Gipson I K, Moccia R, Spurr-Michaud S, Argüeso P, Gargiulo A R, Hill J A, Offner G D, Keutmann H T
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Clin Endocrinol Metab. 2001 Feb;86(2):594-600. doi: 10.1210/jcem.86.2.7174.
The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.
在月经周期中，子宫颈内膜分泌的黏液的物理特性和数量会发生显著变化，以便在月经周期中期排卵时促进精子迁移。黏蛋白是高度糖基化的高分子量蛋白质，是覆盖所有湿润表面上皮（包括子宫颈内膜上皮）的保护性黏液凝胶的主要结构成分。我们之前已经证明，子宫颈内膜上皮表达三种大型凝胶形成黏蛋白的信使核糖核酸（mRNA），分别命名为MUC5AC、MUC5B和MUC6，其中MUC5B的mRNA占主导。由于黏蛋白的蛋白质水平可能在转录后受到调控，因此需要测量整个周期中MUC5B的蛋白质水平，以便与mRNA水平进行关联。黏液分泌物中特定黏蛋白基因产物的测量受到特异性良好、特征明确的抗体的可用性以及黏蛋白分离方案的体积要求的限制，这些方案包括氯化铯密度离心和分级分离。为了测量整个激素周期中宫颈黏液内的MUC5B蛋白质，我们开发了一种针对该黏蛋白的多克隆抗体。该抗体编号为799，针对一种合成肽，该肽模拟MUC5B蛋白质3'区域D4结构域内富含半胱氨酸区域的19个氨基酸片段。它在免疫印迹上能识别天然和变性的MUC5B，可被其肽预吸附，并与表达MUC5B的上皮细胞顶端分泌小泡结合。我们在酶联免疫吸附测定中使用MUC5B抗体以及宫颈黏蛋白标准品和宫颈黏蛋白分离物，以确定在月经周期中采集的人宫颈黏液样本中MUC5B黏蛋白的相对含量。我们证明，与周期早期或晚期的黏液相比，在月经周期中期采集的人宫颈黏液中MUC5B黏蛋白出现峰值。MUC5B含量的这一峰值与月经周期中期发生的黏液特性变化相吻合，表明这种大型黏蛋白可能对精子向子宫的运输很重要。
