1. Donkin et. al. Presented by: Natasha Granneman and Christina Tran
Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans
Donkin et. al.
Presented by: Natasha Granneman and Christina Tran

2. Background/Importance
Obesity has behavioral and heritable causes
Children of obese FATHERS have a higher risk of obesity
Inherited paternal factors?
Socioeconomic status of the father?
Nutritional status and exercise have been shown to alter DNA methylation in somatic tissues
Can obesogenic factors change gamete epigenomes?
Believed to be more than just socioeconomic status because:
-studies in rodents & epidemiological data show that paternal nutritional status can directly affect the health of offspring

3. Background
Epigenetics = anything other than the DNA sequence that influences gene expression
Epigenetic changes can be heritable!

4. Hypotheses
Spermatozoa from obese men have an altered epigenome relative to lean men
Weight loss remodels the epigenome of spermatozoa in human obesity

5. Methods
Lean vs. Obese
Collected single ejaculates from 23 Caucasian males
13 lean, 10 obese
Gastric Bypass induced weight loss
Collected single ejaculates from 6 obese men at 3 time points:
1 week before surgery
1 week after surgery
1 year after surgery

6. Methods Characterized histone positioning
Micrococcal nuclease sequencing (MNase-seq)
Compared small non-coding RNA (sncRNA) expression
sncRNA-seq
Compared DNA methylation patterns
Reduced representation bisulfite Seq (RRBS)
Genotyped all individuals for SNP’s associated with obesity
Infinum CoreExome-24 BeadChip

8. Results
histone positioning was not altered in sperm between lean and obese men
lean and obese men had a 2% genome retention rate
5% of enhancer regions reported in somatic cells retained histones in mature sperm

9. Results Looked at sncRNA content in lean and obese men
only expression levels of piRNA changed significantly
Predicted targets of the differentially expressed piRNAs were in the categories “Chromatin” and “Chemdependancy”
CART = a regulator of food intake involved in obesity
CART
piRNA= known to be predominantly expressed in the germ line because they maintain genomic stability by repressing transposable elements, and they can inhibit gene expression post-transcriptionally

10. Results
appetite control
genes
9,081 genes differentially methylated between lean and obese men
mostly nervous system development genes
274 disease related genes were differentially methylated
regulators of appetite control
genes related to obesity and metabolism
obesity/metabolism
genes
NPY-neuropeptide that increases food intake and fat storage
FTO-more copies → higher obesity risk

11. Results
Genotyped 23 known SNPs associated with obesity to see if epigenetic variation between lean and obese could be due to polymorphisms
polymorphisms could influence methylation of proximal cytosines
Saw that none of the SNPs were within a distance of 1,000 bp of a methylated cytosine
Further analysis of SNPs showed no relationship between the SNP and epigenetic variability
test whether methylation changed due to something environmental or if it's because a change in the sequence (SNP) altered the affinity of that site for methylation

12. Results
Studied sperm DNA methylation profiles in obese men before and after gastric bypass surgery (GBP)
Pre-GBP vs. 1 week after GBP:
1,509 unique genes were differentially methylated
suggests that changes can occur in last stages of sperm maturation
Pre-GBP vs. 1 year after GBP:
3,910 unique genes were differentially methylated
Sperm maturation takes 3 months

13. Results
2,681 of genes with altered DNA methylation between pre-GBP and GBP-1y were also altered in lean vs obese
indicates methylation hotspots that coincide with nutritional intake
Many of these genes can have SNPs associated with severe onset obesity, but none of the participants had an obesity SNP
overlap was not significant to equate the 2 groups as having the same effect on methylation, but can indicate genes that are differentially methylated in response to nutrition
none of the participants had an obesity-carrying SNP, showing gbp induced weight loss modulates the epigenome of sperm in genes that are master regulators of obesity

14. Results
Compared changes in methylation in sperm and somatic tissues (blood and adipose) before and 1 year after GBP
Methylation patterns are unique to each tissue in the 60 CpG sites investigated
Environmental factors associated with GBP induce changes in methylation that are unique to spermatozoa

15. Conclusion
Epigenomes of spermatozoa in lean and obese men differ in DNA methylation patterns and piRNA expression
Methylome of sperm is altered after GBP surgery
the degree to which the sperm epigenome is transmitted to the offspring’s metabolic tissues is unknown
Could not determine which specific factors associated with obesity (BMI, glucose, insulin, LDL, HDL, triglycerides, leptin, etc.) were contributing to changes in methylation

16. Limitations Small sample sizes Some findings were not significant
13 lean, 10 obese, 6 GBP
Some findings were not significant
Genes “differentially methylated” in lean and obese men (NPY, BDNF, CHST8, etc.) did not have statistical significance
Only methylation changes were studied in the GBP cohort
Overlap between methylation changes in Lean vs. Obese cohort and pre-GBP vs. GBP-1y cohort was not significant, but claimed the overlap indicates “hotspots for methylation changes in the genome associated with changes in nutritional intake”

17. References/Additional Reading
Donkin, I., Versteyhe, S., Ingerslev, Lars R., Qian, K., Mechta, M., Nordkap, L., Mortensen, B., Appel, Emil Vincent R., Jørgensen, N., Kristiansen, Viggo B., et al. (2016). Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans. Cell Metabolism, in press
Brøns, C., Jensen, C.B., Storgaard, H., Hiscock, N.J., White, A., Appel, J.S., Jacobsen, S., Nilsson, E., Larsen, C.M., Astrup, A., et al. (2009). Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men. J. Physiol. 587, 2387–2397.
Soubry, A., Murphy, S.K., Wang, F., Huang, Z., Vidal, A.C., Fuemmeler, B.F., Kurtzberg, J., Murtha, A., Jirtle, R.L., Schildkraut, J.M., et al. (2013). Newborns of obese parents have altered DNA methylation patterns at im- printed genes. Int. J. Obes. 39, 650–657.