2018 Fiscal Year Research-status Report
Understanding flagellum formation in mouse spermatozoa
| Project/Area Number |
18K14715
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
CASTANEDA JULIO 大阪大学, 微生物病研究所, 特任助教(常勤) (00791659)
|
|---|
| Project Period (FY) |
2018-04-01 – 2020-03-31
|
| Keywords | spermatogenesis / flagellum / cilia / sperm function / fertility / mice / CRISPR/Cas9 |
|---|
| Outline of Annual Research Achievements |
I am interested in understanding the mechanisms that form and regulate flagella. Flagella are modified cilia, a cellular organelle important for signaling and beating movement in many organs. In fact, defects in cilia formation and regulation lead to syndromic diseases termed ciliopathies that affect multiple organs such as the brain, kidneys, and reproductive system. We use the sperm flagellum of the mouse to understand the mechanisms of flagellum formation and function in mammals. From genetic approaches from our laboratory and literature searches, I have identified 3 key genes that may play a role in flagellum formation and function (Flagellum Proteins 1-3). Using the CRISPR/Cas9 system, I have successfully deleted the coding regions of Flagellum Proteins 1 and 2 in mouse zygotes and generated mosaic founder mice. From these founder mice, I was able to obtain F1 heterozygous mice carrying the deletion. My next step is to breed these F1 mice to generate F2 homozygous null mice to begin to characterize the function of these two proteins in the flagellum. I am also currently in the process of deleting Flagellum Protein 3 using CRISPR/Cas9. This work will provide new insights in the mechanisms that regulate sperm flagellum, and in cilia function.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
I designed guide RNAs to remove the coding region of our 3 genes of interest. Two of the genes were deleted in the first trial to generate mosaic female founder mice. From these female mice, I obtained F1 heterozygous mice. I am currently working on deleting the third gene.
|
| Strategy for Future Research Activity |
My future plan is to mate the F1 heterozygous mice to generate F2 homozygous null males. From the null males, I will determine whether these genes are required for fertility with mating tests. For the mating tests, individual KO males will be paired with wild-type females. Copulatory plugs will be monitored and the number of pups sired will be counted. I will then characterize sperm function to determine if there are any defects in the flagellum of these cells using Computer Assisted Sperm Analysis. I will also look at spermatogenesis via histology and by staining for spermatogenesis markers to see if there are any defects in this process. Lastly, I will insert epitope tags into the endogenous loci of these genes so that we can perform pulldown experiments to understand the function of these genes.
|
