The effect of uteroplacental insufficiency and docosahexaenoic acid supplementation on sex-divergent changes in placental setd8 signalling

Fetal outcomes from pregnancies complicated by uteroplacental insufficiency (UPI) include growth restriction and an increase in neonatal and adult-onset disease. These outcomes are often sex-divergent, with males having worse outcomes than females. Fetal acquisition of long-chain polyunsaturated fatty acids (LCPUFA) is impaired in pregnancies complicated by UPI and is linked to disease outcomes. Fetal acquisition of LCPUFA is mediated by the placenta. PPARγ regulates placental LCPUFA transport through direct control of gene expression, and indirectly by initiating chromatin modifications through Setd8. We have previously shown in our rat model of UPI that the PPARγ-Setd8 axis is present in the placenta and UPI results in sex-divergent changes in fetal serum LCPUFA profiles. Docosahexaenoic acid (DHA) is an LCPUFA ligand of PPARγ and is critical for normal fetal development. We hypothesize that UPI and DHA supplementation will cause sex divergent changes in mRNA levels of Setd8, as well as fatty acid transport and handling genes in association with altered H4k20me along one representative gene. UPI was induced by bilateral uterine artery ligation at embryonic day 19 in pregnant Sprague Dawley rats. Male and female placenta were surgically collected at term (embryonic day 21). mRNA was measured using real-time RT-PCR, and H4K20me on the SLC27A2 gene was measured using ChIP. Oil-Red-O staining was used as an additional qualitative measure of lipid accumulation in the placenta. iv Data are expressed as mean ± standard deviation (SD), *P < .05. In male placenta UPI increases Setd8 and alters mRNA levels of transport, binding, and handling genes. We also performed pilot experiments examining H4K20me along the fatty-acid transport protein 2 gene (gene name SLC27A2). In males, UPI caused a significant increase in H4k20me at the exon 3 region of SLC27A2 (FATP2), but not the promoter, possibly influenced by our small sample size. In contrast, in female placenta, UPI did not affect Setd8 or FATP2 mRNA, or H4K20me along the SLC27A2 (FATP2) gene. The combination of UPI and DHA supplementation, on the other hand, increased Setd8 and FATP2 mRNA, and H4K20me along the SLC27A2 (FATP2) gene in both male and female placenta. Oil-Red-O staining indicates increased lipid droplet formation in the basal zone of the male and female placenta when supplemented with DHA. In conclusion, UPI and DHA supplementation in the rat results in sex-divergent changes in Setd8 and mRNA levels of fatty acid transport and handling genes, as well as H4K20me along the SLC27A2 (FATP2) gene. Given the role of these genes in LCPUFA metabolism in the placenta, sex-divergent changes in gene expression resulting from changes to the PPARγ-Setd8 axis may influence placental handling and transfer of LCPUFA to the fetus.

THE EFFECT OF UTEROPLACENTAL INSUFFICIENCY AND DOCOSAHEXAENOIC ACID SUPPLEMENTATION ON SEX-DIVERGENT CHANGES IN PLACENTAL SETD8 SIGNALLING by Emily Barrett A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science Department of Nutrition and Integrative Physiology The University of Utah August 2019 Copyright © Emily Barrett 2019 All Rights Reserved University of Utah Graduate School STATEMENT OF THESIS APPROVAL The thesis of Emily Barrett has been approved by the following supervisory committee members: Lisa Joss-Moore , Chair 4/16/2019 Date Approved Julie Metos , Member 4/16/2019 Date Approved Kristine Jordan , Member 4/16/2019 Date Approved and by the Department of Scott Summers , Chair of Nutrition and Integrative Physiology and by David B. Kieda, Dean of The Graduate School. ABSTRACT Fetal outcomes from pregnancies complicated by uteroplacental insufficiency (UPI) include growth restriction and an increase in neonatal and adult-onset disease. These outcomes are often sex-divergent, with males having worse outcomes than females. Fetal acquisition of long-chain polyunsaturated fatty acids (LCPUFA) is impaired in pregnancies complicated by UPI and is linked to disease outcomes. Fetal acquisition of LCPUFA is mediated by the placenta. PPARγ regulates placental LCPUFA transport through direct control of gene expression, and indirectly by initiating chromatin modifications through Setd8. We have previously shown in our rat model of UPI that the PPARγ-Setd8 axis is present in the placenta and UPI results in sex-divergent changes in fetal serum LCPUFA profiles. Docosahexaenoic acid (DHA) is an LCPUFA ligand of PPARγ and is critical for normal fetal development. We hypothesize that UPI and DHA supplementation will cause sex divergent changes in mRNA levels of Setd8, as well as fatty acid transport and handling genes in association with altered H4k20me along one representative gene. UPI was induced by bilateral uterine artery ligation at embryonic day 19 in pregnant Sprague Dawley rats. Male and female placenta were surgically collected at term (embryonic day 21). mRNA was measured using real-time RT-PCR, and H4K20me on the SLC27A2 gene was measured using ChIP. Oil-Red-O staining was used as an additional qualitative measure of lipid accumulation in the placenta. Data are expressed as mean ± standard deviation (SD), *P < .05. In male placenta UPI increases Setd8 and alters mRNA levels of transport, binding, and handling genes. We also performed pilot experiments examining H4K20me along the fatty-acid transport protein 2 gene (gene name SLC27A2). In males, UPI caused a significant increase in H4k20me at the exon 3 region of SLC27A2 (FATP2), but not the promoter, possibly influenced by our small sample size. In contrast, in female placenta, UPI did not affect Setd8 or FATP2 mRNA, or H4K20me along the SLC27A2 (FATP2) gene. The combination of UPI and DHA supplementation, on the other hand, increased Setd8 and FATP2 mRNA, and H4K20me along the SLC27A2 (FATP2) gene in both male and female placenta. Oil-Red-O staining indicates increased lipid droplet formation in the basal zone of the male and female placenta when supplemented with DHA. In conclusion, UPI and DHA supplementation in the rat results in sex-divergent changes in Setd8 and mRNA levels of fatty acid transport and handling genes, as well as H4K20me along the SLC27A2 (FATP2) gene. Given the role of these genes in LCPUFA metabolism in the placenta, sex-divergent changes in gene expression resulting from changes to the PPARγ-Setd8 axis may influence placental handling and transfer of LCPUFA to the fetus. iv TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii LIST OF FIGURES ........................................................................................................... vi ACKNOWLEDGMENTS ................................................................................................ vii INTRODUCTION ...............................................................................................................1 Developmental Programming ..............................................................................................1 Placental Sex and LCPUFA Metabolism.............................................................................2 Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) – Setd8 Axis ....................3 Preliminary Data ..................................................................................................................4 Hypothesis............................................................................................................................5 METHODS ..........................................................................................................................7 Rat Model of UPI .................................................................................................................7 Real-Time RT-PCR .............................................................................................................7 Chromatin Immunoprecipitation..........................................................................................8 Oil-Red-O Staining .............................................................................................................8 Statistical Analysis ...............................................................................................................8 RESULTS ............................................................................................................................9 Setd8 ....................................................................................................................................9 FATP, FABP, and PLPN mRNA ........................................................................................9 H4K20me ...........................................................................................................................12 Oil-Red-O Staining ............................................................................................................12 DISCUSSION ....................................................................................................................20 REFERENCES ..................................................................................................................25 LIST OF FIGURES Figures 1. PPARγ-Setd8 axis ............................................................................................................6 2. Setd8 mRNA transcript levels .......................................................................................13 3. FATP mRNA transcript levels .......................................................................................14 4. FABP mRNA transcript levels.......................................................................................15 5. Perilipin mRNA transcript levels ...................................................................................16 6. SLC27A2 (FATP2) ChIP primer and probe sequences and locations ...........................17 7. SLC27A2 (FATP2) H4K20me pilot data ......................................................................18 8. Oil-Red-O staining .........................................................................................................19 ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Lisa Joss-Moore. Your passion for research and life is obvious and inspiring. Thank you for ever being supportive of my responsibilities as a mother while encouraging my scholarly ambitions. You have inspired me to be a better person, a better researcher, and a better dietitian. I would like to thank my committee members, Dr. Julie Metos and Dr. Kristine Jordan. Thank you for your feedback and support. I would also like to thank Haimei Wang for her unflagging support and guidance. The success of this project is in large part due to her guidance, skill, and knowledge of research techniques. Lastly, I would like to thank my mom. Because of you, I was able to dedicate myself to this research and my education while knowing that Eleanor, my daughter, was being lovingly cared for. This project would not have been possible without your constant support and love. INTRODUCTION Developmental Programming The importance of perinatal events in the determination of health in later life was first described by Barker et al. as the developmental origins of health and disease (DOHaD).1 DOHaD begins in utero. The maternal diet, obesity, toxin exposure, pathological conditions of pregnancy, including uteroplacental insufficiency (UPI), and other adverse prenatal events lead to sex-divergent changes in the fetus. These changes contribute to disease onset in both the neonatal and adult periods. In developed countries, preeclampsia is a significant cause of UPI, which results in impaired placental blood flow, compromising transport of oxygen and nutrients to the fetus. UPI leads to organ-specific structural and functional changes, which promote fetal survival in utero. However, UPI can also “program” states of gene expression in offspring through establishing altered epigenetic profiles, via chromatin modification. These chromatin and gene expression changes may predispose the offspring to disease in later life. The disease may involve many organ systems and include type 2 diabetes, cardiovascular disease, and obesity, as well as impaired neurodevelopment.2-4 Importantly, these disease outcomes are often sex-divergent, with males having worse outcomes than females. 2 Placental Sex and LCPUFA Metabolism The placenta provides the interface between the maternal environment and the fetus. It is now appreciated that disruptions to this interface contribute to developmentally programmed sex-divergent disease outcomes.5 The placenta reflects the genetic composition of the fetus and therefore responds to stressors such as UPI in a sexdivergent manner. Sex differences in the placenta are reflected in the unique and sexdivergent changes in placental structure, efficiency, and molecular characteristics induced by UPI. These differences are now being appreciated, with studies showing the placental transcriptome and DNA methylation profile is different between male and female placenta, even in the absence of a perinatal insult.2,6-8 One hypothesis is that the female placenta invests more nutrients into itself, developing a reservoir of nutrients that allows the placenta and fetus to adapt to insults late in gestation. Conversely, the rapid development of the male placenta earlier in pregnancy may enable more nutrients to be invested into the fetus itself but may prevent the male placenta from adapting as successfully as the female placenta to late gestation insults.2 UPI is characterized by impaired placental transport of oxygen and nutrients to the fetus. This impairment is especially significant for the transport of long-chain polyunsaturated fatty acids (LCPUFA). The transport of LCPUFA from maternal to fetal circulation is regulated tightly by the placenta. The placenta also stores LCPUFA for membrane and energy production.9,10 LCPUFA is critical for normal development. Impaired fetal LCPUFA acquisition during development, including impaired acquisition due to UPI, is associated with neonatal and adult disease.11,12 Theoretically, supplementation of LCPUFA could improve or prevent neonatal 3 and adult diseases. However, many studies yield conflicting results of therapeutic maternal LCPUFA supplementation during pregnancy, with studies suggesting positive, negative, and inconclusive outcomes.13-21 A critical control point in the placental handling of LCPUFA is epigenetic regulation of gene transcription, but little is known about the mechanisms regulating LCPUFA metabolism in the placenta in normal pregnancies and pregnancies complicated by UPI. Further complicating investigation, current studies rarely consider the sex of the placenta as a variable even though programming of the placental transcriptome is now accepted to be sex-divergent. Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) -Setd8 Axis PPARγ is a transcription factor activated by LCPUFAs and their metabolites. PPARγ regulates LCPUFA metabolism by indirectly influencing the transcription of suites of fatty acid metabolizing genes via chromatin modifications. Our lab has shown PPARγ upregulates the expression of Setd8 in the placenta, as well in the brain, lung, and other organs, identifying a PPARγ-Setd8 axis involved in transcriptional regulation of LCPUFA metabolism.22, 23 The PPARγ -Setd8 axis, as shown in Figure 1, is an epigenetic pathway activated by LCPUFAs that functions in the placenta and is affected by UPI. The PPARγ -Setd8 axis involves an LCPUFA ligand, such as docosahexaenoic acid (DHA) activating the PPARγ protein. The PPARγ protein then binds to a PPAR response element in the promoter of the Setd8 gene, which is then transcribed and translated into the Setd8 protein. The Setd8 protein is a histone lysine methyltransferase, and once translated is 4 then available to enzymatically place a methyl group on Histone 4 Lysine 20 on suites of long-chain fatty acid metabolizing target genes. The presence of the epigenetic mark H4K20methylation (H4K20me) has the effect of increasing gene transcription, particularly when it is located within the body of the gene.24 Preliminary Data Our lab has previously demonstrated that our rat model of UPI results in offspring that are asymmetrically growth restricted, have sex-divergent metabolic disease onset later in life in various tissues (adipose, lung, liver, and brain), and have metabolic profiles comparable to those expected in a human UPI pregnancy.22,23,25,26 The placental PPARγ-Setd8 axis, when exposed to a UPI insult, corresponds with sex-divergent changes in fetal LCPUFA acquisition. In the rat fetus, UPI reduces serum DHA levels in the male, but not the female. In the placenta supporting male fetuses, UPI increases lipid droplet formation, as well as levels of PPARγ at the Setd8 promoter, mRNA and protein levels of Setd8, and global H4k20me.27 In contrast, in placenta supporting female fetuses, UPI does not affect lipid droplet formation, PPARγ at the Setd8 promoter, mRNA and protein levels of Setd8, or global H4k20me.27 While the data indicate increased flux through the PPARg-Setd8 axis in male UPI placenta, global changes in H4k20me do not indicate which target genes are affected. To identify placental target genes for H4k20me that are affected in UPI pregnancies, in this project we examined the placental fatty acid transport and binding protein gene families FATP and FABP, as well as the Perilipin family. Fatty acid binding proteins genes FABP1, FABP3, and FABP4 are involved in 5 storage of LCPUFA within the placenta. Fatty acid transport proteins FATP1, FATP2, and FATP4, are integral membrane transport proteins. Additionally, FATP2 has intrinsic acyl-CoA synthetase activity, meaning that it can act as a transporter and promote lipid storage.28, 29 Placental FATP2 expression in normal pregnancies is linked to neonatal LCPUFA profiles and infant growth measures.30, 31 Perilipins are important in the formation and function of intracellular lipid droplets, unique organelles that are sites of active neutral lipid metabolism. Intracellular lipid droplets are surrounded by a phospholipid monolayer containing perilipin proteins.32 UPI has been shown to alter expression of Perilipin 2 (PLPN2) in the placenta; however, this study did not consider sex as a variable.33 To date, however, the effects of UPI and maternal DHA supplementation on Setd8 mRNA, and mRNA levels of the fatty acid transport and handling genes, as well as the effects of UPI and DHA on H4k20me is unknown. Hypothesis We hypothesize that UPI and DHA supplementation will cause sex divergent changes in Setd8, and mRNA levels of the fatty acid transport and handling genes in association with altered H4k20me along the SLC27A2 gene (codes for FATP2). We also hypothesize that these changes will be associated with sex-divergent changes in placental lipid droplet formation. 6 A. B. Figure 1. The PPARγ-Setd8 axis. A. Ligand-activated PPARγ protein, a transcription factor, binds to a PPAR response element (PPRE) in the promoter of the Setd8 gene. The Setd8 gene is transcribed and translated to the Setd8 protein. B. The Setd8 protein, a histone lysine methyltransferase, enzymatically places a monomethyl group on Histone 4 Lysine 20 on suites of various LCPUFA metabolizing target genes. The presence of the epigenetic H4K20me mark has the effect of increasing gene transcription. METHODS Rat Model of UPI UPI was induced in pregnant Sprague Dawley rats by bilateral uterine artery ligation. Pregnant rats were randomized to control or UPI groups, as well as regular or DHA supplemented rat chow. Rat chow was supplemented with 0.01% or 0.1% DHA. At embryonic day 19, UPI rat dams underwent bilateral uterine artery ligation to induce UPI. Control rat dams received comparable anesthesia but no surgery. At embryonic day 21, approximately term gestation, pups were delivered by C-section and separated into male or female groups. Placentas were collected at this time. Placenta from male and female rat pups were collected and immediately flash frozen in liquid nitrogen at embryonic day 21 (E21). Placentas supporting female rat pups will be referred to as female placentas and placentas supporting male rat pups as male placentas. Real-Time PCR Real-time reverse transcriptase polymerase chain reaction (RT-PCR) were be used to measure mRNA levels of Setd8, FATP1, FATP2, FATP4, FABP1, FABP3, FABP4, PLPN2, PLPN3, and PLPN5 genes in whole placenta homogenate (UPI, DHA, and control). The following Assay-on demand primer/probe sets were used: Setd8: Rn0146683_g1; FATP1: Rn00585821; SLC27A2 (FATP2): Rn00581971_m1; FATP4: Rn00581971; FABP1: Rn00664587_m1; FABP3: Rn00577366_m1; FABP4: 8 Rn04219585 (exons 1-2); Rn00670361_m1 (exons 2-3); PLPN2: Rn01399516_m1; PLPN3: Rn01527522_m1; PLPN5: Rn01527506_m1. Chromatin Immunoprecipitation Chromatin Immunoprecipitation (ChIP) were used to measure levels of H4K20me. We designed primers and probes, verified specificity using PCR, and measured levels of H4k20me in the promoter and gene body of the SLC27A2 gene (FATP2). We chose to examine SLC27A2 based on its role as both a transporter and a storage gene. Oil-Red-O Staining Oil-Red-O staining was used as an additional qualitative measure of lipid accumulation in the placenta. Frozen placenta sections were fixed, washed twice with 100% propylene glycol (ACROS Organics, Thermo Fisher Scientific NJ), stained with Oil-Red-O (Amresco, Solon, OH), washed with 85% propylene glycol, and stained with Gill #2 modified hematoxylin (Fisher Chemical, Fair Lawn, NJ). All groups were stained and visualized concurrently. Statistical Analysis UPI rat pups were compared to sex-matched control pups. Male and female rats were be considered as separate groups. One-way ANOVA with Fisher's protected leastsignificant difference was used to determine statistically significant differences between groups. Statistical significance was defined as P < .05. RESULTS Setd8 Previous work by our group investigated the effect of UPI and DHA on Setd8 mRNA27. Results from this study confirmed previous results, showing UPI increased Setd8 mRNA levels in male placentas only (Figure 2). Of additional interest was the effect of two doses of DHA (0.1% and 0.01%) on Setd8 mRNA levels. The combination of DHA and UPI normalized Setd8 mRNA levels in male placenta. In females, the combination of UPI and DHA supplementation significantly increased Setd8 mRNA levels. FATP, FABP, and PLPN mRNA To determine potential target genes of the PPARg-Setd8 axis, we measured mRNA transcript levels of placental FATP (FATP1, FATP2, FATP4), FABP (FABP1, FABP3, FABP4), and Perilipin (PLPN2, PLPN3, PLPN5) families. mRNA levels of the FATP family had varying responses to UPI and DHA (Figure 3). UPI did not affect male FATP1 transcription levels, and 0.01% DHA supplementation decreased FATP1 levels to control levels (P < .05), while 0.1% DHA supplementation at increased transcription levels above control levels, to UPI levels (P < .05). In females, UPI decreased FATP1 levels (P < .05), but high DHA (0.1%) supplementation normalized FATP1 transcription levels to the control (P < .05). A significant difference 10 was seen between DHA supplementation levels in females (P < .05). The FATP2 mRNA response was similar to that of Setd8. UPI increased FATP2 mRNA levels in males (P < .05), but not in females. Additionally, 0.01% DHA normalized the FATP2 UPI response in males, with no significant changes between the control male and 0.01% DHA supplemented UPI males. However, 0.1% DHA supplementation increased FATP2 mRNA transcription similarly to UPI response. In females, consistent with the Setd8 response, 0.01% DHA supplementation increased FATP2 mRNA levels (P < .05), while UPI, and UPI 0.1% DHA did not cause any changes relative to control. UPI had no significant effect on FATP4 mRNA transcription levels in males, but a significant difference was seen between control and 0.1% DHA, and 0.01% DHA supplementation males (P < .05). In females, UPI, 0.1% DHA, and 0.01% DHA supplementation had no significant effect on FATP4 transcription levels. Additional sex-divergent results were seen in mRNA transcription levels responses in the FABP family of genes (Figure 4). In males, 0.01% DHA supplementation decreased FABP1 levels below control levels, but UPI and 0.1% DHA had no significant effect (P < .05). In females, 0.1% DHA supplementation increased levels relative to UPI but not control levels of FABP1. Both DHA supplementation levels decreased FABP3 transcription levels relative to control levels in males, but only 0.1% DHA supplementation significantly decreased FABP3 levels relative to UPI. In females, 0.1% DHA increased FABP3 levels as compared to 0.01% DHA supplementation, UPI, and control levels. UPI decreased male FABP4 transcription. 0.01% DHA supplementation, but not 0.1% DHA supplementation, normalized levels to control. In 11 females, both levels of DHA supplementation caused an increase in FABP4 levels above UPI levels and indicated a dose-dependent response. DHA 0.1% resulted in significantly higher FABP4 levels than DHA 0.01%. Sex-divergent results were seen in members of the Perilipin family in response to UPI and DHA supplementation (Figure 5). In males, both levels of DHA supplementation caused a significant decrease in PLPN2 transcription relative to control and UPI. However, in females, only 0.1% DHA supplementation caused a significant increase in PLPN2 transcription levels above control. Additionally, there was a significant difference in levels between the two DHA doses, with 0.1% DHA supplementation causing an increase in transcription levels. UPI caused a significant increase in PLPN3 in males only. PLPN3 levels were decreased with both levels of DHA supplementation relative to control and UPI in males. UPI, 0.01% DHA, and 0.1% DHA supplementation did not cause any significant differences in female PLPN3 levels. In males, UPI caused a significant increase in PLPN5 levels, but 0.1% DHA supplementation normalized these levels to control. A dose-dependent response was seen between DHA supplementation levels. In females, no significant difference was seen between control, UPI, or 0.01% DHA supplementation; however, 0.1% DHA supplementation decreased PLPN5 levels below UPI levels. H4K20me From the group of genes measured that we measured mRNA, we chose to examine H4K20me on SLC27A2 because of its intrinsic lipid transport and storage 12 ability. We designed and tested primers and probes to verify the correct positioning of the amplicon (Figure 6). Next, we conducted a pilot study of H4K20me at both the promoter and exon 3 regions of the SLC27A2 gene to evaluate the effects of UPI and the clinically relevant concentration of DHA (0.01%) on both transcription and elongation. UPI did not alter H4k20me in the promoter region of the SLC27A2 gene in male placenta or female placenta (Figure 7). However, the combination of UPI and DHA significantly increased H4k20me in the promoter region of the SLC27A2 gene in females only (P < .05). Of note, this change was accompanied by large biological variation. Finally, we measured H4K20me in the SLC27A2 Exon 3 region. In male placenta, UPI increased levels of H4K20me in the exon 3 region, and DHA plus UPI was not significantly different from UPI alone. However, the combination of UPI and DHA supplementation caused an increase in H4k20me at exon 3 of the SLC27A2 gene in female placenta while UPI alone was not significantly different from control (P < .05). Oil-Red-O SLC27A2 (FATP2) is implicated both transport and the esterification of fatty acids in the placenta. We performed a qualitative assessment of lipid droplets in the basal zone of the placenta using frozen tissue. Supplementation of 0.01% DHA resulted in a visual increase in lipid droplet formation in the basal zone of the placenta (Figure 8). 13 Female * * Male 0.08 Setd8 mRNA (Relative to GAPDH) * * * * 0.08 0.06 0.06 0.04 0.04 0.02 0.02 0.00 0.00 Control UPI UPI DHA UPI DHA 0.01% 0.1% Control UPI UPI DHA UPI DHA 0.01% 0.1% Figure 2. Setd8 mRNA transcript levels relative to GAPDH (n=6/group). *P < .05 compared to sex-matched control. 14 A. A Male * 0.020 * . FATP1 mRNA (Relative to GAPDH) 0.010 0.010 0.005 0.005 Control UPI DHA UPI DHA 0.01% 0.1% UPI * 0.020 0.015 0.000 * * * Control UPI Male B. 0.015 Female * 0.010 0.010 0.005 0.005 0.000 Control UPI DHA UPI DHA 0.01% 0.1% UPI C. 0.000 * Control 0.03 0.03 0.02 0.02 0.01 0.01 0.00 UPI UPI DHA UPI DHA 0.01% 0.1% 0.05 0.04 Control UPI Female 0.04 0.00 * * Male 0.05 UPI DHA UPI DHA 0.01% 0.1% 0.015 * * FATP4 mRNA (Relative to GAPDH) * 0.015 0.000 FATP2 mRNA (Relative to GAPDH) Female UPI DHA UPI DHA 0.01% 0.1% Control UPI UPI DHA UPI DHA 0.01% 0.1% Figure 3. FATP mRNA transcript levels relative to GAPDH (n=6/group). *P < .05 compared to sex-matched control. A. FATP1 mRNA levels. B. FATP2 (SLC27A2) mRNA levels. C. FATP4 mRNA levels. 15 A. A . FABP1 mRNA (Relative to GAPDH) Female Male 0.0004 0.0004 0.0003 0.0003 0.0002 0.0002 * 0.0001 0.0001 0.0000 0.0000 Control UPI DHA UPI DHA 0.01% 0.1% UPI B. * Control Male UPI DHA UPI DHA 0.01% 0.1% UPI Female 2.0 * 2.0 * * FABP3 mRNA (Relative to GAPDH) 1.5 * 1.5 * 1.0 1.0 0.5 0.5 0.0 Control UPI C. UPI DHA UPI DHA 0.01% 0.1% 0.0 Control UPI DHA UPI DHA 0.01% 0.1% UPI Female Male * FABP4 mRNA (Relative to GAPDH) * 0.04 * * 0.04 * 0.02 0.02 0.00 * 0.06 0.06 Control UPI UPI DHA UPI DHA 0.01% 0.1% 0.00 Control UPI UPI DHA UPI DHA 0.01% 0.1% Figure 4. FABP mRNA transcript levels relative to GAPDH (n=6/group). *P < .05 compared to sex-matched control. A. FABP1 mRNA levels. B. FABP3 mRNA levels. C. FABP4 mRNA levels. 16 A. . PLPN2 mRNA (Relative to GAPDH) A Female Male * 0.4 0.4 * 0.3 * 0.3 * 0.2 0.2 0.1 0.1 0.0 0.0 Control UPI DHA UPI DHA 0.01% 0.1% UPI Control Female Male B. UPI DHA UPI DHA 0.01% 0.1% UPI * 0.06 PLPN3 mRNA (Relative to GAPDH) * * 0.06 0.04 0.04 0.02 0.02 0.00 Control UPI C. UPI DHA UPI DHA 0.01% 0.1% 0.00 Control Male Female * 0.00015 UPI DHA UPI DHA 0.01% 0.1% UPI 0.00015 * * PLPN5 mRNA (Relative to GAPDH) * 0.00010 0.00010 * 0.00005 0.00000 0.00005 Control UPI UPI DHA UPI DHA 0.01% 0.1% 0.00000 Control UPI UPI DHA UPI DHA 0.01% 0.1% Figure 5. Perilipin mRNA transcript levels relative to GAPDH (n=6/group). A. PLPN2. B. PLPN3. C. PLPN5. *P < .05 compared to sex-matched control. 17 Figure 6. SLC27A2 (FATP2) ChIP primer and probe sequences and locations 18 A. Male A Female * 10 . 8 Promoter H4K20me (Percent of Input) * 10 8 6 6 4 4 2 2 0 Control UPI B. UPI DHA 0 Control Male B 20 15 15 Exon 3 H4K20me 10 (Percent of Input) 10 * * * 5 0 UPI DHA Female * 20 . UPI Control 5 UPI UPI DHA 0 Control UPI UPI DHA Figure 7. SLC27A2 (FATP2) H4K20me pilot data (n=3/group) *P < .05 compared to sex-matched control. 0.01% DHA supplementation A. H4K20me at the Promotor region of SLC27A2. B. H4K20me at the Exon 3 region of SLC27A2. 19 A. BA .. B. Figure 8. Oil-Red-O staining. A. Staining in the placental basal layer. Red droplets are lipid droplets. B. Lipid staining in the placental labyrinth layer. Notice the lack of lipid droplets as compared to the basal layer. DISCUSSION The purpose of this study was to investigate the sex-divergent effects of UPI and DHA on the placental PPARγ-Setd8 axis by measuring mRNA transcript levels of Setd8, placental FATP gene family (FATP1, FATP2, FATP4), FABP gene family (FABP1, FABP3, FABP4), and Perilipin gene family (PLPN2, PLPN3, PLPN5) as well as H4K20me of the Promoter and Exon 3 regions of SLC27A2 (FATP2). We demonstrated that UPI and DHA cause sex-divergent changes in Setd8, FATP, FABP, and PLPN mRNA, as well as H4K20me of SLC27A2 (FATP2). We also visualized increased lipid droplet formation in DHA supplemented placenta. Collectively, these data suggest that DHA supplementation may interfere with placental lipid handling in both sexes in the context of UPI, and additional research is needed to elucidate the precise scenarios, including sex and dosage variables, where DHA supplementation may be protective or restorative. To our knowledge, the preliminary data on the PPARγ-Setd8 axis from our group will be the first report of this axis in placental tissue, though it has been previously reported in adipose tissue.12 Thus, identifying PPARγ-Setd8 axis target genes adds to the understanding of the molecular mechanisms of this sex-divergent axis. We demonstrated a significant difference between UPI and control FATP2 mRNA, indicative of sexdivergent flux through the axis. Additionally, increased H4K20me of the SLC27A2 (FATP2) Exon 3 region in UPI males may indicate reprogramming of transcription of the 21 SLC27A2 gene medicated by the PPARγ-Setd8 axis. The sex-divergent effects of DHA warrant further study. DHA caused significant increases in the H4K20me of the SLC27A2 (FATP2) promoter and exon 3 in females while UPI caused a significant increase in the H4K20me of SLC27A2 exon 3 in males. These differing increases indicate a sex-divergent effect of the fatty acid on placental chromatin methylation. In Setd8 and FATP2 mRNA, DHA rescued UPI transcription levels in males but increased transcription levels above UPI and control in females. In males, when UPI significantly affected transcription levels, one of the two DHA doses tended to normalize levels to control, as in FABP4 and PLPN5. Females had a smaller number of significant responses to both UPI and DHA supplementation, but these changes tended to be significantly different from the control and appeared to be more sensitive to DHA supplementation, as in FABP3 and FABP4. These results may begin to explain some of the inconclusive and sex-divergent effects of DHA supplementation. These differing responses to UPI and DHA supplementation, particularly the lack of significantly different transcription levels caused by UPI alone in the female placenta, reinforce the hypothesis that the female placenta may develop by investing more nutrients into the fetus itself, allowing the placenta and fetus to adapt more successfully to late gestation insults than the male placenta. Additionally, the hypothesis of more rapid male placental development and increased tendency to invest more nutrients into the fetus initially may partially explain the more beneficial effects of DHA supplementation on mRNA transcription levels of fatty acid transport and handling genes in the male placenta.2 Our study has strengths and limitations. Our UPI rat model is well-characterized 22 and therefore a strength of our study. Our lab is experienced in performing the bilateral artery ligation surgical procedure to induce UPI in pregnant rats. In our model, UPI is induced late in gestation and therefore excludes maternal adaptations. Additionally, previous work in our lab has demonstrated that our UPI model causes sex-divergent structural and functional changes in rat offspring and programming of adult disease in adipose, lung, and brain, comparable to results seen in human pregnancies complicated by UPI. The dose of DHA used is an additional strength of our study. Our 0.01% dose is a clinically relevant dose of DHA, approximating, by body weight, the dose contained in most prenatal vitamin supplements. A limitation to our study is that molecular measurements of mRNA and H4k20me are done in whole placental tissue, therefore sampling multiple placental cell types. Future studies will attempt to isolate specific cell types within the placenta, furthering our knowledge of specific epigenetic modifications and other molecular mechanisms of LCPUFA metabolism in the placenta. The number of samples considered in the ChIP study of H4K20me on SLC27A2 is another limitation to our study, and can at this time only be considered as pilot data to inform later research. Investigating H4K20me along other fatty acid transport and handling genes will be important to the continued understanding of the PPARg-Setd8 axis. Additionally, we recognize that we have not considered all potential target genes of the PPARg-Setd8 axis. This limitation will provide the basis for future studies. A further limitation is that we did not quantify lipid droplet accumulation. Ongoing studies are quantifying lipid droplets in the basal and labyrinth zones of OTC embedded placental tissue. Future studies are also underway to examine the sex-divergent effects of UPI and 23 DHA supplementation in specific cell types and layers in the placenta. We speculate a sex-based mechanism by which placental LCPUFA is altered in UPI pregnancies in a sex-divergent manner. Studies investigating DHA supplementation in UPI pregnancies, including those complicated by preeclampsia, should consider sex as a variable, due to the differing placental transcriptomes and methylation patterns exhibited in these pregnancies. In conclusion, in male placenta, UPI causes sex-divergent changes in mRNA transcription levels in the fatty acid transport and handling gene families FATP, FABP, and Perilipin. DHA supplementation, both at 0.01% and 0.1% supplementation, similarly causes sex-divergent changes in mRNA transcription levels, though the effects of supplementation are not always restorative of UPI induced changes over control levels. In the male placenta, UPI significantly increases H4k20me in SLC27A2 exon 3. In contrast, in female placenta, UPI does not affect H4k20me at any location on SLC27A2. The combination of UPI and DHA supplementation, on the other hand, increases H4K20me in the SLC27A2 gene in both male and female placenta. 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