The Expression And DNA Methylation Of Sox30 Gene In Testis Development Of Mice And Rats

BackgroundMany studies have shown that the epigenetic modifications play a key role in animal development. Among the epigenetic modifications, DNA methylation is currently the most and in-depth studied. At present, more and more evidences have indicated that DNA methylation maintained the developmental process of organism correctly by regulation the expression of tissue specific genes, and once the DNA methylation changes are abnormal, some diseases will occur frequently. Therefore, to study the epigenetic modification of the development related gene, will help to clarify the pathogenesis of major developmental diseases, and will provide important significance for the understanding of those diseases.Sox30(SRY-box containing gene 30), as a Sox transcription factor and the only member of group H, has been characterized merely in a few species. In the previous studies, Sox30 sequences were not available from the non-mammal animal species, and so many scholars speculated that Sox30 was specific to mammals. In the recent study, it was reported that Sox30 existed widely throughout the animal kingdom rather than specific to mammals. These results mean that Sox30 may be more important than that it was previously thought in animal kingdom. It has been shown that Sox30 gene is expressed in testis germ cells and sertoli cells, suggesting that it may be involved in gonad development, spermatogonial differentiation and spermatogenesis. At present, we have found for the first time that Sox30 is a novel epigenetic silenced tumor gene(regulated by DNA methylation) in lung cancer, and the mechanism of its function is to promote tumor cell apoptosis with inhibiting tumor cell proliferation by transcriptional activation of p53 signaling pathway. However, the specific expression of Sox30 and whether the expression pattern is regulated by DNA methylation in the development of mice and rats are still unclear.Objectives(1) To detect the expression pattern of Sox30 gene in various tissues of adult mice and rats.(2) To detect the expression pattern of Sox30 gene in mouse and rat developmental embryo, and in the developmental ovarian and testicular tissues at different stages.(3) To detect the methylation status of Sox30 gene in various tissues of adult mice and rats.(4) To detect the methylation status of Sox30 gene in mouse and rat embryo and developmental ovarian and testicular tissues at different stages.(5) To analyze the relationship between Sox30 expression and DNA methylation in the developmental testis of mice and rats.Methods(1) The expression and location of Sox30 gene in adult various tissues and developmental testis of mice and rats were detected by reverse transcription-polymerase chain reaction(RT-PCR), quantitative Real-time PCR(RT-q PCR), western blot(WB) and immunohistochemistry(IHC);(2) The DNA methylation status of Sox30 gene in adult various tissues and developmental testis of mice and rats were detected by methylation specific PCR(MSP) and bisulfite sequencing PCR(BSP);(3) The relationship between Sox30 expression and DNA methylation were treated and detected by demethylation experiment with 5-Aza-C treatment.Results(1) The tissue distribution of Sox30 gene in different tissues(including ovary, womb, epididymis, testis, heart, brain, liver, kidney, spleen, pancreas, lung, muscle, intestine, pituitary, blood and hippocampus) from adult mice and rats showed that the expression level of Sox30 gene was the highest in testis, and then in epididymis and lung, and the expression of Sox30 in testis was much higher(nearly one thousand fold change) than that in the epididymis and lung of adult mice. The expression level of Sox30 gene was also the highest in testis, followed by in epididymis and blood, and the expression of Sox30 in testis was much higher than that in epididymis and blood of adult rats. These results indicated that Sox30 gene played an important role in testis development and spermatogenesis of adult mice and rats.(2) The ontogeny analysis of Sox30 in mouse and rat embryo and developmental ovarian and testicular tissues at different stages(including embryonic stage E8.5, E10.5, E12.5, E14.5, E15.5, E19.5 embryo, day after birth 1d, 4d, 7d, 10 d, 15 d, 20 d, 30 d, 40 d, 60 d and 73 d ovarian and testicular tissues) showed that Sox30 expression was low in E8.5, E10.5 and E12.5 embryo, significantly much higher in E14.5 embryo. Sox30 expression increased gradually in testicular tissues in day after birth 1d to 60 d dependent on mouse age, especially in 15 d to 20 d, it was increased sharply, and the expression level reaches the peak in 60 d testes of the developmental mice. Sox30 expression was low in E8.5, E10.5, E12.5, E15.5 and E19.5 embryo, and increased gradually in testes in day after birth 1d to 40 d dependent on rat age, and reached the peak in 60 d teste. Sox30 expression was increased sharply in testes from 15 d to 20 d, and the expression reaches the peak in 40 d testes of the developmental rats. Sox30 expression was very low in ovaries of both mice and rats from 1d to 73 d. These data suggest that Sox30 gene plays a key role in sex differentiation and testis development of mice and rats.(3) The experiment results to ascertain which population of cells expressing Sox30 in testis of mice showed that Sox30 was not only expressed in germ cells, but also expressed in sertoli cells of mouse testis, and the expression of Sox30 was higher in germ cells than that in sertoli cells. The results were further verified by detecting Sox30 expression in separated and purificated germ cells and sertoli cells.These data show that Sox30 plays important roles in the testis development of mouse.(4) The analysis for DNA methylation of Sox30 in different tissues from adult mice showed that Sox30 was in a DNA methylation status in ovary, heart, brain, liver, kidney, spleen, pancreas, muscle and intestine of mice, but in a de-methylation status in epididymis, testis and lung. Considering the distribution of Sox30 expression in the above, the methylation of Sox30 gene was closely related to its expression in adult mouse tissues.(5) The analysis for DNA methylation of Sox30 in developmental testicular tissues at different stages showed that the degree of Sox30 DNA methylation was decreased gradually dependent on age until the de-methylation happens during the development of testis. At the same time, Sox30 expression was increased gradually dependent on age, until the peak is reached. These results indicate that Sox30 expression is significantly affected by the degree of methylation in the embryo and testis development of mouse and rat.(6) To confirm association between Sox30 methylation and its expression, we detected Sox30 expression level and methylation status in GC2(from germ cells), TM3(from leydig cells) and TM4(from sertoli cells) cell lines treated with or without 5-aza-C. The methylation and low expression of Sox30 were found in all three cell lines. After 5-aza-C treatment, de-methylation and re-expression of Sox30 were detected. These data suggest that the expression of Sox30 gene is directly regulated by DNA methylation.ConclusionIn the testis development, Sox30 expression was regulated by DNA methylation strictly, suggesting this regulation mode involved in the testis development in mice and rats. Thus, Sox30 is an important testis development related gene, and worthy for further study. This study will provide evidence for exploration the function and molecular mechanism of Sox30 in testis development.