Synchrotron X-ray diffraction to detect glass or ice formation in the vitrified bovine cumulus-oocyte complexes and morulae

Abstract

Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (-171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.

Figure 1. Custom-designed cryoloop and cryotop using CrystalCap Copper Magnetic.

Each cap possesses a magnetic base which is fixed on the top of cryovial (*) for storage or goniometer head for SXRD analysis. Cryoloop was used for SXRD of solutions (water, TCM-199 and VS2) and cryotop for bovine COCs and morulae.

Figure 2. The end-station of beamline 08ID-1 (A) and close-up of sample position area (B).

1) CCD- X-ray detector. 2) Hutch camera for remote monitoring system. 3) Sample camera. 4) Goniometer head. 5) CrystalCap Copper Magnetic mounted on goniometer head. 6) X-ray tube. 7) Beam stop. 8) Liquid nitrogen supply from CryoJet.

Figure 3. The beamline setup, sample camera and cryoject control tabs of the MxDC software at the 08ID-1 beamline.

A view from sample camera shows two oocytes on cryotop and square in the middle shows the path and size (0.1×0.1 mm) of X-ray beam.

Figure 4. Physical appearance, 2D SXRD and 1D SXRD of Water, TCM-199 and VS2 in cryoloops at 102K.

A) Appearnce of solutions through sample camera. B) Representative 2D SXRD with display of d-spacing (Å). C) Representative 1D SXRD indicating the relative intensities of peaks vs. d-spacing (Å). As d-spacing increases, the diameter of Debye's ring decreases. All peaks in water and TCM-199 corresponded to the hexagonal ice crystals except one peak (*) in TCM-199 which corresponded to NaH₂PO₄.H₂O.

Figure 5. Physical appearance, 2D SXRD and 1D SXRD of bovine COCs in TCM-199 (non-vitrified control) and VS2 (vitrified) on cryotops at 102K.

A) Appearance of COCs through sample camera. B) Representative 2D SXRD with display of d-spacing. C) Representative 1D SXRD indicating the relative intensities of peaks vs. d-spacing. All peaks corresponded to the hexagonal ice crystals except one peak (*) which corresponded to NaH₂PO₄.H₂O.

Figure 6. Ice peaks' intensities in bovine COCs in TCM-199 (non-vitrified control) or VS2 (vitrified).

Each bar represents mean±SEM (N = 5 COCs in each group). Asterisk (*) denotes the significant difference (P<0.05) in ice peak intensity between non-vitrified and vitrified COCs at a given d-spacing. Double asterisks (**) denote the presence of ice peaks in the non-vitrified COCs only.

Figure 7. Physical appearance, 2D SXRD and 1D SXRD of bovine morulae in TCM-199 (non-vitrified control) and VS2 (vitrified) on cryotops at 102K.

A) Appearance of morula through sample camera. B) Representative 2D SXRD with display of d-spacing (Å). C) Representative 1D SXRD indicating the relative intensities of peaks vs. d-spacing (Å). All peaks corresponded to the hexagonal ice crystals except one peak (*) which corresponded to NaH₂PO₄.H₂O.