Yamamura Yasutaka, Aoyama Sho, Oshima Yuki, Kato Takuma, Osawa Noboru, Nakamura Masahisa
Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, Tokyo, Japan.
Zoolog Sci. 2005 Sep;22(9):1045-50. doi: 10.2108/zsj.22.1045.
Sry (sex-determining region on the Y chromosome) is required for testicular differentiation in mammals. In addition to Sry, other genes such as WT1, Fgf9, Dax1, Dmrt1 and Sox9 are widely accepted to be involved in the sex determination in vertebrates. However, the roles of these genes during sex determination still remain unclear in amphibians. This study was undertaken to examine the expression of WT1 and Fgf9 in the developing gonad of amphibians. We first isolated the WT1 cDNA from the frog Rana rugosa. Like WT1 in mice, R. rugosa WT1 showed 2 isoforms; i.e., one had an additional 3 amino acids, KTS, included between the third and fourth zinc fingers. However, 17 amino acids in exon 5 of mammalian WT1 could not be found in R. rugosa WT1, which is also the case in turtle and chicken. The mRNA of both isoforms (+KTS, -KTS) was detected in the lung, kidney and testis, but not in the ovary and muscle of adult frogs. The 2 isoforms were expressed first in the embryos at stage 23. Thereafter, the expressions remained constant in the gonad attached to mesonephros of both sexes during sex determination. We next isolated the R. rugosa Fgf9 cDNA encoding 208 amino acids. The amino acid sequence of Fgf9 had similarity greater than 92% with chicken, mouse and human Fgf9s, suggesting that Fgf9 is highly conserved among vertebrate classes. Fgf9 was expressed in the ovary of an adult frog strongly, but in the lung weakly. In contrast, the Fgf9 mRNA was hardly detected in the kidney, testis and muscle. Moreover, Fgf9 did not show a sexually dimorphic expression pattern during sex determination in R. rugosa. The results, taken together, suggest that both WT1 and Fgf9 are expressed in the indifferent gonad prior to sex determination without any difference in the expression between males and females. Thus, it seems unlikely that they are a key factor to initiate the divergence leading to testicular or ovarian differentiation in R. rugosa.
Sry（Y染色体上的性别决定区域）是哺乳动物睾丸分化所必需的。除了Sry之外，其他基因如WT1、Fgf9、Dax1、Dmrt1和Sox9也被广泛认为参与脊椎动物的性别决定。然而，这些基因在两栖动物性别决定过程中的作用仍不清楚。本研究旨在检测WT1和Fgf9在两栖动物发育性腺中的表达。我们首先从皱皮蛙中分离出WT1 cDNA。与小鼠中的WT1一样，皱皮蛙WT1显示出2种异构体；即，其中一种在第三和第四锌指之间包含另外3个氨基酸KTS。然而，在皱皮蛙WT1中未发现哺乳动物WT1外显子5中的17个氨基酸，乌龟和鸡的情况也是如此。两种异构体（+KTS、-KTS）的mRNA在成年青蛙的肺、肾和睾丸中均有检测到，但在卵巢和肌肉中未检测到。这两种异构体在第23阶段的胚胎中首次表达。此后，在性别决定过程中，两性中与中肾相连的性腺中的表达保持恒定。接下来，我们分离出编码208个氨基酸的皱皮蛙Fgf9 cDNA。Fgf9的氨基酸序列与鸡、小鼠和人类的Fgf9的相似性大于92%，表明Fgf9在脊椎动物类群中高度保守。Fgf9在成年青蛙的卵巢中强烈表达，但在肺中表达较弱。相反，在肾、睾丸和肌肉中几乎未检测到Fgf9 mRNA。此外，在皱皮蛙性别决定过程中，Fgf9未表现出性别二态性表达模式。综合这些结果表明，WT1和Fgf9在性别决定之前的未分化性腺中均有表达，且雄性和雌性之间的表达没有差异。因此，它们似乎不太可能是引发导致皱皮蛙睾丸或卵巢分化差异的关键因素。
