Retinoid signals to control spermatogonial stem cells differentiation

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K16027
• Research Institution: Nagoya City University
• Principal Investigator: シャウキ ホッサム 名古屋市立大学, 医薬学総合研究院(医学), 助教 (70829738)
• Project Period (FY): 2019-04-01 – 2021-03-31
• Keywords: spermatogenesis / Mafb / transcription factor

Outline of Annual Research Achievements

The project aimed to identify the vitamin A downstream targets that lead to induction of spermatogonial stem cell differentiation, which will contribute to treating infertile patients who are not responding to vitamin A treatment itself due to mutated downstream targets. In FY2019, we identified two vitamin A targets; MafB and c-Maf. We then generated the floxed alleles of each gene independently and then lines were mated to obtain double conditional knockout mice, in which Cre recombinase was activated upon tamoxifen treatment. We induced Cre activation at 6 weeks old mice and mice tissues were collected and the deletion of both genes were confirmed. In the second year of this project, we are going to analyze the fertility of the 2 Mo (young) and 8 Mo (old) male mice upon deletions.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The plan of this research are to (1) generate the MafB/c-Maf single and double knockout mice, (2) analyzing whether Mafb/c-Maf compensate each other to fully induce the first spermatogenetic cycle under the control of retinoic acid using the KO mice, (3) rescue vitamin A deficient mouse phenotype by lentivirus overexpressing of Mafb/c-Maf. The first task was achieved during the first year (FY2019) while the second and third tasks are going to be elucidated during the second year (FY2020). the project was progressing rather smoothly in FY2019 to generate the double cKO. However, we noticed that the DKO developed urinary proteinuria after 2 weeks of Cre activation, a sign of kidney dysfunction, therefore we are also aiming to analyze the kidney abnormality caused by Mafb/c-Maf deletion.

Strategy for Future Research Activity

FY2020 is the second year of the research, and we will promote the research as following (1) analyze the physiology and histology of single and double MafB/c-Maf cKO testis, (2) perform fertility test of the KO males, (3) perform RNA-seq from the DKO Sertoli cells to identify the secreted proteins regulated by Maf transcription factors, (4) rescue vitamin A deficient mouse testis by overexpressing of Maf factors. (5) analyze the kidney abnormality caused by double Mafb/c-Maf deletions.

Causes of Carryover

令和元年度に予定したin vitroの実験が一部令和2年度にずれ込んだため。ただし、全体の実験計画に影響はなく、令和2年度末までに、予定の研究を終えることができる見込みである。

Research Products

• [Journal Article] EFCAB2 is a novel calcium-binding protein in mouse testis and sperm. (2019)
  • Author(s): Shawki HH, Ishikawa-Yamauchi Y, Kawashima A, Katoh Y, Matsuda M, Al-Soudy AS, Minisy FM, Kuno A, Gulibaikelamu X, Hirokawa T, Takahashi S, Oishi H.
  • Journal Title: PLoS One Volume: 14 Pages: e0214687
  • DOI: 10.1371/journal.pone.0214687
  • Peer Reviewed / Open Access / Int'l Joint Research