2018, 65 (11), 1111-1120 Original Sex hormones play roles in determining musk composition during the early stages of musk secretion by musk deer (Moschus berezovskii) Mengyuan Fan1) *, Meishan Zhang1) *, Minghui Shi1) *, Tianxiang Zhang1), Lei Qi1), Juan Yu2), Xuxin Li2), Shaobi Lin2), Zhixin Huang2), Shuang Yang1), Juntong Zhou1), Yimeng Li1), Xiaoning Sun1), Muha Cha1), Shanghua Xu1), Yang Liu1), Xiaobing Guo1), Defu Hu1) and Shuqiang Liu1), 2) 1) College of Nature Conservation, Beijing Forestry University, Beijing 100083, People’s Republic of China 2) Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., Fujian 363700, People’s Republic of China Abstract. Musk is a secreted external hormone or information compound that is stored in musk scent glands of the males of species within the family Moschidae, such as Moschus berezovskii. The secretion of musk changes periodically during the courtship and reproduction periods, with the early stage of secretion occurring from May to July, and the maturation stage occurring from August to April of the following year. In this study, we analyzed the dynamic changes in musk components from June to April of the following year. The result showed that musk morphological character, water content, total ion chromatographic pattern, and composition undergo seasonal change. Luminescence immunoassay and radioimmunoassay analyses were performed to determine corresponding fecal hormone levels. The results showed that testosterone, estrogen, and cortisol levels in feces change on a seasonal basis, and are significantly higher in June than in other months (p < 0.01). Correlation analysis showed that the contents of four examined musk components (muscone, cyclopentadecanone, cholesterol, and cholestenol) from June to August were significantly highly negatively correlated with fecal testosterone and estradiol levels (p < 0.01). In contrast, the correlation coefficients were low or not significant from August to April of the following year. These results indicate that testosterone and estradiol may play a major role in determining musk composition during the early stage of musk secretion but not during the course of musk maturation, which suggests that musk secretion may be promoted by increases in sex hormones in June. Key words: Musk secretion, Musk component, Sex hormone, Musk deer MUSK DEER are small Cervidae animals, species of berezovskii), the Alpine musk deer (M. sifanicus), and which include the Chinese forest musk deer (Moschus the Siberian musk deer (M. moschiferus). Due to habitat loss and indiscriminate poaching, wild musk deer popu‐ Submitted May 29, 2018; Accepted Jul. 21, 2018 as EJ18-0211 lations are currently endangered (CITES, Annex II), and Released online in J-STAGE as advance publication Aug. 31, 2018 the species has a National Class I protected status [1]. Correspondence to: Shuqiang Liu, 1 College of Nature Conserva‐ Since the 1950s, musk deer have been artificially bred in tion, Beijing Forestry University, No.35, Qinghua East Road, China in an effort to restore the populations of this mam‐ Haidian District, Beijing 100083 People’s Republic of China; 2 mal. Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., Fujian 363700, People’s Republic of China. Musk, a pheromone that is mainly used for communi‐ E-mail: [email protected] cation, is secreted from the scent glands of mature males Correspondence to: Defu Hu, No. 35, Qinghua East Road, Haidian of different species of musk deer. In the Chinese forest District, Beijing 100083, People’s Republic of China. musk deer, the oblate scent glands protrude from E-mail: [email protected]. between the abdomen and the scrotum [2, 3]. *These authors contributed equally to this work Abbreviations: GC-MS: gas chromatography-mass spectrometry; During the early phases of the musk secretion season, RIA: radioimmunoassay; RLU: relative light units; NSB: nonspe‐ the scent glands gradually enlarge, their openings turn cific binding test; IQR: interquartile range red, and musk fluid volume gradually increases. Behav‐ ©The Japan Endocrine Society 1112 Fan et al. iorally, the deer become excitable and aggressive. During restore musk secretion in old musk deer that have other‐ the peak of musk production period, males stop eating, wise lost this ability [12]. Injecting exogenous luteiniz‐ decrease water intake, and move relatively little. At this ing hormone, luteinizing hormone-releasing hormone, time, the scent glands reach maximum volume, with follicle-stimulating hormone, and human chorionic thickened walls, enlarged subcutaneous blood vessels, gonadotropin can also promote musk secretion during and substantial fluid accumulation. This musk fluid is the non-secretion season, although the effect is not as expelled through frequent contractions of the scent pronounced as that obtained with exogenous testosterone glands. Subsequently, males resume feeding, their scent [13, 14]. Significantly, however, the quantity of musk glands shrink significantly, and the production of musk secretion stimulated in response to injecting with exoge‐ fluid is considerably reduced, during which time it grad‐ nous testosterone or gonadotropic hormones is less than ually becomes brown, reddish-brown, or coffee colored 50% that of the naturally secreted musk [15], which indi‐ as the water content decreases [2, 4]. The musk secretion cates that secretion is not only affected by testosterone or season of musk deer extends from May to July, whereas other gonadotropic hormones but also involves complex during the remainder of the year, no musk is secreted. In physiological reactions regulated by a variety of hor‐ the breeding season, unmated male musk deer can use mones. Moreover, given that musk secretion undergoes musk as a pheromone to attract females [5]. periodic change concomitant with courtship and breed‐ Studies on the quantitation of musk components have ing, it is conceivable that changes in musk composition been limited by the complexity of these substances, and are related to seasonal variations in hormone levels. To those that have been undertaken have not been per‐ date, however, there have been no analyses of the corre‐ formed systematically [6]. To date, there has been no lation between hormone levels and changes in the com‐ systematic analysis that has accurately measured musk position of musk. components across the different periods of musk matura‐ Studies of musk deer hormone are, nevertheless, ham‐ tion. Classic studies on musk have indicated that the pered by difficulties in collecting multiple blood sam‐ major components are muscone, steroids, amino acids, ples, as these animals are highly alert, easily frightened, and polypeptides [7]. Subsequent research has confirmed and avoid humans [16, 17]. In recent years, non-invasive that androstane is one of the important components of sampling methods, such as the collection of feces, urine, musk [8]. Studies have also demonstrated that fatty acids saliva, and other secretions, have broadened the research and esters are other important constituents [9]. However, routes available for the study of wildlife physiology [18]. these studies have focused only on determining the For example, hormone quantitation using fresh fecal composition of mature musk and have not attempted to samples is considered to be a particularly useful techni‐ analyze immature post-secretion musk fluid or its com‐ que [19]. Such non-invasive techniques have also proven ponents. Nevertheless, the appearance and composition to be helpful in study of the Chinese forest musk deer of musk undergo many changes from the onset of secre‐ [20]. tion to final maturation, and the current lack of data on In this study, we analyzed the dynamic changes that the changes in musk components that occur during the occur in musk appearance and composition during the maturation process has hindered an accurate determina‐ course of musk maturation. In addition, we used radioim‐ tion of the dynamic changes in musk during maturation. munoassays and luminescence immunoassays to deter‐ Analysis of the chemical composition of male musk mine hormone levels in feces. Studies have identified 2- deer serum during different periods has shown that the heptanone and squalene as two of the main components content of 17-ketosteroid, a metabolite of testosterone, of pheromones in male rats [21], and that testosterone increases significantly during the incipient stages of plays a role in pheromone regulation [22]. The odorant musk secretion. Removing the testis and epididymis of a components regulated by testosterone and estradiol are normal male musk deer suppresses musk secretion, often active constituents [23]; for example, muscone and whereas the secretion ability can be restored by injection cyclopentadecanone which are ketones, and cholesterol of exogenous testosterone [2]. These findings indicate and cholestenol, which are derivatives of squalene. that the secretion of musk by musk deer is closely related These four components may play roles during male musk to endogenous testosterone levels. Notably, injections of deer breeding similar to those played by 2-heptanone and exogenous testosterone can promote musk secretion dur‐ squalene in male rats. Accordingly, in this study, we ing the non-secretion season [10, 11], and can even analyzed levels of the four musk constituents muscone, Sex hormones play roles in musk 1113 cyclopentadecanone, cholesterol, and cholestenol to into a GC-MS system (Model QP2010; Shimadzu Corp., examine their correlation with hormone levels