Piedrahita J A, Anderson G B
J Reprod Fertil. 1985 Jul;74(2):637-44. doi: 10.1530/jrf.0.0740637.
H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P less than 0.05). Percentage specific killing in each of these sera was 7.8 +/- 4.2, 11.7 +/- 3.0, 26.0 +/- 2.2, 27.7 +/- 3.7 and 39.2 +/- 4.8, respectively (mean +/- s.e.m. with three replicates). The 5 sera and an additional one (4.9 +/- 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P less than 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.
通过向C57BL/6雌性小鼠腹腔内反复注射同基因雄性脾细胞来制备H-Y抗血清。将附睾精子在H-Y抗血清和作为补体来源的豚鼠血清存在的情况下进行孵育。处理后剩余的ATP水平用于计算特异性杀伤量。将具有不同细胞毒性效价的血清用于间接免疫荧光测定，使用异硫氰酸荧光素偶联的山羊抗小鼠IgG（Fc片段特异性）的IgG部分作为二抗。将胚胎分为荧光或非荧光胚胎，转移到假孕受体中，并使其发育至足月。在测试精子细胞毒性的12份血清中，有5份与未免疫的对照血清不同（P小于0.05）。这些血清中每份的特异性杀伤百分比分别为7.8±4.2、11.7±3.0、26.0±2.2、27.7±3.7和39.2±4.8（三次重复的平均值±标准误）。这5份血清和另外一份血清（特异性杀伤为4.9±1.3%）用于胚胎性别鉴定实验。这些血清正确鉴定植入前胚胎性别的准确率分别为60%、46%、74%、73%、74%和48%。特异性精子细胞毒性与雌性非荧光胚胎百分比的相关系数为0.86（P小于0.05），特异性精子细胞毒性与雄性荧光胚胎百分比的相关系数为0.78（无显著性差异）。因此，尽管精子细胞毒性试验对于筛选用于研究植入前胚胎上H-Y抗原表达的抗血清很有用，但将非荧光胚胎分类为雌性比将荧光胚胎分类为雄性更准确。
