Identification, immunolocalization, regulation, and postnatal development of the lipocalin EP17 (epididymal protein of 17 kilodaltons) in the mouse and rat epididymis
Identification, immunolocalization, regulation, and postnatal development of the lipocalin EP17 (epididymal protein of 17 kilodaltons) in the mouse and rat epididymis
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DOI：
10.1210/en.2002-220932
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发表时间：
2003-03-01
期刊：
影响因子：
4.8
通讯作者：
Orgebin-Crist, MC
中科院分区：
文献类型：
--
作者：
Fouchécourt, S;Lareyre, JJ;Orgebin-Crist, MC
小鼠附睾中存在几种脂钙蛋白，它们被认为通过运输亲脂分子而在精子成熟过程中发挥作用。我们以前已经报道了两个Lipocalin基因，mERABP(小鼠附睾维甲酸结合蛋白)和mEP17(小鼠附睾蛋白17 kDa)，它们起源于一个祖先基因，在附睾中特异表达。在本研究中，提出了一种针对重组蛋白的多克隆抗体，以研究mEP17的存在及其调控。MEP17在附睾头的核上区、附睾头的透明细胞和中下端的管腔内均有表达，但在附睾尾的管腔内未检测到mEP17。起始节段和头部组织提取液经高效液相色谱分离。免疫活性的mEP17组分经电泳层析后，用质谱仪分析其免疫反应条带，明确鉴定mEP17。经双向电泳后，mEP17呈现5个22 kDa的斑点，等电点在5.8-6.7之间。N-葡聚糖酶消化产生一个17 kDa的单一斑点，pI为6，预测的质量和pI。在个体发育过程中，mEP17最早在3周龄就检测到，之后逐渐增加。双侧睾丸切除后2d，mEP17消失，睾酮替代治疗后mEP17消失。单侧切除后，mEP17水平仅在切除侧下降。隐睾症或白花丹治疗后，mEP17水平要么显著降低，要么检测不到。这表明mEP17依赖于可能起源于生殖细胞的睾丸因子(S)。综上所述，我们的数据表明mEP17的空间表达、调控和命运不同于高度相关的小鼠附睾维A酸结合蛋白。这表明这两个相关蛋白在小鼠附睾中具有不同的功能。
Several lipocalins are present in the mouse epididymis and are thought to play a role in sperm maturation by transporting lipophilic molecules. We have previously reported that two lipocalin genes, mERABP (mouse epididymal retinoic acid binding protein), and mEP17 (mouse epididymal protein of 17 kDa), derived from an ancestral gene, are specifically expressed in the epididymis. In the present study, a polyclonal antibody was raised against a recombinant protein to investigate the presence and the regulation of mEP17. mEP17 was detected in the supranuclear region of the principal cells of the initial segment, the clear cells of the caput epididymidis, and the lumen of the mid/distal caput but not of the distal epididymis. Initial segment and caput tissue extracts were subjected to HPLC separation. After electrophoresis of the immunoreactive mEP17-enriched fractions, the immunoreactive band was analyzed by mass spectrometry to identified mEP17 unambiguously. After two-dimensional electrophoresis, mEP17 appeared as a train of five 22-kDa spots with a range of pI (isoelectric point) from 5.8-6.7. N-glycanase digestion gave rise to a single spot of 17 kDa and pI 6, the predicted mass and pI.During ontogeny, mEP17 was detected as early as 3 wk of age and increased afterward. After bilateral orchiectomy, mEP17 disappeared 2 d after surgery and was not restored after testosterone replacement. After unilateral orchiectomy, mEP17 levels decreased only in the orchiectomized side. After cryptorchidism or busulfan treatment, mEP17 levels were either greatly diminished or not detected. This suggests that mEP17 is dependent on testicular factor(s) that may have a germ cell origin.Altogether, our data demonstrate that mEP17 spatial expression, regulation, and fate are different from that of the highly related mouse epididymal retinoic acid binding protein. This suggests that these two related proteins exhibit distinct functions in the mouse epididymis.