2020 Fiscal Year Research-status Report
A study of newly-identified testis-specific long non-coding RNAs in mouse spermatogenesis
| Project/Area Number |
20K18103
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| Research Institution | Nippon Medical School |
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Principal Investigator |
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| Project Period (FY) |
2020-04-01 – 2022-03-31
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| Keywords | testis-specific lncRNA / mouse spermatogenesis / long non-coding RNA |
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| Outline of Annual Research Achievements |
Long non-coding RNAs (lncRNAs) are regulators in various cellular process such as spermatogenesis process in male testis. Spermatogenesis means differentiation of male germ cells to produce mature sperm which is important for male reproduction. We histochemically identified new testis-specific lncRNAs (1700101O22Rik lncRNA and 1700108J01Rik lncRNA) (Song and Chaw Kyi-Tha-Thu et al., Histochem Cell Biol 149: 517-527, 2018, co-first author, Grant-in-Aid for Young Scientists B, 2017-2018). Our results showed that these two lncRNAs were localized mainly in the spermatocytes, but not in Sertoli cells. It suggests that these lncRNAs may be involved in cellular process such as cell differentiation or apoptosis during mouse spermatogenesis. The main purpose of this study is to perform functional analysis of these new lncRNAs and its gene regulatory network related to male reproductive system. I tried GFP-tagged lentivirus microinjection into the postnatal mouse testis to induce viral-mediated transfection for gene manipulation of target lncRNAs. However, the viral transfection was not successful and the transfection only occurred in Sertoli cells, in which the target lncRNAs were not expressed. Therefore, I plan to switch my initial proposal method from in vivo functional study to in vitro functional analysis using mouse spermatocyte cell lines.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
In vivo viral transfection method is a challenging technique for me. I needed to do several preliminary practices and skill for the microinjection of rete testis area in postnatal pups. After I could successfully perform the microinjection, I used GFP-tagged lentivirus to induce the viral transfection into my target gene expressed spermatogenetic cells (especially spermatocytes). The main problem of this in vivo functional study is that transfection did not occur in spermatocytes. Therefore, I tried several trials of transfection methods such as using different kinds of virus, and electroporation method with different voltages. Unexpectedly, I could not get an efficient result for transfection of my target cells. This is the main reason why there was a delay in progress of this research.
I changed to a new workplace from 1st April 2021. I hope that I can harmonically perform my duties in both medical education and research. I will try my best to continue my research at current workplace.
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| Strategy for Future Research Activity |
I plan to perform in vitro functional analysis using mouse spermatogenetic cell lines in order to investigate the functions of target lncRNAs in mouse spermatogenesis. I ordered a mouse spermatocyte cell lines which is called GC-2 cell lines (GC-2spd(ts), CRL-2196) from ATCC. I chose the GC-2 cell lines because in situ hybridization results showed that the newly identified lncRNAs (1700101O22Rik lncRNA and 1700108J01Rik lncRNA) were highly expressed in the cytoplasm of round spermatocytes. I have already prepared the culture medium, other reagents for GC-2 cell culture, and lipofectamine 2000 reagents for cell trasnfection. At the same time, I have already designed siRNAs for target lncRNA and ordered two potential siRNAs to knockdown the target lncRNA efficiently. When the GC-2 cell lines arrive, I will start cell seeding, cell passaging, and check the efficiency of siRNAs in GC-2 cell lines. Then, I will transfect the siRNAs into the GC-2 cells and check by quantitative PCR. After successful transfection, I plan to do RNA sequencing to find out the target gene regulatory network related to the testis-specific lncRNAs during mouse spermatogenesis.
After that, I will present my data at both local and international conferences. My goal is to publish new findings, which is helpful for male reproductive system.
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