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Mechanism of palmitic acid-induced deterioration of in vitro development of porcine oocytes and granulosa cells
Theriogenology 141 (2020) 54e61
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Mechanism of palmitic acid-induced deterioration of in vitro development of porcine oocytes and granulosa cells H. Shibahara, A. Ishiguro, Y. Inoue, S. Koumei, T. Kuwayama, H. Iwata* Department of Animal Science, Tokyo University of Agriculture, Funako 1737, Atsugi, Kanagawa, 243-0034, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 April 2019 Received in revised form 19 August 2019 Accepted 5 September 2019 Available online 6 September 2019
The concentration of fatty acids in follicular fluid reflect the physical condition of donors, and palmitic acid (PA) is a major component of follicular fluid. The present study examined the effect of PA on in vitro oocyte growth and investigated the molecular backgrounds of the PA induced-low quality oocytes. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of gilts. The OGCs were cultured for 14 days in a medium containing 0.5 mM PA or vehicle (BSA). PA was found to reduce granulosa cell (GCs) proliferation (0.73 fold) and viability (93.9% vs. 85.8%) and increase lipid content in oocytes and GCs. Oocytes developed in the presence of PA had low developmental ability to the blastocyst stage. In addition, PA affected developmental and epigenetic markers of histone modifications in oocytes; levels of H4K12 acetylation and H3K9 demethylation. PA affected cellular proliferation, apoptosis and endoplasmic reticulum stress markers along with reducing the phosphor-AKT/AKT levels and increasing the expression levels of caspase-3 and CHOP in GCs. Incubation of OGCs with PA increased ceramide content in the GC, and addition of ceramide to the culture medium inhibited GC proliferation. In conclusion, it is suggested that high PA content in the medium reduces viability and proliferation through ceramide accumulation, and PA impaires the developmental ability of oocytes grown in vitro. In addition, high-fat conditions induce changes in the histone modifications of oocytes grown in vitro. © 2019 Published by Elsevier Inc.
Keywords: Early antral follicles Granulosa cells Oocyte growth Palmitic acid
1. Introduction In large animals, oocytes take months to acquire developmental competence, and the quality of the oocytes is profoundly affected by the physiological conditions of donors. Fatty acids in follicular fluid (FF) are important components for oocyte growth [1,2], and physiological conditions, such as obesity in humans and milking in cows profoundly affect the non-esterified fatty acid (NEFA) levels in the follicular fluid (FF), which is associated with low oocyte quality [2e6]. Palmitic acid is a major fatty acid in FFs [1,2] and high concentrations of palmitic and stearic acid in the FF are associated with adverse pregnancy outcomes in humans [7,8]. Consistent with this, fatty acid supplementation during the in vitro oocyte maturation reduced their quality in cows and mice [9e11], and fatty acid in culture milieu induced endoplasmic reticulum stress in bovine cumulus cells [12]. Oocyte growth was reported to be accompanied by increased histone acetylation [13], and high NEFA concentration in oocyte maturation medium were found to affect epigenetic
* Corresponding author. E-mail address: [email protected] (H. Iwata). https://doi.org/10.1016/j.theriogenology.2019.09.006 0093-691X/© 2019 Published by Elsevier Inc.
modification of the oocytes [14]. Furthermore, the oocytes of mice on high fat diet mice had higher histone H3K9 methylation levels [15]. Studies conducted using an in vitro oocyte growth system are beneficial for evaluating the effect of NEFAs on oocyte growth because of the isolation of the oocytes from circulation and the complex interactions among the follicles. To the best of our knowledge, the effect of NEFAs on in vitro oocyte growth has rarely been addressed. In particular, the effects have been evaluated only in mouse oocytes [11], in which medium containing high levels of stearic acid or NEFA showed impaired development of the oocytes, and the high fatty acid culture conditions affected steroid secretion and the gene expression profiles of the granulosa cells. We have found that major fatty acid in porcine FF is palmitic acid (PA) followed by stearic acid and oleic acid [1]. In addition, palmitic acid was reported to induce ceramide accumulation in oocytes, which is a main causal factor associated with the detrimental effect of fatty acids on oocytes including mitochondrial dysfunction [16]. In the present study, oocyte and granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs) of porcine ovaries and cultured for 14 days, followed by maturation and
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parthenogenetic activation. Herein, we examined whether culturing OGCs under high PA conditions affected in vitro oocyte growth, subsequent developmental ability, mitochondrial functions, and histone modifications of the oocytes grown in vitro. In addition, we investigated whether high PA conditions affected granulosa cell proliferation and expression levels of proteins associated with cellular apoptosis, ER stress, and low proliferation, and whether PA induced ceramide accumulation reduced cell viability and proliferation of the granulosa cells. 2. Materials & Methods 2.1. Chemicals and medials All reagents were purchased from Nacalai Tesque (Kyoto, Japan), unless otherwise stated. To culture OGCs, we used a-MEM (SigmaAldrich) supplemented with 10 mM taurine, 0.1 mAU/ml folliclestimulating hormone (Kawasaki Mitaka, Tokyo, Japan), 2% polyvinylpyrrolidone-360 (Sigma-Aldrich), 2 mM hypoxanthine (Sigma-Aldrich), 1% insulin transferrin selenium (Gibco BRL, Paisley, UK), 1 mg/ml 17b-estradiol, 3 mg/ml BSA, and antibiotics. Medium 199 (Gibco) supplemented with 10% porcine follicular fluid (pFF), 0.5 mM L-cysteine, 0.9 mM sodium pyruvate, 1 mM Lglutamine, 10 ng/ml epidermal growth factor, 5% fetal calf serum, 10 IU/ml equine chorionic gonadotropin (ASKA Pharma Co. Ltd, Tokyo, Japan), and 10 IU/ml human chorionic gonadotropin (Fuji Pharma Co. Ltd, Tokyo, Japan) was used as the in vitro maturation (IVM) medium. The in vitro cultures of embryos and oocytes activation were conducted in porcine zygote medium 3 (PZM3) [17]. Palmitic acid was purchased from Wako (Osaka, Japan). 2.2. Collection of OGCs from early antral follicles (EAFs) Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory (at approximately 35 C, in PBS containing antibiotics) within 1 h. Ovarian cortical tissues were excised from the ovarian surface under a stereomicroscope, and OGCs were collected from EAFs (0.5e0.7 mm in diameter). OGCs containing oocytes with diameters ranging from 90 to 100 mm were then selected under a digital microscope (BZ-8000; Keyence, Tokyo, Japan). 2.3. Preparation of palmitic acid Considering that fatty acids bind to albumin in vivo [18], BSA was used as the vehicle. Preparation of the PA solution was conducted according to a previously described protocol [16]. PA was dissolved in ethanol at a concentration of 100 mM, and further diluted with phosphate-buffer saline (PBS) containing 10% BSA to obtain a 10 mM PA-BSA solution. Ethanol-BSA was added to the experimental medium to adjust the vehicle concentration. 2.4. IVG, IVM, activation, and in vitro culture (IVC) OGCs collected from EAFs were individually transferred to a well containing 200 ml of medium (96-well plates, Becton Dickinson) and cultured for 14 days. The bottoms of the culture wells were coated with polyacrylamide gel (PAG) sheets, and OGCs were cultured on the gel. Half of the medium was replaced with fresh medium, and antrum formation and the diameter of OGCs were examined in 4-day intervals. After IVG (14 days), OGCs having an antrum cavity were subjected to IVM for 48 h. In addition, oocyte cumulus cell complexes (COCs) were collected from antral follicles (3e5 mm in diameter) and subjected to IVM. After IVM, oocytes were denuded from surrounding
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granulosa cells (GCs), and parthenogenetically activated in IVC medium containing 10 mg/ml ionomycine for 5 min and incubated for 5 h in PZM3 containing 10 mg/ml cytochalasin B and 10 mg/ml cycloheximide at 38.5 C. After activation, embryos were cultured for 8 days in culture medium, and the rate of blastulation and total blastocyst cell number were determined. Blastocysts were fixed in 4% paraformaldehyde and mounted onto microscope slides using an antifade reagent containing DAPI (ProLong Gold antifade reagent with DAPI; Invitrogen, OR, USA). The total number of cells in the blastocysts was counted using a fluorescence digital microscope (Keyence). In vitro culture of OGCs and oocyte maturation were performed at 38.5 C in an atmospheric condition of 5% CO2 and 95% air, whereas in vitro embryo culture was performed at 38.5 C in an atmospheric condition of 5% O2, 5% CO2, and 90% N2. 2.5. Measurement of survivability and number of the granulosa cells surrounding oocytes grown in vitro After IVG, granulosa cells were enzymatically dispersed (Accumax; Innovative Cell Technologies, San Diego, CA, USA), and GCs were stained with trypan blue. The total number and survival rate were calculated using hematocytometer. 2.6. Measurement of oocyte diameter The diameters of the ooplasms were measured (X and Y axis with a 90 angle) under a digital microscope (Keyence). The averages of the X and Y axis values were calculated as the diameter of the oocytes. 2.7. ATP measurement Oocytes denuded from OGCs were lysed in 50 ml of distilled water and stored at 20 C until analysis. The ATP contents of individual oocytes was determined by measuring luminescence using the ATP assay kit (Toyo-Inc., Tokyo, JAPAN). Luminescence was measured immediately using a Spark multimode microplate reader (Tecan Japan, Kanagawa, Japan). Serial dilutions of the ATP standard ranging from 100 nM to 0.78 nM were prepared, and a standard curve was generated based on the relative light intensities of the serial dilution standards. Sample measurements were performed in duplicate, and the average value was calculated. 2.8. Lipid contents in oocyte Oocytes denuded from OGCs were fixed in 4% paraformaldehyde and subsequently stained with 10 mg/ml Nile Red (Wako, Osaka, Japan) for 10 min. Oocytes were mounted on microscope slides with Pro-Long gold antifade reagent with DAPI (Invitrogen, OR, USA). Fluorescence images of lipid were captured using fluorescence digital microscope (Keyence, Tokyo, Japan), and fluorescence intensities were measured using ImageJ software (NIH, Bethesda, MD, USA). 2.9. Measurement of lipid content in granulosa cells After IVG, five randomly selected OGCs were mixed, and GCs were removed from the OGCs. GCs were enzymatically dispersed and divided into two cellular suspensions. The first suspension was used to count the granulosa cell number in each sample as described above, while the second suspension was used for lipid content measurement using the Lipid Assay Kit (Cosmo Bio AK09F, Tokyo, Japan) following the manufacture's protocols. The values of lipid contents were normalized by the GC number in samples.
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2.10. Measurement of acetylated H4K12 levels in oocytes and ceramide contents in GCs To measure H4K12 acetylation, oocytes were collected from OGCs at the end of the culture period (14 days). Oocytes were fixed in 4% paraformaldehyde, incubated with primary antibody (rabbit polyclonal anti-H4K12, 1:200; Novus International Saint Charles, MO, USA), and incubated with secondary antibody (goat anti-rabbit IgG FITC-conjugate, 1:500; Cell Signaling Technology Inc., Danvers, MA, USA). In addition, to detect ceramide levels in granulosa cells, OGCs were fixed in 4% paraformaldehyde, incubated with the primary antibody (mouse monoclonal Ceramide antibody, 1: 200; Enzo Life Science, Farmingdale, NY, USA) over night, and incubated with the secondary antibody (anti-mouse IgG Alexa Fluor 555, 1:500; Cell Signaling Technology Inc., Danvers, MA, USA) for 1 h. Oocytes and OGCs were mounted on slides with an anti-fade reagent containing DAPI (Invitrogen). The image of oocytes and OGCS were captured under a fluorescence microscope (Keyence Tokyo Japan). Fluorescence intensities of whole oocytes were quantified using ImageJ software (NIH, Bethesda, MD, USA). The fluorescence intensities of four randomly selected region (excluded the oocytes) were quantified using ImageJ software (NIH, Bethesda, MD, USA). The average fluorescence intensity was determined for each OGCs.
Roche) containing protease inhibitors (Complete protease inhibitor cocktail; 1 tablet/10 ml, Roche) and phosphatase inhibitors (PhosSTOP; 1 tablet/10 ml, Roche) for 10 min on ice. Cell lysates were sonicated (TOMY sonicator UD-100; TOMY, Tokyo, Japan) and centrifuged at 15,000 rpm for 20 min to obtain supernatants. The supernatant containing 10 mg of protein was mixed with Laemmli sample buffer containing 2-mercaptoethanol and subsequently boiled at 95 C for 5 min. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Trans-Blot Turbo Mini Transfer Packs; Bio-Rad) using the Trans-Blot Turbo Transfer System (BioRad). Membranes were incubated with each primary antibody (rabbit anti AKT, 1:1000 CST, Cell Signaling Technology, Danvers, MA, USA), phosphate-AKT (1:1000, CST), mouse anti CHOP (1:1000, CST) rabbit anti active caspase-3 (1:1000, Merck Millipore, Billerica, MA, USA) and anti b-actin (1:1000, CST) overnight and then incubated with the secondary antibodies (anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase diluted with Signal Booster B, 1:10000; Beacle, Inc, Kyoto, Japan) for 1 h. Specific bands were detected using Western BLoT Quant HRP Substrate (GE Healthcare, Little Chalfont, UK) and digitized using the ImageQuant LAS 4000 Biomolecular imager and ImageQuant software (GE Healthcare). The expression level of each protein was normalized to the b-actin expression level. 2.12. Data analysis
2.11. Western blotting analysis After IVG, oocytes were removed from OGCs, and granulosa cell mass was incubated in 100 ml of cell lysis buffer (Complete Lysis-M;
All data were analyzed using ANOVA, followed by post-hoc Tukey's test. Percentages were arcsine-transformed before analysis. P < 0.05 was considered significant.
Fig. 1. Effect of PA supplementation on in vitro oocyte growth. A. Representative image of OGCs forming the antrum cavity. B. Rate of antrum formation. C. Diameter of OGCs during the in vitro culture period. D. Number of granulosa cells surrounding the oocytes. E. Survivability of the granulosa cells as determined by trypan blue staining. F. Rate of development to the blastocyst stage of oocytes grown in vitro. All data are presented as mean ± SEM, * significant difference between the control and PA groups (P < 0.05).
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3. Results 3.1. High PA condition impaired the development of OGCs and the developmental ability of the oocytes grown in vitro OGCs cultured in vitro formed antrum like cavities (Fig. 1A and B) and gradually increased in size. As shown in Fig. 1C, a high PA concentration (0.5 mM) reduced the size of OGCs (685.2 ± 22.7 vs. 609.0 ± 13.6 mm, p < 0.05) and number and survivability of the granulosa cells (Fig. 1D and E, BSA vs. PA, 170,000 ± 6530 vs. 123,811 ± 5,473, and 92.6 ± 1.2% vs. 85.0 ± 1.58%, p < 0.05). However, oocyte diameter was comparable among the experimental groups (Vehicle, 108.4 ± 1.2 mm, N. 51 vs. PA, 108.6 ± 1.3 mm, N. 52). When oocytes grown in vitro were subjected to activation following IVM, the rate of development to the blastocyst stage was 1.5% for PA-BSA-oocytes, which was significantly lower than that observed in the control oocytes (Fig. 1F, BSA vs. PA, 9.3 ± 3.6% vs. 1.5 ± 1.5%, p < 0.05). 3.2. PA affects the characteristics of oocytes grown in vitro Supplementation of culture medium with a high PA concentration (0.5 mM) increased the lipid contents in oocytes (Fig. 2A-B,
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1.23-fold, p < 0.01). Oocytes cultured in PA condition had higher ATP contents compared to those cultured without PA (Fig, 2C, BSA vs. PA, 2.1 ± 0.2 vs. 2.9 ± 0.2 pM, p < 0.01). The SN rates reflect the extent of oocyte growth [19]. Our results revealed that the rate of SN was 44.9 ± 8.7 (%) for PA-BSA-oocytes and 59.6 ± 4.5 for control oocytes, and difference was not significant (P ¼ 0.19) (Fig. 2D and E). In the present study, supplementation of the culture medium with 0.5 mM PA reduced H4K12 acetylation levels and increased H3K9 dimethylation levels in oocyte grown in vitro compared to those grown in BSA containing medium (Fig. 3AeD, BSA vs PA; H4K12Ace, 1.00 ± 0.07 vs. 0.62 ± 0.06, and H3K9me2, 1.00 ± 0.02 vs. 1.14 ± 0.03fold, respectively, p < 0.01). 3.3. PA affects protein expression associated with cellular proliferation, apoptosis, and endoplasmic reticulum stress Previous studies showed that supplementation of the culture medium with PA decreased granulosa cell numbers and reduced cell viability. Therefore, we examined the expression levels of proteins related to cellular proliferation, caspase, and endoplasmic reticulum stress. Cells with PA supplementation showed decreased phospho-AKT/AKT levels (0.56 ± 0.04-fold, p < 0.01) and upregulated expression levels of activated-caspase3 and CHOP compared
Fig. 2. Effect of PA supplementation on the quality of oocytes grown in vitro. A-B. Relative lipid content in oocytes and representative images of oocytes stained with Nile red. Lipid content of oocytes developed without PA was defined as 1.0. C. ATP content in oocytes. D-E. Proportion of oocytes with differentially condensed chromatins (SN, surrounded nucleolus, and NSN non-surrounded nucleolus) and representative images of oocytes determined as SN or NSN. Scale bar indicates 100 mm.
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Fig. 3. Effect of PA supplementation on the histone modifications in oocytes. A-B. Relative expression levels of acetylated H4K12. Levels of acetylated H4K12 for oocytes developed without PA was defined as 1.0. Representative images of oocytes stained against Ace-H4K12. C-D. Relative expression levels of dimethyl H3K9. Levels of dimethyl H3K9 in oocytes developed without PA was defined as 1.0. Representative images of oocytes stained against dimethyl-H3K9. All data are presented as mean ± SEM, * indicates a significant difference between the control (BSA) and PA groups (P < 0.05). Scale bar indicates 100 mm.
Fig. 4. Effect of PA on protein expression levels in granulosa cells. A. Relative phospho-AKT/AKT levels and representative images of bands. B. Relative expression levels of activated Caspase-3 and representative image of bands. C. Relative expression levels of activated CHOP and representative image of bands. Expression values in granulosa cells developed without PA were defined as 1.0. * significant difference between the control and PA groups (P < 0.05).
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to cells cultured without PA (BSA) (1.60 ± 0.27-fold and 17.8 ± 0.31fold, respectively, p < 0.05, Fig. 4).
3.4. PA-induced ceramide accumulation reduced proliferation of granulosa cells At the end of the culture period, OGCs cultured with high PA levels showed lower numbers of granulosa cells and high lipid contents (Fig. 1). Therefore, we confirmed low granulosa cell numbers and high lipid content (Fig. 5-A and B, BSA vs. PA, cell number; 60,625 ± 4408 vs. 47,741 ± 4,058, lipid content 1.00 ± 0.13vs. 1.58 ± 0.10, p < 0.01) in oocytes at day 4 of the culture period (Fig. 4-A and B). In addition, OGCs cultured with high PA had high ceramide contents (BSA vs. PA, 1.00 ± 0.05 vs.1.24 ± 0.06, p < 0.05). Furthermore, supplementation of the culture medium with 0, 30, and 50 mM ceramide significantly inhibited granulosa cell proliferation (Fig. 5-E, 0, 30 and 50 mM ceramide, 191,750 ± 13,435, 166,888 ± 9,588, and 151,375 ± 10,864, respectively).
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4. Discussion The present study showed that high PA levels induced lipid accumulation in oocytes and granulosa cells. In high PA conditions, granulosa cell viability and proliferation were reduced and accompanied by greater endoplasmic reticulum stress and apoptosis, and the PA-induced inhibition of cell proliferation was caused by ceramide accumulation. In addition, PA supplementation reduced oocyte quality, causing impaired developmental ability to the blastocyst stage and abnormal histone modifications. Results revealed that oocytes and granulosa cells cultured under high-fat conditions had high lipid contents, and the oocytes showed impaired developmental ability. Lipid content is influenced by physiological conditions, such that oocytes and cumulus cells of mice fed with a high-fat diet contained high levels of lipids [20,21]. Obese mice contained high fatty acid concentrations in circulation [22], and women with high BMIs have high fatty acid concentrations in follicular fluid [4]. Ogawa reported that porcine oocytes and cumulus cells cultured with BSA or FF containing various concentration of NEFAs, FF increased lipid content in oocytes and cumulus
Fig. 5. Effect of PA and ceramide on granulosa cells of OGCs. OGCs were cultured with PA or ceramide for 4 days. A-B. Effect of PA on the number of granulosa cells and lipid content of the granulosa cells. Lipid contents in granulosa cells developed without PA were defined as 1.0. C-D. Relative ceramide content in granulosa cells and representative images of OGCs stained against ceramide. Ceramide content in granulosa cells developed without PA were defined as 1.0. E. Number of granulosa cells of OGCs cultured with 0, 30, and 50 mM for 14 days. * indicates a significant difference between the control (0 mM) and ceramide 50 mM groups (P < 0.05). Scale bar indicates 100 mm.
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cells compared to BSA-counterparts. This increase was found to be dependent on the amount of NEFA added to the culture condition [1]. In addition, supplementation of the maturation medium with PA increased the lipid content in bovine and porcine oocytes and reduced their developmental competence [2,16]. Therefore, high lipid conditions lead to increased lipid accumulation in oocytes and cumulus cells which reduces oocyte competence. Our present study showed that oocytes developed in a high-fat environment have a high ATP content. The ATP content in oocytes is necessary for nuclear maturation [23] and is a marker for their fertilization outcome [24]. By contrast, we previously demonstrated that culturing oocytes with high NEFA concentrations induced lipid accumulation and increased ATP contents but impaired developmental ability [1]. Therefore, it is hypothesized that overaccumulation of lipids in oocytes induces abnormal ATP generation. However, the corresponding molecular mechanism remained to be elucidated. Notably, oocytes developed with high PA concentrations had low H4K12 acetylation levels and high H3K9 demethylation levels. H4K12 Acetylation is high at GV stage oocytes [25] and well reflects oocyte quality [26]. In vitro-grown oocytes surrounded by a greater number of granulosa cells had high H4K12 acetylation levels and good developmental ability [27,28]. The above results indicate that the number of granulosa cells surrounding the oocytes was lower under high PA conditions. Therefore, we hypothesized that low cell viability and a small number of granulosa cells lead to reduced H4K12 acetylation levels. Interestingly, Hou et al. reported that the oocytes of mice on a high fat diet had high levels of demethylated H3K9 [15]. Consistent with the above findings, one study reported that oocytes and embryos cultured under high NEFA concentrations presented epigenetic changes [14]. The results are consistent with those of previous studies, which suggested that high PA concentrations during oocyte growth could lead to epigenetic changes in oocytes. The number of granulosa cells is crucial for follicle development and determines the developmental competence of oocytes [29]. Our findings showed that PA suppressed granulosa cell proliferation and reduced cell viability. Supplementation of the maturation medium of bovine oocytes with high levels of NEFA induced endoplasmic reticulum stress [12]. Consistent with these findings, the granulosa cells of OGCs cultured with high PA showed a low PAKT/AKT ratio and high expression levels of CHOP and Caspase3, which suggests that high PA conditions induce endoplasmic reticulum stress, apoptosis, and low proliferation activity. Pereira et al. reported that high PA concentrations induced ceramide accumulation via serine-palmitoyl transferase [30]. PA supplementation of the oocyte maturation medium induced both lipid and ceramide accumulation and aggravated oocyte quality [16]. In addition, ceramide caused abnormal mitochondrial function in oocytes. Our findings revealed for the first time that PA supplementation of the culture medium induced ceramide accumulation in granulosa cells, and the addition of ceramide reduced granulosa cell proliferation. Based on the above findings, we can conclude that high PA conditions induced lipid accumulation and consequently ceramide accumulation in granulosa cells, which in turn impair granulosa proliferation and proper oocyte growth. In conclusion, high PA conditions lead to lipid and ceramide accumulation in granulosa cells and reduce the viability of granulosa cells. In addition, oocytes grown under high PA conditions have low developmental ability and differential histone modifications. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research C (KAKENHI, grant number: 16K07996).
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oocytes: a study using the mouse ovary-holding stress model. Sci Rep 2016;6. https://doi.org/10.1038/srep28347. 28347. Munakata Y, Ichinose T, Ogawa K, Itami N, Tasaki H, Shirasuna K, et al. Relationship between the number of cells surrounding oocytes and energy states of oocytes. Theriogenology 2016;86:1789e98. https://doi.org/10.1016/ j.theriogenology.2016.05.036. Sugiyama M, Sumiya M, Shirasuna K, Kuwayama T, Iwata H. Addition of granulosa cell mass to the culture medium of oocytes derived from early antral follicles increases oocyte growth, ATP content, and acetylation of H4K12. Zygote 2016;24:848e56. https://doi.org/10.1017/ S0967199416000198. Munakata Y, Ueda M, Kawahara-Miki R, Kansaku K, Itami N, Shirasuna K, et al. Follicular factors determining granulosa cell number and developmental competence of porcine oocytes. J Assist Reprod Genet 2018;35:1809e19. https://doi.org/10.1007/s10815-018-1247-9. Pereira DM, Correia-da-Silva G, Valent~ ao P, Teixeira N1, Andrade PB. Palmitic acid and ergosta-7,22-dien-3-ol contribute to the apoptotic effect and cell cycle arrest of an extract from Marthasterias glacialis L. in neuroblastoma cells. Mar Drugs 2013;12:54e68. https://doi.org/10.3390/md12010054.
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Mechanism of palmitic acid-induced deterioration of in vitro development of porcine oocytes and granulosa cells H. Shibahara, A. Ishiguro, Y. Inoue, S. Koumei, T. Kuwayama, H. Iwata* Department of Animal Science, Tokyo University of Agriculture, Funako 1737, Atsugi, Kanagawa, 243-0034, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 April 2019 Received in revised form 19 August 2019 Accepted 5 September 2019 Available online 6 September 2019
The concentration of fatty acids in follicular fluid reflect the physical condition of donors, and palmitic acid (PA) is a major component of follicular fluid. The present study examined the effect of PA on in vitro oocyte growth and investigated the molecular backgrounds of the PA induced-low quality oocytes. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of gilts. The OGCs were cultured for 14 days in a medium containing 0.5 mM PA or vehicle (BSA). PA was found to reduce granulosa cell (GCs) proliferation (0.73 fold) and viability (93.9% vs. 85.8%) and increase lipid content in oocytes and GCs. Oocytes developed in the presence of PA had low developmental ability to the blastocyst stage. In addition, PA affected developmental and epigenetic markers of histone modifications in oocytes; levels of H4K12 acetylation and H3K9 demethylation. PA affected cellular proliferation, apoptosis and endoplasmic reticulum stress markers along with reducing the phosphor-AKT/AKT levels and increasing the expression levels of caspase-3 and CHOP in GCs. Incubation of OGCs with PA increased ceramide content in the GC, and addition of ceramide to the culture medium inhibited GC proliferation. In conclusion, it is suggested that high PA content in the medium reduces viability and proliferation through ceramide accumulation, and PA impaires the developmental ability of oocytes grown in vitro. In addition, high-fat conditions induce changes in the histone modifications of oocytes grown in vitro. © 2019 Published by Elsevier Inc.
Keywords: Early antral follicles Granulosa cells Oocyte growth Palmitic acid
1. Introduction In large animals, oocytes take months to acquire developmental competence, and the quality of the oocytes is profoundly affected by the physiological conditions of donors. Fatty acids in follicular fluid (FF) are important components for oocyte growth [1,2], and physiological conditions, such as obesity in humans and milking in cows profoundly affect the non-esterified fatty acid (NEFA) levels in the follicular fluid (FF), which is associated with low oocyte quality [2e6]. Palmitic acid is a major fatty acid in FFs [1,2] and high concentrations of palmitic and stearic acid in the FF are associated with adverse pregnancy outcomes in humans [7,8]. Consistent with this, fatty acid supplementation during the in vitro oocyte maturation reduced their quality in cows and mice [9e11], and fatty acid in culture milieu induced endoplasmic reticulum stress in bovine cumulus cells [12]. Oocyte growth was reported to be accompanied by increased histone acetylation [13], and high NEFA concentration in oocyte maturation medium were found to affect epigenetic
* Corresponding author. E-mail address: [email protected] (H. Iwata). https://doi.org/10.1016/j.theriogenology.2019.09.006 0093-691X/© 2019 Published by Elsevier Inc.
modification of the oocytes [14]. Furthermore, the oocytes of mice on high fat diet mice had higher histone H3K9 methylation levels [15]. Studies conducted using an in vitro oocyte growth system are beneficial for evaluating the effect of NEFAs on oocyte growth because of the isolation of the oocytes from circulation and the complex interactions among the follicles. To the best of our knowledge, the effect of NEFAs on in vitro oocyte growth has rarely been addressed. In particular, the effects have been evaluated only in mouse oocytes [11], in which medium containing high levels of stearic acid or NEFA showed impaired development of the oocytes, and the high fatty acid culture conditions affected steroid secretion and the gene expression profiles of the granulosa cells. We have found that major fatty acid in porcine FF is palmitic acid (PA) followed by stearic acid and oleic acid [1]. In addition, palmitic acid was reported to induce ceramide accumulation in oocytes, which is a main causal factor associated with the detrimental effect of fatty acids on oocytes including mitochondrial dysfunction [16]. In the present study, oocyte and granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs) of porcine ovaries and cultured for 14 days, followed by maturation and
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parthenogenetic activation. Herein, we examined whether culturing OGCs under high PA conditions affected in vitro oocyte growth, subsequent developmental ability, mitochondrial functions, and histone modifications of the oocytes grown in vitro. In addition, we investigated whether high PA conditions affected granulosa cell proliferation and expression levels of proteins associated with cellular apoptosis, ER stress, and low proliferation, and whether PA induced ceramide accumulation reduced cell viability and proliferation of the granulosa cells. 2. Materials & Methods 2.1. Chemicals and medials All reagents were purchased from Nacalai Tesque (Kyoto, Japan), unless otherwise stated. To culture OGCs, we used a-MEM (SigmaAldrich) supplemented with 10 mM taurine, 0.1 mAU/ml folliclestimulating hormone (Kawasaki Mitaka, Tokyo, Japan), 2% polyvinylpyrrolidone-360 (Sigma-Aldrich), 2 mM hypoxanthine (Sigma-Aldrich), 1% insulin transferrin selenium (Gibco BRL, Paisley, UK), 1 mg/ml 17b-estradiol, 3 mg/ml BSA, and antibiotics. Medium 199 (Gibco) supplemented with 10% porcine follicular fluid (pFF), 0.5 mM L-cysteine, 0.9 mM sodium pyruvate, 1 mM Lglutamine, 10 ng/ml epidermal growth factor, 5% fetal calf serum, 10 IU/ml equine chorionic gonadotropin (ASKA Pharma Co. Ltd, Tokyo, Japan), and 10 IU/ml human chorionic gonadotropin (Fuji Pharma Co. Ltd, Tokyo, Japan) was used as the in vitro maturation (IVM) medium. The in vitro cultures of embryos and oocytes activation were conducted in porcine zygote medium 3 (PZM3) [17]. Palmitic acid was purchased from Wako (Osaka, Japan). 2.2. Collection of OGCs from early antral follicles (EAFs) Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory (at approximately 35 C, in PBS containing antibiotics) within 1 h. Ovarian cortical tissues were excised from the ovarian surface under a stereomicroscope, and OGCs were collected from EAFs (0.5e0.7 mm in diameter). OGCs containing oocytes with diameters ranging from 90 to 100 mm were then selected under a digital microscope (BZ-8000; Keyence, Tokyo, Japan). 2.3. Preparation of palmitic acid Considering that fatty acids bind to albumin in vivo [18], BSA was used as the vehicle. Preparation of the PA solution was conducted according to a previously described protocol [16]. PA was dissolved in ethanol at a concentration of 100 mM, and further diluted with phosphate-buffer saline (PBS) containing 10% BSA to obtain a 10 mM PA-BSA solution. Ethanol-BSA was added to the experimental medium to adjust the vehicle concentration. 2.4. IVG, IVM, activation, and in vitro culture (IVC) OGCs collected from EAFs were individually transferred to a well containing 200 ml of medium (96-well plates, Becton Dickinson) and cultured for 14 days. The bottoms of the culture wells were coated with polyacrylamide gel (PAG) sheets, and OGCs were cultured on the gel. Half of the medium was replaced with fresh medium, and antrum formation and the diameter of OGCs were examined in 4-day intervals. After IVG (14 days), OGCs having an antrum cavity were subjected to IVM for 48 h. In addition, oocyte cumulus cell complexes (COCs) were collected from antral follicles (3e5 mm in diameter) and subjected to IVM. After IVM, oocytes were denuded from surrounding
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granulosa cells (GCs), and parthenogenetically activated in IVC medium containing 10 mg/ml ionomycine for 5 min and incubated for 5 h in PZM3 containing 10 mg/ml cytochalasin B and 10 mg/ml cycloheximide at 38.5 C. After activation, embryos were cultured for 8 days in culture medium, and the rate of blastulation and total blastocyst cell number were determined. Blastocysts were fixed in 4% paraformaldehyde and mounted onto microscope slides using an antifade reagent containing DAPI (ProLong Gold antifade reagent with DAPI; Invitrogen, OR, USA). The total number of cells in the blastocysts was counted using a fluorescence digital microscope (Keyence). In vitro culture of OGCs and oocyte maturation were performed at 38.5 C in an atmospheric condition of 5% CO2 and 95% air, whereas in vitro embryo culture was performed at 38.5 C in an atmospheric condition of 5% O2, 5% CO2, and 90% N2. 2.5. Measurement of survivability and number of the granulosa cells surrounding oocytes grown in vitro After IVG, granulosa cells were enzymatically dispersed (Accumax; Innovative Cell Technologies, San Diego, CA, USA), and GCs were stained with trypan blue. The total number and survival rate were calculated using hematocytometer. 2.6. Measurement of oocyte diameter The diameters of the ooplasms were measured (X and Y axis with a 90 angle) under a digital microscope (Keyence). The averages of the X and Y axis values were calculated as the diameter of the oocytes. 2.7. ATP measurement Oocytes denuded from OGCs were lysed in 50 ml of distilled water and stored at 20 C until analysis. The ATP contents of individual oocytes was determined by measuring luminescence using the ATP assay kit (Toyo-Inc., Tokyo, JAPAN). Luminescence was measured immediately using a Spark multimode microplate reader (Tecan Japan, Kanagawa, Japan). Serial dilutions of the ATP standard ranging from 100 nM to 0.78 nM were prepared, and a standard curve was generated based on the relative light intensities of the serial dilution standards. Sample measurements were performed in duplicate, and the average value was calculated. 2.8. Lipid contents in oocyte Oocytes denuded from OGCs were fixed in 4% paraformaldehyde and subsequently stained with 10 mg/ml Nile Red (Wako, Osaka, Japan) for 10 min. Oocytes were mounted on microscope slides with Pro-Long gold antifade reagent with DAPI (Invitrogen, OR, USA). Fluorescence images of lipid were captured using fluorescence digital microscope (Keyence, Tokyo, Japan), and fluorescence intensities were measured using ImageJ software (NIH, Bethesda, MD, USA). 2.9. Measurement of lipid content in granulosa cells After IVG, five randomly selected OGCs were mixed, and GCs were removed from the OGCs. GCs were enzymatically dispersed and divided into two cellular suspensions. The first suspension was used to count the granulosa cell number in each sample as described above, while the second suspension was used for lipid content measurement using the Lipid Assay Kit (Cosmo Bio AK09F, Tokyo, Japan) following the manufacture's protocols. The values of lipid contents were normalized by the GC number in samples.
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2.10. Measurement of acetylated H4K12 levels in oocytes and ceramide contents in GCs To measure H4K12 acetylation, oocytes were collected from OGCs at the end of the culture period (14 days). Oocytes were fixed in 4% paraformaldehyde, incubated with primary antibody (rabbit polyclonal anti-H4K12, 1:200; Novus International Saint Charles, MO, USA), and incubated with secondary antibody (goat anti-rabbit IgG FITC-conjugate, 1:500; Cell Signaling Technology Inc., Danvers, MA, USA). In addition, to detect ceramide levels in granulosa cells, OGCs were fixed in 4% paraformaldehyde, incubated with the primary antibody (mouse monoclonal Ceramide antibody, 1: 200; Enzo Life Science, Farmingdale, NY, USA) over night, and incubated with the secondary antibody (anti-mouse IgG Alexa Fluor 555, 1:500; Cell Signaling Technology Inc., Danvers, MA, USA) for 1 h. Oocytes and OGCs were mounted on slides with an anti-fade reagent containing DAPI (Invitrogen). The image of oocytes and OGCS were captured under a fluorescence microscope (Keyence Tokyo Japan). Fluorescence intensities of whole oocytes were quantified using ImageJ software (NIH, Bethesda, MD, USA). The fluorescence intensities of four randomly selected region (excluded the oocytes) were quantified using ImageJ software (NIH, Bethesda, MD, USA). The average fluorescence intensity was determined for each OGCs.
Roche) containing protease inhibitors (Complete protease inhibitor cocktail; 1 tablet/10 ml, Roche) and phosphatase inhibitors (PhosSTOP; 1 tablet/10 ml, Roche) for 10 min on ice. Cell lysates were sonicated (TOMY sonicator UD-100; TOMY, Tokyo, Japan) and centrifuged at 15,000 rpm for 20 min to obtain supernatants. The supernatant containing 10 mg of protein was mixed with Laemmli sample buffer containing 2-mercaptoethanol and subsequently boiled at 95 C for 5 min. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Trans-Blot Turbo Mini Transfer Packs; Bio-Rad) using the Trans-Blot Turbo Transfer System (BioRad). Membranes were incubated with each primary antibody (rabbit anti AKT, 1:1000 CST, Cell Signaling Technology, Danvers, MA, USA), phosphate-AKT (1:1000, CST), mouse anti CHOP (1:1000, CST) rabbit anti active caspase-3 (1:1000, Merck Millipore, Billerica, MA, USA) and anti b-actin (1:1000, CST) overnight and then incubated with the secondary antibodies (anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase diluted with Signal Booster B, 1:10000; Beacle, Inc, Kyoto, Japan) for 1 h. Specific bands were detected using Western BLoT Quant HRP Substrate (GE Healthcare, Little Chalfont, UK) and digitized using the ImageQuant LAS 4000 Biomolecular imager and ImageQuant software (GE Healthcare). The expression level of each protein was normalized to the b-actin expression level. 2.12. Data analysis
2.11. Western blotting analysis After IVG, oocytes were removed from OGCs, and granulosa cell mass was incubated in 100 ml of cell lysis buffer (Complete Lysis-M;
All data were analyzed using ANOVA, followed by post-hoc Tukey's test. Percentages were arcsine-transformed before analysis. P < 0.05 was considered significant.
Fig. 1. Effect of PA supplementation on in vitro oocyte growth. A. Representative image of OGCs forming the antrum cavity. B. Rate of antrum formation. C. Diameter of OGCs during the in vitro culture period. D. Number of granulosa cells surrounding the oocytes. E. Survivability of the granulosa cells as determined by trypan blue staining. F. Rate of development to the blastocyst stage of oocytes grown in vitro. All data are presented as mean ± SEM, * significant difference between the control and PA groups (P < 0.05).
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3. Results 3.1. High PA condition impaired the development of OGCs and the developmental ability of the oocytes grown in vitro OGCs cultured in vitro formed antrum like cavities (Fig. 1A and B) and gradually increased in size. As shown in Fig. 1C, a high PA concentration (0.5 mM) reduced the size of OGCs (685.2 ± 22.7 vs. 609.0 ± 13.6 mm, p < 0.05) and number and survivability of the granulosa cells (Fig. 1D and E, BSA vs. PA, 170,000 ± 6530 vs. 123,811 ± 5,473, and 92.6 ± 1.2% vs. 85.0 ± 1.58%, p < 0.05). However, oocyte diameter was comparable among the experimental groups (Vehicle, 108.4 ± 1.2 mm, N. 51 vs. PA, 108.6 ± 1.3 mm, N. 52). When oocytes grown in vitro were subjected to activation following IVM, the rate of development to the blastocyst stage was 1.5% for PA-BSA-oocytes, which was significantly lower than that observed in the control oocytes (Fig. 1F, BSA vs. PA, 9.3 ± 3.6% vs. 1.5 ± 1.5%, p < 0.05). 3.2. PA affects the characteristics of oocytes grown in vitro Supplementation of culture medium with a high PA concentration (0.5 mM) increased the lipid contents in oocytes (Fig. 2A-B,
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1.23-fold, p < 0.01). Oocytes cultured in PA condition had higher ATP contents compared to those cultured without PA (Fig, 2C, BSA vs. PA, 2.1 ± 0.2 vs. 2.9 ± 0.2 pM, p < 0.01). The SN rates reflect the extent of oocyte growth [19]. Our results revealed that the rate of SN was 44.9 ± 8.7 (%) for PA-BSA-oocytes and 59.6 ± 4.5 for control oocytes, and difference was not significant (P ¼ 0.19) (Fig. 2D and E). In the present study, supplementation of the culture medium with 0.5 mM PA reduced H4K12 acetylation levels and increased H3K9 dimethylation levels in oocyte grown in vitro compared to those grown in BSA containing medium (Fig. 3AeD, BSA vs PA; H4K12Ace, 1.00 ± 0.07 vs. 0.62 ± 0.06, and H3K9me2, 1.00 ± 0.02 vs. 1.14 ± 0.03fold, respectively, p < 0.01). 3.3. PA affects protein expression associated with cellular proliferation, apoptosis, and endoplasmic reticulum stress Previous studies showed that supplementation of the culture medium with PA decreased granulosa cell numbers and reduced cell viability. Therefore, we examined the expression levels of proteins related to cellular proliferation, caspase, and endoplasmic reticulum stress. Cells with PA supplementation showed decreased phospho-AKT/AKT levels (0.56 ± 0.04-fold, p < 0.01) and upregulated expression levels of activated-caspase3 and CHOP compared
Fig. 2. Effect of PA supplementation on the quality of oocytes grown in vitro. A-B. Relative lipid content in oocytes and representative images of oocytes stained with Nile red. Lipid content of oocytes developed without PA was defined as 1.0. C. ATP content in oocytes. D-E. Proportion of oocytes with differentially condensed chromatins (SN, surrounded nucleolus, and NSN non-surrounded nucleolus) and representative images of oocytes determined as SN or NSN. Scale bar indicates 100 mm.
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Fig. 3. Effect of PA supplementation on the histone modifications in oocytes. A-B. Relative expression levels of acetylated H4K12. Levels of acetylated H4K12 for oocytes developed without PA was defined as 1.0. Representative images of oocytes stained against Ace-H4K12. C-D. Relative expression levels of dimethyl H3K9. Levels of dimethyl H3K9 in oocytes developed without PA was defined as 1.0. Representative images of oocytes stained against dimethyl-H3K9. All data are presented as mean ± SEM, * indicates a significant difference between the control (BSA) and PA groups (P < 0.05). Scale bar indicates 100 mm.
Fig. 4. Effect of PA on protein expression levels in granulosa cells. A. Relative phospho-AKT/AKT levels and representative images of bands. B. Relative expression levels of activated Caspase-3 and representative image of bands. C. Relative expression levels of activated CHOP and representative image of bands. Expression values in granulosa cells developed without PA were defined as 1.0. * significant difference between the control and PA groups (P < 0.05).
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to cells cultured without PA (BSA) (1.60 ± 0.27-fold and 17.8 ± 0.31fold, respectively, p < 0.05, Fig. 4).
3.4. PA-induced ceramide accumulation reduced proliferation of granulosa cells At the end of the culture period, OGCs cultured with high PA levels showed lower numbers of granulosa cells and high lipid contents (Fig. 1). Therefore, we confirmed low granulosa cell numbers and high lipid content (Fig. 5-A and B, BSA vs. PA, cell number; 60,625 ± 4408 vs. 47,741 ± 4,058, lipid content 1.00 ± 0.13vs. 1.58 ± 0.10, p < 0.01) in oocytes at day 4 of the culture period (Fig. 4-A and B). In addition, OGCs cultured with high PA had high ceramide contents (BSA vs. PA, 1.00 ± 0.05 vs.1.24 ± 0.06, p < 0.05). Furthermore, supplementation of the culture medium with 0, 30, and 50 mM ceramide significantly inhibited granulosa cell proliferation (Fig. 5-E, 0, 30 and 50 mM ceramide, 191,750 ± 13,435, 166,888 ± 9,588, and 151,375 ± 10,864, respectively).
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4. Discussion The present study showed that high PA levels induced lipid accumulation in oocytes and granulosa cells. In high PA conditions, granulosa cell viability and proliferation were reduced and accompanied by greater endoplasmic reticulum stress and apoptosis, and the PA-induced inhibition of cell proliferation was caused by ceramide accumulation. In addition, PA supplementation reduced oocyte quality, causing impaired developmental ability to the blastocyst stage and abnormal histone modifications. Results revealed that oocytes and granulosa cells cultured under high-fat conditions had high lipid contents, and the oocytes showed impaired developmental ability. Lipid content is influenced by physiological conditions, such that oocytes and cumulus cells of mice fed with a high-fat diet contained high levels of lipids [20,21]. Obese mice contained high fatty acid concentrations in circulation [22], and women with high BMIs have high fatty acid concentrations in follicular fluid [4]. Ogawa reported that porcine oocytes and cumulus cells cultured with BSA or FF containing various concentration of NEFAs, FF increased lipid content in oocytes and cumulus
Fig. 5. Effect of PA and ceramide on granulosa cells of OGCs. OGCs were cultured with PA or ceramide for 4 days. A-B. Effect of PA on the number of granulosa cells and lipid content of the granulosa cells. Lipid contents in granulosa cells developed without PA were defined as 1.0. C-D. Relative ceramide content in granulosa cells and representative images of OGCs stained against ceramide. Ceramide content in granulosa cells developed without PA were defined as 1.0. E. Number of granulosa cells of OGCs cultured with 0, 30, and 50 mM for 14 days. * indicates a significant difference between the control (0 mM) and ceramide 50 mM groups (P < 0.05). Scale bar indicates 100 mm.
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cells compared to BSA-counterparts. This increase was found to be dependent on the amount of NEFA added to the culture condition [1]. In addition, supplementation of the maturation medium with PA increased the lipid content in bovine and porcine oocytes and reduced their developmental competence [2,16]. Therefore, high lipid conditions lead to increased lipid accumulation in oocytes and cumulus cells which reduces oocyte competence. Our present study showed that oocytes developed in a high-fat environment have a high ATP content. The ATP content in oocytes is necessary for nuclear maturation [23] and is a marker for their fertilization outcome [24]. By contrast, we previously demonstrated that culturing oocytes with high NEFA concentrations induced lipid accumulation and increased ATP contents but impaired developmental ability [1]. Therefore, it is hypothesized that overaccumulation of lipids in oocytes induces abnormal ATP generation. However, the corresponding molecular mechanism remained to be elucidated. Notably, oocytes developed with high PA concentrations had low H4K12 acetylation levels and high H3K9 demethylation levels. H4K12 Acetylation is high at GV stage oocytes [25] and well reflects oocyte quality [26]. In vitro-grown oocytes surrounded by a greater number of granulosa cells had high H4K12 acetylation levels and good developmental ability [27,28]. The above results indicate that the number of granulosa cells surrounding the oocytes was lower under high PA conditions. Therefore, we hypothesized that low cell viability and a small number of granulosa cells lead to reduced H4K12 acetylation levels. Interestingly, Hou et al. reported that the oocytes of mice on a high fat diet had high levels of demethylated H3K9 [15]. Consistent with the above findings, one study reported that oocytes and embryos cultured under high NEFA concentrations presented epigenetic changes [14]. The results are consistent with those of previous studies, which suggested that high PA concentrations during oocyte growth could lead to epigenetic changes in oocytes. The number of granulosa cells is crucial for follicle development and determines the developmental competence of oocytes [29]. Our findings showed that PA suppressed granulosa cell proliferation and reduced cell viability. Supplementation of the maturation medium of bovine oocytes with high levels of NEFA induced endoplasmic reticulum stress [12]. Consistent with these findings, the granulosa cells of OGCs cultured with high PA showed a low PAKT/AKT ratio and high expression levels of CHOP and Caspase3, which suggests that high PA conditions induce endoplasmic reticulum stress, apoptosis, and low proliferation activity. Pereira et al. reported that high PA concentrations induced ceramide accumulation via serine-palmitoyl transferase [30]. PA supplementation of the oocyte maturation medium induced both lipid and ceramide accumulation and aggravated oocyte quality [16]. In addition, ceramide caused abnormal mitochondrial function in oocytes. Our findings revealed for the first time that PA supplementation of the culture medium induced ceramide accumulation in granulosa cells, and the addition of ceramide reduced granulosa cell proliferation. Based on the above findings, we can conclude that high PA conditions induced lipid accumulation and consequently ceramide accumulation in granulosa cells, which in turn impair granulosa proliferation and proper oocyte growth. In conclusion, high PA conditions lead to lipid and ceramide accumulation in granulosa cells and reduce the viability of granulosa cells. In addition, oocytes grown under high PA conditions have low developmental ability and differential histone modifications. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research C (KAKENHI, grant number: 16K07996).
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