【作者】 张宁；

【导师】 廖明帜；

【作者基本信息】 西北农林科技大学 ， 生物信息学， 2023， 博士

【摘要】 睾丸是男性生殖系统的重要组成部分,其主要功能是产生精子和雄激素。睾丸发育不全(约占男性1/200-1/500)和男性不育是男性发病率较高的男科疾病。睾丸发育不全和男性不育主要是男性睾丸下降异常和精子发生异常导致,然而调控睾丸发育过程和精子发生的机制还有待研究。睾丸发育和精子发生是时空上精细调控的复杂过程,由多种细胞谱系参与其中。不同谱系细胞之间通过何种方式通讯,来完成其在时空上的精确调度,目前的认知还不足。睾丸的发育过程和男性生殖细胞的命运决定和分化微环境调控机制仍缺乏系统性的研究。因此,阐明正常睾丸发育和精子发生微环境以及细胞通讯的分子机制,不仅有重要科学价值,也能为了解睾丸发育不全和男性不育的发病机制以及临床治疗和诊疗奠定理论基础。多组学测序可以解析组织中细胞群体的异质性、空间位置信息、染色质可及性分布区域和鉴定细胞类型、细胞亚型及空间分布特征,研究微环境和细胞间通讯(互作),绘制细胞发育路径等工作。因此多组学整合成为研究发育生物学问题的重要技术手段。本论文将从以下4方面对睾丸微环境和细胞通讯展开研究:(1)利用空间转录组构建空间细胞通讯数据库,整合血液分泌组构建组织与男性睾丸之间互作(细胞通讯)调控网络。(2)通过scATAC-seq和scRNA-seq数据分析揭示睾丸成形过程中的微环境和染色质可及性图谱,同时构建胚胎发育不同时期睾丸的细胞通讯网络,此外整合空间转录和单细胞转录组数据构建胚胎发育早期睾丸空间细胞通讯图谱。(3)利用青春期睾丸scRNA-seq数据分析揭示精子成熟过程中睾丸微环境变化,利用细胞通讯网络挖掘青春期与精子成熟相关受体配体。(4)利用scRNA-seq数据分析揭示非梗阻性无精症(nonobstructive azoospermia:NOA)患者和正常睾丸的微环境与细胞通讯特点,获得特异性细胞亚型的转录因子调控活性,同时验证内分泌干扰物双酚A(BPA)与NOA的关系。研究结果如下:空间细胞通讯数据库构建及组织与睾丸互作研究:空间细胞通讯数据库从空间揭示组织细胞通讯。通过收集空间转录组数据分析揭示组织空间细胞通讯概况,并整合细胞通讯受体配体及其注释信息,共获得4071对受体配体对,其中人组织鉴定到3191对、小鼠鉴定到3039对。目前整理的空间组织中包括人和小鼠2种12个组织共计66659对受体配体对,其中人组织共有12837对,小鼠组织共有53822对。基于以上数据,构建空间细胞通讯平台,通过进一步整合空间细胞通讯和血液分泌组数据进行人类组织与睾丸的互作研究,共鉴定出383对睾丸与其它组织之间的互作关系对,用于揭示睾丸与其它组织的潜在交互作用。男性性腺形成过程中微环境和细胞通信分析:性腺的形成伴随着睾丸下降,微环境和细胞通讯参与睾丸下降的调控。整合人怀孕7周至19周的男性性腺单细胞转录组(scRNA-seq)和单细胞表观组(scATAC-seq)数据,探究睾丸形成过程中微环境和细胞通讯的变化。结果显示,在7周至19周发育过程中,睾丸组织中占比最大的细胞类型为间质细胞(占比52.2%),且在17周睾丸发育完成时,间质细胞占比最高(87.77%)。在睾丸发育过程中,染色质可及性峰(Peak)主要富集于细胞基因内含子区域。细胞通讯频数在第9周发生最多(共1221对),17周次之(共1207对),其中细胞通讯事件主要发生于配体为间质细胞和受体为内皮细胞的互作。结果表明性腺形成和睾丸下降与间质细胞在微环境的占比存在关联。青春期精子成熟关键受体配体挖掘:精子成熟依赖于成熟的睾丸微环境和细胞通讯。利用青春期男性(7岁、11岁、13岁和14岁)睾丸单细胞转录数据,通过睾丸微环境细胞类型分析发现7岁时睾丸中仅存在精母细胞,而不存在其他生殖细胞;11岁前睾丸中的精子尚未发育成熟。青春期性成熟伴随着睾丸微环境的巨大变化,细胞比例变化大于20%的细胞类型包括4种:内皮细胞、管周肌细胞和间质细胞、以及支持细胞的亚型,伴随着睾丸中精子发育的成熟。通过构建细胞通讯网络,生殖细胞与体细胞构建151对细胞通讯受体和配体对。发现青春期体细胞中支持细胞与生殖细胞通讯最为频繁,存在6对支持细胞与精子特异性的细胞通讯对(DCN＿AQP5、APOA1＿CXCL16、VWF＿AQP5、VWF＿CXCL16、CAV1＿CXCL16、TMSB4X＿CXCL16),并分泌促进精子成熟关键配体。睾丸微环境与男性生殖障碍:睾丸生精微环境中巨噬细胞发育与男性不育。整理正常睾丸与NOA患者的单细胞转录组数据,分析发现巨噬细胞数量存在较大差异。通过伪时间轨迹分析发现NOA睾丸中特异性巨噬细胞亚型炎症功能缺失,而正常睾丸中该细胞亚型较少或不存在。此外,正常人和NOA患者睾丸相同细胞类型的Top转录因子存在差异,BPA通过调控支持细胞配体基因(Dnc、Dusp1、Timp3、Vwf)参与精子成熟过程,同时研究表明BPA可能是引起人NOA的潜在原因。睾丸微环境细胞状态和细胞通讯是引起男性不育的因素之一。综上所述,基于多组学的睾丸微环境和细胞通讯整合研究为男性性腺发育异常和男性不育的研究提供了新的手段和视角,从发育时期和多组学层面系统、全面地阐释了睾丸发育和精子发生的异质性;推断在睾丸发育细胞和精子发生过程中微环境和细胞通讯共同调控了睾丸发育异常和男性不育,从而为睾丸发育异常和男性不育治疗方法提供了新的依据。更多还原

【Abstract】 The testes are an important part of the male reproductive system,and their main function is to produce sperm and androgens.Testicular hypoplasia(about 1/200-1/500 of men)and male infertility are male diseases with a high incidence in men.Testicular hypoplasia and male infertility are mainly caused by abnormal testicular descent and abnormal spermatogenesis in men,however,the mechanisms regulating the process of testicular development and spermatogenesis remain to be investigated.Testicular development and spermatogenesis are complex processes that are finely regulated in space and time and involve multiple cell lineages.The ways through which cells of different lineages communicate with each other to accomplish their precise scheduling in space and time are not well understood.The developmental process of the testis and the mechanisms regulating the fate determination and differentiation microenvironment of male germ cells still lack systematic studies.Therefore,elucidating the molecular mechanisms of the microenvironment of normal testicular development and spermatogenesis as well as cell communication not only has important scientific value,but also can lay the theoretical foundation for understanding the pathogenesis of testicular dysplasia and male infertility as well as clinical treatment and therapy.Multi-omics sequencing can resolve heterogeneity,spatial location information,chromatin accessibility distribution regions and identify cell types,cell subtypes and spatial part characteristics in tissue cell populations,study microenvironment and intercellular communication(interactions),and map cell developmental pathways,among other tasks.Thus multi-omics integration becomes an important technical tool to study developmental biology problems.In this thesis,we will investigate the testis microenvironment and cellular communication from the following aspects:(1)constructing a spatial cellular communication database using the spatial transcriptome,and constructing a regulatory network of interactions(cellular communication)between tissues and male testes in combination with the blood secretome.(2)To reveal the microenvironment and staining accessibility during testis formation by scATAC-seq and scRNA-seq,and to construct the cellular communication network of testis at different stages of embryonic development,in addition to integrating spatial transcription and single cell transcriptome data to construct a spatial cellular communication map of testis at 14 weeks of embryonic development.(3)Using pubertal testis scRNA-seq data to reveal changes in testis microenvironment during sperm maturation,and mining receptor ligands associated with sperm maturation during puberty by constructing cellular communication networks.(4)To use scRNA-seq data to reveal the microenvironmental and cellular communication characteristics of non-obstructive azoospermia(NOA)patients and normal testes,analyze the transcription factor regulatory activity of specific cellular subtypes,and also verify the relationship between endocrine disruptor bisphenol A and NOA.The results of the study were as follows:Spatial cell communication database construction and tissue-testis interactions study: spatial cell communication database from spatial response to tissue cell communication.By collecting spatial transcriptome data to reveal tissue spatial cell communication profiles and integrating cell communication receptor ligands and their annotation information,a total of 4071 receptor ligand pairs were obtained,among which 3191 pairs were identified in human tissues and 3039 pairs in mice.A total of 66659 pairs of receptor ligands were collated from12 tissues of 2 species,human and mouse,of which 12837 pairs were identified from human tissues and 53822 pairs from mouse tissues.Based on the above data,a spatial cell communication platform was constructed,and a total of 383 pairs of interactions between testis and other tissues were identified by further integrating spatial cell communication and blood secretome data for the study of interactions between body tissues and testis,which was used to reveal the potential interactions between testis and other tissues.Analysis of microenvironment and cellular communication during male gonad formation: gonad formation is accompanied by testicular descent,and microenvironment and cellular communication are involved in the regulation of testicular descent.Single-cell transcriptome(scRNA-seq)and single-cell epigenome(scATAC-seq)data of male gonads from 7 weeks to 19 weeks of human pregnancy were integrated to explore changes in microenvironment and cell communication during testis formation.The results showed that the cell type with the largest proportion of testicular tissue during development from 7 weeks to 19 weeks was mesenchymal cells(52.2%),and the highest proportion of mesenchymal cells(87.77%)was observed at the completion of testis development at 17 weeks.During testis development,chromatin accessibility peaks(Peaks)were mainly enriched in cytogenetic intronic regions.The highest frequency of cell communication occurred at week 9(1221 pairs in total),followed by week 17(1207 pairs in total),where cellular communication events occurred mainly in ligands as mesenchymal and receptors as endothelial cell interactions.The results suggest a correlation between gonad formation and testicular descent and the percentage of mesenchymal cells in the microenvironment.Key receptor-ligand mining for sperm maturation during puberty: sperm maturation is dependent on a mature testicular microenvironment and cellular communication.Single-cell transcriptional data from testes of adolescent males(7,11,13 and 14 years old)were collected,and analysis of cell types in the testicular microenvironment revealed that only spermatocytes and no other germ cells were present in the testes at age 7;spermatozoa in the testes were immature by age 11.Sexual maturation during puberty is accompanied by dramatic changes in the testicular microenvironment,and cell types with a change in cell proportions greater than 20% include four types: endothelial cells,peritubular myocytes and mesenchymal cells,and subtypes of supporting cells,when mature spermatozoa are present in the testis.By constructing a cellular communication network,germ cells construct 151 pairs of cellular communication receptor and ligand pairs with somatic cells.Six pairs of cellular communication pairs(DCN＿AQP5,APOA1＿CXCL16,VWF＿AQP5,VWF＿CXCL16,CAV1＿CXCL16,TMSB4X＿CXCL16)were found to be the most frequent communication between supporting cells and germ cells in the somatic cells during puberty,and secreted key ligands that promote sperm maturation.Testicular microenvironment and male reproductive disorders: macrophage development in the testicular spermatogenic microenvironment and male infertility.Singlecell transcriptome data from normal testes and NOA patients were collated and analyzed to reveal the existence of different subtypes of macrophages.Pseudo-temporal trajectory analysis identified the presence of a specific subtype of inflammatory function enriched deficiency in NOA testes,while this cell subtype was less or absent in normal testes.In addition,Top transcription factors of the same cell type in normal and NOA patient testes differed,and bisphenol A(BPA)regulation of supporting cell ligand genes(Dnc,Dusp1,Timp3,Vwf)was involved in sperm maturation,while studies suggest that BPA may be a potential cause of NOA in humans.The cellular state and cellular communication of the testicular microenvironment are among the factors causing male infertility.In summary,the integration of testicular microenvironment and cellular communication based on multi-omics provides new tools and perspectives for the study of male gonadal abnormalities and male infertility,and systematically and comprehensively elucidates the heterogeneity of testicular development and spermatogenesis from the developmental period and multi-omics level;inferring that microenvironment and cellular communication jointly regulate testicular abnormalities and male infertility during testicular development cells and spermatogenesis.This provides a new basis for the treatment methods of testicular development abnormalities and male infertility.更多还原