| Summary: | 碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 99 === In zebrafish, the sex chromosome has not been found and little is known about the mechanisms about sex determination and the sexually dimorphic gonad differentiation. Most of studies about gonad differentiation focus on the stages after three weeks when oocytes are already formed. However, recent study in our lab shows that the earliest sign of sexual dimorphism starts at 12 dpf when male and female gonads start to have different sizes. This indicates that this earlier period is the critical stage for gonad development, and that at this time the growth rate of germ cells and the expression of certain genes should start to be different.
I examined the expression of two genes in the gonad by in situ hybridization. One is dmrt1a, which is an evolutionarily conserved gene involved in sex determination and sex differentiation in many species. I found that it started to be expressed in zebrafish gonad at 10 dpf, while dimorphic expression level was observed after 10 dpf. Furthermore, the expression of dmrt1a was always weaker in 17α-methyltestosterone-treated gonads. The other one is gsdf, which is a novel member of TGF-β superfamily. It started to be expressed at 8 dpf, while dimorphic expression level was observed after 12 dpf. The expression levels of gsdf were always weaker in 17α-methyltestosterone-treated gonads. Moreover, in dnd morphants, in which germ cells were depleted, the expression levels of gsdf were also weaker. This indicates that germ cells may affect the expression of gsdf during early gonadal development.
To understand the relationship between germ cell number and gonad development, I examined the number of germ cell by counting vasa-positive germ cells, and found germ cell number started to increase at 8 dpf. In order to trace germ cells in individual larva, mCherry-nanos 3’UTR transgenic fish with red fluorescent germ cells were generated. Germ cell proliferation was also observed after 8 dpf by counting the number of red fluorescent germ cells. Furthermore, larvae could be segregated into two groups according to the number of red fluorescent germ cells after 8 dpf. One group has higher germ cell number, while the other group has lower germ cell number, likely representing the female and male groups. These results indicate that during this critical period, the growth rates of germ cells are dimorphic and certain genes are dimorphically expressed between male and females. These differences may further lead to the development toward sexual dimorphic gonad differentiation pathway in each individual larva
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