Expression Of Heat Shock Genes In Mouse Spermatogenic Ce

热激基因在小鼠生精细胞中的表达

• 批准号: 6838563
• 负责人: EDWARD MITCHELL EDDY
• 金额: --
• 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/6838563
• 关键词: DNA footprinting; apoptosis; cell cycle proteins; gel mobility shift assay; gene expression; gene induction /repression; gene targeting; genetically modified animals; heat shock proteins; laboratory mouse; male reproductive system; molecular chaperones; protein denaturation; protein folding; reproductive development; spermatogenesis; stress proteins; tissue /cell culture; yeast two hybrid system

There are two unique members of the hsp70 heat-shock protein family in male germ cells. The goals of these studies are to identify the mechanisms regulating their expression and to determine the roles of these proteins in germ cells. The HSP70 proteins are molecular chaperones that assist in the folding of nascent polypeptides and assembly of multimeric complexes, and in the refolding of denatured proteins following heat shock and other stresses. The genes for most HSP70 proteins are expressed constitutively (Hsc70, Hsp78) or in response to stress (Hsp70-1 and Hsp70-3), while Hsp70-2 and Hsc70t are expressed in response to developmental cues and only in male germ cells. Permanent cell lines are not available for promoter analysis studies of gene expression in male germ cells and reliable methods for transfecting primary cultures of germ cells have not been developed. We therefore used transgenic mice to delimit the upstream regulatory region required for developmental expression of Hsp70-2. Different Hsp70-2 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 unknown proteins present in germ cell nuclei bind to specific sequences in these regions. HSP70-2 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 Hsp70-2 gene resulted in developmental arrest and apoptosis of pachytene spermatocytes at the G2/M-phase transition of meiosis I. Since this event requires cyclin B1-dependent Cdc2 kinase activity, we further hypothesized that HSP70-2 is a chaperone required for Cdc2 activation. Although Cdc2 was present in the testis of Hsp70-2 knockout mice, it did not form a heterodimer with cyclin B1 and lacked kinase activity. Addition of recombinant HSP70-2 protein to a homogenate of testis from Hsp70-2 knockout mice restored the ability of Cdc2 to form a heterodimer with cyclin B1 and to become an active kinase, confirming that HSP70-2 is a chaperone for Cdc2. However, the effect is leaky and a few germ cells avoid apoptosis, undergo one or sometimes both meiotic divisions and begin acrosome formation. This indicates that some developmental events that normally occur in spermatids do not require completion of meiosis. Similar results occur in Drosophila with mutations in the Cdc25 or Cdc2 genes, suggesting that other kinases may partially compensate in the absence of Cdc2 kinase activity. The HSC70T protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSP70-2, we hypothesized that HSC70T is a chaperone for unique proteins involved in post-meiotic germ cell development or sperm function. However, male Hsc70t knockout mice have normal fertility and there are no apparent changes in testis morphology or in sperm numbers and motility. The absence of an overt phenotype suggested that other HSP70 proteins might compensate for the absence of HSC70T, but no changes in their mRNA or protein levels were found by northern or western blot analysis. However, closer examination found that sperm from Hsc70t-/- mice incubated for longer than 30 minutes in vitro because immotile, while those from wild-type retained motility for several hours. ATP levels of sperm from Hsc70t-/- mice is about 1% of that in wild-type sperm and cAMP levels are about half. Preliminary results indicate that conversion of lactate to pyruvate is compromised, suggesting that LDH-C function is altered or that NADH levels are reduced in sperm from Hsc70t-/- mice. Yeast two-hybrid screens identified a BAG-domain protein that binds to HSC70T and HSP70-2 and is expressed predominantly in testis. The protein binds through its BAG domain to the N-terminal ATPase domain of HSC70T and HSP70-2 and inhibits their ATPase activity. The human homolog was reported to bind to the death domain on some tumor necrosis factor-" receptors (TNFR). It was named "silencer of death domain" (SODD) and found to bind to HSC70. SODD was hypothesized to regulate apoptosis by suppressing signal transduction events downstream of death-domain containing proteins such as TNFR1. However, we made the significant finding that TNFR1 has an atypical ATPase domain to which SODD binds. Recent studies indicate that SODD modulates TNFR1 function by inhibiting the ATPase activity required for receptor clustering and activation of the apoptosis cascade.

在雄性生殖细胞中有两个独特的热休克蛋白家族成员。这些研究的目的是确定调节其表达的机制，并确定这些蛋白质在生殖细胞中的作用。HSP 70蛋白是分子伴侣，其有助于新生多肽的折叠和多聚体复合物的组装，以及热休克和其他应激后变性蛋白的重折叠。大多数HSP 70蛋白的基因组成型表达（Hsc 70，Hsp 78）或响应于应激（Hsp 70 -1和Hsp 70 -3），而Hsp 70 -2和Hsc 70 t仅在雄性生殖细胞中响应于发育线索表达。永久性细胞系不能用于雄性生殖细胞基因表达的启动子分析研究，也没有开发出可靠的生殖细胞原代培养物分离方法。因此，我们使用转基因小鼠来界定Hsp 70 -2发育表达所需的上游调控区。将不同的Hsp 70 -2基因启动子片段与Lacz报告基因连接，并在转基因小鼠睾丸中测定β-半乳糖苷酶的表达。发现翻译起始位点的604 bp内的序列是正确表达所需的。用体外方法进一步检查该区域。足迹分析确定了两个结构域保护免受DNA酶消化的生殖细胞核蛋白，称为框1（bp -555和-503之间）和框2（bp -346和-335之间）。这些结构域含有转录因子结合基序簇。凝胶位移和超位移分析表明，生殖细胞核中存在的几个已知的转录因子和未知的蛋白质结合到这些区域的特定序列。HSP 70 -2在雄性生殖细胞发育的减数分裂阶段合成，我们假设它是参与减数分裂的蛋白质的伴侣。这是使用基因敲除方法证实的。Hsp 70 -2基因的破坏导致粗线期精母细胞在减数分裂I的G2/M期转变时的发育停滞和凋亡。由于这一事件需要细胞周期蛋白B1依赖的Cdc 2激酶活性，我们进一步假设，热休克蛋白70 -2是一个伴侣所需的Cdc 2激活。虽然Cdc 2存在于Hsp 70 -2基因敲除小鼠的睾丸中，但它不与细胞周期蛋白B1形成异源二聚体，并且缺乏激酶活性。将重组HSP 70 -2蛋白加入HSP 70 -2基因敲除小鼠睾丸匀浆中，可恢复Cdc 2与细胞周期蛋白B1形成异源二聚体并成为活性激酶的能力，证实HSP 70 -2是Cdc 2的伴侣。然而，这种作用是有漏洞的，少数生殖细胞避免了凋亡，经历了一次或有时两次减数分裂并开始顶体形成。这表明精子细胞中通常发生的一些发育事件并不需要减数分裂的完成。类似的结果发生在果蝇突变的Cdc 25或Cdc 2基因，表明其他激酶可能部分补偿Cdc 2激酶活性的情况下。HSC 70 T蛋白仅存在于精子细胞中，在雄性生殖细胞发育的减数分裂后阶段。通过与HSP 70 -2类比，我们假设HSC 70 T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。然而，雄性Hsc 70 t基因敲除小鼠具有正常的生育能力，睾丸形态或精子数量和活力没有明显变化。没有明显的表型表明，其他HSP 70蛋白可能会补偿HSC 70 T的缺乏，但没有发现它们的mRNA或蛋白质水平的变化由北方或Western印迹分析。然而，更仔细的检查发现，来自Hsc 70 t-/-小鼠的精子在体外孵育超过30分钟，因为不动，而来自野生型的精子保持活力数小时。Hsc 70 t-/-小鼠精子的ATP水平约为野生型精子的1%，cAMP水平约为野生型精子的一半。初步结果表明，乳酸转化为丙酮酸是妥协，这表明LDH-C功能被改变或NADH水平降低精子从Hsc 70 t-/-小鼠。酵母双杂交筛选鉴定了一种结合HSC 70 T和HSP 70 -2的BAG结构域蛋白，该蛋白主要在睾丸中表达。该蛋白通过其BAG结构域与HSC 70 T和HSP 70 -2的N-末端ATP酶结构域结合并抑制它们的ATP酶活性。据报道，该人类同源物与某些肿瘤坏死因子受体（TNFR）的死亡结构域结合。它被命名为“死亡结构域沉默者”（SODD），并被发现与HSC 70结合。假设SODD通过抑制包含死亡结构域的蛋白质（如TNFR 1）下游的信号转导事件来调节细胞凋亡。然而，我们发现TNFR 1有一个与SODD结合的非典型ATP酶结构域。最近的研究表明，SODD通过抑制受体聚集和凋亡级联激活所需的ATP酶活性来调节TNFR 1功能。