Rivkin Eugene, Eddy Edward M, Willis William D, Goulding Euginia H, Suganuma Ryota, Yanagimachi Ryuzo, Kierszenbaum Abraham L
Department of Cell Biology and Anatomical Sciences, The Sophie Davis School of Biomedical Education, The City University of New York Medical School, 138th Street and Convent Avenue, Harris Hall 306, New York, NY, USA.
Mol Reprod Dev. 2005 Oct;72(2):259-71. doi: 10.1002/mrd.20335.
Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out. While both heterozygous (K9(+/neo)) and homozygous (K9(neo/neo)) mutant mice expressed the wild-type form of K9 protein, the expression profile of the wild-type K9 in K9(neo/neo) mutants was modified. In addition, the open reading frame of the aberrant mRNA terminated at the exon 6/intron 6 splice site, resulting in a truncated K9 protein. Both K9(+neo) and K9(neo/neo) male mice displayed spermatids with ectopic manchette. Coiled tails were seen in maturing spermatids and epididymal sperm of mutant mice and sperm with deformed tails displayed forward motility. A predominant sperm anomaly was residual cytoplasm at the end of the mitochondria-containing middle piece tail segment. The residual cytoplasm displayed vesicles with random in situ motion, suggesting a transport impediment toward the distal end of the sperm tail. All mutant mice were fertile. Surprisingly, in oocyte nuclear injection experiments using K9(neo/neo) sperm donor, 76% of the resulting animals displayed a deletion of the neo(r) gene from the intron 6 of the mutated K9 allele. Results of this study support the view that intron 6 influences the transcriptional efficiency of the K9 gene by decreasing production of wild-type K9 and changing the expression of K9 proteins.
角蛋白9（K9）是在发育中的精子细胞中发现的精子环周环的组成成分之一，但主要在脚垫表皮（人类表皮中的手掌和脚底）中表达。作为确定K9蛋白在精子发育中功能的第一步，我们通过同源重组靶向载体产生了一只突变小鼠，该靶向载体包含破坏的K9基因，其中neo(r)基因插入到内含子6中。这种插入导致了两种K9 mRNA的表达：一种野生型K9 mRNA，其中带有neo(r)基因的内含子6被完全剪接出去；另一种突变形式，其中neo(r)基因和外显子7之间的内含子6只有一部分被剪接出去。虽然杂合子（K9(+/neo)）和纯合子（K9(neo/neo)）突变小鼠都表达野生型形式的K9蛋白，但K9(neo/neo)突变体中野生型K9的表达谱发生了改变。此外，异常mRNA的开放阅读框在外显子6/内含子6剪接位点处终止，导致产生截短的K9蛋白。K9(+neo)和K9(neo/neo)雄性小鼠的精子细胞都显示出异位的精子环周环。在突变小鼠成熟的精子细胞和附睾精子中可见卷曲的尾巴，尾巴变形的精子具有向前运动能力。一个主要的精子异常是在含线粒体的中段尾部末端有残留细胞质。残留细胞质显示出带有随机原位运动的小泡，表明向精子尾部远端的运输受到阻碍。所有突变小鼠都具有生育能力。令人惊讶的是，在使用K9(neo/neo)精子供体的卵母细胞核注射实验中，76%的后代动物显示突变的K9等位基因内含子6中的neo(r)基因缺失。这项研究的结果支持这样一种观点，即内含子6通过减少野生型K9的产生和改变K9蛋白的表达来影响K9基因的转录效率。
