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Endocrinology, doi:10.1210/en.2007-1570
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Endocrinology Vol. 149, No. 5 2646-2656
Copyright © 2008 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor-{gamma} Agonist Rosiglitazone Reverses the Adverse Effects of Diet-Induced Obesity on Oocyte Quality

Cadence E. Minge, Brenton D. Bennett, Robert J. Norman and Rebecca L. Robker

School of Paediatrics and Reproductive Health, Discipline of Obstetrics & Gynaecology, The University of Adelaide, Adelaide, South Australia 5005, Australia

Address all correspondence and requests for reprints to: Dr. R. Robker, Discipline of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, South Australia 5005, Australia. E-mail: rebecca.robker{at}adelaide.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obesity and its physiological consequences are increasingly prevalent among women of reproductive age and are associated with infertility. To investigate, female mice were fed a high-fat diet until the onset of insulin resistance, followed by assessments of ovarian gene expression, ovulation, fertilization, and oocyte developmental competence. We report defects to ovarian function associated with diet-induced obesity (DIO) that result in poor oocyte quality, subsequently reduced blastocyst survival rates, and abnormal embryonic cellular differentiation. To identify critical cellular mediators of ovarian responses to obesity induced insulin resistance, DIO females were treated for 4 d before mating with an insulin-sensitizing pharmaceutical: glucose and lipid-lowering AMP kinase activator, 5-aminoimidazole 4-carboxamide-riboside, 30 mg/kg·d; sodium salicylate, I{kappa}K inhibitor that reverses insulin resistance, 50 mg/kg·d; or peroxisome proliferator activated receptor-{gamma} agonist rosiglitazone, 10 mg/kg·d. 5-aminoimidazole 4-carboxamide-riboside or sodium salicylate treatment did not have significant effects on the reproductive parameters examined. However, embryonic development to the blastocyst stage was significantly improved when DIO mice were treated with rosiglitazone, effectively repairing development rates. Rosiglitazone also normalized DIO-associated abnormal blastomere allocation to the inner cell mass. Such improvements to oocyte quality were coupled with weight loss, improved glucose metabolism, and changes in ovarian mRNA expression of peroxisome proliferator activated receptor-regulated genes, Cd36, Scarb1, and Fabp4 cholesterol transporters. These studies demonstrate that peri-conception treatment with select insulin-sensitizing pharmaceuticals can directly influence ovarian functions and ultimately exert positive effects on oocyte developmental competence. Improved blastocyst quality in obese females treated with rosiglitazone before mating indicates that peroxisome proliferator activated receptor-{gamma} is a key target for metabolic regulation of ovarian function and oocyte quality.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A GROWING BODY of evidence shows that female fertility is significantly compromised under conditions of overweight and obesity (1, 2, 3). Overweight women experience longer times to conception than women with moderate body weights (4), indicating that reproductive function is impaired even at the earliest, preimplantation stages. However, due to the difficulty of examining preimplantation processes in humans, the exact nature of the lesions is unclear. Although a disproportionate percentage of women undergoing assisted reproduction technologies are overweight or obese, there is some conflicting evidence regarding oocyte quality of obese women undergoing in vitro fertilization (1, 5, 6, 7, 8) due to relatively small numbers of highly heterogeneous patients, and major differences in technical and analytical methodologies. The present study has used a mouse model to determine more precisely whether defects in oocyte developmental quality and/or early embryo development occur in response to obesity.

Our studies used a well-characterized model of obesity in which a high-fat diet (HFD) in C57BL/6 mice causes excessive weight gain and hyperinsulinemia, metabolic parameters implicated in poor reproductive outcomes (9, 10, 11, 12). It is evident that insulin resistance occurring from obesity is key in the development of female reproductive dysfunction. Hyperinsulinemia can interfere directly with ovarian cell function or be indirectly associated with other hormonal conditions detrimental to optimal fertility (13, 14, 15, 16).

For our studies we treated mice with specific insulin-sensitizing and plasma glucose-reducing pharmaceuticals to reverse the effects of obesity/hyperinsulinemia and identify the signaling pathways responsible for disruption of preimplantation events.

5-aminoimidazole 4-carboxamide-riboside (AICAR) is an adenosine analog that acts through stimulation of AMP kinase activity (17). In this effect, AICAR is similar in mechanism to metformin, although it is recognized as more specific in action (18, 19). AMP kinase itself phosphorylates and inactivates a number of key biosynthetic enzymes (20, 21, 22, 23), consequently inhibiting glycogen synthesis, fatty acid synthesis, and isoprenoid/sterol synthesis. AICAR administration to rats increases the activity of the insulin receptor signaling (24), and increases glucose uptake (25), via up-regulated translocation of glucose transporter 4 to the plasma membrane (26, 27).

Closely related to aspirin, sodium salicylate is a nonsteroidal antiinflammatory drug with two distinct molecular modes of action; at low doses sodium salicylate inhibits the classical nonsteroidal antiinflammatory drug targets, cyclooxygenase-1 (28, 29, 30) and -2 (28, 31), thus blocking prostaglandin formation, but at high doses sodium salicylate blocks the action of nuclear factor {kappa}B (32) and its upstream activator I{kappa}B kinase-β (33). Via reduced signaling through the I{kappa}B kinase-β pathway, sodium salicylate treatment lowers blood glucose concentrations (34), improves insulin resistance (35), and restores normal insulin sensitivity in mice with diet-induced obesity (DIO) (36).

Rosiglitazone (Avandia; GlaxoSmithKline, Middlesex, UK) is an insulin-sensitizing agent of the thiazolidinedione class of drugs that also includes pioglitazone (Actos; Takeda Pharmaceutical Co. Ltd. (Osaka, Japan)/Eli Lilly and Co., Indianapolis, IN) (37). The thiazolidinediones are highly selective and potent agonists for the nuclear receptor peroxisome proliferator activated receptor-{gamma} (PPARG) (38), strongly implicated in female reproduction (39). After rosiglitazone activation of PPARG, a heterodimeric complex with the retinoid X receptor forms and binds to PPAR response elements (PPREs) located in promoter regions of target genes (40), thus altering transcription. Recent investigation using genome-wide screening has produced a comprehensive list of genes containing PPREs that are potentially regulated by the PPARG/rosiglitazone complex (41). Among these genes are many related to lipid metabolism, including those involved in fatty acid transport and lipid clearance from the circulation (apolipoproteins and lipoprotein lipase), fatty acid transport through plasma membranes [CD36 and scavenger receptor class B, member 1 (SCARB1)], fatty acid oxidation (acyl-coenzyme A oxidase), mitochondrial uncoupling [uncoupling proteins (UCPs)-1, UCP-2, and UCP-3], lipogenesis (acetyl-coenzyme A carboxylase, fatty acid synthase), and transcription factors involved in lipid metabolism control (sterol-regulatory element-binding protein 1) (reviewed in Ref. 42), as well as other genes related to glucose metabolism (43). A number of studies has reported on the effects of rosiglitazone treatment in obese rodents, frequently reporting changes in body weight, plasma lipid profile, blood glucose levels, and circulating insulin levels (44, 45).

The effects of DIO on aspects of female reproductive function (ovulation, fertilization, and embryonic development to the blastocyst stage) were assessed. Obese animals were also treated with one of the three insulin sensitizers. Comparing the effects of each drug on metabolic status and ovarian gene expression, as well as upon later reproductive outcomes, identified the pathway most important for these processes. To focus on the peri-conception effects of these drugs, the treatment time frame was limited to 4 d immediately before ovulation and mating (Fig. 1Go), thereby restricting systemic effects of persistent treatment yet elucidating acute effects on the ovarian follicle and oocyte. After indication of mating, all oocytes were isolated from the oviducts and monitored in vitro, enabling evaluation of oocyte health and precise temporal assessments of developmental competence.


Figure 1
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  		FIG. 1. The experimental treatment protocol. Female mice were fed a CD or HFD for 16 wk starting at 5 wk of age. Four days before pairing with a male, mice fed the HFD were treated with AICAR, sodium salicylate, rosiglitazone, or vehicle via ip injection once daily. Mice were inspected for evidence of mating (vaginal plug) at 0800 h each morning, and deemed to be at d-1 pregnancy. Zygotes were removed from the oviduct at 1300 h and maintained in in vitro culture until d 5, when a differential stain was performed on blastocysts.

 
By comparing the effects of each drug on ovarian gene expression, oocyte health, and early embryonic developmental competence in DIO mice, we have identified cellular pathways affected by insulin resistance that are important in regulating oocyte potential.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diet
All mice were obtained from the University of Adelaide Laboratory Animal Services, Adelaide, Australia. The animal ethics committees of both The Queen Elizabeth Hospital and The University of Adelaide approved all experiments, and the animals were handled in accordance with the Australian Code of Practices for the Care and Use of Animals for Scientific Purposes. All mice had free access to water and food, and were maintained at The Queen Elizabeth Hospital animal house at 24 C on a 14-h light, 10-h dark illumination cycle. Five-week-old female C57BL/6 mice were housed in groups of five and fed either a HFD containing 22% fat (0.15% cholesterol), 19% protein, and 49.5% carbohydrate (SF00–219; Specialty Feeds, Glen Forrest, Australia), or a matched control diet (CD) containing only 6% fat, 19% protein, and 64.7% carbohydrate (SF04–057 Specialty Feeds) for 16 wk. Body weights were determined weekly. Male mice were maintained on standard rodent chow, were 10- to 16-wk old at mating, and were proven fertile.

Insulin sensitizer treatment
Female mice fed the HFD were randomly allocated to the HFD plus vehicle group, HFD plus AICAR group, HFD plus sodium salicylate group, or HFD plus rosiglitazone group (all groups n = 15). Four days before the conclusion of the 16-wk feeding period, they were injected ip once daily with 30 mg/kg·d AICAR (Toronto Research Chemicals, Ontario, Canada; in 0.9% saline), 50 mg/kg·d sodium salicylate [Sigma-Aldrich, St. Louis, MO; in sterile water (46)], 10 mg/kg·d rosiglitazone [Avandia, in 10% dimethylsulfoxide (47)], or vehicle (10% dimethylsulfoxide). Body weight was recorded immediately before the first dosage of drug or vehicle (d –4) and immediately before the final dose of drug or vehicle (d –1) to indicate body weight flux resulting from drug administration.

Tissue and zygote collection
After 16-wk CD, or 16-wk HFD, including 4-d drug vehicle treatment, two female mice (mixed experimental groups) were caged with one male mouse for a maximum of eight nights. Females were checked daily at 0800 h for the presence of a postcoital vaginal plug. On the day of the presence of a vaginal plug, females were deemed to be at d-1 pregnancy, and tissues were collected at 1300 h that day. Any females in which a plug was not observed over the 8-d period of cohabitation (10 of 75 mice) were housed individually for a further 7 d to allow conceptions that may have occurred to progress to a developmental stage in which visual inspection of the uterine horns could confirm pregnancy. From these, one female was found to have implantation sites, and one was found to have implantation and resorption sites, indicating that two plugs had been missed out of 75 animals. The number of days postcoitus was estimated. Before killing by cervical dislocation, blood was collected, allowed to clot at room temperature, and centrifuged at 4000 rpm for 10 min and serum removed. Ovulated oocytes were collected immediately from the oviductal ampulla into G-MOPS media and treated with hyaluronidase (0.5 mg/ml; Sigma-Aldrich; bovine testes, type IV) to facilitate removal of the surrounding cumulus cells. Abdominal and retroperitoneal adipose tissue, ovary, and liver were dissected, weighed, and snap frozen in liquid nitrogen.

Metabolite and endocrine measurements
Samples for analysis of fed plasma insulin levels were analyzed by a Sensitive Rat Insulin RIA Kit (LINCO Research, Inc., St. Charles, MO), with a sensitivity of 0.02 ng/ml and an intraassay coefficient of 3.13%. Fed blood glucose levels and the circulating lipid profile of each mouse were determined from samples of serum using a Roche Cobas Mira automated sample system (Roche, Castle Hill, Australia). Cholesterol levels were measured using the Cholesterol (CHOL-PAP) assay kit (Roche), and the mean coefficient of variation was less than 2.7%. Triglycerides were measured using the triglycerides (TRIG) assay kit (Roche), and the mean coefficient of variation was less than 2.6%. Each of these also used the Calibrator for Automated Systems and the Precinorm U and Precipath U Quality Controls (Roche). Free fatty acids were measured using the NEFA-C Free Fatty Acid assay kit (NovoChem, Victoria, Australia) and quality controls: QCS 1 and 2 (Bio-Rad Laboratories Pty., Ltd., New South Wales, Australia). The mean coefficient of variation was less than 4.6%. All assays have been validated for use in the mouse. Mice were not fasted to avoid detrimental physiological impact of short-term starvation on hormone production, oocyte quality, or fertilized zygote survival.

mRNA preparation and real-time RT-PCR
Total cellular RNA was isolated from the liver and ovary using a Tri Reagent (Sigma-Aldrich) protocol. RNA concentration and purity were determined using NanoDrop Spectrophotometer (NO-100; Biolab, Victoria, Australia). Five hundred nanograms of RNA were reverse transcribed using random primers (Roche) and a Superscript II RNase H– Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA) preamplification system for first-strand cDNA synthesis according to the manufacturer’s instructions. For each RT, a control was performed in which all incubations and buffers were identical, but no Superscript RT enzyme was added, verifying the absence of contaminating genomic DNA in PCR. cDNA templates were then subjected to fluorometric semiquantitative real-time PCR using the Corbett Rotor-Gene 6000 (Corbett Life Sciences, Sydney, Australia) real-time rotary analyzer with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Ribosomal protein L19 was used as an internal control for every sample. All primers were designed using Primer Express software and synthesized by GeneWorks (Thebarton, South Australia, Australia): ribosomal protein L19, forward 5'-TTCCCGAGTACAGCACCTTTGAC-3', reverse 5'-CACGGCTTTGGCTTCATTTTAAC-3'; Pparg, forward 5'-CCACTATGGAGTTCATGCTTGTG-3', reverse 5'-TTTGTGGA-TCCGGCAGTTAAG-3'; Cd36, forward 5'-TCATGCCAGTCGGAGA-CATG-3', reverse 5'-TGGTGCCTGTTTTAACCCAGTT-3'; Scarb1, forward 5'-GGTCCTCAACGGCCAGAAG-3', reverse 5'-CACGGTGTCGTTGTCATTGAA-3'; and Fabp4, forward 5'-TGATGCCTTTGTGGGAACCT-3', reverse 5'-ATCCTGCCACTTTCCTTGT-3'.

In vitro embryo culture
Ovulated and denuded oocytes were maintained for 48 h in G1 media (48, 49), with assessment for fertilization indicated by first cleavage division occurring after 24 h. Two-cell embryos were transferred to EDTA-free G2 media (49, 50), shown previously to provide an optimum environment for growth of the post-compaction embryo. The development of fertilized oocytes was assessed on d 3 at 0900 h, on d 4 at 1600 h and on d 5 at 0900 h by assessors blinded to maternal treatment group. Embryos were indicated as "on-time" when normal cell numbers and morphology were observed (i.e. d 3, four to eight cells; d 4, morula-blastocyst; and d 5, expanded or hatching blastocyst), fragmentation was less than 10%, and the zona pellucida was intact (until d 5). Embryos were categorized as not "on-time" if any of these developmental targets were not achieved. In addition, embryos exhibiting slow/ceased cell division events (indicating cellular arrest) or overtly accelerated divisions (indicating insufficient or incomplete processing of cellular division) were duly noted as such and consequently categorized as not "on-time" for the remainder of the culturing period, even if their developmental progress later appeared normal.

Differential nuclear staining
Expanded and hatching blastocysts surviving at d-5 culture were subjected to a differential staining protocol for identification of cells within the fetal precursor-inner cell mass (ICM) and placental precursor-trophectoderm (TE) layer following the methods of Hardy et al. (51). A blinded assessor then counted red (TE) and blue (ICM) fluorescent cells on an Olympus VANOX AHBT-3 photomicroscope (Faulding Imaging, Mulgrave North, Victoria, Australia).

Statistical analysis
Values are reported as mean ± SEM. Statistical differences were determined by ANOVA and {chi}2 analysis using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL). One-way ANOVA across CD and HFD ± drug treatments was used, with post hoc analyses of significance made by Tukey’s test. A Student’s t test was used in the case of comparison of normalized gene expression between CD or HFD and specific drug treatments. For all analyses, P < 0.05 was defined as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin sensitizer influences on metabolic and endocrine measurements
Mice fed a HFD gained significantly more weight than those on a CD over the course of 16 wk (Fig. 2AGo). Animals on the HFD were assigned to four different groups, and at the initiation of treatment regimens, body weight was not significantly different between HFD experimental groups (Fig. 2BGo). Over the 4 treatment days, body weight was maintained in both CD animals ({Delta}weight = 0.36 ± 0.35 g), as well as in the HFD plus vehicle group ({Delta}weight = –0.26 ± 0.26 g) (Fig. 2CGo). Treatment with AICAR or sodium salicylate had no effect on body weight compared with vehicle-treated animals, however, treatment with rosiglitazone caused significant weight loss ({Delta}weight = –3.27 ± 0.21 g, P < 0.001 compared with all other groups).


Figure 2
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  		FIG. 2. Body weights of mice after feeding with a CD or HFD and in response to treatment with insulin sensitizers or vehicle. A, Weight gain of mice fed a CD or HFD for the 16 wk preceding drug administration. B, Body weight of the CD and four HFD groups immediately before drug administration. C, Change in body weight after 3-d drug or vehicle treatment. Bars and data points indicate mean ± SEM (n = 15). *, P < 0.05; **, P < 0.001. Different letters indicate statistically different means, P < 0.05. Rosi, Rosiglitazone; SS, sodium salicylate.

 
None of the treatments reduced adipose tissue mass, otherwise elevated by consumption of HFD (Fig. 3AGo). Rosiglitazone treatment reduced liver mass (Fig. 3BGo; P = 0.048 compared with HFD plus vehicle), indicating that the significant weight loss observed with this treatment is likely due to fat mobilization from the liver.


Figure 3
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  		FIG. 3. Tissue weights of mice fed a CD or HFD and in response to treatment with insulin sensitizers or vehicle. Adipose tissue (abdominal plus retroperitoneal) weight (A) and liver weight (B). Bars indicate mean ± SEM (n = 15). Different letters indicate statistically different means, P < 0.05. Rosi, Rosiglitazone; SS, sodium salicylate.

 
Blood collected at postmortem was analyzed for circulating lipids, glucose, and insulin. Circulating total cholesterol was not significantly different in HFD plus vehicle mice (Fig. 4AGo) or affected by treatment with insulin sensitizers. Circulating free fatty acids were significantly increased by HFD feeding but were not altered in mice treated with insulin sensitizers compared with vehicle (Fig. 4BGo). Triglyceride levels (Fig. 4CGo) were not affected by HFD or insulin sensitizers, however, rosiglitazone treatment resulted in the lowest levels (P = 0.047, compared with HFD plus AICAR). Nonfasting blood glucose and insulin levels were not significantly elevated in response to HFD feeding. Blood glucose levels in HFD fed mice were not significantly altered by treatment with AICAR or sodium salicylate (Fig. 5AGo). However, rosiglitazone significantly lowered blood glucose levels compared with vehicle-treated mice (P = 0.028 vs. HFD plus vehicle). Circulating insulin was significantly reduced by both sodium salicylate and rosiglitazone delivery (Fig. 5BGo), compared with vehicle treatment (HFD plus vehicle vs. HFD plus sodium salicylate or rosiglitazone; P = 0.021 and P = 0.032, respectively). All blood samples were obtained in the nonfasted state to avoid acute effects of fasting on the fertilized oocytes. Previous cohorts of mice, which were fasted, showed significantly elevated cholesterol, free fatty acids, blood glucose, as well as significant hyperinsulinemia (data not shown) in HFD compared with CD treatments.


Figure 4
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  		FIG. 4. Circulating lipids in mice fed a CD or HFD and in response to treatment with insulin sensitizers or vehicle. A, Total cholesterol. B, Free fatty acids. C, Triglycerides. Bars indicate mean ± SEM (n = 15). Different letters indicate statistically different means, P < 0.05. Rosi, Rosiglitazone; SS, sodium salicylate.

 

Figure 5
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  		FIG. 5. Circulating glucose (A) and insulin (B) in mice fed a CD or HFD and in response to treatment with insulin sensitizers or vehicle. Measurements were obtained under nonfasted conditions. Bars indicate mean ± SEM (n = 15). Different letters indicate statistically different means, P < 0.05. Rosi, Rosiglitazone; SS, sodium salicylate.

 
Modulation of ovarian gene expression
To determine if drug treatment was affecting ovarian gene expression, real-time RT-PCR was performed on ovarian RNA collected from d-1 pregnant animals (Fig. 6Go). Hepatic RNA was used for comparison as a control tissue in which the effects of each insulin sensitizer have been better characterized. Specifically, the transcription of genes with previously documented nutritional and hormonal regulation was investigated. Expression of the rosiglitazone receptor Pparg was not found to be different between CD and HFD fed mice, in either hepatic or ovarian samples (Fig. 6Go, A and B), although expression tended to be higher in ovaries from HFD plus rosiglitazone animals. Rosiglitazone treatment increased expression of scavenger receptor Cd36 mRNA within the liver of HFD fed mice (Fig. 6CGo). The HFD tended to increase ovarian expression of the Cd36, which was moderately lower in both HFD plus AICAR and HFD plus sodium salicylate ovaries (Fig. 6DGo). Treatment with rosiglitazone increased expression of Cd36 when compared with CD ovaries (P = 0.037). Consistent with previous reports (52), hepatic expression of scavenger receptor, Scarb1, was not influenced by diet (Fig. 6EGo). It was also unaffected by administration of any insulin-sensitizing drug within the liver. However, rosiglitazone lowered ovarian expression of Scarb1 by 61% compared with HFD plus vehicle-treated ovaries (P = 0.003; Fig. 6FGo). Hepatic expression of intracellular lipid transporter Fabp4 (also known as adipocyte P2) was affected by rosiglitazone in a similar way to Cd36, with an up-regulation of transcription (P = 0.0005; Fig. 6GGo). This effect was mirrored within the ovary, although to a greater extent (7.6-fold increase, compared with 3.4-fold increase in the liver, Fig. 6HGo). In general, ovarian expression of each gene reflected the same pattern of changes as those observed in the liver.


Figure 6
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  		FIG. 6. Gene expression in liver (A, C, E, and G) and ovary (B, D, F, and H) from mice fed a CD or HFD and in response to treatment with insulin sensitizers or vehicle. Genes analyzed were Pparg (A and B), Cd36 (C and D), Scarb1 (E and F), and Fabp4 (G and H). Each is normalized to ribosomal L19 and expressed as fold change from the CD group. Bars indicate mean ± SEM (n = 15). *, P < 0.05. **, P < 0.001. Rosi, Rosiglitazone; SS, sodium salicylate.

 
Incidence and rate of ovulation
Reproductive parameters were next assessed: onset to the mating event, ovarian weight, incidence of anovulation, and ovulation rate (Table 1Go). The number of days to plug tended to be longer in the HFD group, with HFD plus vehicle females requiring 3.00 ± 0.59 d compared with CD females requiring 1.93 ± 0.4. Ovarian tissue weight was also increased by the HFD (P = 0.049 vs. CD). Surprisingly, in several mice zero oocytes were present in both oviducts, with this incidence of anovulation highest (six of 15 animals) in response to the HFD. In mice that did ovulate, the HFD increased the ovulation rate (7.0 ± 0.6 oocytes) compared with CD (4.9 ± 0.9 oocytes; P = 0.036). Serum progesterone, although not analyzed in this series of experiments, was measured in previous cohorts of mice and was not influenced by the HFD (data not shown).


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  		TABLE 1. Ovulation incidence and rate in response to HFD ± insulin sensitizers

 
None of these outcomes (Table 1Go) was significantly affected by treatment with the insulin sensitizers with the exception that HFD mice treated with sodium salicylate required an extended period of time before mating, 3.92 ± 0.40 d (P < 0.044 vs. CD, HFD plus AICAR and HFD plus rosiglitazone groups). Sodium salicylate actions associated with the inhibition of cyclooxygenases required for ovulation may be responsible for this observation. Interestingly, with each parameter rosiglitazone treatment resulted in outcomes most similar to those exhibited by CD mice.

Early embryo development
Embryonic development in vitro was assessed by blinded, daily evaluation and scoring for correct morphology of the fertilized oocytes. Cleavage rates were not affected by treatments (data not shown). HFD fed mice produced embryos with reduced on-time progression to all developmental milestones assessed (Fig. 7Go): the four to eight cell stage (P = 0.0001), morula/early blastocyst stage (P = 0.002), and the expanded/hatching blastocyst stage (P = 0.002), compared with embryos produced by CD fed females. Neither AICAR nor sodium salicylate administration affected embryo on-time development. However, fertilized oocytes obtained from HFD plus rosiglitazone animals demonstrated significantly improved developmental potential compared with HFD plus vehicle oocytes at the four to eight cell stage (P = 0.001), at the morula/compacted blastocyst stage (P = 0.0003), and at the expanded/hatching blastocyst stage (P = 0.004). Overall, there was no discernable difference in developmental dynamics of oocytes obtained from CD animals, and oocytes obtained from obese, HFD animals that had been treated with preovulatory rosiglitazone.


Figure 7
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  		FIG. 7. Percentage of cleaved oocytes that reached the four to eight cell, morula/blastocyst and expanded blastocyst/hatching blastocyst stages on time. Data show the percentage of fertilized zygotes to pass each developmental stage with the actual number of zygotes indicated in the table below. Each female contributed between one and 11 oocytes. *, P < 0.05; **, P < 0.001. Rosi, Rosiglitazone; SS, sodium salicylate.

 
Blastomere differentiation
All embryos surviving to d-5 culture were subjected to a differential staining protocol to permit counting of cells constituting the TE layer and the ICM. Embryos derived from HFD fed mice had higher numbers of cells allocated into the TE layer and slightly reduced cell numbers within the ICM. Consequently, the proportion of ICM, as a percentage of the total embryonic cell number, is smaller (Fig. 8Go). Both AICAR and rosiglitazone administration significantly reduced the number of cells within the TE layer (P = 0.047 HFD plus vehicle vs. HFD plus AICAR; P = 0.007 HFD plus vehicle vs. HFD plus rosiglitazone; Fig. 8AGo). None of the drug treatments had a dramatic influence on the number of cells within the ICM (Fig. 8BGo). However, there was a statistically significant increase in the percentage of cells contained within the ICM (P = 0.033 HFD plus vehicle vs. HFD plus rosiglitazone; Fig. 8CGo).


Figure 8
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  		FIG. 8. Cellular composition of embryos. Number of TE (A) and ICM cells (B) per embryo. C, Percentage of ICM per embryo. Bars indicate mean ± SEM (n = 21–38 blastocysts per group). Different letters indicate statistically different means, P < 0.05. Rosi, Rosiglitazone; SS, sodium salicylate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report that a HFD leading to obesity leads to reproductive defects that are initiated within the ovarian environment and manifest as impaired development of the early embryo. Such perturbations of blastocyst development are increasingly understood to result in suboptimal fetal growth as well as to contribute to the fetal origins of adult disease (53, 54). Our findings support evidence of altered granulosa cell phenotype and ovarian dysfunction under conditions of gene mutation-induced obesity (55, 56, 57, 58) but expand our understanding to show it persists in wild-type populations in which caloric intake is excessive. The finding that a preovulatory ovarian environment placed within the context of a metabolically compromised individual produces oocytes with a severely impaired capacity to support normal embryo development is particularly alarming as rates of human obesity in developed nations continue to increase.

We have further demonstrated that the PPARG pathway, potentially operating at the ovarian level, is intrinsically involved in this interaction. Rosiglitazone is uniquely able to overcome the negative influence of HFD consumption on embryonic on-time development. Rosiglitazone treatment also increased the percentage of inner mass cells per embryo, a measurement previously confirmed as indicative of improved fetal outcomes (54, 59). Neither AICAR nor sodium salicylate was able to induce such profound effects, even though they exhibited some expected systemic effects, such as lowering the level of circulating insulin. This suggests that either the metabolic consequences of rosiglitazone, including reducing plasma triglycerides and lowering elevated blood glucose, or the direct molecular targets of rosiglitazone within ovarian cells are responsible for the observed effects.

The molecular action of rosiglitazone is well established; upon entry into the cell via transmembrane diffusion (as a small and lipophilic molecule), rosiglitazone binds to PPARG, stimulating formation of regulatory complexes that either up- or down-regulate transcription of target genes. Among the comprehensive list of PPARG-regulated genes are many genes principally involved in lipid uptake and metabolism, glucose uptake and metabolism, and immune cell responses (41). Systemically insulin resistance in peripheral tissues is ameliorated, and circulating levels of lipids are lowered (60, 61). Within ovarian tissue, PPARG is most highly expressed in the granulosa cells and luteal cells, in the ovaries of rodents and ruminants (62, 63, 64, 65). In addition, ovarian macrophages, which surround ovarian follicles and release pro-inflammatory cytokines, have high levels of transcript and protein expression (65). Within the oocyte itself, PPARG expression seems to be dependent upon species. Although moderate expression has been reported in ruminants (66), oocyte PPARG expression is low within rodents (63, 65, 67). Therefore, it is likely that modulation of somatic ovarian cell functions is mediating the improved outcomes observed when mice are treated with rosiglitazone.

In support of this, we identified significant, and specific, modulation of ovarian gene expression, namely, up-regulation of Cd36 and Fabp4, and suppression of Scarb1, genes known to possess PPARG response elements (PPREs) in proximal promoter regions (68, 69, 70) and to be regulated in response to PPARG activation (71, 72, 73). Within the ovary, high levels of CD36 protein is found in granulosa cells of preantral and early antral follicles, and also within the vascular thecal layer; SCARB1 expression is strongly associated with the high-density lipoprotein (HDL)-cholesterol ester requirement for production of steroid hormones such as androgen (estradiol precursor) and progesterone, and fatty acid binding protein 4 is found predominately within granulosa cells of follicles undergoing atresia (74). Fatty acid binding protein 4 is known to solubilize lipophilic fatty acids facilitating their intracellular transport, in particular, positioning lipid ligands of PPARG in close proximity to this nuclear receptor allowing selective enhancement of PPARG transcriptional activity (75). Both CD36 and SCARB1 are involved in selective cholesterol ester uptake from HDL lipoproteins, and SCARB1 is additionally able to bind and uptake unmodified low-density lipoprotein. By up-regulating Cd36 expression and down-regulating Scarb1 expression, rosiglitazone may specifically increase HDL-uptake potential and minimize the possibility of native low-density lipoprotein-cholesterol ester uptake within the ovary. At the time of sample collection (d-1 pregnancy), ovarian function comprises the establishment of luteal activity, which includes increased cholesterol uptake. Rosiglitazone-induced modifications to transport protein, and subsequently cholesterol availability, would dramatically influence such functions as progesterone production initiates. Furthermore, activity and expression of key molecular regulators of steroidogenesis, including steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase, are also both regulated by PPARG activation (76, 77). Precisely how these functions would benefit ovarian follicular function to improve oocyte quality remains to be determined, and close inspection of hormonal synthesis in response to this treatment and implications for oocyte developmental competence is required.

Overall, PPARG activation may present a promising option in the in vitro fertilization setting if pharmaceutical alternatives are required to overcome the influence of a suboptimal maternal metabolic profile on oocyte health. Although currently listed as a Pregnancy Category C drug (not tested for use during pregnancy), recent studies have reported no adverse effects of maternal treatment with rosiglitazone during the peri-ovulatory period on embryonic or fetal outcomes (78, 79). In addition, alternative activators of PPARG, including many of the naturally occurring, fatty acid-based ligands, may prove useful in circumventing the limitations of rosiglitazone.

In conclusion, this study has demonstrated that rosiglitazone, either through systemic improvements to specific metabolic parameters or by directly modulating PPARG-regulated gene expression in ovarian cells, is able to reverse deficits in oocyte quality brought about by DIO, such that early embryonic developmental competence is greatly improved. It emphasizes the important contribution of PPARG-controlled genes in optimal ovarian biology that are consequently key mediators of female reproductive potential.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 14, 2008

Abbreviations: AICAR, 5-Aminoimidazole 4-carboxamide-riboside; CD, control diet; DIO, diet-induced obesity; HDL, high-density lipoprotein; HFD, high-fat diet; ICM, inner cell mass; PPARG, peroxisome proliferator activated receptor-{gamma}; PPRE, peroxisome proliferator-activated receptor response element; SCARB1, scavenger receptor class B, member 1; TE, trophectoderm; UCP, uncoupling protein.

Received November 15, 2007.

Accepted for publication February 6, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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