The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells
The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells
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DOI：
10.1093/humrep/det043
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发表时间：
2013-06-01
影响因子：
6.1
通讯作者：
Wilkinson, M. F.
中科院分区：
文献类型：
--
作者：
Song, H. W.;Anderson, R. A.;Wilkinson, M. F.
什么样的人体组织和细胞类型表达X连锁生殖同源盒（RHOX）基因簇？RHOX同源框基因和蛋白质选择性地表达于卵巢和睾丸的生殖细胞中，RHOX同源框转录因子由X连锁基因簇编码，其成员选择性地表达于小鼠和大鼠的雄性和雌性生殖道中。Rhox基因经历了强大的选择压力，迅速演变，使其不确定是否保持其生殖组织为中心的表达模式在人类，我们在本报告中解决的问题。我们检查了11个胎儿和8成人组织中的所有成员的人类RHOX基因簇的表达。我们分析的重点是胎儿睾丸，我们评估了16个不同的样本，从8到20周的妊娠。我们还分析了胎儿睾丸、成人睾丸和成人卵巢的固定切片，以确定RHOX基因编码蛋白的细胞类型特异性表达模式，我们使用定量逆转录聚合酶链反应分析来检测人RHOX基因的表达。我们制备了RHOX蛋白的抗血清，并利用它们进行了RHOXF 1和RHOXF 2/2B蛋白表达的Western印迹、免疫组织化学和免疫荧光分析，我们发现RHOXF 1和RHOXF 2/2B基因在睾丸中高度表达，而在大多数其他器官中表现出低表达或检测不到表达。使用RHOXF 1和RHOXF 2/2B特异性抗血清，我们发现RHOXF 1和RHOXF 2/2B主要在成年睾丸的生殖细胞中表达。早期生殖细胞（精原细胞和早期精母细胞）表达RHOXF 2/2B，而晚期生殖细胞（粗线期精母细胞和圆形精子细胞）表达RHOXF 1。RHOXF 1和RHOXF 2/2B在人胎儿睾丸的精子原细胞前表达。与此相一致，RHOXF 1和RHOXF 2/2B mRNA表达增加，在第二个三个月期间，在胎儿睾丸发育过程中，当生殖细胞分化成前精原细胞。在人类成年卵巢中，我们发现RHOXF 1和RHOXF 2/2B主要在卵母细胞中表达，尽管RHOX基因在除睾丸外的所有19种人类组织中的平均表达水平较低或检测不到，但RHOX基因仍可能在这些非睾丸组织中的一小部分细胞中高度表达。作为一个恰当的例子，我们发现，RHOX蛋白在人卵巢内的卵母细胞中高度表达，尽管在整个卵巢中RHOX mRNA的水平较低，RHOX转录因子的细胞类型特异性和发育调控的表达模式表明，它们在人胎儿生殖细胞发育，精子发生和卵子发生过程中执行调节功能。我们的研究结果还提高了调节RHOX基因水平可以纠正某些人类不育症的可能性，并且其编码的蛋白质是避孕药物设计的候选靶点。这项工作得到了美国国立卫生研究院赠款R 01-HD 053808和-HD 45595（M.F.W.）的支持，U 54-HD 012303（至S.S.和R.J.C.），和K12-HD 001259（至L.C.L.），和医学研究理事会赠款G1100357/1（给R.A.A.）。没有一位作者声明存在利益冲突。
What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster?The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis.The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report.We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes.We used quantitative reverse transcriptionpolymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression.We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes.While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary.The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.This work was supported by the National Institutes of Health grants R01-HD053808 and -HD45595 (to M.F.W.), U54-HD012303 (to S.S. and R.J.C.), and K12-HD001259 (to L.C.L.), and Medical Research Council grant G1100357/1 (to R.A.A.). None of the authors declared a conflict of interest.