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Influence of proton magic angle spinning nuclear magnetic resonance spectroscopy on in vitro mouse embryo development
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Thefiogenobgy INFLUENCE OF PROTON MAGIC ANGLE SPINNING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY ON IN VITRO MOUSE EMBRYO DEVELOPMENT J.T. Lyman1, D.K. Ohz, G.L. Torres 3, S-J Choi 1, R.L. Magin2'3, M.B. Wheeler1 1 Department of Animal Sciences, University of Illinois, Urbana-Champaign, 61801 2 Department of Biophysics, University of Illinois, Urbana-Champaign 3 Department of Bioengineering, University of Illinois, Chicago
Efforts have been made by our group to develop an embryo analysis and transport system capable of detecting and sorting embryos. One potential analysis which could be performed on the embryos in this system is proton magic angle spinning nuclear magnetic resonance spectroscopy (1H MAS NMR). Little work has been done demonstrating the effects of 1H MAS NMR on mammalian embryos. ~H MAS NMR involves spinning of the sample at an angle of 54° 44', application of radiofrequency pulses, and the presence of a high magnetic field (11.75 T). In addition, deuterated water (D20) is used as a medium for the NMR analysis to minimize the large signal from the water. One potential hazard of this technique is the use of D20 in the analysis medium. D20 has been shown in other species to arrest mitosis, and would likely have an effect on the capability of the embryos to survive tH MAS NMR. Superovulated ICR mice were mated with B6SJL/F1 males to produce 2-cell embryos, which were collected into complete PBS with 0.4%BSA. The PBS was used to minimize contamination of the sample with any organic molecules which would appear on the spectra generated through the 1H MAS NMR. After collection in the PBS, the embryos were randomly assorted into 4 groups. The control embryos were immediately placed in M16 with 0.4% BSA and cultured at 37°C for 96 hours. The three treatment groups were washed into PBS with 0.4%BSA made with D20. The embryos were then either subjected to NMR (NMR group), placed in NMR carriers (at room temperature) but not used in the magnet (SHAM), or left in the D20 PBS (at 37°C) for the duration of the NMR analysis (2 hours) on the other embryos (D20). The IH MAS NMR was performed at 21 °C on a 500 MHz NMR spectrometer with a spinning frequency of 2 KHz. Spectra were acquired with a simple water suppression pulse sequence (presat-90-acquire) and an acquisition time of 28 minutes. At the end of the analysis on the NMR group, all three treatment groups were placed in M16 and cultured for 96 hours. All groups were examined every 24 hours for development. Each experiment was replicated three times. Data were analyzed using ANOVA and least square means for comparisons between groups. The control group (44/53, 83%) had higher (p<.001) development to blastocysts at 96 hours than any of the treatment groups. There was no difference betweenthe NMR (24/44, 54.5%) and SHAM (19/40, 47.5%) groups, though both NMR and the SHAM groups had higher (p<.05) development than the D20 (11/40, 27.5%) group. It appears that lowering the temperature of the sample while the embryos are exposed to D20 provides some attenuation of the injurious effects. From this we conclude that though the NMR group did have lower development rates than the control, the NMR procedure itself does not appear to be the cause. The DzO in the analysis medium seems to be the detrimental factor causing poor development following NMR. Therefore, further investigation alternative media for NMR is necessary. A second possibility may be to attempt to decrease the temperature of the magnet lower than room temperature to further attenuate the deleterious effects of the DzO.
Thefiogenobgy INFLUENCE OF PROTON MAGIC ANGLE SPINNING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY ON IN VITRO MOUSE EMBRYO DEVELOPMENT J.T. Lyman1, D.K. Ohz, G.L. Torres 3, S-J Choi 1, R.L. Magin2'3, M.B. Wheeler1 1 Department of Animal Sciences, University of Illinois, Urbana-Champaign, 61801 2 Department of Biophysics, University of Illinois, Urbana-Champaign 3 Department of Bioengineering, University of Illinois, Chicago
Efforts have been made by our group to develop an embryo analysis and transport system capable of detecting and sorting embryos. One potential analysis which could be performed on the embryos in this system is proton magic angle spinning nuclear magnetic resonance spectroscopy (1H MAS NMR). Little work has been done demonstrating the effects of 1H MAS NMR on mammalian embryos. ~H MAS NMR involves spinning of the sample at an angle of 54° 44', application of radiofrequency pulses, and the presence of a high magnetic field (11.75 T). In addition, deuterated water (D20) is used as a medium for the NMR analysis to minimize the large signal from the water. One potential hazard of this technique is the use of D20 in the analysis medium. D20 has been shown in other species to arrest mitosis, and would likely have an effect on the capability of the embryos to survive tH MAS NMR. Superovulated ICR mice were mated with B6SJL/F1 males to produce 2-cell embryos, which were collected into complete PBS with 0.4%BSA. The PBS was used to minimize contamination of the sample with any organic molecules which would appear on the spectra generated through the 1H MAS NMR. After collection in the PBS, the embryos were randomly assorted into 4 groups. The control embryos were immediately placed in M16 with 0.4% BSA and cultured at 37°C for 96 hours. The three treatment groups were washed into PBS with 0.4%BSA made with D20. The embryos were then either subjected to NMR (NMR group), placed in NMR carriers (at room temperature) but not used in the magnet (SHAM), or left in the D20 PBS (at 37°C) for the duration of the NMR analysis (2 hours) on the other embryos (D20). The IH MAS NMR was performed at 21 °C on a 500 MHz NMR spectrometer with a spinning frequency of 2 KHz. Spectra were acquired with a simple water suppression pulse sequence (presat-90-acquire) and an acquisition time of 28 minutes. At the end of the analysis on the NMR group, all three treatment groups were placed in M16 and cultured for 96 hours. All groups were examined every 24 hours for development. Each experiment was replicated three times. Data were analyzed using ANOVA and least square means for comparisons between groups. The control group (44/53, 83%) had higher (p<.001) development to blastocysts at 96 hours than any of the treatment groups. There was no difference betweenthe NMR (24/44, 54.5%) and SHAM (19/40, 47.5%) groups, though both NMR and the SHAM groups had higher (p<.05) development than the D20 (11/40, 27.5%) group. It appears that lowering the temperature of the sample while the embryos are exposed to D20 provides some attenuation of the injurious effects. From this we conclude that though the NMR group did have lower development rates than the control, the NMR procedure itself does not appear to be the cause. The DzO in the analysis medium seems to be the detrimental factor causing poor development following NMR. Therefore, further investigation alternative media for NMR is necessary. A second possibility may be to attempt to decrease the temperature of the magnet lower than room temperature to further attenuate the deleterious effects of the DzO.