Verhage H G, Boomsma R A, Mavrogianis P A, Li W, Fazleabas A T, Jaffe R C
Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago 60680.
Biol Reprod. 1989 Aug;41(2):347-54. doi: 10.1095/biolreprod41.2.347.
The major objective of this study was to make a polyclonal antibody to a previously described group of progesterone (P)-dependent low molecular weight secretory proteins of the cat uterus. Proteins present in uterine flushings obtained from a P-treated cat were partially purified using Sephadex G-75, separated on two-dimensional polyacrylamide gels, and transferred to nitrocellulose membranes. The region containing the polypeptides were cut out, solubilized in dimethyl sulfoxide, mixed with Freund's adjuvant, and injected at 2-wk intervals into a male rabbit. The antiserum used in this study was obtained 8 wk after the initial injection and crossreacted with antigens on Western blots of uterine flushings and uterine culture medium obtained from ovariectomized estradiol (E2)-primed cats treated with P, and from pregnant preimplantation animals. Polypeptides in three molecular weight regions crossreacted with the antisera (Mr approximately equal to 28,000, pI 5.5 6.0; Mr approximately equal to 36,000, pI 6.0 6.5; Mr approximately equal to 41,000, pI 5.5 6.0), and each region consisted of several isoelectric variants. The Mr approximately equal to 28,000 proteins were the dominant form observed in culture media, and the Mr approximately equal to 36,000 proteins were the major form present in uterine flushings. The antigens were not detected in uterine flushings or culture medium obtained from ovariectomized, E2-treated, and estrous animals. The antigens were also absent in serum and other reproductive and nonreproductive tract tissues. Immunocytochemical analysis demonstrated that antigen staining was limited to the epithelial cells of the deeper uterine glands of the P-dominated animal. Immunoperoxidase staining was diffuse throughout the cytoplasm of these epithelial cells. Thus, the epithelial cells of the uterine glands in the P-dominated cat synthesize and secrete a complex group of P-dependent, uterine-specific proteins that may have potential functional significance during early blastocyst development and implantation.
本研究的主要目的是制备针对猫子宫中先前描述的一组孕酮（P）依赖性低分子量分泌蛋白的多克隆抗体。从经P处理的猫获得的子宫冲洗液中的蛋白质，先用葡聚糖凝胶G-75进行部分纯化，然后在二维聚丙烯酰胺凝胶上分离，并转移至硝酸纤维素膜上。切下含有多肽的区域，溶解于二甲基亚砜中，与弗氏佐剂混合，每隔2周注射到一只雄性兔子体内。本研究中使用的抗血清在初次注射后8周获得，并与从经P处理的去卵巢雌二醇（E2）预处理的猫以及妊娠植入前动物获得的子宫冲洗液和子宫培养基的蛋白质免疫印迹上的抗原发生交叉反应。三个分子量区域的多肽与抗血清发生交叉反应（分子量约为28,000，等电点5.5 - 6.0；分子量约为36,000，等电点6.0 - 6.5；分子量约为41,000，等电点5.5 - 6.0），每个区域由几个等电变体组成。分子量约为28,000的蛋白质是在培养基中观察到的主要形式，而分子量约为36,000的蛋白质是子宫冲洗液中的主要形式。在去卵巢、E2处理和发情动物的子宫冲洗液或培养基中未检测到这些抗原。在血清以及其他生殖和非生殖 tract 组织中也不存在这些抗原。免疫细胞化学分析表明，抗原染色仅限于以P为主导的动物较深子宫腺的上皮细胞。免疫过氧化物酶染色弥漫于这些上皮细胞的整个细胞质中。因此，以P为主导的猫子宫腺的上皮细胞合成并分泌一组复杂的P依赖性、子宫特异性蛋白质，这些蛋白质在早期胚泡发育和植入过程中可能具有潜在的功能意义。
