Molecular chaperones in male infertility

1.通过Northan杂交分析发现，在32 ℃至39 ℃的温度范围内，人细胞中Hsp 110家族成员Apg-1和Hsp 110均能被诱导表达，而Apg-2则不能被诱导表达。应用荧光原位杂交技术将人Apg-1和Apg-2基因分别定位于4 q28和5q23.3-q31.1。通过基因组克隆，证实了人类中不存在p52和Hsp 70 RY这两个基因。2.发现Apg-1 mRNA和蛋白在人睾丸中的分化生殖细胞中有表达，尤其是在粗线期后的精母细胞中。Apg-1 mRNA在支持细胞综合征睾丸组织中未检测到，提示Apg-1可能在生殖细胞中特异表达。我们发现Apg-1蛋白在浓缩、纯化的精液样品的精子部分中。提示Apg-1可能参与人类的受精过程。在男性不育患者中，Apg-1蛋白表达水平不均匀，在无精子症患者中，未检测到Apg-1蛋白。3.从小鼠体内克隆了Rbm 3基因，并制备了其多克隆抗体。Rbm 3是冷休克蛋白Cirp的一个蛋白质，被认为是PNA分子伴侣之一。Rbm 3在小鼠和人的细胞中被~（32）C诱导表达，是一种冷休克蛋白。Rbm 3在睾丸支持细胞中组成型表达，实验性隐睾降低Rbm 3的表达。4.从多种逆转录病毒LTR和PGK启动子中筛选出在小鼠生殖细胞系GC 1和GC 2中表达量较高的启动子。我们发现，与造血干细胞相同的杂合载体可有效地转移到这些生殖细胞系中。

1.We found that Apg-1, one of Hsp110 family, and Hsp110 were nduced by a temperature shift from 32C to 39C in human cell lines, but Apg-2couldn't be induced under such conditions, by Northan blot analysis. The human Apg-1 and Apg-2 genes were mapped to the chromosomal loci 4q28 and 5q23.3-q31.1, respectively, by fluorescence in-situ hybridization. We proved that p52, and Hsp70RY, which were reported as similar genes in human did not exist by genomic gene cloning.2.We found Apg-1 mRNA and protein expressed in differentiating germ cells, especially from after pachytene spermatocytes in human testis. We did not detect Apg-1mRNA in Sertoli-cell only syndrome testis, so Apg-1 might express in germ cells specifically. We found Apg-1 proteins in sperm fraction of concentrated, purified semen samples. It suggests Apg-1 might Involved in human fertilization process. In male infertility cases, the protein expression levels were uneven, andin azospermia samples, Apg-1 couldn't be detected. Further analysis is needed.3.We cloned Rbm3 from mice and made the polyclonal antibody. Rbm3 is a paralog of Cirp, which is one of cold shock proteins and is suggested to be one of PNA chaperones. Rbm3 was induced by 32C in mouse and human cells, It proved to be one of cold shock proteins. It expressed in sertoli cells constitutively, and expression of Rbm3 decreased by experimental cryptorchidism.4.We selected the promoter which showed higher expression level of transgenes into mouse GC1 and GC2 germ cell lines, from various retrovirus LTR and PGK promoters. We found that the hybrid vectors same as for hematopoietic stem cells were effective to transfer into these germ cell lines.