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Research Article |
Research Centre for Reproductive Health,3 Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia
Area of Biochemistry,4 School of Veterinary Sciences, University of Buenos Aires, C1427CWO Buenos Aires, Argentina
Department of Physiology and Pharmacology,5 School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
ABSTRACT
Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.
cumulus, cumulus cells, embryo, early development, glucose, glucosamine, hexosamine, oocyte, oocyte development
INTRODUCTION
The extent of glucose metabolism during maturation of the mammalian oocyte is known to influence several aspects of oocyte maturation and is related to oocyte developmental capacity [1–4]. For example, oocytes with improved developmental capacity, such as those derived from adult donors and in vivo matured oocytes, have increased glucose metabolism via glycolysis and pentose phosphate pathway activity compared to oocytes with compromised development, such as those derived from prepubertal donors or in vitro maturation (IVM) [5, 6]. During IVM, lactic acid is the primary product of glucose metabolism by the bovine cumulus-oocyte complex (COC), indicating that glycolysis is the predominant fate of glucose [7]. However, over a 24-h IVM period, a considerable proportion of glucose is increasingly used by the somatic compartment for the production of extracellular matrix to facilitate FSH-stimulated mucification [8]. Furthermore, glucose concentration during IVM can significantly alter the kinetics of meiotic maturation [9].
The hexosamine biosynthesis pathway is the metabolic pathway that enables glucose to be used for glycosaminoglycan synthesis, such as hyaluronic acid. In nonreproductive somatic cells, this accounts for 1%–3% of glucose metabolism [10]. The rate-limiting enzyme regulating the hexosamine pathway is glutamine-fructose-6-phosphate transaminase 1 (GFPT1), which converts fructose-6-phosphate into glucosamine-6-phosphate (GlcN6P). GFPT1 is an enzyme that is allosterically regulated by the hexosamine pathway end product, UDP-N-acetyl glucosamine [11]. The hexosamine pathway is readily upregulated by the addition of glucosamine (GlcN), which can be transported by the facilitated glucose transporters and is also readily phosphorylated to GlcN6P, thereby bypassing the rate-limiting enzyme GFPT1 [12]. In a previous study, we demonstrated that glucosamine addition to IVM medium reduced the uptake of glucose by bovine COCs and reduced the incorporation of glucose into the hyaluronic acid-rich matrix produced by FSH-induced cumulus expansion during bovine in vitro maturation [8]. We therefore postulated that glucosamine addition to IVM medium would be preferentially utilized for hyaluronic acid synthesis, enabling glucose to be used in other metabolic pathways, thereby improving oocyte developmental competence. Here we describe the effect of glucosamine supplementation during IVM in both cattle and pig COCs on oocyte developmental competence and initiate experiments examining the influence of the hexosamine biosynthesis pathway on oocyte developmental competence.
MATERIALS AND METHODS
Unless specified, all reagents were purchased from Sigma. COC handling medium for both bovine and porcine studies was Hepes-buffered Tissue Culture Medium 199 (TCM199; ICN Biochemicals) supplemented with 0.5 mM sodium pyruvate and 4 mg/ml fatty acid-free BSA (ICPbio Ltd.).
LY294002 was dissolved in 100% methanol (16.27 mM, stored at –80°C), wortmannin was dissolved in dimethyl sulfoxide (DMSO; 1 mM, stored at –20°C) and benzyl-2-acetamido-2-deoxy-
-D-galactopyranoside (BADGP) was dissolved in methanol (100 mM, stored at –20°C). The final concentrations of methanol were 0.3% and 2% for LY294002 and BADGP, respectively, and 0.1% DMSO for wortmannin in maturation drops.
Bovine and Porcine Oocyte Collection
Bovine ovaries from cycling cows were transported from a local abattoir in saline (30–35°C). Follicles of nonatretic appearance and 3–8 mm in size were aspirated using an 18-gauge needle and 10-ml syringe. COCs with multiple intact (
3) cell layers and even cytoplasm were collected from aspirated fluids and were washed twice in handling medium and once in corresponding maturation media.
Prepubertal porcine ovaries were transported from a local abattoir in saline (30–35°C). Ovaries were washed thoroughly in saline (37°C). Antral follicles between 3 and 6 mm in diameter were aspirated using a 21-gauge needle and vacuum. COCs were recovered from the aspirated follicular fluid and selected based on similar parameters to those for bovine COCs, and were washed three times in Hepes-buffered TCM-199 and once in IVM medium.
Bovine Oocyte Nuclear Maturation and Effect of Glucosamine
The base medium for bovine oocyte maturation was bicarbonate-buffered TCM199 supplemented with 0.5 mM sodium pyruvate and 4 mg/ml fatty acid-free BSA (B-TCM199) or Bovine VitroMat (Cook Australia), a defined medium based on the composition of follicular fluid from antral follicles, containing 5.6 mM glucose, nonessential and essential amino acids, and 4 mg/ml fatty acid-free BSA [10]. All bovine IVM treatments were supplemented with 100 mIU/ml FSH (Puregon; Organon) and, 100 mIU/ml hCG (Pregnyl; Organon). To determine the influence of glucosamine supplementation during IVM on the rate of meiotic resumption (germinal vesicle breakdown [GVBD]) and completion (metaphase II [MII]), bovine COCs were cultured in groups of 10 in 100 µl of Bovine VitroMat (Cook Australia) ± 5 mM glucosamine. Pre-equilibrated culture drops were overlaid with mineral oil and cultured in 6% CO2 in humidified air at 39°C. After 6 h or 24 h of culture, COCs were mechanically denuded in handling medium containing 50 IU/ml hyaluronidase and fixed for assessment of nuclear maturation using 1% w/v orcein in 45% acetic acid. Oocytes were classified as germinal vesicle (GV), GVBD, metaphase I (MI) or MII. This experiment was replicated 8 times with 10 COCs used per treatment group.
Glucosamine Supplementation and Bovine Embryo Development
The influence of glucosamine supplementation during IVM on bovine oocyte developmental capacity was assessed by culturing groups of 10 COCs in 100 µl of either 1) B-TCM199, 2) –GlcN (VitroMat) or 3) + GlcN (VitroMat + 5 mM glucosamine). Pre-equilibrated drops were overlaid with mineral oil and cultured in 6% CO2 in humidified air at 39°C. After 24 h of culture, COCs were fertilized from a single sire of proven fertility with either 1 x 106 or 5 x 106 motile spermatozoa/ml prepared using a discontinuous (45%/90%) Percoll gradient (Amersham Biosciences). The latter high spermatozoa concentration was used to assess if glucosamine supplementation altered matrix formation and required increased number of spermatozoa in the insemination period. Fertilization was performed using in vitro fertilization medium (Bovine VitroFert; Cook Australia; supplemented with 10 µM heparin, 200 µM penicillamine, and 100 µM hypotaurine). Presumptive zygotes were denuded of their cumulus vestments using a finely drawn glass pipette and washed twice in Bovine VitroCleave (Cook Australia). Five presumptive zygotes were transferred into 20-µl microdrops of pre-equilibrated Bovine VitroCleave overlaid with mineral oil and cultured for five days (Day 1 to Day 5) at 39°C in a 7% O2, 6% CO2, balanced N2 atmosphere. On Day 5, embryos were transferred to 20-µl microdrops of pre-equilibrated Bovine VitroBlast (Cook Australia) overlaid with mineral oil and cultured to Day 8. Five replicate experiments were performed with 40 COCs used per treatment group.
Glucosamine Dose-Response and Bovine Embryo Development
To determine the effects of different glucosamine concentrations on bovine oocyte developmental capacity, groups of 10 COCs were cultured in 100 µl of VitroMat (Control), or VitroMat supplemented with 0.5, 1, 2.5 or 5 mM glucosamine. Pre-equilibrated drops were overlaid with mineral oil and COCs were cultured in 6% CO2 in humidified air at 39°C. After 24 h, COCs were fertilized with 1 x 106 spermatozoa/ml and then cultured to the blastocyst stage, as described above. Five replicate experiments were performed with 30 COCs used per treatment.
Porcine Oocyte Maturation
Porcine IVM medium consisted of B-TCM199 (as described above) supplemented with 8.2 mg/ml insulin, 10 µg/ml epidermal growth factor (EGF), 75 mIU/ml FSH (Bioniche Animal Health), and 1.8 mIU/ml LH (Bioniche Animal Health), and did not contain follicular fluid. Porcine COCs were cultured in groups of 10 in 100 µl of pre-equilibrated IVM medium (control) or IVM medium containing 1 mM or 2.5 mM glucosamine, and were cultured at 38.5°C in 5%CO2 in humidified air. After 46 h of culture, COCs were exposed to 50 IU/ml hyaluronidase in IVM media for 1 min, and gently aspirated using a fine bore glass pipette to remove all cumulus cells. Oocytes were fixed and stained for the assessment of nuclear maturation using 1% orcein in 45% acetic acid, and were classified as GV, MI, or MII. Approximately 50 oocytes were matured per treatment, in three replicate experiments (approximately 150 per treatment).
Porcine Embryo Development
Denuded oocytes were incubated in pre-equilibrated Tyrodes albumin lactate pyruvate supplemented with polyvinyl alcohol (TALP-PVA) until fertilization. Commercially prepared semen (Sabor) was washed three times in sperm preincubation media and incubated at 39°C in 6% CO2 in humidified air for 30–40 minutes at a concentration of 1 x 108 sperm/ml. Incubated spermatozoa were diluted to a concentration of 5 x 106 sperm/ml in sperm preincubation media, and 10 µl was added per 90 µl TALP-PVA drop containing 15 denuded oocytes. The oocytes and sperm (final concentration 5 x 105 sperm/ml) were cocultured for 30 min at 39°C in 6% CO2 in humidified air, then oocytes and zona-attached sperm were transferred to fresh 100 µl drops TALP-PVA and incubated overnight.
At the end of this incubation period (20–21 h) oocytes were washed twice in NCSU23 culture media, transferred to 50 µl drops of NCSU23 overlaid with oil (20 oocytes/50-µl drop) and incubated in 5% O2, 5% CO2, and 90% N2 at 39°C. On Day 2 of culture, cleavage rate was assessed; on Day 5, 10% filtered fetal calf serum was added to the culture drops; and on Day 7, developmental stage was recorded for all groups. Approximately 50 oocytes were fertilized per treatment group, in three replicate experiments.
Inhibition of O-Linked Glycosyltransferase and Bovine Embryo Development
To determine whether O-linked glycosylation was involved in the inhibition of embryo development following glucosamine exposure during IVM, groups of 10 bovine COCs were incubated for 24 h in 100 µl of Bovine VitroMat in the presence of 1 mM glucosamine and 0.5, 1, and 2 mM of the OGT inhibitor BADGP. A further control containing no glucosamine or BADGP (or methanol) was also included. A separate study demonstrated that the inclusion of 2% methanol during IVM in the presence or absence of glucosamine had no effect on subsequent development (data not shown). Following IVM, mature oocytes were fertilized and cultured to the blastocyst stage as described above.
Immunohistochemical Localization of O-Linked Glycosylation in Bovine COCs
Bovine COCs were fixed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 1 h at 25°C, washed with PBS, and kept in PBS containing 0.2% PFA before further processing for immunofluorescence as previously described [13]. Briefly, whole COCs were adhered on Cell-Tak (Collaborative Biomedical Products) -coated coverslips before localization of immunoreactivity using a monoclonal antibody against O-linked N-acetyl glucosamine (RL2, Abcam). Following application of the primary antibody (1:200 at 25°C for 2 h), embryos were washed in PBS and exposed for 1 h at 25°C to Texas-red conjugated goat anti-rabbit IgG (Calbiochem-Novachem) diluted 1:100 with PBS. Coverslips were mounted on cavity slides in glycerol following brief exposure to 2.5, 5, 10, 20, 50, and 70% v/v glycerol in PBS and examined using a Zeiss LSM 510 meta confocal spectral imaging system with a Zeiss Axioplan 2 MOT upright optical microscope equipped with a Plan-Apochromat 63x oil objective.
Involvement of the Phosphatidylinositol 3-Kinase Pathway in Bovine Oocyte Developmental Competence
To determine whether the perturbation in bovine oocyte developmental capacity after glucosamine supplementation during IVM was caused by a downregulation of the phosphatidylinositol 3-kinase (PI3K) pathway, COCs were cultured in the presence of either the specific PI3K inhibitor LY294002 or the nonspecific kinase inhibitor wortmannin. Groups of 10 COCs were cultured in 100 µl of either Bovine VitroMat, VitroMat + 25 or 50 µM of LY294002, or VitroMat + 0.1 or 1 µM wortmannin. Pre-equilibrated drops were overlaid with mineral oil and COCs were cultured in 6% CO2 in humidified air at 39°C. After 24 h, 20 COCs per treatment groups were mechanically denuded in handling medium containing 50 IU/ml hyaluronidase and fixed for assessment of nuclear maturation. The remaining COCs were fertilized with 1 x 106 spermatozoa/ml and cultured as previously described. Four replicates were performed using LY294002 and five replicates were performed using wortmannin.
Statistical Analyses
Embryo development and nuclear maturation data were arcsine transformed. Data were analyzed using ANOVA followed by an all pair-wise multiple comparisons Tukey test, performed using SigmaStat version 2.0 (SPSS Inc.). A polynomial regression analysis was conducted on development rates within the BADGP treatments to assess for a dose-linear effect. Probabilities less than 0.05 were considered significantly different.
RESULTS
Glucosamine Supplementation and Nuclear Maturation in Bovine and Porcine Oocytes
The influence of glucosamine supplementation during IVM on bovine oocyte nuclear maturation was determined by assessing meiotic progression after 6 h (coinciding with the resumption of meiosis) and 24 h. Glucosamine supplementation did not affect the resumption of meiosis, with similar proportions of oocytes undergoing GVBD by 6 h of culture (Fig. 1A, no GlcN = 14% and +GlcN = 19% GVBD).
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By 24 h, the majority of COCs had completed nuclear maturation to the MII stage, regardless of the presence or absence of glucosamine (Fig. 1B, no GlcN = 87% and +GlcN = 81% MII). There was a suggestion that oocytes matured in glucosamine were slightly delayed in meiotic progression, because more COCs matured in the presence of glucosamine were at the MI stage after 24 h, compared to COCs cultured in the absence of glucosamine (Fig. 1B, P < 0.05). In contrast, no significant differences in meiotic kinetics of porcine oocytes were observed following maturation in 0, 1.25, or 2.5 mM glucosamine for 46 h (84 vs. 81 vs. 88% MII, respectively).
Glucosamine Supplementation and Bovine Embryo Development
An initial experiment was conducted to assess sperm penetration at 2 concentrations (1 x 106 and 5 x 106 sperm/ml) of the cumulus matrix in response to altered glycosaminoglycan synthesis levels, even though there was no evidence of altered cumulus expansion (data not shown). There were no significant differences in cleavage rates of bovine embryos between all treatment groups (Table 1). However, significantly fewer oocytes matured in Bovine VitroMat + 5 mM glucosamine and fertilized in 5 x 106 sperm/ml reached the 4-cell embryo stage than oocytes in any other treatment group (P < 0.05). At Day 8 of embryo culture, significantly fewer blastocysts developed from oocytes that were matured in glucosamine (main effect: no GlcN = 29.7 ± 2.3% vs. +GlcN = 3.2 ± 2.0%, P < 0.001). The concentration of spermatozoa used to fertilize the oocytes did not affect blastocyst development.
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Based on this initial finding, we investigated the influence of maturing bovine COCs in Bovine VitroMat with different concentrations of glucosamine on subsequent embryo development. Embryo cleavage rates ranged from 68.3 ± 2.6 to 82.5 ± 3.3%, and there was no variation detected between treatment groups for the proportion of cleaved zygotes and embryos 4 cells (Table 2). However, the addition of 0.5 mM and 1 mM glucosamine to Bovine VitroMat approximately halved blastocyst rates compared to oocytes matured in media without glucosamine (P < 0.05), with the addition of 2.5 and 5 mM glucosamine to IVM being the most detrimental to subsequent embryo development (Table 2).
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Porcine Embryo Development
To determine if the effect of glucosamine is restricted to ruminant embryos, we assessed if porcine oocytes matured in the presence of glucosamine were similarly affected. No difference was observed for early cleavage rates on Day 2 (Fig. 2). However, a significant decrease in blastocyst development at Day 7 was observed (P < 0.05) between treatments, with pair-wise comparisons revealing that the maturation in 2.5 mM glucosamine was significantly lower than control medium (Fig. 2).
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O-Linked Glycosylation Inhibition During Bovine IVM
Analysis of variance revealed that all treatment groups containing glucosamine had significantly lower rates of blastocyst development than the control treatment, regardless of the presence or absence of BADGP (P < 0.01, Fig. 3A). However, polynomial regression revealed a significant (P < 0.05) linear increase in development to the blastocyst stage with increasing levels of the O-linked glycosyltransferase (OGT) inhibitor, BADGP, suggesting that a recovery in development can be achieved by the inhibition of O-linked glycosylation (Fig. 3A). Furthermore, RL2 immunohistochemistry revealed that significant O-linked glycosylation was evident in the cumulus cells of matured bovine COCs, with little to no staining detectable in the oocyte (Fig. 3B). No visible differences were observed between COCs incubated in the presence or absence of glucosamine, suggesting that O-linked glycosylation occurs readily in glucose-containing medium. In contrast, the addition of BADGP (2 mM) appeared to decrease the level of RL2 staining (Fig. 3B).
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PI3K Pathway Inhibition During Maturation Does Not Affect Oocyte Developmental Competence
The PI3K pathway is an important regulatory pathway for numerous cellular functions, and it was recently reported that pancreatic ß-cells cultured in the presence of glucosamine had compromised activation of PI3K, leading to impaired insulin-stimulated protein synthesis [14]. To determine whether glucosamine potentially acted by downregulating the PI3K pathway during IVM, bovine COCs were cultured in the presence of LY294002, a specific PI3K inhibitor, or wortmannin, a less specific inhibitor.
After 24 h of culture in the presence of LY294002 or wortmannin, significantly fewer COCs completed nuclear maturation compared to the control and methanol groups (Fig. 4, A and C). However, neither LY294002 nor wortmannin affected blastocyst development at either concentrations (Fig. 4, B and D).
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DISCUSSION
Glucosamine is an alternative substrate to glucose for extracellular matrix synthesis during cumulus expansion [15, 16] and its supplementation during IVM reduces glucose consumption by bovine COCs [8]. The aim of this study was to determine the effects of glucosamine supplementation on oocyte developmental capacity.
Despite the addition of glucosamine having no effect on oocyte nuclear maturation, the developmental capacity of COCs to form blastocyst stage embryos was severely diminished in both bovine and porcine oocytes when matured in concentrations of 2.5 mM GlcN or more. This is manifested as a detrimental effect on developmental competence following early embryo development, because cleavage rates were comparable to those of oocytes matured without glucosamine in both species. The majority of embryos arrested precompaction, coinciding with embryonic genome activation in bovine embryos [17]. The detrimental affects of glucosamine were not caused by batch toxicity or media acidification, because separate sources of pH-neutralized glucosamine yielded the same effect in inhibiting bovine developmental competence (data not shown), and the results have been repeated by us in two separate laboratories.
The Hexosamine Biosynthesis Pathway
The hexosamine biosynthesis pathway has come under considerable recent attention because of its role in regulating the activity of many signaling pathways, including a variety of transcription factors (for reviews, see [18, 19]; Fig. 5), and is now referred to as an energy-sensing pathway, coupling ligand-induced receptor signaling with energy homeostasis. This is because of O-linked glycosylation, involving UDP-N-acetyl glucosamine, the end product of the hexosamine pathway (Fig. 5). In some cases, serines and threonines that can be phosphorylated (reviewed in [19, 20]) are alternatively O-linked glycosylated, thus influencing signaling activity. This has been described as the yin-yang relationship between phosphorylation and O-linked glycosylation. In the presence of excess UDP-N-acetyl glucosamine and OGT, signaling peptides requiring phosphorylation can be inhibited by O-linked glycosylation [21]. Our data suggests that O-linked glycosylation occurs extensively within cumulus cells during in vitro maturation (and therefore, during cumulus expansion) of bovine COCs in the presence of glucose (regardless of the presence or absence of glucosamine). We further tested whether glycosylation regulates bovine oocyte developmental competence, to assess whether this is a potential mechanism of glucosamine-induced inhibition of oocyte developmental capacity, by incubating COCs in the presence of glucosamine and the OGT inhibitor BADGP [22]. Although no attempt was made to quantify the level of O-linked glycosylation, BADGP appeared to decrease the level of RL-2 staining, suggesting that inhibition took place. In contrast, there appeared no difference in RL-2 intensity with the inclusion of glucosamine. This absolute change in staining level is not unsurprising, because OGT activity is regulated by a complex and not fully understood mechanism that appears to operate at two different levels. Although high levels of cellular UDP-GlcNAc enhance its activity by increasing its affinity for protein acceptor substrates and therefore glycosylation [23], its basal activity is modulated by its own intrinsic O-glycosylation/phosphorylation status [24]. Therefore, similarly to the substrates that it glycosylates, OGT's activity can be decreased in the glycosylated state. This provides a mode of regulation that ultimately results in a finely balanced equilibrium wherein even small deviations in O-linked glycosylation would result in a perturbed cellular signaling, and may explain why no obvious increase in O-linked glycosylation was apparent in the presence of glucosamine.
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Overall, our data revealed that glucosamine-induced inhibition was at least partially reversed by an inhibitor of OGT activity, BADGP, suggesting that O-linked glycosylation of signaling molecules within cumulus cells during maturation is a possible contributing mechanism responsible for the decrease in oocyte developmental competence.
The best-described example of protein signaling disruption via glucosamine is in the field of insulin resistance, because O-linked glycosylation of the insulin receptor substrate downregulates insulin signaling and decreases activity of the PI3K/AKT1 signaling pathway (Fig. 5) [14, 22]. Glucosamine addition therefore acts as a hyperglycemic mimetic and has been used in the diabetes field for induction of insulin resistance within pancreatic ß-cells [14, 22] and in localized muscular perfusions, mediated by an upregulated hexosamine biosynthesis pathway (see for example [25]). Colton et al. [26] investigated the influence of diabetic conditions on oocyte maturation and demonstrated that COCs derived from induced-diabetic mice have altered meiotic kinetics in both spontaneous and ligand-induced nuclear maturation, suggesting that, in part, an uncoupling of cell-cell communication between the cumulus cells and oocyte occurs under these conditions [26]. Other alterations in oocyte development and meiotic progression were also observed by Chang et al. [27]. We specifically tested whether the action of glucosamine on bovine oocyte developmental competence was via inhibition of the PI3K/AKT1 pathway by incubation of bovine COCs in the presence of well-characterized PI3K/AKT1 inhibitors. The results here indicate that although the PI3K/AKT1 pathway affects the kinetics of meiosis during in vitro maturation (and agrees with the published data by [28, 29]), it does not contribute to subsequent developmental competence.
The action of an upregulated hexosamine pathway, particularly by the inclusion of glucosamine is known to cause other cellular stress mechanisms, such as depletion of ATP levels [12, 30, 31] and oxidative stress [32, 33], the latter via a depletion of reduced glutathione by inhibition of the pentose-phosphate pathway [33], a key pathway in determining developmental competence in the oocyte [34, 35]. Further investigations are required to assess the effect of glucosamine addition on ATP and reactive oxygen species (ROS) generation within cumulus cells and oocytes.
The concentration of glucosamine that lead to the lowest blastocyst rate resulted in the highest degree of cumulus expansion in rodent COCs [16]. Although cumulus expansion is important for ovulation and postovulatory events [15, 36], our data suggests that oversupply of substrate for cumulus expansion, such as follicular hyperglycemia (possibly as a result of diabetes), or reduced stimulation for cumulus expansion (possibly as a result of poor oocyte-cumulus cell signaling), could significantly impede oocyte developmental capacity. Interestingly, inhibition of the hexosamine pathway by the GFPT1 inhibitor 6-diazo-5-oxo-L-norleucine (DON) does not appear to significantly affect oocyte developmental competence (C. Gutnisky, G. Dalvit, L. Pintos, J.G. Thompson, M. Beconi, and P. Cetica, personal communication), although, as expected, the presence of DON significantly inhibited FSH-stimulated cumulus expansion in bovine COCs.
In conclusion, despite glucosamine having no effect on oocyte nuclear maturation, supplementation during IVM leads to perturbed oocyte cytoplasmic maturation, demonstrated by decreased development to the blastocyst stage. Our data suggest that upregulation of O-linked glycosylation, most likely in the cumulus, is an explanation for this effect, but does not appear to include inhibition of the PI3K/AKT1 pathway.
ACKNOWLEDGMENTS
The authors would like to thank Chris Kraft for ovary collections, Sam Schulz, Kyleen Catanzariti and Tamer Hussein for assistance with ovary aspirations and Alex Harvey and Chris Grupen for additional assistance.
FOOTNOTES
1 Supported by an Australia Research Council (SPIRT, C00107702 to M.S.-M.) and Cook Australia Pty Ltd (to M.S.-M.) and partially supported as part of the NICHD National Cooperative Program on Female Health and Egg Quality under cooperative agreement U01 HD044644 (to J.G.T.). 
2 Correspondence: Jeremy G. Thompson, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd., Woodville, SA 5011, Australia. FAX: 61 8 8222 7521; jeremy.thompson{at}adelaide.edu.au
Received: 13 October 2005.
First decision: 21 November 2005.
Accepted: 23 January 2006.
REFERENCES
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