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.

Musk secretion weight comparison

The weight of musk that the selected muskrats secreted during the breeding and non-breeding seasons was recorded and summed for the two seasons (Fig. 1a). The total weight produced in the breeding season (17.36 ± 1.67 g in March and April) was significantly higher than that in the non-breeding season (2.16 ± 0.40 g in October and November), (p < 0.01).

The seasonal profile of musk gland and testis morphology

The lengths of musk glands during the two seasons were significantly different (Fig. 1c). It is obvious that the musk gland in the breeding (38.3 ± 4.0 mm) was much longer than in the non-breeding season (15.3 ± 6.7 mm, p < 0.01). Similar changes occurred in the testis (Fig. 1b), which was much larger in the breeding (20.6 ± 2.5 mm) than in the non-breeding season (10.7 ± 1.9 mm, p < 0.01). Morphological observations during the breeding and non-breeding seasons of musk glands and testis are compared in Fig. 1d, e. The seasonal differences were significant. Musk glands and testes were larger in the breeding season. The musk gland cells and epithelial cells become smaller in the non-breeding season. In the testis, the Leydig cells produce testosterone. The seminiferous tubules and Leydig cells were smaller in the non-breeding season. There were fewer developed spermatids in the non-breeding season.

Immunohistochemical expression of AR in the musk gland

Androgen receptor was detected in the musk gland during the breeding and non-breeding seasons by immunohistochemistry (Fig. 2a, c), and localized mainly in the glandular cells. The positive section of breeding season was more significant. The distribution of AR in muskrat musk glands was similar to that in musk deer (Fig. 2b) and in the small Indian civet (Fig. 2d).

Protein expression of AR in the musk gland

According to the Western blotting results (Fig. 2e), the positive bands of AR were shown at 92 kDa. We considered GAPDH as a reference which were shown at 37 kDa. The comparison of AR protein level in non-breeding and breeding season is significant (Fig. 2f).

Immunohistochemical detection of StAR, P450scc and 3β-HSD in the testis

StAR, P450scc and 3β-HSD were detected in testis in the breeding and non-breeding seasons (Fig. 3a–f). Positive reaction for StAR, P450scc and 3β-HSD was observed mainly in the Leydig cells in both seasons. These three factors were significantly more highly expressed in the breeding than in the non-breeding season.

The immunodetection rate was calculated as the percentage of positive Leydig cells among the total number of Leydig cells. The immunodetection rates of StAR, P450scc and 3β-HSD were all reduced in the non-breeding season (Fig. 3g).

Serum testosterone concentration

The concentrations of testosterone in the serum of muskrats in the breeding and non-breeding seasons were measured using an ELISA kit. There was a significant decrease in serum testosterone from the breeding (1.874 ± 0.167 ng/ml in April) to the non-breeding season (0.198 ± 0.0673 ng/ml in November, p < 0.01) (Fig. 3h).

AR mRNA levels in the musk gland

The expression levels of androgen receptor were assayed in 4 musk glands from 4 individuals from the breeding and non-breeding seasons (April and November) (Fig. 4a). The expression levels of AR in the breeding were significantly higher than in the non-breeding season (p < 0.01).

The expression of AR mRNA of breeding sample is stronger than that of non-breeding season sample (Fig. 4c). The comparison of AR mRNA level in non-breeding and breeding season is significant (Fig. 4d).

The antibodies used in this study were all polyclonal antibodies. Comparing the RNA-sequencing analysis results with the mRNA found in NCBI gene bank, we found that the CDS of androgen receptor mRNA from muskrat had 94.4% similarity with the CDS from rat (Rattus norvegicus). It is considered that the androgen receptors of rat and muskrat has high similarity (Fig. 4b).