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Mouse dynein axonemal intermediate chain 2: cloning and expression
1School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Abstract:
Ovarian follicular development is a complex process. Investigation of the mechanisms regulating the initiation of follicular growth, and the growth and differentiation of preantral follicles is of great interest. In an effort to clone follicular development-related genes, we selected a partial cDNA fragment by differential display reverse-transcription PCR using total RNA extracted from 5-day-old and 10-day-old mouse ovaries, and its open reading frame was obtained by rapid amplification of cDNA ends. Sequencing showed that the fragment is the mouse dynein axonemal intermediate chain 2 gene (Dnaic2), which has an 87% homology with human DNAI2, a candidate gene for primary ciliary dyskinesia. Northern and western analyses indicate that Dnaic2 produces an approximate 3 kb mRNA that is translated into an approximate 70 kDa protein. The mRNA is predominantly expressed in mouse ovary, testis, and lung. In mouse ovaries, Dnaic2 mRNA was detected at high levels in vivo on day 10, with a subsequent decrease on days 15 and 20, in adult and old ovaries. However, Dnaic2 expression was weak on day 5. Dnaic2 protein was localized on the surface of the oocyte. No obvious fluorescence signal was detected in primordial and primary follicles, while strong signals were detected on the oocyte surface of secondary and antral follicles, in particular for secondary follicles in day 10. These data suggest that Dnaic2 plays a role in ovarian follicular development.
Insights
Mouse dynein axonemal intermediate chain 2 (Dnaic2) gene expression is crucial for ovarian follicular development. Dnaic2 protein is found on oocyte surfaces, particularly in developing follicles, suggesting a key regulatory role.
Area of Science:
- Reproductive Biology
- Molecular Genetics
- Cell Biology
Background:
- Ovarian follicular development is a complex biological process.
- Understanding genes regulating follicular growth and differentiation is critical.
- Previous research has focused on identifying key genes involved in these processes.
Purpose of the Study:
- To identify novel genes involved in ovarian follicular development.
- To clone and characterize a gene potentially regulating follicular growth.
- To investigate the expression pattern and localization of the identified gene in the mouse ovary.
Main Methods:
- Differential display reverse-transcription PCR was used to identify candidate genes.
- Rapid amplification of cDNA ends was employed to obtain the full open reading frame.
- Northern and western blot analyses were performed to assess gene and protein expression.
- Immunofluorescence was used to determine the protein's subcellular localization within ovarian follicles.
Main Results:
- A mouse dynein axonemal intermediate chain 2 gene (Dnaic2) was identified, showing homology to human DNAI2.
- Dnaic2 mRNA is predominantly expressed in the ovary, testis, and lung.
- Ovarian Dnaic2 expression levels varied with age, peaking around day 10 and decreasing thereafter.
- Dnaic2 protein was localized to the oocyte surface, with increased presence in secondary and antral follicles.
Conclusions:
- Dnaic2 is expressed in a stage-specific manner during ovarian follicular development.
- The localization of Dnaic2 protein on the oocyte surface suggests a role in oocyte-related processes.
- These findings indicate that Dnaic2 plays a significant role in regulating ovarian follicular development.
