Metabolic Differences in Bovine Cumulus-Oocyte ... - CiteSeerX

BIOLOGY OF REPRODUCTION (2012) 87(4):87, 1–8 Published online before print 15 August 2012. DOI 10.1095/biolreprod.112.102061

Metabolic Differences in Bovine Cumulus-Oocyte Complexes Matured In Vitro in the Presence or Absence of Follicle-Stimulating Hormone and Bone Morphogenetic Protein 151 Melanie L. Sutton-McDowall,2,3 David G. Mottershead,3 David K. Gardner,4 Robert B. Gilchrist,3 and Jeremy G. Thompson3 3

The Robinson Institute, Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, South Australia, Australia 4 Department of Zoology, University of Melbourne, Melbourne, Victoria, Australia INTRODUCTION

Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% % 6 7.4% %; with FSH and rhBMP15, 51.5% % 6 5.4% %; P , 0.05). Glucose uptake and lactate production was significantly increased by greater than 2-fold with FSH (P , 0.05), whereas rhBM15 supplementation did not increase these levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P , 0.05). Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD++) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD++:NAD(P)H). The significant increase in FAD++ and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.

Paracrine- and gap junction-mediated bidirectional communication between the oocyte and its cumulus vestment is critical for optimal developmental competence [1–3]. The role of cumulus cells is to provide the oocyte with both smallmolecular-weight compounds essential for development, such as nutrients for energy production and factors involved in meiotic control [4], as well as yet-to-be-identified paracrine factors. The oocyte secretes growth factors (oocyte-secreted factors [OSFs]) that regulate the activity of cumulus cells, such as differentiation from other follicular cell types [5, 6], proliferation [7–9], prevention of apoptosis [10], steroidogenesis [11], and cumulus expansion [12, 13]. Whereas the secretome of the oocyte is not fully defined, members of the transforming growth factor beta superfamily, bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), have been identified as critical OSFs [14]. Important species-specific differences exist in the relative participation of these factors, as demonstrated by genedeficiency studies, species-specific oocyte expression levels, and differing degrees of protein biopotency. For example, the expression ratio of GDF9 to BMP15 is 5.19:1 in mouse oocytes, compared to 0.25:1 in bovine oocytes, with a similarly low ratio for all mono-ovular species examined [15]. Moreover, mouse GDF9 is secreted in an active form, whereas human GDF9 (and, possibly, GDF9 in most mono-ovular species) is secreted in a latent form [16]. The addition of these growth factors during in vitro oocyte maturation (IVM), either via conditioned media [17], coculture with denuded oocytes (DOs) [18, 19], or recombinant proteins [19, 20], improves oocyte developmental competence, as is evident by improved embryo development [18, 19] and subsequent fetal survival following embryo transfer [20]. In line with the more prominent role for BMP15 in cow oocytes, supplementation with a partially purified recombinant ovine BMP15 during cattle IVM significantly increased blastocyst development, whereas recombinant mouse GDF9 supplementation was less effective [18, 19]. The two cellular compartments of COCs have different carbohydrate metabolic profiles; the oocyte itself has a low capacity to metabolize glucose [21] compared to cumulus cells, which metabolize 23-fold more glucose (per ml tissue/h) than the oocyte [22]. This is facilitated by the expression of a highaffinity glucose transporter (SLC2A4 [23, 24]) in the cumulus cells, whereas the oocyte has low activity of key enzymes involved in glycolysis, such as phosphofructokinase [25]. In return, the oocyte can readily metabolize pyruvate via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation and consumes 3-fold more oxygen per ml tissue/h than

bone morphogenetic protein 15, cumulus-oocyte complex, FSH, growth factors, in vitro oocyte maturation (IVM), metabolism, oocyte maturation 1 Supported by the National Health and Medical Research Council Australia Project (1008137) and Development (1017484) grants and a collaborative research grant from Cook Medical (Eight Mile Plains, QLD, Australia). 2 Correspondence: E-mail: [email protected]

Received: 17 May 2012. First decision: 26 June 2012. Accepted: 3 August 2012. Ó 2012 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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ABSTRACT

SUTTON-MCDOWALL ET AL. Groups of 10 COCs were cultured in 100 ll of pre-equilibrated IVM media overlaid with paraffin oil (Merck) at 38.58C in 6% CO2 in humidified air for 23 h.

cumulus cells [22]. Hence, a key role of cumulus cells during oocyte maturation is to provide the oocyte with substrates for the TCA cycle and oxidative phosphorylation [26]. Recently, it has been demonstrated that the oocyte orchestrates cumulus cell metabolism, in particular the expression of key glycolytic genes within the cumulus cells that have low expression levels within the oocyte [27]. Mouse oocytectomized complexes (OOXs; i.e., COCs in which the oocyte has been surgically removed) have significantly lower mRNA expression of key glycolytic enzymes, such as phosphofructokinase, lactate dehydrogenase, and pyruvate kinase [27]. Metabolic activity through glycolysis and the TCA cycle, along with mRNA expression of enzymes, was restored to levels comparable to those of COCs when OOXs were cocultured with fully grown DOs. Specifically, BMP15 and fibroblast growth factor 8 appear to be critical OSFs in mediating mouse cumulus cell glycolytic metabolism [28]. Conversely, we saw no significant differences in glucose and lactate metabolism within bovine OOXs compared to intact COCs or OOXs plus DOs [29]. However, these experiments were performed in the presence of follicle-stimulating hormone (FSH), a stimulator of glucose metabolism, which may have masked the influences of OSFs. We hypothesized that the increase in oocyte developmental competence observed in COCs cultured in the presence of recombinant human BMP15 (rhBMP15) would be reflected by changes in oocyte and cumulus cell metabolism. Furthermore, we hypothesized that these changes in metabolism are mediated via cumulus cells. The aim was, first, to determine the developmental competence of bovine COCs cultured in the presence of absence of rhBMP15, FSH (a known stimulator of carbohydrate metabolism), or both and, second, to relate this to metabolic outputs, such as glucose uptake and lactate production, energy production, and mitochondrial activity.

In Vitro Embryo Production and Blastocyst Cell Numbers

MATERIALS AND METHODS Unless specified, all chemicals were obtained from Sigma-Aldrich.

Glucose, Lactate, ATP, and ADP Levels After 23 h of culture, COCs were removed. Half were mechanically denuded of their cumulus vestments to generate cumulus-devoid oocytes (CDOs), and groups of five COCs/CDOs were snap-frozen in 10 ll of wash medium. Spent media were also snap-frozen, and COCs/CDOs and spent media were stored at 808C. Glucose and lactate concentrations in 50 ll of spent media (derived from 10 COCs cultured in 100 ll) were determined using a Hitachi 912 chemical analyser (F. Hoffmann-La Roche Ltd.), and six media samples were measured per treatment group. Glucose uptake and lactate production were expressed as pmol COC1 h1. ATP/ADP levels were determined using a commercial colorimetric kit (ApoSENSOR ADP/ATP kit; Biovision) per the manufacturer’s instructions. Eight groups of COCs/CDO were measured per treatment group (five COCs/CDO per well).

Oocyte Collection and Culture Immature cow COCs were aspirated from abattoir-derived ovaries using an 18-gauge needle attached to a 10-ml syringe. Compact COCs with intact cumulus vestments greater than three cell layers and ungranulated ooplasms were selected in undiluted follicular fluid, then washed briefly once in VitroWash (IVF Vet Solutions) with 4 mg/ml of fatty acid-free bovine serum albumin (FAF BSA; ICPbio Ltd.) and once in corresponding IVM media. Base IVM medium was bicarbonate-buffered TCM199 (ICN Biochemicals) with 4 mg/ml of FAF BSA. IVM medium contained 5.6 mM glucose, 0.5 mM pyruvate, 0.68 mM glutamine, and no lactate. Treatment groups were as follows: 1) without FSH and rhBMP15, which used base IVM medium; 2) with FSH, which used base IVM medium plus 0.1 IU/ml of FSH (Puregon; Organon); 3) with rhBMP15, which used base IVM medium plus 100 ng/ml of rhBMP15 (a concentrated preparation of human recombinant BMP15 pro/ mature complex produced in 293T cells [30, 31]); and 4) with FSH and rhBMP15, which used base IVM medium plus 0.1 IU/ml of FSH plus 100 ng/ ml of rhBMP15.

Autofluorescence After 0 or 23 h of culture, live and intact COCs were washed briefly once in wash medium and transferred into 5 ll of VitroWash plus 4 mg/ml of FAF

TABLE 1. Embryo development of COCs matured with or without FSH and rhBMP15. Percentage of blastocyst/cleaved* FSH (0.1 IU/ml) þ þ

rhBMP15 (100 ng/ml) þ þ

Cleavage (% 2 cells/total)* 79.6 86 77.8 77.7

6 6 6 6

a

1.8 3.7a 3.6a 2.7a

Total 28.4 44.4 41.0 51.5

6 6 6 6

Expanded b

7.4 3.9bc 2.9bc 5.4c

22.3 25.3 25.9 33.2

6 6 6 6

Hatched d

5.5 1.2d 4.7d 5.6d

10.6 11.2 17.1 19.6

6 6 6 6

3.1e 1.9e 2.5e 2.7e

* Data represent the mean 6 SEM. a–e Different superscript letters within columns indicate significant differences (P , 0.05).

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The developmental competence of COCs following IVM in the presence of absence of FSH, rhBMP15, or both was determined by assessing the rate of embryo development following in vitro fertilization (IVF) and embryo culture, as previously described [18]. After 23 h of IVM, COCs were washed briefly once in wash medium (VitroWash plus 4 mg/ml of FAF BSA), once in IVF medium (VitroFert [IVF Vet Solutions] plus 4 mg/ml of FAF BSA plus 10 IU/ ml of heparin), and then transferred into pre-equilibrated IVF drops (40–50 COCs in 500 ll of IVF medium) overlaid with mineral oil. Thawed sperm from a single sire with proven fertility was prepared using a discontinuous Percoll gradient (45%:90%; GE Healthcare) and added to IVF wells with COCs at a final concentration of 1 3 106 sperm/ml. After 24 h of culture (Day 1), presumptive zygotes were mechanically denuded of cumulus cells using a finely pulled glass pipette, and groups of five presumptive zygotes were transferred into 20 ll of cleavage medium (VitroCleave [IVF Vet Solutions] plus 4 mg/ml of FAF BSA) and cultured at 38.58C in 6% CO2, 7% O2, nitrogen balance. On Day 5 of culture, embryos were transferred into blastocyst medium (VitroBlast [IVF Vet Solutions] plus 4 mg/ml of FAF BSA), and embryo development was assessed on Day 8. Five replicate experiments were performed with 40–50 COCs per treatment per replicate. Blastocysts were collected, and trophectoderm (TE) and inner cell mass (ICM) cell numbers were determined using a published protocol [32]. The zona pellucida was removed by incubating embryos in 0.5% pronase at 378C. Embryos were washed briefly in wash medium minus protein and cultured in 10% 2,4,6-trinitrobenzene sulfonic acid for 10 min at 48C in the dark. Embryos were then incubated in anti-2,4-dinitrophenol (1:10) for 10 min at 378C, followed by a 10-min incubation in complement (2 lg/ml of propidium iodine:guinea pig serum, 1:1). Embryos were transferred to and incubated in 25 lg/ml of Hoechst 33342 (bisbenzimide) in ethanol at 48C overnight. Following a brief wash in 100% ethanol, embryos were transferred in minimum ethanol to 3-ll drops of 100% glycerol on microscope slides, after which a coverslip was added and slight pressure applied to the coverslip. Staining was visualized using an epifluorescence microscope (excitation, 340–380 nm; emission, 440– 480 nm), and the TE cells (pink) and ICM cells (blue) within the embryo were counted. Five embryos per treatment, from three replicates, were collected and stained.

COC METABOLISM 6 FSH 6 rhBMP15 TABLE 2. ICM, TE, and total cell numbers of Day 8 embryos following oocyte maturation with or without FSH and rhBMP15. FSH (0.1 IU/ml) þ þ

rhBMP15 (100 ng/ml) þ þ

ICM* 38.5 41.1 35.5 36.9

6 6 6 6

TE*

2.7a 3.5a 3.1a 2.3a

70.6 97.4 99.4 76.3

6 6 6 6

Total cells*

7.2b 14.1b 9.7b 7.1b

109.1 138.6 134.9 113.3

6 6 6 6

ICM:total cells*

8.6c 15.9c 10.6c 9.2c

0.37 0.31 0.27 0.33

6 6 6 6

0.02d 0.02df 0.02ef 0.01df

* Data represent the mean 6 SEM. a–f Different superscript letters within columns indicate significant differences (P , 0.05). conditions as described above. Ten COCs/DOs were measured per treatment group and time point.

BSA overlaid with mineral oil in glass-bottom confocal dishes (Cell E&G). The fluorescent intensity of oxidized flavin adenine dinucleotide (FADþþ) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) were determined using green (excitation, 473 nm; emission, 490–590 nm) and blue (excitation, 405 nm; emission, 420–520 nm) filters, respectively. Images were captured at 403 magnification using a Fluoview FV10i confocal microscope (Olympus), and the microscope and image settings remained constant. Mean fluorescence intensity within the image of the oocyte was measured using Image J software (National Institutes of Health). Fluorescence intensity measures were normalized to a fluorescence standard (Inspeck; Molecular Probes). Autofluorescence was also determined in DOs after 23 h of culture. Before IVM, cumulus cells were mechanically removed, and groups of 10 DOs were cultured in the presence or absence of rhBMP15, FSH, or both under the same

Statistical Analyses

RESULTS Oocyte Developmental Competence (In Vitro Embryo Production) At the completion of IVM (23 h), COCs cultured in the presence of FSH had visibly greater cumulus expansion compared to those cultured in the presence of rhBMP15 and in the control (without rhBMP15 and FSH) group. Despite the lack of expansion in the groups with no FSH, no significant differences were found in cleavage rates (Table 1). On Day 8 of embryo development, the combination of FSH and rhBMP15 supplementation during IVM yielded the highest blastocyst rate (P , 0.05) (Table 1). However, no differences were found in the proportion of expanded and hatched blastocysts between treatment groups. Cell allocation within expanded blastocysts was determined. No differences were found in the number of ICM and TE cells within blastocysts between treatment groups. However, the ICM:total cell ratio was significantly lower in the blastocysts from the with-rhBMP15 group compared to the control group (P , 0.05) (Table 2), and this was the result of a trend for TE cell numbers to be lower in the group cultured with rhBMP15 (P ¼ 0.065). Glucose and Lactate Metabolism Glucose is the preferred energy substrate of intact COCs during maturation, with a large proportion of consumed glucose metabolized via glycolysis, producing lactate. Hence, glucose consumption and lactate production of COCs cultured TABLE 3. Linear regression analyses of the relationship between glucose consumption and lactate production of COCs cultured for 23 h with or without FSH and rhBMP15. FSH (0.1 IU/ml) þ þ

FIG. 1. Glucose consumption (A) and lactate production (B) of COCs after 23 h of culture in the presence or absence of FSH, rhBMP15, or both. Bars represent the mean þ SEM, and different lowercase letters indicate significant differences (P , 0.05).

3

rhBMP15 (100 ng/ml)

Slope

r2

P value

þ þ

2.1 1.75 1.66 1.74

0.935 0.992 0.872 0.953

0.002 ,0.001 0.006 0.001

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Statistical differences between treatment groups were determined using a general linear model. Proportional data (embryo development) were arcsine transformed before statistical analyses. Post-hoc analyses were performed using a Bonferroni test. Linear regression analyses were used to determine the relationship between glucose and lactate metabolism. P-values of less than 0.05 were deemed to be significantly different. Standard curves for the ATP/ADP assay were generated using known concentrations and linear regression analyses. Standard curves with r2 values of greater than 0.95 were used. All statistical analyses were performed using PASW Statistics (version 18; IBM, Inc.) software.

SUTTON-MCDOWALL ET AL.

in the presence or absence of FSH, rhBMP15, or both at the cessation of IVM (23 h) was determined. A significant interaction was found between FSH and rhBMP15 in regards to glucose consumption and lactate production (P , 0.05) (Fig. 1). Both glucose consumption and lactate production levels were highest in the group cultured with both FSH and rhBMP15, with a 2.5-fold increase in glucose consumption compared to those cultured without FSH and rhBMP15 (1498.6 6 87.2 vs. 589.9 6 31.1 pmol COC1 h1) (Fig. 1A). However, the presence of rhBMP15 alone did not appear to have an effect on glucose and lactate metabolism compared to the control group (without FSH and rhBMP15 vs. with rhBMP15). The relationship between glucose consumption and lactate production by the COC was based on the metabolism of one molecule of glucose via glycolysis producing two molecules of lactate (lactate:glucose ratio). The presence of rhBMP15 alone decreased the lactate:glucose ratio (Table 3), indicating these COCs had the least lactate production for every glucose molecule consumed, suggesting an alternative fate for the glycolytic intermediates under these conditions (P , 0.001) (Table 3). Regardless of the presence or absence of rhBMP15, FSH stimulation resulted in the greatest glucose uptake and highest lactate:glucose ratio (Table 3), indicating a large proportion of FSH-stimulated glucose consumption in these COCs was metabolized via glycolysis.

ATP and ADP Production Both ATP and ADP concentrations were measured in intact COCs and in oocytes that were cultured as COCs (CDOs) after 23 h of culture in the presence or absence of FSH, rhBMP15, or both. ATP is formed from ADP, and regardless of the presence or absence of FSH, rhBMP15 supplementation significantly reduced ADP levels within the COCs (P , 0.05) (Fig. 2B). Furthermore, the ATP:ADP ratio was 2.5-fold higher in the presence of FSH and rhBMP15 compared to the group cultured without FSH and rhBMP15 (P , 0.05) (Fig. 2C). No significant differences in the ATP levels were found between treatment groups, but a trend was observed for increased ATP levels in COCs cultured with FSH and rhBMP15 (P ¼ 0.05). Intraoocyte ATP and ADP levels in oocytes that were cultured as COCs (CDOs) were also determined, and no significant differences were found between treatments (P . 0.05) (Fig. 2). Autofluorescence after 23 h of IVM Mitochondrial and cytoplasmic REDOX state in the presence or absence or rhBMP15, FSH, or both was also determined by measuring the intensity of two endogenous fluorophores—namely, FAD þþ and NAD(P)H (mitochondrial and cytoplasmic localization) autofluorescence [33]. Using confocal microscopy, the autofluorescence of intact COCs and DOs can be determined as an indicator of cellular REDOX state [34]. After 23 h of culture, COCs cultured with rhBMP15 4

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FIG. 2. ATP (A), ADP (B), and ATP:ADP ratio (C) of COCs and COC-derived DOs (CDOs) after 23 h of culture in the presence of absence of FSH, rhBMP15, or both. Bars represent the mean þ SEM, and different lowercase letters indicate significant differences (P , 0.05).

COC METABOLISM 6 FSH 6 rhBMP15

but without FSH had noticeably higher levels of FAD þþ and NAD(P)H fluorescence (P , 0.05) (Figs. 3B and 4, E and F), but in the presence of FSH and rhBMP15, this was reduced to levels comparable to the group cultured without FSH and rhBMP15 (Fig. 3B). The ratio of intensities of FAD þþ:NAD(P)H (i.e., REDOX ratio) was determined, and regardless of treatment, no significant differences in the REDOX ratio, indicating that the presence of rhBMP15 without FSH did not alter the REDOX state within the oocyte, with increased levels of FAD þþ balanced by increased levels of NAD(P)H (Fig. 3D). Autofluorescence was also measured in DOs after 23 h of culture in the presence of absence of FSH, rhBMP15, or both, and no significant differences were found in FAD þþ and NAD(P)H intensity or cellular REDOX state (P . 0.05) (Fig. 3, A and C).

major OSF expressed and secreted by cow oocytes [15], and the addition of recombinant BMP15 during cow oocyte maturation significantly improves developmental competence [18, 19]. Our earlier study [38] revealed little influence of endogenous OSFs on cumulus cell metabolism during IVM, but this result may have been masked by the presence of FSH [39]. The aim of the present study was not to measure the influence of endogenous OSFs during maturation but, rather, to dissect the influences of exogenous rhBMP15 supplementation and FSH (a stimulator of metabolism) on cow COC developmental competence and metabolism. Oocyte developmental competence was affected by the presence of either FSH or rhBMP15, with the two having a significant additive effect to increase development to more than 50% of cleavage-stage embryos. To our knowledge, this is the first report of improved oocyte development competence using a highly purified OSF. Previous reports from our laboratory used partially purified recombinant BMP15 and/or GDF9 preparations [18–20], and further recent reports have used native OSFs by coculturing with DOs [17, 40, 41]. However, this increase in competence was not paralleled by an increase in cell numbers, in that blastocysts from the group cultured in the presence of both FSH and rhBMP15 had cell numbers similar to controls. We suggest these different recombinant OSF

DISCUSSION Supplementation with FSH during IVM is known to have a significant influence on cumulus cell metabolic activity [35]. In addition, OSFs regulate cumulus cell behavior, especially metabolic activity, which in turn increases oocyte developmental competence [36, 37]. BMP15 has been identified as a 5

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FIG. 3. Autofluorescence (FADþþ and NAD(P)H) and FADþþ:NAD(P)H ratio of DOs and COCs after 23 h of culture in the presence of absence of FSH, rhBMP15, or both. A) Autofluorescence of DOs. B) Autofluorescence of oocytes within COCs. C) FADþþ:NAD(P)H ratios of DOs. D) FADþþ:NAD(P)H ratios of oocytes within COCs. Bars represent the mean þ SEM, and different lowercase letters indicate significant differences (P , 0.05).


Downloaded from www.biolreprod.org. FIG. 4. Autofluorescence (FADþþ and NAD(P)H) within the oocytes of cumulus oocyte complexes (COCs) following 23 h of culture without FSH and rhBMP15 (A and B), with FSH (C and D), with rhBMP15 (E and F), and with FSH and rhBMP15 (G and H). A, C, E, and G represent FADþþ, and B, D, F, and H represent NAD(P)H.

lism such that, in general, there appears to be a poor relationship between the metabolic outputs measured and oocyte developmental competence. For example, glucose consumption and lactate production by COCs was stimulated by FSH, indicating stimulation of glycolytic activity within the COC. In contrast, rhBMP15 supplementation decreased the

preparations during IVM have differing abilities to program subsequent embryo developmental mitogenesis. Table 4 summarizes the effects of the presence or absence of FSH, rhBMP15, or both during IVM on the metabolic outputs measured. Treating COCs with FSH or rhBMP15 improved development but differentially affected COC/oocyte metabo6

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COC METABOLISM 6 FSH 6 rhBMP15 TABLE 4. Changes in parameters relative to the control group (without FSH and rhBMP15). Treatment* Parameter Blastocyst development Glucose uptake Lactate production Conversion from glucose to lactate ATP (COC) ADP (COC) ATP:ADP (COC) ATP (CDO) ADP (CDO) ATP:ADP (CDO) FADþþ (CDO) NAD(P)H (CDO) FADþþ:NAD(P)H (CDO) FADþþ (DO) NAD(P)H (DO) FADþþ:NAD(P)H (DO)

FSH

rhBMP15

FSH þ rhBMP15

" " " " #

" # # # ## " " "

"" " " " ## ""

* " ¼ increase; "" ¼ large increase; ¼ no change; # ¼ decrease; ## ¼ large decrease.

proportion of glucose consumed to lactate produced via glycolysis but increased intraoocyte activity through the electron-transport chain/oxidative phosphorylation, as indicated by higher FAD þþ intensity within oocytes of COCs (Table 4). Furthermore, a significant effect of rhBMP15 supplementation on energy production was found, with the presence of rhBMP15 reducing ADP levels and increasing the ATP:ADP ratio within COCs, reflective of increased metabolic activity and cell proliferation. These data demonstrate that FSH and rhBMP15 stimulate different metabolic pathways in COCs—namely, glycolysis in cumulus cells and oxidative phosphorylation in oocytes, respectively. Perhaps unexpectedly, these two protein ligands interact when together to balance the metabolic change induced by the other protein. This returns the metabolic state to ‘‘equilibrium’’ within the COC when stimulated by both of these promoters of oocyte competence. We have previously reported that glucose, lactate, pyruvate, and oxygen metabolism by cow cumulus cells at different stages of maturation were not affected by endogenous OSFs, with no significant differences seen between the activities of COCs, OOXs, and OOXs and DOs [38]. This agrees with the present study, in which FSH stimulation increases total glucose consumption and lactate production at the completion of IVM, regardless of rhBMP15 treatment. In contrast, Sugiuria et al. [27, 28] saw significant changes in the mRNA expression of glycolytic enzymes and glycolytic activity in mouse OOX vs. OOX plus native or recombinant OSF. The present data align themselves to this observation, in as much as in the absence of FSH, exogenous BMP15 has a significant metabolic influence. However, as we have not used an OOX model here, it remains to be determined if endogenous OSFs influence glycolytic metabolism in cattle COCs. The present data reveal that exogenous rhBMP15 certainly affects oocyte metabolism, but this effect can only be due to the influence of rhBMP15 on cumulus cells. Differences exist between the two studies, the most pertinent being that the mouse experiments were conducted using phosphodiesterase inhibitors that arrest maturation [27, 28], compared to a spontaneous maturation system used in the previous [38] and current studies.

ACKNOWLEDGMENT The authors thank the members of the oocyte biology and early development groups (UoA) and SEMEX Australia for the kind donation of bull sperm.

REFERENCES 1. Eppig JJ. Intercommunication between mammalian oocytes and companion somatic cells. Bioessays 1991; 13:569–574. 2. Albertini DF, Combelles CM, Benecchi E, Carabatsos MJ. Cellular basis for paracrine regulation of ovarian follicle development. Reproduction 2001; 121:647–653. 3. Gilchrist RB, Ritter LJ, Armstrong DT. Oocyte-somatic cell interactions during follicle development in mammals. Anim Reprod Sci 2004; 82–83: 431–446. 4. Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its

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Regardless of treatment, we observed the majority of glucose consumed was metabolized via glycolysis, as indicated by the levels of lactate production. However, treatment with rhBMP15 alone (with rhBMP15 but without FSH) resulted in a proportion of glucose being metabolized via a nonglycolytic pathway. We propose that rhBMP15 (without FSH) stimulates a preference for the flow of cumulus cell-derived glycolytic intermediates for oxidative phosphorylation within the oocyte, leading to reduced lactate production by surrounding cumulus cells. This is supported by the increased oocyte FAD þþ levels observed in rhBMP15-treated COCs. FAD þþ is exclusively found within the mitochondria and is involved in the electrontransport chain. A REDOX ratio can be determined by FAD þþ:NAD(P)H, with high/increasing ratios indicating increased oxidative metabolic activity within cells [42]. Nicotinamide adenine dinucleotide (NADH) is found in both the cytoplasm and the mitochondria and functions as an reducing agent in numerous metabolic pathways, such as glycolysis, beta-oxidation, the TCA cycle, and oxidative phosphorylation. In contrast, nicotinamide adenine dinucleotide phosphate (NADPH) is found in smaller quantities within the cytoplasm and is reduced within the oxidative phase of the pentose phosphate pathway and by isocitrate dehydrogenase within the TCA cycle. The major role of NADPH is in the reduction of oxidized glutathione (GSSG to GSH), an important antioxidant. Both NADH and NADPH are indistinguishable in regards to their autofluorescence. We measured FAD þþ and NAD(P)H within the oocyte of COCs and DOs after 23 h of culture in the presence or absence of FSH, rhBMP15, or both and saw significantly higher levels of both FAD þþ and NAD(P)H intensity in the oocytes of COCs cultured with rhBMP15 alone. However, this increase in intensity in response to rhBMP15 was not seen in DOs, indicating that cumulus cells mediate the increase in FAD þþ and NAD(P)H. However, regardless of treatment, no change was observed in the REDOX ratio, demonstrating the importance of maintaining a metabolic equilibrium of REDOX throughout development [43]. In conclusion, FSH and rhBMP15 supplementation during IVM mediates changes in energy production, mitochondrial activity, and REDOX state via different metabolic pathways within the two cellular components of the COC, which is largely mediated through cumulus cells. These effects of FSH and rhBMP15 on COC and oocyte metabolism lead to improvement in oocyte developmental competence. The present results suggest that using metabolic measures of COCs to determine oocyte developmental competence can be significantly influenced by the presence of growth factor ligands that act on independent pathways.


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