Animals
Three adult male muskrats were obtained in the breeding season (April) and three in the non-breeding season (November) from Xinji Jinmu Muskrat Breeding Farm, Hebei, China. The six individuals were similar in size and weight. The musk glands were collected from one adult male musk deer dead of an accident (in July) at Fengxian Musk Deer Breeding Farm, Shanxi, China. The musk gland samples of the small Indian civet were collected from one adult male individual dead of an accident (in June) at Hengshan Wild Animal Breeding Farm, Anhui, China. All animals were treated in accordance with the National Animal Welfare Legislation. All experimental procedures were carried out in accordance with the guidelines established by the Beijing Forestry University. After fixation, the musk glands and testes were kept in 70% alcohol until used for immunohistochemistry. The lengths of the muskrats’ musk glands were measured and recorded. The contralateral musk gland of each muskrat was divided into small pieces and some of them were kept in liquid nitrogen for RNA-seq analysis and Western blotting test. The rest of the pieces were kept in ribonuclease inhibitor at 4 °C. Blood was centrifuged at 3000g for 20 min to separate serum from blood cells, which were collected and stored at −20 °C until used for hormone analysis.
Musk secretion weight measurement
Ten adult male muskrats were selected for musk secretion measurement. The total musk weight in the breeding season of the selected muskrats was recorded, beginning on March 1. The measurement was made 3 times per half month. Measurements during the non-breeding season were taken in the same way, beginning on October 15.
Histology
The musk gland and testis samples were dehydrated in an ethanol series and embedded in paraffin wax. Serial sections (4–6 μm) were mounted on slides coated with APES (3-aminopropyl-triethoxysilane). Some sections were stained with haematite hematoxylin (Solarbio) for observations of general histology.
Immunohistochemistry
Serial sections of musk gland were incubated with primary polyclonal antibody (200 μg/ml, 1:200 dilution) against AR (Abcam) for 12 h at 4 °C. Serial sections of testis were incubated with primary polyclonal antibody (200 μg/ml, 1:200 dilution) against StAR (Santa Cruz Biotechnology), P450scc (Abcam) or 3β-HSD (Abcam) for 12 h at 4 °C. The sections were then incubated with a second antibody, goat anti-rabbit IgG conjugated to biotin and to peroxidase with avidin, using a rabbit ExtrAvidin staining kit (ZSGB-BIO), followed by visualizing with 0.5 mg 3,3-diaminobenzidine (Solarbio) solution in 1 ml of 0.05 M Tris–HCl buffer, pH 7.6, plus 0.4 μl H2O2.
Western blotting
The musk gland tissues were kept at −80 °C. The samples were from three individuals in April and another three in November. Take approximately 0.1 g tissue from each individuals. Homogenize the tissue in a homogenizer containing 300 μl of 10 mg/ml PMSF stock and incubated on ice for 30 min while maintaining the temperature at 4 °C throughout all the procedures. Take 20 μl protein sample mixed with 5 μl loading buffer (final concentration: 32 mM Tris–HCl, pH 6.8, 12.5% glycerol (v/v), 1% SDS, and 31 μM β-mercaptoethanol) and denature it at 100 °C for 5 min. Separate the samples and marker (Fermentas, 10–170 kDa) on 12% polyacrylamide gels, and transferred onto PVDF membranes. The membranes were blocked in 5% non-fat dry milk and incubated with primary antibodies (rabbit anti-rat AR, 200 μg/ml, 1:2000 dilution) at room temperature for 60 min, washed in 0.1% Tween-20 containing buffer. Secondary incubation of the membrane was then carried out using a 1 mg/ml, 1:40000 dilution of goat anti-rabbit IgG tagged with alkaline phosphatase for 60 min.
Hormone measurements
Serum testosterone was assayed by use of a testosterone ELISA kit (BNIBT). The operation was conducted according to the specification.
RNA isolation and reverse transcription
The musk gland tissues were kept in RNA Fixer (Biomarker technologies, China) at 4 °C. The samples were from three individuals in April and another three in November. Total RNA was isolated using Trizol reagent (Qiagen, USA) according to manufacturer recommendations. RNA of checked quality was reverse transcribed into complementary DNA (Omniscript RT Kit, Qiagen, USA) following the manufacturer’s protocol.
RT-PCR
The PCR conditions were 94 °C for 3 min, followed by 33 cycles at 94 °C for 30 s, 55 °C for 20 s, and 72 °C for 20 s using a melting curve program (increasing the temperature from 55 to 95 °C at 0.5 °C per 10 s) and continuous fluorescence measurement. The PCR primers used in this experiment were 5′-gagacagagtggacgggat-3′ and 5′-ggaggttacaccaaaggg-3′. Transcription of GAPDH gene was used as a reference. PCR products were electrophoresed on 1.0% agarose gels.
RNA-seq analysis
Total RNA was isolated from musk gland tissues using Trizol reagent (Qiagen, USA), the quality of RNAs was determined by gel electrophoresis and spectrophotometry. Approximately 20 μg of total RNAs from two individuals in each season (April and November) was used for Illumina sequencing at Biomarker technologies (Beijing, China). All procedures for cDNA library construction were performed via the standard Illumina sample preparation protocol. Sequencing of the purified libraries were carried out on an Illumina GA-II (Illumina Inc., USA).
Statistical analysis
In this study, the statistical comparisons were made with the Student t test and One-way analysis of variance.
