Mouse Spermatogenic Cells Heat Shock Genes Expression

工作概述：hsp70热休克蛋白家族在男性生殖细胞中有两个独特的成员。这些研究的目的是确定调节其表达的机制，并确定这些蛋白在生殖细胞中的作用。HSP70蛋白是一种分子伴侣蛋白，帮助新生多肽的折叠和多聚体复合物的组装，以及在热休克和其他应激后变性蛋白质的再折叠。大多数HSP70蛋白的基因是组成性表达（Hsc70、Hsp78）或应激反应（HSP70 -1和HSP70 -3），而HSP70 -2和Hsc70t是根据发育线索表达的，仅在男性生殖细胞中表达。永久细胞系无法用于男性生殖细胞基因表达的启动子分析研究，也没有可靠的方法来转染生殖细胞原代培养物。因此，我们使用转基因小鼠来划定Hsp70-2发育表达所需的上游调控区。将不同的Hsp70-2基因启动子片段连接到Lacz报告基因上，并在转基因小鼠睾丸中检测β -半乳糖苷酶的表达。结果发现，正确表达需要翻译起始位点604bp以内的序列。用体外方法进一步检查该区域。足迹分析确定了两个不被生殖细胞核蛋白酶切的区域，称为框1（在bp -555和-503之间）和框2（在bp -346和-335之间）。这些结构域包含转录因子结合基序簇。凝胶移位和超移位分析表明，生殖细胞核中存在的几种已知转录因子和未知蛋白与这些区域的特定序列结合。HSP70-2是在男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。通过基因敲除方法证实了这一点。Hsp70-2基因的破坏导致粗线精母细胞在减数分裂的G2/ m期发生发育停滞和凋亡。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激活所需的伴侣蛋白。尽管Cdc2存在于Hsp70-2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HSP70-2蛋白加入HSP70-2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，这种作用是有漏洞的，少数生殖细胞避免凋亡，进行一次或有时同时进行减数分裂并开始顶体形成。这表明通常发生在精细胞中的一些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，这表明其他激酶可能部分补偿了Cdc2激酶活性的缺失。HSC70T蛋白仅存在于精子中，在男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基因敲除小鼠生育能力正常，睾丸形态、精子数量和活力均无明显变化。显性表型的缺失表明，其他HSP70蛋白可能弥补了HSC70T的缺失，但northern或western blot分析未发现其mRNA或蛋白水平的变化。然而，更仔细的检查发现，Hsc70t-/-小鼠的精子在体外培养超过30分钟后就失去了运动能力，而野生型小鼠的精子则保持了几个小时的运动能力。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，Hsc70t-/-小鼠精子中乳酸到丙酮酸的转化受到损害，这表明ldl - c功能发生改变或NADH水平降低。酵母双杂交筛选鉴定出一种bag结构域蛋白，该蛋白与HSC70T和HSP70-2结合，主要在睾丸中表达。该蛋白通过BAG结构域与HSC70T和HSP70-2的n端atp酶结构域结合，抑制其atp酶活性。据报道，人类同源物与一些肿瘤坏死因子受体（TNFR）的死亡结构域结合。它被命名为“死亡域沉默者”（SODD），并被发现与HSC70结合。假设SODD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，我们发现TNFR1有一个非典型的atp酶结构域，SODD与之结合。最近的研究表明，SODD通过抑制受体聚集和细胞凋亡级联激活所需的atp酶活性来调节TNFR1的功能。意义：生精细胞含有两种独特的HSP70蛋白，但特别容易受到高温和环境因子的损伤，必须保持在比体温低几度的环境中才能发育。这表明HSP70-2和HSC70T的进化目的不仅仅是为了防止热休克或应激，而是在精子发生中具有独特的伴侣作用。确定这些作用，确定它们的功能是如何调节的，并了解对损害敏感的机制，可能会导致预防、诊断或治疗生殖毒物或其他不育原因的影响的新策略，或导致男性避孕药的未来发展。

Summary of work: 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. Significance: Spermatogenic cells contain two unique HSP70 proteins, but are particularly susceptible to damage by elevated temperature and environmental agents and must be maintained a few degrees below body temperature to develop. This suggests that HSP70-2 and HSC70T have evolved for purposes other than to protect against heat shock or stress and have unique chaperone roles in spermatogenesis. Identifying these roles, determining how their function is regulated, and learning the mechanisms which are sensitive to damage may lead to novel strategies for prevention, diagnosis or treatment of the effects of reproductive toxicants or other causes of infertility, or to the future development of a male contraceptive.