A research group comprising Assistant Professor Toshinori Nakagawa and Professor Shosei Yoshida at the Division of Germ Cell Biology, National Institute for Basic Biology, in collaboration with Professor Takashi Nagasawa at the Graduate School of Frontier Biosciences, Osaka University, Dr. Benjamin Simons at the University of Cambridge, and others, reported that mammalian sperm stem cells continue producing sperm while transitioning between undifferentiated and differentiated states. On investigating the gene expression of the sperm stem cell population, the researchers discovered Plvap1 and Sox3 genes, which continually interconvert with each other. This was evidenced by tracking the fate of cells in which these genes were expressed. It is expected to be widely useful for elucidating the properties of stem cells. The results were published in the October 19, 2021 issue of Cell Reports, an international scientific journal.
Sperm stem cells are the source of sperm and can continue producing the differentiated cells capable of forming sperm, as well as producing sperm stem cells themselves. Previously, the research group has reported that sperm stem cells are a cell population that expresses the GFRα1 gene. Although undifferentiated, these cells are diverse in morphology and gene expression. However, the details of these phenomena were unclear.
To investigate further, the research group conducted a detailed analysis of the sperm stem cell population (GFRα1-positive cells) expressing the GFRα1 gene. First, sperm stem cells were divided into three categories according to the degree of GFRα1 expression using a fluorescence-activated cell sorter, and the genes in each cell population were analyzed. Among the GFRα1-positive populations, Plvap was expressed in a population with more morphologically immature characteristics, while Sox3 was expressed in a population with more differentiated characteristics. On performing an in-depth examination, Plvap and Sox3 were found to be exclusively expressed by each other. The researchers observed that GFRα1-positive cells could be classified into three types: X cells ( Plvap +), Y cells ( Plvap-, Sox3-), and Z cells ( Plvap-, Sox +).
Next, the researchers investigated how X cells and Y cells differentiate into sperm. Specifically, the researchers produced genetically modified mice expressing tamoxifen-operated Cre recombinase in Plvap-positive cells and mice expressing tamoxifen-operated Cre recombinase in Sox3-positive cells. The fate of Plvap-expressing cells and Sox3-expressing cells was tracked.
The sperm stem cells appeared to maintain a non-uniform population on the whole, while transitioning between X and Z states. Furthermore, the frequency of sperm stem cells transitioning between X, Y, and Z was analyzed by incorporating the follow-up results into a mathematical model. Accordingly, the rate of transition per day between these X, Y, Z states in sperm differentiation was 6% for X → Y, 9% for Y → Z, 21% for Z → sperm, and the transition was more likely to occur in the order of X → Y → Z. In the undifferentiated direction, the rate of transition was 2% for Z → Y and 4% for Y → X was 4%, which was in contrast to that observed for differentiation. Moreover, these transitions were irregular and occurred stochastically.
These properties work as a mechanism to suppress the load of DNA replication and the accumulation of mutations or to potentially produce sperm if Y or Z cells are alive even if X cells die owing to cell death. Similar mechanisms may also exist in other stem cells. According to Assistant Professor Nakagawa, "This study revealed that sperm stem cells behave flexibly and dynamically rather than staying in a fixed state. In the future, we plan to eliminate any potential bias and examine the state and properties of stem cells in an in-depth manner by comprehensively investigating gene expression at the single-cell level. We believe that these studies will help clarify the mechanism underlying sperm stem cell transition."
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