Yeung C H, Barfield J P, Cooper T G
Institute of Reproductive Medicine of the University, D-48129 Münster, Germany.
Biol Reprod. 2005 Nov;73(5):1057-63. doi: 10.1095/biolreprod.105.044123. Epub 2005 Jul 20.
As with other mammalian species, human spermatozoa experience a decrease in extracellular osmolarity in cervical mucus upon ejaculation, which requires the efflux of osmolytes and water to counteract swelling that hinders mucus penetration. Recent evidence for the operation of K+ channels in the process of volume regulation suggests parallel involvement of Cl-/anion channels for electro-neutrality as in somatic cells. This was studied using ejaculated spermatozoa washed at seminal osmolality and incubated for 30 min in a medium of mucus osmolality in the presence of Cl- channel blockers. Increases in cell size measured as laser forward-scatter by flow cytometry were detected in the presence of 100 microM 5-nitro-2(3-phenylpropylamino) benzoic acid, 400 microM diisothiocyanato-stilbene-2,2'-disulphonic acid, and 20 microM tamoxifen. No volume changes were found with 400 microM 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid, 200 microM verapamil, or niflumic acid, whereas 1 mM niflumic acid induced shrinkage. Among the candidate channel proteins, Western blotting revealed the presence of ClC-3 (CLCN3) at 87 kDa, but the absence of ClC-2 (CLCN2) from sperm proteins in all samples tested. ICln (CLNS1A) was found in only one of eight samples. Immunocytochemistry localized CLCN3 to the sperm tail. To confirm molecular identities, sperm mRNA was extracted and checked for quality by the presence of protamine 2 transcripts and the absence of sperm DNA and leukocyte mRNA using reverse transcription-polymerase chain reaction. Transcripts of Clcn3 were found in all samples and that of Clns1a in some but not all samples. Clcn3 was therefore considered the most likely candidate of Cl- channel involved in volume regulation of human sperm.
与其他哺乳动物物种一样，人类精子在射精后会经历宫颈黏液中细胞外渗透压的降低，这需要渗透溶质和水的流出以抵消阻碍黏液穿透的肿胀。最近关于钾离子通道在体积调节过程中作用的证据表明，氯离子/阴离子通道也像在体细胞中一样参与其中以维持电中性。本研究使用在精液渗透压下洗涤并在存在氯离子通道阻滞剂的黏液渗透压培养基中孵育30分钟的射出精子进行。在存在100微摩尔5-硝基-2(3-苯基丙基氨基)苯甲酸、400微摩尔二异硫氰酸根合芪-2,2'-二磺酸和20微摩尔他莫昔芬的情况下，通过流式细胞术测量的激光前向散射检测到细胞大小增加。在存在400微摩尔4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸、200微摩尔维拉帕米或尼氟灭酸时未发现体积变化，而1毫摩尔尼氟灭酸会导致细胞收缩。在候选通道蛋白中，蛋白质印迹法显示存在87 kDa的氯离子通道蛋白3(CLCN3)，但在所有测试样本的精子蛋白中未检测到氯离子通道蛋白2(CLCN2)。仅在八个样本中的一个中发现了氯离子细胞内通道蛋白(CLNS1A)。免疫细胞化学将CLCN3定位到精子尾部。为了确认分子身份，提取精子mRNA，并使用逆转录-聚合酶链反应通过鱼精蛋白2转录本的存在以及精子DNA和白细胞mRNA的缺失来检查质量。在所有样本中都发现了Clcn3的转录本，在一些但不是所有样本中发现了Clns1a的转录本。因此，Clcn3被认为是参与人类精子体积调节的氯离子通道的最可能候选者。
