Johanna Bennemann
Experimental studies to investigate the impact of different oxygen tensions during in vitro maturation on global DNA methylation of in vitro fertilized
bovine zygotes
The aim of the present study was to investigate the effect of different oxygen tensions during in vitro maturation of bovine oocytes on global DNA methylation of in vitro produced zygotes.
In preliminary experiments an immunocytochemical protocol for visualization and quantitative analysis of global 5-methylcytosine methylation in in vitro produced bovine zygotes was established. Zygotes as well as two-, four- and eightcell-embryos derived from in vitro production were used. Optimum binding of the antibodies to methylated cytosines requires digestion of zona pellucida. Evaluation of adequate incubation time of the embryos in acidic Tyrode’s solution targeted a preferably complete digestion of the zona pellucida and simultaneous specific staining of DNA methylation. Dilution series of primary and secondary antibody aimed to identify optimal concentrations of the immunoglobulins for visualization of 5-methylcytosine at the best. Furthermore, each run contained a primary and secondary negative control and an isotype control.
Thereafter, a method for staining the total embryonic DNA amount was established.
The fluorescence signal of DNA was used for normalization of 5-methylcytosine fluorescence signal in the context of quantification of DNA methylation. The identification of an adequate hydrochloric acid concentration was of importance, causing a denaturation of DNA which was essential for the binding of the primary antibody to 5-methylcytosine, but which was also suitable for visualization of the entire amount of nucleic acids by staining with a fluorescent dye. DAPI, Propidiumiodid, Hoechst 33258, Hoechst 33342, Ethidium Homodimer-1 and Ethidium Homodimer-2 were tested.
The main experiments were conducted to investigate the effect of oxygen tension (5 % and 20 %) during bovine oocyte in vitro maturation on global DNA methylation of in vitro produced zygotes. For this purpose one half of the embryos underwent the immunocytochemical staining and the other half was stained with Hoechst 33342 to measure the total amount of DNA. Quantitative analysis of fluorescence signals was performed with the ImageJ program. The fluorescence intensities of immunocytochemically stained pronuclei served as a measure for the degree of DNA methylation, those obtained pronuclei stained with Hoechst 33342 were indicators for the amount of DNA. In total, quantitative analysis of fluorescence signals were assessed in1261 zygotes derived from 9 IVP-sessions.
The following results could be obtained:
1. Complete digestion of the zona pellucida was not possible with the tested incubation times in acidic Tyrode’s solution. Exposure of the embryos to the acidic environment for too long impeded the generation of specific fluorescence signals of methylated DNA. Incubating the zygotes in acidic Tyrode’s solution for two minutes at 37°C was suitable for partial digestion of zona pellucida and simultaneous optimal immunocytochemical image acquisition of 5-methylcytosine.
2. Dilution series resulted in optimal concentrations of primary and secondary antibody, reduced unspecific binding of the immunoglobulins and enabled specific presentation of 5-methylcytosine in DNA.
3. Controls in each run were essential for validity and specificity of results. Primary negative control served to exclude unspecific binding of secondary antibody.
Secondary negative control made sure that autofluorescence of the embryos didn’t influence results. Isotype control excluded unspecific binding of primary antibody.
4. Following denaturation, visualization of total DNA amount of zygotes with fluorescent dyes DAPI, Hoechst 33258, Hoechst 33342, Ethidium Homodimer-1 and Ethidium Homodimer-2 was not possible.
5. Following denaturation, visualization of total DNA amount of zygotes with Propidiumiodid was not reproducible.
6. In comparison to COC matured at 20 % oxygen, cumulus-oocyte-complexes matured at 5 % oxygen displayed a more compact, less expanded cumulus.
7. Fertilisation rates were reduced following in vitro maturation at 5 % oxygen compared to maturation at 20 % oxygen.
8. Normalized 5-methylcytosine signals differed significantly between maternal and paternal pronuclei of oocytes matured in vitro at 5 % oxygen. Paternal pronuclei showed a significantly greater signal.
9. Normalized 5-methylcytosine signals did not differ significantly between maternal and paternal pronuclei of oocytes matured in vitro at 20 % oxygen.
10. There was a significant difference of normalized fluorescence signals of 5-methylcytosine between maternal pronuclei of oocytes matured at 5 % oxygen and 20 % oxygen. Following maturation at 20 % oxygen, maternal pronuclei of zygotes displayed a significant greater fluorescence signal.
11. Maturation at 5 % oxygen and 20 % oxygen did not cause significant differences of normalized fluorescence signals of paternal pronuclei.
12. There was a significant difference in normalized relative methylation of paternal pronuclei subsequent to maturation in vitro at 5 % and 20 % oxygen. Oocytes
matured in vitro at 5 % oxygen displayed a significant greater normalized relative methylation level.
The obtained results hypothesize an increased resistance of bovine zygotes’ zona pellucida to digestion with acidic Tyrode’s solution by in vitro culture. Furthermore this study shows the necessity of using accurately tested antibody concentrations as well as negative controls in each run for immunocytochemical visualization of 5-methylcytosine. Failure in using selected fluorescent dyes for measuring total DNA amount following denaturation suggests single stranded DNA status caused by denaturation to be the cause for non-specific binding of theses dyes to DNA.
Cumulus expansion to a lesser extent as well as decreased fertilization rates following in vitro maturation at 5 % oxygen compared to maturation in vitro at 20 % oxygen hypothesizes an impaired fertilization prozess caused by reduced oxygen tension during in vitro maturation.
Furthermore results demonstrate a reduction of global methylation level of the maternal genome in in vitro produced bovine zygotes derived from in vitro maturation at 5 % oxygen. To detect whether the decrease in global methylation of the maternal genome of zygotes is associated with changes in gene expression and whether it affects the development of individuals, further analyses for identification of involved sequences and gene expression studies are required.