Expression Analysis Of Five Related Genes In The Development Of Giant Salivary Gland

Gonadal development is one of the hotspots in the developmental biology of today.The studies on the related genes of sex determination and gonadal development have had a deeper understanding in mammals and fish,while the research on amphibians is very limited.This study was conducted to examine the expression of five sex-related genes in gonadal development of amphibians.In this study,Chinese giant salamander(Andrias davidianus),a unique Urodele Amphibian in China,was selected as the study object.And based on the differentially expressed genes screened from the high-throughput transcriptome sequencing data of gonads in Chinese giant salamander,five sex-related genes were selected as the target genes:Dmrtl,Fgf9,Fsh-R,Wnt4 and Foxl2.The semi-quantitative RT-PCR was performed to detect the expression characteristics of five target genes in testis or ovary of the 24 months old Chinese giant salamander.And In situ hybridization was used to identify the localization of Dmrtl and Fgf9 in the testis and ovary of the 18 and 24 months old Chinese giant salamander.It aims to explore the role and molecular mechanisms of the five sex-related genes in gonadal development of the Chinese giant salamander,and accumulate basic biological data for building the gene regulatory network related to sex determination and differentiation and discussing gonadal development of the Chinese giant salamander,as well as provide a theoretical basis for the racial continuation of this ancient species and sustainable and healthy development of the Chinese giant salamander breeding industry.The main results and conclusions are as follows:1.The partial cDNA sequence of Dmrtl,Fgf9,Fsh-R,Wnt4 and Foxl2 were frist amplified in the Chinese giant salamander.2.RT-PCR results showed that Dmrtl was expressed in the testis and ovary of 24 months old Chinese giant salamander,but the expression of testis was significantly higher than that of ovary.And the results of in situ hybridization showed that,for the testis,the strong positive expression of Dmrtl mRNA in 18 months old was mainly in the low differentiated spermatogonia and somatic cells,and the positive expression was mainly in the somatic cells of the connective tissue in the middle part of the testis and the spermatogonia of the seminiferous lobules.The strong positive expression of Dmrtl mRNA in 24 months old was mainly in the spermatogonia and somatic cells of immature region,and the positive expression was mainly in spermatogonia and interstitial cells,and no positive expression was found on spermatozoa.For the ovary,the positive expression of Dmrtl mRNA in 18 months old was mainly expressed in the follicular cells,the weak positive expression of Dmrtl mRNA in 24 months old was also mainly expressed in the follicular cells and a very weak expression around the oocyte nucleus.That is to say,the positive expression of Dmrtl mRNA in the testis of 18 and 24 months old were stronger than that of ovary.The results revealed that Dmrtl gene plays an important role in the regulation of testis development and spermatogenesis of the Chinese giant salamander,especially plays an important role in the proliferation of spermatogonia and the maintenance of testis function,and may play a certain role in the oogenesis.3.RT-PCR results showed that Fgf9 was expressed in ovary and testis of 24 months old Chinese giant salamander,but the expression in ovary was stronger than that in testis.And the results of in situ hybridization showed that,for the ovary,the positive expression of Fgf9 mRNA in 18 months old was mainly in follicular cells,and the strong positive expression in 24 months old also mainly in follicular cells,and weakly expressed around the oocyte nucleus.For the testis,the strong positive expression of Fgf9 mRNA in 18 months old was mainly in the somatic cells of connective tissue in the upper part of the testis,and the positive expression was mainly in spermatogonia of seminiferous lobules.The positive expression of Fgf9 mRNA in 24 months old was mainly in the spermatogonia and interstitial cells,and no positive expression was found on spermatozoa.That is,with the gonadal development of the Chinese giant salamander,the expression of Fgf9 in the testis was gradually decreasing,while the expression in the ovary was gradually increasing.Based on the above results,the Fgf9 gene is closely related to the gonadal development of the Chinese giant salamander,and is not only closely related to the ovary development of the Chinese giant salamander,but also may be related to the testis development.4.The results of paraffin section showed that,there were no spermatozoa in the 18 months old Chinese giant salamander,but the seminiferous lobules of mature region gathered more clusters of spermatozoa in 24 months old.And the follicular cells of ovary in the 24 months old Chinese giant salamander were significantly larger than those in 18 months old.In addition,RT-PCR results showed that Fsh-R was expressed stronger in testis,while the ovarian expression is weaker.Based on the above results,the Fsh-R gene has a stimulating effect on the spermatogenesis of the Chinese giant salamander,and may also has a certain role for promoting the ovarian follicle development.5.The results of RT-PCR showed that both Wnt4 and Foxl2 were highly expressed in ovary of 24 months old,and almost none expression was found in testis.Based on this,Wnt4 and Foxl2 genes may be involved in regulating ovary development of the Chinese giant salamander.