2018 Fiscal Year Research-status Report
Discovery of genes essential for male fertility using CRISPR/Cas9-based genome editing technology
| Project/Area Number |
18K16735
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| Research Institution | Osaka University |
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Principal Investigator |
Lu Yonggang 大阪大学, 微生物病研究所, 特任研究員(常勤) (00817033)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | Sperm / Spermatogenesis / CRISPR/Cas9 / Testis / Genome editing / Mouse model / Male fertility / Reproductive biology |
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| Outline of Annual Research Achievements |
In the current project, to screen for candidate genes that may have essential role in male fertility, the applicant generated more than 10 knockout mice lacking testis-enriched genes using CRISPR/Cas9 system. About half of the knockout male mice show normal fecundity, suggesting the genes are dispensable for male reproduction, while the other ones exhibit inability to sire offspring after mating with wildtype females for more than two months.
The genes that are not essential for male fertility were summarized with other dispensable testis-enriched genes in a manuscript and have been submitted to a peer-reviewed international journal (currently under review). This paper is expected to be published around mid 2019.
The genes that are indispensable for male fertility are currently under phenotypic and mechanistic studies. The genes were found to play important roles in sperm-oocyte fusion, acrosome reaction, sperm motility and spermiogenesis, respectively. We believe that these genes will be strong candidates for targets of non-hormonal male contraceptives, biomarkers for diagnosis of idopathic male infertiltiy and keys for acquiring profound insights into the underlying mechanisms of important events in reproduction. We expect to publish the mechanisitic studies of at least two essential genes and share the outcomes with researchers in international conferences in mid 2020.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In the original application for the Kakenhi, the applicant proposed to conduct several rounds of in vivo screening for essential male factors by continuously generating knockout mice using CRISPR/Cas9 system. During 2018, the applicant generated more than 10 knockout mouse lines in two rounds of screening and discovered that about half of the mouse lines show phenotype in male mice. Therefore, instead of continuing to knockout more genes, the applicant decides to focus on the genes that are confirmed to be indispensable for underpinning the normal male fertility.
About the status of studying the functions of these male factors, the applicant finished the majority of the phenotypic analyses and started to conduct mechanistic studies to uncover the molecular network underlying essential reproductive events, such as acrosome reaction, capacitation, sperm-oocyte fusion, spermatogenesis and spermiogenesis.
The overall progress of the entire project is rather optimistic. However, the mechanistic studies are usually more difficult and complex compared with phenotypic analyses. Using the research techniques and knowledge background, the applicant will endeavour to solve the research questions and produce satisfactory publications with indepth insights into the mechanisms underpinning male fertility.
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| Strategy for Future Research Activity |
For studying the essential male factors, the applicant will try the best to uncover the molecular mechanisms using the following strategies.
1) Although male infertility consists of an extreme heterogeneous spectrum of phenotypic presentations, we constantly find same or similar phenotypes in previous studies in male reproduction. Regardless of the depth or reliability of the knowledge provided by those articles, we can make rather rational hypotheses and determine our research direction based on the information. 2) Since the current technologies possessed by our laboratory are sometimes not enough to solve the research questions, we will continuously introduce new techniques to our lab, such as BioID and yeast two-hybrid to study interacting proteins. In case of lacking important instruments, we also have trustworthy collaborators to cope with such difficult circumstances. 3) Apart from analyzing the underlying mechanisms of the essential factors, we also look into the potential of these genes as targets of non-hormonal male contraceptives and biomarkers for diagnosis of idopathic male infertiltiy. We collaborate with experts in designing small molecules targeting and inhibiting specific proteins that are important for male fertility. We also have collaborators who can help us to screen for homozygous mutantions of genes of interest in humans.
We plan to submit at least two papers on the essential male factors in 2020.
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