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Mouse Sertoli cells isolation by lineage tracing and sorting
Helena D Zomer1, Prabhakara P Reddi1
1Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois.
Molecular Reproduction and Development
|August 1, 2020
Summary
Researchers developed a new method using lineage tracing and fluorescence-activated cell sorting (FACS) to isolate pure mouse Sertoli cells, overcoming challenges in obtaining uncontaminated primary cultures for spermatogenesis research.
Area of Science:
- Reproductive Biology
- Cell Biology
- Genetics
Background:
- Sertoli cells are crucial for spermatogenesis, supporting germ cell development.
- Obtaining pure Sertoli cell cultures is difficult due to cellular heterogeneity and contamination.
- Existing isolation methods yield enriched, not pure, Sertoli cell populations.
Purpose of the Study:
- To develop a novel method for isolating pure mouse Sertoli cells.
- To utilize lineage tracing and fluorescence-activated cell sorting (FACS) for enhanced purity.
- To establish a reliable technique for studying Sertoli cell function.
Main Methods:
- Generated transgenic mice expressing tdTomato specifically in Sertoli cells using the Amh-Cre line.
- Isolated primary Sertoli cell cultures from prepubertal and adult mice.
- Employed fluorescence microscopy, flow cytometry, and FACS for cell identification and sorting.
- Validated cell purity using markers like vimentin and α-smooth muscle actin (α-SMA).
Main Results:
- Initial cultures showed ~79% tdTomato+ cells, with vimentin present in most cells and α-SMA in contaminating cells.
- FACS sorting resulted in virtually 100% tdTomato+ cells, confirming high purity.
- Vimentin was confirmed as a marker for Sertoli cells, while α-SMA identified peritubular myoid cells.
- Prepubertal mice provided a higher yield of Sertoli cells compared to adults.
Conclusions:
- Lineage tracing combined with FACS is an effective strategy for obtaining pure murine Sertoli cell populations.
- This method addresses the limitations of current protocols for Sertoli cell isolation.
- The purified Sertoli cells are suitable for detailed functional studies in reproductive biology.
