Introduction
Mammalian oocytes develop within follicles that continue to increase in size. Once the follicle reaches the antral follicle (AF) stage with a certain size, the oocytes acquire developmental competence. However, the number of follicles that successfully reach this size is limited, with almost small follicles regressing during development in the ovaries. Given appropriate culture conditions, oocytes in these small follicles can grow to full size and produce live offspring in mice and bovine (Eppig & O'Brien, Reference Eppig and O'Brien1996; Hirao et al., Reference Hirao, Itoh, Shimizu, Iga, Aoyagi, Kobayashi, Kacchi, Hoshi and Takenouchi2004). Therefore, oocytes in small immature follicles are a useful resource for embryo production. However, currently available culture systems are inadequate and provide an extremely low success rate of growing oocyte in vitro (Kubo et al., Reference Kubo, Cayo-Colca and Miyano2015).
The oocyte is surrounded by numerous granulosa cells (GCs) that proliferate to a great extent (Salfi et al., Reference Salfi, Ventura and Caraceni1979) during follicle development. The GCs support oocyte growth via complex interactions involving autocrine and paracrine factors as well as transportation of energy substrates across the gap junction (Gilula et al., Reference Gilula, Epstein and Beers1978; Brower & Schultz, Reference Brower and Schultz1982; Gougeon, Reference Gougeon1996; Sutton-McDowall et al., Reference Sutton-McDowall, Gilchrist and Thompson2010). Owing to their low phosphofructokinase and hexokinase activity, oocytes are believed to directly use only a small quantity of glucose (Tsutsumi et al., Reference Tsutsumi, Yano, Satoh, Mizuno and Kato1990; Cetica et al., Reference Cetica, Pintos, Dalvit and Beconi2002) and provision of energy substrates from GCs that actively uptake glucose is important for energy status of oocytes (Biggers et al., Reference Biggers, Whittingham and Donahue1967; Sugiura & Eppig, Reference Sugiura and Eppig2005; Sutton-McDowall et al., Reference Sutton-McDowall, Gilchrist and Thompson2010). However, in vitro culture conditions differ from those in vivo. When oocyte–granulosa cell complexes (OGCs) are cultured in vitro, the number of GCs increases only up to one-tenth of that seen in vivo (Oi et al., Reference Oi, Tasaki, Munakata, Shirasuna, Kuwayama and Iwata2015). Therefore, we hypothesized that modifying the culture system to enhance GC proliferation may improve oocyte grown in vitro. Insulin and insulin-like growth factor-1 are known to promote GC proliferation in the follicle (Peluso et al., Reference Peluso, Delidow, Lynch and White1991; Kadakia et al., Reference Kadakia, Arraztoa, Bondy and Zhou2001). Additionally, insulin supplementation in culture media has been reported to promote GC proliferation (Savion et al., Reference Savion, Lui, Laherty and Gospodarowicz1981; Hein et al., Reference Hein, Panzani, Rodríguez, Salvetti, Díaz, Gareis, Benítez, Ortega and Rey2015). Furthermore, it has been reported that GC proliferation is regulated by PI3K/PTEN/AKT pathway, which is a downstream pathway of insulin (Goto et al., Reference Goto, Iwase, Harata, Takigawa, Suzuki, Manabe and Kikkawa2009; Makker et al., Reference Makker, Goel and Mahdi2014). Moreover, recent RNA-sequencing of GCs showed that upregulation of HIF1α, up-stream factor of glucose metabolism and/or insulin signaling (Shiratsuki et al., Reference Shiratsuki, Hara, Munakata, Shirasuna, Kuwayama and Iwata2016), and glycolysis are major events in porcine follicle development (Munakata et al., 2016a) and the results suggest that exogenous insulin and glucose may enhance glycolysis and induce GC proliferation and hence help in in vitro OGCs development.
Oocytes of cows and pigs have a large number of lipid droplets in their cytoplasm, which increases during oocyte growth (Fair et al., Reference Fair, Hulshof, Hyttel, Greve and Boland1997; Prates et al., Reference Prates, Nunes and Pereira2014). As lipid is an energy substrate for the oocyte during maturation and embryo development (Ferguson & Leese, Reference Ferguson and Leese2006; Dunning et al., Reference Dunning, Cashman, Russell, Thompson, Norman and Robker2010; Dunning et al., Reference Dunning, Akison, Russell, Norman and Robker2011), the lipid content in oocytes relates to high developmental ability to form blastocysts in cows (Jeong et al., Reference Jeong, Cho, Lee, Deb, Lee, Kwon and Kong2009). In this context, we previously reported that the number of GCs surrounding the oocyte positively relates to the lipid and adenosine triphosphate (ATP) content in oocytes grown in vitro and in vivo (Munakata et al., 2016b). Thus, we hypothesized that an optimal combination of exogenous insulin and glucose can improve the developmental ability of oocytes grown in vitro as a result of enhanced GC proliferation and oocyte energy status in terms of lipid and ATP content.
In the present study, we examined the effect of supplementing the culture medium with a combination of insulin and glucose on proliferation of GCs and oocyte development.
Materials and methods
Reagents and media
Unless otherwise stated, all drugs were purchased from Nacalai Tesque (Kyoto, Japan). The medium used for in vitro growth (IVG) of OGCs (IVG medium) was described in previous report (Itami et al., 2015a). In brief, medium used for IVG was α-MEM (Sigma Aldrich, St. Louis, MO, USA), supplemented with insulin transferrin and selenium (ITS). We cultured OGCs individually using 96 well culture plate and the amount of IVG medium per 1 well was 200 μl. The combination of insulin and glucose content in the IVG medium used is presented in Table 1. As the number of OGCs collected from early antral follicles (EAFs) was insufficient to allocate all possible combinations, we used two paired combinations. The in vitro maturation medium (IVM medium) consisted of Medium 199 (Gibco) supplemented with 10% v/v porcine follicle fluid, 0.5 mM l-cysteine, 0.9 mM sodium pyruvate, 1 mM l-glutamine, 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). Porcine follicle fluid was collected from antrum follicles (3–6 mm in diameter), centrifuged (10 000 g for 5 min), and stored at −20°C until use. Finally, porcine zygote medium 3 (PZM3, Yoshioka et al., Reference Yoshioka, Suzuki, Tanaka, Anas and Iwamura2002) was used for in vitro embryo culture (IVC medium).
Grouping the conditions of culture medium
Collection of OGCs, IVG, IVM, activation, and IVC
Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and were transported to the laboratory in phosphate-buffered saline (PBS) containing antibiotics at 37°C within 1 h. OGCs were collected from EAFs (500–700 µm) under a stereomicroscope using an 18-G needle connected to a syringe. The OGCs were cultured in IVG medium for 14 days. Every 4 days, half of the medium was replaced with fresh medium, at which point the OGCs were examined for antrum formation to determine antrum formation rate. At the end of the IVG period (14 days), OGCs with an antrum cavity were used for experiment or subsequent IVM culture for 44 h. Oocyte activation was conducted as previously described (Itami et al., 2015b). In brief, oocytes were denuded from granulosa cells and treated with 10 µM ionomycin for 5 min followed by 6 h of incubation in IVC medium containing 10 µM cytochalasin B and 10 µM cycloheximide. After activation, parthenogenetic embryos were cultured for 7 days and the ratio of blastulation and total cell number of the blastocyst were determined. We stained blastocyst by DAPI and observed using fluorescence microscopy and determined total cell number. In vitro culture of OGCs and IVM were performed at 38.5°C in an atmosphere of 5% CO2 and 95% air. In vitro embryo culture was performed at 38.5°C in an atmosphere of 5% O2, 5% CO2 and 90% N2.
Measurement of oocyte diameter and GC number of OGCs
To obtain oocyte diameter and total cell number of the OGCs, oocytes were denuded from the surrounding cells using a narrow pulled glass pipette and the GCs were dispersed in cell dispersion cocktail (Accumax, Innovation Cell Technology, Inc. San Diego, CA, USA). Total GC number was calculated using the volume of the cellular suspension and cellular concentration was determined by hematocytometer. Oocyte x and y axes diameters were measured under a digital microscope (BZ-8000; Keyence, Tokyo, Japan) and the average was defined as a diameter of the oocyte.
Lipid staining with Nile Red
Oocytes were denuded from GCs using a narrow pulled glass pipette and fixed in 4%paraformaldehyde. Neutral lipid in oocyte cytoplasm was stained by Nile Red (Wako, Tokyo, Japan) according to the manufacturer protocol. After staining, fluorescence of the oocyte was examined under a digital fluorescence microscope (Keyence) and the fluorescence intensity was pixel transformed using ImageJ software (NIH: National Institute of Health, New York, MD, USA).
Assessment of ATP content in oocyte
Oocytes were lysed in 50 μl distilled water and stored at −20°C until analysis. ATP content of individual oocytes was determined by measuring luminescence generated by ATP-dependent luciferin–luciferase reactions as previously described (Iwata et al., Reference Iwata, Goto, Tanaka, Sakaguchi, Kimura, Kuwayama and Monji2011).
Measurement of glucose concentration in IVG medium
The media used for glucose measurement were collected at 8 or 14 days of culture periods and stored at −20°C until analysis. Glucose concentration in IVG medium was measured using LabAssay Glucose (Mutarotase-GOD method, Wako) according to the manufacturer's protocol.
Experimental design
Experiment 1: Comparison of OGCs and oocyte development at high and low insulin concentrations with low glucose concentration
Firstly, we examined the effect of insulin in medium containing 5.5 mM glucose on development of OGCs and oocytes. Twenty OGCs were individually cultured under two conditions: medium containing 10 or 20 μg/ml insulin with 5.5 mM glucose (see Table 1, Group 1 versus Group 2). As described above, control medium was supplemented with ITS which contained 10 μg/ml insulin, thus this concentration was defined as low insulin group (Group 1). In the control medium is During IVG (day 8) and at the end of the culture period (14 days), ATP and lipid content, and oocyte diameter were measured, and the number of GCs surrounding the oocyte was determined. At day 8, all OGCs were analysed, since oocyte degeneration was not observed, whereas at the end of the culture period OGCs having antrum cavity were selected, considering that OGCs without antrum contain degenerated oocytes.
Experiment 2: Effect of various insulin and glucose concentration on the glucose content in IVG medium
Next, we examined glucose concentration in the medium after 8 and 14 days of OGCs incubation. OGCs were cultured in a medium containing selective combinations of insulin and glucose: Group1, 5.5 mM glucose and 10 μg/ml insulin; Group 2, 5.5 mM glucose and 20 μg/ml insulin; Group 3, 11 mM glucose and 20 μg/ml insulin. The medium was sampled on day 8 and 14, and glucose concentration of the randomly selected samples were measured.
Experiment 3: Comparison of OGCs and oocyte development at high and low glucose concentrations with high insulin concentration
Here, we examined the effect of glucose (5.5 or 11 mM) combined with high insulin (20 μg/ml) (See Table 1, Group 2 versus Group 3) on OGCs and oocyte development. Twenty OGCs were collected at the end of the culture period and ATP and lipid content, diameter of oocytes grown in vitro, and number of GCs surrounding the OGCs were determined.
Experiment 4: Comparison of OGCs and oocyte development at high and low glucose concentrations with low insulin concentration
Here, we examined the effect of glucose (5.5 or 11 mM) combined with 10 μg/ml insulin (See Table 1, Group 1 versus Group 4) on OGCs and oocyte development. Twenty OGCs were collected and at the end of the culture period and content of ATP and lipid, diameter of oocytes, and number of GCs surrounding the OGCs were determined.
Experiment 5: Comparison of lipid content in oocytes cultured in medium containing 20 μg/ml insulin and 11 mM glucose versus that containing 10 μg/ml insulin and 5.5 mM glucose
To determine whether high insulin and glucose conditions enhanced lipid content of oocytes, we directly compared Group 1 and Group 3, based on pairwise comparison. Twenty OGCs were cultured under 2 conditions; Group1: 5.5 mM glucose and 10 μg/ml insulin, Group 3: 11 mM glucose and 20 μg/ml insulin, At the end of culture period, lipid content of oocytes was determined.
Experiment 6: Effect of the combination of high insulin and glucose concentration on the rate of blastocyst formation after parthenogenetical activation
Here, we cultured OGCs under three culture conditions (Groups 1, 2 and 3), and compared the developmental rate to the blastocyst stage following IVM and activation. As development of OGCs and energy status of the oocytes did not differ between Group 1 and Group 4, and considering our previous report regarding developmental rate to the blastocyst stage (Tasaki et al., Reference Tasaki, Munakata, Arai, Murakami, Kuwayama and Iwata2015), in this experiment, we compared Groups 1–3. Twenty OGCs were cultured under the three conditions: Group 1, 10 μg/ml insulin and 5.5 mM glucose; Group 2, 20 μg/ml insulin and 5.5 mM glucose; and Group 3, 20 μg/ml insulin and 11 mM glucose. At the end of the culture period, OGCs with an antrum cavity were subjected to IVM, while the oocytes were activated and cultured to determine their developmental ability to the blastocyst stage.
Statistical analysis
All data obtained were analysed using Student's t-test. Glucose concentration in the medium and oocyte developmental rate were assessed using a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Developmental rates were arcsine-transformed prior to the statistical analysis. Differences with P-values <0.05 were considered statistically significant.
Results
Experiment 1: In spite a temporary improvement of oocyte growth, low quality of oocytes were obtained with medium containing 5.5 mM glucose and high insulin concentration
High insulin concentration (20 μg/ml) improved antrum formation of the OGCs at day 8 of culture period (Fig. 1 A) with greater numbers of GCs and higher ATP content in the oocytes compared with the results obtained with lower insulin concentration (10 μg/ml), (Table 2). At the end of the culture period (day 14), high insulin concentration caused an increase in GC number; however, lipid and ATP content in the oocytes was significantly low compared with that in oocytes cultured with low insulin concentration (Table 3).
Comparison of OGCs development under high and low insulin concentrations with low glucose concentration. OGCs were cultured in media containing 10 μg/ml insulin and 5.5 mM glucose (Group 1) or 20 μg/ml insulin and 5.5 mM glucose (Group 2). (A) Antrum formation rate of OGCs during 14 days of culture periods. (B, C) Representative images of Group 1 OGCs with antrum cavity (B: day 8; C: day 14. Black bar indicates 100 μm). Data are presented as the mean ± standard error of the mean (SEM). Statistical significance is presented as *: P < 0.05.
Effect of insulin concentration on OGCs development at day 8
a,b P < 0.05. Data are shown as mean ± standard error of the mean (SEM).
OGCs numbers using each experiment between Groups 1 and 2 were described below.
Diameter: 46 versus 53. Cell number: 45 versus 53. Lipid: 46 versus 53. ATP: 58 versus 56.
Effect of insulin concentration on OGCs development at day 14
a,b P< 0.05. Data are shown as mean ± standard error of the mean (SEM).
OGCs numbers using each experiment between Groups 1 and 2 were described below.
Diameter: 54 versus 55. Cell number: 54 versus 60. Lipid: 38 versus 34. ATP: 61 versus 60.
Experiment 2: Combination of high insulin and 5.5 mM glucose resulted in exhaustion of glucose in the medium
At day 14, the combination of 20 μg/ml insulin and 5.5 mM glucose (Group 2) reduced glucose concentration in IVG medium to 1.2 mM which was significantly different from that observed for Group 1 (10 μg/ml insulin and 5.5 mM glucose). Conversely, the combination of 20 μg/ml insulin and 11 mM glucose (Group 3) maintained glucose concentration at 5.6 mM which was significantly higher than that observed for the other groups (Group 1 and 2), (Fig. 2).
Effect of various insulin and glucose concentrations on glucose content in the medium. OGCs were cultured in media containing selective combinations of insulin and glucose: Group 1, 10 μg/ml insulin and 5.5 mM glucose; Group 2, 20 μg/ml insulin and 5.5 mM glucose; Group 3, 20 μg/ml insulin and 11 mM glucose. Two dotted lines indicate the concentration of glucose: low, 5.5 mM and high, 11 mM. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance is presented as **: P < 0.01 and ***: P < 0.001.
Experiment 3: High concentration of glucose improved quality of oocytes cultured in medium containing high concentration of insulin
Adding high glucose concentration to the medium with high insulin concentration (Group 3) increased lipid and ATP content in the oocytes as well as GC number in the OGCs compared with the results observed for OGCs cultured with high insulin and low glucose concentrations (Group 2), (Table 4). High insulin condition enhance antrum formation of the OGCs at day 4 of culture period but identical at the other culture period (Fig. 3).
Effect of glucose concentration in high insulin medium on OGCs development at day 14
a,b P < 0.05. Data are shown as mean ± standard error of the mean (SEM).
OGCs numbers using each experiment between Group 2 and 3 were described below.
Diameter: 73 versus 72. Cell number: 73 versus 72. Lipid: 51 versus 50. ATP: 45 versus 50.
Comparison of OGCs development at high or low glucose concentrations with high insulin concentration. OGCs were cultured in media containing 5.5 mM glucose and 20 μg/ml insulin (Group 2) or 11 mM glucose and 20 μg/ml insulin (Group 3). Antrum formation rate of OGCs during 14 days of culture periods. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance is presented as *: P < 0.05.
Experiment 4: High concentration of glucose did not affect development of OGCs cultured in medium containing 10 μg/ml insulin
No difference was observed in either lipid and ATP content in oocytes or GC numbers of OGCs between the two culture conditions (Groups 1 and 4), (Table 5). Additionally antrum formation was also similar between the two culture conditions (Fig. 4).
Effect of glucose concentration alone on OGCs development at day 14
Data are shown as mean ± standard error of the mean (SEM).
OGCs numbers using each experiment between Groups 1 and 4 were described below.
Diameter: 71 versus 69. Cell number: 73 versus 70. Lipid: 51 versus 44. ATP: 54 versus 65.
Comparison of OGCs development at high or low glucose concentrations with low insulin concentration, OGCs were cultured in media containing 5.5 mM glucose and 10 μg/ml insulin (Group 1) or 11 mM glucose and 10 μg/ml insulin (Group 4). Antrum formation rate of OGCs during 14 days of culture periods. Data are presented as the mean ± standard error of the mean (SEM).
Experiment 5: High concentration of insulin and glucose increased lipid content in oocytes
The medium containing 20 μg/ml insulin and 11 mM glucose (Group 3) showed an increase in lipid content in oocytes compared with that for Group 1 (10 μg/ml insulin and 5.5 mM glucose), (Fig. 5). The lipid content in oocytes was the highest for Group 3.
Comparison of lipid content in oocytes cultured in medium containing 11 mM glucose and 20 μg/ml insulin versus that containing 5.5 mM glucose and 10 μg/ml insulin. (A) Comparison of lipid content in oocytes from OGCs cultured in medium containing 5.5 mM glucose and 10 μg/ml insulin (Group 1) versus that containing 11 mM glucose and 20 μg/ml insulin (Group 3). (B, C) Representative images of oocytes stained by Nile Red. [(B) Group 1, (C) Group 3)]. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance is presented as *P < 0.05.
Experiment 6: Medium containing high concentrations of insulin and glucose showed improved oocyte developmental competence after activation
Medium containing 20 μg/ml insulin and 11 mM glucose (Group 3) showed the highest developmental rate to the blastocyst stage, whereas the medium containing 20 μg/ml insulin and 5.5 mM glucose (Group 2) showed the lowest developmental rate (Table 6).
Effect of combinations of insulin and glucose of developmental competence of oocyte grown in vitro
a,b P < 0.05. Data are shown as mean ± standard error of the mean (SEM).
Discussion
The results of this study showed that insulin enhances proliferation of GCs of OGCs derived from EAFs, but does not affect developmental ability of oocytes, unless supplemented with additional glucose (11 mM). Additionally, supplementation with both insulin and glucose increases GC number as well as ATP and lipid content in the oocytes and enhances developmental ability of oocytes to the blastocyst stage.
The low quality of in vitro-grown oocytes is due to improper culture milieu and extremely low number of GCs surrounding the oocytes under in vitro culture conditions (Oi et al., Reference Oi, Tasaki, Munakata, Shirasuna, Kuwayama and Iwata2015). Recent comprehensive gene expression analysis has shown that follicle development associates with upregulation of HIF1A, vascular endothelial growth factor (VEGF), and glycolytic activity (Munakata et al., 2016a). In line with these findings, it has been reported that GC proliferation depends on VEGF-mediated activation of PI3K/AKT pathway (Irusta et al., Reference Irusta, Abramovich, Parborell and Tesone2010). Furthermore, considering the reports, activation of AKT/mTOR pathway via certain cytokines is a possible means to enhance GC proliferation of OGCs and hence improve oocyte development in vitro. In line with this, Serafim et al. (Reference Serafim, Silva, Duarte, Araújo, Silva, Lima, Chaves, Campello, Silva and Figueiredo2013) reported that FSH, insulin enhanced in vitro follicular growth and survivability. Therefore, we examined the effect of supplementing the culture medium with insulin on OGC development in the present study. In experiment 1, addition of 20 μg/ml insulin increased GC proliferation and ATP content in oocytes at day 8 of culture period, however ATP and lipid content in the oocytes decreased significantly at the end of culture period even though the number of GCs was still significantly greater than that cultured in medium containing with low insulin. Insulin stimulates AKT/mTOR pathway and enhances cellular proliferation (Kido et al., Reference Kido, Nakae and Accili2001), while activation of mTOR signaling induces follicular development and increases ovary volume in vitro (Cheng et al., Reference Cheng, Kim, Li and Hsueh2015). Likewise, inhibition of AKT activity increases apoptosis rate in rat GCs (Choi et al., Reference Choi, Jo, Lee and Choi2013). Furthermore, insulin enhances glucose uptake by human GCs (Purcell et al., Reference Purcell, Chi, Lanzendorf and Moley2012). Therefore, we speculated that insulin may upregulate GCs proliferation and glucose utilization, which would exhaust glucose content in the culture medium, which in turn would result in low ATP and lipid content in the oocytes. As expected, we found significantly low glucose concentration in the group where the medium contained high insulin concentration (Group 2) compared with that containing low insulin concentration (Group 1). Furthermore, additional glucose supplementation of the medium containing high insulin concentration allowed for the maintenance of high glucose concentration up to 14 days of culture period (Group 3, Fig. 2). Further, we examined the effect of the combination of high insulin (20 μg/ml) and high glucose (11 mM) concentrations on OGCs and oocyte development, and found significantly higher GC number as well as ATP and lipid content in oocytes. Thus indicates that additional glucose ameliorates starvation of oocytes and increases provision of energy substrate to the oocytes. In addition, we previously reported (Tasaki et al., Reference Tasaki, Munakata, Arai, Murakami, Kuwayama and Iwata2015) and confirmed in this study (Experiment 4) that oocyte developmental ability as well as ATP and lipid content in oocytes did not change when OGCs were cultured in a medium supplemented with glucose alone (Group 4). To the best of our knowledge, this is the first report demonstrating that modification of culture conditions in terms of both insulin and glucose concentration increases lipid and ATP content of oocytes. In this study, oocytes grown in the medium containing both high insulin and glucose concentrations were found to have high developmental ability (Table 6). This is consistent with previous reports where oocytes with high lipid and ATP content have been shown to exhibit high developmental ability (Jeong et al., Reference Jeong, Cho, Lee, Deb, Lee, Kwon and Kong2009; Sato et al., Reference Sato, Itami, Tasaki, Takeo, Kuwayama and Iwata2014). In this context, presence of sufficient glucose is required for the insulin-mediated enhanced viability of cancer cells, which depends on glycolysis (Zhang et al., Reference Zhang, Fei, Li, Zhang, Sun, Zhu, Wang and Sun2016). Thus, insulin-induced proliferation of GCs requires sufficient glucose in the culture milieu.
Lipids are known to accumulate into lipid droplets during oocyte growth; however, the factors affecting this accumulation have not yet been clarified. Recently, Munakata et al. (2016b) have shown that the number of GCs closely relates to the lipid and ATP content in oocyte cultured under both in vivo and in vitro conditions. Notably, insulin potentially affects both oocytes and GCs owing to the presence of receptors on both cells (Acevedo et al., Reference Acevedo, Ding and Smith2007). Although the molecular background underlying lipid accumulation in oocytes is still unclear, insulin-induced proliferation of GCs is a causal factor for greater lipid accumulation in oocytes grown in vitro.
In conclusion, supplementation of the culture medium with both high insulin and glucose concentrations enhances GC proliferation and oocyte energy status, which in turn, improves the developmental ability of oocytes.
Financial support
This study was supported by the Grant-in-Aid for JSPS Fellows (KAKENHI, grant No. JP16J07329) from the Japan Society for the Promotion of Science.
Statement of interest
The authors have no conflicts of interest to declare.
