The production of sperm by spermatogenesis comes from spermatogonial stem cells, or SSCs, which undergo several morphological and functional transformations in their differentiation. But what is the fate of cells selected for differentiation (producing sperm) versus those selected for self-renewal (producing more SSC)? How do these processes affect the structure of their valuable genetic cargo? A mistake in this process can lead to male infertility, so accuracy is key.
Scientists believe that dramatic alterations in 3D chromatin structure occur throughout these processes, but this has never really been identified. Yi Zheng, Lingkai Zhang, Long Jin, Pengfei Zhang and their colleagues from Northwest A&F University in Shaanxi, China, and Sichuan Agricultural University in Sichuan, China, investigate this phenomenon in a recent paper from the Journal of Biological Chemistry to discover exactly how the structure of chromatin changes.
Exploring chromatin dynamics requires very high resolution data. However, as Zheng said, “With the rapid development of omics techniques, it is now possible to study this topic in much more detail.”
That didn’t mean things were easy, though.
“We found that this resolution required an input of about 20 million cells,” Zheng said. “That means hundreds of mice would have to be sacrificed.”
To avoid this, they switched to a larger model organism – the pig. Even still, collecting samples required an abundance of patience – 14 months. “It took almost a year longer than expected,” Zheng said. “The two populations of rare cells (undifferentiated and differentiating spermatogonia) must come from different ages of pigs (90 days and 150 days, respectively) and be enriched by different and laborious methods.”
Once these samples were collected, the researchers assembled an advanced bioinformatics pipeline for data analysis, incorporating a novel technique, high-throughput chromosome conformation capture, as well as RNA sequencing and chromatin immunoprecipitation. . “As we used a new bioinformatics technique, learning and building the pipeline was quite tricky and time-consuming,” Zheng said.
Their patience was rewarded. The data indicated that chromatin architecture was weakened when an SSC was chosen for differentiation. “Spermatogonial differentiation is, in essence, a transitional process that gradually prepares the genome for subsequent meiotic events,” Zheng said.
Their search for high-resolution data also had the benefit of visualizing how transcriptional regulation works during this process. Each scale of chromatin structural variation during differentiation appears to play a discrete role in dynamic gene expression. All of this combines to provide essential insight into the mechanics of CSS development.
After all that, it seems like everyone was won over by the humble pig. “I would like to point out that the value of pigs as a model species is grossly underestimated,” Zheng said. “Pigs share more similarities with humans in terms of anatomy, physiology and genetics than mice, and pigs are increasingly being used in translational studies in hopes of moving xenotransplantation to the clinic, as organ sources. I am committed to establishing a stable and long-term culture system for porcine spermatogonial stem cells.
Spermatogonial stem cells undergo several structural changes to begin the process of spermatogenesis. These changes are related to significant alterations in the structure of chromatin to prepare it for later steps towards sperm production.