|
内容記述 |
Mammalian sex is determined by differentiation of the gonad (testis or<br />ovary), which governs phenotypic sex through the production of hormones. Initially,<br />embryonic gonads develop as bipotential gonad, and its sexual differentiation is<br />genetically controlled: depending on the presence or absence of the Y chromosome.<br />In the developing testis, male-specific patterning of the vasculature is induced by<br /><i>Sry</i>. Following the expression of <i>Sry</i>, endothelial cells are recruited vigorously to<br />the testis from mesonephros and a large artery is formed at the coelomic surface at<br />around embryonic day (E) 12.5 (coelomic vessel). Thereafter, the vessel branches<br />from the coelomic vessel and extends progressively between testicular cords. In<br />contrast, no such active cell migration is observed in the developing fetal ovary.<br />This male-specific vascular system that develops during fetal life is required for<br />efficient export of testosterone from the testis to masculinize the embryo. <br /> Forkhead (Fox) transcription factors carry a winged helix DNA-binding<br />domain that share homology with their founding member forkhead protein in<br /><i>Drosophila</i>. Phylogenic analysis of the forkhead domain consisting of<br />highly-conserved 100 amino acids led to placement of the family members into 20<br />subclasses, FoxA to FoxS. Fox proteins bind to consensus sequences, RYMAAYA<br />(R=A or G; Y=C or T; M=A or C), as a monomer. Regions other than the conserved<br />domain vary in terms of sequence and function. Some members act as<br />transcriptional activators while others as repressors. Probably as transcriptional<br />regulators, Fox genes are thought to play a variety of roles in fetal and adult tissues<br />and mutations in <i>FOX</i> genes have been linked to human diseases. <i>Fkhl18</i>, a<br />member of the <i>Fox</i> family, was originally identified by low-stringency screening of<br />mouse and human genomic libraries. <i>Fkhl18</i> has low homology to other members of<br />the Fox family, and is categorized under the <i>FoxS</i> subclass. However, its expression<br />and function remain to be examined. In the present study, she demonstrated that<br /><i>Fkhl18</i> was expressed in periendothelial cells and Sertoli cells of the developing<br />fetal testis. She then generated the <i>Fkhl18</i> knockout (KO) mouse to examine the<br />physiological function of the gene product. Interestingly, the KO fetuses displayed<br />affected testicular vasculature, suggesting that <i>Fkhl18</i> was involved in<br />development of the fetal testis vasculature system.<br /> <i>Fkhl18</i> KO mice displayed the following testicular abnormalities during<br />fetal life; 1) accumulation of blood cells in the central part of the fetal testis, 2)<br />presence of gaps, measuring 100-400 nm in diameter, between endothelial cells,<br />allowing leakage of injected carbon ink from the testicular vessels, and 3) aberrant<br />apoptosis of periendothelial cells. These features strongly suggest the importance of<br /><i>Fkhl18</i> expression in the periendothelial cells for development of the testicular<br />vascular system through direct and indirect regulation of the functions of<br />periendothelial and endothelial cells, respectively. The indirect function of <i>Fkhl18</i><br />indicates a functional interaction between endothelial and periendothelial cells.<br />The importance of interactions between the two cell types for vascular maturation<br />has been examined by gene knockout studies of signal transductions: <br /><i>angiopoietin-1/TIE-2 receptor and platelet-derived growth factor (PDGF)-BB/<br />PDGF receptor β (PDGFRβ)</i>. Unlike these KO mice, recruitment of eriendothelial<br />cells did not seem to be affected in the fetal testes of <i>Fkhl18</i> KO mice. Interestingly,<br />however, marked apoptosis of periendothelial cells was observed; with resultant<br />focal and transient loss of periendothelial cells. Since the expression of <i>Fkhl18</i> was<br />not detected in endothelial cells, the structural defect induced in endothelial cells<br />possibly resulted from weakened interaction with the affected or decreased <br />periendothelial cells.<br /> To further investigate the molecular mechanisms underlying proapoptotic<br />effect of <i>Fkhl18</i>, she demonstrated electrophoretic mobility shift assay (EMSA) and<br />reporter gene assay. Reporter gene assays revealed that Fkhl18 suppressed<br />transcription mediated by FoxO3a and FoxO4. Since EMSA showed that Fkhl18 had<br />ability to bind to consensus DNA binding sequence for <i>Fox</i>, it potentially repressed<br />transcription by competing for binding sites with other Fox proteins. Considering<br />the suppressive function of Fkhl18, it is interesting to note that <i>FoxOs</i> mediate<br />proapoptotic gene expression. For example, overexpression of <i>FoxOs</i> resulted in<br />apoptosis through direct induction of proapoptotic genes: <i>tumor necrosis<br />factor-related apoptosis-inducing ligand (TRAIL)</i> in prostate cancer, <i>bim (Bcl-2<br />interacting mediator of cell death)</i>, and <i>FasL</i>. Based on the results published so far, <br />she hypothesized that the marked apoptosis of periendothelial cells in <i>Fkhl18</i> KO<br />testes was caused by defective proapoptotic gene transcription, which was normally<br />attenuated by <i>Fkhl18</i>. As expected, <i>Fkhl18</i> suppressed transcription from <i>FasL</i> gene<br />promoter in cultured smooth muscle cells prepared form bovine blood vessels. <br /> In the present study, she focused on the function of <i>Fkhl18</i> during blood<br />vessel formation of the fetal testis; blood vessel development in the ovary remains to<br />be investigated. Likewise, she has not examined whether the blood vessels in tissues<br />other than the gonads are affected by <i>Fkhl18</i>. Considering that <i>Fkhl18</i> is expressed<br />in periendothelial cells of other tissues, the defects seen in the fetal testis could be<br />also seen in other tissues. However, obvious accumulation of blood cells was not<br />observed in any tissues other than the testis, strongly arguing against a major defect<br />of blood vessel development in these tissues. Together with the highest expression of<br /><i>Fkhl18</i> in the developing testis, it is conceivable that <i>Fkhl18</i> plays a unique role in<br />the development of the testicular vasculature system. |
要旨・審査要旨 (310.0 kB)
