McBride Deborah S, Boisvert Chantale, Bleau Gilles, Kan Frederick W K
Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Biol Reprod. 2004 Jan;70(1):198-203. doi: 10.1095/biolreprod.103.020305. Epub 2003 Oct 1.
The oviduct contributes to the reproductive environment by secreting various factors, including a family of glycoproteins termed oviductins. Although many studies have demonstrated that ovarian hormones modulate oviductin gene expression in several mammalian species, there has been controversy surrounding the regulation of golden hamster oviductin. The current study was undertaken to investigate the transcriptional and translational modifications of hamster oviductin during the estrous cycle. First, we verified that hamster oviductin mRNA expression remains constant throughout the estrous cycle by semiquantitative reverse transcription polymerase chain reaction. We then developed a polyclonal antibody against recombinant hamster oviductin (rhaOvm). The anti-rhaOvm antibody was subsequently used in conjunction with quantitative immunocytochemistry to investigate the oviductin levels in the hamster oviduct during the estrous cycle. Quantification of immunolabeling revealed a high, consistent level of glycoprotein throughout the estrous cycle. Therefore, it appears that the production of oviductin is not regulated differentially during the estrous cycle. Size variations in hamster oviductin expression were also investigated by Western blot analysis. The oviduct contains several forms of oviductin at each stage of the estrous cycle, the native glycosylated form(s) of 160-350 kDa, and several precursor forms of 70-100 kDa. Although variations in the intensities of the polydispersed band were not evident during the estrous cycle, additional bands ranging from 90 to 100 kDa were detected in the estrus, metestrus, and diestrus 1 stages. The results from the present investigations suggest that whereas ovarian hormones do not appear to influence the hamster oviductin mRNA and protein expressions, glycosylation of hamster oviductin appears to be differentially regulated during the estrous cycle.
输卵管通过分泌多种因子，包括一类名为输卵管蛋白的糖蛋白家族，来构建生殖环境。尽管许多研究表明，卵巢激素可调节多种哺乳动物物种中输卵管蛋白基因的表达，但关于金黄仓鼠输卵管蛋白的调控一直存在争议。本研究旨在调查仓鼠输卵管蛋白在发情周期中的转录和翻译修饰。首先，我们通过半定量逆转录聚合酶链反应验证了仓鼠输卵管蛋白mRNA表达在整个发情周期中保持恒定。然后，我们制备了一种针对重组仓鼠输卵管蛋白（rhaOvm）的多克隆抗体。随后，该抗rhaOvm抗体与定量免疫细胞化学结合使用，以研究发情周期中仓鼠输卵管内的输卵管蛋白水平。免疫标记定量显示，在整个发情周期中糖蛋白水平高且一致。因此，输卵管蛋白的产生在发情周期中似乎没有差异调节。我们还通过蛋白质印迹分析研究了仓鼠输卵管蛋白表达的大小变化。在发情周期的每个阶段，输卵管中都含有几种形式的输卵管蛋白，天然糖基化形式为160 - 350 kDa，还有几种70 - 100 kDa的前体形式。尽管在发情周期中多分散条带强度的变化不明显，但在发情期、动情后期和间情期1阶段检测到了90至100 kDa的额外条带。本研究结果表明，虽然卵巢激素似乎不影响仓鼠输卵管蛋白的mRNA和蛋白质表达，但仓鼠输卵管蛋白的糖基化在发情周期中似乎受到差异调节。
