Expression Of Heat Shock Genes In Mouse Spermatogenic Cells

我们已经确定了Hspa2基因的最小上游调控区域，该区域决定了该基因的生殖细胞类型和阶段特异性表达。不同的Hspa2基因启动子片段连接到Lacz报告基因，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至少一种新蛋白与这些区域的特定序列结合。一种针对未知蛋白的抗体被生成，ChIP实验证实了它与启动子的关联，并绘制了其结合位点。在这些研究中发现的启动子区域被用来产生一种转基因小鼠，该小鼠在粗线精母细胞中表达cre重组酶，可用于在生精细胞减数分裂期间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蛋白。通过基因敲除方法证实了这一点。Hspa2基因的破坏导致粗线精母细胞在减数分裂i的G2/ m期转变时发育停滞和凋亡。我们随后发现Hspa2作为CDC2的伴侣，是CDC2/cyclin 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，它与主要的精细胞dna包装蛋白、过渡蛋白1和2密切相关。这表明HSPA2也作为过渡蛋白的伴侣，参与了发生在精子中的核凝聚过程。

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.