Prematuration with Cyclic Adenosine Monophosphate Modulators ...

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Jun 25, 2014 - Hai-Tao Zeng,3,4 Dulama Richani,3 Melanie L. Sutton-McDowall,3 Zi Ren,3,5 Johan ... Stokes,7 Robert B. Gilchrist,3 and Jeremy G. Thompson2,3 ...... Webb RJ, Marshall F, Swann K, Carroll J. Follicle-stimulating hormone.
BIOLOGY OF REPRODUCTION (2014) 91(2):47, 1–11 Published online before print 25 June 2014. DOI 10.1095/biolreprod.114.118471

Prematuration with Cyclic Adenosine Monophosphate Modulators Alters Cumulus Cell and Oocyte Metabolism and Enhances Developmental Competence of In VitroMatured Mouse Oocytes1 Hai-Tao Zeng,3,4 Dulama Richani,3 Melanie L. Sutton-McDowall,3 Zi Ren,3,5 Johan E.J. Smitz,6 Yvonne Stokes,7 Robert B. Gilchrist,3 and Jeremy G. Thompson2,3 3

Research Centre for Reproductive Health, Robinson Institute, and Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, South Australia, Australia 4 Center for Reproductive Medicine, Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China 5 Center for Reproductive Medicine, General Hospital of Guangdong, Guangzhou, People’s Republic of China 6 Follicle Biology Laboratory, Center for Reproductive Medicine and Medical School, Free University Brussels (VUB), Brussels, Belgium 7 School of Mathematical Science, The University of Adelaide, Adelaide, South Australia, Australia

Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and folliclestimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/ cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulusoocyte complexes (COCs) were subjected to a 1 h prematuration phase 6 the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM 6 FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P , 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio,

ATP, cAMP, FSH, glycolysis, IVM, mitochondria, oocyte metabolism

INTRODUCTION In vitro maturation (IVM) of mammalian oocytes is an important assisted reproductive technology and research tool in reproductive and developmental biology. IVM is widely used to generate mature oocytes for a range of applications, including human infertility treatment, embryo production for livestock artificial breeding programs, and cloning, stem cell, and transgenic technologies [1]. Despite its clinical and financial advantages over the commonly used reproductive technique of in vitro fertilization (IVF), a major drawback of IVM is its significantly lower efficiency compared with conventional IVF, including decreased preimplantation embryo development, pregnancy, and live birth rates [2–4]. Over the past decade, new insights have emerged surrounding the intricate bidirectional communication axes between the oocyte, follicular somatic cells, and the endocrine system. These new insights include: oocyte regulation of granulosa and cumulus cell differentiation and function through the secretion of soluble paracrine growth factors [5]; identification of the significant role epidermal growth factor (EGF)-like peptides, the EGF receptor, and their intracellular second messengers play in mediating the ovulatory luteinizing hormone (LH) signal through the follicle [6]; and new perspectives on the participation of cyclic nucleotides, phosphodiesterases, and gap-junctions in the regulation of oocyte meiotic maturation [7–10]. The second messenger cyclic adenosine monophosphate (cAMP) plays an important role in the control of meiotic progression in mammalian, and some invertebrate, oocytes [11–13]. Cyclic AMP is synthesized within the oocyte and additionally by the mural granulosa and cumulus cells of the follicle and is supplied to the oocyte via gap-junctions [14–17].

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Supported by Cook Medical with the Cook Medical Adelaide Fellowship awarded to H.-T.Z. and Cook Medical collaborative research grants awarded to The University of Adelaide and the Free University of Brussels. This work was also supported by Australian National Health and Medical Research Council (NHMRC) Project Grants awarded to R.B.G. and J.E.J.S. (APP1007551) and J.G.T., R.B.G., and M.S.M. (APP1008137), NHMRC Senior Research Fellowships (APP1023210, APP627007), the National Natural Science Foundation of China (30901605, 81000248), and by the Belgian Institute for the Promotion of Innovation in Science and Industry (IWT-070719). 2 Correspondence: Jeremy Thompson, School of Paediatrics and Reproductive Health, Faculty of Health Sciences, The University of Adelaide, Level 2, Medical School South, Frome Road, Adelaide, SA 5005, Australia. E-mail: [email protected] Received: 9 February 2014. First decision: 17 March 2014. Accepted: 13 June 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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and glutathione levels (P , 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes.

ABSTRACT

ZENG ET AL.

the glycolytic and hexosamine biosynthesis metabolic pathways [38], and conditions that trigger meiotic maturation are associated with an increased flux through the pentose phosphate pathway [46]. A comprehensive examination of COC and oocyte metabolism in mouse COCs during IVM in comparison to in vivo matured oocytes has yet to be conducted. This study aims to remedy this by examining extensively the metabolic profile of COCs and oocytes in addition to the in vitro developmental outcomes of in vitro matured oocytes. The study included IVM conditions that combined prematuration of COCs with IBMX and forskolin to modulate cAMP/cGMP levels as well as FSH during IVM, and compared many of the measured parameters within in vivo matured oocytes. Furthermore, oocyte meiotic stability (correct pairing, recombination, and segregation of chromosomes) and cumulus expansion were also examined. We hypothesized that the metabolic profile of COCs and oocytes will alter as a consequence of treatments known to enhance developmental competence.

Within the oocyte, cAMP both inhibits and induces oocyte meiotic maturation. A moderate concentration of cAMP is maintained within the oocyte to maintain meiotic arrest; however, the ovulatory LH surge induces an acute and transient increase in oocyte cAMP that triggers meiotic resumption [18, 19]. In contrast, oocytes that are removed from their antral follicle and matured in vitro exhibit a rapid drop in cAMP and cyclic guanosine monophosphate (cGMP), which leads to activation of the oocyte phosphodiesterase 3 (PDE3), a potent cAMP hydrolyzing enzyme [7, 10, 19]. This loss of cyclic nucleotides leads to protein kinase A inactivation within the oocyte, which culminates in meiotic resumption [7, 10]. IVM cumulus-oocyte complexes (COCs) are known to suffer a significant loss in cAMP and, as such, the oocytes undergo spontaneous maturation and consequently exhibit decreased developmental competence [19]. It is well documented that the artificial modulation of cAMP within IVM COCs through the use of pharmacological agents enhances oocyte developmental competence as evidenced by improved subsequent developmental outcomes, including embryo and fetal development [12, 19–29]. Prevention of cAMP degradation in IVM COCs increases oocyte developmental competence by prolonging gap-junctional communication and hence the exchange of regulatory molecules and metabolites between the oocyte and its surrounding cumulus cells [12, 19, 25, 30, 31]. Cyclic AMP modulation during IVM has been achieved through the use of nonspecific PDE inhibitors such as 3isobutyl-1-methylxanthine (IBMX), which also prevents the hydrolysis of cGMP, or agents that increase COC cAMP such as dibutyryl cAMP or forskolin (an adenylate cyclase activator that stimulates cAMP synthesis) [12, 19–21, 29, 32, 33]. We have developed a new IVM system that modulates cAMP prior to maturation [12, 19]. This system involves prematuration of COCs with IBMX and forskolin to generate a large (.100fold) and rapid increase in specifically cAMP levels (whilst simultaneously maintaining cGMP levels) that mimics the spike seen in vivo in response to the ovulatory LH surge [14, 18, 19, 34]. Importantly, this prevents spontaneous meiotic resumption and slows the associated loss of oocyte-cumulus gap-junctional communication during maturation. Metabolism of the mature COC is a dynamic process during oocyte maturation, with bovine COCs consuming 2-fold more glucose, oxygen, and pyruvate than immature COCs [35, 36]. COCs preferentially use glucose as an energy substrate [37]. Oocytes have a poor capacity for glucose uptake and hence rely on glucose metabolism by the cumulus cells, which provide the oocyte with energy substrates such as pyruvate and lactate via gap-junctions [38]. Hence, the premature cessation of gapjunctional communication that occurs in standard IVM COCs likely affects its metabolic activity; there is abundant evidence that COC metabolism is an important contributing factor to oocyte developmental competence. Glucose is predominantly metabolized via glycolysis for ATP synthesis and the production of metabolites including pyruvate and lactate [37, 39]; pyruvate is involved in ATP synthesis as it is metabolized via the tricarboxylic acid (TCA) cycle that is coupled with mitochondrial oxidative phosphorylation [40]. FSH is routinely used during IVM because it improves oocyte developmental competence relative to spontaneous maturation [41] and stimulates cumulus expansion [42]. FSH has a major impact on metabolism of cumulus cells during IVM by acting on several metabolic pathways. A series of studies detailing the influence of energy substrates on oocyte maturation have shown that FSH induction of meiotic maturation, but not spontaneous maturation, requires the presence of glucose [43–45]. FSH is known to stimulate both

MATERIALS AND METHODS

Oocyte Collection and Culture Immature female 129/Sv inbred mice (21–24 days of age) were used and housed in a temperature- and light-controlled environment. For IVM, immature COCs were collected 46 h after administration of 5 international units of equine chorionic gonadotrophin (eCG) (Folligon; Intervet). For in vivo oocyte maturation, 5 international units of human chorionic gonadotrophin (hCG) (Organon) was administered 46 h following eCG priming, and COCs were collected 16 h later. COCs were freed from antral follicles into HEPES-buffered alpha minimal essential medium (aMEM) (12000-022; Invitrogen) supplemented with 3 mg/ml bovine serum albumin (BSA) (ICP Biologica) and their respective treatments. Treatment groups were: 1) IVM without FSH (spontaneous maturation); 2) IVM with FSH (Puregon); 3) prematuration with cAMP-elevating agents (defined below) þ IVM without FSH; 4) prematuration with cAMP-elevating agents þ IVM with FSH. The 1 h prematuration phase (termed pre-IVM treatment) involved incubation of COCs in HEPES-aMEM 6 the adenylate cyclase activator forskolin (50 lM) and the PDE inhibitor IBMX (50 lM) at 378C for 1 h in atmospheric O2. At the end of the pre-IVM phase, COCs were washed twice in bicarbonate-buffered aMEM containing 3 mg/ml fatty acid-free (FAF) BSA, 1 mg/ml fetuin, and their respective IVM treatments, and transferred to drops of the same medium and cultured for 16 h at 378C with 5% CO2 in air. Data presented represents the mean of four replicate experiments.

Cumulus Expansion Assessment IVM COCs were cultured for 16 h in media containing BSA. In vivo COCs were collect 16 h post-hCG administration. Blinded scoring by an experienced assessor of the cumulus expansion index was then performed using the reported scoring system [47]. At least 109 COCs were assessed per treatment group over four replicate experiments.

IVF and Embryo Culture The effect of treatments on oocyte developmental competence was assessed by examining the capacity of the oocyte to support preimplantation embryo development following IVM. IVF and embryo culture were performed as previously described [19]. All the media used were from the Vitro Research Media series generously donated by Cook Medical (Brisbane, Australia). Data presented represent the mean of four replicate experiments. At least 193 oocytes were used per treatment group over three replicate experiments.

Blastocyst Differential Staining On Day 6 of embryo culture, blastocysts and hatching blastocysts were differentially stained for inner cell mass (ICM) and trophectoderm (TE) cells as

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Mice were maintained in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and with the approval of the University of Adelaide Animal Ethics Committee. Unless otherwise stated, all the reagents used were purchased from Sigma Aldrich.

EFFECT OF cAMP AND FSH ON IVM COC METABOLISM oocytes from each treatment were collected in 10 ll of ice-cold distilled water and snap-frozen and stored at 808C until analysis. ATP and ADP content in oocytes was measured using the bioluminescence ApoSENSOR ATP Assay Kit (BioVision) according to the manufacturer’s instructions. A duplicated fivepoint ATP (Roche Diagnostics) standard curve (0, 0.5, 1, 2, and 3 lM) was used to calculate sample concentrations. ATP and ADP were measured in COCs from three replicates with at least five samples per treatment group per replicate experiment.

previously described by Hardy et al. [48]. Briefly, zona pellucidae were dissolved by incubation in 0.5% pronase at 378C. Blastocysts were then transferred to 10 mM tri-nitrobenzenesulfonic acid for 10 min at 48C, followed by incubation in 0.1 mg/ml of anti-dinitrophenyl-BSA for 10 min at 378C, then 5 min at 378C in guinea pig serum containing 10 lg/ml of propidium iodide. Blastocysts were then stained overnight in 6 lg/ml of bisbenzimide in ethanol at 48C. Blastocysts were washed in 100% ethanol and mounted on siliconized slides in glycerol drops. The number of pink (i.e., TE) and blue (i.e., ICM) fluorescent cells was assessed, blinded to treatment group, using a fluorescent microscope and Hg lamp, with an ultraviolet excitation wavelength dichroic mirror at 4003 magnification (TE 2000-E; Nikon; excitation 340–380 nm, emission 440–480 nm). At least 29 blastocysts were stained per treatment group over three replicate experiments.

Autofluorescence Detection of Reduced Nicotinamide Adenine Dinucleotide (Phosphate) and Oxidized Flavin Adenine Dinucleotide

For staining of a-tubulin, oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Following permeabilization with 0.5% Triton X-100 at room temperature for 20 min, oocytes were blocked in 1% BSA-supplemented PBS for 1 h and incubated overnight at 48C with 1:200 anti-a-tubulin antibody. Following three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each, oocytes were incubated with 1:100 fluorescein isothiocyanate-conjugated immunoglobulin G for 1 h at room temperature. Oocytes were then washed in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 and were costained with Hoechst 33258 (10 lg/ml in PBS). Oocytes were then mounted on glass slides and examined using a FluoView FV10i Confocal Microscope (Olympus). At least 55 oocytes were examined per treatment group over three replicate experiments. Spindle morphology was classified as normal when there was a barrel-shaped structure with slightly elongated poles, organized microtubules, and the chromosomes were arranged in a compact metaphase plate at the equator of the spindle. Spindle structure was recorded as abnormal when there was a reduction in the longitudinal dimension of the spindle, complete absence or remnant of dispersing spindle, or chromosomes displaced from the plane of the metaphase plate.

JC-1 Staining The mitochondrial membrane potential sensitive fluorescent dye, JC-1 (Molecular Probes), was used to measure the activity of oocyte mitochondria [52]. Following 16 h IVM, oocytes were denuded and incubated for 25 min in HEPES-buffered aMEM containing their respective treatments and 2 lM JC-1 dye. JC-1 exhibits potential-dependent accumulation in mitochondria where it aggregates as J-aggregates in high-polarized mitochondria (dWm  140 mV) that fluoresce red. JC-1 accumulates as multimers in low-polarized mitochondria (dWm  100 mV) that fluoresce green. The red:green fluorescence intensity ratio is used as an indicator of relative mitochondrial polarization, and thus mitochondrial activity, whereby an increased ratio indicates increased mitochondrial activity [53]. Oocyte fluorescence was observed using a narrow green filter (490–540 nm) and a narrow red filter (570–620nm), using a FluoView FV10i Confocal Microscope. Laser power and photomultiplier settings were kept constant for all the experiments. A single optical scan through the center of the oocyte was used for the analysis. The images were processed, and fluorescence intensities in oocytes were measured and analyzed using the accompanying software of the FluoView FV10i Confocal Microscope. Three replicate experiments were performed using 10–20 oocytes per treatment group per replicate.

Glucose and Lactate Quantification Following 16 h of IVM, spent COC culture media were collected, snapfrozen in liquid nitrogen, and stored at 808C until analysis. Glucose and lactate concentrations in 50 ll of spent media samples (derived from 15 COCs cultured in 100 ll media drops) were measured using a Hitachi 912 chemical analyzer (F. Hoffmann-La Roche Ltd.). A minimum of 15 media samples were measured per treatment group over three replicate experiments. Glucose consumption and lactate production are expressed as pmol/COC/h.

Oxygen Consumption Assay COC oxygen consumption was assayed from 0–4 h of IVM as previously described [49]. This method is based on the fluorescent properties of pyrene, an oil-soluble compound that is excited at 340 nm and whose fluorescence dissipates in a linear fashion in the presence of increasing O2 concentration. Five microliter PCR-grade micropipettes (Drummond Scientific) and stainless steel plungers were used. One microliter of 1 mM pyrene, dissolved in mineral oil, was drawn into the micropipette, followed by 2 ll of HEPES-buffered maturation media containing five COCs. An airtight seal was made at the open end of the chamber, and the plunger was fixed using sealing wax. Negative control (0% O2) chambers were constructed in a similar manner, but with 0.1 M sodium L-ascorbate and sodium hydroxide in HEPES-buffered maturation media (equilibrated overnight to remove O2) replacing the media; positive controls (20% O2) were constructed with media alone. The fluorescence emission of pyrene at the pyrene-media and the pyrene-plunger interfaces were measured using a fluorophotometric-inverted microscope (Leica). Fluorescence measurements were taken at intervals of 30 min over a 4 h period. Chambers were maintained at 378C between measurements. The amount of oxygen consumed by each COC was determined assuming a linear relation between fluorescence and oxygen concentration and then using a mathematical model that describes the diffusion gradient of O2 in mineral oil as a function of O2 depletion in the adjacent medium containing the COCs [50]. Three replicate experiments were conducted representing five to eight samples per treatment group per replicate experiment.

Reduced Glutathione Measurement A membrane-permeable fluorescence probe, monochlorobimane (MCB) (Molecular Probes), is used as a sensitive and specific probe to analyze intracellular reduced glutathione (GSH) content in living cells [54, 55]. Following 16 h IVM, oocytes were denuded and exposed to 100 lM MCB in VitroWash medium containing 3 mg/ml FAF-BSA and their respective treatments for 30 min. Excitation and emission wavelengths of 458 and 475 nm, respectively, were used to detect the fluorescent GSH-bimane adduct. Laser power and photomultiplier settings were kept constant for all the experiments. A single optical scan through the center of the oocyte was used for the analysis. Images were processed, and oocyte fluorescence intensities were measured and analyzed using the accompanying software of the FluoView FV10i Confocal Microscope. Three replicate experiments were performed using 10–20 oocytes per treatment group per replicate.

Measurement of Intracellular Reactive Oxygen Species Intracellular reactive oxygen species (ROS) production was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), a nonfluorescent probe for intracellular ROS detection, which is converted to the fluorescent compound 2,7-dichlorofluorescein when oxidized by intracellular ROS [56]. This probe is highly reactive with hydrogen peroxide and has been used in evaluating ROS generation in mammalian cells [57]. Following 16 h IVM, oocytes were denuded and exposed to 10 lM DCFH-DA in VitroWash media containing 3 mg/ml of FAF-BSA and their respective treatments for 30 min.

ATP/ADP Assay To determine ATP and ADP levels within MII oocytes, COCs were denuded of cumulus cells following 16 h IVM. For each sample preparation, 10

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After 16 h of culture, live intact COCs were denuded and oocytes were transferred on to glass-bottom confocal dishes (Cell E&G) in 5 ll of VitroWash medium containing 3 mg/ml FAF-BSA and their respective treatments, and overlaid with mineral oil. The fluorescent intensities of oxidized flavin adenine dinucleotide (FAD) 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 [51]. Images were captured at 903 magnification using a FluoView FV10i confocal microscope (Olympus); microscope and image settings were kept constant across replicate experiments. Mean fluorescence intensities within the images of the oocytes were quantified using Image J software (National Institutes of Health). Fluorescence measurements were performed on at least five oocytes per treatment group over three replicate experiments.

Immunofluorescent Microscopy

ZENG ET AL. Oocyte fluorescence intensity was measured at excitation and emission wavelengths of 500 and 529 nm, respectively. The arbitrary units, relative fluorescence units, were based directly on fluorescence intensity. Three replicate experiments were performed where five oocytes were examined per treatment group per replicate.

However, despite this, blastocyst cell numbers from combined pre-IVM treatment þ FSH IVM COCs remained significantly lower than those from blastocysts from in vivo matured COCs (P , 0.05).

Statistical Analyses

FSH Exhibited a More Profound Effect on Glucose Metabolism than Pre-IVM Treatment

Statistical analyses were conducted using the Statistical Package for Social Sciences 18.0 (SPSS). Treatment effects on spindle morphology were assessed by chi-square test. For all the other data, one-way ANOVA followed by either Dunnett or Bonferroni multiple-comparison post hoc tests were used to identify individual differences between means. Developmental data (including proportion of MII oocytes) was transformed by arcsine prior to ANOVA. All the values are presented as means with their corresponding standard error of the mean (SEM). P , 0.05 was considered statistically significant.

RESULTS Combined Pre-IVM Treatment and IVM with FSH Promoted the Greatest Cumulus Expansion As expected, cumulus expansion did not occur in the absence of pre-IVM treatment or FSH during IVM (Fig. 1, A and B). Pre-IVM treatment with forskolin and IBMX significantly increased cumulus expansion compared to control (1.07 6 0.02 vs. 2.06 6 0.07, respectively; P , 0.05), however cumulus was significantly less expanded than with standard IVM with FSH (3.58 6 0.06, P , 0.05). Interestingly, pre-IVM treatment and FSH during IVM had a significant interactive impact on cumulus expansion whereby expansion was significantly greater than with either treatment alone and was statistically similar to in vivo (3.81 6 0.04, P , 0.05). As well as deficient cumulus expansion, a significant reduction in first polar body extrusion (Fig. 1C) and an increase in abnormal spindle structure (Fig, 1, D and E) was observed in the absence of pre-IVM treatment or FSH during IVM following 16 h IVM (P , 0.05). No significant differences in the levels of polar body extrusion and abnormal spindle rate were observed between the pre-IVM treatment and/or IVM with FSH (P . 0.05).

Combined Pre-IVM Treatment and IVM with FSH Yielded Greater Oocyte Energy Production and Oxidative Metabolism than Individual Treatment The ATP and ADP levels were measured in denuded oocytes that were either matured in vivo or cultured as COCs for 16 h 6 FSH 6 pre-IVM treatment (Fig. 5, A and B). Oocyte ATP levels were similar across all the treatment groups with the exception of the combined pre-IVM treatment þ FSH IVM group, which exhibited significantly higher ATP level than the those without a pre-IVM treatment (P , 0.05; Fig. 5A). Corresponding ADP levels yielded by this combined treatment group were lowest amongst all the groups and significantly lower than both FSH treatment alone and in vivo derived COCs (P , 0.05; Fig. 5B). A significant increase (P , 0.05) in the ratio of ATP:ADP, a measure of energy demand, was higher in the combined pre-IVM treatment þ FSH IVM group than all the other IVM treatment groups (P , 0.05) except for the pre-IVM treatment alone (Fig. 5C). Oxygen consumption by intact IVM COCs was measured over 0–4 h. FSH, but not pre-IVM treatment, enhanced oxygen consumption (Fig. 5D). A significant interaction between preIVM treatment and IVM with FSH was found in regards to oxygen consumption (P , 0.05) with the combined pre-IVM treatment and FSH IVM stimulating more consumption than any other treatment. Oocyte mitochondrial activity, as measured by red:green fluorescence JC-1 staining, was significantly higher in oocytes matured in vivo compared with all the IVM treatments (Fig. 5, E and F). Amongst IVM groups, FSH significantly upregulated mitochondrial activity compared with control both in the presence or absence of pre-IVM treatment.

Combined Pre-IVM Treatment and FSH During IVM Yielded the Greatest Subsequent Blastocyst Yield and Quality The effects of either pre-IVM treatment and/or IVM with FSH on subsequent embryo development were determined. Fertilization, cleavage, blastocyst, and hatching blastocyst rates yielded from in vivo matured COCs were significantly higher than from any IVM treatment (Fig. 2; P , 0.05). Unsurprisingly, COCs matured spontaneously (no FSH or pre-IVM treatment), yielded the lowest fertilization, cleavage, blastocyst, and hatching blastocyst rates of all the treatments (P , 0.05). IVM COCs matured with FSH IVM 6 pre-IVM treatment yielded the second highest fertilization and cleavage rates, which were significantly higher than those derived from COCs matured in the absence of FSH (P , 0.05). Pre-IVM treatment significantly increased fertilization and cleavage rates compared to control (spontaneous IVM) regardless of the presence of FSH during IVM (P ,0.05). Most notably, combined preIVM treatment þ FSH IVM yielded significantly higher blastocyst and hatching blastocyst rates compared to IVM with FSH (no pre-IVM) (P , 0.05; Fig. 2, C and D), although the rates were not as high as those from in vivo matured COCs. Blastocysts derived from COCs subjected to combined preIVM treatment þ FSH IVM were also of greater quality than those from COCs subjected to only one treatment or no treatment. This is evidenced by significantly higher numbers of ICM cells and a higher proportion of ICM (Fig. 3; P , 0.05).

Intraoocyte Redox State Was Altered During IVM In order to evaluate the reduction-oxidation of IVM oocytes, the antioxidant GSH was measured using MCB, which forms a fluorescent adduct following an enzyme-catalyzed reaction 4

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Glucose is the preferred energy substrate of COCs during maturation with a large proportion of consumed glucose metabolized via glycolysis, producing lactate. Hence, glucose consumption and lactate production of IVM COCs subjected to pre-IVM treatment and/or IVM with FSH were measured following 16 h of IVM. As expected, COC glucose consumption and lactate production were significantly affected by FSH during IVM with levels significantly higher than in all the treatments devoid of FSH. Intriguingly, pre-IVM treatment did not affect glucose consumption but significantly (P , 0.05) increased lactate production compared to no treatment (spontaneous maturation) (Fig. 4B), with the highest lactate production occurring within the combined prematuration treatment and maturation with FSH. One possible source of this additional lactate is the cytoplasmic conversion of glycolysis-derived pyruvate to lactate to regenerate oxidized NAD þ and maintain the cytoplasmic redox state. Alternatively, aMEM medium contains 1 mM pyruvate, which may also participate in this reaction.

EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

Downloaded from www.biolreprod.org. FIG. 1. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on COCs maturation. Following 16 h IVM or in vivo maturation, cumulus expansion (A and B) was assessed. IVM oocytes were then denuded and fixed for detection of oocyte maturation rate (C) and spindle morphology of mature oocyte (D and E). Data represent the mean 6 SEM of three independent experiments: cumulus expansion rate (A) and percentage of MII oocytes (C). Differences in percentage of abnormal spindles (D) was determined by chi-square analysis. Bars not sharing a common letter are significantly different (P , 0.05). Original magnification 310 (B) and 390 (E).

with GSH [58]. Individually, pre-IVM treatment and FSH IVM significantly increased oocyte MCB fluorescence compared to control (P , 0.05), however, a significant interaction between pre-IVM treatment and FSH IVM was observed (P , 0.05), which yielded significantly higher MCB fluorescence than either treatment alone and was statistically similar to that of oocytes matured in vivo (Fig. 6A).

Mitochondrial and cytoplasmic oocyte redox state was determined by measurement of the autofluorescence of two endogenous fluorophores, FAD (mitochondrial localization) and NAD(P)H (mitochondrial and cytoplasmic localization) following 16 h post-hCG administration or 16 h IVM [59, 60]. FAD fluorescence was highest in the FSH IVM group and lowest in vivo (Fig. 6, B and F; P , 0.05). NAD(P)H 5

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IBMX and the adenylate cyclase activator forskolin followed by a prolonged IVM phase in the presence of FSH and the PDE3-specific inhibitor cilostamide. SPOM generated increased embryo yield from cattle and mouse oocytes and improved fetal survival following mouse embryo transfer [19]. The individual contributions of components of the SPOM system on developmental competence and other aspects of COC biochemistry, however, are yet to be described, although in a follow-up study we demonstrated that the pre-IVM phase alone improves oocyte developmental competence via mechanisms related to oocyte energy metabolism [20]. This study further examines the impact of the IBMX/forskolin prematuration and its relationship with FSH during maturation, on oocyte developmental competence, meiotic stability, and metabolic impact, and compares most measures to in vivo matured oocytes. As glucose uptake, lactate production, and O2 consumption measurements are dependent on differences in concentration over a specific incubation period, it was not feasible to conduct these with in vivo matured oocytes. The LH surge in vivo and FSH in vitro activate the Gs proteins on granulosa and cumulus cells, which then stimulate

autofluorescence of in vivo matured oocytes was significantly lower than that of all IVM treatments (Fig. 6, C and F; P , 0.05). IVM treatments recorded similar NAD(P)H autofluorescence except for combined pre-IVM treatment þ FSH IVM, which stimulated significantly lower fluorescence than control and FSH IVM groups (P , 0.05). FSH stimulated the greatest FAD:NAD(P)H ratio, which was significantly higher than control and in vivo groups (Fig. 6, D and F; P , 0.05). DCFHDA fluorescence, indicative of ROS production, in vivo and during IVM in the absence of FSH was similar; FSH 6 preIVM treatment significantly increased DCFH-DA fluorescence compared to in vivo maturation (Fig, 6, E and F; P , 0.05). DISCUSSION We and others have previously reported that modulation of cAMP levels within mammalian COCs during IVM can substantially improve oocyte developmental competence in several species, including human [12, 19–29]. Our laboratory recently described a combinational IVM system, termed simulated physiological oocyte maturation (SPOM) [19], that involves COC prematuration with the general PDE inhibitor 6

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FIG. 2. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on embryo development. In vitro fertilization (A), Day 2 cleavage rate (B), Day 6 blastocyst rates (blastocysts per cleaved embryos) (C), and Day 6 hatching blastocyst rates (hatching blastocysts per cleaved embryos) (D) were measured following 16 h of IVM or 16 h following hCG. Data represent the mean 6 SEM of three independent experiments representing at least 40 oocytes per replicate. Bars not sharing a common letter are significantly different (P , 0.05).

EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

64]. Individually, both pre-IVM treatment and FSH were equally able to enhance blastocyst development relative to control. However, in combination, they were able to further enhance development, albeit not to the capacity of in vivo matured oocytes; this effect was also evident in blastocyst quality (as evidenced by ICM and total cell numbers). These data demonstrate that combined treatment significantly increases oocyte developmental competence over either treatment alone. It is well established that elevated cAMP levels during IVM enhance and prolongs gap-junctional communication between the oocyte and cumulus cells [12, 14, 15, 19, 31], and because both these treatments elevate and maintain cAMP, prolonged gap-junctional communication is a likely mechanism by which these treatments increase oocyte developmental competence and alter oocyte metabolism. Glucose is a pivotal substrate for COC metabolism and can be metabolized via glycolysis, the pentose phosphate pathway, and the hexosamine biosynthesis pathway [38]. The glycolytic pathway accounts for the majority of glucose metabolized by the COC and facilitates energy production in the form of ATP as well as the production of metabolites readily utilized by the oocyte, most notably pyruvate and lactate [65, 66]. In the

membrane-associated adenylate cyclase to elevate intracellular cAMP levels [61, 62]. Cyclic AMP further activates protein kinases to induce cumulus expansion. Both the pre-IVM treatment and FSH stimulated cumulus expansion; however, the pre-IVM treatment alone was significantly poorer at eliciting expansion. Combined pre-IVM treatment with FSH in IVM led to the greatest IVM cumulus expansion, which was comparable to that seen in vivo. Although both treatments elicit an increase in cyclic nucleotides, the differential levels of cumulus expansion suggest that there are differences in cumulus expansion stimulation that warrant further investigation. Fertilization rates across treatments paralleled the degree of cumulus expansion, which is unsurprising given that expansion facilitates fertilization [63]. Both pre-IVM treatment and FSH improved meiotic competence and reduced spindle abnormalities relative to the control (spontaneous IVM), and meiotic competence was comparable to that of in vivo matured oocytes, suggesting that a large pulse of cAMP (and maintaining cGMP levels) prior to IVM is sufficient to achieve full meiotic competence. Our results confirm the benefit of combining the pre-IVM treatment in conjunction with FSH during maturation [19–21, 7

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FIG. 3. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on embryo quality. Embryo quality at Day 6 was examined by evaluation of the number of inner cell mass (ICM) cells (A), trophectoderm (TE) cells (B), total cells (C), and the ratio of cell number of ICM to total blastocyst cells (D). Data represent the mean 6 SEM of three independent experiments with at least 193 embryos examined per treatment group. Bars not sharing a common letter are significantly different (P , 0.05).

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FIG. 4. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on glucose metabolism. COC glucose consumption (A) and lactate production (B) were measured following the first 4 h of IVM. Data represent the mean 6 SEM of three independent experiments representing at least 15 media samples per treatment group; different letters at the top of the bars indicate significant difference (P , 0.05).

bovine COC, glucose uptake and lactate production is reported to occur approximately in a 1:2 relationship. Furthermore, FSH stimulates glucose uptake in COCs, but this increase is associated with up-regulation of the hexosamine biosynthesis pathway, the pathway that provides the substrate for hyaluronic acid production [63]. In mouse COCs, this relationship appears to be markedly different. Glucose consumption and lactate production does not occur in a 1:2 ratio [67], and prematuration treatment-stimulated cumulus expansion was not associated with an increase in glucose consumption (current study). This suggests that the pre-IVM treatment of COCs with IBMX and forskolin improved embryo yields by altering the flux of glucose through glycolysis. Moreover, oxygen consumption by COCs was increased by both treatments in combination, demonstrating that this model induces increased demand for oxidative metabolism and ATP production. Together, these data point to a much greater dependency of mouse cumulus cells for oxidative metabolism compared with the bovine COC and, together with hexosamine biosynthesis metabolic activity, a significant amount of glucose provides cumulus cells and the oocyte with metabolic intermediates, including ATP. An increase in ATP generation would then meet the large demand 8

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for cAMP synthesis stimulated by the combined prematuration and FSH treatments. This study examined several indicators of energy production, mitochondrial activity, and redox potential within the oocyte because the oocyte is highly dependent on oxidative metabolism for developmental competence [68, 69]. We demonstrated that ATP levels in oocytes did not vary between control and treatments with the exception that more ATP was found in oocytes subjected to combined pre-IVM and FSH treatment, as previously demonstrated [20]. This pattern was also observed in ADP levels, leading to a significant shift in the ATP:ADP ratio by this treatment. An increased ATP:ADP ratio is indicative of increased energy demand, met through increased oxidative metabolic activity. Higher ATP levels are associated with increased developmental potential in oocytes and embryos [70–73]; our data agrees with this principle, yet it is noteworthy that the in vivo matured oocyte ATP level was not different from most of the IVM treatments. The functional capacity of mitochondria for ATP production was examined by measuring mitochondrial activity; FSH stimulation was associated with increased activity, regardless of prematuration. Unsurprisingly, this result was mirrored by FAD/NAD(P)H and ROS levels within the oocyte. Nevertheless, in vivo matured oocytes exhibited significantly higher mitochondrial activity than all the other treatments and lower ROS levels, suggesting that the efficiency of ATP production is greatest in these oocytes. These results reveal that the energy production efficiency and activity of metabolic pathways utilized during IVM to produce highly competent oocytes remain dissimilar to their in vivo counterparts. Increasing b-oxidation within IVM oocytes by the addition of fatty acids and L-carnitine, an essential cofactor for fatty acid transport into the mitochondria, may further increase mitochondrial efficiency for ATP production [74]. An approximation of the redox state of the mitochondrial matrix space can be determined from the redox ratio, which refers to the ratio of oxidized FAD to reduced NAD(P)H [75]. This fluorescence measurement indicates relative changes in the oocyte’s redox without the use of exogenous stains or dyes. The redox ratio is sensitive to changes in the cellular metabolic rate and oxygen supply [75]. An increase in the redox ratio is indicative of an increased oxidized state [75, 76]. The current study demonstrated that cellular metabolic activity of the oocyte was greatly altered by FSH exposure in IVM with an increased oocyte redox ratio and by a larger ATP:ADP ratio, while oocytes matured in vivo had a relatively quiet oxidation state, but contained more high membrane potential mitochondria, as determined by JC-1 fluorescence. This adds to the evidence that treatment strategies that improve oocyte competence during IVM remain suboptimal compared with in vivo maturation. Autofluorescence cannot distinguish NADH and NADPH because their emission spectra are nearly identical [75]. NADH is found in both the cytoplasm and the mitochondria and functions as a reducing agent in numerous metabolic pathways, such as glycolysis, b-oxidation, the TCA cycle, and oxidative phosphorylation. 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. An important role for NADPH is the reduction of oxidized glutathione to GSH. We measured the intensity of MCB within oocytes after 16 h IVM and observed significantly higher MCB intensity following either pre-IVM treatment or FSH. GSH is synthesized largely by cumulus cells because denuding oocytes greatly reduces intraoocyte GSH levels [77] and represents an important defense molecule against oxidative stress [77]. Several studies have demonstrated

EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

FIG. 6. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on oocyte redox state. A) The relative level of monochlorobimane (MCB), a measure of GSH, was evaluated following 16 h IVM or 16 h following hCG. The relative autofluorescence of FAD (B) and NAD(P)H (C) were also measured. D) The ratio of FAD:NAD(P)H was determined as a measure of redox state. E) Intracellular ROS production was examined following 16 h IVM or 16 h following hCG using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), a nonfluorescent probe for intracellular ROS detection. F) Representative images of oocyte fluorescence for each parameter explored. Original magnification 390. Data represent the mean 6 SEM of three replicate experiments representing at least five oocytes per replicate experiment. Bars not sharing a common letter are significantly different (P , 0.05).

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FIG. 5. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on COC energy metabolism. Oocyte ATP (A) and ADP (B) content were measured following 16 h of IVM or 16 h following hCG. The ATP:ADP ratio was calculated as an indicator of energy demand (C). COC oxygen consumption (D) following the first 4 h of IVM was measured. Oocyte mitochondrial activity following 16 h IVM or 16 h following hCG was evaluated by mitochondrial membrane potential sensitive JC-1 staining (E and F), where a green fluorescence is emitted as JC-1 monomers in low-polarized mitochondria and a red fluorescence as J-aggregates in highly polarized mitochondria. Original magnification 390 (F). Data represent the mean 6 SEM of three independent experiments representing the mean of at least five samples per replicate experiment. Bars not sharing a common letter are significantly different (P , 0.05).

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