【作者】 刘宇；

【导师】 张迎梅；

【作者基本信息】 兰州大学 ， 生物学·动物学， 2025， 硕士

【摘要】 重金属污染能够显著改变基因组内DNA甲基化水平,对基因组稳定性和基因表达造成不同程度的影响,这种影响能够持续多代,而生殖腺精巢是DNA甲基化水平受环境胁迫影响最为敏感且转录水平较无规律的组织之一。本课题组前期研究发现,在环境重金属胁迫下,雀形目鸟类树麻雀(Passer montanus)的精子发生了快速适应性进化,这种进化可能受到PIM基因家族成员PIM1的调控,而且在精巢中还发现了大量组织特异表达的复制基因家族,但驱动PIM1的差异表达以及这些基因家族的组织特异性表达的遗传机制尚不明确,环境污染导致树麻雀精子快速适应性进化的潜在分子机制也有待深入探究。基于课题组前期研究结果,本研究继续选择约有70年环境重金属污染历史的白银地区(Baiyin,BY)和被列为饮用水保护区相对无污染的刘家峡地区(Liujiaxia,LJX)作为研究样地,以两地繁殖前期的雄性树麻雀作为研究对象,检测两地树麻雀精巢重金属水平、身体状况、精子长度和精子速度等指标,分析各项指标于两地的差异性;对精巢和肌肉组织进行甲基化测序,并对精巢进行转录组测序,对比分析DNA甲基化对树麻雀复制基因家族精巢特异表达可能的调控作用,进而确定DNA甲基化对两地树麻雀生殖相关基因表达的调控;结合两地树麻雀精子形态学特征,探究调控精子快速适应性进化新的潜在分子机制。本研究主要结果如下:1.环境重金属在精巢组织的积累及其对雄性树麻雀成鸟身体状况与精子运动能力的影响。BY雄性树麻雀精巢重金属镉(Cadmium,Cd)浓度显著高于LJX(P<0.05),身体状况显著低于LJX(P<0.05),精子曲线运动速度(Curvilinear Velocity)、直线运动速度(Straight Line Velocity)和平均路径速度(Average Path Velocity)显著高于LJX(P<0.05),精子总长和鞭毛长度极显著高于LJX(P<0.001),精子的头/鞭毛比显著低于LJX(P<0.05)。表明环境重金属胁迫对雄性树麻雀身体状况产生显著不利影响,Cd能够进入精巢组织且持续积累,但精子运动能力出现适应性增强。2.树麻雀甲基组特征分析。树麻雀基因组整体胞嘧啶的甲基化水平为5.91%,CG环境下为52.96%。利用DNA甲基化数量性状位点(Methylation Quantitative Trait Loci)关联探究的甲基化区域调控的差异甲基化基因(Differentially Expressed Gene,DMG)占BY和LJX总差异甲基化基因1%。表明环境重金属胁迫是造成两地树麻雀精巢组织差异甲基化以及发挥甲基化调控的主要影响因素。3.PIM1的DNA甲基化调控。BY树麻雀精巢中PIM1表达显著升高,在其启动子区域存在长137 bp,包含17个低甲基化的Cp G位点,在肌肉中这些位点没有出现差异甲基化的情况,精巢中由这些位点组成的低甲基化区域可能与调控PIM1在精巢中表达显著升高有关。表明DNA甲基化极有可能是调控与树麻雀精子快速适应性进化相关基因PIM1表达升高的主要因素。4.DNA甲基化对顶体反应分子的调控。BY树麻雀精巢中表达显著上调的SPATA16基因是顶体形成过程中重要的调控基因,其基因体(Genebody)位置发现了4个高甲基化位点;ACR顶体酶蛋白基因编码的顶体蛋白酶是辅助精子穿透卵细胞外层的关键物质,该基因的基因体位置存在显著高甲基化的差异甲基化区域。表明DNA甲基化可能通过影响SPATA16及ACR顶体蛋白酶基因调节顶体内相关蛋白的活性和功能,进而改变精子顶体的生化特性与受精能力,实现其在顶体反应中的调控作用。5.差异甲基化基因与雄性生殖功能调控密切相关。精巢中数百个DMGs的富集结果主要包含细胞周期和增殖的精密调控、细胞外信号和基质重构、环境信号响应和分子合成与修饰,显示DNA甲基化水平的改变可能通过调控这些关键信号通路中基因的表达水平,影响细胞增殖、分化以及细胞外基质的动态重构,进而可能改变细胞对环境信号的响应。表明精巢差异甲基化基因可能调控精巢发育、影响精子形成及受精功能关键通路。6.DNA甲基化对复制基因精巢特异表达模式的潜在调控作用。树麻雀基因组内8个显著扩张的复制基因家族的启动子与7个基因体甲基化水平在精巢组织中出现了显著变化:精巢中5个复制基因家族的启动子显著低甲基化、3个显著高甲基化(P<0.05);4个基因家族的基因体区域显著低甲基化、3个显著高甲基化(P<0.05)和1个基因家族的基因体区域甲基化水平之间没有显著差异性。相同的基因家族在不同组织间的启动子DNA甲基化显著差异,表明DNA甲基化对其精巢特异表达具有潜在的调控作用。综上,本研究发现树麻雀精巢差异甲基化基因主要受环境重金属胁迫影响,且能够调控关键基因PIM1在精巢的显著高表达;研究还发现精巢调控顶体反应的基因SPATA16及ACR受到DNA甲基化的调控,并且SPATA16基因表达显著上调,虽然没有观察到顶体结构显著改变,但影响顶体功能的分子途径可能发生了变化;DMGs的基因本体论(Gene Ontology)富集与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes)通路其对生殖相关基因网络的影响,调控精子生成,以应对环境重金属胁迫。此外,DNA甲基化可能参与调控复制基因家族的精巢特异表达,其能够提供更多的遗传资源,加速精子的适应性进程。本研究结果可为探究DNA甲基化在雄性生殖中的调控作用以及环境胁迫下其适应性的调控策略提供参考。更多还原

【Abstract】 Heavy metal pollution can profoundly alter genome-wide DNA methylation levels,thereby affecting genomic stability and gene expression to varying degrees.These effects can persist across multiple generations.Among all tissues,the testicular gonad is particularly sensitive to environmental stressors in terms of DNA methylation changes and exhibits notably irregular transcriptional responses.Our previous work showed that under heavy metal stress,the sperm of the tree sparrow(Passer montanus),a member of the order Passeriformes,undergoes rapid adaptive evolution,which may be regulated by PIM1,a member of the PIM gene family.We also identified numerous tandemly duplicated gene families that are expressed in a tissue-specific manner in the testis.However,the genetic mechanisms driving the differential expression of PIM1and the testis-specific expression of these duplicate gene families remain unclear,as do the molecular pathways underlying heavy metal–induced rapid sperm adaptation in this species.Building on these earlier findings,we selected two study sites:Baiyin(BY),which has experienced approximately 70 years of environmental heavy metal contamination,and Liujiaxia(LJX),a designated drinking-water protection zone with relatively low pollution.We captured adult male tree sparrows from both sites during the pre-breeding season to compare testicular heavy metal concentrations,body condition,sperm length,and sperm velocity.We conducted whole‐genome bisulfite sequencing on testis and pectoral muscle tissues,and transcriptome sequencing on testis samples.By integrating DNA methylation and gene expression data,we assessed how methylation may regulate testis-specific expression of duplicate gene families and modulate reproductive‐related genes in both populations.Finally,we correlated these molecular findings with sperm morphological traits to explore novel mechanisms of rapid sperm adaptation.The main findings are as follows:1.Accumulation of environmental heavy metals in testicular tissue and their effects on the body condition of adult male tree sparrows and on sperm motility.Testicular cadmium(Cd)levels in BY males were significantly higher than in LJX males(P<0.05),and BY birds exhibited poorer body condition(P<0.05).Paradoxically,BY males displayed enhanced sperm performance:curvilinear velocity,straight-line velocity,and average path velocity were all significantly greater than in LJX males(P<0.05),while total sperm length and flagellum length were markedly increased(P<0.001),and the head-to-flagellum length ratio was significantly reduced(P<0.05).These findings indicate that environmental heavy metal stress exerts a significantly adverse effect on the body condition of male tree sparrows;Cd is capable of penetrating testicular tissue and continuously accumulating there,yet sperm motility exhibits adaptive enhancement.2.Methylome characteristics of tree sparrow.The overall cytosine methylation level in the tree sparrow genome was 5.91%,with 52.96%methylation in CG contexts.Through methylation quantitative trait loci apping,methylation‐regulated differentially methylated genes(DMGs)accounted for 1%of all DMGs between BY and LJX testes.This suggests that environmental heavy metal stress is the primary driver of differential testicular methylation and its regulatory effects.3.DNA methylation regulation of PIM1.PIM1 expression was significantly elevated in BY testes.In its promoter region,we identified a 137‐bp low‐methylation region containing 17 hypomethylated Cp G sites;these sites showed no differential methylation in muscle tissue.This testis‐specific hypomethylated region likely underlies the marked upregulation of PIM1 in testes,indicating that DNA methylation is a key factor in regulating this gene during rapid sperm adaptation.4.Methylation control of acrosomal reaction genes.SPATA16,a gene critically involved in acrosome formation,was significantly upregulated in BY testes and harbored four hypermethylated Cp G sites within its gene body.Likewise,the gene encoding the acrosomal protease acrosin(ACR)-essential for sperm penetration of the egg’s extracellular layers—contained a significantly hypermethylated region within its gene body.These results indicate that DNA methylation may modulate the activity and function of acrosomal proteins,thereby altering sperm biochemical properties and fertilization capacity.5.Differentially methylated genes are closely associated with regulation of male reproductive function.Enrichment analysis of hundreds of testicular DMGs revealed overrepresentation related to precise regulation of the cell cycle and proliferation,extracellular signaling and matrix remodeling,environmental signal response,and molecular synthesis and modification.This suggests that changes in DNA methylation levels may influence spermatogenesis and fertilization by modulating gene expression in these key signaling pathways,thereby potentially altering cellular proliferation,differentiation,and dynamic remodeling of the extracellular matrix,as well as cellular responses to environmental cues.Indicating that testicular DMGs may regulate testis development and affect critical pathways of sperm formation and fertilization function.6.Potential methylation‐based regulation of testis‐specific expression in duplicate gene families.Among eight significantly expanded duplicated gene families in the tree sparrow genome,promoter methylation levels changed significantly in testes for seven gene bodies:five families exhibited promoter hypomethylation and three exhibited promoter hypermethylation(P<0.05);within gene bodies,four families showed significant hypomethylation,three showed significant hypermethylation(P<0.05),and one family showed no significant difference.The same gene families displayed significant promoter methylation differences between tissues,indicating that DNA methylation may underlie their testis‐specific expression.In summary,this study demonstrates that environmental heavy metal stress drives differential DNA methylation in tree sparrow testes,significantly upregulating PIM1;reveals that DNA methylation regulates acrosomal reaction genes SPATA16 and ACR—accompanied by significant upregulation of SPATA16 expression,albeit without observable structural changes in the acrosome,suggesting altered molecular pathways affecting acrosome function;shows that DMG enrichment in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways impacts reproductive gene networks and regulates spermatogenesis in response to heavy metal stress;and suggests that DNA methylation may participate in regulating testis‐specific expression of duplicated gene families,providing additional genetic resources to accelerate sperm adaptive evolution.These results offer a valuable reference for exploring the epigenetic regulation of male reproduction and adaptive strategies under environmental stress.更多还原