Expression Heat Shock Genes In Mouse Spermatogenic Cells

在雄性生殖细胞中，HSP70热休克蛋白家族有两个独特的成员。这些研究的目标是确定它们的表达调节机制，并确定这些蛋白质在生殖细胞中的作用。HSP70蛋白是一种分子伴侣，它帮助新生多肽的折叠和多聚体复合体的组装，以及在热休克和其他应激后变性蛋白质的重新折叠。大多数HSP70蛋白的基因是结构性表达的(Hsc70，Hsp78)或对应激反应(Hsp70-1和Hsp70-3)，而Hsp70-2和Hsc70t表达是对发育线索的响应，并且只在雄性生殖细胞中表达。永久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方法来转染生殖细胞的原代培养。因此，我们使用转基因小鼠来界定Hsp70-2发育表达所需的上游调控区域。将不同的Hsp70-2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，生殖细胞核中存在的几个已知转录因子和未知蛋白质与这些区域的特定序列结合。HSP70-2是在雄性生殖细胞发育的减数分裂阶段合成的，我们推测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。HSP70-2基因的突变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，由于这一事件需要细胞周期蛋白B1依赖的CDc2激酶活性，我们进一步假设HSP70-2是CDc2激活所需的伴侣。虽然在Hsp70-2基因敲除小鼠的睾丸中存在Cdc2，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HSP70-2蛋白加入到Hsp70-2基因敲除小鼠的睾丸匀浆中，恢复了CDc2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，证实了HSP70-2是CDc2的伴侣。然而，这种影响是有漏洞的，少数生殖细胞避免了凋亡，经历了一个或两个减数分裂，并开始形成顶体。这表明，一些通常发生在精子细胞中的发育事件不需要完成减数分裂。在CDC25或CDC2基因突变的果蝇中也出现了类似的结果，这表明在缺乏CDc2激酶活性的情况下，其他激酶可能会部分补偿。 HSC70T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HSP70-2的类比，我们假设HSC70T是参与减数分裂后生殖细胞发育或精子功能的独特蛋白的伴侣。然而，雄性Hsc70t基因敲除小鼠的生育力正常，睾丸形态、精子数量和活力没有明显变化。没有明显的表型表明，其他HSP70蛋白可能弥补了HSC70T的缺失，但Northern或Western印迹分析没有发现它们的mRNA或蛋白水平的变化。然而，更仔细的检查发现，来自Hsc70T/-小鼠的精子在体外孵化超过30分钟，因为它们是静止的，而来自野生型的精子保持了几个小时的活力。Hsc70t-/-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，乳酸到丙酮酸的转化受到影响，这表明Hsc70T-/-小鼠精子中的LDH-C功能发生了改变，或者NADH水平降低。此外，最近的研究表明，与野生型小鼠的精子相比，缺乏HSC70T蛋白的精子在获能条件下无法实现高度激活的运动能力。 酵母双杂交筛选发现了一种BAG结构域蛋白，它与HSC70T和HSP70-2结合，主要在睾丸中表达。该蛋白通过其BAG结构域与HSC70T和HSP70-2的N端ATPase结构域结合，并抑制其ATPase活性。人类的同系物被报道与某些肿瘤坏死因子受体(TNFR)上的死亡结构域结合。它被命名为死亡结构域的沉默(Silencer of Death Domainer，SODD)，并被发现与HSC70结合。SODD被假设通过抑制含有死亡结构域的蛋白(如TNFR1)下游的信号转导事件来调节细胞凋亡。然而，我们有一个重要的发现，TNFR1具有一个与SODD结合的非典型的ATPase结构域。最近的研究表明，SODD通过抑制受体聚集和激活凋亡级联所需的ATPase活性来调节TNFR1的功能。

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. In addition, recent studies indicate that sperm lacking the HSC70T protein fail to achieve hyperactivated motility under capacitation conditions, compared to sperm from wild type 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.