EXPRESSION OF HEAT SHOCK GENES IN MOUSE SPERMATOGENIC CELLS

这些研究的目的是确定hsp70热休克蛋白家族的两个独特成员在男性生殖细胞中的表达调控和作用。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中基因表达的启动子分析研究，也没有可靠的转染原代细胞的方法。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。足迹分析鉴定出两个不受生殖细胞核蛋白dna酶酶切的结构域，称为框1(在bp ？555和？503)和2号盒子(在bp ？346英镑和335英镑)。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。此外，杂合子雄性的生育能力中度降低，提示HSP70-2在生殖细胞发育中存在剂量效应，纯合子雄性的出生数量少于预期，提示HSP70-2在精子功能或胚胎发育中起作用。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的独特蛋白的伴侣。尽管雄性Hsc70t基因敲除小鼠的生育能力略有下降，但睾丸形态、精子数量和活力没有明显变化。显性表型的缺失表明，精子中存在的其他HSP70蛋白可能弥补了HSC70T的缺失，但northern或western blot分析未发现mRNA或蛋白水平的变化。该基因还映射到小鼠17号染色体上MHC的一个区域，该区域与过敏性睾丸炎的易感性有关。然而，在分析HSC70T在这种睾丸自身免疫性疾病中的可能作用时，未能发现野生型和HSC70T敲除小鼠之间的差异（合作者Tung）。缺乏HSC70T是否会增加精细胞或精子对热、氧化或化学应激的敏感性仍有待确定。酵母双杂交筛选鉴定出一种独特的蛋白（称为蛋白D1），它与HSC70T和HSP70-2结合，主要在睾丸中表达。HSC70T和D1还结合仅在减数分裂后生殖细胞中表达的j结构域蛋白MSJ-1（合作者Berruti）。j结构域蛋白结合到HSP70蛋白的c端区域，并调节它们与其他蛋白相互作用的特异性。D1与HSC70T和HSP70-2的n端atp酶结构域结合，但不与HSC70结合。最近报道的一种与D1具有显著相似性的人类蛋白与一些tnf受体的死亡结构域结合，并被命名为死亡结构域沉默者（SODD）。它还结合HSC70，并被假设通过抑制死亡结构域蛋白下游的信号转导事件来调节细胞凋亡。目前的研究正在确定D1是否调节HSC70T和MSJ-1的相互作用或伴侣活性，D1和SODD蛋白之间的关系以及D1在生殖细胞凋亡中的作用将被研究。

The goals of these studies are to determine the regulation of expression and roles of two unique members of the hsp70 heat-shock protein family in male 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 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. In addition, heterozygous males have moderately reduced fertility, suggesting a dosage effect of HSP70-2 in germ cell development, and fewer homozygous males are born that expected, suggesting a role for HSP70-2 in sperm function or embryonic development. 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. Although male Hsc70t knockout mice have slightly reduced fertility, there are no apparent changes in testis morphology, or sperm numbers and motility. The absence of an overt phenotype suggested that other HSP70 proteins present in spermatids might compensate for the absence of HSC70T, but no changes in mRNA or protein levels were found by northern or western blot analysis. The gene also maps to a region of the MHC on mouse chromosome 17 linked to susceptibility to allergic orchitis. However, analysis of the possible role of HSC70T in this testicular autoimmune disease failed to detect differences between wild-type and Hsc70t knockout mice (collaborator Tung). It remains to be determined if lack of HSC70T increases the sensitivity of spermatids or sperm to the effects of thermal, oxidative, or chemical stresses. Yeast two-hybrid screens identified a unique protein (referred to as protein D1) that binds to HSC70T and HSP70-2 and is expressed predominantly in testis. HSC70T and D1 also bind MSJ-1, a J-domain protein expressed only in post-meiotic germ cells (collaborator Berruti). J-domain proteins bind to a C-terminal region of HSP70 proteins and modulate the specificity of their interactions with other proteins. D1 binds to the N-terminal ATPase domain of HSC70T and HSP70-2 but not HSC70. A recently reported human protein with significant similarity to D1 binds to the death domain on some TNF receptors and was named silencer of death domain (SODD). It also binds HSC70 and was hypothesized to regulate apoptosis by suppressing signal transduction events downstream of death-domain proteins. Current studies are determining if D1modulates HSC70T and MSJ-1 interactions or chaperone activities and the relationship between the D1 and the SODD protein and the role of D1 in germ cell apoptosis will be examined.