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The effects of carbon-13 incorporation into preimplantation mouse embryos on development before and after implantation
Life Scieacea Vol . 19, pp . 633-640, 1976 . Printed in the U .S .A .
Pergamon Presa
THE EFFECTS OF CARBON-13 INCORPORATION INTO PREIMPLANTATION MOUSE EMBRYOS ON DEVELOPMENT BEFORE AND AFTER IMPLANTATION Horst Spielmann, Hans Georg Eibs, Dagmar Nagel and Charles T . Gregg + Pharmakologisches Institut der Freien Universitât Berlin, West Germany, Abteilung Embryonalpharmakologle and Los Alamos Scientific Laboratory , University of California Los Alamos, New Mexico, USA . (Received in final form June 7, 1976) SUMMARY Pretmplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled ' a C-glucose . The ' °C content of the blastocysts was 20 atom $ according to incorporation studies with "`C-glucose . No embryotoxic effects of carbon=l3 incorporation could be detected on the basis of these criteria of normal development : the percentage of embryos reaching the blastocyst stage during the culture period ; the number of cells in these blastocysts ; and the development after transplantation to pseudopregnant foster mothers . The increasing diagnostic use of stable isotopes In situations where the radiation from radioactive tracers is unacceptable, for example in metabolic studies on children and pregnant women, enhances the importance of studies on biological isotope effects in sensitive mammalian systems (2,3,4) . In a previous study where mice were fed a diet of 80 atom $ "C yeast, 60$ of the body carbon was replaced by ' 3 C, but there was no apparent toxic effect on the living animals and no tissue damage was seen either at autopsy or histologically (5) . In additional experiments pregnant mice received uniformly labelled ' 3 C-glucose (U-' 3 C-glucose) by stomach tube during organogenesis . The ' 3C enrichment in fetal tissues after this treatment was 2-5 atom $ 'jC, and no alterations of normal development due to stable isotope enrichment were found (6) . In recent years simple methods for the in vitro culture of early mammalian embryos have been developed (7) . In the present investigation ' aC from culture media containing U- "C-glucose was incorporated Into preimplantation mouse embryos . Glucose as the only energy source does not support the development of mouse embryos before the 8-cell stage (8) . 8-cell mouse embryos were, ~herefore, cu~~ured to the blastocyst stage in media with U-' 3 C-glucose or a C-glucose ( C Indicates natural carbon isotope abundance, t .e . about 1 .1 atom $ 'aC) . The effects of the stable Isotope on embryonic development in vita and after transplantation to foster mothers were studied since this combined in vitz+o and in vivo model system of early mammalian development is sensitive to various embryotoxic treatments (9,10,11) . * A preliminary account of these studies has appeared elsewhere (1) .
633
13C Tozicity in Preimplantation Embryos
634
Vol . 19, No . 5
MATERIALS AND METHODS
In vitro
Culture Conditions
Eight-cell mouse embryos were obtained by flushing the oviducts of female mice of the strain NMRI (breeder : Schwencke â Co ., Nauheim, West Germany) 60 hours after copulation . The standard culture medium was prepared according to Whitten (12) and contained either 0 .3$ bovine serum albumin (W-BSA) or 0 .3$ fatty acid free BSA (W-ff-BSA) . Since BSA and also ff-BSA can be used as energy sources by 8-cell mouse embryos (8), albumin in Whltten's medium was replaced, in a few experiments, by the polysaccharide Flcoll (Pharmacta, Sweden) at a concentration of 0 .5$ (W-FIC) . The culture medium with glucose (concentration 5 .5 mM) as the only energy source was prepared according to Donahue (13) . It was used with 0 .3$ BSA (DONH-8SA), with ff-BSA (DONH-ffBSA), and with 0 .5$ Ficoll (DONW-FIC) . The embryos were cultured according to Brinster's method (14) in 5$ COz in air under paraffin oil (Uvasol : Merck A .G ., Darmstadt, West Germany) In tissue culture dishes (Falcon Plastics, Oxnard, California, USA) . In this system 8-cell mouse embryos developed to the morula stage (2614 cells/embryo to the NMRI strain (15) after 24 hours in culture and reached the blastocyst stage (35-86 cells/embr~o (15) ) after 48 hours as described by previous investigators (7) . The ~ C content of the U-' 3 C-glucose in the in vitro culture experiments was 86 atom $ ~ 3C . Determination of the Cell Number of the Embryos The cell number of the blastocysts was determined at the end of the culture period according to Tarkowski's method (16) .
vitro
in
U- "'C-Glucose Incorporation Studies Embryos were cultured in media containing U-~ ° C-glucose (Amersham-Buchler GmbH, Braunschweig, West Germany) with a specific activity of 3 .0 mC/mmole . After incubation, the embryos were rapidly washed through ten 0 .1 ml drops of culture medium under paraffin oil . For determination of precursor uptake, washed embryos in groups of 20-40 were taken up to 20 ul of the final wash solution and placed in scintillation counting vials containing 0 .1 ml water and 1 .0 ml of the tissue soluültzer Soluene 350 (Packard Instrument Company Inc ., Zurich, Switzerland) was added . When mixing of the water and solubillzer was complete, 10 ml of a toluene PPO-POPOP scintillation counting mixture were added, and the vials were counted to a TRI-CARB liquid scintillation spectrometer (Model 3380 :Packard, Zurich, Switzerland) . Blanks were determined by counting aliquots of the final wash solutions under Identical conditions . The amount of intracellular substrate, expressed as pg atoms of carbon/embryo/hour, was calculated from the counts in the embryos, corrected for background,and the specific acttvtty of the Incubation medium . Transplantation of Blastocysts to Pseudopreyna nt Foster Mothers Normal mouse blastocysts obtained from the uteri of female mice on day 3 of pregnancy (day 0 = the day following the overnight mating period) and also blastocysts which had developed from 8-cell embryos during a 48 hour in vitro culture period were surgically transferred in groups of 5 to each uterine horn of pseudopregnant females on day 2 of pseudopregnancy . Pseudopregnancy was induced by mating normal females with vasectomtzed males . The foster mothers were sacrificed on day 17 of pregnancy and the success rate of the transplantations was determined by the number of resorbed and live embryos . The embryos were weighed, carefully inspected for growth retardation and malformations, and stained for skeletal abnormalities with Alizarin Red S according to Lorke (17) .
vol . 19, No . 5
13C Tozicity in Preimplantation 13mbryoe
635
RESULTS
in vitro
Influence of U-' aC-gl ucose on
development
The first step of differentiation during embryogenesis of the mouse is blastulation, which is characterized by the formation of the blastocyst cavity and the segregation of the embryonic cells into trophoblast and Inner cell mass . The influence of U-~ 3 C-glucose was studied in media containing energy sources other than glucose and in media with glucose as the only energy source . The substances in Whitten's medium that support cleavage of 8-cell mouse embryos are the carbohydrates pyruvate, lactate, and glucose, and also BSA (8) .Table 1 indicates that blastulation of 8-cell embryos was not inhibited when BSA in Whitten's medium was replaced by ff-BSA or even FIC, which does not support cleavage (8) . In the medium with glucose as the only carbohydrate (DDNH), the replacement of B5A by ff-BSA and by FIC did not affect blastulation . Finally the in vitro development of 8-cell embryos to the blastocyst stage was not modified in the presence of U-~ 3 C-glucose even when this substrate was the only energy source In the medium (DDNH-FIC) . TABLE 1
In-vtitx~o
Culture of 8-Cell Mouse Embryos to the Blastocyst Stage (48 hours) in Media containing na C- or U- na C-glucose
Medium
Glucose
B
M
deg
Number of Determinations (Experiments)
W-BSA W-ff-BSA W-FIC
na C ..
DDNH-BSA DDNH-ff-BSA
w
DDNH-FIC W-BSA
U-' 3 C
94$
5$
1$
340 (15)
3$
3$
98 (5)
loo$
~
0
60 (4)
100$
17
p
93$
5$
2$
55 (4)
97$
2$
1$
163 (5)
97$
3$
0
86 (5)
2$
96 (5) 54 (4)
94$
45 (5)
W-ff .-BSA
"
92$
6$
w-Flc
"
loo$
DDNH-ff .-BSA
"
94$
0 4$
0 2$
45 (4)
94$
2$
4$
163 (5)
DONH-FIC
For abbreviations used for the Media see text ; develop-. mental stage of the embryos : B-blastocysts, M~norulae (retarded for 24 hours), deg-degenerated . The cell number of normal blastocysts of the NMRI strain increases from 3514 cells/embryo to early blastocysts up to 86±16 cells/embryo in late blastocysts at the time of implantation (15) . For a better evaluation of the in vitro development the cell number of the embryos was, therefore, determined at the end of the incubation period . According to the results in TABLE 2 the
636
13 C Toxicity is Preimplantation Embryos
Vol . 19, No . 5
cell number of the blastocysts was significantly higher after incubation in the optimal medium (W-BSA) than after culture in the medium with glucose as . Since the cell number is identical in inthe only energy source (DONH-FIC) na C-glucose or U-' 3 C-glucose, no inhibitory effects cubations carried out with of the stable isotope on embryonic development were detected during the in vitro culture period . TABLE 2 Cell Number of Preimplantation Mouse Embryos after
in~Vitro
Culture
from the 8-Cell to the Blastocyst Stage .
Medium
Glucose
Number of Celis per Embryo (± S .D .)
Number of Determinations (Experiments)
1, W-BSA
na C-glucose
80 .1± 21 .7
34 (6)
2, W-BSA
U-~ 3 C-glucose
78 .3 ± 22 .4
22 (4)
3 . DONH-FIC
na C-glucose
53 .6 ± 19 .6
22 (4)
4, DONH-FIC
U-' 3 C-glucose
61 .8 ± 18 .8
33 (6)
1 . is significantly different from 3 . (p < 0 .001), but there is no significant difference between 1 . and 2 . nor between 3 . and 4 . The number of experiments is in parentheses . Carbon- 1 3 Enr ichment During in vitro Culture The incorporation of ~ °C from U- "`C-glucose into whole embryos was determined during the 24 hour period between the 8-cell and the morula stage, and also during the second 24 hour period between the morula and the blastocyst stage . The latter embryos had been cultured in unlabelled medium prior to the incubation with U-~ ° C-glucose . The incorporation of "'C from U- "`C-glucose into whole embryos during the in vitro culture (TABLE 3) was identical in the optimal medium (W-BSA) and in the poorest medium (DONH-FIC) . TABLE 3 Carbon Accumulated From Glucose By Preimplantation Mouse Embryos Cultured
in vitro
Brinster (1970) 8-cell to morula morula to blastula
For 24 Hours
0 u r W-BSA .
r e s u 1 t s DONH-FIC .
-
1 .9 (4)
2 .3 (6)
7 .0
7,2 (4)
6 .8 (10)
Labelled substrate was U- "'C-glucose, specific activity 3 .0 mC/mmole . Carbon accumulated is given in pg atoms of carbon/embryo/h . The number of experiments is given in parentheses .
13C Todcity is Preimplaatation Embryos
Vol . 19, No . 5
Since our data are in good agreement with a previous report (18), the incorporation of ' 3 C from U-' 9 C-glucose into preimplantatton mouse embryos was calculated from our ' ~C incorporation rates . The result is presented in TABLE 4 . TABLE 4 Carbon-13 Content of Mouse Embryos After in
vitro
Culture
For 48 Hours From The 8-Cell To The Blastocyst Stage In Medium DONH-FIC Containing U-' 3C-Glucose C-incorporated-first 24 hours + second 24 hours C-incorporated 24 x 2 .3 pg atoms + 24 x 6 .8 pg atoms C-incorpôrated- 214 pg atoms
2568 pg C- 2 .6ng C
C-content
- 50$ of dry weight ( 23 ng of protein/embryo (18) )
C-content
- 11 .5 ng/embryo
2 .6 ng out of 11 .5 ng a 22 .6$
The "C-content of the glucose is 86 atom $, therefore, the ' 3C-content of the embryos is 19 .4 atom $ . Using the dry weight value of mouse blastocysts reported by Brinster (19) about 20$ of the carbon of the embryos was replaced by ' 3C at the end of the 48 hour in vitro culture period . TABLE 5 Success Rate of Blastocyst Transplantations
A development before transfer
in vivo
B culture medium (glucose)
-
C
D
total number of embryos
E
Implantation sites ($ of C)
F
living embryos ($ of C)
resorbed embryos ($ of C)
400
52
38
14
W-~SA (n C)
75
53
27
26
W-BSA (u- "c)
75
52
25
27
in vitro
DRNH-FIC ( aC)
80
47
21
26
in vitro
DONH-FIC (U-'~C)
80
45
19
26
in vitro in vitro
638
13C Tozicity in Preimplaatation Embryos
Vol. 19, No . 5
Development of Cul tured Blastocysts After Transplantation
Preimplantation mouse embryos may develop in vitro without visible and measurable retardation, but they may be Incapable of in vivo development following transplantation to pseudopregnant foster mothers (9,11,20) . Blastocysts that had been cultured in vitro from 8-cell embryos were, therefore, transplanted to pseudopregnant foster mothers . The success rate of these transplantations is given in TABLE 5 and also the results from transfers of normal blastocysts . Experiments in which the foster mothers were not found pregnant at term were excluded from the calculations . After transplantation the percentage of implantations (living and resorbed embryos) was identical with that of normal blastocysts and also with blastocysts that had been cultured In the optimal medium (W-BSA) . The difference between these two ,groups was the lower percentage of living fetuses resulting from in vitro development . In contrast to these two groups the percentage of both implantation sites and living fetuses was reduced after transfer of blastocysts cultured in medium DONH-FIC . It is of particular Importance that there was no difference in the development after transfer when blastocysts had been cultured in media containing either n C-glucose or U-' 3 C-glucose . From the determination of the wet weight of the embryos on day 17 of pregnancy, there was no Indication of developmental retardation in any of the groups of transferred embryos of TABLE 5 . In the 44 living fetuses which had developed to the blastocyst stage in media containing U- "C-glucose (both in W-BSA and in DONH-FIC), no signs of malformations could be found in any organ or in the skeleton . DISCUSSION In contrast to the striking biological effects of deuterium enrichment in living cells (21), the stable isotopes ' 9 C and ' 8 0 do not exhibit toxic isotope effects even in mammalian systems (5,22) . In early studies with deuterium on the toxicity of stable isotopes in mammals Thomson (21) found that all fetuses died at levels of deuterium enrichment which did not affect the longevity of adult animals . This strong evidence for the higher sensitivit~r of fetal systems to stable isotopes and the increasing clinical use of ' C led to the present invesitgatton . Whole animal feeding experiments are limited by the large quantities of isotopic compounds required and the correspondingly small numbers of animals which can be used . Therefore, ' 3C was incorporated into prelrt~lantatton mouse embryos during an in vitro culture for 48 hours under conditions allowing the subsequent in vivo development after transfer to foster mothers . A very similar approach was used by Fisher and Smithberg (9) to test the toxicity of trypan blue on preimplantation mouse embryos . During a 48 hour in vitro culture period in the presence of the drug these investigators observed a dose-dependent reduction of the number of embryos reaching the blastocyst stage . After transplantation of treated blastocysts to pseudopregnant hosts, the frequency of implants developing into fetuses was significantly reduced for all treated embryos . Snow (23) cultured pretmplantation mouse embryos from the 2-cell to the blastocyst stage in the presence of 3H-thymidtne ( 3 H-Tdr) . The percentage of embryos forming blastocysts and the cell number were dependent on the 31i-Tdr concentration in the medium . The cells most susceptible to $ H-Tdr damage were those of the inner cell mass of the blastocyst . An analysis of the further development of these blastocysts showed that the cells were capable of further differentiation but only as invasive trophoblast cells with no trace of fetal tissue (10) . In a similar teratological study, Bell and Glass (11) incubated blastocysts with increasing concentrations of actinomycin D for 2 hours . After transfer to recipient animals, they found a significant dose dependent
Vol . 19, No . 5
13 C Toaicity in Preimplantation ]3mbryos
639
decrease in implantation rate and a total inhibition at the highest concentrations . In all these investigations on embryotoxic effects of treatment during preimplantation development in vitro, the three parameters that we used in the present study were sensitive enough to detect differences between treated and untreated embryos . The rate of embryo development to the blastocyst sta~e as well as the cell number of the blastocysts was not influenced by the U-' Cglucose to the culture medium . Furthermore, the implantation rate and the percentage of blastocysts developing into living fetuses was not influenced by stable isotope enrichment . Malformations among fetuses developing from treated and transferred preimplantation embryos have never convincingly been reported so far (20) and could, therefore, not be expected as the result of the stable isotope treatment . Our estimation of the ' 3 C content of the blastocysts at the end of the culture period is based on our ' ° C-incorporation results, which are identical with those of a recent report of Brinster (18), although in an earlier report Brinster measured an incorporation of carbon from glucose into mouse embryos between the 8-cell and the blastocyst stage that was three times higher (24) . In the same investigation he found that 85$ of the total carbon accumulated from glucose by the embryos is incorporated into macromolecules like proteins and glycogen . More recently Pike and Wales (25) measured the amount of carbon incorporated into embryonic proteins from glucose during the same period and reported values that are identical to our total incorporation values . Our own data on total carbon incorporation from U-' ° C-glucose, therefore, seem to be rather low, and the ' 3C-content of our blastocysts is probably higher than calculated from our results . In accordance with previous investigations there is no indication in the present study of toxic isotope effects of ;3C enrichment in mammalian embryos . The level of stable Isotope enrichment achieved in our system (20 atom $ ' 3 C) is considerably higher than the accumulation of about 2-5 atom $ ' 3 C reached in mouse embryos during the stage of organogenesis after application of U-' 3C-glucose to the mothers (ti) . It is of particular importance that the ' 3C level of the embryos In our study is 10-fold higher than usually achieved in clinical use (4) . ACKNOWLEDGEMENTS This work is being performed under the Joint auspices of the U .S . Energy Research and Development Administration, the Deutsche Forschungsgemeinschaft by grants awarded to Sonderforschungsbereich 29, and the Scientific Affairs devislon, N .A .T .O . REFERENCES D . NAGEL, and C .T . GREGG, New Approaches to the Evaluation of Abnormal Dnbryonic Development, D . Neubett and H .J . Merke r,
1 . H . SPIELMANN, H .G . EIBS,
eds ., pp . 408-417, Georg Thieme Publishers, Stuttgart (1975) . 2 . M .G . HORNING, W .G . STILLWELL, J . NOWLiN, K . LERTRATANANGKOON, D . CARROL, I . DZIDIC, R .N . STILLWELL, E .C . HORNING, and R . M . HILL, Pt~ooeeding of
the First International Conference on Stable Isotopes in Chemistry, Biology and Medicine, P . D . Klein and S .V . Peterson, eds ., pp . 299 - 309, National Technical field, Va . (1973) .
Information Service, U .S . Dept . of Commerce, Spring-
640
13C Toaicity in Preimplantation Embryos
Vol . 19, No . 5
3 . L . SWEETMAN, W .L . NYHAN, P . D . KLEIN, and P . SZCZEPANIK, Proceedings of
the First International Conference on Stable Isotopes in Chemistry, Biology and Medicine, P . D . Klein and S .V . Peterson, eds ., pp . 404-409,
4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
National Technical Information Service, U .S . Dept . of Cortmerce, Springfield, Va . (1973) . C .T . GREGG, Europ . J. clin . Pharrracol . 7 315-319 (1974) . C .T . GREGG, J .Y . HUTSON, J . R . PRINE, D .G . OTT, and J . E, FURCHNER, Life Sci . 13 775-782 (1973) . C .T . ~FEGG, D . SMITH, D .G . OTT, J . MALANIFY, B .B . McINTEER, N . MATAWIYOFF, and D . NEUBERT, Abstrricts of the Ninth International Congress of Biochemistry, Stockholm, Sweden (1973) . J . D . RIGGERS, W . K . WHITTEN, and D .G . WHITTINGHAM, Methods in M~renaZian EhrbryoZogy, J .C . Daniel, ed ., pp . 86-116, W .H . Freeman and Company, San Francisco (1971) . R .L . BRINSTER aQd J .L . THOMSON, ExpZ . Cell Res . 42 308 - 315 (1966) . D .L . FISHER and M . SMITHBERG, Teratology 6 159 - 1 (1972) . J .D . ANSELL and M .H .L . SNOW, J. E>nbryoZ . exp. Morph. 33 177 - 185 (1975) " P .S . BELL and R .H . GLASS, FerttiZ. Steril . 26 449-454 (1975) " W . K . WHITTEN, Advances in the Biosciences b, G . Rasps, ed ., pp . 129 - 139, Pergamon Press, New York-Vieweg Verlag, Brâunschweig (1971) . R .P . DONAHUE, J. Exp. Zoo Z . 169 237 - 249 (1968) . R .L . BRINSTER, E~.pZ. CeZZ Res. 32 205-208 (1963) " J . SCHIFFNER and H . SPIELMANN, J'. Reprod . Fert . 47 145-147 (1976) . A . K . TARKOWSKI, Cytogenetics 5 394-400 (1966) . D . LORKE, Naunyn-Schmiedebergs Arch . exp . Path . u. Pharmak . 25 0 360-382 (1965) . R . L . BRINSTER, MarmrtZian Repr~oduetion,H . Giblan and E .J . Plotz, eds ., pp . 229-263, Springer Verlag, Berlin, Heidelberg, New York (1970) . R . L . BRINSTER, J. Reprod . Fert . 13 413-420 (1967) . H . SPIELMANN, Current Topfes in Pathology, VoZ . Developmental Biology and Pathology, A . Gropp and K . Benirschke, eds ., Springer Verlag, Berlin, Heidelberg, New York (1976), in press J .F . THOMSON, Biological Effeeta of Deuterium, The MacMillan Company New York (1963) " D . SAMUEL, D . WOLF, A . MESHORER, and I . WASSERMAN, Proceedings of the
First International Conference on Stable Isotopes in Biology, Chemistry and Medicine, P .D . Klein and S .V . Peterson, eds ., pp . 203-209, National
Technical Information Service, U .S . Dept . of Conmerce, Springfield, Va ., (1973) . 23 . M .H .L . SNOW, J. F.7nbryoZ . exp. Morph. 2 9 Got-615 (1973) . 24 . R .L . BRINSTER, F.tpZ . Cell Rea. 58 153=158 (1969) . 25 . I .L . PIKE and R .G . WALES, unpublished data cited in : R .G . WALES, Biol . Reprod. 12 66-81 (1975) "
Pergamon Presa
THE EFFECTS OF CARBON-13 INCORPORATION INTO PREIMPLANTATION MOUSE EMBRYOS ON DEVELOPMENT BEFORE AND AFTER IMPLANTATION Horst Spielmann, Hans Georg Eibs, Dagmar Nagel and Charles T . Gregg + Pharmakologisches Institut der Freien Universitât Berlin, West Germany, Abteilung Embryonalpharmakologle and Los Alamos Scientific Laboratory , University of California Los Alamos, New Mexico, USA . (Received in final form June 7, 1976) SUMMARY Pretmplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled ' a C-glucose . The ' °C content of the blastocysts was 20 atom $ according to incorporation studies with "`C-glucose . No embryotoxic effects of carbon=l3 incorporation could be detected on the basis of these criteria of normal development : the percentage of embryos reaching the blastocyst stage during the culture period ; the number of cells in these blastocysts ; and the development after transplantation to pseudopregnant foster mothers . The increasing diagnostic use of stable isotopes In situations where the radiation from radioactive tracers is unacceptable, for example in metabolic studies on children and pregnant women, enhances the importance of studies on biological isotope effects in sensitive mammalian systems (2,3,4) . In a previous study where mice were fed a diet of 80 atom $ "C yeast, 60$ of the body carbon was replaced by ' 3 C, but there was no apparent toxic effect on the living animals and no tissue damage was seen either at autopsy or histologically (5) . In additional experiments pregnant mice received uniformly labelled ' 3 C-glucose (U-' 3 C-glucose) by stomach tube during organogenesis . The ' 3C enrichment in fetal tissues after this treatment was 2-5 atom $ 'jC, and no alterations of normal development due to stable isotope enrichment were found (6) . In recent years simple methods for the in vitro culture of early mammalian embryos have been developed (7) . In the present investigation ' aC from culture media containing U- "C-glucose was incorporated Into preimplantation mouse embryos . Glucose as the only energy source does not support the development of mouse embryos before the 8-cell stage (8) . 8-cell mouse embryos were, ~herefore, cu~~ured to the blastocyst stage in media with U-' 3 C-glucose or a C-glucose ( C Indicates natural carbon isotope abundance, t .e . about 1 .1 atom $ 'aC) . The effects of the stable Isotope on embryonic development in vita and after transplantation to foster mothers were studied since this combined in vitz+o and in vivo model system of early mammalian development is sensitive to various embryotoxic treatments (9,10,11) . * A preliminary account of these studies has appeared elsewhere (1) .
633
13C Tozicity in Preimplantation Embryos
634
Vol . 19, No . 5
MATERIALS AND METHODS
In vitro
Culture Conditions
Eight-cell mouse embryos were obtained by flushing the oviducts of female mice of the strain NMRI (breeder : Schwencke â Co ., Nauheim, West Germany) 60 hours after copulation . The standard culture medium was prepared according to Whitten (12) and contained either 0 .3$ bovine serum albumin (W-BSA) or 0 .3$ fatty acid free BSA (W-ff-BSA) . Since BSA and also ff-BSA can be used as energy sources by 8-cell mouse embryos (8), albumin in Whltten's medium was replaced, in a few experiments, by the polysaccharide Flcoll (Pharmacta, Sweden) at a concentration of 0 .5$ (W-FIC) . The culture medium with glucose (concentration 5 .5 mM) as the only energy source was prepared according to Donahue (13) . It was used with 0 .3$ BSA (DONH-8SA), with ff-BSA (DONH-ffBSA), and with 0 .5$ Ficoll (DONW-FIC) . The embryos were cultured according to Brinster's method (14) in 5$ COz in air under paraffin oil (Uvasol : Merck A .G ., Darmstadt, West Germany) In tissue culture dishes (Falcon Plastics, Oxnard, California, USA) . In this system 8-cell mouse embryos developed to the morula stage (2614 cells/embryo to the NMRI strain (15) after 24 hours in culture and reached the blastocyst stage (35-86 cells/embr~o (15) ) after 48 hours as described by previous investigators (7) . The ~ C content of the U-' 3 C-glucose in the in vitro culture experiments was 86 atom $ ~ 3C . Determination of the Cell Number of the Embryos The cell number of the blastocysts was determined at the end of the culture period according to Tarkowski's method (16) .
vitro
in
U- "'C-Glucose Incorporation Studies Embryos were cultured in media containing U-~ ° C-glucose (Amersham-Buchler GmbH, Braunschweig, West Germany) with a specific activity of 3 .0 mC/mmole . After incubation, the embryos were rapidly washed through ten 0 .1 ml drops of culture medium under paraffin oil . For determination of precursor uptake, washed embryos in groups of 20-40 were taken up to 20 ul of the final wash solution and placed in scintillation counting vials containing 0 .1 ml water and 1 .0 ml of the tissue soluültzer Soluene 350 (Packard Instrument Company Inc ., Zurich, Switzerland) was added . When mixing of the water and solubillzer was complete, 10 ml of a toluene PPO-POPOP scintillation counting mixture were added, and the vials were counted to a TRI-CARB liquid scintillation spectrometer (Model 3380 :Packard, Zurich, Switzerland) . Blanks were determined by counting aliquots of the final wash solutions under Identical conditions . The amount of intracellular substrate, expressed as pg atoms of carbon/embryo/hour, was calculated from the counts in the embryos, corrected for background,and the specific acttvtty of the Incubation medium . Transplantation of Blastocysts to Pseudopreyna nt Foster Mothers Normal mouse blastocysts obtained from the uteri of female mice on day 3 of pregnancy (day 0 = the day following the overnight mating period) and also blastocysts which had developed from 8-cell embryos during a 48 hour in vitro culture period were surgically transferred in groups of 5 to each uterine horn of pseudopregnant females on day 2 of pseudopregnancy . Pseudopregnancy was induced by mating normal females with vasectomtzed males . The foster mothers were sacrificed on day 17 of pregnancy and the success rate of the transplantations was determined by the number of resorbed and live embryos . The embryos were weighed, carefully inspected for growth retardation and malformations, and stained for skeletal abnormalities with Alizarin Red S according to Lorke (17) .
vol . 19, No . 5
13C Tozicity in Preimplantation 13mbryoe
635
RESULTS
in vitro
Influence of U-' aC-gl ucose on
development
The first step of differentiation during embryogenesis of the mouse is blastulation, which is characterized by the formation of the blastocyst cavity and the segregation of the embryonic cells into trophoblast and Inner cell mass . The influence of U-~ 3 C-glucose was studied in media containing energy sources other than glucose and in media with glucose as the only energy source . The substances in Whitten's medium that support cleavage of 8-cell mouse embryos are the carbohydrates pyruvate, lactate, and glucose, and also BSA (8) .Table 1 indicates that blastulation of 8-cell embryos was not inhibited when BSA in Whitten's medium was replaced by ff-BSA or even FIC, which does not support cleavage (8) . In the medium with glucose as the only carbohydrate (DDNH), the replacement of B5A by ff-BSA and by FIC did not affect blastulation . Finally the in vitro development of 8-cell embryos to the blastocyst stage was not modified in the presence of U-~ 3 C-glucose even when this substrate was the only energy source In the medium (DDNH-FIC) . TABLE 1
In-vtitx~o
Culture of 8-Cell Mouse Embryos to the Blastocyst Stage (48 hours) in Media containing na C- or U- na C-glucose
Medium
Glucose
B
M
deg
Number of Determinations (Experiments)
W-BSA W-ff-BSA W-FIC
na C ..
DDNH-BSA DDNH-ff-BSA
w
DDNH-FIC W-BSA
U-' 3 C
94$
5$
1$
340 (15)
3$
3$
98 (5)
loo$
~
0
60 (4)
100$
17
p
93$
5$
2$
55 (4)
97$
2$
1$
163 (5)
97$
3$
0
86 (5)
2$
96 (5) 54 (4)
94$
45 (5)
W-ff .-BSA
"
92$
6$
w-Flc
"
loo$
DDNH-ff .-BSA
"
94$
0 4$
0 2$
45 (4)
94$
2$
4$
163 (5)
DONH-FIC
For abbreviations used for the Media see text ; develop-. mental stage of the embryos : B-blastocysts, M~norulae (retarded for 24 hours), deg-degenerated . The cell number of normal blastocysts of the NMRI strain increases from 3514 cells/embryo to early blastocysts up to 86±16 cells/embryo in late blastocysts at the time of implantation (15) . For a better evaluation of the in vitro development the cell number of the embryos was, therefore, determined at the end of the incubation period . According to the results in TABLE 2 the
636
13 C Toxicity is Preimplantation Embryos
Vol . 19, No . 5
cell number of the blastocysts was significantly higher after incubation in the optimal medium (W-BSA) than after culture in the medium with glucose as . Since the cell number is identical in inthe only energy source (DONH-FIC) na C-glucose or U-' 3 C-glucose, no inhibitory effects cubations carried out with of the stable isotope on embryonic development were detected during the in vitro culture period . TABLE 2 Cell Number of Preimplantation Mouse Embryos after
in~Vitro
Culture
from the 8-Cell to the Blastocyst Stage .
Medium
Glucose
Number of Celis per Embryo (± S .D .)
Number of Determinations (Experiments)
1, W-BSA
na C-glucose
80 .1± 21 .7
34 (6)
2, W-BSA
U-~ 3 C-glucose
78 .3 ± 22 .4
22 (4)
3 . DONH-FIC
na C-glucose
53 .6 ± 19 .6
22 (4)
4, DONH-FIC
U-' 3 C-glucose
61 .8 ± 18 .8
33 (6)
1 . is significantly different from 3 . (p < 0 .001), but there is no significant difference between 1 . and 2 . nor between 3 . and 4 . The number of experiments is in parentheses . Carbon- 1 3 Enr ichment During in vitro Culture The incorporation of ~ °C from U- "`C-glucose into whole embryos was determined during the 24 hour period between the 8-cell and the morula stage, and also during the second 24 hour period between the morula and the blastocyst stage . The latter embryos had been cultured in unlabelled medium prior to the incubation with U-~ ° C-glucose . The incorporation of "'C from U- "`C-glucose into whole embryos during the in vitro culture (TABLE 3) was identical in the optimal medium (W-BSA) and in the poorest medium (DONH-FIC) . TABLE 3 Carbon Accumulated From Glucose By Preimplantation Mouse Embryos Cultured
in vitro
Brinster (1970) 8-cell to morula morula to blastula
For 24 Hours
0 u r W-BSA .
r e s u 1 t s DONH-FIC .
-
1 .9 (4)
2 .3 (6)
7 .0
7,2 (4)
6 .8 (10)
Labelled substrate was U- "'C-glucose, specific activity 3 .0 mC/mmole . Carbon accumulated is given in pg atoms of carbon/embryo/h . The number of experiments is given in parentheses .
13C Todcity is Preimplaatation Embryos
Vol . 19, No . 5
Since our data are in good agreement with a previous report (18), the incorporation of ' 3 C from U-' 9 C-glucose into preimplantatton mouse embryos was calculated from our ' ~C incorporation rates . The result is presented in TABLE 4 . TABLE 4 Carbon-13 Content of Mouse Embryos After in
vitro
Culture
For 48 Hours From The 8-Cell To The Blastocyst Stage In Medium DONH-FIC Containing U-' 3C-Glucose C-incorporated-first 24 hours + second 24 hours C-incorporated 24 x 2 .3 pg atoms + 24 x 6 .8 pg atoms C-incorpôrated- 214 pg atoms
2568 pg C- 2 .6ng C
C-content
- 50$ of dry weight ( 23 ng of protein/embryo (18) )
C-content
- 11 .5 ng/embryo
2 .6 ng out of 11 .5 ng a 22 .6$
The "C-content of the glucose is 86 atom $, therefore, the ' 3C-content of the embryos is 19 .4 atom $ . Using the dry weight value of mouse blastocysts reported by Brinster (19) about 20$ of the carbon of the embryos was replaced by ' 3C at the end of the 48 hour in vitro culture period . TABLE 5 Success Rate of Blastocyst Transplantations
A development before transfer
in vivo
B culture medium (glucose)
-
C
D
total number of embryos
E
Implantation sites ($ of C)
F
living embryos ($ of C)
resorbed embryos ($ of C)
400
52
38
14
W-~SA (n C)
75
53
27
26
W-BSA (u- "c)
75
52
25
27
in vitro
DRNH-FIC ( aC)
80
47
21
26
in vitro
DONH-FIC (U-'~C)
80
45
19
26
in vitro in vitro
638
13C Tozicity in Preimplaatation Embryos
Vol. 19, No . 5
Development of Cul tured Blastocysts After Transplantation
Preimplantation mouse embryos may develop in vitro without visible and measurable retardation, but they may be Incapable of in vivo development following transplantation to pseudopregnant foster mothers (9,11,20) . Blastocysts that had been cultured in vitro from 8-cell embryos were, therefore, transplanted to pseudopregnant foster mothers . The success rate of these transplantations is given in TABLE 5 and also the results from transfers of normal blastocysts . Experiments in which the foster mothers were not found pregnant at term were excluded from the calculations . After transplantation the percentage of implantations (living and resorbed embryos) was identical with that of normal blastocysts and also with blastocysts that had been cultured In the optimal medium (W-BSA) . The difference between these two ,groups was the lower percentage of living fetuses resulting from in vitro development . In contrast to these two groups the percentage of both implantation sites and living fetuses was reduced after transfer of blastocysts cultured in medium DONH-FIC . It is of particular Importance that there was no difference in the development after transfer when blastocysts had been cultured in media containing either n C-glucose or U-' 3 C-glucose . From the determination of the wet weight of the embryos on day 17 of pregnancy, there was no Indication of developmental retardation in any of the groups of transferred embryos of TABLE 5 . In the 44 living fetuses which had developed to the blastocyst stage in media containing U- "C-glucose (both in W-BSA and in DONH-FIC), no signs of malformations could be found in any organ or in the skeleton . DISCUSSION In contrast to the striking biological effects of deuterium enrichment in living cells (21), the stable isotopes ' 9 C and ' 8 0 do not exhibit toxic isotope effects even in mammalian systems (5,22) . In early studies with deuterium on the toxicity of stable isotopes in mammals Thomson (21) found that all fetuses died at levels of deuterium enrichment which did not affect the longevity of adult animals . This strong evidence for the higher sensitivit~r of fetal systems to stable isotopes and the increasing clinical use of ' C led to the present invesitgatton . Whole animal feeding experiments are limited by the large quantities of isotopic compounds required and the correspondingly small numbers of animals which can be used . Therefore, ' 3C was incorporated into prelrt~lantatton mouse embryos during an in vitro culture for 48 hours under conditions allowing the subsequent in vivo development after transfer to foster mothers . A very similar approach was used by Fisher and Smithberg (9) to test the toxicity of trypan blue on preimplantation mouse embryos . During a 48 hour in vitro culture period in the presence of the drug these investigators observed a dose-dependent reduction of the number of embryos reaching the blastocyst stage . After transplantation of treated blastocysts to pseudopregnant hosts, the frequency of implants developing into fetuses was significantly reduced for all treated embryos . Snow (23) cultured pretmplantation mouse embryos from the 2-cell to the blastocyst stage in the presence of 3H-thymidtne ( 3 H-Tdr) . The percentage of embryos forming blastocysts and the cell number were dependent on the 31i-Tdr concentration in the medium . The cells most susceptible to $ H-Tdr damage were those of the inner cell mass of the blastocyst . An analysis of the further development of these blastocysts showed that the cells were capable of further differentiation but only as invasive trophoblast cells with no trace of fetal tissue (10) . In a similar teratological study, Bell and Glass (11) incubated blastocysts with increasing concentrations of actinomycin D for 2 hours . After transfer to recipient animals, they found a significant dose dependent
Vol . 19, No . 5
13 C Toaicity in Preimplantation ]3mbryos
639
decrease in implantation rate and a total inhibition at the highest concentrations . In all these investigations on embryotoxic effects of treatment during preimplantation development in vitro, the three parameters that we used in the present study were sensitive enough to detect differences between treated and untreated embryos . The rate of embryo development to the blastocyst sta~e as well as the cell number of the blastocysts was not influenced by the U-' Cglucose to the culture medium . Furthermore, the implantation rate and the percentage of blastocysts developing into living fetuses was not influenced by stable isotope enrichment . Malformations among fetuses developing from treated and transferred preimplantation embryos have never convincingly been reported so far (20) and could, therefore, not be expected as the result of the stable isotope treatment . Our estimation of the ' 3 C content of the blastocysts at the end of the culture period is based on our ' ° C-incorporation results, which are identical with those of a recent report of Brinster (18), although in an earlier report Brinster measured an incorporation of carbon from glucose into mouse embryos between the 8-cell and the blastocyst stage that was three times higher (24) . In the same investigation he found that 85$ of the total carbon accumulated from glucose by the embryos is incorporated into macromolecules like proteins and glycogen . More recently Pike and Wales (25) measured the amount of carbon incorporated into embryonic proteins from glucose during the same period and reported values that are identical to our total incorporation values . Our own data on total carbon incorporation from U-' ° C-glucose, therefore, seem to be rather low, and the ' 3C-content of our blastocysts is probably higher than calculated from our results . In accordance with previous investigations there is no indication in the present study of toxic isotope effects of ;3C enrichment in mammalian embryos . The level of stable Isotope enrichment achieved in our system (20 atom $ ' 3 C) is considerably higher than the accumulation of about 2-5 atom $ ' 3 C reached in mouse embryos during the stage of organogenesis after application of U-' 3C-glucose to the mothers (ti) . It is of particular importance that the ' 3C level of the embryos In our study is 10-fold higher than usually achieved in clinical use (4) . ACKNOWLEDGEMENTS This work is being performed under the Joint auspices of the U .S . Energy Research and Development Administration, the Deutsche Forschungsgemeinschaft by grants awarded to Sonderforschungsbereich 29, and the Scientific Affairs devislon, N .A .T .O . REFERENCES D . NAGEL, and C .T . GREGG, New Approaches to the Evaluation of Abnormal Dnbryonic Development, D . Neubett and H .J . Merke r,
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13C Toaicity in Preimplantation Embryos
Vol . 19, No . 5
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