Batova I N, Kyurkchiev S D, Kehayov I R
Department of Immunobiology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Anim Reprod Sci. 1997 Sep;49(1):55-69. doi: 10.1016/s0378-4320(97)00036-5.
Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.
对去膜的公猪精子头部在高盐 - 尿素、蛋白酶解和DNase I切割条件下进行差异提取，这些条件模拟了促进受精精子细胞核在卵细胞质中体内解聚的条件。研究了精子特有的蛋白质亚群，这些蛋白质保留在残余的耐盐核结构中，该结构在操作上被定义为精子核基质。通过多价抗血清，与公猪肝脏的体细胞型去组蛋白染色质相比，使用ELISA和微量补体结合试验评估精子核蛋白复合物的免疫特异性。为了定义具有免疫特异性的精子DNA相关蛋白，通过将用公猪精子蛋白/DNA复合物免疫的淋巴细胞进行融合来产生杂交瘤。选择了单克隆抗体（Mab 1A8、1B3、2B5、2H5和3A4），这些抗体识别精子DNA紧密结合蛋白复合物中的蛋白质部分，该部分对DNase I切割具有抗性，而对高浓度蛋白酶消化敏感。未记录到抗体与体细胞染色质有明显反应，且与单链DNA无明显结合。通过蛋白质印迹法检测到，Mab 3A4识别出残余精子核结构中一种表观分子量为27 kDa的多肽。对DNase I消化的精子核部分的反应性增强（Mab 2H5除外）表明，被蛋白质保护免受核酸酶消化的DNA可能对免疫反应性和完整的抗原完整性至关重要，以及同源蛋白对抗原性和免疫特异性的DNA结合依赖性。预计所鉴定的假定精子特异性蛋白在合子染色质的结构重排中发挥作用。
