Molecular chaperones in male infertility

1.通过Northan blot分析，我们发现Hsp110家族成员Apg-1和Hsp110可以在32℃到39℃的温度下诱导，而apg -2不能在此温度下诱导。利用荧光原位杂交技术，将人类Apg-1和Apg-2基因分别定位于染色体4q28和5q23.3-q31.1位点。我们通过基因组基因克隆证明了p52和Hsp70RY这两个在人类中报道的相似基因不存在。我们发现Apg-1 mRNA和蛋白在人类睾丸中分化的生殖细胞中有表达，尤其是在粗线后精细胞中。我们未在仅支持细胞综合征睾丸中检测到Apg-1mRNA，因此Apg-1可能在生殖细胞中特异性表达。我们在浓缩纯化的精液样本中发现了Apg-1蛋白。提示Apg-1可能参与人类受精过程。在男性不育病例中，蛋白表达水平不均匀，在无精子症样本中，无法检测到Apg-1。需要进一步的分析。我们从小鼠中克隆Rbm3，制备了该多克隆抗体。Rbm3是Cirp的类似物，Cirp是冷休克蛋白之一，被认为是PNA伴侣蛋白之一。32C诱导小鼠和人细胞产生Rbm3，证实其为冷休克蛋白之一。Rbm3在支持细胞中组成性表达，实验隐睾导致Rbm3的表达降低。我们从各种逆转录病毒LTR和PGK启动子中筛选出转基因在小鼠GC1和GC2生殖细胞系中表达水平较高的启动子。我们发现与造血干细胞相同的杂交载体可以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.