Abstract
Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes. The current structural models of the mammalian SC are based on electron microscopy, superresolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. To further analyze the structure of the SC, without chemical fixation, we have adapted a cryo-fixation method for electron microscopy where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation, cryo-substitution, and electron tomography. In parallel, we performed conventional chemical fixation and electron tomography on mouse seminiferous tubules to compare the SC structure obtained with the two fixation methods. We found several differences in the structure and organization of the SC in cryo-fixed samples when compared to chemically preserved samples. We found the central region of the SC to be wider and the transverse filaments to be more densely packed in the central region of the SC.
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Abbreviations
- CE:
-
Central element
- CR:
-
Central region
- EM:
-
Electron microscopy
- ET:
-
Electron tomography
- FACS:
-
Fluorescence-activated cell sorting
- FS:
-
Freeze-substitution
- HPF:
-
High-pressure freezing
- LEs:
-
Lateral elements
- nda:
-
Neighbor density analysis
- SC:
-
Synaptonemal complex
- TFs:
-
Transverse filaments
- 3D:
-
Three-dimensional
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Acknowledgements
We would like to thank the Electron Tomography Facility at Karolinska Institute for providing its services.
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This work was supported by grants from the Swedish Research Council (2017–01853 to C.H) and the Hospital Infantil de México Federico Gómez (HIM/2018/079 SSA 1518 and HIM/2021/061 to A.H.H).
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A.H.H and C.H conceived the experiments. A.H.H designed and performed most of the experiments. R.O performed sample sectioning and data analysis. S.M performed data acquisition and tomogram generation. C.H, O.M.E, and A.H.H contributed with reagents, equipment, and funding. A.H.H and C.H supervised the experiments and data analyses. AHH and C.H wrote the manuscript. All the authors discussed the data and reviewed the manuscript.
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Ortiz, R., Echeverría, O.M., Masich, S. et al. A cryo-fixation protocol to study the structure of the synaptonemal complex. Chromosome Res 30, 385–400 (2022). https://doi.org/10.1007/s10577-022-09689-2
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DOI: https://doi.org/10.1007/s10577-022-09689-2