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Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova
SmallRuminantResearch, 7 (1992) 347-352
347
Elsevier Science Publishers B.V., Amsterdam
Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova
J.E. Butler and J.E. Williams Department of Animal Science, University of Idaho, Moscow, ID 83843, USA (Accepted 3 July 1991 )
ABSTRACT Butler, J.E. and Williams, J.E., 1992. Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova. Small Rumin. Res., 7:347-352. Using a noninvasive, fluorometric assay, glucose uptake was measured in individual preimplantation sheep embryos and unfertilized ova. Glucose uptake remained low through the morula stage of development. By the late morula stage, glucose uptake began to increase and was significantly higher by the blastocyst stage (P< 0.08 ). This pattern was similar to that reported in mice.
INTRODUCTION
The ability to maintain embryos in vitro is central to the ovine application of technologies such as embryo transfer, in vitro maturation and fertilization, embryo cloning and genetic engineering. A better understanding of the metabolic needs of early embryos from livestock species is essential in improving in vitro culture conditions. Considerable knowledge of the metabolism of preimplantation embryos in laboratory species has been gained. However, little information of the metabolic events during early embryonic development in farm animals exists. The limited number of embryos that can be obtained from livestock donors even with the use of superovulation, cost and difficulty of obtaining oviductal embryos from livestock, and the need to use groups of embryos in order to obtain accurate results are factors contributing to the scarcity of information in livestock embryo metabolism. Recent techniques allow the measurements of substrate usage by individual embryos (Leese and Barton, 1984; O'Fallon and Wright, 1986; Wales, 1986 ), but only limited use has been made of them with livestock embryos. The objective of this study was to determine patterns of glucose uptake by early embryos and unfertilized sheep ova. 0921-4488/92/$05.00 © 1992 Elsevier Science Publishers B.V. All fights reserved.
348
J.E. BUTLER AND J.E. WILLIAMS
MATERIALS AND METHODS
Materials Mature white-face ewes from the U.S. Sheep Experiment Station, Dubois, Idaho were used. Animals were cared for at the University of Idaho Sheep Research Center in accordance with U.S. Public Health Service guidelines. Follicle stimulating hormone (FSH) was obtained from Schering Corp. (Kenilworth, N J, USA). Vaginal pessaries for estrous synchronization were purchased from Tuco Products Inc. (Orangeville, Ont., Canada). Paraffin oil was obtained from Fisher Scientific (Kent, WA, USA); all other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals in the preparation of culture media were of tissue culture grade. All enzymes in the determination of glucose uptake were of assay grade. Embryo recovery Embryos were surgically recovered from superovulated donor ewes at 1, 1.5, 2, 3, 5, 6, and 7 d after mating to rams of known fertility. Unfertilized ova were collected from unmated ewes in the morning following the end of behavioral estrus. Ewes were synchronized with vaginal pessaries and super ovulated by twice daily injecting decreasing amounts of FSH (Bondioli and Wright, 1980). Embryos were recovered following midventral laparotomy (Anderson et al., 1981 ) in Dulbecco's phosphate buffered saline containing 25 mg/1 kanamycin sulfate and 1% heat-treated newborn calf serum. Embryo culture Embryos were cultured in Whitten's medium (Whitten and Biggers, 1968 ), modified to contain 1.0 mM glucose, 1.0 mM sodium pyruvate and 5 mg/ml bovine serum albumin. Prior to measurement of glucose uptake, embryos were washed three times in culture medium and graded. Embryos that had gross morphological defects or were intermediate in their stage of development (e.g., three-ceU embryos) were excluded from the study. Individual embryos were placed in 62 nl drops of culture medium on siliconized microscope slides maintained under a layer of saline-equilibrated paraffin oil. Methods for the manufacture of micropipets and the pipetting of microdrops has been described previously (Mroz and Lechene, 1980; Butler et al., 1988 ). In addition to the drops containing embryos, each slide also contained two control drops without embryos. It was determined that approximately 3 nl were transferred to each drop along with the embryo, resulting in a final drop volume of 65 nl. Due to the fact that Whitten's medium has bicarbonate as its primary buffer, drops were prepared the previous afternoon and allowed to equilibrate overnight in an atmosphere of 5% CO2 in air. Embryos were then cultured for 3 hrs at 37 °C in a 5% CO2 atmosphere. At the end of the incubation period, a
NONINVASIVEMEASUREMENTOFGLUCOSEUPTAKE
349
5.8 nl sample of medium was removed from each drop and assayed to determine glucose content.
Glucose assay The assay was based on the technique of Mroz and Lechene (1980) for measurement of glucose in picoliter samples, as modified and validated for use with individual embryos (Butler et al., 1988). The 5.8 nl samples from the culture drops were added to 47 nl assay drops, also maintained under paraffin oil on a siliconized slide. Composition of the assay drops was 3.7 mM MgSOa'7H20, 0.6 mM nicotinamide adenine dinucleotide phosphate (NADP), 0.5 mM adenosine 5'-triphosphate (ATP), 0.5 mM dithiothreitol, 12 U/ml hexokinase (E.C. 2.7.1.1 ) (Sigma type VII), and 6 U/ml glucose-6phosphate dehydrogenase (E.C. 1.1.1.49) (Sigma type IX) in 50 mM EPPS buffer (N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid). After addition of the sample, assay drops were incubated at 37 °C for 15 minutes to allow the reaction to go to completion. Glucose concentration was determined fluorometricaUy by the stoichiometric production of dihydronicotinamide adenine dinucleotide phosphate (NADPH) in the coupled reaction: hexokinase
Glucose + ATP
, glucose-6-phosphate + ADP glucose-6-phosphate
Glucose-6-phosphate + NADP
,6-P-gluconate dehydrogenase
+NADPH+H + NADPH production was measured using a Nikon Diaphot microscope equipped for quantitative fluorometry. Readings obtained were stable for at least 30 min. Two measurements were made on each assay drop and the values averaged. A standard curve was included in each assay. Glucose uptake was calculated by subtracting the amount of glucose determined for each culture drop from the average glucose content of the two control drops that did not contain embryos.
Statistical analysis Data were analyzed following log transformation using the general linear models procedure of the Statistical Analysis System program (SAS, 1985 ). Analysis of variance revealed donor ewe to be a significant source of variation (P< 0.01 ). To account for this, the statistical model was modified to include ewe-within-stage of embryonic development [ ewe (stage) ] as an error term. Differences in glucose uptake by stage of development were then determined using Fisher's protected least significant difference test.
350
J.E. BUTLERAND J.E. WILLIAMS
TABLE1 Least squares means and standard errors (SEM) for glucose uptake by sheep unfertilized ova and embryos. Units are in p m o l / e m b r y o / 3 h Stage
Unfertilized ova l-cell embryos 2-cell embryos 4-cell embryos 8-cell embryos Early morula embryos 2 Late morula embryos 3 Blastocyst embryos Expanded blastocysts
Number
Uptake I
Donors
Embryos
Mean
SEM
5 4 4 4 5 4 5 8 5
38 42 18 21 34 45 16 22 18
14.00" 7.07 a 15.55 ab 13.79 a 16.18 "b 12.90 a 27.70 a~ 37.85 ~ 46.81 c
7.29 5.96 9.36 8.29 6.90 5.90 12.14 9.62 11.68
Walues with different superscripts are significantly different ( P < 0.08 ). 2Collected on day 5 (estrus = day 0). 3Collected on day 6. RESULTS
The increase in fluorescence observed with increasing amounts of glucose was highly linear. The mean coefficient of determination r 2 for 34 standard curves was 0.99. Accuracy of the assay was assessed using the control drops included in each assay. Mean glucose concentration in control drops was 1.05 mM (SEM = 0.015 ) compared to the actual concentration of 1.00 mM. Because of the variation due to donor ewe, statistically significant differences in glucose uptake with stage of development could only be established at P < 0.08. Least squares means and standard errors for glucose uptake are in Table 1. Mean glucose uptake for unfertilized ova was 14.0 pmol/embryo/3 h. There was an apparent drop in glucose uptake at the one-cell stage but this decrease was not statistically significant ( P = 0 . 6 8 ) . Uptake values at the twocell stage returned to approximately those seen for unfertilized ova. Uptake then remained relatively constant up to the late morula stage. Glucose uptake began to increase at the late morula stage and was significantly higher ( P < 0.08 ) by the expanded blastocyst stage. DISCUSSION
The majority of data on substrate usage by preimplantation mammalian embryos comes from work in mice. Brinster (1969) measured the accumulation of [U-14C]-glucose by preimplantation mouse embryos. He reported that glucose metabolism was constant up to the morula stage after which it increased through the blastocyst stage. In other experiments with mice, Gard-
NONINVASIVEMEASUREMENT OF GLUCOSE UPTAKE
3 51
ner and Leese (1986) employed the same methods used in this paper and reported a similar pattern with the exception that the rise in glucose uptake did not appear until the blastocyst stage. The limited data available on glucose usage by embryos from livestock species indicate that the pattern of glucose metabolism in these embryos is similar to that in mice. Flood and Wiebold ( 1988 ) measured the metabolism of [ 5-3H]-glucoseby porcine embryos and found a significant increase in glucose metabolism at the compacted morula stage. Pike ( 1981 ) measuring the incorporation of [U-14H]-glucose into the acid-soluble glycogen pool in sheep embryos found an increase in label incorporation between the 8-cell and morula stages that continued through the blastocyst stage. These data correspond well with the present study. As with mice, glucose uptake in sheep embryos was low until the late morula stage and then showed a steady increase through the expanded blastocyst stage. Taken together, these data suggest that embryos from livestock use only limited amounts of glucose until the late morula or blastocyst stages. Indirect evidence that preimplantation ovine embryos metabolize only limited amounts of glucose comes from the work of Thompson et al. (1989) who showed that the presence or absence of glucose in synthetic oviductal medium had no effect on the development of sheep embryos in vitro. The reason for low glucose utilization in early embryos is thought to be due to an inhibition of the Embden-Myerhof pathway. Barbehenn et al. (1974) reported that in mice the inhibition site was at the enzyme phosphofructokinase (PFK). Further evidence for PFK as the control site comes from the finding that prior to the blastocyst stage, mouse embryos have a high ATP/ ADP ratio which is known to inhibit PFK activity (Leese et al., 1984). Although this study as well as others indicate that the glycolytic pathway is blocked in early embryos from livestock species, the site of blockage may differ from that in mice. Based on the production of 3H20 from [ 5-3H I-glucose, Rieger and Guay (1988) concluded that the block in bovine embryos occurred further down the glycolytic pathway at pyruvate kinase rather than at PFK. The present data also indicate that the amount of glucose used by ovine embryos is greater than used by mouse embryos. Using the same technique, Gardner and Leese (1986) reported glucose uptake of 4 pmol per mouse embryo per h at the blastocyst stage. Extrapolating their data to the 3 h time period used in this study, the total uptake would be 12 pmol/embryo/3 h or approximately 30% of the uptake observed with sheep blastocysts in this study. Embryos from livestock species are being increasingly used in techniques to improve animal production. However, very little is known about the metabolic events of early development in farm animals. This study characterized the pattern of glucose uptake by sheep embryos from the unfertilized ova to the expanded blastocyst stage of development. Further work needs to be done to determine the possible effects of interactions between culture media corn-
352
J.E. BUTLER AND J.E. WILLIAMS
ponents as well as the potential role of oviductal and/or uterine factors in controlling embryo metabolism. A better understanding of the metabolic needs of developing embryos may lead to improved conditions for maintaining livestock embryos in vitro. ACKNOWLEDGEMENTS
The authors acknowledge the U.S. Sheep Experiment Station, Dubois, Idaho, USA for providing sheep used in this experiment. This work was supported by NIH grant HD24556 to J.E.B.
REFERENCES Anderson, G.B., Bradford, G.E. and Cupps, P.T., 1981. Length of gestation in ewes carrying lambs of two different breeds. Theriogenology, 16:119-129. Barbehenn, E.K., Wales, R.G. and Lowry, O.H., 1974. The explanation for the blockade of glycolysis in early mouse embryos. Proc. Natl. Acad. Sci. USA, 71: 1056-1069. Bondioli, K.R. and Wright, R.W., 1980. Superovulation of progestin synchronized ewes. Theriogenology, 13: 89. Brinster, R.L., 1969. Incorporation of carbon from glucose and pyruvate into the preimplantation mouse embryo. Expt. Cell. Res., 58:153-158. Butler, J.E., Lechene, C. and Biggers, J.D., 1988. Noninvasive measurement of glucose uptake by two populations of murine embryos. Biol. Reprod., 39: 779-786. Flood, M.R. and Wiebold, J.L., 1988. Glucose metabolism by preimplantation pig embryos. J. Reprod. Fert., 84: 7-12. Gardner, D.K. and Leese, H.J., 1986. Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos. Hum. Reprod., 1: 25-27. Leese, H.J. and Barton, A.M., 1984. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J. Reprod. Fert., 72: 9-13. Leese, H.J., Biggers, J.D., Mroz, E.A. and Lechene, C., 1984. Nucleotides in a single mammalian ovum or preimplantation embryo. Anal. Biochem., 140: 443-448. Mroz, E.A. and Lechene, C., 1980. Fluorescence analysis of picoliter samples. Anal. Biochem., 102: 90-96. O'Fallon, J.V. and Wright, R.W., Jr., 1986. Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos. Biol. Reprod., 34: 58-64. Pike, I.L., 1981. Comparative studies of embryo metabolism in early pregnancy. J. Reprod. Fert. Suppl., 29:203-213. Rieger, D. and Guay, P., 1988. Measurement of the metabolism of energy substrates in individual bovine blastocysts. J. Reprod. Fert., 83: 585-591. SAS, 1985. SAS User's Guide: Statistics, Version 5. SAS Institute, Cary NC, pp. 433-506. Thompson, J.G.E., Parton, G.A.J., Cruickshank, G.W., Smith, J.F. and Wales, R.G., 1989. Development of sheep preimplantation embryos in media supplemented with glucose and acetate. Theriogenology, 32: 323-330. Wales, R.G., 1986. Measurement of metabolic turnover in single mouse embryos. J. Reprod. Fert., 76: 717-725. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fert., 17:399-401.
347
Elsevier Science Publishers B.V., Amsterdam
Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova
J.E. Butler and J.E. Williams Department of Animal Science, University of Idaho, Moscow, ID 83843, USA (Accepted 3 July 1991 )
ABSTRACT Butler, J.E. and Williams, J.E., 1992. Noninvasive measurement of glucose uptake by preimplantation sheep embryos and unfertilized ova. Small Rumin. Res., 7:347-352. Using a noninvasive, fluorometric assay, glucose uptake was measured in individual preimplantation sheep embryos and unfertilized ova. Glucose uptake remained low through the morula stage of development. By the late morula stage, glucose uptake began to increase and was significantly higher by the blastocyst stage (P< 0.08 ). This pattern was similar to that reported in mice.
INTRODUCTION
The ability to maintain embryos in vitro is central to the ovine application of technologies such as embryo transfer, in vitro maturation and fertilization, embryo cloning and genetic engineering. A better understanding of the metabolic needs of early embryos from livestock species is essential in improving in vitro culture conditions. Considerable knowledge of the metabolism of preimplantation embryos in laboratory species has been gained. However, little information of the metabolic events during early embryonic development in farm animals exists. The limited number of embryos that can be obtained from livestock donors even with the use of superovulation, cost and difficulty of obtaining oviductal embryos from livestock, and the need to use groups of embryos in order to obtain accurate results are factors contributing to the scarcity of information in livestock embryo metabolism. Recent techniques allow the measurements of substrate usage by individual embryos (Leese and Barton, 1984; O'Fallon and Wright, 1986; Wales, 1986 ), but only limited use has been made of them with livestock embryos. The objective of this study was to determine patterns of glucose uptake by early embryos and unfertilized sheep ova. 0921-4488/92/$05.00 © 1992 Elsevier Science Publishers B.V. All fights reserved.
348
J.E. BUTLER AND J.E. WILLIAMS
MATERIALS AND METHODS
Materials Mature white-face ewes from the U.S. Sheep Experiment Station, Dubois, Idaho were used. Animals were cared for at the University of Idaho Sheep Research Center in accordance with U.S. Public Health Service guidelines. Follicle stimulating hormone (FSH) was obtained from Schering Corp. (Kenilworth, N J, USA). Vaginal pessaries for estrous synchronization were purchased from Tuco Products Inc. (Orangeville, Ont., Canada). Paraffin oil was obtained from Fisher Scientific (Kent, WA, USA); all other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals in the preparation of culture media were of tissue culture grade. All enzymes in the determination of glucose uptake were of assay grade. Embryo recovery Embryos were surgically recovered from superovulated donor ewes at 1, 1.5, 2, 3, 5, 6, and 7 d after mating to rams of known fertility. Unfertilized ova were collected from unmated ewes in the morning following the end of behavioral estrus. Ewes were synchronized with vaginal pessaries and super ovulated by twice daily injecting decreasing amounts of FSH (Bondioli and Wright, 1980). Embryos were recovered following midventral laparotomy (Anderson et al., 1981 ) in Dulbecco's phosphate buffered saline containing 25 mg/1 kanamycin sulfate and 1% heat-treated newborn calf serum. Embryo culture Embryos were cultured in Whitten's medium (Whitten and Biggers, 1968 ), modified to contain 1.0 mM glucose, 1.0 mM sodium pyruvate and 5 mg/ml bovine serum albumin. Prior to measurement of glucose uptake, embryos were washed three times in culture medium and graded. Embryos that had gross morphological defects or were intermediate in their stage of development (e.g., three-ceU embryos) were excluded from the study. Individual embryos were placed in 62 nl drops of culture medium on siliconized microscope slides maintained under a layer of saline-equilibrated paraffin oil. Methods for the manufacture of micropipets and the pipetting of microdrops has been described previously (Mroz and Lechene, 1980; Butler et al., 1988 ). In addition to the drops containing embryos, each slide also contained two control drops without embryos. It was determined that approximately 3 nl were transferred to each drop along with the embryo, resulting in a final drop volume of 65 nl. Due to the fact that Whitten's medium has bicarbonate as its primary buffer, drops were prepared the previous afternoon and allowed to equilibrate overnight in an atmosphere of 5% CO2 in air. Embryos were then cultured for 3 hrs at 37 °C in a 5% CO2 atmosphere. At the end of the incubation period, a
NONINVASIVEMEASUREMENTOFGLUCOSEUPTAKE
349
5.8 nl sample of medium was removed from each drop and assayed to determine glucose content.
Glucose assay The assay was based on the technique of Mroz and Lechene (1980) for measurement of glucose in picoliter samples, as modified and validated for use with individual embryos (Butler et al., 1988). The 5.8 nl samples from the culture drops were added to 47 nl assay drops, also maintained under paraffin oil on a siliconized slide. Composition of the assay drops was 3.7 mM MgSOa'7H20, 0.6 mM nicotinamide adenine dinucleotide phosphate (NADP), 0.5 mM adenosine 5'-triphosphate (ATP), 0.5 mM dithiothreitol, 12 U/ml hexokinase (E.C. 2.7.1.1 ) (Sigma type VII), and 6 U/ml glucose-6phosphate dehydrogenase (E.C. 1.1.1.49) (Sigma type IX) in 50 mM EPPS buffer (N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid). After addition of the sample, assay drops were incubated at 37 °C for 15 minutes to allow the reaction to go to completion. Glucose concentration was determined fluorometricaUy by the stoichiometric production of dihydronicotinamide adenine dinucleotide phosphate (NADPH) in the coupled reaction: hexokinase
Glucose + ATP
, glucose-6-phosphate + ADP glucose-6-phosphate
Glucose-6-phosphate + NADP
,6-P-gluconate dehydrogenase
+NADPH+H + NADPH production was measured using a Nikon Diaphot microscope equipped for quantitative fluorometry. Readings obtained were stable for at least 30 min. Two measurements were made on each assay drop and the values averaged. A standard curve was included in each assay. Glucose uptake was calculated by subtracting the amount of glucose determined for each culture drop from the average glucose content of the two control drops that did not contain embryos.
Statistical analysis Data were analyzed following log transformation using the general linear models procedure of the Statistical Analysis System program (SAS, 1985 ). Analysis of variance revealed donor ewe to be a significant source of variation (P< 0.01 ). To account for this, the statistical model was modified to include ewe-within-stage of embryonic development [ ewe (stage) ] as an error term. Differences in glucose uptake by stage of development were then determined using Fisher's protected least significant difference test.
350
J.E. BUTLERAND J.E. WILLIAMS
TABLE1 Least squares means and standard errors (SEM) for glucose uptake by sheep unfertilized ova and embryos. Units are in p m o l / e m b r y o / 3 h Stage
Unfertilized ova l-cell embryos 2-cell embryos 4-cell embryos 8-cell embryos Early morula embryos 2 Late morula embryos 3 Blastocyst embryos Expanded blastocysts
Number
Uptake I
Donors
Embryos
Mean
SEM
5 4 4 4 5 4 5 8 5
38 42 18 21 34 45 16 22 18
14.00" 7.07 a 15.55 ab 13.79 a 16.18 "b 12.90 a 27.70 a~ 37.85 ~ 46.81 c
7.29 5.96 9.36 8.29 6.90 5.90 12.14 9.62 11.68
Walues with different superscripts are significantly different ( P < 0.08 ). 2Collected on day 5 (estrus = day 0). 3Collected on day 6. RESULTS
The increase in fluorescence observed with increasing amounts of glucose was highly linear. The mean coefficient of determination r 2 for 34 standard curves was 0.99. Accuracy of the assay was assessed using the control drops included in each assay. Mean glucose concentration in control drops was 1.05 mM (SEM = 0.015 ) compared to the actual concentration of 1.00 mM. Because of the variation due to donor ewe, statistically significant differences in glucose uptake with stage of development could only be established at P < 0.08. Least squares means and standard errors for glucose uptake are in Table 1. Mean glucose uptake for unfertilized ova was 14.0 pmol/embryo/3 h. There was an apparent drop in glucose uptake at the one-cell stage but this decrease was not statistically significant ( P = 0 . 6 8 ) . Uptake values at the twocell stage returned to approximately those seen for unfertilized ova. Uptake then remained relatively constant up to the late morula stage. Glucose uptake began to increase at the late morula stage and was significantly higher ( P < 0.08 ) by the expanded blastocyst stage. DISCUSSION
The majority of data on substrate usage by preimplantation mammalian embryos comes from work in mice. Brinster (1969) measured the accumulation of [U-14C]-glucose by preimplantation mouse embryos. He reported that glucose metabolism was constant up to the morula stage after which it increased through the blastocyst stage. In other experiments with mice, Gard-
NONINVASIVEMEASUREMENT OF GLUCOSE UPTAKE
3 51
ner and Leese (1986) employed the same methods used in this paper and reported a similar pattern with the exception that the rise in glucose uptake did not appear until the blastocyst stage. The limited data available on glucose usage by embryos from livestock species indicate that the pattern of glucose metabolism in these embryos is similar to that in mice. Flood and Wiebold ( 1988 ) measured the metabolism of [ 5-3H]-glucoseby porcine embryos and found a significant increase in glucose metabolism at the compacted morula stage. Pike ( 1981 ) measuring the incorporation of [U-14H]-glucose into the acid-soluble glycogen pool in sheep embryos found an increase in label incorporation between the 8-cell and morula stages that continued through the blastocyst stage. These data correspond well with the present study. As with mice, glucose uptake in sheep embryos was low until the late morula stage and then showed a steady increase through the expanded blastocyst stage. Taken together, these data suggest that embryos from livestock use only limited amounts of glucose until the late morula or blastocyst stages. Indirect evidence that preimplantation ovine embryos metabolize only limited amounts of glucose comes from the work of Thompson et al. (1989) who showed that the presence or absence of glucose in synthetic oviductal medium had no effect on the development of sheep embryos in vitro. The reason for low glucose utilization in early embryos is thought to be due to an inhibition of the Embden-Myerhof pathway. Barbehenn et al. (1974) reported that in mice the inhibition site was at the enzyme phosphofructokinase (PFK). Further evidence for PFK as the control site comes from the finding that prior to the blastocyst stage, mouse embryos have a high ATP/ ADP ratio which is known to inhibit PFK activity (Leese et al., 1984). Although this study as well as others indicate that the glycolytic pathway is blocked in early embryos from livestock species, the site of blockage may differ from that in mice. Based on the production of 3H20 from [ 5-3H I-glucose, Rieger and Guay (1988) concluded that the block in bovine embryos occurred further down the glycolytic pathway at pyruvate kinase rather than at PFK. The present data also indicate that the amount of glucose used by ovine embryos is greater than used by mouse embryos. Using the same technique, Gardner and Leese (1986) reported glucose uptake of 4 pmol per mouse embryo per h at the blastocyst stage. Extrapolating their data to the 3 h time period used in this study, the total uptake would be 12 pmol/embryo/3 h or approximately 30% of the uptake observed with sheep blastocysts in this study. Embryos from livestock species are being increasingly used in techniques to improve animal production. However, very little is known about the metabolic events of early development in farm animals. This study characterized the pattern of glucose uptake by sheep embryos from the unfertilized ova to the expanded blastocyst stage of development. Further work needs to be done to determine the possible effects of interactions between culture media corn-
352
J.E. BUTLER AND J.E. WILLIAMS
ponents as well as the potential role of oviductal and/or uterine factors in controlling embryo metabolism. A better understanding of the metabolic needs of developing embryos may lead to improved conditions for maintaining livestock embryos in vitro. ACKNOWLEDGEMENTS
The authors acknowledge the U.S. Sheep Experiment Station, Dubois, Idaho, USA for providing sheep used in this experiment. This work was supported by NIH grant HD24556 to J.E.B.
REFERENCES Anderson, G.B., Bradford, G.E. and Cupps, P.T., 1981. Length of gestation in ewes carrying lambs of two different breeds. Theriogenology, 16:119-129. Barbehenn, E.K., Wales, R.G. and Lowry, O.H., 1974. The explanation for the blockade of glycolysis in early mouse embryos. Proc. Natl. Acad. Sci. USA, 71: 1056-1069. Bondioli, K.R. and Wright, R.W., 1980. Superovulation of progestin synchronized ewes. Theriogenology, 13: 89. Brinster, R.L., 1969. Incorporation of carbon from glucose and pyruvate into the preimplantation mouse embryo. Expt. Cell. Res., 58:153-158. Butler, J.E., Lechene, C. and Biggers, J.D., 1988. Noninvasive measurement of glucose uptake by two populations of murine embryos. Biol. Reprod., 39: 779-786. Flood, M.R. and Wiebold, J.L., 1988. Glucose metabolism by preimplantation pig embryos. J. Reprod. Fert., 84: 7-12. Gardner, D.K. and Leese, H.J., 1986. Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos. Hum. Reprod., 1: 25-27. Leese, H.J. and Barton, A.M., 1984. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J. Reprod. Fert., 72: 9-13. Leese, H.J., Biggers, J.D., Mroz, E.A. and Lechene, C., 1984. Nucleotides in a single mammalian ovum or preimplantation embryo. Anal. Biochem., 140: 443-448. Mroz, E.A. and Lechene, C., 1980. Fluorescence analysis of picoliter samples. Anal. Biochem., 102: 90-96. O'Fallon, J.V. and Wright, R.W., Jr., 1986. Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos. Biol. Reprod., 34: 58-64. Pike, I.L., 1981. Comparative studies of embryo metabolism in early pregnancy. J. Reprod. Fert. Suppl., 29:203-213. Rieger, D. and Guay, P., 1988. Measurement of the metabolism of energy substrates in individual bovine blastocysts. J. Reprod. Fert., 83: 585-591. SAS, 1985. SAS User's Guide: Statistics, Version 5. SAS Institute, Cary NC, pp. 433-506. Thompson, J.G.E., Parton, G.A.J., Cruickshank, G.W., Smith, J.F. and Wales, R.G., 1989. Development of sheep preimplantation embryos in media supplemented with glucose and acetate. Theriogenology, 32: 323-330. Wales, R.G., 1986. Measurement of metabolic turnover in single mouse embryos. J. Reprod. Fert., 76: 717-725. Whitten, W.K. and Biggers, J.D., 1968. Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fert., 17:399-401.