Integrating Transcriptome And Metabolome To Explore The Mechanism Of Musk Secretion In Muskrat(Ondatra Zibethicus)

Muskrat(Ondatra zibethicus)has a wide distribution of resources,strong adaptability,and rapid reproduction.The muskrat scent secreted by adult males during the fragrance secretion period has similar chemical components and pharmacological effects to musk.The anatomical location,morphological structure,and activity of the muskrat’s scent glands are similar to those of the forest musk deer(Moschus berezovskii),and the ability of both to secrete scent is closely related to the level of sex hormones in the body.Studying the aroma secretion mechanism of muskrats,with their abundant resources,will help explore the mechanism of musk secretion,improve the yield and quality of musk and protect musk resources.The objective of this study was to investigate the mechanism and regulatory pathways of muskrat fragrance secretion to enhance the yield and quality of muskrat fragrance in production.To achieve this,we selected the fragrance glands and testes of 2-year-old muskrats in both non-perfumed and perfumed stages as our research subjects.We employed transcriptomics and metabolomics to conduct bioinformatics analyses of differential genes and metabolites,identifying key candidate genes,metabolites,and pathways regulating incense secretion.We also investigated the preliminary effects of key genes on muskrat fragrance secretion to reveal the underlying mechanism of muskrat incense secretion.The main findings of this study are as follows:1.Histological and morphological studies revealed significant differences in the tissue structure of gland and testis tissue between the secreting and non-secreting phases.During the perfuming stage,the length,width,thickness,and weight of gland and testicle tissue in muskrats were significantly higher than those in the non-perfuming stage(P <0.001).Adenoid cells,elliptic or fusiform nuclei,stromal cells,and epithelial cells were more developed in the secreting stage compared to the non-secreting stage.In the testis during the perfuming stage,spermatogenic cells,including mature spermatozoa,were observed,while only spermatogonocytes and primary spermatocytes were observed during the non-perfuming stage.2.Transcriptome sequencing analysis of aromatic-gland and testis tissues in muskrats during the perfumed and non-perfumed stages revealed 1,068 differentially expressed genes annotated in the aromatic-gland tissues.Of these,563 genes were significantly up-regulated and 505 genes were significantly down-regulated during the perfumed stage.In muskrat testis,there were 12,107 differentially expressed genes,with 7,249 genes significantly up-regulated and 4,858 genes significantly down-regulated during the odorant period.Fifteen candidate genes related to muskrat perfume secretion were screened in scent gland tissues,including Acacb,Fads2,Alox,Pla2g4,Calm,Msmo1,Cacna1h,Cacna1s,Cyp2j,Krt19,Akr1b,Aldh9a1,Aldh3,Adh1 and Lipa.In testicular tissue,seven key genes regulating the synthesis and function of sex hormones in muskrats were obtained,including Itpr1,Plcb2,Prkca,Srd5a Sult2b,Cyp11a and Hsd17b1.3.Metabolomics sequencing and analysis of scent gland and testis tissues during the musk-secreting and non-musk-secreting periods revealed a total of 1,451 metabolites in the testis and scent gland tissues.In the scented gland tissues,248 metabolites were significantly different during different periods.In the testis,there were 184 metabolites with significant differences during different periods.Among them,the level of the metabolite muscone was significantly up-regulated in the scent gland tissue during the secretion period.4.Transcriptomics and metabolomics correlation analysis of the scent gland suggested that cholesterol metabolism,unsaturated fatty acid synthesis,and fatty acid metabolism pathways are important regulatory pathways for musk secretion.At the same time,during the scenting period,the expression levels of phosphatidylethanol,dehydroepiandrosterone sulfate,and testosterone in the testis were significantly increased.The expression level of 11-deoxycortisol in the scent gland decreased during the scenting period,while the levels of arachidonic acid,cholesterol,dehydroepiandrosterone,cortisol,androstane-17-one,and estriol were significantly increased.At the metabolite level,it was shown that the testis regulates the secretion of muskrat in the scent gland tissue through candidate genes(Itpr1,Plcb2,Prkca,Hsd11b2 and Cyp11a).In conclusion,this study used metabolomics and transcriptomics for association analysis to jointly explore the mechanism of muskrat incense secretion and the regulation mechanism of the testis on incense gland tissue at the gene transcription and metabolism levels.The key genes affecting the synthesis and secretion of the key components and sex hormones of muskrat were screened and identified.This study provides a scientific basis for using muskrat as a model animal to replace forest musk deer,an endangered animal,and further supports the exploration of other odorous glandular mammals,such as civets and beavers,was further accelerated.