Abstract
Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus–oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.
Introduction
In vitro maturation (IVM) of cumulus–oocyte complexes (COCs) is a key component of assisted reproduction in cattle, humans, and other species (Gilchrist et al. 2008), although its widespread use is hindered because developmental competence of the oocyte is markedly compromised by IVM in cattle, sheep, mice, and humans (Thompson et al. 1995, Child et al. 2002, Rizos et al. 2002, Vanhoutte et al. 2009). Clearly, more detailed information is required on the cascade of molecular events that lead to COC maturation for the improvement of IVM protocols. The preovulatory LH surge induces COC expansion in vivo (reviewed by Richards et al. (2002)), and studies on mice and rats indicate that the effect of LH on the COC is mediated by the epidermal growth factor (EGF)-like family members, amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC), that are secreted by mural granulosa cells and act upon cumulus cells (Park et al. 2004, Ashkenazi et al. 2005, Conti et al. 2006). FSH can also stimulate the expression/synthesis of EGF-like growth factors in murine and bovine cumulus cells (Downs & Chen 2008, Caixeta et al. 2012). EGF-like growth factors are synthesized as transmembrane precursors and must undergo proteolytic cleavage (‘shedding’) by members of the disintegrin and metalloproteinase (ADAM) family (reviewed by Ben-Ami et al. (2006)). Once released, the EGF-like factors activate the EGF receptor (EGFR) on cumulus cells and stimulate the expression of genes necessary for cumulus expansion, including prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)), pentraxin 3 (PTX3), and hyaluronan synthase 2 (HAS2) (Ashkenazi et al. 2005, Conti et al. 2006, Shimada et al. 2006, Su et al. 2010).
HAS2 is necessary for the synthesis of hyaluronic acid, the major component of cumulus extracellular matrix, and reduction of HAS2 by siRNA in the mouse COC inhibited expansion (Sugiura et al. 2009). Hyaluronic acid is produced by glucose metabolism via the hexosamine biosynthetic pathway in cumulus cells, for which the rate-limiting step is the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the enzymes glucosamine:fructose-6-PO4 transaminases (GFPTs). The final step in this pathway leads to hyaluronic acid production, catalyzed by HAS2. Although the hexosamine biosynthetic pathway is critical for expansion, most of the glucose consumed by cumulus cells is metabolized via the glycolytic pathway by the rate-limiting enzyme phosphofructokinase (PFK) for the production of energy (reviewed by Sutton-McDowall et al. (2010)).
Cumulus expansion and glucose metabolism are modulated in part by oocyte-secreted factors (OSFs) including bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 8 (FGF8) (Valve et al. 1997, Juengel et al. 2004, Gilchrist et al. 2008). BMP15 treatment during IVM stimulated cumulus expansion in mice (Yoshino et al. 2006) and blastocyst production in cattle (Hussein et al. 2006, 2011). Exogenous BMP15 in IVM may improve oocyte developmental competence by enhancing oocyte oxidative phosphorylation, mediated via the cumulus cells (Sutton-McDowall et al. 2012). In mice, BMP15 and FGF8 together, but not separately, increased abundance of mRNA encoding glycolytic enzymes PFKP and lactate dehydrogenase A (LDHA), as well as glycolytic activity in cumulus cells (Sugiura et al. 2007). However, FGF8, either alone or in combination with BMP15 or growth differentiation factor 9, did not alter competence of mouse cumulus cells to undergo expansion (Sugiura et al. 2010). Another FGF of interest is FGF10, which was localized to the bovine oocyte, and its receptors (FGFR1B and FGFR2B) to cumulus cells (Buratini et al. 2007, Cho et al. 2008). Interestingly, supplementation of IVM medium with FGF10 enhanced cumulus expansion and embryo development in cattle (Zhang et al. 2010), although the mechanism of action is mostly unknown.
The objective of this study was to examine the roles of FGF10 and BMP15 in the preovulatory/expansion cascade in cattle. We first tested the hypothesis that BMP15, alone or in combination with FGF10, enhances cumulus expansion in cattle. Then, to gain insight into the mechanisms of action of BMP15 and FGF10 in cumulus expansion, we tested the hypothesis that these growth factors regulate glucose metabolism and the expression of genes in the ovulatory cascade necessary for expansion (ADAM10, ADAM17, AREG, EREG, BTC, EGFR, PTGS2, PTX3, TNFAIP6, and HAS2).
Materials and methods
Unless specified, all chemicals and reagents were purchased from Sigma.
In vitro maturation
Ovaries of adult cows (predominantly Nellore, Bos indicus; around 90 ovaries per collection) were obtained from an abattoir local to the Sao Paulo State University campus in Botucatu and transported to the laboratory in saline solution (0.9% NaCl) containing antibiotics (penicillin G, 100 IU/ml, and streptomycin, 100 μg/ml) at 35–37 °C. COCs were aspirated from 3–8 mm diameter follicles with an 18 gauge needle and pooled in a 15 ml conical tube. After sedimentation, COCs were recovered and selected using a stereomicroscope. Only COCs with homogenous cytoplasm and at least five compact layers of cumulus cells were used. COCs were washed and transferred in groups of 20 to a 100 μl drop of maturation medium, TCM199, containing Earle's salts supplemented with 1 μg/ml porcine FSH (equivalent to 0.002 IU; Folltropin-V Bioniche Animal Health, Belleville, ON, Canada), 10 IU/ml LH (Lutropin-V, Bioniche Animal Health), 22 μg/ml sodium pyruvate, 75 μg/ml amicacin, 4 mg/ml BSA, and growth factors (see below). Drops were covered with mineral oil and incubated at 38.5 °C in 5% CO2 in humidified air.
The effects of graded doses of recombinant human BMP15 (R&D Systems, Minneapolis, MN, USA; 0, 10, 50, and 100 ng/ml; four replicates/dose) or recombinant human FGF10 (R&D Systems; 0, 0.5, 10, and 50 ng/ml; four replicates/dose) on cumulus expansion were tested after 22 h of culture. To test potential synergism between BMP15 and FGF10 in the regulation of cumulus expansion, an additional experiment was performed with minimally effective doses of BMP15 alone (100 ng/ml), FGF10 alone (10 ng/ml), and BMP15 (100 ng/ml) plus FGF10 (10 ng/ml; all treatments were done in four replicates). Cumulus expansion was visually assessed according to a subjective scoring system. Grades 1–3 were attributed to increasing degrees of expansion (1 – poor expansion, characterized by a few morphological changes compared with before maturation; 2 – partial expansion, characterized by fair expansion but notable clusters lacking expansion; 3 – complete or nearly complete expansion; Zhang et al. (2010)).
To test the effects of BMP15 and FGF10 and of their combination on gene expression, maturation medium was supplemented with growth factors as described above (four replicates). All treatments were tested after 4, 12, and 22 h of culture; these time points were chosen based on a previous time-course study (Caixeta et al. 2012). Different times of culture were assessed in different experiments and thus treatments were compared within each time point but not between time points.
Gene expression analysis
After culture, cumulus cells and oocytes were mechanically separated from all 20 COCs cultured per group by repeated pipetting in PBS. Cumulus cells were transferred to 1.5 ml tubes, washed twice by centrifugation for 5 min at 700 g, and 350 μl of the RNA extraction lysis buffer was added to the cell pellets. Samples were stored at −80 °C until RNA extraction.
Total RNA was extracted from cumulus cells using the RNeasy kit (Qiagen) as recommended by the manufacturer. After purification, RNA samples were eluted in 30 μl of RNAse-free water. Total RNA concentrations were measured by spectrophotometer using a NanoDrop ND 1000 (Thermo Scientific, Wilmington, DE, USA). Total RNA (100 ng/reaction) was incubated with DNAse I to prevent interference of DNA contamination with the PCR analysis (1 U/μg; Invitrogen) and then reverse transcribed using Oligo-dT primers and Omniscript reverse transcriptase (Qiagen). The reagents were incubated at 37 °C for 60 min and then at 93 °C for 3 min for enzyme inactivation.
Relative real-time RT-PCR analysis was performed with an ABI 7500 thermocycler using Power Sybr Green PCR Master Mix (Applied Biosystems). The final volume of the PCR mix was 25 μl and thermocycling conditions were 95 °C for 10 min (1 cycle), denaturing at 95 °C for 10 s followed by annealing for 1 min (40 cycles). The primer sequences, amplicon sizes, and annealing temperatures for each target gene are given in Table 1. Reactions were optimized to provide maximum amplification efficiency for each gene. The specificity of the PCR products was assessed by melting curve analyses and amplicon size was determined by electrophoresis in 2% agarose gels.
Information of specific primers used for amplification in real-time PCR.
| Genes | Primer sequence | Fragment size (bp) | Annealing temperature (°C) | Reference |
|---|---|---|---|---|
| CYC-A | F: 5′-GCCATGGAGCGCTTTGG-3′ | 65 | 60 | Machado et al. (2009) |
| R: 5′-CCACAGTCAGCAATGGTGATCT-3′ | ||||
| GAPDH | F: 5′-GGCGTGAACCACGAGAAGTATAA-3′ | 119 | 62 | Machado et al. (2009) |
| R: 5′-CCCTCCACGATGCCAAAGT-3′ | ||||
| H2AFZ | F: 5′-GAGGAGCTGAACAAGCTGTTG-3′ | 74 | 60 | Machado et al. (2009) |
| R: 5′-TTGTGGTGGCTCTCAGTCTTC-3′ | ||||
| AREG | F: 5′-CTTTCGTCTCTGCCATGACCTT-3′ | 100 | 60 | Portela et al. (2011) |
| R: 5′-CGTTCTTCAGCGACACCTTCA-3′ | ||||
| EREG | F: 5′-ACTGCACAGCATTAGTTCAAACTGA-3′ | 100 | 60 | Portela et al. (2011) |
| R: 5′-TGTCCATGCAAACAGTAGCCATT-3′ | ||||
| BTC | F: 5′-GCCCCAAGCAGTACAAGCAT-3′ | 100 | 59 | AF140597 |
| R: 5′-GCCCCAGCATAGCCTTCATC-3′ | ||||
| EGFR | F 5′-AAAGTTTGCCAAGGGACAAG-3′ | 253 | 53 | Caixeta et al. (2009) |
| R: 5′-AAAGCACATTTCCTCGGATG-3′ | ||||
| PTGS2 | F: 5′-AAGCCTAGCACTTTCGGTGGAGAA-3′ | 168 | 60 | NM_174445.2 |
| R: 5′-TCCAGAGTGGGAAGAGCTTGCATT-3′ | ||||
| HAS2 | F: 5′-ACACAGACAGGCTGAGGACAACTT-3′ | 133 | 60 | NM_174079.2 |
| R: 5′-AAGCAGCTGTGATTCCAAGGAGGA-3′ | ||||
| PTX3 | F: 5′-CCTCAGCTATCGGTCCATAA-3′ | 294 | 54 | Caixeta et al. (2009) |
| R: 5′-ATTGAAGCCTGTGAGGTCTGC-3′ | ||||
| TNFAIP6 | F: 5′-GCAAAGGAGTGTGGTGGTGTGTTT-3′ | 135 | 60 | BC151789.1 |
| R: 5′-ACTGAGGTGAATGCGCTGACCATA-3′ | ||||
| ADAM10 | F: 5′-ACCCCCCAAAGTCTCTCACA-3′ | 210 | 60 | Li et al. (2009b) |
| R: 5′-AATCATGCGGAGATCCAAAGTT-3′ | ||||
| ADAM17 | F: 5′-TGGGATGTGAAGATGTTGCTAGA-3′ | 105 | 60 | Portela et al. (2011) |
| R: 5′-ATCCAAGTGTTCCCATATCAAAATC-3′ | ||||
| GFPT1 | F: 5′-GTTGAAACATGGCCCTCTGGCTTT-3′ | 117 | 60 | NM_001109961.1 |
| R: 5′-TGCCTAGCAACCACTTGCTGTAGA-3′ | ||||
| GFPT2 | F: 5′-GCCTTGTACCCAAGTGCTTTGCTT-3′ | 123 | 60 | NM_001076883.1 |
| R: 5′-TGCACGGTATTGGAAGAGTCTGCT-3′ | ||||
| GLUT1 | F: 5′-CAGGAGATGAAGGAGGAGAGC-3′ | 258 | 59 | BC119940.1 |
| R: 5′-CACAAATAGCGACACGACAGT-3′ | ||||
| GLUT4 | F: 5′-ATTGTGGCCATCTTTGGCTTCGTG-3′ | 160 | 60 | AY458600.1 |
| R: 5′-AACCCATGCCGATGATGAAGTTGC-3′ | ||||
| PFKP | F: 5′-TCAGAGAACCGTGCCTGGAAGAAA-3′ | 112 | 59 | NM_001193220.1 |
| R: 5′-TGACCACAAGCTCCTTGATCTGCT-3′ | ||||
| LDHA | F: 5′-TCTGGATTCAGCTCGCTTCCGTTA-3′ | 147 | 60 | BC146210.1 |
| R: 5′-TTCTTCAGGGAGACACCAGCAACA-3′ |
F, forward primer; R, reverse primer.
To select the most stable housekeeping gene, cyclophilin-A (CYCA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone H2AFZ (H2AFZ) amplification profiles were compared using the geNorm applet for Microsoft Excel (medgen.ugent.be/genorm; Vandesompele et al. (2002)); the most stable housekeeping gene was CYCA.
The relative expression values for each gene were calculated using the ΔΔCt method with efficiency correction and using one control sample as calibrator (Pfaffl 2001). Mean efficiency values for each gene were calculated from the amplification profile of individual samples with LinRegPCR software (Ramakers et al. 2003). Each sample was run in duplicate.
Glucose metabolism
Glucose and lactate concentrations were determined in spent media (including media that were not used for IVM), following 22-h culture with and without BMP15 (100 ng/ml) and FGF10 (10 ng/ml). At the completion of culture, spent media were collected, snap frozen with liquid nitrogen, and stored at −80 °C. Glucose and lactate levels were measured using a Hitachi 912 chemical analyzer (F. Hoffmann-La Roche Ltd.) from three experimental replicates. To determine glucose uptake, the measured glucose concentration was subtracted from the concentration of glucose in media blanks (drops of media, cultured without cells). The base medium (TCM199) did not contain lactate. Glucose uptake and lactate production were expressed as pmol/COC per h (Sutton-McDowall et al. 2012).
Statistical analysis
Cumulus expansion data were transformed to radians using arcsine transformation, and gene expression data were transformed to base 10 logarithms when not normally distributed. The effects of treatments with BMP15 and/or FGF10 on cumulus cell expansion, gene expression, and glucose and lactate levels were tested by ANOVA, and means were compared with the Tukey–Kramer HSD test. The analyses were performed with JMP software (SAS Institute, Cary, NC, USA) and the results are presented as means±s.e.m. Differences were considered significant when P<0.05.
Results
Effects of BMP15 and FGF10 on cumulus expansion
COCs were exposed to graded doses of BMP15 and FGF10 and cumulus expansion was recorded. The percentage of grade 3 COCs (complete or nearly complete cumulus expansion) was increased by BMP15 at 100 ng/ml (Fig. 1A) and by FGF10 at 10 ng/ml (Fig. 1B). A combination of BMP15 with FGF10 did not further enhance cumulus expansion compared with either BMP15 or FGF10 alone (Fig. 1C).
Effects of BMP15 (A), FGF10 (B), and the combination of both (C) on cumulus expansion. COCs were cultured with increasing doses of BMP15 and FGF10, and with a combination of BMP15 (100 ng/ml) and FGF10 (10 ng/ml). After culture for 22 h, the degree of expansion was classified as grade 1 (poor expansion, few morphological changes), 2 (partial expansion), or 3 (complete expansion). Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from four independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of BMP15 (A), FGF10 (B), and the combination of both (C) on cumulus expansion. COCs were cultured with increasing doses of BMP15 and FGF10, and with a combination of BMP15 (100 ng/ml) and FGF10 (10 ng/ml). After culture for 22 h, the degree of expansion was classified as grade 1 (poor expansion, few morphological changes), 2 (partial expansion), or 3 (complete expansion). Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from four independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of BMP15 (A), FGF10 (B), and the combination of both (C) on cumulus expansion. COCs were cultured with increasing doses of BMP15 and FGF10, and with a combination of BMP15 (100 ng/ml) and FGF10 (10 ng/ml). After culture for 22 h, the degree of expansion was classified as grade 1 (poor expansion, few morphological changes), 2 (partial expansion), or 3 (complete expansion). Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from four independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of BMP15 and FGF10 on cumulus expansion-related gene expression
To gain insight into the mechanisms by which BMP15 and FGF10 enhance cumulus expansion, we examined whether these factors regulate expression of key genes involved in the ovulatory cascade. BMP15 caused a dose-dependent increase in ADAM10, ADAM17, AREG, and EREG mRNA levels at 12 h of culture and of ADAM17, AREG, EREG, PTGS2, PTX3, and TNFAIP6 mRNA levels at 22 h of culture (Fig. 2). BMP15 had no effect on BTC and EGFR mRNA abundance (data not shown).
Effects of graded doses of BMP15 on ADAM10/17, AREG, EREG, PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of BMP15 on ADAM10/17, AREG, EREG, PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of BMP15 on ADAM10/17, AREG, EREG, PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
FGF10 did not affect abundance of mRNA encoding ADAM10, ADAM17, EGF-like factors, or EGFR (data not shown) but stimulated PTGS2 mRNA levels by 4 h of culture, which was also observed at 12 and 22 of culture (Fig. 3). FGF10 increased PTX3 mRNA levels at 12 and 22 h and TNFAIP6 mRNA levels only at 22 h (Fig. 3). BMP15 and FGF10 in combination did not alter abundance of mRNA of any of these genes compared with each growth factor alone at 22 h of culture (data not shown).
Effects of graded doses of FGF10 on PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of FGF10 for 4, 12, and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of FGF10 on PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of FGF10 for 4, 12, and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of FGF10 on PTGS2, PTX3, and TNFAIP6 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of FGF10 for 4, 12, and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of FGF10 and BMP15 on glucose metabolism
FGF10 and BMP15 alone each stimulated COC glucose uptake and the combination of the two did not further increase uptake (Fig. 4). In contrast, neither growth factor alone or in combination increased COC lactate production (Fig. 4). BMP15 increased abundance of mRNA encoding GLUT1 and GFPT1 at 22 h of culture, increased GFPT2 mRNA levels at 12 and 22 h, and increased HAS2 mRNA levels at 12 h (Fig. 5). Addition of FGF10 increased GLUT1, GLUT4, and GFPT1 at 12 h but had no effect on HAS2 or GFPT2 mRNA abundance (Fig. 6).
Effects of BMP15 and FGF10 on glucose metabolism in cumulus cells. (A and B) COCs were cultured for 22 h with BMP15 (100 ng/ml), FGF10 (10 ng/ml), or both together, and glucose uptake and lactate production were measured at the end of culture. Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from three independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of BMP15 and FGF10 on glucose metabolism in cumulus cells. (A and B) COCs were cultured for 22 h with BMP15 (100 ng/ml), FGF10 (10 ng/ml), or both together, and glucose uptake and lactate production were measured at the end of culture. Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from three independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of BMP15 and FGF10 on glucose metabolism in cumulus cells. (A and B) COCs were cultured for 22 h with BMP15 (100 ng/ml), FGF10 (10 ng/ml), or both together, and glucose uptake and lactate production were measured at the end of culture. Asterisks denote means that were significantly different from control values (P<0.05). Data were derived from three independent replicates for each treatment.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of BMP15 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of BMP15 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of BMP15 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of FGF10 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of FGF10 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
Effects of graded doses of FGF10 on GFPT1, GFPT2, GLUT1, GLUT4, and HAS2 mRNA abundance in cumulus cells. COCs were cultured with increasing doses of BMP15 for 12 and 22 h. Messenger RNA abundance was measured by real-time PCR. Data are presented as mean values (±s.e.m.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P<0.05). Data were derived from four independent replicates for each time point.
Citation: REPRODUCTION 146, 1; 10.1530/REP-13-0079
The potential interaction of FGF10 and BMP15 on glucose metabolic enzyme genes was tested at 22 h of culture. BMP15 alone increased GFPT2 mRNA abundance and there was no further change in the presence of both BMP15 and FGF10. Neither BMP15 nor FGF10, alone or together, altered GFPT1, PFKP, or LDHA mRNA levels (data not shown).
Discussion
Expansion of the cumulus is enhanced by OSFs, including BMP15 and FGF10 (Yoshino et al. 2006, Zhang et al. 2010), but the mechanisms of action of these growth factors have not been explored. There is also evidence of synergy between BMPs and FGFs, as BMP15 and FGF8 synergize to increase glucose metabolism in mouse cumulus cells (Sugiura et al. 2007). In the present study, we provide evidence that BMP15 and FGF10 enhance cumulus expansion in vitro through modulation of PTGS2 mRNA abundance and/or upstream genes and that both factors enhance glucose metabolism through the hexosamine pathway. However, we found no evidence for synergy between BMP15 and FGF10 in the regulation of expansion, gene expression, or glucose metabolism of cumulus cells from in vitro-matured bovine COCs.
BMP15 enhanced cumulus expansion in vitro in cattle in the present study, in agreement with a previous study in mice (Yoshino et al. 2006); this is consistent with data showing that BMP15 improves oocyte competence and early embryo development in cattle (Hussein et al. 2006, 2011), as increased cumulus expansion has been previously associated with improved blastocyst rates (Furnus et al. 1998). The relatively low proportion of COCs reaching full expansion in the control group in the present study is probably due to the absence of serum in the culture medium, as we aimed to assess the effects of OSFs under defined culture conditions. In association with this enhancement in cumulus expansion, BMP15 stimulated the expression of genes critical for this process. BMP15 increased abundance of mRNA encoding AREG, EREG, PTGS2, PTX3, and TNFAIP6 as previously observed in the mouse (Yoshino et al. 2006, Li et al. 2009a), although BMP15 had no effect on BTC mRNA levels in cattle unlike the situation in mice (Yoshino et al. 2006); this is probably because of the marked species differences in the regulation of BTC expression (Caixeta et al. 2012). Most interesting however, is the stimulatory action of BMP15 on mRNA expression of ADAM10 and ADAM17 in the present study. To our knowledge, this has not been previously described in any species and suggests that BMP15 acts at a major upstream event that influences not only EGF-like growth factor shedding but also proteolytic processing of matrix components such as versican that is required for ovulation (Ben-Ami et al. 2006, Russell & Robker 2007).
In vitro cumulus expansion of bovine COCs was also enhanced by FGF10, confirming a previous report (Zhang et al. 2010). However, in contrast to BMP15, FGF10 did not alter levels of RNA encoding ADAM10, ADAM17, or EGF-like factors but did affect the expression of downstream genes in our study. PTGS2 expression was the first stimulated by FGF10, which occurred at 4 h of culture. Next was PTX3, which was increased by FGF10 at 12 h, followed by TNFAIP6 expression, which was upregulated at 22 h of culture. TNFAIP6 was reported to be a target of PG action in mice and pigs, and either silencing of PTGS2 or disruption of PG signaling severely impaired TNFAIP6 expression (Ochsner et al. 2003, Takahashi et al. 2006, Yamashita et al. 2011). Therefore, the effects of FGF10 on TNFAIP6 expression are probably mediated through increased PTGS2.
In our study, both BMP15 and FGF10 increased glucose uptake by COCs but did not alter lactate production or the expression of the glycolytic enzymes PFKP and LDHA, suggesting that, unlike the combination of BMP15 plus FGF8 in the mouse (Sugiura et al. 2007), BMP15 plus FGF10 do not increase glycolysis in the bovine COCs. The increase in glucose uptake observed here differs from our recent results suggesting that BMP15 (alone and in the presence of FSH) did not significantly alter uptake (Sutton-McDowall et al. 2012); however, culture conditions differed with respect to the FSH and BMP15 used. Our previous study used a pro-mature form of the BMP15 protein, consisting of the ∼42 kDa BMP15 proregion complexed with the ∼17 kDa mature region, whereas the current study uses the smaller mature region only of BMP15 (∼17 kDa monomer). Also previously, we used 0.1 IU FSH whereas in the present study we used 0.002 IU, and FSH has been shown to increase glucose uptake (Sutton-McDowall et al. 2012). Therefore, it is likely that the substantially higher levels of FSH may mask the potentially beneficial effects of BMP15 on cumulus metabolism. Interestingly, in our study, BMP15 and FGF10 increased expression of mRNA encoding GFPT1 and GFPT2, key enzymes involved in directing glucose metabolism down the hexosamine pathway. In a previous study, the inhibition of GFPT activity attenuated cumulus expansion in bovine COCs (Gutnisky et al. 2007). Conversely, in our study, increased GFPT expression could probably result in greater amounts of precursor for hyaluronic acid synthesis, especially in the presence of increased HAS2 and therefore enhanced cumulus expansion. An increase in glucose uptake without increased lactate production has been observed in the latter stages of IVM in cattle, which is consistent with increased hyaluronic acid synthesis by cumulus cells at this time (Sutton-McDowall et al. 2010), although it is also possible that part of the increased metabolism of glucose may be directed toward oxidative phosphorylation (Sutton-McDowall et al. 2012).
In summary, this study presents novel findings from functional and gene expression studies that allow a better understanding of the actions of OSFs in the regulation of COC maturation in cattle, reinforcing previous evidence that BMP15 and FGF10 may be useful to improve IVM protocols (Hussein et al. 2006, 2011, Zhang et al. 2010). For the first time, we have demonstrated that BMP15 enhances cumulus expansion in cattle and provided evidence that the mechanisms by which BMP15 and FGF10 enhance cumulus expansion involve an increase in glucose uptake by cumulus cells associated with upregulation of the expression of key genes involved in hyaluronic acid production. Both growth factors also increased abundance of mRNA encoding another enzyme critical for ovulation/fertilization, PTGS2, although they seem to act at different steps in the ovulatory cascade; whereas BMP15 appears to act upon ADAM10, ADAM17, AREG, and EREG, FGF10 appears to act more directly on PTGS2 and downstream genes of the cumulus expansion cascade.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), and National Health and Medical Research Council of Australia (Grant 1008137).
Acknowledgements
The authors thank Drs Rubia B da Silva, Anthony C S Castilho, and Felipe M Dalanezi for their technical assistance.
References
Ashkenazi H, Cao X, Motola S, Popliker M, Conti M & Tsafriri A 2005 Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146 77–84. (doi:10.1210/en.2004-0588)
Ben-Ami I, Freimann S, Armon L, Dantes A, Ron-El R & Amsterdam A 2006 Novel function of ovarian growth factors: combined studies by DNA microarray, biochemical and physiological approaches. Molecular Human Reproduction 12 413–419. (doi:10.1093/molehr/gal045)
Buratini J Jr, Pinto MG, Castilho AC, Amorim RL, Giometti IC, Portela VM, Nicola ES & Price CA 2007 Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in bovine follicles. Biology of Reproduction 77 743–750. (doi:10.1095/biolreprod.107.062273)
Caixeta ES, Ripamonte P, Franco MM, Buratini J & Dode MA 2009 Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence. Reproduction, Fertility, and Development 21 655–664. (doi:10.1071/RD08201)
Caixeta ES, Machado MF, Ripamonte P, Price C & Buratini J Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle Reproduction, Fertility, and Development 2012 [in press] doi:10.1071/RD12125)
Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B & Tan SL 2002 A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstetrics and Gynecology 100 665–670. (doi:10.1016/S0029-7844(02)02193-2)
Cho JH, Itoh T, Sendai Y & Hoshi H 2008 Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles. Molecular Reproduction and Development 75 1736–1743. (doi:10.1002/mrd.20912)
Conti M, Hsieh M, Park JY & Su YQ 2006 Role of the epidermal growth factor network in ovarian follicles. Molecular Endocrinology 20 715–723. (doi:10.1210/me.2005-0185)
Downs SM & Chen J 2008 EGF-like peptides mediate FSH-induced maturation of cumulus cell-enclosed mouse oocytes. Molecular Reproduction and Development 75 105–114. (doi:10.1002/mrd.20781)
Furnus CC, de Matos DG & Moses DF 1998 Cumulus expansion during in vitro maturation of bovine oocytes: relationship with intracellular glutathione level and its role on subsequent embryo development. Molecular Reproduction and Development 51 76–83. (doi:10.1002/(SICI)1098-2795(199809)51:1<76::AID-MRD9>3.0.CO;2-T)
Gilchrist RB, Lane M & Thompson JG 2008 Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14 159–177. (doi:10.1093/humupd/dmm040)
Gutnisky C, Dalvit GC, Pintos LN, Thompson JG, Beconi MT & Cetica PD 2007 Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development. Reproduction, Fertility, and Development 19 488–497. (doi:10.1071/RD06134)
Hussein TS, Thompson JG & Gilchrist RB 2006 Oocyte-secreted factors enhance oocyte developmental competence. Developmental Biology 296 514–521. (doi:10.1016/j.ydbio.2006.06.026)
Hussein TS, Sutton-McDowall ML, Gilchrist RB & Thompson JG 2011 Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM. Reproduction, Fertility, and Development 23 576–584. (doi:10.1071/RD10323)
Juengel JL, Bodensteiner KJ, Heath DA, Hudsona NL, Moeller CL, Smith P, Galloway SM, Davis GH, Sawyer HR & McNatty KP 2004 Physiology of GDF9 and BMP15 signalling molecules. Animal Reproduction Science 82–83 447–460. (doi:10.1016/j.anireprosci.2004.04.021)
Li Q, Rajanahally S, Edson MA & Matzuk MM 2009a Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15. Molecular Human Reproduction 15 779–788. (doi:10.1093/molehr/gap062)
Li Q, Jimenez-Krassel F, Ireland JJ & Smith GW 2009b Gene expression profiling of bovine preovulatory follicles: gonadotropin surge and prostanoid-dependent up-regulation of genes potentially linked to the ovulatory process. Reproduction 137 297–307. (doi:10.1530/REP-08-0308)
Machado MF, Portela VM, Price CA, Costa IB, Ripamonte P, Amorim RL & Buratini J Jr 2009 Regulation and action of fibroblast growth factor 17 in bovine follicles. Journal of Endocrinology 202 347–353. (doi:10.1677/JOE-09-0145)
Ochsner SA, Russell DL, Day AJ, Breyer RM & Richards JS 2003 Decreased expression of tumor necrosis factor-α-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 144 1008–1019. (doi:10.1210/en.2002-220435)
Park JY, Su YQ, Ariga M, Law E, Jin SL & Conti M 2004 EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303 682–684. (doi:10.1126/science.1092463)
Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29 2002–2007. (doi:10.1093/nar/29.9.e45)
Portela VM, Zamberlam G, Gonçalves PBD, de Oliveira JFC & Price CA 2011 Role of angiotensin II in the periovulatory epidermal growth factor-like cascade in bovine granulosa cells in vitro. Biology of Reproduction 85 1167–1174. (doi:10.1095/biolreprod.111.094193)
Ramakers C, Ruijter JM, Deprez RH & Moorman AF 2003 Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience Letters 339 62–66. (doi:10.1016/S0304-3940(02)01423-4)
Richards JS, Russell DL, Ochsner S & Espey LL 2002 Ovulation: new dimensions and new regulators of the inflammatory-like response. Annual Review of Physiology 64 69–92. (doi:10.1146/annurev.physiol.64.081501.131029)
Rizos D, Ward F, Duffy P, Boland MP & Lonergan P 2002 Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Molecular Reproduction and Development 61 234–248. (doi:10.1002/mrd.1153)
Russell DL & Robker RL 2007 Molecular mechanisms of ovulation:co-ordination through the cumulus complex. Human Reproduction Update 3 289–312. (doi:10.1093/humupd/dml062)
Shimada M, Hernandez-Gonzalez I, Gonzalez-Robayna I & Richards JS 2006 Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Molecular Endocrinology 20 1352–1365. (doi:10.1210/me.2005-0504)
Su YQ, Sugiura K, Li Q, Wigglesworth K, Matzuk MM & Eppig JJ 2010 Mouse oocytes enable LH-induced maturation of the cumulus–oocyte complex via promoting EGF receptor-dependent signaling. Molecular Endocrinology 24 1230–1239. (doi:10.1210/me.2009-0497)
Sugiura K, Su YQ, Diaz FJ, Pangas SA, Sharma S, Wigglesworth K, O'Brien MJ, Matzuk MM, Shimasaki S & Eppig JJ 2007 Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells. Development 134 2593–2603. (doi:10.1242/dev.006882)
Sugiura K, Su YQ & Eppig JJ 2009 Targeted suppression of Has2 mRNA in mouse cumulus cell–oocyte complexes by adenovirus-mediated short-hairpin RNA expression. Molecular Reproduction and Development 76 537–547. (doi:10.1002/mrd.20971)
Sugiura K, Su YQ, Li Q, Wigglesworth K, Matzuk MM & Eppig JJ 2010 Estrogen promotes the development of mouse cumulus cells in coordination with oocyte-derived GDF9 and BMP15. Molecular Endocrinology 24 2303–2314. (doi:10.1210/me.2010-0260)
Sutton-McDowall ML, Gilchrist RB & Thompson JG 2010 The pivotal role of glucose metabolism in determining oocyte developmental competence. Reproduction 139 685–695. (doi:10.1530/REP-09-0345)
Sutton-McDowall ML, Mottershead DG, Gardner DK, Gilchrist RB & Thompson JG 2012 Metabolic differences in bovine cumulus–oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15. Biology of Reproduction 87 1–8. (doi:10.1095/biolreprod.112.102061)
Takahashi T, Morrow JD, Wang H & Dey SK 2006 Cyclooxygenase-2-derived prostagrandin E2 directs oocyte maturation by differentially influencing multiple signaling pathways. Journal of Biological Chemistry 281 37117–37129. (doi:10.1074/jbc.M608202200)
Thompson JG, Gardner DK, Pugh PA, McMillan WH & Tervit HR 1995 Lamb birth-weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biology of Reproduction 53 1385–1391. (doi:10.1095/biolreprod53.6.1385)
Valve E, Penttila TL, Paranko J & Härkönen P 1997 FGF-8 is expressed during specific phases of rodent oocyte and spermatogonium development. Biochemical and Biophysical Research Communications 232 173–177. (doi:10.1006/bbrc.1997.6256)
Vandesompele J, Preter KD, Pattyn F, Poppe B, Roy NV, Paepe AD & Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3 1–11. (doi:10.1186/gb-2002-3-7-research0034)
Vanhoutte L, Nogueira D, Dumortier F & De Sutter P 2009 Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor. Human Reproduction 24 1946–1959. (doi:10.1093/humrep/dep104)
Yamashita Y, Okamoto M, Kawashima I, Okazaki T, Nishimura R, Gunji Y, Hishinuma M & Shimada M 2011 The positive feedback loop between prostaglandin E2 and EGF-like factors is essential for the sustainable activation of MAPK3/1 in cumulus cells during in vitro maturation of porcine cumulus–oocyte complexes. Biology of Reproduction 85 1073–1082. (doi:10.1095/biolreprod.110.090092)
Yoshino O, McMahon HE, Sharma S & Shimasaki S 2006 A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse. PNAS 103 10678–10683. (doi:10.1073/pnas.0600507103)
Zhang K, Hansen PJ & Ealy AD 2010 Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro. Reproduction 140 815–826. (doi:10.1530/REP-10-0190)
