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. 2010 Jun 23;151(8):4039–4046. doi: 10.1210/en.2010-0098

Diet-Induced Obesity Model: Abnormal Oocytes and Persistent Growth Abnormalities in the Offspring

Emily S Jungheim 1, Erica L Schoeller 1, Kerri L Marquard 1, Erica D Louden 1, Jean E Schaffer 1, Kelle H Moley 1
PMCID: PMC2940512  PMID: 20573727

Abstract

Associations between maternal obesity and adverse fetal outcomes are well documented, but the mechanisms involved are largely unknown. Most previous work has focused on postconceptional events, however, our laboratory has shown pre- and periconceptional aberrations in maternal glucose metabolism have adverse effects on oocytes and embryos that carry on to the fetus. To demonstrate effects of maternal obesity in the pre- and periconceptional periods, we compared reproductive tissues from diet-induced obese female mice to those of control mice. Ovaries were either stained for follicular apoptosis or dissected and evaluated for oocyte size and meiotic maturation. Mice were also mated and followed for reproductive outcomes including preimplantation embryonic IGF-I receptor (IGF-IR) immunostaining, midgestation fetal growth, and midgestational placental IGF receptor 2 (Igf2r) mRNA. Delivered pups were followed for growth and development of markers of metabolic syndrome. Compared with controls, obese mice had significantly more apoptotic ovarian follicles, smaller and fewer mature oocytes, decreased embryonic IGF-IR staining, smaller fetuses, increased placental Igf2r mRNA, and smaller pups. All weaned pups were fed a regular diet. At 13 wk pups delivered from obese mice were significantly larger, and these pups demonstrated glucose intolerance and increased cholesterol and body fat suggesting early development of a metabolic-type syndrome. Together, our findings suggest maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception.

Oocytes and embryos show aberrations in the model of maternal obesity that may carry on to the fetus and offspring, underscoring the importance of optimal maternal weight and nutrition in the pre- and peri-conceptual stages of development.

The prevalence of obesity among reproductive women is on the rise (1). Despite health risks associated with obesity, many affected young women are asymptomatic as they have not yet developed conditions associated with long-standing obesity. Unfortunately, it may not be until after these women conceive and present for prenatal care that they are counseled of the risks obesity poses on their pregnancies, including increased risks of miscarriage, gestational diabetes, and preeclampsia (2,3). Of further concern, there is emerging data that the morbidity of obesity is not limited to the mother. Congenital anomalies are increased among offspring, fetal growth is often affected, and epidemiologic and experimental data suggest maternal obesity also increases risks among offspring for developing obesity and insulin resistance later in life as a result of “nutritional programming” during gestation (3,4). While efforts are being made to increase awareness of the risks associated with obesity in pregnancy, the mechanisms involved are largely unknown (5).

Efforts to improve fetal outcomes among obese women often focus on postconceptional interventions like limiting weight gain and monitoring for gestational diabetes, however the preimplantation stage of development represents a particularly vulnerable time in embryonic development when aberrations in maternal physiology can have both immediate and long-term consequences (6,7,8). This is demonstrated clinically in cases of poorly controlled diabetes mellitus where high glucose levels at the time of conception result in miscarriage or fetal malformation despite optimal control of glucose levels postconceptionally (9). Experimental models from our laboratory suggest adverse outcomes in both type 1 and type 2 diabetic pregnancies are due in part to adverse effects of these diseases on normal insulin signaling and glucose uptake in preovulatory follicles, oocytes, and the preimplantation embryo (6,9,10,11,12,13,14,15,16,17). Given that obesity is also a condition marked by abnormalities in circulating levels of substrates for energy production, we hypothesized that it too may have adverse effects on pre- and periconceptional reproductive tissues that would persist as growth and metabolic abnormalities in the offspring. Based on relevant findings from our previous work in maternal models of diabetes, we sought to determine whether maternal obesity was associated with: 1) apoptosis of granulosa cells in preovulatory ovarian follicles, 2) decreased oocyte size and meiotic maturation, and 3) decreased IGF-IR receptor (IGF-IR) expression, consistent with insulin resistance in preimplantation embryos. In addition, we followed fetal growth and placental IGF receptor 2 (Igf2r) mRNA at midgestation as it has been suggested that aberrations in embryonic allelic expression Igf2r may affect fetal growth and we hypothesized that such an aberration could account for altered fetal growth in our model (18). Finally, we evaluated long-term outcomes for offspring including weight, glucose tolerance, cholesterol, and body fat.

Materials and Methods

Animals and diet

All animal experiments were approved by our Animal Studies Committee. Female C57BL/6J mice (National Cancer Institute, Frederick, MD) 3–4 wk of age were housed five per cage and given access to water and fed either a high-fat diet (HFD) containing 35.8% fat, 20.7% protein, and 35% carbohydrates, or a matched control diet containing 4.8% fat, 73.9% carbohydrate, and 14.8% protein (A1N-76A; TestDiet, Richmond, IN) ad libitum. After 16 wk of feeding, the mice were weighed and their blood was drawn for measurement of circulating serum free fatty acids [NEFA-HR (2) kit, Wako Diagnostics, Richmond, VA], and for fasting glucose (Hemocue B glucose analyzer, Stockholm, Sweden).

Apoptosis of ovarian follicles, and oocyte size and maturation

After 16 wk of feeding, eight female mice were superovulated with 10 IU pregnant mare serum gonadotropin (PMSG) (Sigma, St. Louis, MO) (19,20,21) and killed 48 h later. Ovaries collected from these mice were dissected for oocytes. Another cohort of mice received an injection of 5 IU human chorionic gonadotropin (hCG) 48 h after PMSG and killed 6 h later. Ovaries from these mice were either flash frozen in liquid nitrogen or dissected for oocytes. A total of eight ovaries from eight animals were flash frozen and sectioned into 11-μm sections. Ten slides per ovary were analyzed. Apoptosis was determined by TUNEL assay and visualized by laser scanning confocal microscope as described below. In ovaries not prepared for follicular studies, oocytes were isolated from antral follicles and observed for maturation and size. Cryopreserved ovarian sections were fixed in 3% paraformaldehyde, permeabilized with 0.1% Tween 20, and incubated at 37 C in the dark with fluorescein-labeled dUTP and terminal transferase (TUNEL, Cell Death in Situ Kit; Roche Molecular Biochemicals, Mannheim, Germany). Nuclear DNA counterstaining was performed by incubating the sections in 4-μm To-Pro-3-iodide (Molecular Probes, Eugene, OR) for 20 min. Confocal immunofluorescent microscopy of stained ovarian sections was performed with an Olympus laser-scanning microscope. Levels of apoptosis were calculated as the number of TUNEL-positive follicles per total number of follicles.

Oocytes were assessed using Hoffman optics on an inverted Nikon microscope for completion of meiosis I or germinal vesicle breakdown (GVBD). Maturation was expressed as a percentage of oocytes that achieved GVBD divided by the total number of oocytes. Oocyte diameters (excluding the zona pellucida) were recorded at ×200 magnification with an eyepiece graticule (×200 magnification). The volume of each oocyte was calculated based on the formula for the volume of a sphere [(4/3)(πr3)] as previously described (11).

Embryo recovery and IGF-IR expression

A cohort of female mice were also housed with C57BL/6J males of proven fertility and checked daily for vaginal plugging as evidence of mating. Mated female mice were not superovulated. Once mated, females were placed in separate cages and maintained on respective diets until embryonic d 4 at which time the mice were killed and embryos were recovered from their uterine horns as previously described (16) and analyzed for IGF-IR expression as described previously (17) by confocal immunofluorescent microscopy using a polyclonal antibody against the IGF-IR (catalog no. 3027, lot no. 1; Cell Signaling Technology, Danvers, MA). Briefly, embryos were fixed in 3% paraformaldehyde for 30 min and permeabilized with 0.1% Tween 20 for another 30 min. The embryos were then blocked for 1 h with 20% normal goat serum (Pierce) in PBS with 2% BSA (PBS/BSA) followed by three 10-min washes with PBS/BSA. After blocking, the embryos were incubated for 40 min in 20 μg/ml IGF-IR antibody in PBS/BSA followed again by three 10-min washes with PBS/BSA. The embryos were then incubated with the secondary antibody, Alexa Fluor 488 goat antirabbit IgG (Molecular Probes) at a concentration of 2 μg/ml for 30 min. Nuclear DNA counterstaining was performed by incubating the embryos in 4 μm To-Pro-3-iodide for another 20 min followed by three final washes in PBS/BSA. The embryos were mounted under coverslips in drops of Vectashield (Vector Laboratories, Burlingame, CA). Confocal immunofluorescent microscopy was performed with an Olympus laser-scanning microscope. Relative staining was determined by blinded observers as previously described (22,23,24,25,26,27).

Fetal growth and RT-PCR analysis of placental Igf2r at embryonic d 14.5

A cohort of mated mice was also killed on embryonic d 14.5 (e14.5). Fetuses were collected and evaluated for crown-rump length as previously described (16). Placentas were collected at e14.5 for Igf2r mRNA measurement. Total cellular RNA was extracted from frozen fetal placentas homogenized in TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. The samples were DNase treated using DNA-free (Ambion). After quantitation with the NanoDrop (Thermo Fischer Scientific), 5ug of RNA was reverse-transcribed using SuperScriptIII reverse polymerase (Invitrogen) and oligo(deoxythymidine) primers according a protocol previously established in the lab. The following primer sets for the Igf2r gene were used: F: 5′-TGG-CTT-GTA-TTC-CTT-CTG-TAG-3′, R: 5′-AGT-TGT-CTC-CTT-CCT-CTC-TGA-3′ (28). Real-time PCR reactions were carried out in 96-well plates using ABI Prism 7700 Sequence Detection System (Applied Biosystems). Each reaction was carried out in a total of 25 μl using 50ng of cDNA with 300 nm forward and reverse primer concentrations and 12.5 μl of 2× Power SYBR Green (PE Applied Biosystems). Each sample was loaded in triplicate and the reactions were carried out for 40 cycles (95 C for 15 sec, 60 C for 1 min) after 10 min at 95 C. Housekeeping gene, GAPDH, was used as an endogenous control gene as GAPDH expression has been shown not to vary in response to obesity (29).

Offspring growth and metabolic studies

A final cohort of mated mice was allowed to deliver and suckle their pups while on their respective diets. Offspring were weaned, placed on control diets, and followed through 13 wk of life. Offspring were weighed at day of life 18, 25 (also day of weaning), 35, and 91 (13 wk of life). Percent body fat was determined by dual x-ray absorptiometry (DXA) scan at 6.5 and 10 wk of life. Two-hour ip glucose tolerance tests (GTT) were performed at 13 wk of life, and serum cholesterol was quantified at 10 wk.

Statistics

Student’s t test or ANOVA with Bonferroni post hoc test were used in the analyses. Results are expressed as means and sem. Significance was defined as P < 0.05.

Results

Apoptosis of ovarian follicles, and oocyte size and maturation

Body weights (P < 0.001), fasting serum glucose (P < 0.001), and free fatty acid levels (P < 0.03) were significantly higher in mice fed a HFD compared with controls (Table 1). Ovaries were collected and sectioned as described above from animals that were primed with PMSG, given hCG 48 h later, and then killed 6 h after hCG. The percentage of TUNEL-positive, nuclei-containing follicles was significantly higher in the obese group than in the nonobese group (59.64% vs. 17.64, P < 0.01). Zero hours after hCG injection, control oocytes were larger than oocytes from obese mice (334.247 μm3, n = 65 vs. 314.12 μm3, n = 64, P < 0.01). Six hours after hCG injection, control oocytes were still larger than oocytes from obese mice (278.75 μm3, n = 85 vs. 251.49 μm3, n = 52, P < 0.01). Oocytes from obese mice were also found to have a delay in maturation as measured by GVBD. Zero hours after hCG injection 7.81% of obese oocytes (n = 64) had reached GVBD, whereas 13.85% of control oocytes (n = 65) had completed this maturational stage (P < 0.01). Similarly, at 6 h 9.62% of obese oocytes (n = 52) had completed GVBD and 29.41% of control oocytes (n = 85) had (P < 0.01) (Table 2).

Table 1.

Characteristics of female mice on a regular diet vs. a high-fat diet after 16 wk of feeding

Regular diet (n = 10) High-fat diet (n = 10)
Body weight (g) 22.04 ± 0.8 46.92 ± 1.8a
Glucose 97 ± 8.9 145.7 ± 3.6a
FFA 0.43 ± 0.06 0.90 ± 0.17b
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Results are expressed as means ± sem. 

a

P < 0.001 vs. control. 

b

P < 0.03 vs. control. 

Table 2.

Follicular apoptosis, oocyte size, and oocyte maturation in mice fed a regular diet vs. high-fat diet

Regular diet High-fat diet
Percentage TUNEL-positive follicles 17.6 ± 1.2 59.64 ± 2.2
Average oocyte size 0 h after hCG 334.25 ± 14.3 μm3 (n = 65) 314.12 ± 14.2 μm3 (n = 64)
Average oocyte size 6 h after hCG 278.75 ± 10.5 μm3 (n = 85) 251.49 ± 11.7 μm3 (n = 52)
Percentage of oocytes reaching GVBD 0 h after hCG 13.85 7.81
Percentage of oocytes reaching GVBD 6 h after hCG 29.41 9.62
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Results are expressed as means ± sem unless otherwise specified. All results were significant at P < 0.01 vs. control. 

Embryonic IGF-IR staining

Two independent blinded observers scored embryos from high-fat fed mothers and mothers on control diet on a scale of 1–3 for staining of IGF-IR with 3 being the highest. These scores revealed a relative decrease in IGF-IR staining in embryos from mothers on the HFD compared with those on the regular diet (P < 0.001) (Fig. 1).

Figure 1.

Figure 1

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IGF-I receptor expression is decreased in blastocysts recovered from female mice on a HFD (n = 10 mice) vs. a regular-fat diet (n = 8 mice) (P < 0.001). A, Relative IGF-I receptor expression was determined by two independent and blinded observers. B, Confocal image of IGF-I receptor protein. Blue channel, Nuclear staining; green channel, IGF-I receptor labeling.

Placental Igf2r mRNA and fetal size at embryonic d 14.5

Placentas of fetuses collected from obese mice demonstrated a 3-fold increase in Igf2r mRNA compared with the placentas of fetuses collected from mice on the control diet (3.2 ± 0.54 fold vs. control; P < 0.001). Fetuses from the mothers on a HFD were significantly smaller than fetuses from mothers on a regular diet as measured by crown-rump length (P < 0.001) (Fig. 2).

Figure 2.

Figure 2

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Smaller fetuses in mice fed a HFD. A, Crown-rump length at embryonic day 14.5 (e14.5) is significantly lower in the fetuses collected from the mice on a HFD (n = 65 pups) vs. the regular diet (n = 75 pups) (P < 0.0001). B, Representative fetuses from each group.

Offspring growth, glucose tolerance, percent body fat, and cholesterol

Offspring of obese mice were smaller at day of life 18 than offspring of controls, but they demonstrated catch-up growth, and their weights eventually surpassed those of the control offspring by day of life 25 (Fig. 3A). This increased weight continued through 13 wk of life when the studies were completed.

Figure 3.

Figure 3

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Adverse metabolic parameters in pups from high-fat fed mice. A, Average weight of pups from embryonic day 14.5 (e14.5) to 13 wk of age (p91) (weight at e14.5 time point n = 9 pups from 3 control dams, n = 7 from 3 HFD dams; weight at p18–p91 time points n = 7 pups from 3 control dams; n = 6 pups from 3 HFD dams). *, P < 0.01; **, P < 0.001. B, GTT at 13 wk of age [Control diet n = 3 experiments with different mice (n = 4 pups from 2 control dams, n = 4 pups from 2 HFD dams); HFD n = 3 experiments with different mice (n = 4 pups from 2 control dams, n = 4 pups from 2 HFD dams); *, P < 0.01]. C, Percent body fat by DXA in 6.5-wk and 10-wk-old offspring from mice fed a HFD (n = 6 females; n = 3 males from 3 HFD dams) vs. control diet (n = 4 females and n = 4 males from 3 control dams). *, P < 0.05; **, P < 0.001; ^, P < 0.0001. D, Total serum cholesterol (mg/dl) in 10-wk-old offspring of age from mice fed a HFD (n = 6 females; n = 3 males from 3 HFD dams) vs. control diet (n = 4 females and n = 4 males from 3 control dams). *, P < 0.0001.

The offspring exposed to a HFD during oogenesis and during gestation also manifested other signs of metabolic syndrome besides increased weight as they aged, however this phenotype was more pronounced in the male pups. At 13 wk of age, the male offspring demonstrated impaired glucose tolerance as measured by oral GTT (n = 4, HFD offspring; n = 4 control diet offspring; *, P < 0.01) (Fig. 3B). In addition at 6.5 wk of life, the percent body fat by DXA was significantly higher in both females and males compared with offspring from mothers fed a regular diet was significantly (females, n = 6 high fat vs. n = 4 control; *, P < 0.05) (males, n = 3 high fat vs. n = 4 control; **, P < 0.001) (Fig. 3C). By 10 wk of age, however, the difference between the same females became nonsignificant, whereas the difference between the same set of males remained significant (^, P < 0.0001). Finally, at 10 wk of age, serum cholesterol levels were significantly higher in the male offspring from the high-fat fed mothers (*, P < 0.0001) (Fig. 3D).

Discussion

Our experiments demonstrate the adverse effects of diet-induced maternal obesity on oocyte maturation and embryonic IGF-IR expression, which manifest later as differences in offspring size, growth patterns, and metabolic parameters. The metabolic differences, specifically percent body fat, growth rate, glucose intolerance and elevated cholesterol levels, suggest that the offspring, males in particular, are predisposed to develop metabolic syndrome. Why males are more affected is not entirely clear, however other experimental models have shown that C57BL/6J male mice are more susceptible to features of metabolic syndrome than C57BL/6J females (8,30). Our model may be useful in future studies providing further insight into the pathophysiology of human reproduction in the setting of obesity and type 2 diabetes mellitus where glucose levels and free fatty acid levels are elevated. Because both maternal obesity and insulin resistance are much more prevalent in pregnancy today, and because obesity in childhood has become equally more evident, the question raised is whether a diet-related transgenerational phenomenon is occurring, which predisposed the offspring to metabolic syndrome and obesity. Further work is needed to determine the mechanism by which maternal obesity has its effects on the offspring in this model, however our findings suggest the effects of preconceptional obesity and periconceptional obesity are significant. Three possible mechanisms we demonstrate here include aberrations in oocyte development, decreased IGF-IR expression in the preimplantation blastocyst, and/or increased midgestational placental transcription of Igf2r—a maternally expressed gene known to influence placental and fetal size (31).

Recently, Minge et al. (32) demonstrated poor postovulatory oocyte quality and abnormal ovarian gene expression in a similar model of murine diet-induced obesity. Fertilized oocytes resulted in blastocysts with reduced survival rates and abnormal embryonic cellular differentiation. In our studies, we demonstrate that apoptosis is specifically increased in the preovulatory ovarian follicle in diet-induced obesity. Oocytes resulting from these follicles are smaller than controls and display decreased maturation. Our findings suggest that the ovarian follicular environment is altered in obese mice, which is supported by recent studies demonstrating altered metabolites, hormones in gene expression in ovarian follicular fluid from obese women undergoing in vitro fertilization (33). The oocyte and ovarian follicular abnormalities that we demonstrate here are also similar to those seen in murine models of type I diabetes mellitus (11). In a study by Wyman et al. (12), it was recently demonstrated that when transferred into nondiabetic gestational carriers, one-cell zygotes from these type I diabetes mellitus models result in growth restricted offspring.

We have previously demonstrated the importance of embryonic IGF-IR expression and function in achieving optimal reproductive outcome including embryonic implantation and fetal growth (16,17,34). The IGF-IR is critical to normal insulin signaling and glucose transport in the preimplantation blastocyst (17)—a stage in development in which glucose is the predominant energy source (35,36). In this current study, we demonstrate a decrease in IGF1 receptor at the blastocyst stage of development. We hypothesize that the high long-chain saturated-fat diet, a fat known to cause insulin resistance and cell death, was an inciting factor in the decreased expression (37,38,39). Alternatively, elevated circulating free fatty acids resulting from maternal obesity may have affected Igf1r expression in the blastocysts in our model as other models of obesity-related type 2 diabetes have shown that elevated free fatty acid levels can result in subsequent insulin resistance (40). In addition, we found a 3-fold increase in Igf2r mRNA levels in placentas taken from obese mothers at midgestation compared with nonobese mice. Normally, Igf2r is expressed only from the maternal allele. However, our results suggest biallelic expression Igf2r in the placentas collected from the obese mice. Previous studies have demonstrated that biallelic expression of Igf2r leads to fetal growth restriction (41), likely by targeting the ligand IGF2 for lysosomal degradation (42,43). We suspect exposure to high-fat conditions during early development and up to midgestation of the embryo results in alterations of gene expression—possibly due to improper maintenance of parental imprinting markers or alterations in histone structures during the preimplantation period and/or later during midgestation in the placenta.

The adverse effects of obesity on reproductive outcome are well documented, as are the effects of adverse maternal nutrition on the long-term outcomes for offspring (3,8,44,45,46). Our model demonstrates that the effects may begin as early as pre- and periconception, however further work delineating the mechanisms is necessary including embryo transfer studies of control embryos into obese recipients and embryos from obese mice transferred into control recipients to determine if these effects are isolated to preimplantation events. Moreover, these changes may contribute to lasting morbidity in offspring of obese mothers, underscoring the importance of optimal maternal weight and nutrition before conception.

Acknowledgments

We thank Dennis Oakley for his technical assistance in obtaining confocal images.

Footnotes

This research was supported by National Institutes of Health (NIH) Grants T32-HD-07440-07 and K12HD063086-01, funding through Washington University’s Clinical Nutrition Research Unit Pilot and Feasibility Program (P30DK056341 from the National Institute of Diabetes and Digestive and Kidney Diseases), and funding from the American Society for Reproductive Medicine/Ortho Research Grant in Reproductive Medicine (all to E.S.J.); NIH Grants RO1-DK070351 and U01HD044691 (to K.H.M.) and NIH Neuroscience Blueprint Core Grant NSO57105 to Washington University; P60 DK20570 (Diabetes Research and Training Center) (to J.E.S.); and a Children’s Discovery Institute grant (to J.E.S. and K.H.M.).

Disclosure Summary: The authors have nothing to declare.

First Published Online June 23, 2010

For editorial see page 3475

Abbreviations: DXA, X-ray absorptiometry; GTT, glucose tolerance tests; GVBD, germinal vesicle breakdown; hCG, human chorionic gonadotropin; HFD, high-fat diet; IGF-IR, IGF-I receptor; Igf2r, IGF receptor 2; PMSG, pregnant mare serum gonadotropin.

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