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Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes
Exposure to lipid-rich follicular fluid is associated with endoplasmic reticulum stress and impaired oocyte maturation in cumulus-oocyte complexes Xing Yang, M.D., Ph.D.,a,b Linda L. Wu, Ph.D.,b Lindsay R. Chura,b Xiaoyan Liang, M.D.,a Michelle Lane, Ph.D.,b Robert J. Norman, M.D.,b and Rebecca L. Robker, Ph.D.b a Reproductive Medical Center, Sixth Affiliated Hospital of Sun Yan-sen University, Guangzhou, People's Republic of China; and b Robinson Institute, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia, Australia
Objective: To determine whether the high lipid content of human follicular fluid influences oocyte maturation. Design: Mouse oocytes as substitutes for human oocytes were exposed to follicular fluids of differing lipid content with outcome monitoring. Setting: Private infertility clinic and university laboratory. Patient(s): Seventy-four women seeking assisted reproduction, and gonadotropin-stimulated mice. Intervention(s): Assay of follicular fluids for triglyceride and free fatty acids, and stimulation of mouse cumulus-oocyte complexes (COCs) to maturity in vitro in the presence of lipid-rich or lipid-poor follicular fluid. Main Outcome Measure(s): Oocyte lipid content, expression of endoplasmic reticulum stress marker genes, and oocyte maturation assessed in mouse COCs exposed to lipid-rich follicular fluid were compared with complexes exposed to lipid-poor follicular fluid and complexes matured in vivo. Result(s): Follicular fluids were obtained from women of known body mass index undergoing oocyte aspiration at a private infertility clinic, and the follicular fluids were assayed for triglyceride and free fatty acids; those with the highest and lowest levels of these lipids were selected. The mouse COCs exposed to lipid-rich follicular fluid during their maturation had increased oocyte lipid content, induction of endoplasmic reticulum stress markers, and impaired oocyte nuclear maturation. Conclusion(s): Increased body mass index is associated with elevated triglycerides and free fatty acids in ovarian follicular fluid. Maturation within this lipid-rich environment is detrimental to oocytes. (Fertil SterilÒ 2012;97:1438–43. Ó2012 by American Society for Reproductive Medicine.) Key Words: ATF4, ATF6, endoplasmic reticulum stress, free fatty acids, GRP78, lipotoxicity, obesity, triglyceride
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besity in women is associated with reduced conception rates (1–3), even when oocytes are fertilized in vitro (4–8). Studies in female mice more clearly demonstrate that obesity induced by a high-fat diet affects oocyte quality, resulting in oocytes that when fertilized in vivo are slower to develop to blastocysts and exhibit a skewed trophectoderm and
inner cell mass ratio (9). Oocytes from obese mice also have increased lipid content (10), alterations in mitochondrial activity (10, 11), and evidence of endoplasmic reticulum stress (10); these cellular responses likely contribute to the increased granulosa cell apoptosis (10, 12) and decreased rates of oocyte nuclear maturation (12) and fertilization (10). This accumulating evidence of
Received September 30, 2011; revised and accepted February 23, 2012; published online March 20, 2012. X.Y. has nothing to disclose. L.L.W. has nothing to disclose. L.R.C. has nothing to disclose. X.L. has nothing to disclose. M.L. has nothing to disclose. R.J.N. has nothing to disclose. R.L.R. has nothing to disclose. Supported by grants from the National Health and Medical Research Council of Australia (to R.J.N. and R.L.R.) and the National Natural Science Foundation of China (grant no. 30973202 to X.Y.). Reprint requests: Rebecca L. Robker, Ph.D., School of Paediatrics and Reproductive Health, University of Adelaide, Medical School North, Level 2, Adelaide, South Australia, 5005, Australia (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.034 1438
associations between diet-induced obesity and altered oocyte quality in mice does not, however, demonstrate whether similar cellular changes occur in the oocytes of obese women. Further, it is currently unclear whether the effect of obesity on oocytes is instigated during their growth and folliculogenesis or is a result of acute events immediately before conception. Periconception oocyte maturation is initiated when a large antral follicle receives the luteinizing hormone (LH) surge. While the cumulus cells surrounding the oocyte rapidly produce the viscous extracellular matrix necessary for ovulation and fertilization, the oocyte resumes meiosis and extrudes its first polar body. Physiologically this process occurs within the confines of the ovarian follicular VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® environment wherein the cumulus-oocyte complex (or COC) is surrounded by follicular fluid. With obesity however, ovarian follicular fluid is altered, having significantly higher levels of leptin (13), triglycerides (14), and markers of oxidative stress (15) than follicular fluid of moderate weight women. The impact that these and other alterations in the follicular environment of obese women may have on these final stages of oocyte maturation is not known. To directly determine whether lipids within the ovarian follicular environment near the time of conception influence oocyte maturation, we used mouse oocytes as surrogates for human oocytes and stimulated them to mature in vitro in the presence of lipid-rich follicular fluid or lipid-poor follicular fluid. We assessed oocyte lipid content, endoplasmic reticulum stress markers, and oocyte maturation. The results confirm that the ovarian follicular environment contains more lipids with increasing body mass index (BMI) and demonstrate that maturation within such a lipid-rich environment is detrimental to oocytes.
MATERIALS AND METHODS All chemicals were purchased from Sigma-Aldrich (SigmaAldrich Pty. Ltd.) unless otherwise stated.
Ovarian Follicular Fluid Ethics approval was obtained from the Women's and Children's Hospital, Adelaide, South Australia. Ovarian follicular fluid was obtained from women (n ¼ 74) seeking assisted reproduction at a private clinic in South Australia. Participant body mass index (BMI ¼ Weight [kg]/Height [m2]) was recorded when known: moderate (BMI ¼ 20–24.9 kg/m2; n ¼ 21), overweight (BMI ¼ 25–29.9 kg/m2; n ¼ 20), and obese (BMI R30 kg/m2; n ¼ 22). The primary infertility etiologies of the patients varied and included male factor, tubal disease, anovulation, unexplained infertility, and in some cases more than one cause or both male and female factor infertility. Women with symptoms of polycystic ovary syndrome (PCOS) according to either National Institutes of Health or Rotterdam criteria were specifically excluded. There were no differences in the proportions of infertility diagnoses across the BMI groups. All patients were administered daily recombinant follicle-stimulating hormone (FSH, 150–300 IU) from the start of their menstrual cycle for 8 to 10 days until a minimum of three follicles of more than 18 mm diameter was observed by transvaginal ultrasound scan. A gonadotropin-releasing hormone (GnRH) antagonist (Orgalutron) was used from day 6 of the cycle to block ovulation. Women were then administered human chorionic gonadotropin (hCG, Ovidrel 250 mg), and transvaginal follicular aspiration was performed 36 hours after hCG administration. Blood-free follicular aspirates were centrifuged at 4,000 rpm for 10 minutes, and the follicular fluid was removed and stored at 80 C. Levels of follicular fluid triglyceride and free fatty acids were measured using the Roche Hitachi 912 Chemistry Analyzer as per the manufacturer's instructions, and using quality VOL. 97 NO. 6 / JUNE 2012
controls QCS 1 and 2 (Bio-Rad). The mean coefficient of variation for each assay was less than 4.6%.
Isolation and In Vitro Maturation of Mouse Cumulus-Oocyte Complexes Experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Immature COCs were isolated from 23-day-old mice (CBAXC57Bl/6F1) by puncturing the antral follicles of ovaries collected 44 hours after IP injection of 5 IU equine chorionic gonadotropin (eCG, Gestyl; Professional Compounding Centre of Australia). Mature COCs were dissected from oviducts of mice 44 hours after eCG administration and 13 hours after IP administration of 5 IU hCG (Pregnyl; Organon). The COCs were collected in HEPES-buffered a-minimal essential medium (MEM; GIBCO, Invitrogen Australia Pty. Ltd.) and cultured in groups of 20 at 37 C in 6% CO2/94% air in 100 mL drops of bicarbonatebuffered a-MEM, supplemented with 50% (v/v) human follicle fluid and overlaid with sterile paraffin oil (Merck). Maturation was stimulated by treatment with 50 mIU/mL of recombinant human FSH and 10 ng/mL of epidermal growth factor (EGF). Cumulus expansion according to the 0–4 scale (16) and the presence or absence of germinal vesicle breakdown (GVBD) and first polar body extrusion were evaluated after 16 hours of culture.
Lipid Droplet Staining Lipophilic dye BODIPY 493/503 (Invitrogen), which stains intracellular neutral lipids, was used to localize the lipid droplets in oocytes, as described previously described elsewhere (17). Briefly, COCs were paraformaldehyde-fixed, stained in BODIPY 493/503, and imaged using a Leica SP5 spectral scanning confocal microscope, with identical conditions throughout all experiments. Fluorescence in the oocytes was calculated by Analysis Pro Software (Olympus Australia Pty. Ltd.).
RNA Isolation and Real-Time RT-PCR Total RNA was isolated from COCs (R35 COCs per RNA sample) using RNeasy Micro Kit (Qiagen Pty Ltd.), with 600 ng reverse transcribed using random primers (Roche) and Superscript III Reverse Transcriptase (Invitrogen). Quantitect Primer Assays (Qiagen) Plin2, Atf4, Atf6, Grp78, and ribosomal protein L19 (internal control) were used. The PCR analysis was performed in triplicate using the Rotor-Gene 6000 (Corbett Research) analyzer with SYBR Green PCR Master Mix (Applied Biosystems). Data were analyzed using the 2DDCT method, for quantification relative to a calibrator sample, and were expressed as the relative fold change.
Statistical Analysis Statistical analysis was performed using Graph Pad Prism version 5.01 (GraphPad Software Inc.). Statistically significant correlations between BMI and follicular fluid metabolites (n ¼ 64 to 74, as indicated) were detected by Spearman (two-tailed) 1439
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY correlation. Comparisons between the selected follicular fluid samples (n ¼ 8 lipid rich and n ¼ 8 lipid poor) are reported as mean standard error of the mean (SEM) of each group and were analyzed by unpaired two-tailed t-test as indicated. In vitro maturation experiments were conducted in a parallel treatment design whereby groups of COCs were subjected to either lipid-rich or lipid-poor maturation treatment and were compared with ovulated (in vivo) COCs. This experimental design was repeated three times with biologically independent maturation treatments. Outcome parameters of oocyte lipid content, COC gene expression, cumulus expansion rates, and oocyte maturation rates are reported as mean SEM of each measure from three independent experiments and were analyzed by one-way analysis of variance (ANOVA) with the Newman-Keuls multiple comparison test as indicated. P< .05 was considered statistically significant.
RESULTS Increasing BMI in women (n ¼ 64) was positively correlated with increasing levels of triglyceride (r ¼ 0.44; P¼ .0003) and free fatty acids (r ¼ 0.25; P¼ .04) in ovarian follicle fluid. To examine how the increased lipid content of follicle fluid of obese women may impact oocyte maturation, the most lipid-rich and lipid-poor follicular fluid samples were sought. The levels of triglyceride and free fatty acids in follicular fluid were positively correlated (P< .001), and the samples that were relatively high or relatively low in these two measures were identified (Fig. 1; squares and triangles, respectively). The lipid-rich samples (n ¼ 8) were from obese women (BMI 32.6 1.7), and, as expected, they had statistically significantly elevated levels of free fatty acids (0.38 0.04 meq/L) and triglycerides (0.24 0.015 mmol/L) compared with the lipid-poor samples (n ¼ 8); the latter were from nonobese women (BMI 24.8 1.0) and contained statistically significantly lower levels of lipids (free fatty acids: 0.16 0.02 meq/L; P¼ .0001; triglycerides: 0.09 0.004 mmol/L, P< .0001 by t-test compared with lipid-rich samples). Equal volumes of two or three patient samples of each follicular fluid type (lipid rich or lipid poor) were pooled as depicted in Figure 1 to generate three entirely distinct paired pools of follicular fluid. Each pair of lipidrich and lipid-poor samples (i.e., same colors in Fig. 1) were then used in independent experiments for in vitro maturation of mouse COCs and for comparison with ovulated COCs collected in parallel to each in vitro experiment. Immature COCs, isolated from preovulatory follicles of mice treated with eCG, were stimulated to mature for 16 hours in either lipid-rich follicular fluid (high-lipid FF) or lipid-poor follicular fluid (low-lipid FF) and were compared with those matured in vivo (i.e., isolated from oviducts after ovulation). The COCs were stained with neutral lipid dye, and the lipid content within the oocytes was determined. Oocytes matured in lipid-rich follicular fluid contained statistically significantly more neutral lipid than the oocytes matured in lipidpoor follicular fluid or those matured in vivo (Fig. 2A). Expression of perilipin-2 (Plin2/ADRP), a lipid droplet protein expressed in the COC that coats lipid droplets in mouse oocytes, was also statistically significantly higher in COCs matured in lipid-rich follicular fluid than in COCs matured in lipid-poor follicular fluid or in vivo (see Fig. 2B). Expression 1440
FIGURE 1
Identification of lipid-rich and lipid-poor follicular fluids. Levels of triglyceride and free fatty acids in follicular fluid are statistically significantly correlated (P<.001 by Spearman correlation). Samples with the highest levels of these lipids (squares; n ¼ 8) and the lowest levels (triangles; n ¼ 8) were identified, and equal volumes from two or three women (those with identical symbols) were pooled. Each color indicates samples that were used in independent experiments (n ¼ 3) for the in vitro maturation of mouse cumulus-oocyte complexes. Yang. Lipid-rich follicle fluid impacts oocytes. Fertil Steril 2012.
of endoplasmic reticulum stress markers Atf4, Atf6, and Grp78 were also statistically significantly increased in COCs matured in lipid-rich follicular fluid compared with the COCs matured in lipid-poor follicular fluid (see Fig. 2C–2E), similar to the COCs from mice with diet-induced obesity (10). Maturation of the COCs was assessed by measuring the cumulus expansion score, oocyte germinal vesicle breakdown (GVBD), and oocyte polar body extrusion. Cumulus expansion scores were not statistically significantly different between the treatment groups (in vivo: 3.33 0.16; low-lipid follicular fluid: 3.67 0.11; high-lipid follicular fluid: 3.50 1.11; n R 17 COCs per group). The GVBD rates were R94% in all experiments and were not affected by culture with either of the follicular fluids compared with the in vivo matured COCs. However, maturation in lipid-rich follicular fluid dramatically decreased oocyte maturation to metaphase II (MII), assessed as polar body formation, to approximately 25% of in vivo rates (see Fig. 2F).
DISCUSSION These results show that the hyperlipidemia associated with obesity in women extends directly into the ovarian follicular microenvironment, with increasing BMI associated with markedly increased triglyceride and free fatty acid levels in follicle fluid. Maturation of COCs in this lipid-rich environment promoted lipid accumulation and increased the lipid droplet protein mRNA expression in mouse COCs exposed to the environment in vitro. This disrupted physiologic maturation environment was detrimental to oocytes, leading to the induction of multiple endoplasmic reticulum stress markers and impaired maturation. These results provide insight into cellular alterations that might mechanistically contribute to obesity-associated impairments in oocyte maturation and developmental competence. VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility®
FIGURE 2
Effects of maturation in lipid-rich follicular fluid (FF) on mouse cumulus-oocyte complexes (COCs). (A) Oocyte neutral lipid content is increased in COCs matured in lipid-rich follicular fluid (high-lipid FF) compared with COCs matured in lipid-poor follicular fluid (low-lipid FF). Mean SEM; n ¼ 18–22 COCs per treatment group. Expression of lipid droplet protein (B) perilipin-2 and the endoplasmic reticulum stress markers (C) Atf4, (D) Atf6, and (E) Grp78 is increased in COCs matured in high-lipid FF. Mean SEM; n ¼ 3 independent experiments using different follicular fluid pools and R35 COCs per treatment group. (F) Oocyte maturation to MII is impaired in COCs matured in high-lipid FF. Mean SEM; n ¼ 3 independent experiments using different follicular fluid pools and R17 COCs per treatment group. For each assay, ovulated COCs obtained from the mouse oviduct (in vivo matured) were also included. Different letters indicate significant differences by one-way analysis of variance (ANOVA). A,B,C,E P<.05; D P<.01; F P<.0001. Yang. Lipid-rich follicle fluid impacts oocytes. Fertil Steril 2012.
It was predictable that exposure to lipid-rich follicular fluid led to lipid accumulation in oocytes because maturation in fetal calf serum also leads to increased oocyte neutral lipid content compared with maturation in serum-free conditions (17, 18). The increased expression of lipid droplet protein perilipin-2/ADRP in the COC may further reflect the expansion of lipid stores because perilipin-2/ADRP expression generally correlates with intracellular levels of neutral lipid and is also inducible by exposure to fatty acids (19). Somewhat surprising was the dramatic detrimental effect that lipidrich follicular fluid had on oocyte maturation, which may indicate compatibility issues in this nonphysiologic oocyte maturation system using tissues from different species. However, our demonstration that lipid-rich follicle fluid impairs oocyte maturation is in general agreement with recent reports that fatty acid content of follicle fluid in women is associated with poor COC morphology (20) and that treatment of bovine COCs with increasing doses of specific fatty acids impairs oocyte maturation and subsequent embryo development (21–24). The poor maturation rates we observed in the oocytes matured in lipid-rich follicle fluid precluded their use for in vitro fertilization and for any further developmental assessments that could have provided important additional information about oocyte quality. Our work is the first to demonstrate that the detrimental effects of lipids on oocytes may be mediated via the induction VOL. 97 NO. 6 / JUNE 2012
of endoplasmic reticulum stress pathways. Endoplasmic reticulum stress occurs with obesity (25) and in cells exposed to high levels of free fatty acids (26), activating the unfolded protein response (or UPR). This response to endoplasmic reticulum stress is characterized by the induction of a cohort of genes, including the markers Atf4, Atf6, and Grp78, which counteract cellular stress by slowing protein production and up-regulating protein-folding chaperones (27). If the UPR is unable to resolve endoplasmic reticulum stress, additional lipotoxicity pathways are initiated, potentially culminating in apoptotic cell death. That lipid-rich follicle fluid induces endoplasmic reticulum stress markers in COCs suggests that endoplasmic reticulum stress and lipotoxicity may be occurring in the ovaries of obese women. Supporting this, expression of the endoplasmic reticulum stress marker ATF4 is increased in the granulosa cells of obese women (10). It is interesting that the COCs exposed to follicle fluid that was lipid poor had a lower expression of Grp78 than the ovulated COCs matured in vivo. Unlike endoplasmic reticulum stress markers ATF4 and ATF6, which act as transcription factors, GRP78 has additional roles beyond that of an endoplasmic reticulum stress-induced chaperone protein, and it localizes to the cell surface, mitochondria, or nucleus under different conditions to mediate diverse functions (28). Grp78 is known to be transcriptionally regulated by changes to glucose availability, oxygen tension, 1441
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY and pH; thus, the lower levels in COCs matured in lipid-poor follicle fluid may be a reflection of a fundamental difference in one or more of these parameters under in vitro compared with in vivo maturation conditions. Overall though, each of the three endoplasmic reticulum stress markers examined exhibited a higher expression in the COCs matured in lipid-rich compared with lipid-poor in vitro conditions, indicating that exposure to high levels of lipids in follicle fluid is indeed inducing endoplasmic reticulum stress and potentially lipotoxicity responses. It is likely that the observed changes in gene expression reflect alterations in cumulus cells, as oocytes are transcriptionally inactive at this stage and because cumulus cell mRNA comprises a disproportionate majority of the sample. However, whether endoplasmic reticulum stress responses are occurring in the oocyte, cumulus cells, or both cannot be definitively determined from our current study and remains an important question to resolve. This study focused specifically on the effects of excess triglycerides and free fatty acids because [1] our previous study found that triglyceride was elevated in the follicular fluid of obese women (14), [2] free fatty acids are potent inducers of endoplasmic reticulum stress, which we have found is induced in COCs from obese mice (10) and in COCs treated with high doses of palmitic acid (29), and [3] these lipids are used for beta-oxidation, which is important for oocyte developmental competence (30). It should be noted however that these experiments using human follicular fluid cannot determine whether the detrimental effects observed are due to high triglyceride and free fatty acid content per se or due to other potential mediators such as cholesterol esters and phospholipids, which are even more abundant in follicular fluid. Indeed, in our patient cohort the level of cholesterol in follicular fluid was also tightly linked with triglyceride levels (r ¼ 0.54; P< .0001) and free fatty acid levels (r ¼ 0.62; P< .0001 by Spearman correlation), and thus is an additional potential contributor to the observed effects on oocyte neutral lipid content and nuclear maturation. Similarly, insulin levels in follicular fluid also tended to be related to triglycerides (r ¼ 0.27; P¼ .02) and free fatty acids (r ¼ 0.30; P¼ .01). Further, there may be other nonlipid confounders such as protein levels, which can degrade into urea and ammonia in vitro and thereby indirectly cause detrimental effects on oocyte quality (31). It is likely that additional factors also associate with high lipid content in follicle fluid and contribute to the dramatic effects on oocyte maturation; for instance, inflammatory mediators and oxidative stress are elevated in the follicle fluid of obese women (14, 15). Thus, the mechanisms linking lipids, endoplasmic reticulum stress, and oocyte quality still need to be clarified before we can determine the fundamental causes of poor fertility reported in obese women and elucidate the contextual signals that influence oocyte developmental competence precisely at the time of conception. Our study shows that lipid levels in follicular fluid vary markedly between women and that differing levels of these lipids, and presumably many additional cofactors, in follicular fluid can exert dramatically different effects. The impact of different follicular maturation environments on human oocytes remains to be directly determined; however, the use 1442
of mouse oocytes indicates that specific follicular fluid environments can acutely increase oocyte lipid content, induce endoplasmic reticulum stress in COCs, and profoundly impact oocyte maturation. Acknowledgments: The authors thank Brenton D. Bennett for assistance, and the patients and staff of Repromed for their participation.
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Objective: To determine whether the high lipid content of human follicular fluid influences oocyte maturation. Design: Mouse oocytes as substitutes for human oocytes were exposed to follicular fluids of differing lipid content with outcome monitoring. Setting: Private infertility clinic and university laboratory. Patient(s): Seventy-four women seeking assisted reproduction, and gonadotropin-stimulated mice. Intervention(s): Assay of follicular fluids for triglyceride and free fatty acids, and stimulation of mouse cumulus-oocyte complexes (COCs) to maturity in vitro in the presence of lipid-rich or lipid-poor follicular fluid. Main Outcome Measure(s): Oocyte lipid content, expression of endoplasmic reticulum stress marker genes, and oocyte maturation assessed in mouse COCs exposed to lipid-rich follicular fluid were compared with complexes exposed to lipid-poor follicular fluid and complexes matured in vivo. Result(s): Follicular fluids were obtained from women of known body mass index undergoing oocyte aspiration at a private infertility clinic, and the follicular fluids were assayed for triglyceride and free fatty acids; those with the highest and lowest levels of these lipids were selected. The mouse COCs exposed to lipid-rich follicular fluid during their maturation had increased oocyte lipid content, induction of endoplasmic reticulum stress markers, and impaired oocyte nuclear maturation. Conclusion(s): Increased body mass index is associated with elevated triglycerides and free fatty acids in ovarian follicular fluid. Maturation within this lipid-rich environment is detrimental to oocytes. (Fertil SterilÒ 2012;97:1438–43. Ó2012 by American Society for Reproductive Medicine.) Key Words: ATF4, ATF6, endoplasmic reticulum stress, free fatty acids, GRP78, lipotoxicity, obesity, triglyceride
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besity in women is associated with reduced conception rates (1–3), even when oocytes are fertilized in vitro (4–8). Studies in female mice more clearly demonstrate that obesity induced by a high-fat diet affects oocyte quality, resulting in oocytes that when fertilized in vivo are slower to develop to blastocysts and exhibit a skewed trophectoderm and
inner cell mass ratio (9). Oocytes from obese mice also have increased lipid content (10), alterations in mitochondrial activity (10, 11), and evidence of endoplasmic reticulum stress (10); these cellular responses likely contribute to the increased granulosa cell apoptosis (10, 12) and decreased rates of oocyte nuclear maturation (12) and fertilization (10). This accumulating evidence of
Received September 30, 2011; revised and accepted February 23, 2012; published online March 20, 2012. X.Y. has nothing to disclose. L.L.W. has nothing to disclose. L.R.C. has nothing to disclose. X.L. has nothing to disclose. M.L. has nothing to disclose. R.J.N. has nothing to disclose. R.L.R. has nothing to disclose. Supported by grants from the National Health and Medical Research Council of Australia (to R.J.N. and R.L.R.) and the National Natural Science Foundation of China (grant no. 30973202 to X.Y.). Reprint requests: Rebecca L. Robker, Ph.D., School of Paediatrics and Reproductive Health, University of Adelaide, Medical School North, Level 2, Adelaide, South Australia, 5005, Australia (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.034 1438
associations between diet-induced obesity and altered oocyte quality in mice does not, however, demonstrate whether similar cellular changes occur in the oocytes of obese women. Further, it is currently unclear whether the effect of obesity on oocytes is instigated during their growth and folliculogenesis or is a result of acute events immediately before conception. Periconception oocyte maturation is initiated when a large antral follicle receives the luteinizing hormone (LH) surge. While the cumulus cells surrounding the oocyte rapidly produce the viscous extracellular matrix necessary for ovulation and fertilization, the oocyte resumes meiosis and extrudes its first polar body. Physiologically this process occurs within the confines of the ovarian follicular VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® environment wherein the cumulus-oocyte complex (or COC) is surrounded by follicular fluid. With obesity however, ovarian follicular fluid is altered, having significantly higher levels of leptin (13), triglycerides (14), and markers of oxidative stress (15) than follicular fluid of moderate weight women. The impact that these and other alterations in the follicular environment of obese women may have on these final stages of oocyte maturation is not known. To directly determine whether lipids within the ovarian follicular environment near the time of conception influence oocyte maturation, we used mouse oocytes as surrogates for human oocytes and stimulated them to mature in vitro in the presence of lipid-rich follicular fluid or lipid-poor follicular fluid. We assessed oocyte lipid content, endoplasmic reticulum stress markers, and oocyte maturation. The results confirm that the ovarian follicular environment contains more lipids with increasing body mass index (BMI) and demonstrate that maturation within such a lipid-rich environment is detrimental to oocytes.
MATERIALS AND METHODS All chemicals were purchased from Sigma-Aldrich (SigmaAldrich Pty. Ltd.) unless otherwise stated.
Ovarian Follicular Fluid Ethics approval was obtained from the Women's and Children's Hospital, Adelaide, South Australia. Ovarian follicular fluid was obtained from women (n ¼ 74) seeking assisted reproduction at a private clinic in South Australia. Participant body mass index (BMI ¼ Weight [kg]/Height [m2]) was recorded when known: moderate (BMI ¼ 20–24.9 kg/m2; n ¼ 21), overweight (BMI ¼ 25–29.9 kg/m2; n ¼ 20), and obese (BMI R30 kg/m2; n ¼ 22). The primary infertility etiologies of the patients varied and included male factor, tubal disease, anovulation, unexplained infertility, and in some cases more than one cause or both male and female factor infertility. Women with symptoms of polycystic ovary syndrome (PCOS) according to either National Institutes of Health or Rotterdam criteria were specifically excluded. There were no differences in the proportions of infertility diagnoses across the BMI groups. All patients were administered daily recombinant follicle-stimulating hormone (FSH, 150–300 IU) from the start of their menstrual cycle for 8 to 10 days until a minimum of three follicles of more than 18 mm diameter was observed by transvaginal ultrasound scan. A gonadotropin-releasing hormone (GnRH) antagonist (Orgalutron) was used from day 6 of the cycle to block ovulation. Women were then administered human chorionic gonadotropin (hCG, Ovidrel 250 mg), and transvaginal follicular aspiration was performed 36 hours after hCG administration. Blood-free follicular aspirates were centrifuged at 4,000 rpm for 10 minutes, and the follicular fluid was removed and stored at 80 C. Levels of follicular fluid triglyceride and free fatty acids were measured using the Roche Hitachi 912 Chemistry Analyzer as per the manufacturer's instructions, and using quality VOL. 97 NO. 6 / JUNE 2012
controls QCS 1 and 2 (Bio-Rad). The mean coefficient of variation for each assay was less than 4.6%.
Isolation and In Vitro Maturation of Mouse Cumulus-Oocyte Complexes Experiments were approved by the University of Adelaide Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Immature COCs were isolated from 23-day-old mice (CBAXC57Bl/6F1) by puncturing the antral follicles of ovaries collected 44 hours after IP injection of 5 IU equine chorionic gonadotropin (eCG, Gestyl; Professional Compounding Centre of Australia). Mature COCs were dissected from oviducts of mice 44 hours after eCG administration and 13 hours after IP administration of 5 IU hCG (Pregnyl; Organon). The COCs were collected in HEPES-buffered a-minimal essential medium (MEM; GIBCO, Invitrogen Australia Pty. Ltd.) and cultured in groups of 20 at 37 C in 6% CO2/94% air in 100 mL drops of bicarbonatebuffered a-MEM, supplemented with 50% (v/v) human follicle fluid and overlaid with sterile paraffin oil (Merck). Maturation was stimulated by treatment with 50 mIU/mL of recombinant human FSH and 10 ng/mL of epidermal growth factor (EGF). Cumulus expansion according to the 0–4 scale (16) and the presence or absence of germinal vesicle breakdown (GVBD) and first polar body extrusion were evaluated after 16 hours of culture.
Lipid Droplet Staining Lipophilic dye BODIPY 493/503 (Invitrogen), which stains intracellular neutral lipids, was used to localize the lipid droplets in oocytes, as described previously described elsewhere (17). Briefly, COCs were paraformaldehyde-fixed, stained in BODIPY 493/503, and imaged using a Leica SP5 spectral scanning confocal microscope, with identical conditions throughout all experiments. Fluorescence in the oocytes was calculated by Analysis Pro Software (Olympus Australia Pty. Ltd.).
RNA Isolation and Real-Time RT-PCR Total RNA was isolated from COCs (R35 COCs per RNA sample) using RNeasy Micro Kit (Qiagen Pty Ltd.), with 600 ng reverse transcribed using random primers (Roche) and Superscript III Reverse Transcriptase (Invitrogen). Quantitect Primer Assays (Qiagen) Plin2, Atf4, Atf6, Grp78, and ribosomal protein L19 (internal control) were used. The PCR analysis was performed in triplicate using the Rotor-Gene 6000 (Corbett Research) analyzer with SYBR Green PCR Master Mix (Applied Biosystems). Data were analyzed using the 2DDCT method, for quantification relative to a calibrator sample, and were expressed as the relative fold change.
Statistical Analysis Statistical analysis was performed using Graph Pad Prism version 5.01 (GraphPad Software Inc.). Statistically significant correlations between BMI and follicular fluid metabolites (n ¼ 64 to 74, as indicated) were detected by Spearman (two-tailed) 1439
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY correlation. Comparisons between the selected follicular fluid samples (n ¼ 8 lipid rich and n ¼ 8 lipid poor) are reported as mean standard error of the mean (SEM) of each group and were analyzed by unpaired two-tailed t-test as indicated. In vitro maturation experiments were conducted in a parallel treatment design whereby groups of COCs were subjected to either lipid-rich or lipid-poor maturation treatment and were compared with ovulated (in vivo) COCs. This experimental design was repeated three times with biologically independent maturation treatments. Outcome parameters of oocyte lipid content, COC gene expression, cumulus expansion rates, and oocyte maturation rates are reported as mean SEM of each measure from three independent experiments and were analyzed by one-way analysis of variance (ANOVA) with the Newman-Keuls multiple comparison test as indicated. P< .05 was considered statistically significant.
RESULTS Increasing BMI in women (n ¼ 64) was positively correlated with increasing levels of triglyceride (r ¼ 0.44; P¼ .0003) and free fatty acids (r ¼ 0.25; P¼ .04) in ovarian follicle fluid. To examine how the increased lipid content of follicle fluid of obese women may impact oocyte maturation, the most lipid-rich and lipid-poor follicular fluid samples were sought. The levels of triglyceride and free fatty acids in follicular fluid were positively correlated (P< .001), and the samples that were relatively high or relatively low in these two measures were identified (Fig. 1; squares and triangles, respectively). The lipid-rich samples (n ¼ 8) were from obese women (BMI 32.6 1.7), and, as expected, they had statistically significantly elevated levels of free fatty acids (0.38 0.04 meq/L) and triglycerides (0.24 0.015 mmol/L) compared with the lipid-poor samples (n ¼ 8); the latter were from nonobese women (BMI 24.8 1.0) and contained statistically significantly lower levels of lipids (free fatty acids: 0.16 0.02 meq/L; P¼ .0001; triglycerides: 0.09 0.004 mmol/L, P< .0001 by t-test compared with lipid-rich samples). Equal volumes of two or three patient samples of each follicular fluid type (lipid rich or lipid poor) were pooled as depicted in Figure 1 to generate three entirely distinct paired pools of follicular fluid. Each pair of lipidrich and lipid-poor samples (i.e., same colors in Fig. 1) were then used in independent experiments for in vitro maturation of mouse COCs and for comparison with ovulated COCs collected in parallel to each in vitro experiment. Immature COCs, isolated from preovulatory follicles of mice treated with eCG, were stimulated to mature for 16 hours in either lipid-rich follicular fluid (high-lipid FF) or lipid-poor follicular fluid (low-lipid FF) and were compared with those matured in vivo (i.e., isolated from oviducts after ovulation). The COCs were stained with neutral lipid dye, and the lipid content within the oocytes was determined. Oocytes matured in lipid-rich follicular fluid contained statistically significantly more neutral lipid than the oocytes matured in lipidpoor follicular fluid or those matured in vivo (Fig. 2A). Expression of perilipin-2 (Plin2/ADRP), a lipid droplet protein expressed in the COC that coats lipid droplets in mouse oocytes, was also statistically significantly higher in COCs matured in lipid-rich follicular fluid than in COCs matured in lipid-poor follicular fluid or in vivo (see Fig. 2B). Expression 1440
FIGURE 1
Identification of lipid-rich and lipid-poor follicular fluids. Levels of triglyceride and free fatty acids in follicular fluid are statistically significantly correlated (P<.001 by Spearman correlation). Samples with the highest levels of these lipids (squares; n ¼ 8) and the lowest levels (triangles; n ¼ 8) were identified, and equal volumes from two or three women (those with identical symbols) were pooled. Each color indicates samples that were used in independent experiments (n ¼ 3) for the in vitro maturation of mouse cumulus-oocyte complexes. Yang. Lipid-rich follicle fluid impacts oocytes. Fertil Steril 2012.
of endoplasmic reticulum stress markers Atf4, Atf6, and Grp78 were also statistically significantly increased in COCs matured in lipid-rich follicular fluid compared with the COCs matured in lipid-poor follicular fluid (see Fig. 2C–2E), similar to the COCs from mice with diet-induced obesity (10). Maturation of the COCs was assessed by measuring the cumulus expansion score, oocyte germinal vesicle breakdown (GVBD), and oocyte polar body extrusion. Cumulus expansion scores were not statistically significantly different between the treatment groups (in vivo: 3.33 0.16; low-lipid follicular fluid: 3.67 0.11; high-lipid follicular fluid: 3.50 1.11; n R 17 COCs per group). The GVBD rates were R94% in all experiments and were not affected by culture with either of the follicular fluids compared with the in vivo matured COCs. However, maturation in lipid-rich follicular fluid dramatically decreased oocyte maturation to metaphase II (MII), assessed as polar body formation, to approximately 25% of in vivo rates (see Fig. 2F).
DISCUSSION These results show that the hyperlipidemia associated with obesity in women extends directly into the ovarian follicular microenvironment, with increasing BMI associated with markedly increased triglyceride and free fatty acid levels in follicle fluid. Maturation of COCs in this lipid-rich environment promoted lipid accumulation and increased the lipid droplet protein mRNA expression in mouse COCs exposed to the environment in vitro. This disrupted physiologic maturation environment was detrimental to oocytes, leading to the induction of multiple endoplasmic reticulum stress markers and impaired maturation. These results provide insight into cellular alterations that might mechanistically contribute to obesity-associated impairments in oocyte maturation and developmental competence. VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility®
FIGURE 2
Effects of maturation in lipid-rich follicular fluid (FF) on mouse cumulus-oocyte complexes (COCs). (A) Oocyte neutral lipid content is increased in COCs matured in lipid-rich follicular fluid (high-lipid FF) compared with COCs matured in lipid-poor follicular fluid (low-lipid FF). Mean SEM; n ¼ 18–22 COCs per treatment group. Expression of lipid droplet protein (B) perilipin-2 and the endoplasmic reticulum stress markers (C) Atf4, (D) Atf6, and (E) Grp78 is increased in COCs matured in high-lipid FF. Mean SEM; n ¼ 3 independent experiments using different follicular fluid pools and R35 COCs per treatment group. (F) Oocyte maturation to MII is impaired in COCs matured in high-lipid FF. Mean SEM; n ¼ 3 independent experiments using different follicular fluid pools and R17 COCs per treatment group. For each assay, ovulated COCs obtained from the mouse oviduct (in vivo matured) were also included. Different letters indicate significant differences by one-way analysis of variance (ANOVA). A,B,C,E P<.05; D P<.01; F P<.0001. Yang. Lipid-rich follicle fluid impacts oocytes. Fertil Steril 2012.
It was predictable that exposure to lipid-rich follicular fluid led to lipid accumulation in oocytes because maturation in fetal calf serum also leads to increased oocyte neutral lipid content compared with maturation in serum-free conditions (17, 18). The increased expression of lipid droplet protein perilipin-2/ADRP in the COC may further reflect the expansion of lipid stores because perilipin-2/ADRP expression generally correlates with intracellular levels of neutral lipid and is also inducible by exposure to fatty acids (19). Somewhat surprising was the dramatic detrimental effect that lipidrich follicular fluid had on oocyte maturation, which may indicate compatibility issues in this nonphysiologic oocyte maturation system using tissues from different species. However, our demonstration that lipid-rich follicle fluid impairs oocyte maturation is in general agreement with recent reports that fatty acid content of follicle fluid in women is associated with poor COC morphology (20) and that treatment of bovine COCs with increasing doses of specific fatty acids impairs oocyte maturation and subsequent embryo development (21–24). The poor maturation rates we observed in the oocytes matured in lipid-rich follicle fluid precluded their use for in vitro fertilization and for any further developmental assessments that could have provided important additional information about oocyte quality. Our work is the first to demonstrate that the detrimental effects of lipids on oocytes may be mediated via the induction VOL. 97 NO. 6 / JUNE 2012
of endoplasmic reticulum stress pathways. Endoplasmic reticulum stress occurs with obesity (25) and in cells exposed to high levels of free fatty acids (26), activating the unfolded protein response (or UPR). This response to endoplasmic reticulum stress is characterized by the induction of a cohort of genes, including the markers Atf4, Atf6, and Grp78, which counteract cellular stress by slowing protein production and up-regulating protein-folding chaperones (27). If the UPR is unable to resolve endoplasmic reticulum stress, additional lipotoxicity pathways are initiated, potentially culminating in apoptotic cell death. That lipid-rich follicle fluid induces endoplasmic reticulum stress markers in COCs suggests that endoplasmic reticulum stress and lipotoxicity may be occurring in the ovaries of obese women. Supporting this, expression of the endoplasmic reticulum stress marker ATF4 is increased in the granulosa cells of obese women (10). It is interesting that the COCs exposed to follicle fluid that was lipid poor had a lower expression of Grp78 than the ovulated COCs matured in vivo. Unlike endoplasmic reticulum stress markers ATF4 and ATF6, which act as transcription factors, GRP78 has additional roles beyond that of an endoplasmic reticulum stress-induced chaperone protein, and it localizes to the cell surface, mitochondria, or nucleus under different conditions to mediate diverse functions (28). Grp78 is known to be transcriptionally regulated by changes to glucose availability, oxygen tension, 1441
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY and pH; thus, the lower levels in COCs matured in lipid-poor follicle fluid may be a reflection of a fundamental difference in one or more of these parameters under in vitro compared with in vivo maturation conditions. Overall though, each of the three endoplasmic reticulum stress markers examined exhibited a higher expression in the COCs matured in lipid-rich compared with lipid-poor in vitro conditions, indicating that exposure to high levels of lipids in follicle fluid is indeed inducing endoplasmic reticulum stress and potentially lipotoxicity responses. It is likely that the observed changes in gene expression reflect alterations in cumulus cells, as oocytes are transcriptionally inactive at this stage and because cumulus cell mRNA comprises a disproportionate majority of the sample. However, whether endoplasmic reticulum stress responses are occurring in the oocyte, cumulus cells, or both cannot be definitively determined from our current study and remains an important question to resolve. This study focused specifically on the effects of excess triglycerides and free fatty acids because [1] our previous study found that triglyceride was elevated in the follicular fluid of obese women (14), [2] free fatty acids are potent inducers of endoplasmic reticulum stress, which we have found is induced in COCs from obese mice (10) and in COCs treated with high doses of palmitic acid (29), and [3] these lipids are used for beta-oxidation, which is important for oocyte developmental competence (30). It should be noted however that these experiments using human follicular fluid cannot determine whether the detrimental effects observed are due to high triglyceride and free fatty acid content per se or due to other potential mediators such as cholesterol esters and phospholipids, which are even more abundant in follicular fluid. Indeed, in our patient cohort the level of cholesterol in follicular fluid was also tightly linked with triglyceride levels (r ¼ 0.54; P< .0001) and free fatty acid levels (r ¼ 0.62; P< .0001 by Spearman correlation), and thus is an additional potential contributor to the observed effects on oocyte neutral lipid content and nuclear maturation. Similarly, insulin levels in follicular fluid also tended to be related to triglycerides (r ¼ 0.27; P¼ .02) and free fatty acids (r ¼ 0.30; P¼ .01). Further, there may be other nonlipid confounders such as protein levels, which can degrade into urea and ammonia in vitro and thereby indirectly cause detrimental effects on oocyte quality (31). It is likely that additional factors also associate with high lipid content in follicle fluid and contribute to the dramatic effects on oocyte maturation; for instance, inflammatory mediators and oxidative stress are elevated in the follicle fluid of obese women (14, 15). Thus, the mechanisms linking lipids, endoplasmic reticulum stress, and oocyte quality still need to be clarified before we can determine the fundamental causes of poor fertility reported in obese women and elucidate the contextual signals that influence oocyte developmental competence precisely at the time of conception. Our study shows that lipid levels in follicular fluid vary markedly between women and that differing levels of these lipids, and presumably many additional cofactors, in follicular fluid can exert dramatically different effects. The impact of different follicular maturation environments on human oocytes remains to be directly determined; however, the use 1442
of mouse oocytes indicates that specific follicular fluid environments can acutely increase oocyte lipid content, induce endoplasmic reticulum stress in COCs, and profoundly impact oocyte maturation. Acknowledgments: The authors thank Brenton D. Bennett for assistance, and the patients and staff of Repromed for their participation.
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