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Updated: Jun 16, 2026
Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment
Published on: September 17, 2019
Microgravity promotes differentiation and meiotic entry of postnatal mouse male germ cells
Manuela Pellegrini1, Sara Di Siena, Giuseppina Claps
1Department of Public Health and Cellular Biology, University of Rome Tor Vergata, Rome, Italy.
Abstract:
A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.
Insights
Simulated microgravity using a rotary cell culture system promotes mouse spermatogonia differentiation. This system enables mitotic spermatogonia to enter meiosis without external factors, advancing male germ cell research.
Area of Science:
- Reproductive Biology
- Cell Biology
- Developmental Biology
Background:
- Spermatogenesis requires mitotic spermatogonia to enter meiosis, a process difficult to study in vitro.
- Previous research indicated simulated microgravity (SM) supports meiotic progression in pachytene spermatocytes.
Purpose of the Study:
- To investigate if simulated microgravity (SM) in a rotary cell culture system (RCCS) can induce differentiation and meiotic entry in mouse mitotic spermatogonia.
- To explore the molecular pathways involved in SM-induced germ cell differentiation.
Main Methods:
- Culturing isolated mouse spermatogonia under SM using RCCS.
- Monitoring meiotic entry via synaptonemal complex 3 protein (Scp3) organization and pro-meiotic gene expression.
- Assessing the role of the phosphatidyl inositol 3 kinase (PI3K) pathway using a specific inhibitor.
Main Results:
- Spermatogonia under RCCS conditions entered meiosis (leptotene stage), evidenced by Scp3 organization and gene upregulation.
- SM activated the PI3K pathway, inducing DNA replication and preleptotene stage characteristics.
- A PI3K inhibitor blocked SM-induced meiotic entry.
- SM also promoted differentiation in undifferentiated spermatogonia, increasing Kit and Stra8 expression.
Conclusions:
- Simulated microgravity (SM) via RCCS promotes postnatal male germ cell differentiation and entry into meiosis.
- This system offers a novel in vitro tool for studying the mitosis-to-meiosis transition in mammals.
- The PI3K pathway is crucial for SM-induced meiotic entry.
