Determiningtheeffectofcholinesupplementationand

assessingthecorrelationofsexonmurinefetal
development

Kali Blain
Packer Collegiate Institute
February 23, 2017

Dr. Xinyin Jiang

Brooklyn College

Abstract

Choline is an essential nutrient that lowers the risks of certain diseases such as

cardiovascular disease, non-alcoholic fatty liver disease, and neurological disorders. Its ability to

donate a methyl groupone carbon and three hydrogencontributes to a large portion of the

bodys DNA methylationthe addition of a methyl group to a genetic sequencewhich helps

regulate gene expression. However, many people, especially pregnant women, are deficient in

chlorine. This leads to a higher risk for gestational diabetes, which affects both mother and child.

The offspring of such women have poorer physical development and are usually fatter. This

study will look into the effects of choline supplementation on the fetal development of mice. The

fetuses of four maternal groups, consisting high-fat, high-fat choline supplementation, low-fat

control, and low-fat control choline supplementation, respectively, will be analyzed for physical

development halfway through gestation. The gender of each fetus will also be determined to

assess any possible correlation between the sex of the fetus and cholines effects on the mices

development. Results from this study will provide information on cholines effect on fetal

development and any differences in the male and female development. Possible revisions to the

adequate intake, especially during pregnancy, may be considered.

Introduction

Gestational diabetes mellitus (GDM) is the onset of glucose intolerance during pregnancy

in women who have no prior history of diabetes mellitus. There are many causes of GDM, such

as genetics, diet, and other environmental factors. While GDM typically goes away after

gestation, the disease has various negative health effects on both the mother and child. Women
with GDM have a higher risk of preeclampsia, miscarriage, and preterm birth. GDM also is

linked to offspring with higher body mass index (BMI) and an increase in future health risks

(Jiang et al., 2014).

One factor that contributes to GDM is the alteration of glucose transporters which

facilitate the transport of glucose across the cell membrane and help stabilize blood-glucose

levels. Alteration of these glucose transporters can lead to the destabilization of blood-glucose

levels and glucose metabolism (Jiang et al., 2016).

Choline, an essential vitamin found in most animal productssuch as eggs, meat, milk,

and peanutsplays an important role in cellular functions (Jiang et al., 2014). It serves as a

major methyl donor which contributes to the DNA methylation of glucose transporter genes

(Obeid et al., 2013). It is shown that without substantial choline supply, DNA methylation is

decreased, negatively impacting the function of glucose transporter genes (Karnieli et al., 1990).

Choline intake has also been shown to reduce the risk of other ailments such as non-alcoholic

fatty liver disease (NAFLD), neurological disorders, cardiovascular disease, and even reverse the

effects of NAFLD (Ziesel et al., 2009). Possible implications of choline supplementation include

the prevention and possible reversal of gestational diabetes.

Cholines importance in the body is not widely known, and thus many people are often

deficient, leading to an increased risk of certain diseases. Even more, less is known about how

choline affects fetal development, so not many people are aware that it is necessary. The current

adequate intake (AI) for choline are as such: 550 mg/day for men and lactating womenfor

choline is found in high concentrations in breast milk450 mg/day for pregnant women, and

425 mg/day for non-pregnant women (Ziesel et al., 2009).

 This experiment looks into the effect choline has on fetal development in mice and

whether or not the sex of the fetuses correlates to the development. Further research on choline

and possible revision of the AI may help prevent GDM.

Methodology

Twenty-four female mice were started on diets of either a high-fat or a low-fat control.

The fat content is The high-fat diet consisted of 60% fat, and the low-fat control diet contained

10% fat. All of the diets contained 2 millimolars (mM) of choline per 1000 kilocalories. Six mice

from both the high-fat and low-fat control diets were then selected to receive additional choline

supplementation of 25mM.

Four weeks into the experimental diets, the mice were impregnated, and the diets were

continued until embryonic day 12.5 (mid-gestation). On embryonic day 12.5 the mice were

sacrificed, with tissue and serum samples from the mother collected and stored in -80C for other

work in the study. The fetuses were also dissected out of the womb, and a visual of each fetus

was taken on a 1x1 centimeter grid for further analysis. Fetal tail and liver samples were also

collected for analysis and stored at -80C until use.

A measurement from the crown (top of the head) to the rumpcommonly referred to as a

crown-rump length (CRL)for each fetus will be taken from the photographs of the fetuses

using the electronic measuring system ImageJ. Abnormalities and approximate size are recorded

for later interpretation.

DNA will be extracted from fetal liver and tail samples using the GeneJet DNA

Purification Kit protocol and supplies. The extracted DNA is then going to be stored in -20C

awaiting analysis or immediately sequenced using qPCR. The DNA will be sequenced for the
MYOG gene, located on the X chromosome, and/or the sry gene, located on the Y chromosome.

The results of the qPCR tests will identify whether the samples were from male or female fetuses

since dissection occurred too early for visual confirmation.

Predictions

The two parts of the experimentthe CRL and the gender typingallow for trends to be

made about cholines effects on the physical development of fetal murine mice as well as any

correlation between the sex of the fetuses and their development. Since choline is so crucial to

fetal development in mammals, superior development, shown by larger fetuses, is expected in the

fetuses belonging to mothers in the choline supplementation group compared to those from the

choline control group. Not enough previous data is available yet to make educated predictions

about the correlation between sex and cholines effect on fetal development.

References

Jiang, X., Greenwald, E., & Jack-Roberts, C. (2016). Effects of Choline on DNA Methylation
and Macronutrient Metabolic Gene Expression in In Vitro Models of Hyperglycemia.
Nutrition and Metabolic Insights, 9, 1117. http://doi.org/10.4137/NMI.S29465

M, K. (2016). Regulation of glucose transporters in diabetes. - PubMed - NCBI .

Ncbi.nlm.nih.gov. Retrieved 6 December 2016, from
https://www.ncbi.nlm.nih.gov/pubmed/2210626

Obeid, R. (2013). The Metabolic Burden of Methyl Donor Deficiency with Focus on the Betaine
Homocysteine Methyltransferase Pathway. Nutrients, 5(9), 3481-3495.
doi:10.3390/nu5093481
Zeisel, S. H., & da Costa, K.-A. (2009). Choline: An Essential Nutrient for Public Health.
Nutrition Reviews, 67(11), 615623. http://doi.org/10.1111/j.1753-4887.2009.00246.x