Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed C. F.
Fagbohun
mouse
oocytes
and S. M. Downs
Biology Department, Marquette University, Milwaukee, WI53233,
USA
Summary. In this study, the effect of different energy sources used in Eagle's minimum
essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5\m=.\5mmol 1\m=-\1), pyruvate (0\m=.\23mmol 1\m=-\1) and glutamine (2 mmol 1\m=-\1) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 \g=m\mol dibutyryl cAMP 1 \m=-\1 during 17\p=n-\18h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99\p=n-\100%germinal vesicle breakdown); all other combinations resulted in \m=le\54%germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (\m=le\10%GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (\m=ge\86% viability in pyruvate-containing medium; \m=le\35%viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (\m=ge\89%germinal vesicle breakdown) and viability (\m=ge\91%).The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5\p=n-\33%germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon d-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon d-glucose. Uptake and metabolism of d-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of d-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of d-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) d-mannose, a glycolysable sugar, was as effective as d-glucose in supporting the ligand effects. Thus, while pyruvate appeared to be most effective in supporting the spontaneous maturation of denuded oocytes, the presence of d-glucose is important in cumulus cell-enclosed oocytes for both the maintenance of meiotic arrest and ligand-induced meiotic maturation.
Keywords: glucose; oocyte maturation; mouse "Current address: West Michigan USA. (Corresponding author.
Reproductive Institute, P.C.,
885 Forest Hills Avenue, Grand
Rapids,
MI 49506,
Introduction The isolated oocyte-cumulus cell complex has been a popular model system for studying the control of meiotic maturation in mammalian oocytes. Removal of oocytes from Graafian follicles releases them from inhibitory constraints that originate in the somatic compartment of the follicle and leads to spontaneous germinal vesicle breakdown in culture. Supplementation of medium with agents that stimulate the production of cAMP prevents the resumption of maturation (Tsafriri et al., 1982; Eppig & Downs, 1984). This inhibitory influence can, in turn, be abolished by the addition of stimulatory ligands such as follicle-stimulating hormone (FSH), epidermal growth factor (EGF) and mitogenic lectins that induce germinal vesicle breakdown despite the continued presence of inhibitory agents within the medium (Dekel & Beers, 1978; Downs et al., 1988; Fagbohun & Downs, 1990). Previous studies have addressed the energy requirements of mammalian oocytes undergoing spontaneous meiotic maturation in culture. In mice, it was shown that denuded, cumulus cell-free oocytes do not exhibit spontaneous maturation when glucose is the sole energy source, but do undergo germinal vesicle breakdown in the presence of pyruvate or oxaloacetate (Biggers et al., 1967). Cumulus cell-enclosed oocytes, however, resume nuclear maturation in glucose-containing medium (Biggers et al., 1967), because cumulus cells metabolize glucose to pyruvate that is then made available to the oocyte (Donahue & Stern, 1968; Leese & Barton, 1985). It is also well established that cumulus cells mediate the stimulatory action of a number of ligands in promoting germinal vesicle breakdown in cultured oocytes (Downs et al., 1988; Fagbohun & Downs, 1990). It is therefore likely that modulation of both the type of energy source and its concentration within culture medium plays a vital role in the meiotic response of the oocyte in vitro. Studies from this laboratory involving in vitro oocyte maturation have used Eagle's minimum essential medium (MEM) containing D-glucose, pyruvate and glutamine as potential energy sources. The present study was carried out to determine the relative importance of each of these components in meiotic maturation of the mouse oocyte in culture, with particular emphasis on ligand-stimulated germinal vesicle breakdown. The data demonstrate that in the absence of cumulus cells, pyruvate is indeed the important energy source in supporting both meiotic maturation and oocyte viability. However, in cumulus cell-enclosed oocytes, glucose is the critical energy source, particularly for induction of meiotic maturation by FSH and Concanavalin A (ConA).
Materials and Methods
Experimental protocols and culture conditions SJL/J F, female mice, 19-22 days old, were used in all experiments. Mice were primed with 5iu serum gonadotrophin (Diosynth, Inc., Chicago, IL) and killed by cervical dislocation 48 h later. Ovaries were placed in medium containing 200 pmol 3-isobutyl-l-methylxanthine 1" ' (IBMX: Aldrich Chemical Co. Inc., Milwaukee, WI) to maintain meiotic arrest throughout the isolation procedure. Large antral follicles were punctured with sterile needles to release the cumulus cell-enclosed oocytes. Denuded oocytes were obtained by repeated pipetting of the cumulus cell-enclosed oocytes with sterile pasteur pipettes. Unless otherwise specified, cumulus cell-enclosed oocytes and denuded oocytes were washed in four changes of IBMX-free medium before transfer to 1 ml of test medium in stoppered borosilicate culture tubes, and cultured at 37°C for various periods. Tubes were gassed with a humidified mixture of 5% 02, 5% C02 and 90% N2. For the cumulus expansion experiment, cumulus cell-enclosed oocytes were isolated in inhibitor-free medium, washed and then transferred to 3 ml of appropriate test medium in 35 mm Petri dishes (Falcon no. 1008, Falcon Plastics, Los Angeles, CA). These were placed in modular incubator chambers (Billups-Rothenberg, Del Mar, CA), gassed and cultured in a water-jacketed incubator for 17-18 h at 37°C. The degree of expansion was assessed according to a subjective scale as described by Fagbohun & Downs (1990). The culture medium used for all these studies was Eagle's minimum essential medium (MEM) with Earle's salts and antibiotics. The concentrations of energy substrates were 5-5 mmol D-glucose C'; 0-23 mmol pyruvate 1_1 and 2 mmol glutamine 1_1. For oocyte maturation experiments, the medium was supplemented with 3 mg crystallized lyophilized bovine serum albumin ml"1 (ICN ImmunoBiologicals, Lisle, IL) and cumulus expansion experiments were carried out in MEM containing 5% fetal bovine serum (FBS: Hyclone Laboratories, Inc., Logan, Utah). For the maturation kinetics experiment denuded oocytes were cultured in the same energy supplement variations but this time C57BL/6J
pregnant mares'
in 100 µ drops of medium under oil, and were examined after 3, 6, 12 and 21-22 h of culture. At each time point the number of dead oocytes was determined and the nuclear status in the remaining viable oocytes was assessed.
Chemicals All hexoses, dibutyryl cAMP, phloretin, and ConA were purchased from Sigma Chemical Co. (St Louis, MO). Biological grade ovine FSH-16 (oFSH) was generously provided by the National Hormone and Pituitary Program of the NIDDK (Bethesda, MD). Stock solutions of ConA and FSH were prepared in phosphate-buffered saline con¬ bovine serum albumin ml ' and stored at 20°C. ConA was used at concentrations of 10 pg ml ' and taining 3 mg 25 pg ml" ' for oocyte maturation and cumulus expansion experiments, respectively. FSH was used at a concentration of 0-1 pg ml~'. These concentrations were previously determined to be efficient in producing the optimal stimulatory response of the oocyte-cumulus cell complex. All other agents were prepared fresh before each experiment. "
~
—
Uptake of radiolabelled sugars and ConA Uptake of tritiated D-glucose (66-6 Ci mmol"'), 2-deoxyglucose (6-3 Ci mmol"') and 3-O-methylglucose (79-0 Ci mmol"1) by oocyte-cumulus cell complexes was assayed during a 3 h culture period by the addition of 10 pCi of the respective radiolabelled sugar in 1 ml medium. All sugars were obtained from New England Nuclear (Boston, MA). For each treatment group, 20 complexes were washed through four changes of medium without radiolabel and
transferred to a vial for scintillation counting. An equal volume of medium from the last wash dish served as a blank. Tissue was solubilized with NaOH, neutralized with HC1 and assayed by scintillation spectroscopy. For measurement of ConA uptake, complexes were treated in identical fashion except that medium contained 2 pCi of the tritiated lectin (24-5 Ci mmol" '; Amersham Corp., Arlington Heights, IL).
Oocyte assessment and statistical analysis Each oocyte maturation experiment was performed three or more times with at least 40 oocytes per group per the data reported as mean percentage germinal vesicle breakdown + sem. Oocytes were assessed for maturation at the end of the culture period by scoring them for germinal vesicle breakdown, the first observable manifestation of maturation. All frequencies were subjected to arcsin transformation before statistical analysis. Differences between two groups were compared statistically by Student's t test. When more than two groups were involved, data were compared by analysis of variance (anova) followed by Duncan's multiple range test. A value
Fagbohun
mouse
oocytes
and S. M. Downs
Biology Department, Marquette University, Milwaukee, WI53233,
USA
Summary. In this study, the effect of different energy sources used in Eagle's minimum
essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5\m=.\5mmol 1\m=-\1), pyruvate (0\m=.\23mmol 1\m=-\1) and glutamine (2 mmol 1\m=-\1) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 \g=m\mol dibutyryl cAMP 1 \m=-\1 during 17\p=n-\18h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99\p=n-\100%germinal vesicle breakdown); all other combinations resulted in \m=le\54%germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (\m=le\10%GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (\m=ge\86% viability in pyruvate-containing medium; \m=le\35%viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (\m=ge\89%germinal vesicle breakdown) and viability (\m=ge\91%).The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5\p=n-\33%germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon d-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon d-glucose. Uptake and metabolism of d-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of d-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of d-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) d-mannose, a glycolysable sugar, was as effective as d-glucose in supporting the ligand effects. Thus, while pyruvate appeared to be most effective in supporting the spontaneous maturation of denuded oocytes, the presence of d-glucose is important in cumulus cell-enclosed oocytes for both the maintenance of meiotic arrest and ligand-induced meiotic maturation.
Keywords: glucose; oocyte maturation; mouse "Current address: West Michigan USA. (Corresponding author.
Reproductive Institute, P.C.,
885 Forest Hills Avenue, Grand
Rapids,
MI 49506,
Introduction The isolated oocyte-cumulus cell complex has been a popular model system for studying the control of meiotic maturation in mammalian oocytes. Removal of oocytes from Graafian follicles releases them from inhibitory constraints that originate in the somatic compartment of the follicle and leads to spontaneous germinal vesicle breakdown in culture. Supplementation of medium with agents that stimulate the production of cAMP prevents the resumption of maturation (Tsafriri et al., 1982; Eppig & Downs, 1984). This inhibitory influence can, in turn, be abolished by the addition of stimulatory ligands such as follicle-stimulating hormone (FSH), epidermal growth factor (EGF) and mitogenic lectins that induce germinal vesicle breakdown despite the continued presence of inhibitory agents within the medium (Dekel & Beers, 1978; Downs et al., 1988; Fagbohun & Downs, 1990). Previous studies have addressed the energy requirements of mammalian oocytes undergoing spontaneous meiotic maturation in culture. In mice, it was shown that denuded, cumulus cell-free oocytes do not exhibit spontaneous maturation when glucose is the sole energy source, but do undergo germinal vesicle breakdown in the presence of pyruvate or oxaloacetate (Biggers et al., 1967). Cumulus cell-enclosed oocytes, however, resume nuclear maturation in glucose-containing medium (Biggers et al., 1967), because cumulus cells metabolize glucose to pyruvate that is then made available to the oocyte (Donahue & Stern, 1968; Leese & Barton, 1985). It is also well established that cumulus cells mediate the stimulatory action of a number of ligands in promoting germinal vesicle breakdown in cultured oocytes (Downs et al., 1988; Fagbohun & Downs, 1990). It is therefore likely that modulation of both the type of energy source and its concentration within culture medium plays a vital role in the meiotic response of the oocyte in vitro. Studies from this laboratory involving in vitro oocyte maturation have used Eagle's minimum essential medium (MEM) containing D-glucose, pyruvate and glutamine as potential energy sources. The present study was carried out to determine the relative importance of each of these components in meiotic maturation of the mouse oocyte in culture, with particular emphasis on ligand-stimulated germinal vesicle breakdown. The data demonstrate that in the absence of cumulus cells, pyruvate is indeed the important energy source in supporting both meiotic maturation and oocyte viability. However, in cumulus cell-enclosed oocytes, glucose is the critical energy source, particularly for induction of meiotic maturation by FSH and Concanavalin A (ConA).
Materials and Methods
Experimental protocols and culture conditions SJL/J F, female mice, 19-22 days old, were used in all experiments. Mice were primed with 5iu serum gonadotrophin (Diosynth, Inc., Chicago, IL) and killed by cervical dislocation 48 h later. Ovaries were placed in medium containing 200 pmol 3-isobutyl-l-methylxanthine 1" ' (IBMX: Aldrich Chemical Co. Inc., Milwaukee, WI) to maintain meiotic arrest throughout the isolation procedure. Large antral follicles were punctured with sterile needles to release the cumulus cell-enclosed oocytes. Denuded oocytes were obtained by repeated pipetting of the cumulus cell-enclosed oocytes with sterile pasteur pipettes. Unless otherwise specified, cumulus cell-enclosed oocytes and denuded oocytes were washed in four changes of IBMX-free medium before transfer to 1 ml of test medium in stoppered borosilicate culture tubes, and cultured at 37°C for various periods. Tubes were gassed with a humidified mixture of 5% 02, 5% C02 and 90% N2. For the cumulus expansion experiment, cumulus cell-enclosed oocytes were isolated in inhibitor-free medium, washed and then transferred to 3 ml of appropriate test medium in 35 mm Petri dishes (Falcon no. 1008, Falcon Plastics, Los Angeles, CA). These were placed in modular incubator chambers (Billups-Rothenberg, Del Mar, CA), gassed and cultured in a water-jacketed incubator for 17-18 h at 37°C. The degree of expansion was assessed according to a subjective scale as described by Fagbohun & Downs (1990). The culture medium used for all these studies was Eagle's minimum essential medium (MEM) with Earle's salts and antibiotics. The concentrations of energy substrates were 5-5 mmol D-glucose C'; 0-23 mmol pyruvate 1_1 and 2 mmol glutamine 1_1. For oocyte maturation experiments, the medium was supplemented with 3 mg crystallized lyophilized bovine serum albumin ml"1 (ICN ImmunoBiologicals, Lisle, IL) and cumulus expansion experiments were carried out in MEM containing 5% fetal bovine serum (FBS: Hyclone Laboratories, Inc., Logan, Utah). For the maturation kinetics experiment denuded oocytes were cultured in the same energy supplement variations but this time C57BL/6J
pregnant mares'
in 100 µ drops of medium under oil, and were examined after 3, 6, 12 and 21-22 h of culture. At each time point the number of dead oocytes was determined and the nuclear status in the remaining viable oocytes was assessed.
Chemicals All hexoses, dibutyryl cAMP, phloretin, and ConA were purchased from Sigma Chemical Co. (St Louis, MO). Biological grade ovine FSH-16 (oFSH) was generously provided by the National Hormone and Pituitary Program of the NIDDK (Bethesda, MD). Stock solutions of ConA and FSH were prepared in phosphate-buffered saline con¬ bovine serum albumin ml ' and stored at 20°C. ConA was used at concentrations of 10 pg ml ' and taining 3 mg 25 pg ml" ' for oocyte maturation and cumulus expansion experiments, respectively. FSH was used at a concentration of 0-1 pg ml~'. These concentrations were previously determined to be efficient in producing the optimal stimulatory response of the oocyte-cumulus cell complex. All other agents were prepared fresh before each experiment. "
~
—
Uptake of radiolabelled sugars and ConA Uptake of tritiated D-glucose (66-6 Ci mmol"'), 2-deoxyglucose (6-3 Ci mmol"') and 3-O-methylglucose (79-0 Ci mmol"1) by oocyte-cumulus cell complexes was assayed during a 3 h culture period by the addition of 10 pCi of the respective radiolabelled sugar in 1 ml medium. All sugars were obtained from New England Nuclear (Boston, MA). For each treatment group, 20 complexes were washed through four changes of medium without radiolabel and
transferred to a vial for scintillation counting. An equal volume of medium from the last wash dish served as a blank. Tissue was solubilized with NaOH, neutralized with HC1 and assayed by scintillation spectroscopy. For measurement of ConA uptake, complexes were treated in identical fashion except that medium contained 2 pCi of the tritiated lectin (24-5 Ci mmol" '; Amersham Corp., Arlington Heights, IL).
Oocyte assessment and statistical analysis Each oocyte maturation experiment was performed three or more times with at least 40 oocytes per group per the data reported as mean percentage germinal vesicle breakdown + sem. Oocytes were assessed for maturation at the end of the culture period by scoring them for germinal vesicle breakdown, the first observable manifestation of maturation. All frequencies were subjected to arcsin transformation before statistical analysis. Differences between two groups were compared statistically by Student's t test. When more than two groups were involved, data were compared by analysis of variance (anova) followed by Duncan's multiple range test. A value
