalized based on that of GAPDH. All data were obtained using three replicates, and the results are presented as mean±SD. The bar chart superscripts indicate significant differences (^*** P<0.001), and an unpaired t-test was used for analysis. AR: androgen receptor; Cav-1: Caveolin-1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; mRNA: messenger RNA; qRT-PCR: quantitative real-time polymerase chain reaction; SD: standard deviation; Sox9: sex-determining region Y box protein 9.

Fig. 9. Protein expression of Sox9, Cav-1, and AR in the abdominal skin, back skin, and musk gland tissues. (a) Western blot for detecting Sox9, Cav-1, and AR protein expression, with β-actin as the internal reference for protein detection. (b‒d) Quantitative analyses of Sox9 (b), Cav-1 (c), and AR (d) proteins. All samples had three replicates, and the results are presented as mean±SD. The bar chart superscripts indicate significant differences (^* P<0.05, ^*** P<0.001), and an unpaired t-test was used for analysis. AR: androgen receptor;Cav-1: Caveolin-1; SD: standard deviation; Sox9: sex-determining region Y box protein 9.

Fig. 10. Schematic representation of the relationships among the back skin, abdominal skin, and musk gland of forest musk deer (FMD) at the tissue, cell, and gene levels. AR: androgen receptor; AS: abdominal skin; BS: back skin; Cav-1: Caveolin-1; HE: hematoxylin-eosin; MG: musk gland; RNA-seq: RNA sequencing; SG: sebaceous gland; Sox9: sex-determining region Y-box protein 9.