TOPIC CHOSEN:
Effects of Paternal Alcohol Consumption on Fetal Development
SPECIFIC QUESTION:
Are Stfmb2, Snrpn-Ube3a, Dlk-Dio3 differentially methylated in the sperm DNA of CD1 mice treated with ethanol compared to saline?
HYPOTHESIS:
Stfmb2, Snrpn-Ube3a, Dlk-Dio3 are differentially methylated in the sperm DNA of CD1 mice treated with ethanol compared to saline.
EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):
It has been observed by Lee et al.(2013) that preconception consumption of ethanol by male mice results in skull malformation. Knezovich & Ramsay (2012) found that transgenerational toxic effects are due to epigenetic mutations in sperm DNA caused by alcohol. Laufer et al. (2013) found three imprinting control regions (ICRs), Sfmbt2, Snrpn-Ube3a, Dlk-Dio3, that are differentially methylated when the fetus is exposed to alcohol. These three ICRs are related to the development of the brain (Laufer and Singh, 2012).
PREDICTION(S):
If these imprinting control regions are differentially methylated, then there would be a possibility that these epigenetic mutations will be passed on to the offspring. This may be a reason why Lee et al. (2013) see skull malformations in the fetuses.
EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):
Treat CD1 mice (postnatal day 49) with a 4 g/kg EtOH with 0.9% saline oral administration once in the morning and once in the evening, for a 7 week period. Control mice would be fed a saline solution. (adapted from Lee et al. 2013)
Mature sperm from the experimental and control group is harvested and DNA is extracted. Using oxidative bisulfite sequencing (OxBS-seq), methylation in the three loci will be analysed. This method is able to distinguish between 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) (Booth et al., 2013). Knezovich and Ramsay (2012) used a method that could not differentiate between these two bases and proposed that this led to the observation of very little demethylation of the sperm DNA. Look for differential methylation (only 5-mC) in the 3 loci between the control group and the experimental group.
LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of an annotated bibliography):
Laufer et al. 2013 – found three differentially methylated ICRs when the fetus were treated with alcohol that are related to development of the brain
Laufer & Singh 2012 – more in depth review of Sfmbt2, Snrpn-Ube3a, and Dlk-Dio3
Knezovich & Ramsay 2012 – found epigenetic mutations in sperm DNA, will be using an adapted experimental from this paper
Lee et al. 2013 – found skull malformations in mice fetuses when the fathers are fed alcohol
Liu et al. 2009 – found alcohol to changes the DNA methylation in mouse embryos during early development of neurons
Booth et al. 2013 – paper about oxidative bisulfite sequencing
HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?
If we can find a direct link in between how transgenetic toxic effects of alcohol are passed on to offsprings, such as epigenetic mutations in the sperm DNA, it will no longer be just a correlation and people will pay more attention to the consumption of alcohol if they are planning to have offsprings.
POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):
Blog posts, posters, word of mouth
ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:
None yet! Sorry!
ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:
Does the topic need to be more specific? Would “Effect of Paternal Alcohol Consumption on the Epigenome of Sperm” be better?
Is the length of this experiment adequate? Should I include looking for differentially methylated at these 3 loci in genome of the fetus similar to Knezovich and Ramsay (2012) looked at in the offspring?
One Comment
Since you ask… yes, I think that for your topic, saying “Effect of Paternal Alcohol Consumption on the Epigenome of Sperm” would be more descriptive of what your question and experiment are actually about.
In your intro, make sure to really put your experiment and question in the context of what is already known by actually pointing out, very explicitly, what it is that we still don’t know (i.e. the connection that you are trying to make).
Your experiment is fine and yes, i would include checking the methylation state of the three loci in the offspring,too (that would make a really nice story, plus it would confirm the previous results). Things hat you will need to think about will be how many mice you will have in your experimental and control group, how many offsprings you will test from each mouse, and how you will evaluate the difference in methylation for your sperm (chances are that not every single sperm will be methylated at those three loci exactly in the same way, so you will need to decide on how you will measure the difference).