Gene Expression In Spermatogenic Cells

生精细胞中的基因表达

• 批准号: 8734111
• 负责人: EDWARD MITCHELL EDDY
• 金额: $ 208.44万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8734111
• 关键词: Adult; Alleles; Area; CDC2 Protein Kinase; Cells; Chimera organism; Chromatin; Chromosomes; Coculture Techniques; Comet Assay; Complex; DNA; Deletion Mutagenesis; Development; Embryo; Enzymes; Event; Excision; Female; Fertility; Flagella; GDNF gene; Gene Expression; Gene Targeting; Genes; Genetic Crossing Over; Genetic Programming; Germ Cells; Germ Lines; Glycolysis; Goals; Haploidy; Histones; Human; Immune Sera; In Vitro; Infertility; Intracytoplasmic Sperm Injections; Kinetochores; Learning; Mammals; Meiosis; Meiotic Prophase I; Messenger RNA; Metaphase; Microarray Analysis; Mitosis; Morphology; Mouse Strains; Mus; Mutation; Oocytes; Organelles; Partner in relationship; Phase; Physical condensation; Primary Cell Cultures; Process; Protamines; Proteins; Quantitative Trait Loci; Regulation; Research; Resolution; Role; Sampling; Scaffolding Protein; Services; Shapes; Signal Transduction; Site; Spatial Distribution; Sperm Count Procedure; Sperm Motility; Sperm Tail; Spermatids; Spermatogenesis; Spermatogenic Cell; Staging; Stem cells; Structural Protein; System; Testing; Testis; Transcript; Transplantation; Two-Hybrid System Techniques; Yeasts; cell motility; cyclin B1; embryonic stem cell; human PRM2 protein; knockout gene; male; mutant; novel; offspring; protamine 1; protein function; protein protein interaction; pup; scaffold; self-renewal; sperm cell; transmission process

These studies will identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells and that ultimately define the conditions responsible for male fertility. The approaches currently being applied are to identify genes expressed specifically in male germ cells, use the gene knockout approach to define the roles of the proteins they encode, employ yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essential for development of the male gamete, and prepare antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and selected genes are being studied that encode proteins whose functions are essential for novel aspects of gamete development and/or function. (1) Role of peritubular myoid cells in spermatogonial stem cell self-renewal in the testis niche: We hypothesized that T-regulated GDNF expression in PM cells is required for SSC renewal. This hypothesis was tested using an adult mouse PM cell primary culture system. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when SSCs were isolated and co-cultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number of SSC-derived colonies was increased significantly by in vitro T treatment. These results strongly suggest that T-dependent regulation of GDNF expression in PM cells has a significant role in defining the microenvironment of the niche and influencing SSC self-renewal. (2) Regulation of meiotic progression in spermatogenesis: The mechanisms regulating transition from prophase I to metaphase I of meiosis during spermatogenesis are uncertain. Previous studies have suggested that proteins other than or in addition to CDK1 and Cyclin B1 may regulate this process during spermatogenesis. Conditional gene targeting approaches are being used to determine if these proteins are essential for the progression of meiosis in male germ cells. (3) The role of novel scaffold proteins and glycolytic enzymes in sperm motility: The flagellum is one of the most complex of all cell organelles and many of its proteins are encoded by genes expressed during post-meiotic haploid phase of spermatogenesis. We have used a variety of approaches to identify components of the fibrous sheath, a key cytoskeletal component of the sperm flagellum. These included structural proteins, signal transduction anchoring proteins, and glycolytic enzymes, most of which are encoded by genes only expressed in male germ cells. This led us to hypothesize thatmammalian sperm require ATP produced by glycolysis for motility and the fibrous sheath is an essential scaffold for the glycolytic enzymes. This hypothesis has been confirmed using gene targeting and current studies are determining how the different glycolytic enzymes and structural components of the fibrous sheath are assembled and how their functions are regulated. (4) Strain-specific modifier gene for male fertility in mice heterozyogus for a targeted mutation in protamine 2 (Prm2) gene: Chromatin condensation following meiosis in the male germ line involves removal of most histones and their replacement with transition proteins and then protamines. Mice and humans have two protamines (PRM1 and PRM2), while most other mammals have only PRM1. These genes are expressed only in post-meiotic male germ cells. Cell cleavage is incomplete in male germ cells and they share the mRNAs and proteins for PRM1 and PRM2. We previously generated chimeras with ES cells derived from 129 strain mice with a mutation in the Prm2 gene and were unable to achieve germ line transmission, even though half of the sperm contained an intact Prm2 allele. Using ICSI, we found that sperm from mice heterozygous for a mutation in Prm2 were unable to produce viable embryos beyond early cleavage stages and comet assays indicated that the sperm DNA was fragile and prone to fragmentation. We hypothesized that disruption of chromatin integrity in heterozygous mice was due to haploinsufficiency in PRM1 and PRM2 levels in spermatids with either a mutant or an intact allele. To allow further studies, mice were generated with a floxed Prm2 gene, the gene was disrupted in oocytes, and colonies of C57BL/6 and 129S6 strain mice were generated and maintained by transmitting the mutation through females. While heterozygous C57BL/6 males were fertile, heterozygous 129S6 males were infertile. By qPCR analysis, the Prm2 mRNA levels in the testes of heterozygous C57BL/6 and 129S6 mice were half the levels of their WT littermates. The morphology and motility of sperm from C57BL/6 males were indistinguishable from WT sperm, but sperm from 129S6 males had reduced motility and many were seen by SEM to be abnormal in shape and organization. This suggested that a 129 strain-specific modifier gene (or genes) causes the infertility. To identify the modifier gene, F2 male mice were generated by crossing WT C57BL/6 male mice with heterozygous 129S6 females and intercrossing their offspring to maximize meiotic cross-over events. The F2 males were test-mated for three months and total pups sired, average pups per litter, sperm numbers, and sperm percent motility were determined for each male. DNA samples from these male were subjected to QTL analysis by JAX Services, using a low-resolution SNP panel (6-8 strain-specific SNPs per chromosome). No significant QTLs were detected for sperm numbers or sperm motility, but significant QTLs for average pups per sire and number of pups per sire were identified on chr4 and chr11. The stronger and more promising of the QTLs was in the interval between 42.44 and 83.48 Mb from the kinetochore on chr11. The candidate QTL interval on chr11 contains around 600 genes. Significant differences were detected by eQTL microarray analysis between the levels of mRNA from genes in this interval in the testes of heterozygous C57BL/6 and 129S6 mice.

这些研究将确定和描述控制男性生殖细胞发育和功能的内在遗传程序的关键组成部分，并最终确定导致男性生育能力的条件。目前应用的方法是鉴定在男性生殖细胞中特异性表达的基因，使用基因敲除方法来确定它们编码的蛋白质的作用，使用酵母双杂交试验和删除诱变来鉴定对男性配子发育至关重要的蛋白质-蛋白质相互作用，并制备抗血清来确定特定基因产物的时空分布。许多基因仅在男性生殖细胞中表达，目前正在研究的一些基因编码的蛋白质，其功能对配子发育和/或功能的新方面至关重要。(1)小管周围肌样细胞在睾丸壁龛中精原干细胞自我更新中的作用：我们假设t调节的GDNF在PM细胞中的表达是SSC更新所必需的。用成年小鼠PM细胞原代培养系统验证了这一假设。我们发现T在PM细胞中诱导GDNF mRNA和蛋白水平的表达。此外，当分离ssc并与有或没有T的PM细胞共培养并移植到生殖细胞枯竭小鼠的睾丸时，体外T处理显著增加了ssc衍生的菌落数量。这些结果强烈表明，t依赖性调节PM细胞中GDNF表达在确定微环境和影响SSC自我更新方面具有重要作用。(2)精子发生过程中减数分裂过程的调控：精子发生过程中减数分裂前期向中期过渡的调控机制尚不明确。先前的研究表明，在精子发生过程中，CDK1和Cyclin B1以外或以外的蛋白质可能调节这一过程。条件基因靶向方法被用来确定这些蛋白质是否对男性生殖细胞减数分裂的进展至关重要。(3)新型支架蛋白和糖酵解酶在精子运动中的作用：鞭毛是所有细胞器中最复杂的细胞器之一，其许多蛋白是由精子发生后减数分裂单倍体阶段表达的基因编码的。我们使用了多种方法来鉴定纤维鞘的成分，这是精子鞭毛的关键细胞骨架成分。这些蛋白质包括结构蛋白、信号转导锚定蛋白和糖酵解酶，其中大多数是由只在男性生殖细胞中表达的基因编码的。这使我们假设哺乳动物精子的运动需要糖酵解产生的ATP，而纤维鞘是糖酵解酶必不可少的支架。这一假设已经通过基因靶向得到证实，目前的研究正在确定不同的糖酵解酶和纤维鞘的结构成分是如何组装的，以及它们的功能是如何被调节的。(4)小鼠杂合子精蛋白2 （Prm2）基因靶向突变的雄性生殖能力的菌株特异性修饰基因：雄性生殖系减数分裂后的染色质凝聚涉及去除大多数组蛋白并将其替换为过渡蛋白和精蛋白。老鼠和人类有两种蛋白（PRM1和PRM2），而大多数其他哺乳动物只有PRM1。这些基因仅在减数分裂后的男性生殖细胞中表达。在男性生殖细胞中，细胞分裂是不完全的，它们共享PRM1和PRM2的mrna和蛋白质。我们之前用129株Prm2基因突变的小鼠的胚胎干细胞产生嵌合体，即使一半的精子含有完整的Prm2等位基因，也无法实现生殖系传播。通过ICSI，我们发现Prm2突变的杂合小鼠精子在卵裂早期后无法产生可存活的胚胎，彗星试验表明精子DNA是脆弱的，容易破碎。我们假设杂合小鼠中染色质完整性的破坏是由于突变等位基因或完整等位基因的精子中PRM1和PRM2水平的单倍不足。为了进行进一步的研究，我们用一个固定的Prm2基因产生小鼠，在卵母细胞中破坏该基因，通过在雌性中传递突变产生并维持C57BL/6和129S6品系小鼠的菌落。杂合的C57BL/6雄性不育，而杂合的129S6雄性不育。通过qPCR分析，杂合子C57BL/6和129S6小鼠睾丸中Prm2 mRNA水平为WT同窝小鼠的一半。C57BL/6雄性精子的形态和活力与WT精子难以区分，而129S6雄性精子的活力下降，许多精子在形状和组织上都有异常。这表明129株特异性修饰基因（或多个基因）导致不育。为了鉴定修饰基因，将WT C57BL/6雄性小鼠与杂合的129S6雌性小鼠杂交，产生F2雄性小鼠，并使其后代杂交，以最大化减数分裂杂交事件。研究人员对F2只雄鼠进行了3个月的交配测试，并对每只雄鼠的产仔总数、每窝平均产仔数、精子数量和精子活力进行了测定。这些男性的DNA样本由JAX Services进行QTL分析，使用低分辨率SNP面板（每条染色体6-8个菌株特异性SNP）。在chr4和chr11上未检测到与精子数量或精子活力相关的显著qtl，但在chr4和chr11上检测到与平均每胎仔数和每胎仔数相关的显著qtl。这些qtl在chr11上距着丝点42.44 ~ 83.48 Mb之间较强。chr11上的候选QTL区间包含约600个基因。通过eQTL微阵列分析，在杂合子C57BL/6和129S6小鼠的睾丸中，这段时间基因的mRNA水平存在显著差异。