Engineering Immortalized Bovine Granulosa Cells Using a Triple-Gene Approach: Mutant CDK4, Cyclin D1, and TERT

Area of Science:

• Reproductive Biology
• Cell Biology
• Livestock Science

Background:

• Advancing cattle reproductive technologies is crucial for enhancing livestock productivity and genetic potential.
• Primary bovine granulosa cells (bGCs) have limited in vitro proliferation, hindering research into follicular development mechanisms.
• Bovine granulosa cells are vital for oocyte development, producing essential hormones and growth factors.

Purpose of the Study:

• To overcome the limited proliferative capacity of primary bGCs.
• To establish a stable cell model for investigating follicular development and reproductive biotechnologies in cattle.
• To enable large-scale studies on factors influencing oocyte development and follicular growth.

Main Methods:

• Co-expression of human mutant cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and telomerase reverse transcriptase (TERT) in bGCs using lentiviral vectors.
• Assessment of proliferative lifespan, cell cycle activity, and telomerase activity of the immortalized cells (bGCs-K4DT).
• Evaluation of bGC-specific marker (aromatase) expression in the immortalized cell line.

Main Results:

• The immortalized bGCs-K4DT exhibited extended proliferative capacity, exceeding 100 population doublings without senescence.
• Transduced cells showed enhanced cell cycle activity and increased telomerase activity compared to parental bGCs.
• Aromatase expression was retained in bGCs-K4DT, although at reduced levels, indicating partial preservation of granulosa cell characteristics.

Conclusions:

• The immortalized bGC model (bGCs-K4DT) provides a robust platform for studying bovine follicular development.
• This cell line facilitates research into bioactive components, such as platelet-rich plasma (PRP), for follicular activation and growth.
• The development supports advancements in livestock reproductive technologies and cattle breeding programs.