Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: implication for the site of action of antispermatogenic agents
Expression of the cystic fibrosis transmembrane conductance regulator in rat spermatids: implication for the site of action of antispermatogenic agents
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DOI：
10.1093/molehr/7.8.705
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发表时间：
2001-08-01
影响因子：
4
通讯作者：
Wong, PYD
中科院分区：
文献类型：
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作者：
Gong, XD;Li, JCH;Wong, PYD
为了确定囊性纤维化跨膜电导调节因子(CFTR)在睾丸中是否有功能表达，我们对大鼠睾丸生精细胞CFTRmRNA、蛋白和通道活性进行了分析。逆转录-聚合酶链式反应检测到CFTRmRNA在成熟但未成熟大鼠的睾丸中表达。用CFTR特异性抗体进行的Western印迹分析显示，生精细胞膜提取液中有免疫反应。免疫组织化学研究定位于圆形和细长精子细胞中，但不存在于完全发育的精子中。采用全细胞膜片钳技术，在圆形精子细胞上记录到由胞内cAMP(100mU/L)激活的内向电流。该电流呈线性IN关系，并可被氯通道阻断剂二苯胺-2-羧酸盐(DPC)所抑制。将大鼠生殖细胞Cf，Trc DNA导入人胚胎肾(HEK)293细胞，可产生cAMP激活的具有CFTR特征的氯电流。抗生精药1-(2，4-二氯苯基)-吲唑-3-羧酸、氯硝胺(500mU/L)和1-(2，4-二氯苯基)吲唑-3-丙烯酸(250mU/L)可完全阻断该电流。综上所述，这些结果提供了CFTR在精子发生过程中在精子细胞中差异表达的证据。我们推测，CFTR可能与水通道蛋白相互作用，导致胞浆体积收缩，这是精子发生的一个基本特征。
To establish whether cystic fibrosis transmembrane conductance regulator (CFTR) is functionally expressed in the testis, we subjected spermatogenic cells from rat testes to analysis of CFTR mRNA, protein and channel activity. CFTR mRNA was detected in the testes of mature but not immature rats using reverse transcription-polymerase chain reaction analysis. Western blot analysis performed with a CFTR specific antibody revealed immunoreactivity in the membrane extract of spermatogenic cells. Immunohistochemical studies localized CFTR in round and elongated spermatids, but not in the fully developed spermatozoa. Using a whole-cell patch clamp technique, we recorded an inward current activated by intracellular cAMP (100 mu mol/l) in round spermatids. The current displayed a linear IN relationship and was inhibited by diphenylamine-2-carboxylate (DPC), a chloride channel blocker. Transfection of the rat germ cell CF, TR cDNA into human embryonic kidney (HEK) 293 cells caused the expression of a cAMP-activated chloride current with CFTR characteristics. The current was completely blocked by the antispermatogenic agents 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid, lonidamine (500 mu mol/l) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid, AF2785 (250 mu mol/l). These results taken together provide evidence that CFTR is differentially expressed in spermatids during spermiogenesis. We speculate that CFTR may interact with aquaporin to bring about cytoplasmic volume contraction which is an essential feature of spermiogenesis.