Deciphering the cellular and molecular mechanisms underlying mammalian sperm-egg fusion
| Project/Area Number |
22K15103
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 44010:Cell biology-related
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| Research Institution | Osaka University |
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Principal Investigator |
Lu Yonggang 大阪大学, 免疫学フロンティア研究センター, 特任助教(常勤) (00817033)
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| Project Period (FY) |
2022-04-01 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2022)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2023: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
Fiscal Year 2022: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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| Keywords | Fertilization / Sperm-egg fusion / Spermiogenesis / piRNA biogenesis / Male infertility / Male reproduction / Genome editing / Cell biology / Sperm |
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| Outline of Research at the Start |
The study is subdivided into three major sections: 1) large-scale screening of molecules implicated in the sperm-egg fusion using the CRISPR/Cas9-based knockout strategy in mice; 2) in-depth functional analyses of the gamete fusion-required proteins using genetics, proteomics, and cell biology techniques; 3) detailed investigation of the gamete fusion machinery from different perspectives, such as species-specific recognition at sperm-egg fusion and the plasma membrane block to polyspermy.
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| Outline of Annual Research Achievements |
In the fiscal year of 2022, I published three research articles and one review article as the first author.
In Tang, Lu et al. 2022 PNAS, we resolved the crystal structure of human TMEM95 by X-ray crystallography and generated monoclonal antibodies against human TMEM95 and IZUMO1. To investigate the molecular function of TMEM95, we exploited the nature that human sperm can fertilize zona-free golden hamster eggs. Using Fab fragments of the antibodies, we found that IZUMO1 Fabs ablate sperm-egg binding, in corroboration with our recent finding in mice and rats (Matsumura et al. 2022 Front Cell Dev Biol). In contrast, TMEM95 Fabs do not affect sperm-egg binding, but reduce sperm-egg fusion, suggesting that TMEM95 plays a role distinct to IZUMO1. Further, we engineered TMEM95-Fc fusion protein and discovered that the recombinant protein binds the hamster egg plasma membrane, indicating that TMEM95 has an egg receptor.
In Lu et al. 2023 PNAS, we discovered that 1700029I15Rik, a type II transmembrane protein localized to the ER, regulates the biosynthesis of acrosomal membrane glycoproteins required for sperm-egg interaction. This pathway is independent from Calmegin-mediated biosynthesis of sperm plasma membrane proteins, suggesting that acrosomal and plasma membrane proteins are processed in a spatiotemporal manner.
Additionally, in Lu et al. (2023) Andrology, we reported that ADAD2, in association with other RNA-binding proteins (e.g., MIWI, RNF17), regulates piRNA biogenesis in testis. Moreover, ADAD2 forms novel granules with and stabilizes RNF17 in pachytene spermatocytes.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Despite the difficulty in the mechanistic studies of sperm-egg fusion in mammals, in the fiscal year of 2022, I have successfully published two PNAS papers that 1) elucidated the detailed mechanism underlying TMEM95-mediated sperm-egg plasma membrane fusion and 2) uncovered a novel pathway underpinning the biosynthesis of multiple acrosomal membrane proteins required for sperm-egg interaction. These high-impact works 1) significantly improve our understanding of mammalian fertilization and sperm-egg fusion; 2) provide novel strategies and methodologies for the functional analyses of other sperm fusion factors; 3) facilitate diagnosis of idiopathic male infertility; and 4) support rational development of non-hormonal male contraceptives.
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| Strategy for Future Research Activity |
In this year, I will 1) screen sperm-egg fusion-related factors, as well as the bona fide fusogen, through in silico structural analysis and CRISPR/Cas9-mediated gene knockout in mice; 2) survey the egg surface receptor of TMEM95 by affinity purification or library screening; 3) study how the other known sperm fusion factors facilitate gamete fusion and whether they have binding partners on the egg plasma membrane; and 4) investigate how a multitude of sperm acrosomal membrane proteins (and egg receptors) collectively underpin sperm-egg fusion.
In addition, I will further elucidate the molecular function of 1700029I15Rik (I15Rik) by 1) investigating whether depletion of I15Rik impairs the expression or functionality of key catalytic subunits of oligosaccharyltransferase (OST), such as STT3A and STT3B; 2) identifying substrate proteins that transiently interact with I15Rik by proximity labeling; 3) interrogating the feasibility of using I15Rik as a target for non-hormonal male contraceptive.
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Report
(1 results)
Research Products
(6 results)
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[Journal Article] ADAD2 functions in spermiogenesis and piRNA biogenesis in mice2023
Author(s)
Lu Yonggang、Nagamori Ippei、Kobayashi Hisato、Kojima‐Kita Kanako、Shirane Kenjiro、Chang Hsin‐Yi、Nishimura Toru、Koyano Takayuki、Yu Zhifeng、Casta?eda Julio M.、Matsuyama Makoto、Kuramochi‐Miyagawa Satomi、Matzuk Martin M.、Ikawa Masahito
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Journal Title
Andrology
Volume: 11
Issue: 4
Pages: 698-709
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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