小白鼠貯精囊自體抗原的研究

Abstract

以純系小白鼠貯精囊分泌蛋白，同時對成熟的 ICR 公鼠或母鼠進行自體免疫，由西方墨點法分析顯示，在此分泌液中僅有一蛋白質可被來自公鼠或母鼠的抗血清所辨識。此一自體免疫抗原可從貯精囊分泌液中被純化，其核心蛋白的一級結構已由cDNA篩選及蛋白質定序而建立，經鑑定為一新的19 kDa醣蛋白(Yu, 1993)，我們將之命名為貯精囊自體抗原（SVA）。經由西方墨點法及北方墨點法分析，顯示貯精囊是唯一表現此自體抗原的器官，其基因表現受雄性素所調控。經由進一步的研究證實，SVA可與鋅離子結合，其結合常數在生理條件下是（1.35±0.04) × l0（5）M-1。 其次，利用組織切片法，我們發現SVA分佈於貯精囊中一次及二次皺褶的表皮細胞內，它可與副睪尾部的精子結合。以125I-SVA進行結合反應，經Scatchard Plot分析得一上凹曲線，推測其與精子表面有兩個結合位置，其中高親和力之結合常數為(5.36 士 0.02) × l0(6)M-l，結合量為 7.33×10(7)分子/精子；低親和力之結合常數為(5.05 ± 0.25) × 10(5)M-1，結合量為 2.35 × 10(8)分子/精子。我們在實驗中利用125I 標示的SVA (125I-SVA)與薄層層析覆蓋法進行分析，証明SVA結合於精子上的磷脂質—phosphatidylcholine (PC）及sphingomyelin (SPM)，而利用擾動螢光光譜分析，可以計算出SVA 與 PC或 SPM的結合常數，分別是(1.08 土 0.02)×l0(4)M-1，或(3.43 ± 0.03) ×10(5)M-1。 精子可藉由 BSA 將其表面的鋅離子去除，增加細胞膜的流動性，以達到體外獲能作用的目的。實驗結果顯示，SVA 可抑制由 BSA 所誘發之精子凝集(aggregation)、進行獲能作用（capacitation）及移除鋅離子的能力；而此抑制精子進行獲能作用的能力，可藉由 BSA 蛋白濃度的提高而被克服。

A saline extract of protein in mouse seminal vesicle secretion was used to immunize mature mice (ICR) of both sexes. Results of Western analyses for the secretory proteins indicated that only one minor protein component could be recognized by the autoantisera prepared from either autoimmunization of male mice or isoimmunization of female mice. The autoantigen (SVA) was purified from mouse seminal vesicle secretion and identified to be a noble 19 kDa glycoprotein. The primary structure of its core protein has been established by cDNA cloning and protein sequencing(Yu, 1993). Results of Western and Northern analyses for various tissue indicated that the seminal vesicle is the sole organ to produce SVA. The gene expression of SVA was shown to be androgen dependent. We showed further that SVA is able to complex with Zn2+ and the association constant for the complex formation was determinated to be (1.35 ± 0.04)×10(5)M-1 at pH 7.4. By immunohistochemical method, I found that SVA could be immunolocalized in the primary and secondary folders of epithelial cells in seminal vesicles and it could bind to spermatozoa collected from caudal epididymus. The analysis of the equilibrium binding data using 125I-SVA revealed a non-linear curve in the Scatchard plot. Presuming two type of SVA-binding sites on the sperm surface, we estimated a high-affinity site With Ka of (5.36 ± 0.02) × 10(6)M-1 and binding capacity of 7.33 × 10(7) sites/ cell and a low-affinity site with Ka of(5.05 ± 0.25) ×10(5) M-1 and binding capacity of 2.35 × 10(8) sites / cell. Using TLC-overlay technique, I demonstrated the specific binding of SVA to the major sperm membrane phospholipid, namely phosphatidyicholine (PC) and sphingomyelin (SPM) on the spermatozoa. This was confirmed also by the analysis of the SVA fluorescence perturbed by phospholipid. The association constant for the complex formation of SVA With SPM or PC was estimated to be ( 3.43 ± 0.03 ) ×10(5)M-1 or (1.08 ± 0.02) ×10(4)M-1. It has long known the removal of zinc from the cell surface and the increase in the plasma membrane fluidity in the in vitro capacitation achieved by incubating mammalian spermatozoa in a bovine serum albumin (BSA) containing medium. Our results proved that the BSA-induced sperm aggregation and capacitation and the ability of BSA to remove zinc ion in the sperm cell can be suppressed by SVA.