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Updated: Aug 7, 2026
In Vivo Microinjection and Electroporation of Mouse Testis
Published on: August 23, 2014
[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells]
Chun-Lin Wu1, Hong-Gang Li2,3, Cheng-Liang Xiong2,3
1Department of Obstetrics and Gynecology, Wuhan First Hospital, Wuhan, Hubei 430022, China.
Objective:
To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs.
Methods:
Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR.
Results:
Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes.
Conclusion:
Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.
Insights
Epididymosomes successfully delivered epididymis-specific mRNAs, Adam7 and Crisp1, into N2a and TM4 cells. These findings suggest epididymosomes can transfer genetic material, potentially influencing cellular function.
Area of Science:
- Reproductive Biology
- Cell Biology
- Molecular Biology
Context:
- Epididymosomes are vesicles released by the epididymis, carrying specific mRNAs and proteins.
- Understanding epididymosome cargo delivery is crucial for exploring their role in sperm maturation and function.
- Previous research has identified epididymis-specific genes like Adam7 and Crisp1, but their intercellular transfer mechanisms remain unclear.
Purpose:
- To investigate the delivery of mouse epididymis-specific mRNAs (Adam7 and Crisp1) into N2a and TM4 cells.
- To establish an experimental foundation for studying the functional roles of epididymal mRNAs.
- To confirm the presence and uptake of epididymosomes by recipient cells.
Summary:
- Epididymis-specific genes Adam7 and Crisp1 were detected in mouse and human sperm, but not testes.
- Fluorescently labeled mouse epididymosomes were shown to fuse with and be ingested by N2a and TM4 cells.
- RT-PCR confirmed the presence of Adam7 and Crisp1 mRNAs in N2a and TM4 cells post-incubation, and Western blot detected CRISP1 protein.
Impact:
- Demonstrates that epididymosomes can act as carriers for epididymis-specific mRNAs into somatic cells.
- Suggests potential for Crisp1 mRNA translation into functional proteins within recipient cells.
- Provides a basis for future research into the functional significance and therapeutic applications of epididymosome-mediated gene transfer.
