Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 151--164 (1973) 9 by Springer-Verlag 1973

Changes in the Respiratory Activity of Different Tissues of Rat and Mouse Embryos during Development* H o r s t S p i e l m a n n a n d I n g r i d Liicke Pharmakologisches Irmtitut der Freien Universit/it Berlin Abt. "Embryonal-Pharmakologie" Received February 1, 1973

Summary. Whole naked mouse and rat embryos have identical respiratory rates during organogenesis when judging from the dry weight basis. This way of plotting enables to distinguish between a developmental retardation for hours and days and a significant developmental reduction on the basis of the two parameters. This allows to draw conclusions in experimental tetratology at this very early stage which cannot be drawn from an age-dependent Qo, plot. The respiratory capacity of mouse embryos on day 9 and rat embryos on day 10 + 16 h is significantly lower than on the following day. Since whole rat embryos exceed the limiting thickness on day 13 of gestation, it is suggested to perform measurements of the Qo, on day 14 and 15 with isolated organs (hearts and livers) and from day 16 to term with liver slices of rat embryos applying the Warburg method. The Qo~ of isolated hearts and livers on day 14 to 16 and of liver slices from day 16 to term is at least 10, which is comparable to adult liver slices. The succinate-dependent respiratory activity can be measured with these embryonic tissue preparations on day 14 and later on, it increases considerably on day 19 in liver slices and reaches the values of adult liver tissue at term. This developmental change was confirmed by measurements in the presence of the uncoupler 2,4-dinitrophenol and the inhibitor rotenone. Key words: Experimental Teratology--Developmental Biochemistry--Embryonic Energy Metabolism -- Respiratory Inhihitors. T h r o u g h o u t t h e l a s t d e c a d e t h e field o f d e v e l o p m e n t a l biology h a s become of g r e a t e r i m p o r t a n c e since m i c r o t e c h n i q u e s h a v e been d e v e l o p e d which allow a n e l u c i d a t i o n os b i o c h e m i c a l p a t h w a y s even if o n l y v e r y small a m o u n t s o f tissue are available. A m o n g o t h e r fields of i n t e r e s t t h e m a m m a l i a n e m b r y o n i c e n e r g y m e t a b o l i s m has i n t e n s i v e l y been s t u d i e d b y different groups. I t is h o p e d t h a t b e t t e r i n f o r m a t i o n on t h i s special topic m a y allow a b e t t e r u n d e r s t a n d i n g of n o r m a l a n d * This work was supported by grants from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29, "Embryonale Entwicklung und Differenzierung (Embryonal-Pharmakologie)". 11"

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abnormal development. The biochemical analysis of the mechanisms which control the normal development of mammalian embryos may give rise to new concepts in teratology. The species in which most of the investigations on mammalian embryonic metabolism between organogenesis and term have been performed is the rat. The first experiments have been carried out on whole rat embryos during the late stage of organogenesis (Negelein, 1925; Kleiber, 1943). Predominantly the respiratory capacity and the rate of glycolysis of the embryos were studied using the Warburg technique. These experiments have been repeated and extended during the last few years (I~etzloff et al., 1968; De Plaen, 1970; Neubert et al., 1971, a, b ; Spielmann and Lficke, 1971). Investigations on the development of mitochondrial enzymes during fetal development have usually been performed on the last few days before and the first week after term (Ballard and Hanson, 1967 ; Schreiber et al., 1970 ; Levy and Toury, 1970; Jacovcic et al., 1971 ; Hommes et al., 1971, a, b.), since, with conventional methods, for the preparation of isolated mitochondria and even for experiments with homogenates a comparatively large amount of embryonic tissue is required. A few groups have overcome these difficulties and have been able to get information on enzymes of the energy metabolism already during organogenesis using both isolated mitochondria (Bass, 1970; Bass and Schmidt, 1971) and homogenates (Aksu et al., 1968; Maekler, 1970; Oerter and Bass, 1972). Furthermore, a method for a successful in-vitro culture of whole rat embryos has been developed by New (1966) and--using the incorporation of radioactively labelled substrates--some studies on reactions of the energy metabolism have been performed in a similar system with 10- to 12-day old embryos (Tanimura and Shepard, 1970). Recently the preparation of intact fetal liver cells has been reported by Hommes et al. (1971b). These authors measured the oxygen consumption of the isolated ceils polarographically. We have been able to show that the respiratory capacity (---- Qo2 -- ~zl of oxygen consumed per hour and mg dry weight) of whole rat embryos on day 11 and 12 of gestation--in contrast to the results published by previous investigators--is comparable with adult tissues (Spielmann and Lficke, 1971). Moreover, these studies revealed that wrong data regarding the respiratory capacity must be obtained if measurements of the Qo~ are performed at 37 oC using the Warburg technique with whole rat embryos older than day 12. In this paper the previous data are extended to mouse embryos, and the applicability of this method in experimental teratology during organogenesis will be discussed. Furthermore, an attempt is made to overcome the difficulties, which arise from the

Changes in the Respiratory Activity of Different Tissues

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l i m i t i n g thickness (Warburg, 1926) b y using whole e m b r y o n i c organs (heart, liver) as well as liver slices i n order to get more i n f o r m a t i o n o n the r e s p i r a t o r y capacity of e m b r y o n i c tissues u p to term. The influence of different s u b s t r a t e s a n d inhibitors was studied.

Materials and Methods Wistar rats of the strain SW 69 (bred by Winkelmann, Kirehborchen, Germany) were kept under a reverse day/night cycle. Males and females were mated for 2 h (from 8.00 am to 10.00 am). The presence of sperms in vaginal smears indicated day 0 of pregnancy. Mice of the strain NMRI (purchased from Schwenke Co., Nauheim, Germany) were kept under a normal day/night cycle. Animals weighing 30 g were mated for 2 h (7.00 am to 9.00 am) and the 24 h period following the mating and the detection of vaginal plugs was called day 0 of pregnancy. Both rats and mice were fed Altromin R and tap water ad libitnm. The preparation of the whole mouse embryo and the incubation procedure were identical with the experiments carried out with whole rat embryos which have previously been described in detail (Spielmann and Lficke, 1971). For a sucessful Qol determination we needed 10 rat embryos per incubation flask on day 10 % 16 h and 8 mouse embryos on day 9 of gestation. The preparation of organs of the rat embryos was performed at room temperature in a Krebs-Ringer phosphate solution of pH 7.4. The Qo, determinations of the hearts were performed with 6 to 8 organs per Warburg flask on day 14, with 6 hearts on day 15 and 16, with 4 organs on day 17, and 2--3 hearts on day 18 or 19 of gestation. The ventricles were incised in order to favour an optimal gas exchange. In the experiments with livers we needed 4 livers per flask on day 14, or 2 organs on day 15 and 2 lobes of one liver on day 16. The livers were either intact or the parenchyme was incised with dissecting knives. The incisions were performed to check whether the thickness of the organs was already limiting the method (Warburg, 1926). From day 16 of gestation up to term we were able to prepare liver slices. A tissue amount of about 1 mg dry weight was generally necessary to grant the measurement of reproducible Oo, values. All experiments were performed with an oxygen gas phase. Hepes buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), retenone and 2,4-dinitrophenol were purchased from the Sigma Chemical Co., St. Louis, USA. Statistical comparisons of experimental means were made using Student's t-test according to P~tau (1943), differences were assumed to be significant at p ~ 0.0027.

Results

1. Qo~ and Dry Weight o/ Whole Rat and Mouse Embryos during the Late Stage o/ Organogenesis (Fig. 1) On d a y 10 of gestation mouse e m b r y o s have the highest r e s p i r a t o r y a c t i v i t y which is n o t significantly different from the Qo2 m e a s u r e d with r a t e m b r y o s on d a y 11 a n d 12. The Qo2 of mouse e m b r y o s on d a y 9 a n d

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% -12

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day of gestation Fig. 1. Respiratory capacity and dry weight of whole rat and mouse embryos during the l~te phase of organogenesis. Qo, = ~I O~ • h-~ • mg dry weigh%-~. The figures beside the Qo, values indicate the number of determinations which is identical for the dry weight determinations on the same day of gestation. All data represent average values and standard deviation

11 is significantly lower than on day t0 (p < 0.001). But on day 11 of gestation mouse embryos seem to exceed the limiting thickness in the same way as rat embryos on day 13. A determination of the Qo~ of whole naked mouse embryos is therefore impossible with embryos which are older than day 10 of gestation. Mouse embryos on day 9 and rat embryos on day I0-~ 16 h have a Qo2 which is significantly lower than that of embryos on the following day of gestation (p < 0.0027). According to Fig. 1 rat embryos seem to have similar dry weight and Qo~ values as mouse embryos which are 11/~ to 2 days younger.

Changes in the Respiratory Activity of Different Tissues

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l~ig.2. Relationship between Qo, and dry weight of whole rat and mouse embryos. The average values measured with mouse embryos are plotted into the standard deviation of rat embryos. The numbers indicate the days of gestation. 10.7 is equal to day 10 -~ 16 h

2. Relationship between Dry Weight and Qo2 /or Whole Rat and Mouse Embryos during Organogenesis (Fig. 2) I t was of interest to compare the Qo~ of rat and mouse embryos of the same size--not the same gestational age. We, therefore, plotted the Qo~-values versus the dry weight of the embryos. This furthermore has the advantage of eliminating variations in the size of the embryos at a given developmental age, which--especially with mouse embryos is not negligible. This plot shows that average-Qo:-values of the mouse embryos on different days of gestation lie within the standard deviation of the values measured with rat embryos. This indicates that on the dry weight basis whole rat and mouse embryos have identical Qo2-values at a given stage of development.

3. The Qo~ against Dry Weight Plot as a Tool in Experimental Teratology (Fig. 2) Furthermore, plotting the Qo~-data versus dry weight of the rat embryos allows us to distinguish between a simple development retardation for several hours or days and a significant reduction of one of the two parameters, either Qo2 or dry weight of whole embryos. For example, a group of rat embryos on day 11 of gestation which may be represented by the point x z in Fig. 2 has ~ significantly reduced dry weight but also a slightly reduced Qo2. Taking the standard deviation for rat embryos into account it appears that the embryos seem to be "normal embryos",

156

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Fig. 3. Qo, of different embryonic rat liver preparations in vitro between day 14 and 16 of g e s t a t i o n . intact livers, - - - - i n c i s e d livers. All values are means of at least 5 determinations, the standard deviation is indicated

which are a few hours younger t h a n day 11. Another group of rat embryos which m a y be represented b y x~ can, however, not be considered as just retarded although the dry weight is the same as for the x z group. The data compiled in Fig. 2 also indicate t h a t the limiting thickness apparently is exceeded if the embryos have a dry weight of more than 1.5--2.0 mg. I f 12-day rat embryos are used with air as gas phase or 13-day old embryos even in an oxygen p h a s e - - a n experimental setup some previous investigators have u s e d - - a retardation would result in an increase in the apparent Qor

4. Qo~ o] Embryonic Liver Tissue o] Rat Embryos between Day 14 and 16 o/Gestation (Fig. 3) On day 14 of gestation both intact and incised livers have a Qo~ of at least 10 which is significantly higher t h a n t h a t which can be measured with whole embryos, which of course exceed the limiting thickness. The Qo~ of incised livers on day 14 is moderately increased by the substrates glucose and succinate when comparing it with the respiratory activity

Changes in the Respiratory Activity of Different

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Qo2 15.

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Fig.4. Qo, of embryonic rat hearts in vitro between day 14 and 18. Details of measurement and plotting are the same as in Fig. 3

in a medium without any substrates (p < 0.01). There are no similar differences measurable with the intact livers. On d a y 15 and 16 all Qoz-values decrease when compared with the previous day. The reason for this is t h a t the liver preparations on d a y 15 and 16 exceed the limiting thickness. No final conclusions can therefore be drawn from Fig. 3 on the effect of the two substrates glucose and sueeinate on the Qo2 of liver tissue of 15 and 16 day old embryos.

5. Qo~ o/Embryonic Rat Hearts between Day 14 and 18 o/Gestation (Fig. 4) The Qo2 of the embryonic hearts decreases with increasing gestational age. The addition of suecinate has a moderate effect on all days (p < 0.05).

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The values in the succinate medium are higher than those found with the incised livers on the same days.

6. The Influence o/ Succinate on the Respiratory Capacity o] Di//erent Embryonic Tissues o/ the Rat (Fig. Sa) All embryonic preparations which were tested seem to nave a Qo~ of at least 10 throughout all days of gestation in a medium containing glucose. The Qo, is identical in a glucose medium with and without succinate when using whole embryos on day 11 and 12, dissected parts on day 12 and 13, and isolated livers on day 14 and 15. The Qo2 is higher after the addition of succinate to the glucose medium at the p ~ 0.01 level for liver slices on day 16, 17, and 18. A remarkably increased respiratory rate in a medium containing glucose and succinate is found with fetal liver slices on day 19 (p ~ 0.001). I t reaches values comparable to adult tissue at term.

7. Influence ol 2,4-Dinitrophenol on the Qo 2 o/Embryonic Tissues o/the Rat (Figs. 5b and 5c) The difference between the Qo~ of adult tissues in glucose medium with and without succinate is more striking after the addition of the uncoupler 2,4-dinitrophenol. In order to get more information on the activity of the succinate-metabolizing system in rat embryos in the late stage of organogenesis we performed experiments as described in the previous section in a medium containing 50 9M 2,4-dinitrophenol. The uncoupler increased the Qo~ of whole embryos on day 11 and 12. On these two days we could not find an effect of succinate which is measurable with liver preparations from day 14 on. Liver slices of embryos of d a y 19 have an uncoupled respiratory rate which is higher than that of whole embryos on day 11 and 12; it reaches the values of adult tissue at term. 2,4-dinitrophenol caused an extremely high Qo~ of about 30 in isolated rat hearts on day 14 in a medium with glucose or succinate (Fig. 5 c). The Qo~ of the heart tissue under the influence of the uncoupler decreases with increasing gestational age.

8. E//ect o/ Rotenone on the Respiration o/ Embryonic Rat Tissues in Media Containing Succinate or Glucose (Table 1) Rotenone inhibits the oxidation of NAD-linked substrates without inhibiting the oxidation of succinate (Lindahl et al., 1961 ; Ernster et al. 1963 ; Nenbert, 1963), this is demonstrated for adult liver slices in Table 1. Rotenone has a dose dependent effect on the Qo~ of adult liver slices in

160

H. Spielmann and I. Liicke:

Table 1. Effect of rotenone on the glucose- and succinate-dependent respiration. Lncubation medium: Krebs-ringer-phosphate solution, 10 mM Hepes buffer, pH 7.4. Average values 4- standard deviation, number of determinations in brackets. Rot. ~ rotenone, Glue. = glucose, Succ. ~ succinate Tissue preparation

Incubation medium

Qo~

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5 mlVIGlue. 5 mM Glue. 5 mM Glue. 5 mM Gluc. 20 mM Suce. 20 mlYlSuec. 20 ~ Suce. 20 mM Succ.

1.5 4- 1.0 (3) 2.9 -4- 0.4 (3) 5.4 4- 0.5 (3) 10.4 4- 2.1 (5) 21.9 4- 0.9 (3) 22.2 4- 1.5 (3) 23.5 4- 1.9 (3) 22.0 4- 1.6 (24)

10 ~ Rot., 1 ~M Rot., Rot., 10 ~M Rot., 1 t~M Rot., Rot.,

5 mM Glue. 5 mM Glue. 5 mM Glue. 20 mM Succ. 20 m_~ Succ. 20 mM Succ.

0 (3) 1.0 4- 0.5 (3) 11.0 4- 1.0 (12) 2.0 -~ 0.6 (3) 7.0 4- 0.5 (3) 9.1 4- 0.9 (3)

0.1 ~dV[Rot., 5 ram Glue. 0.05 ~ Rot., 5 mM Glue. 0.01 izM Rot., 5 r a ~ Glue. Rot., 5 mM Glue. 0.1 y~I l~ot., 20 m_~ Suet. 0.05 ~ Rot., 20 mM Suce. 0.01 ~ Rot., 20 mM Succ. Rot., 20 mM Succ.

O (3) 2.6 4- 1.4 (6) 6.3 i 1.2 (3) 12.0 4- 1.0 (35) 0 (3) 4.1 4- 1.4 (8) 6.5 4- 1.2 (3) 10.1 4- 0.25 (4)

Incised embryonic livers, day Incised embryonic livers, day Incised embryonic livers, day Incised embryonic livers, day Incised embryonic livers, day Incised embryonic livers, day Whole embryos, day Whole embryos, day Whole embryos, day Whole embryos, day Whole embryos, day Whole embryos, day Whole embryos, day Whole embryos, day

12 12 12 12 12 12 12 12

14 14 14 14 14 14

a glucose m e d i u m . I n a m e d i u m containing succinate, however, t h e Qo2 is u n c h a n g e d b y t h e s a m e r o t e n o n e concentrations. Similar e x p e r i m e n t s were p e r f o r m e d w i t h e n b r y o n i c tissues in o r d e r t o g e t f u r t h e r i n f o r m a t i o n on d e v e l o p m e n t a l changes of t h e r o t e n o n e - i n s e n s i t i v e - s u c c i n a t e - d e p e n d e n t r e s p i r a t o r y a c t i v i t y . Significantly lower i n h i b i t o r c o n c e n t r a t i o n s h a v e a dose d e p e n d e n t effect on t h e Qo2 of whole r a t e m b r y o s on d a y 12 of g e s t a t i o n in m e d i a containing glucose or succinate. A t t h e t e s t e d inh i b i t o r c o n c e n t r a t i o n s no differences of t h e Qo~-values in t h e glucose or succinate m e d i a could be d e t e r m i n e d w i t h these embryos. T h e s a m e result was f o u n d w i t h isolated h e a d s or bodies of r a t e m b r y o s on d a y 12 of gestation, these p r e p a r a t i o n s u s u a l l y f a v o u r a b e t t e r diffusion o f b o t h s u b s t r a t e a n d inhibitor. I n e x p e r i m e n t s w i t h isolated livers of r a t e m b r y o s on d a y 14 r o t e n o n e decreased t h e Qo2 in b o t h glucose a n d succinate media, t h e i n h i b i t i o n in glucose media, however, was stronger t h a n in m e d i a containing suc-

Changes in the Respiratory Activity of Different Tissues

161

cinate. The effective inhibitor concentrations in these experiments were between those inhibiting the adult liver tissue and the 12 day embryos. Discussion The age-dependent Qo~ curves of whole rat and mouse embryos during organogenesis seem to run parallel. The weight-dependent Qo~ curve is identical for both species. Since mouse embryos are two days in advance when judging from the dry weight value and comparing them with rat embryos, they are also two days in advance when judging from the Qo~ value. Bass and Sehmidt (1971) were able to measure cytochrome oxidase activity in rat embryos on day 11 and 12 of gestation which was very low, whereas the activity of the dehydrogenases was comparatively high. Cytoehrome oxidase may, therefore, be the rate limiting enzyme of the respiratory chain at this stage of development and may, therefore, be responsible for the decreased Qo2 value of whole rat embryos on day 10 ~ 16 h when compared with embryos of day 11 and 12. Mouse embryos may be a better model to study tetratological alterations of the respiratory activity of whole embryos since their Qo, values arc significantly different on day 9 and 10. In experimental teratology the Qo2 against the dry weight plot seems to be more useful than the age-dependent Qo~ plot. Netzloff et al. (1968), for example, used the age-dependence in order to detect alterations of the respiratory metabolism in rat embryos. They did not realize that the higher @o2 values of their embryos on day 13 and 14 after treatment of the mother with a PGA-deficient diet was caused by retardation of the embryos. A treatment of the pregnant rat on day 9 or 10 with 2-dcoxyglucose or sodium fluoroacetate, however, led to a significant reduction of both dry weight and Qo2 of the embryos on day 11 (Spielmann et al., 1972), e.g. point x 2 in Fig.2. In our determinations with whole rat embryos on day 11 and 12, whole hearts on day 14 to 16, and even liver slices from day 16 to term the Qo~ of the embryonic tissues in a glucose medium was about 10, which is very similar to many adult tissues. This finding is in good agreement with the results reported by most of the investigators who used the Warburg technique in order to determine the embryonic respiratory activity (Barrels, 1970). Hommes et al. (1971 b) found a more than 10 times lower respiratory activity using a preparation of isolated liver cells from rat embryos at the end of gestation in a medium conraining 2 mM glucose. At present, we are unable to explain this difference. Embryonic liver cells m a y be more sensitive against the described lysosyme treatment during the isolation procedure than adult cells.

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Netzloff et al. (1968) excluded all experiments from their determinations in which the embryos showed no heart beat at the end of the incubation period. As previously reported we found no significantly different Qo~ values in embryos on day 11 and 12 with and without a beating heart (Spielmann and Lficke, 1971). Therefore, in our studies the isolated hearts on day 14 and 15 were not examined for beating. Their respiratory rate is significantly higher than that of whole embryos on the same days, which in our opinion is the result of a different thickness of these two tissue preparations. A measurable activity of the enzyme succinate dehydrogenase has been reported both by Aksu et al. (1968) and by Bass and Schmidt (1971) for subcellular fractions of rat embryos on day 12 of gestation. Using our tissue preparations of whole rat embryos and organs we have not earlier been able to determine a succinate dependent respiratory activity than on day 14 of gestation with heart tissue. On day t2 of pregnancy succinate did not increase the Qo~ of both whole embryos and of dissected parts of the embryos, which might suggest t h a t the lacking effect of succinate on this day is not due to a reduced diffusion of the substrate into the whole embryos. The Qo~ is not glucose or succinate dependent in our determinations on day 11 and 12 whereas it is glucose and succinate dependent in the experiments with isolated organs on day 14 and later on. The experiments with the uncoupler 2,4-dinitrophenol and the inhibitor rotenone led to the same results. When using these two drugs we could not measure differences in the Qo~ values in media containing glucose only or both glucose and succinate any earlier than on day i4:. The sharp increase of the succinate dependent respiratory activity on day 19 observed in our determinations with liver slices is in good agreement with the results reported by Jacovcie et al. (1971). An increase of the flavoprotcin content on day 19 and 20 in embryonic rat liver mitochondria was described by De Vries e~ al. (1968). This strongly supports our data. IIommes et al. (1971 b), however, even one day before birth could hardly detect any stimulating effect of succinate on isolated fetal rat liver cells. Since liver cells make up more than 800/0 of the liver tissue in fetal rat liver on day 19 of gestation according to ttommes e~ al. (1971 b) and since the blood has been eluted out of our slices during the preparation procedure, we suggest that the increased suceinate dependent respiratory activity on this day of gestation in our determinations is in facf, caused by an increased succinate dehydrogenase activity of the fetal rat liver cells. Measurements of the respiratory capacity with different embryonic tissue preparations seem to be a very useful tool in mammalian developmental biochemistry, ff one is aware of the limits of the method and, therefore, changes the preparation technique at different developmental

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stages as d e s c r i b e d above. T h e m e t h o d allows some i n f o r m a t i o n on t h e d e v e l o p m e n t of t h e e n e r g y m e t a b o l i s m a n d its m o d i f i a c t i o n b y drugs as d e s c r i b e d h e r e a n d in p r e v i o u s p a p e r s (Spielmann a n d Liicke, 1971; S p i e l m a n n et al., 1972).

References Aksu, 0., Mackler, B., Shepard, R. H., Lemire, R. J. : Studies on the development of congenital anomalies in embryos of riboflavin-deficient, galactoflavin fed rats. IL RoIe of the terminal electron transport systems. Tetratology 1, 93--102 (1968). Ballard, F. J., Hanson, R. W. : Changes in lipid synthesis in rat liver during development. Biochem. J. 102, 952--958 (1967). Barrels, If.: Prenatal Respiration; Frontiers of Biology, Vol. 17. AmsterdamLondon: North-Holland Publ. Comp. 1970. Bass, R. : Respiration and oxydative phosphorylation of mitochondrial fractions isolated from rat embryos. Symposium: Metabolic Pathways in Mammalian Embryos during Organogenesis and its Modification by Drugs, Berlin 1970. Bass, R., Sehmidt, C.: Respiration and oxidative phosphorylation in embryomitochondria (rats), Naunyn-Schmiedebergs Arch. Pharmak., Suppl. to Vol. 270, R 6 (1971). Ernster, L., Dauner, G., Azzone, G. F. : Differential effects of rotenone and amytal on mitochondrial electron and energy transfer. J. biol. Chem. 288, 1124 (1963). Ifommes, F . A . , Luit-De Haan, G.; Richters, A. R. : The development of some Krebs-cycle enzymes in rat liver motochondria. Biol. Neonat. (Basel) 17, 15--23 (197i a). Ifommes, F. A., Oudman-Richters, A. R. ; Molenaar, I. : The preparation of isolated fetal liver cells. Biochim. biophys. Aeta (Amst.) 244, 191--199 (1971b). Jacovcic, S., Haddock, J., Getz, G. S., Rabinowitz, M., Swift, H.: Mitochondrial development in liver of foetal and newborn rats. Biochem. J. 121, 341--347 (1971). Kleiber, M., Cole, H. If., Smith, A. H. : Metabolic rate of rat fetuses in vitro. J. cell. comp. Physiol. 22, 167--176 (1943). Levy, M., Toury, R. : Etude de l'EvoIution des Activit6s des Enzymes Mitochondriaux de l'Hepatocyte au Cours du Development du Rat. Biochim. biophys. Aeta (Amst.) 216, 318--327 (1970). Lindah], P. E., 0berg, K. E. : The effect of rotenone on respiration and its point of attack. Exp. Cell Res. 28, 228 (1961). Maekler, B. : Studies of mitochondrial energy systems during embryogenesis in the rat. Symposium: Metabolic Pathways in Mammalian Embryos during Organogenesis and its Modification by Drugs, Berlin 1970. Negelein, E. : ?2bet die glycolytische Wirkung des embryonalen Gewebes. Biochem. Z. 165, 122--133 (1925). Netzloff, M.L., Johnson, E.M., Kaplan, S.: Respiratory changes observed in abnormally developing rat embryos. Teratology 1, 375--386 (1968). NEUBEaT, D.: Einflul3 yon Pharmaka auf energieliefernde Reaktionen des Stoffwechsels. Naunyn-Sehmiedebergs Arch. exp. Path. Pharmak. 246, 101--132 (1963). Neubert, D., Merker, H,-J., KShlcr, E., ]~'owke, R., Barrach, If.-J. : Biochemical aspects of teratology, Advances in the Biosciences, Vol. 6, pp. 574--621, OxfordEdinburg-New u Pergamon Press, Vieweg 1971a.

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