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. 2011 Jun 8;85(4):678–683. doi: 10.1095/biolreprod.111.092148

Preimplantation Exposure of Mouse Embryos to Palmitic Acid Results in Fetal Growth Restriction Followed by Catch-Up Growth in the Offspring1

Emily S Jungheim 3, Erica D Louden 3, Maggie M-Y Chi 3, Antonina I Frolova 3, Joan K Riley 3, Kelle H Moley 3,,4,2,
PMCID: PMC3184288  PMID: 21653893

Abstract

Free fatty acids (FFAs) are energy substrates for many cell types, but in excess, some FFAs can accumulate in nonadipose cells, inducing apoptosis. Also known as lipotoxicity, this phenomenon may play a role in the development of obesity-related disease. Obesity is common among reproductive age women and is associated with adverse pregnancy and fetal outcomes; however, little is known about the effects of excess FFAs on embryos and subsequent fetal development. To address this knowledge gap, murine blastocysts were cultured in excess palmitic acid (PA), the most abundant saturated FFA in human serum, and ovarian follicular fluid. Targets susceptible to aberrations in maternal physiology, including embryonic IGF1 receptor (IGF1R) expression, glutamic pyruvate transaminase (GPT2) activity, and nuclei count, were measured. PA-exposed blastocysts demonstrated altered IGF1R expression, increased GPT2 activity, and decreased nuclei count. Trophoblast stem cells derived from preimplantation embryos were also cultured in PA. Cells exposed to increasing doses of PA demonstrated increased apoptosis and decreased proliferation. To demonstrate long-term effects of brief PA exposure, blastocysts cultured for 30 h in PA were transferred into foster mice, and pregnancies followed through Embryonic Day (ED)14.5 or delivery. Fetuses resulting from PA-exposed blastocysts were smaller than controls at ED14.5. Delivered pups were also smaller but demonstrated catch-up growth and ultimately surpassed control pups in weight. Altogether, our data suggest brief PA exposure results in altered embryonic metabolism and growth, with lasting adverse effects on offspring, providing further insight into the pathophysiology of maternal obesity.

Keywords: developmental biology, developmental origins of health and disease, early development, embryo, embryo culture, metabolism

Preimplantation exposure to excess palmitic acid results in metabolic changes in the embryo, increased apoptosis, and decreased cellular proliferation of embryonic stem cells culminating in altered growth both in the fetus and postnatally.

INTRODUCTION

It is well established that obese women are at increased risk for adverse pregnancy outcomes including gestational diabetes and preeclampsia [1, 2]. It is also well established that children born to obese women are at increased risk for fetal malformations and high birth weight [3–7]. Of further concern is the fact that there is mounting evidence that children born to obese women are at increased risk for obesity-related disease, a risk thought to be due in part to fetal programming in response to an aberrant maternal environment [1, 8–11]. As a matter of disease prevention, obese women traditionally have been counseled to limit weight gain during pregnancy [2, 7]. Unfortunately this measure may be too late in preventing the adverse effects of maternal environment on the offspring, as susceptible developmental events may have already occurred. Support for this is demonstrated clinically in another example of aberrant preconception physiology, pregestational diabetes. In cases of poorly controlled pregestational diabetes, high glucose levels at the time of conception result in miscarriage or fetal malformation despite optimal glucose control after clinical recognition of pregnancy [12]. Further support is found in experimental animal models of diabetes from our laboratory, where preimplantation embryos exposed to excess glucose exhibit mitochondrial dysfunction, insulin signaling abnormalities, and increased apoptosis. These embryonic abnormalities manifest later as fetal growth disturbances and malformations [13–17].

Like diabetes, obesity is a condition marked by aberrations in maternal hormonal milieu and metabolic homeostasis that have the potential to affect early reproductive events. In addition to abnormal insulin signaling and elevated glucose levels, obesity is often marked by elevated free fatty acid (FFA) levels [18–20]. Studies have shown that specific saturated FFAs like palmitic acid (PA), a fatty acid produced by adipose tissue and acquired through diet, may play a role in development of obesity-related disease in the nonpregnant state [21]. In excess, PA can cause insulin resistance and mitochondrial dysfunction with subsequent apoptosis in various nonadipose cells including pancreatic beta cells [22] and cardiac myocytes [23], a phenomenon known as lipotoxicity [23]. PA has been isolated from uterine fluid in mammalian species [24, 25], but little is known of its effects on the preimplantation embryo and fetal development, despite the fact that the preimplantation embryo is capable of metabolizing PA [26–28].

Recently we developed a reproductive model of diet-induced obesity characterized by elevated serum FFA and glucose levels [9]. In this murine model, preimplantation embryos demonstrate decreased IGF1 receptor expression, and the resulting fetuses are growth restricted. Accelerated growth patterns and metabolic parameters consistent with the development of a metabolic syndrome are seen in delivered pups, despite placement on a normal diet after weaning, suggesting that exposure to some component of maternal obesity during development contributes to lasting morbidity in the offspring. Given the findings of our work with this in vivo model and our previous work demonstrating the adverse effects of excess glucose on preimplantation embryos and subsequent fetal development, we were interested in focusing on the short- and long-term effects of isolated preimplantation embryonic exposure to excess FFAs. Specifically, we investigated the effects of PA exposure for 30 h on blastocyst stage embryos and on IGF1 receptor (IGF1R) expression and the activity of glutamic pyruvate transaminase 2 (GPT2), an enzyme sensitive to changes in insulin signaling and available glucose [29, 30]. We also assessed cell count in exposed embryos. After noting fewer cells in embryos exposed to excess PA, we used a stem cell model of blastocyst trophectoderm to measure proliferation and apoptosis in response to increasing doses of PA. To determine if the effects of brief exposure to excess PA in the preimplantation stage of embryonic development had long-term effects on the offspring, we transferred PA-exposed blastocysts to recipient mice and followed the growth of the fetus and resulting pups at several time points.

MATERIALS AND METHODS

In Vitro Embryo Recovery and Embryo Culture

Institutional approval was obtained for all experiments performed. All mice were properly treated in accordance with the National Research Council's Guide for Care and Use of Laboratory Animals. Female B6×SJL F1 mice 3–4 weeks old (The Jackson Laboratory, Bar Harbor, ME) were superovulated with pregnant mare serum gonadotropin (Sigma Chemical Co., St. Louis, MO) and human chorionic gonadotropin (Sigma). The females were mated with B6×SJL F1 males of proven fertility and sacrificed 72 h later by cervical dislocation. Morulae were obtained by flushing dissected uterine horns and ostia as previously described [17] and immediately placed in human tubal fluid (HTF; Irvine Scientific, CA) containing 0.25% bovine serum albumin (BSA, fraction V; Sigma) and cultured at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 overnight. Resulting blastocysts were cultured under oil in groups of 20–30 for 30 h under one of the following conditions: i) control HTF with 0.25% BSA; or ii) a 200 μM PA solution in HTF with 0.25% BSA (Sigma). PA-containing medium was prepared as described previously [31]. Briefly, a solution of 20 mmol/L PA in 0.01 N NaOH was incubated for 30 min at 70°C. A 200-μl aliquot of the PA-NaOH solution was mixed with 200 μl of 30% BSA and filter sterilized with 20 ml of HTF medium. These conditions were based on a review of pertinent literature demonstrating total FFA serum levels of 200-2000 μM in normal and pathologic states [22, 31–34] and measurement of the in vivo physiologic concentrations of saturated fatty acids in the human reproductive tract from our previous study of follicular fluid and serum FFA levels in women undergoing in vitro fertilization (follicular saturated fatty acids, 20–169 μM; serum saturated fatty acids, 43–383 μM) [35].

Visualization of IGF1R Expression in Embryos by Confocal Microscopy

Blastocysts from the two culture conditions were fixed on slides in 3% paraformaldehyde (Sigma) for 20 min and permeabilized in a 0.1% Tween-20 (Sigma) solution for 20 min. The embryos were blocked with 20% normal donkey serum in PBS with 2% BSA (Sigma) at room temperature for 60 min in a humidified chamber. Slides were washed three times with PBS/BSA, followed by 1-h incubation with antibody against IGF1R (Upstate Cell Signaling, Lake Placid, NY). Three washes with PBS/BSA were performed, followed by a 20-min incubation with an appropriate fluorescein-tagged secondary antibody. The embryos were washed three more times in PBS/BSA, and nuclei were stained with To-Pro-3-iodide (Molecular Probes, Eugene, OR) for twenty min. Three final washes with PBS/BSA were performed, and the embryos were mounted in VectaShield (Vector Labs, Burlingame, CA), covered with cover slips, and sealed. The slides were examined with an Olympus laser-scanning microscope. Relative IGF1R staining intensity was quantified by two independent and blinded observers as previously described [9]. Briefly, the observers assigned scores to embryos cultured in control vs. those cultured in PA-containing medium on a scale of 1 to 3 for intensity of IGF1R staining of the entire embryo and for IGF1R staining of the basolateral membrane.

GPT2 Analysis in Cultured Embryos

Blastocysts from each of the culture conditions were freeze dried as previously described [29, 36, 37]. Briefly, blastocysts from each culture condition were individually transferred with 0.5–1 μl of culture medium onto a glass slide with a braking pipette, dipped into isopentane followed by liquid nitrogen, and then freeze dried in a glass vacuum tube placed in a cryostat at −35°C. Specimens were transferred to individual wells drilled into microscope slides and stored at −70°C. GTP2 levels were measured in frozen blastocysts by using previously described microfluorometric cycling enzymatic assays [29, 37] and were expressed as picomoles per hour.

Embryo Cell Counts

For this experiment, collected morulae were cultured for 30 h in control medium or 200 μM PA. Resulting blastocysts were fixed on slides, and their cellular nuclei were stained with To-Pro-2 iodide. Stained embryos were subsequently mounted, covered, and sealed as described above. A Z-series consisting of eight sections was taken for each embryo, using the laser-scanning microscope. Nuclei count per embryo was determined by adding the number of nuclei counted from each section and dividing the sum by 8.

Trophoblast Stem Cell Culture

Trophoblast stem (TS) cells were obtained from murine C57BL/6J embryos, as described previously [38, 39], and plated at 4 × 105 cells in 6-well plates and grown overnight at 37°C and 5% CO2. The cells were then cultured for 30 h under the following conditions: i) control TS medium [40]; ii) 200 μM PA TS medium (Sigma); iii) 300 μM PA TS medium; or iv) 400 μM PA TS medium.

PA-containing medium was prepared using methods similar to those described above for preparation of the embryo culture medium by adding either 200 μl, 300 μl, or 400 μl of the PA-NaOH solution with TS medium containing BSA. The resulting mixture was filter sterilized.

Evaluation of TS Cell Proliferation by BrdU Uptake

TS cells were plated at a density of 20 000 cells per well in 96-well plates, grown overnight at 37°C and 5% CO2, and then cultured for 30 h in the culture conditions described above. Bromodeoxyuridine (BrdU) reagent obtained from the BrdU cell proliferation assay (Chemicon International, Temecula, CA) was added for the final 18 h of culture. Optical density reflecting BrdU uptake was measured on a spectrophotometer following the manufacturer's instructions.

Evaluation of TS Cell Apoptosis by Transferase-Mediated dUTP Nick-End Labeling Assay

TS cells cultured under the conditions described above were analyzed for apoptosis by using terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling (TUNEL) assay as described previously [41]. Briefly, TUNEL assay was performed using In Situ Cell Death Detection kit, TMR (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. TUNEL staining of the TS cells was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). An arbitrary gate was drawn, and the percentage of apoptotic cells within the gate was determined using Cell Quest software (BD Biosciences).

Embryo Transfer Studies

Unhatched cultured blastocysts from the culture conditions described above were transferred into separate pseudo-pregnant ICR female mice 3.5 days postcoitum [42]. Ten blastocysts were transferred into each uterine horn for each recipient mouse. One group of recipients was sacrificed on Embryonic Day 14.5, and fetuses were collected and evaluated for crown–rump length, whereas another group of recipients was left to deliver. Offspring were weighed and weaned at Day of Life (DOL) 18. They were followed for growth by weight at DOL 25, 35, and 50.

Statistical Methods

Blastocyst IGF1R staining, GPT2 levels, and nuclei counts were compared using two-tailed Student t-test or χ2 analysis where appropriate. Fetal crown–rump lengths and weights of the offspring from the transfer experiments were compared using two-tailed Student t-test. Apoptosis and proliferation in TS cells cultured under the different conditions were compared using ANOVA. Results are expressed as means ± SE. Significance was defined as a P value of <0.05.

RESULTS

IGF1R Expression Is Altered in Embryos Exposed to Excess PA

Intensity of IGF1R expression in embryos cultured in control medium (n = 4) vs. that of embryos cultured in excess PA (n = 4) was not different (1.75 vs. 2.5, P = 0.1). On the other hand, in evaluating specific staining patterns, we found that IGF1R staining in the basolateral membrane of PA-exposed embryos was significantly increased over that of embryos cultured in control medium, suggesting increased membrane expression of IGF1R in the embryos exposed to PA (1 vs. 2.5, P = 0.01) (Fig. 1).

FIG. 1.

FIG. 1.

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IGF1R staining in embryos cultured in control medium vs. 200 μM PA is shown. Overall intensity levels of IGF1R staining in the embryos were no different; however, IGF1R expression in the basolateral membrane of the embryonic cells was more intense in embryos exposed to PA (1 vs. 2.5, P = 0.01). Fluorescence was detected with laser-scanning confocal immunofluorescence microscopy (Nikon Eclipse E800 model; Nikon Instruments Corp., Melville, NY). Original magnification ×63.

GPT2 Activity Is Increased in Embryos Exposed to PA

Given the altered pattern of IGF1R expression demonstrated by embryos exposed to excess PA, we sought to determine if downstream targets of the IGF1 signaling pathway were affected in PA-exposed blastocysts. We chose to do this through measurement of GPT2 activity, as we had shown previously that GPT2 activity was associated with abnormal insulin signaling [29].

In this study, embryos cultured in PA (n = 12) demonstrated GPT2 activity that was increased compared to that of embryos cultured in control medium (n = 12) (PA, 0.311 ± 0.01 pmol/h, vs. control, 0.29 ± 0.017 pmol/h [P < 0.05]) (Fig. 2).

FIG. 2.

FIG. 2.

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GPT2 activity in embryos cultured in control medium vs. that in 200 μM PA is shown. GPT2 activity was increased in embryos cultured in 200 μM PA. Values are means ± SEM from at least three independent experiments. *P < 0.05.

Embryo Nuclei Count Is Decreased in Embryos Exposed to PA

More nuclei were present in embryos cultured in control medium (107.35 ± 12.12 nuclei, n = 23) than in embryos cultured in PA (90.52 ± 11.04 nuclei, n = 21 [P < 0.0001]).

Cellular Proliferation in TS Cells Is Decreased after Exposure to PA

Given that we detected fewer nuclei in embryos exposed to PA, we sought to determine if this could be attributed to decreased cellular proliferation. Cellular proliferation in TS cells cultured in PA was decreased compared to that in control medium, as measured by optical density with the BrdU uptake assay (control, 2.97 ± 0.11; 200 μM PA, 2.64 ± 0.15 [P < 0.01]; 300 μM PA, 2.69 ± 0.03 [P < 0.01]; 400 μM PA, 2.67 ± 0.05 [P < 0.01]; n = 3).

Apoptosis Is Increased in TS Cells Exposed to PA

Given previous work demonstrating that apoptosis resulted from exposure of nonadipose cells to excess lipid, we also sought to determine if apoptosis was increased in TS cells exposed to excess PA. We found that apoptosis increased in a dose-dependent manner; however, only the cells exposed to the highest concentration, 400 μM PA, demonstrated significantly more apoptosis than cells grown in control medium (control, 100 geometric mean fluorescence intensity; 200 μM PA, 109.6 (7.14; 300 μM PA, 114.2 (7.4; 400 μM PA, 171 (14.8211935 [P < 0.01]; n = 3) (Fig. 3).

FIG. 3.

FIG. 3.

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Apoptosis is increased in TS cells exposed to increasing concentrations of PA (n = 3). Control, 100; 200 μM PA, 109.6 ± 7.14; 300 μM PA, 114.2 ± 7.4; 400 μM PA, 171 ± 14.82. Values are means ± SEM from at least three independent experiment. *P < 0.05.

Fetuses from Blastocysts Exposed to PA in the Preimplantation Stage of Development Are Growth-Restricted

Fetuses resulting from preimplantation embryos exposed to excess PA and transferred into control mice were significantly smaller than those resulting from embryos exposed to control conditions (1.02 ± 0.02 cm, n = 37 control fetuses in six recipients vs. 0.96 ± 0.12 cm, n = 25 PA-exposed fetuses in six recipients, P < 0.002) (Fig. 4). Implantation rates were not different between the two groups (73% in controls vs. 65% in PA-exposed, P = 0.21).

FIG. 4.

FIG. 4.

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Smaller fetuses resulted from embryos cultured in 200 μM PA than from embryos cultured in control medium. A) Crown–rump length at Embryonic Day 14.5 (e14.5) is significantly shorter in fetuses from embryos cultured in 200 μM PA than from those cultured in control medium. Values are means ± SEM from at least three independent experiments. B) Representative fetuses from each group are shown. Original magnification ×20.

Offspring Weight and Growth

Weight data were collected at DOL 18, 25, 35, and 50. At DOL 18, offspring derived from embryos cultured in excess PA were significantly smaller than offspring derived from embryos cultured in control medium (PA-exposed embryos, 4.2 ± 0.4 g, n = 14, vs. controls, 6.1 ± 0.2 g, n = 11; P < 0.0001), a finding consistent with fetal growth restriction. By DOL 25, however, the growth-restricted mice caught up in size, and by DOL 50, they were significantly larger (PA-exposed offspring, 28.6 ± 2.1 g, n = 13, vs. controls, 23 ± 1.9 g, n = 10; P < 0.0001) (Fig. 5).

FIG. 5.

FIG. 5.

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Smaller pups resulting from embryos cultured in 200 μM PA vs. control medium demonstrated catch-up growth followed by increased body weight by DOL 50. Weights are shown at DOL 18 (n = 11 control, n = 14 PA), 25 (n = 11 control, n = 13 PA), 35 (n = 10 control, n = 13 PA), and 50 (n = 10 control, n = 13 PA). Values are means ± SEM from at least three independent experiments. *P < 0.0001.

DISCUSSION

The experiments outlined above provide novel insight into potential mechanisms involved in the development of adverse fetal outcomes in the setting of maternal obesity. In our model, metabolic sequelae of PA exposure in preimplantation embryos include altered IGF1R expression and increased GPT2 activity. These metabolic changes are associated with fewer cellular nuclei in PA-exposed embryos, possibly the result of both increased apoptosis and decreased cellular proliferation in the embryo, as suggested by our experiments in which TS cells were exposed to increasing concentrations of PA. In other words, preimplantation embryos demonstrate altered metabolic and growth patterns in response to exposure to excess PA. In our transfer experiments, fetuses resulting from these embryos were growth restricted in utero and early in postnatal life compared to embryos cultured in control medium, despite postimplantation exposure to the same maternal environment. Later, however, offspring derived from PA-exposed blastocysts ultimately caught up in growth and surpassed that of control offspring, despite being fed the same postnatal diet. This aberrant growth pattern has been associated with fetal programming and the development of adult onset diabetes and obesity in both human epidemiologic studies and in experimental animal studies [1, 43]. The novel aspect of this study is that unlike most previous work that has examined postimplantation events and postnatal growth [43], this study focuses on exposure in just the preimplantation stage of development, prior to maternal recognition of pregnancy.

The preimplantation stage of development is a critical time in the growth of an organism, marked by increased energy demands and sensitivity to aberrations in availability of maternal nutrients. This sensitivity has been demonstrated in experimental models of maternal diabetes from our laboratory, in which normal insulin signaling and energy availability in the preimplantation embryo is impaired in response to elevated glucose levels. In these models, even brief preimplantation exposure to an aberrant maternal environment results in changes in embryonic metabolism with subsequent adverse fetal outcomes in the long term [13, 16, 17, 44].

In the present study, IGF1R expression at the basolateral membrane is increased in PA-exposed blastocysts, along with increased GPT2 activity, suggesting altered embryonic insulin signaling through the IGF1R and altered energy substrate metabolism. GPT2 activity is critical to maintaining flux of amino acids and pyruvate through the tricarboxylic acid cycle, and it is sensitive to alterations in insulin signaling and decreased availability of glucose [29, 30, 45]. While metabolic changes like the ones seen in our PA-exposed embryos could affect long-term outcomes merely by influencing preimplantation cellular proliferation and apoptosis through altered energy availability, another possibility is that these metabolic changes are indicative of global changes in the organism's epigenetic regulation of metabolism or aberrant programming of mitochondrial function, as suggested by authors working with other in vivo models of maternal obesity and high fat feeding [10, 11, 43].

A recently published study by McCurdy et al. [11] demonstrated that high fat feeding in primates during pregnancy resulted in increased expression of multiple fetal genes involved in the gluconeogenic pathway. Further work in the same model demonstrated alterations in histone modification of several fetal genes important to metabolism, including the Gpt2 gene [10]. Pertinent to our model, the increase in GPT2 activity seen in the blastocyst could be indicative not only of altered metabolic function but also of histone modification of the Gpt2 gene. Further studies at this stage of development need to be done to investigate this possibility and to investigate other genes that may be altered in response to excess PA.

Many links have recently been made between elevated levels of PA and cellular dysfunction, insulin resistance, and cellular death in a number of different tissue types including skeletal tissue, muscle, cardiac muscle, and pancreatic beta cells [19, 22, 23]. This phenomenon, known as lipotoxicity, is characterized by lipid accumulation in nonadipose tissues, resulting in increased endoplasmic reticulum stress and subsequent cell death [23, 34]. While other studies have demonstrated embryos are capable of metabolizing PA [28], this is the first study, to our knowledge, that demonstrates the acute adverse effects of excess PA on preimplantation embryos that subsequently manifest long-term as effects such as fetal growth restriction and abnormal growth patterns in the offspring.

We believe the metabolic alterations we detected in PA-exposed embryos along with altered cellular proliferation and growth inferred by our TS cell experiments culminated in fetal growth restriction followed by postnatal catch-up growth. This latter finding is of particular concern as epidemiological [46–48] and experimental models [49] have linked this type of growth pattern in the offspring to adult onset or type 2 diabetes and cardiovascular disease. On the other hand, in our model, we explored only the effects of PA on TS cells. It is possible that PA exposure has differential effects on the embryonic trophectoderm and the inner cell mass. If that is the case, it is possible that all postnatal effects seen in our model are the result of altered placentation. Further work with differential staining of the embryos and work with embryonic stem cells may aid in determining whether this is the case.

Regardless of the mechanisms involved in the adverse effects of PA in this model, our findings suggest links between elevated FFA levels in obese women and adverse outcomes in pregnancy are worth exploring. Further studies of circulating fatty acid levels in obese women along with studies of fatty acid levels in the reproductive tracts of these women are worthwhile but difficult given that many women who would qualify for such studies are often trying to conceive. We propose using in vivo animal models to direct future human study and to inform novel interventions. These interventions may include alterations in maternal diet and fatty acid intake, weight loss prior to pregnancy, possible medical therapies aimed at correcting FFA metabolism in nonadipose cells, and improvement of pregnancy outcomes and offspring well being among obese individuals.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Leon Carayonnaopoulos for the generous gift of trophoblast stem cells and Dennis Oakley for technical assistance in obtaining confocal images.

Footnotes

1

Supported by National Institutes of Health (NIH) grants T32-HD-07440-07 and K12HD-063086-01, an American Society for Reproductive Medicine/Ortho Research Grant in Reproductive Medicine to E.S.J., NIH grants RO1-DK070351 and U01HD-044691 to K.H.M., and NIH Neuroscience Blueprint Core Grant NSO57105 to Washington University.

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