Molecular Regulation Of Reproductive Suppression In Male Plateau Zokor(Eospalax Baileyi)

Plateau zokor(Eospalax baileyi)is endemic species of the Qinghai-Tibet Plateau,and its population density has increased under the influence of climate change and human activities,accelerating the degradation of grasslands and seriously affecting their production and ecological functions.After killing the plateau zokor,its population can quickly recover.Revealing the reproductive basis of plateau zokor population disaster is the scientific basis for its effective management.Reproductive suppression refers to the inhibition or damage of normal reproductive development in animals due to the impact of specific environmental factors or internal conditions,resulting in reduced or lost reproductive capacity.reproductive suppression is an adaptive strategy that affects the success and efficiency of reproduction,which in turn influence survival and evolution.In field study,it was found that some adult plateau zokors were not involved in reproduction and reproduction was inhibited in adult males.To clarify the molecular regulation mechanism of reproductive suppression in plateau zokors,this study analyzed the testicular morphology,physiological hormone and transcriptomic characteristics of normal reproductive individuals and reproductively suppressed individuals by means of histomorphology,immunohistochemistry,animal physiology and molecular biology,using non-breeding season plateau zokors as control,screened differentially expressed m RNA,mi RNA and pi RNA in testes,and explored key genes and signaling pathways related to testicular development and spermatogenesis.We combined bioinformatics to analyze the mi RNA–m RNA and pi RNA–m RNA interaction networks and understand the relevant regulatory patterns.The differentially expressed m RNA,mi RNA and pi RNA were verified by q PCR and the target genes were verified by double luciferase.The main research results are as follows:1.Reproductive suppression was observed in male plateau zokor.The proportion of reproductively suppressed individuals in undisturbed populations ranged from 5% to 5.40%.The proportion of reproductively suppressed individuals decreased or even disappeared after artificial hunting interference.The average body weight and testis weight of reproductively suppressed individuals were smaller than those of normal reproductive individuals(P<0.05).The disappearance of reproductive suppression under artificial hunting interference led to the rapid recovery of plateau zokor populations,which may be one of the reasons for the plateau zokor disaster.2.Serum testosterone levels were reduced in reproductively suppressed and non-breeding season individuals(P<0.05).In normal reproductive individuals,there were spermatogonia,primary spermatocytes,secondary spermatocytes,spermatocytes and spermatozoa in the testicular varicocele,whereas in reproductively suppressed and non-breeding season individuals,there were only spermatogonia and Sertoli cells in the varicocele.The expression of spermatogonia-specific genes was not significantly different in normal,suppressed and non-breeding season individuals,whereas the expression of specific genes was downregulated in spermatocytes and spermatozoa in suppressed and non-breeding season individuals.3.There were 12975(up-regulated 5014,down-regulated 7961),5672(up-regulated 2369,down-regulated 3303)and 15327(up-regulated 8365,down-regulated 6962)differentially expressed m RNAs;138(up-regulated 68,down-regulated 70),70(up-regulated 31,down-regulated 39),167(up-regulated 90,down-regulated 77)differentially expressed mi RNAs;17421(up-regulated 14030,down-regulated 3211),11478(up-regulated 6234,down-regulated 5244),97580(up-regulated 86433,down-regulated 11147)differentially expressed pi RNAs identified in the testes of suppressed vs.normal individuals,suppressed vs.non-breeding season individuals,and normal vs.non-breeding season individuals,respectively.The results of q PCR validation of 9 randomly selected m RNAs,9mi RNAs and 6 pi RNAs indicated that the transcriptome data were reliable.4.The Top 10 differentially expressed m RNAs revealed that AMH and its transcription factors were significantly up-regulated in the testes of suppressed individuals and non-breeding season individuals(P<0.05).The GO and KEGG enrichment analysis of differentially expressed m RNAs revealed that the genes down-regulated in the testes of suppressed individuals and non-breeding season individuals were mainly involved in biological processes related to spermatogenesis,meiosis and metabolism;they were significantly enriched in oocyte meiosis,progesterone-mediated oocyte maturation,glycerolipid metabolism,and AMPK signaling pathway related to testicular cell development and spermatogenesis.The genes up-regulated in the testes of suppressed individuals and non-breeding season individuals were mainly involved in apoptosis,cell adhesion,and response to estradiol;they were significantly enriched in Hippo signaling pathway,relaxin signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway.5.Based on mi RNA–m RNA and pi RNA–m RNA interaction network analysis,we found that the target genes of differentially expressed mi RNAs and pi RNAs are mainly involved in biological processes such as cell differentiation,lipid metabolic processes,multicellular biological development,sperm flagellar viability,sperm axoneme components,and metabolic pathways,chemokine signaling pathway,Ras signaling pathway,relaxin signaling pathway,MAPK signaling pathway,and inositol phosphate metabolism.The mi RNA–m RNA regulatory network of genes related to testis development and spermatogenesis was constructed and analyzed by q PCR and dual luciferase assay,which revealed that UBB gene was mainly expressed in primary spermatocytes during meiosis and mir-24-3p could regulate the expression of UBB.In summary,high AMH levels caused low testosterone,resulting in delayed testicular development in plateau zokors,which led to the physiological suppression of reproduction.Based on m RNA and s RNA expression profiling,it was shown that reproductively suppressed individuals and non-breeding season individuals exhibited signs of spermatogenesis being blocked at the meiotic stage at the transcriptome level,leading to impaired spermatogenesis in reproductively suppressed individuals,which may be the reason for the inability of reproductively suppressed individuals to participate in reproduction.These findings enrich the understanding of reproductive suppression in solitary mammals,fill a gap in the transcriptomics of reproductively suppressed animals,and provide a theoretical basis for further understanding the mechanisms of testicular development and spermatogenesis in plateau zokors and other reproductively suppressed animals,and provide a reference for scientific prevention and control of rodent infestation.