Klinefelter Gary R, Welch Jeffrey E, Perreault Sally D, Moore Harry D, Zucker Robert M, Suarez Juan D, Roberts Naomi L, Bobseine Kathy, Jeffay Susan
US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North Carolina 27711, USA.
J Androl. 2002 Jan-Feb;23(1):48-63. doi: 10.1002/jand.2002.23.1.48.
We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization.
我们之前证实，精子膜蛋白SP22的水平与暴露于附睾和睾丸毒物的大鼠附睾尾部精子的生育力高度相关，并且睾丸特异性SP22转录本在减数分裂后生殖细胞中表达。在本研究中，制备了多克隆和单克隆抗体，以研究SP22在睾丸和附睾中的表达，并确定SP22在生育力中是起巧合作用还是因果作用。用全长重组大鼠SP22（rSP22）在绵羊中制备多克隆抗血清。用rSP22免疫小鼠并用天然SP22加强免疫，产生杂交瘤克隆；阳性克隆用于腹水生产。免疫印迹表明，亲和纯化的抗rSP22免疫球蛋白（Ig）和腹水Ig分别识别变性和天然的SP22。对189个氨基酸的SP22序列进行线性表位作图，发现抗rSP22 Ig识别3个不同的肽序列，腹水Ig识别1个序列。圆形精子细胞的细胞质以及伸长/伸长精子细胞的头部用抗rSP22和腹水抗体进行免疫染色。分离的睾丸网精子在细胞质滴上显示离散染色，而当精子到达附睾头时，头部赤道段出现染色。有趣的是，清亮细胞沿附睾长度进行免疫染色。腹水Ig和抗SP22 Ig均识别大鼠、仓鼠、公牛、兔子和人类精子头部的赤道段。腹水Ig和亲和纯化的抗rSP22 Ig在体内均显著抑制大鼠附睾尾部精子的生育力，以及体外附睾尾部精子的受精率。此外，亲和纯化的抗rSP22显著抑制完整透明带和去透明带仓鼠卵母细胞的体外受精，表明SP22可能在受精的透明带穿透和膜融合步骤中起作用。
