Related Experiment Video
Updated: Oct 12, 2025
A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model
Published on: February 6, 2018
Study on cryopreservation of mouse single seminiferous tubule
Shuyong Zhang1, Yingying Guo1, Liangyu Zhao2
1Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
Abstract:
Cryopreservation of single seminiferous tubule is significant for fertility preservation, especially for male patients with cryptorchidism, Y-chromosome deletion and orchitis. However, there are few studies on cryopreservation of single seminiferous tubule. This study proposes several improved strategies for cryopreservation of single seminiferous tubule in mice. First, single seminiferous tubule was cryopreserved with modified slow freezing and vitrification methods. The results showed that the apoptosis negative rates of spermatogenic cells in single seminiferous tubule with modified slow freezing method were significantly higher than vitrification with plastic slide. Then, plastic slide and two metal vitrification carriers with high thermal conductivity, copper mesh and aluminum foil, were used to vitrify single seminiferous tubule. The metal carriers could improve the outcome of vitrification than plastic slide. The apoptosis negative rates of spermatogenic cells in the aluminum foil group was significantly higher than that in the copper mesh group. Finally, single seminiferous tubule was perfused with CPAs by micro-injection technique and vitrified. The results of cryo-microscopy experiments showed that the ice crystals formed inside the injected seminiferous tubule was reduced during the cooling process, the apoptosis negative rate of spermatocytes, spermatids and Sertoli cells were significantly higher than that of the non-injection group, indicating that the injection technique can effectively improve the effect of vitrification. This study has potential clinical value for in-vitro culture of spermatogonial stem cells and autologous testicular tissue grafting in patients with azoospermia.
Insights
Improved cryopreservation of single seminiferous tubules enhances fertility preservation. Modified slow freezing and vitrification techniques, especially using metal carriers and micro-injection, significantly reduce cell apoptosis.
Area of Science:
- Reproductive Biology
- Cryobiology
- Cell Biology
Background:
- Fertility preservation is crucial for males with conditions like cryptorchidism, Y-chromosome deletion, and orchitis.
- Cryopreservation of single seminiferous tubules is vital but understudied.
- Developing effective cryopreservation methods for seminiferous tubules is essential.
Purpose of the Study:
- To investigate and improve cryopreservation strategies for single mouse seminiferous tubules.
- To compare the efficacy of modified slow freezing and vitrification methods.
- To evaluate the impact of different vitrification carriers and a micro-injection technique.
Main Methods:
- Single seminiferous tubules were cryopreserved using modified slow freezing and vitrification.
- Vitrification was performed on plastic slides and metal carriers (copper mesh, aluminum foil).
- Cryoprotective agents (CPAs) were perfused via micro-injection into seminiferous tubules before vitrification.
Main Results:
- Modified slow freezing showed higher survival rates than standard vitrification on plastic slides.
- Metal carriers improved vitrification outcomes compared to plastic slides, with aluminum foil being superior to copper mesh.
- Micro-injection of CPAs significantly reduced ice crystal formation and improved cell survival (spermatocytes, spermatids, Sertoli cells).
Conclusions:
- Modified slow freezing and vitrification using metal carriers enhance seminiferous tubule cryopreservation.
- Micro-injection technique effectively improves vitrification outcomes by minimizing intracellular ice.
- These advancements hold significant potential for clinical applications in fertility preservation and azoospermia treatment.
