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Experimental Cell Research91 (1975) 113-l 18
CARBOHYDRATE AND RABBIT
AND
AMINO
AMMONIA
FOLLICULAR IN-HA
ACID
REQUIREMENTS
PRODUCTION
OOCYTES
MATURED
OF IN VITRO
BAE and R. H. FOOTE
Department of Animal Science, Cornell University, Ithaca, N.Y. 14850, USA
SUMMARY Rabbit follicular oocytes were cultured at 37°C for 18-24 h in a basic salt medium containing 0.4% bovine serum albumin (BSA), carbohydrates and amino acids in various combinations. Osmolarity of the medium was maintained at 308 mOsm. The carbohydrates, pyruvate, lactate and glucose were all about equally beneficial, but not essential for rabbit oocyte maturation. Glutamine and proline, but not methionine or phenylalanine stimulated oocyte development. Glutamine stimulated more follicular oocytes to develop to prophase and metaphase II than did any of the three carbohydrates tested alone or in combination. Ammonia production after 24 h of culture was highest in media containing glutamine (15.2 pg/ml) but this was not inhibitory to maturation. Negligible amounts of ammonia were found with the other amino acids added. With 0, 0.08, 0.4, 2, 10 and 50 mM of glutamine in the basic salt medium, plus 0.4 % BSA, but without carbohydrates, 30, 73, 70, 71, 59 and 45 % of the follicular oocytes developed to the prophase or metaphase II stage. It is concluded that the optimum level of glutamine ranged from 0.08 to 2 mM and that no carbohydrate need be added to the medium for culturing oocytes when glutamine is included.
Requirements for resumption of meiosis in the oocyte prior to ovulation and after variable periods of ‘storage’ in the dictyate condition are not well understood. Chang [l] found that rabbit serum added to Ringer’s solution permitted rabbit follicular oocytes cultured in vitro to develop to metaphase II, but no studies in the rabbit with defined media have been reported. The rabbit is a good model for these investigations because, as an induced ovulator, accurately timed events in vivo can be compared with studies in vitro. Kane & Foote [2] reported that pyruvate increased the proportion of rabbit zygotes which developed into blastocysts. Brinster [3] suggested that rabbit embryos might use nitrogenous compounds as a source of energy.
Kane [4] showed that a high proportion of zygotes develop into hatched blastocysts in a complex defined medium containing a variety of amino acids, and that the inclusion of simple carbohydrates as energy substrates had no effect. Mouse oocytes will develop to metaphaseII in defined media containing pyruvate, oxaloacetate [SJor lactate under aerobic conditions if NAD is added to the medium [6]. The amino acids requirement is not well defined. Cumulus cells recently have been reported not to improve mouse oocyte maturation in vitro [7] despite earlier reports to the contrary [8-g]. Hamster oocytes can undergo maturation when cultured in a carbohydrate-containing salt solution without bovine serum albumin Exptl Cell Res 91 (1975)
114 Bae and Foote (BSA) providing that isoleucine, glutamine, phenylalanine and methionine are included [lo]. Composition of media for culturing embryos of several species has been reviewed by Gwatkin [l 11. All media given contain some simple carbohydrate. Little attention has been given to the possibility that the oocyte might utilize a non-carbohydrate source of energy for development. With amino acids in the medium ammonia formation is expected. Ammonia toxicity for the living cell is well recognized in vitro and in vivo [12-141. There has been no attempt to investigate the toxicity of ammonia for the mammalian oocytes, eggs and embryos, although many researchers have used various amino acids in culture media. The present investigations were undertaken to elucidate carbohydrate and amino acid requirements for oocyte maturation and to determine ammonia production during culture. MATERIALS
AND
METHODS
Animals. All rabbits used in the present experiments were virgin Dutch belted does raised in our colony. They were 4 to 7 months old when sacrificed by disarticulation to avoid possible effects of anesthesia on the ovaries. Experimental design. Several experiments were conducted to compare the carbohydrates most frequently used for culture of mammalian oocytes and embryos (pyruvate, lactate and glucose) along with the amino acids, glutamine, methionine, phenylalanine, and proline. Each experiment had its own control and was replicated several times. Each replicate consisted of pooled oocytes obtained at one time from 3-4 donors and which were distributed to all treatments. Oocyte recovery from animals. Ovaries were removed immediately after killing by opening the abdomen, freeing the ovaries and washing them 3 times in basic medium without BSA. Thev were transferred to a plastic culture dish containing basic medium under a 5 ml laver of mineral oil (viscositv 1251135). The medium-used for washing and handling oocytes before incubation did not contain any carbohydrate or amino acids. Follicles ranging from 1 mm in diameter up to preovulatory graafian follicles were ruptured with a sharp needle under a stereomicroscope. Oocytes with attached cumulus cells released from the follicles were sucked into a finely drawn pipette Exptl
Cell Res 91 (1975)
Table 1. Composition of the media Concentration Component Basic NaCl
g/l
medium
5.54-6.96 KC1 0.356 Ca Lactate. 5H,0b 0.527 CaClz*2H,0b 0.256 KH,PO, 0.162 MgS04. 7HZ0 0.293 NaHCO, 2.106 Antibiotics: PenicillinG Streptomycin Bovine serum albumin 4.000 Carbohydrates,
acids,
94.7-l 19.10a 4.78 1.71 1.71 1.19 1.19 25.07 100 U/ml 50 iuglml 0.4 %
when added
(tables 2-5) Na pyruvate 0.028 Na lactate (60 % syrup) 3.68 ml Glucose 1.OOo Amino
mM
0.25 21.55 5.56
when added
(tables 3-5)’ L-glutamine L-methionine L-phenylalanine L-proline L-glutamine (table 6)
1.460 0.447 0.049 0.115 0.017-7.307
10.00
3.00 0.30 1.00 0.08-50
a NaCl was varied to maintain the medium at an osmotic concentration of 308 mOsm. b Calcium was supplied from various sources depending upon whether or not the medium contained lactate. ’ When all 4 amino acids were used each was added at l/4 the level shown. and transferred to a round bottom glass dish containing the medium under an oil layer. By repeated suction and expulsion of the oocytes the mucous layer around the cumulus cells and oocytes was removed. Oocytes were washed another 3 times in the medium with 0.1 % BSA, and pooled until incubation. Oocytes appearing to be degenerating, when viewed microscopically, were discarded from the pool and the remainder prepared for incubation. Preparation of culture medium. All the components of the basic medium, except NaHCO, and BSA, were dissolved in advance in water redistilled twice through glass. Just before use, NaHCO,, the three carbohydrates and amino acids. as desired. and BSA were added to the basic medium’in that order (table 1). All culture media had 100 % COz gas bubbled through it briefly to adjust the final pH to about 7.0. The media was sterilized by filtration with Millipore filters with 0.22 or 0.45 Mum diameter nores. All filtered media were placed ‘in sterile b&ties and sealed with serum rubber stoppers under pressure until used.
Culturing of rabbit follicular Table
oocytes
115
2. Effect of somecarbohydrates on maturation of oocytes Meiotic phase of development, % & S.E.a Meiosis II No. of oocytes cultured
Media 1. 2. 3. 4. 5.
Basic medium f Pyruvate (P) + Lactate (L) + Glucose (G) +P+L+G
45b 44: :z
Meiosis I (
Experimental Cell Research91 (1975) 113-l 18
CARBOHYDRATE AND RABBIT
AND
AMINO
AMMONIA
FOLLICULAR IN-HA
ACID
REQUIREMENTS
PRODUCTION
OOCYTES
MATURED
OF IN VITRO
BAE and R. H. FOOTE
Department of Animal Science, Cornell University, Ithaca, N.Y. 14850, USA
SUMMARY Rabbit follicular oocytes were cultured at 37°C for 18-24 h in a basic salt medium containing 0.4% bovine serum albumin (BSA), carbohydrates and amino acids in various combinations. Osmolarity of the medium was maintained at 308 mOsm. The carbohydrates, pyruvate, lactate and glucose were all about equally beneficial, but not essential for rabbit oocyte maturation. Glutamine and proline, but not methionine or phenylalanine stimulated oocyte development. Glutamine stimulated more follicular oocytes to develop to prophase and metaphase II than did any of the three carbohydrates tested alone or in combination. Ammonia production after 24 h of culture was highest in media containing glutamine (15.2 pg/ml) but this was not inhibitory to maturation. Negligible amounts of ammonia were found with the other amino acids added. With 0, 0.08, 0.4, 2, 10 and 50 mM of glutamine in the basic salt medium, plus 0.4 % BSA, but without carbohydrates, 30, 73, 70, 71, 59 and 45 % of the follicular oocytes developed to the prophase or metaphase II stage. It is concluded that the optimum level of glutamine ranged from 0.08 to 2 mM and that no carbohydrate need be added to the medium for culturing oocytes when glutamine is included.
Requirements for resumption of meiosis in the oocyte prior to ovulation and after variable periods of ‘storage’ in the dictyate condition are not well understood. Chang [l] found that rabbit serum added to Ringer’s solution permitted rabbit follicular oocytes cultured in vitro to develop to metaphase II, but no studies in the rabbit with defined media have been reported. The rabbit is a good model for these investigations because, as an induced ovulator, accurately timed events in vivo can be compared with studies in vitro. Kane & Foote [2] reported that pyruvate increased the proportion of rabbit zygotes which developed into blastocysts. Brinster [3] suggested that rabbit embryos might use nitrogenous compounds as a source of energy.
Kane [4] showed that a high proportion of zygotes develop into hatched blastocysts in a complex defined medium containing a variety of amino acids, and that the inclusion of simple carbohydrates as energy substrates had no effect. Mouse oocytes will develop to metaphaseII in defined media containing pyruvate, oxaloacetate [SJor lactate under aerobic conditions if NAD is added to the medium [6]. The amino acids requirement is not well defined. Cumulus cells recently have been reported not to improve mouse oocyte maturation in vitro [7] despite earlier reports to the contrary [8-g]. Hamster oocytes can undergo maturation when cultured in a carbohydrate-containing salt solution without bovine serum albumin Exptl Cell Res 91 (1975)
114 Bae and Foote (BSA) providing that isoleucine, glutamine, phenylalanine and methionine are included [lo]. Composition of media for culturing embryos of several species has been reviewed by Gwatkin [l 11. All media given contain some simple carbohydrate. Little attention has been given to the possibility that the oocyte might utilize a non-carbohydrate source of energy for development. With amino acids in the medium ammonia formation is expected. Ammonia toxicity for the living cell is well recognized in vitro and in vivo [12-141. There has been no attempt to investigate the toxicity of ammonia for the mammalian oocytes, eggs and embryos, although many researchers have used various amino acids in culture media. The present investigations were undertaken to elucidate carbohydrate and amino acid requirements for oocyte maturation and to determine ammonia production during culture. MATERIALS
AND
METHODS
Animals. All rabbits used in the present experiments were virgin Dutch belted does raised in our colony. They were 4 to 7 months old when sacrificed by disarticulation to avoid possible effects of anesthesia on the ovaries. Experimental design. Several experiments were conducted to compare the carbohydrates most frequently used for culture of mammalian oocytes and embryos (pyruvate, lactate and glucose) along with the amino acids, glutamine, methionine, phenylalanine, and proline. Each experiment had its own control and was replicated several times. Each replicate consisted of pooled oocytes obtained at one time from 3-4 donors and which were distributed to all treatments. Oocyte recovery from animals. Ovaries were removed immediately after killing by opening the abdomen, freeing the ovaries and washing them 3 times in basic medium without BSA. Thev were transferred to a plastic culture dish containing basic medium under a 5 ml laver of mineral oil (viscositv 1251135). The medium-used for washing and handling oocytes before incubation did not contain any carbohydrate or amino acids. Follicles ranging from 1 mm in diameter up to preovulatory graafian follicles were ruptured with a sharp needle under a stereomicroscope. Oocytes with attached cumulus cells released from the follicles were sucked into a finely drawn pipette Exptl
Cell Res 91 (1975)
Table 1. Composition of the media Concentration Component Basic NaCl
g/l
medium
5.54-6.96 KC1 0.356 Ca Lactate. 5H,0b 0.527 CaClz*2H,0b 0.256 KH,PO, 0.162 MgS04. 7HZ0 0.293 NaHCO, 2.106 Antibiotics: PenicillinG Streptomycin Bovine serum albumin 4.000 Carbohydrates,
acids,
94.7-l 19.10a 4.78 1.71 1.71 1.19 1.19 25.07 100 U/ml 50 iuglml 0.4 %
when added
(tables 2-5) Na pyruvate 0.028 Na lactate (60 % syrup) 3.68 ml Glucose 1.OOo Amino
mM
0.25 21.55 5.56
when added
(tables 3-5)’ L-glutamine L-methionine L-phenylalanine L-proline L-glutamine (table 6)
1.460 0.447 0.049 0.115 0.017-7.307
10.00
3.00 0.30 1.00 0.08-50
a NaCl was varied to maintain the medium at an osmotic concentration of 308 mOsm. b Calcium was supplied from various sources depending upon whether or not the medium contained lactate. ’ When all 4 amino acids were used each was added at l/4 the level shown. and transferred to a round bottom glass dish containing the medium under an oil layer. By repeated suction and expulsion of the oocytes the mucous layer around the cumulus cells and oocytes was removed. Oocytes were washed another 3 times in the medium with 0.1 % BSA, and pooled until incubation. Oocytes appearing to be degenerating, when viewed microscopically, were discarded from the pool and the remainder prepared for incubation. Preparation of culture medium. All the components of the basic medium, except NaHCO, and BSA, were dissolved in advance in water redistilled twice through glass. Just before use, NaHCO,, the three carbohydrates and amino acids. as desired. and BSA were added to the basic medium’in that order (table 1). All culture media had 100 % COz gas bubbled through it briefly to adjust the final pH to about 7.0. The media was sterilized by filtration with Millipore filters with 0.22 or 0.45 Mum diameter nores. All filtered media were placed ‘in sterile b&ties and sealed with serum rubber stoppers under pressure until used.
Culturing of rabbit follicular Table
oocytes
115
2. Effect of somecarbohydrates on maturation of oocytes Meiotic phase of development, % & S.E.a Meiosis II No. of oocytes cultured
Media 1. 2. 3. 4. 5.
Basic medium f Pyruvate (P) + Lactate (L) + Glucose (G) +P+L+G
45b 44: :z
Meiosis I (
