Transcriptional and Translational Heterogeneity among Neonatal Mouse Spermatogonia1

Abstract

Spermatogonial stem cells (SSCs) are a subset of undifferentiated spermatogonia responsible for ongoing spermatogenesis in mammalian testes. Spermatogonial stem cells arise from morphologically homogeneous prospermatogonia, but growing evidence suggests that only a subset of prospermatogonia develops into the foundational SSC pool. This predicts that subtypes of undifferentiated spermatogonia with discrete mRNA and protein signatures should be distinguishable in neonatal testes. We used single-cell quantitative RT-PCR to examine mRNA levels of 172 genes in individual spermatogonia from 6-day postnatal (P6) mouse testes. Cells enriched from P6 testes using the StaPut or THY1(+) magnetic cell sorting methods exhibited considerable heterogeneity in the abundance of specific germ cell and stem cell mRNAs, segregating into one somatic and three distinct spermatogonial clusters. However, P6 Id4-eGFP(+) transgenic spermatogonia, which are known to be enriched for SSCs, were more homogeneous in their mRNA levels, exhibiting uniform levels for the majority of genes examined (122 of 172). Interestingly, these cells displayed nonuniform (50 of 172) expression of a smaller cohort of these genes, suggesting there is substantial heterogeneity even within the Id4-eGFP(+) population. Further, although immunofluorescence staining largely demonstrated conformity between mRNA and protein levels, some proteins were observed in patterns that were disparate from those detected for the corresponding mRNAs in Id4-eGFP(+) spermatogonia (e.g., Kit, Sohlh2, Stra8), suggesting additional heterogeneity is introduced at the posttranscriptional level. Taken together, these data demonstrate the existence of multiple spermatogonial subtypes in P6 mouse testes and raise the intriguing possibility that these subpopulations may correlate with the development of functionally distinct spermatogenic cell types.

On the origin of sperm epigenetic heterogeneity

“…It is important to note that there are clear evidences that spermatogonia might also have a degree of constitutive heterogeneity at the gene expression and epigenetic level, at least in mouse models, that allows the cells to retain a certain degree of plasticity (Hammoud et al 2015, Hermann et al 2015. This is in accordance with data from other cell types indicating that epigenetic heterogeneity is usually connected to transcriptional heterogeneity.…”

“…This is, presumably, a mechanism that enables spermatogonia to react to stimuli and interact with the stem cell niche differently and might be linked to decision-making pathways in these cells, i.e. either to replenish the testicular stem cell pool or to progress through spermatogenesis (Hammoud et al 2015, Hermann et al 2015, Krieger & Simons 2015. Whether such constitutive spermatogonial heterogeneity is also present in humans (and if this mechanism is perturbed in infertile patients) remains to be explored.…”

ID4 levels dictate the stem cell state in mouse spermatogonia

“…The development of tools to isolate populations enriched or even purified for different spermatogonial subtypes, such as the Id4-eGfp transgenic mouse model, has refined the ability to explore SSCs specifically. Using these tools, a recent study profiled single ID4-EGFP + spermatogonia from P6 mice to identify bimodal expression of a group of genes known to regulate spermatogonial functions, and identified clusters of cells expressing different levels of select cell surface markers including TSPAN8 (Hermann et al, 2015). In a follow-up study, the population of ID4-EGFP + spermatogonia expressing a high level of TSPAN8 (TSPAN8-Hi) was found to have potent SSC activity by transplantation analyses, whereas the ID4-EGFP + population expressing low levels of TSPAN8 (TSPAN8-Lo) possessed significantly less stem cell capacity (Mutoji et al, 2016).…”

Adult Human Mesenchymal Stem Cell Differentiation at the Cell Population and Single-Cell Levels Under Alternating Electric Current

“…15 Briefly, individual hMSCs were captured on a C1 Integrated Fluidic Circuit (17-25 mm cells) using the Fluidigm C1 Single-Cell Autoprep System, stained using LIVE/DEAD Cell Viability/ Cytotoxicity Kit (Life Technologies), and imaged using an AxioImager M1 microscope (Zeiss).…”

“…Preamplified cDNA harvested from the C1 was then used for high-throughput qRT-PCR measurement of each amplicon using a BioMark HD system as described in literature reports, [15][16][17] with modifications. Briefly, a 2.25 mL aliquot of each amplified cDNA was mixed with 2.5 mL of 2X SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and with 0.25 mL of 20X DNA Binding Dye Sample Loading Reagent (Fluidigm).…”