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Expression Of Heat Shock Genes In Mouse Spermatogenic Cells

热休克基因在小鼠生精细胞中的表达

基本信息

批准号：
8553742
负责人：
EDWARD MITCHELL EDDY
金额：
$ 22.03万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/8553742
关键词：
Antibodies Apoptosis Backcrossings Binding Binding Sites Biological Assay Boxing CDC2 gene Cell Nucleus Complex DNA Packaging Deoxyribonucleases Development Digestion Elements Fertility Gel Gene Expression Gene Mutation Genes Genetic Germ Cells Goals Heat shock proteins Heat-Shock Proteins 70 Heat-Shock Response In Vitro Incubated Knockout Mice LacZ Genes Maps Meiosis Methods Mitosis Molecular Chaperones Morphology Mus Nuclear Nuclear Proteins Nucleic Acid Regulatory Sequences Phase Phase Transition Phenotype Physical condensation Process Promoter Regions Protein Family Proteins Reporter Genes Role Site Sperm Count Procedure Spermatids Spermatocytes Spermatogenic Cell Staging Stress Testis Transgenic Mice Translations beta-Galactosidase cell type cyclin B1 knockout gene male member novel polypeptide promoter recombinase sperm cell sperm function transcription factor transition protein 1

项目摘要

We have identified the minimal upstream regulatory region of the Hspa2 gene required to determine the germ cell-type and stage-specific expression of the gene. Different Hspa2 gene promoter fragments were ligated to the Lacz reporter gene and beta-galactosidase expression determined in the testes of transgenic mice. It was found that sequences within 604 bp of the translation start site are required for correct expression. This region was examined further with in vitro methods. Footprint analysis identified two domains protected from DNase digestion by germ cell nuclear proteins, referred to as box 1 (between bp -555 and -503) and box 2 (between bp -346 and -335). These domains contain clusters of transcription factor binding motifs. Gel shift and super-shift analyses indicated that several known transcription factors and at least one novel protein present in germ cell nuclei bind to specific sequences in these regions. An antibody was generated against the unknown protein and ChIP assays have confirmed its association with the promoter and mapped its binding site. The promoter region identified in these studies was used to generate a transgenic mouse that expresses the cre recombinase in pachytene spermatocytes that can be used to produce a conditional gene mutation during meiosis in spermatogenic cells. The HSPA2 protein is synthesized during the meiotic phase of male germ cell development and we hypothesized that it is a chaperone for proteins involved in meiosis. This was confirmed using the gene knockout approach. Disruption of the Hspa2 gene resulted in developmental arrest and apoptosis of pachytene spermatocytes at the G2/M-phase transition of meiosis I. We subsequently found that HSPA2 serves as a chaperone for CDC2 and is required for assembly of the CDC2/cyclin B1 meiosis promoting complex. More recent studies identified an unexpected role for HSPA2, a tight association with the major spermatid DNA-packaging proteins, transition proteins 1 and 2. This suggests that HSPA2 also serves as a chaperone for the transition proteins and participates in the process of nuclear condensation that occurs in spermtids. The HSPA1L protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSPA2, we hypothesized that HSP1L is a chaperone for unique proteins involved in post-meiotic germ cell development or sperm function. Male Hsp1l knockout mice have normal fertility and there are no apparent changes in testis morphology or in sperm numbers. Efforts to replicate earlier evidence that sperm from Hspa1l-/- mice incubated for longer than 30 minutes in vitro became immotile were unsuccessful. Backcrossing studies to determine if the phenotype observed earlier was due to mixed genetic background did not support this conclusion. The lack of an identifiable phenotype in the absence of HSPA1L protein may be due to the high level of HSPA2 protein in spermatids serving a redundant role.

我们已经确定了Hspa2基因的最小上游调节区，这是确定该基因的生殖细胞类型和阶段特异性表达所必需的。将不同的Hspa2基因启动子片段连接到LacZ报告基因上，检测转基因小鼠睾丸中β-半乳糖苷酶的表达。研究发现，正确表达需要翻译起始点604个碱基以内的序列。用体外方法对该区域进行了进一步的检测。足迹分析确定了两个被生殖细胞核蛋白保护而不被DNA酶消化的结构域，称为Box 1(在BP-555和-503之间)和Box 2(在BP-346和-335之间)。这些结构域包含转录因子结合基序簇。凝胶漂移和超漂移分析表明，几个已知的转录因子和至少一个存在于生殖细胞核中的新蛋白与这些区域的特定序列结合。针对未知蛋白产生了抗体，芯片检测证实了它与启动子的关联，并绘制了它的结合部位。这些研究中确定的启动子区域被用来产生在粗线期精母细胞中表达cre重组酶的转基因小鼠，该重组酶可用于在生精细胞减数分裂过程中产生条件性基因突变。HSPA2蛋白是在雄性生殖细胞发育的减数分裂阶段合成的，我们假设它是参与减数分裂的蛋白质的伴侣。使用基因敲除方法证实了这一点。Hspa2基因的缺失导致粗线期精母细胞在减数分裂I的G2/M期转变为发育停滞和凋亡。我们随后发现HSPA2作为CDC2的伴侣，是CDC2/细胞周期蛋白B1减数分裂促进复合体组装所必需的。最近的研究发现了HSPA2的一个意想不到的作用，它与精子细胞的主要DNA包装蛋白--过渡蛋白1和2密切相关。这表明HSPA2也是过渡蛋白的伴侣，并参与精子的核凝聚过程。 HSPA1L蛋白只存在于精子细胞中，处于雄性生殖细胞发育的减数分裂后阶段。通过与HSPA2的类比，我们假设HSP1L是参与减数分裂后生殖细胞发育或精子功能的独特蛋白质的伴侣。雄性Hsp1基因敲除小鼠生育能力正常，睾丸形态和精子数量没有明显变化。复制早期证据的努力失败了，这些证据表明，Hspa11-/-小鼠的精子在体外孵化超过30分钟后变得不活动。回交研究确定早先观察到的表型是否是由于混合遗传背景造成的，但并不支持这一结论。在缺乏HSPA1L蛋白的情况下缺乏可识别的表型可能是由于精子细胞中高水平的HSPA2蛋白起着多余的作用。