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Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye
Published on: January 14, 2021
Differential regulation of gene expression in mouse spermatogonial cells after blocking c-kit-SCF interaction with
Arun P Sikarwar1, Murali K Rambabu, K V R Reddy
1Department of Immunology, National Institute for Research in Reproductive Health, Parel, Mumbai, India - 400 012.
Abstract:
c-Kit, the gene product of the W locus is a receptor tyrosine kinase that regulates the survival, growth and differentiation of spermatogonial cells (SGCs). Stem cell factor (SCF), the gene product of the steel (Sl) locus is the ligand for c-kit. Normal function of SGCs requires cross-talk between c-kit and SCF through which the receptor-ligand pair regulates the functions of SGCs. The implications of cross-talk between c-kit and SCF in regulating SGC function remains unclear due to the molecular complexity of this interaction. In the present study, we analyzed the interactions between c-kit and SCF in mouse primary SGCs after blocking the c-kit expression by c-kit siRNA and its effect on cell fate were determined using cDNA Expression Array and Real-time PCR. Immunofluorescence (IF) and western blot studies revealed that c-kit protein was detected in SGCs and knocked down to undetectable levels at 24 hr post transfection with 10 nM concentration of c-kit siRNA. We further demonstrated that expression of various genes involved in cell signaling, cell differentiation, apoptosis and cell cycle pathways was altered. SGC functions are affected by SCF signaling through c-kit receptor and this signaling appears to be important to maintain balance between cell proliferation and apoptosis along with the modulation of inflammatory responses of SGCs. To the best of our knowledge, this is the first report that identifies the putative molecular pathways in murine SGCs in response to specific blocking of c-kit-SCF interactions by siRNA. In conclusion, the present study may provide useful insights into siRNA function and hopefully aid in understanding the involvement of c-kit in the early events of SGC activities and spermatogenesis in mice.
Insights
Blocking c-kit expression in mouse spermatogonial stem cells (SGCs) with siRNA altered gene expression in cell signaling, differentiation, and apoptosis. This highlights c-kit
Area of Science:
- Reproductive Biology
- Molecular Cell Biology
- Spermatogenesis Research
Background:
- c-Kit receptor tyrosine kinase and its ligand, Stem Cell Factor (SCF), are crucial for spermatogonial stem cell (SGC) survival, growth, and differentiation.
- The precise molecular mechanisms of c-Kit/SCF cross-talk in regulating SGC function remain incompletely understood.
Purpose of the Study:
- To investigate the role of c-Kit in mouse primary SGCs by blocking its expression using siRNA.
- To elucidate the downstream molecular pathways affected by the inhibition of c-Kit/SCF interactions.
Main Methods:
- Utilized c-Kit siRNA to knockdown c-Kit expression in primary mouse SGCs.
- Employed Immunofluorescence (IF) and Western Blot to confirm c-Kit protein knockdown.
- Analyzed gene expression changes using cDNA Expression Array and Real-time PCR.
Main Results:
- Successfully reduced c-Kit protein to undetectable levels in SGCs within 24 hours post-transfection with c-Kit siRNA.
- Observed significant alterations in the expression of genes related to cell signaling, differentiation, apoptosis, and cell cycle.
- Demonstrated that SCF signaling via c-Kit is vital for balancing SGC proliferation and apoptosis, and modulating inflammatory responses.
Conclusions:
- This study provides the first identification of molecular pathways in murine SGCs affected by siRNA-mediated blocking of c-Kit/SCF interaction.
- Findings suggest c-Kit is integral to early SGC activities and spermatogenesis, influencing cell fate and inflammatory modulation.
- The results offer insights into siRNA applications and the fundamental role of c-Kit in male germ cell development.
