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Biochemical studies of the developing mammalian fetus
Biochemical
Studies Mammalian
of the Developing Fetus’
I. Urea Cycle Enzymes ALFRED
L. KENSAP?
:-\sD PHILIP
P. Corm
INTRODUCTION
The evolution of an adult animal from an embryo is accompanied by a complex series of biochemical changes including enzyme synthesis and activation to provide pathways for the specialized biochemical functions of the adult organism (Boell, 1955; Steinbach and Moog, 195s). During this developmental period there is a rapid exchange of water and metabolites between the mother and fetus. The fetal monkey (macaque) exchanges 1 liter of water per hour with the mother at term (Plentl, 1957). Following delivery, the oral intake of water will average less than 20 ml in a comparable period of time. This fiftyfold decrease in exchangeable water makes possible the elimination of nitrogen by different methods and appears to recapitulate the evolutionary historv of nitrogen cscretion as has~l on the availability of water. The evolutionary adaptations for nitrogen excretion as expressed bv Baldwin (1937, 1952) permit animals which live entirely in fresh water to excrete most of their nitrogen as ammonia since, with a plentiful water supply, ammonia can 1~ removed efficiently and rapidly and its toxicitv thus be minimized. Urea, whicll is less toxic than ammonia and is very sohtble in water, is the p’zcretion product under conditions of relative water shortage. Uric acid.
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L. KENNAN
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PHILIP
P, COHEN
which is almost insoluble in water and may be excreted in supersaturated solutions, would be expected to evolve as the nitrogen excretory end product under circumstances of limited water supplies or where the animal would have occasion to be away from the water for extended periods. In the passage from aquatic to terrestrial living, detoxication of ammonia requires not only a more efficient biosynthetic mechanism, but also a more efficient excretion system for the nitrogenous end products, Among land vertebrates, detoxication is usually accomplished by the predominant formation of either urea or uric acid. Ureotelism, a state in which the metabolic end product of protein nitrogen is predominantly urea, occurs in Amphibia and Mammalia; and uricotelism, a state in which the metabolic end product of protein nitrogen is predominantly uric acid, is found in the Sauropsida (snakes, lizards, and birds). Needham (1931, 1942) has suggested that the embryo excretes ammonia first and eventually develops a mechanism for nitrogen excretion characteristic of the species, recapitulating its evolution. The tadpole has been shown to change from ammonotelism to ureotelism during metamorphosis (Brown and Cohen, 1958; Brown et al., 1959). The placental mammals have not been systematically studied as to mode of nitrogen excretion during development nor as to the enzymes available for ammonia fixation. Since the mammal is ureotelic, it would be anticipated that during embryogenesis development of the enzymes for urea biosynthesis would take place in preparation for the nonaquatic environment after birth. A systematic study of nitrogen metabolism in fetal tissues was undertaken for the purpose of (1) investigating the activation of the enzymes necessary for ammonia fixation and urea synthesis; (2) assigning where possible these functions to the fetal liver and/or membranes; and (3) correlating the activity of the different enzymes with the stages of pregnancy and birth. Ammonia can be fixed as glutamate and glutamine, but the latter two compounds are not normally quantitatively important as urinary components of nitrogen excretion in mammals. Interest in this general problem dates back to the nineteenth century, but only in recent years could definitive enzyme studies be carried out (Brown and Cohen, 1959). There is extensive literature on studies carried out in lower forms to establish the mode of excretion (ammonia, urea, or uric acid) and to assign evolutionary
FETAL
UREA
CYCLE
ENZYMES
513
and taxonomic significance to these findings, but studies in the mammalian embryo and fetus have been limited for the most part to levels of ammonia and/or urea in their tissues, or in a few instances, to enzymes known to be involved in ammonia production or uptake. In the human species, observations have been made at different stages of pregnancy in an attempt to correlate the urea concentration of the amniotic fluid with that of the mother’s serum ( Friedburg, 1955). These studies revealed that there was some accumulation of urea in the amniotic fluid during the final four months of pregnancy. A thorough study of maternal and fetal serum concentrations of nonprotein nitrogen and urea revealed no differences in the human (Slemons and Morriss, 1916) and rat (Daly, et al., 1947) but suggested that the fetal serum had a higher concentration in animals with a thicker placental membrane (reviewed by Needham, 1931). Recently studies in the sheep and goat (Bella, 1958) have shown that the nonprotein nitrogen and urea levels were in general higher in fetal than in maternal plasma. In some instances the differences were quite marked. The ammonia and urea content of human placentas expelled at term has been determined (Hammett, 1918a, b). A wide, variation was found, but the placentas from pregnant patients with toxemia contained on the average more urea. Human fetal liver of three to four months’ gestation was studied with the tissue slice technique employed by Krebs and Henseleit ( Manderscheid, 1933). 1Jrtxa was formed under these conditions. Study of the human placenta (Luschinsky, 19.51) has shown that glutaminase activity decreased as pregnancy progressed. The fetal membranes of pigs at 46 and 90 days contain enzymes which are thought to be involved in regulation of the H+ concentration (carbonic anhydrase) and ammonia (glutaminase) in urine of adult animals (Radde and McCance, 1959). Studies of the formation of glntamint and asparagine in the placentas and fetal livers of rats and rabbits have shown that glutamine is synthesized in the fetal livers of both animals and in the placenta of the rabbit (Shmerling and Mo);ilevskaya, 1954). Arginase and the urea-synthesizing activity \vere low(lr in fetal liver when compared to adult tissue of the rat (Greenstein, 1954). Arginase has been studied in the livers of white rats from birth to maturity (Lightbody, 1938); changes in activity were noted at birth and were found to be affected bv diet and sexual maturity. The kidneys of the fetal rat and guinea pig were found to 1~~pra;:-
514
ALFRED
L. KENNAN
AND
PHILIP
P. CONES
tically devoid of arginase activity (Nataf and Sfez, 1955). In the guinea pig, this activity increased in the maternal kidney and decreased in the placenta (Nataf, 1953a, b). The availability of assay procedures for the urea cycle enzymes (shown in reactions 1-4) has made it possible to study this aspect of fetal development and further has permitted a comparison of the enzyme activities of maternal and fetal liver and fetal membranes. (1) Carbamyl phosphate synthetase 0 ACglut. II HCOI- + NHJ+ + 2 ATP ------) HzNCwP + 2 ADP + Pi ivrg++ (earbamyl phosphate) (2) Ornithine transcarbamylast: 0 HeNCmP + ornithinc + citrulline + P, (3) Arginine synthetase system (a) citrulline + aspartate + ,4TP -
-+ argininosuccinate + PPi + AMP
(b) argininosuccinate ---f arginine + fumarate (4) Arginase arginine + Hz0 -----f ornithine + urea
Reactions (3a) and (3b) were measured as a single over-all system. Animals of two placental types, the rat (hemochorial) and pig (epitheliochorial) were used in the present study. It is appreciated that these two animals are not comparable in the sense that maturation has become equivalent before birth, but rather, the data only show the time of acquisition of the urea-synthesizing enzymes in animals with these two placental types. PROCEDURES
Rats at different stages of pregnancy were stunned and decapitated. The fetuses with their membranes were removed and a portion of each was weighed and homogenized. In the earlier stages, the livers from several specimens were pooled to provide adequate material.
FETAL
UREA CYCLE ETZYMES
515
A portion of the maternal liver was taken in each experiment to provide a control, Rats were allowed to deliver and individual livers examined at selected intervals up to 21 days for enzymatic development postpartum, Several litters were studied in this way. Pig embryos and fetuses were available from a local abattoir and were obtained approximately 30 minutes after the sows were slaughtered. The crown-rump length of the animal was taken for age identification, and the livers were frozen with dry ice. A portion of the sow’s liver was taken concurrently for a control specimen. Selected embryos, fetuses, and piglets (21 days, 105 days, and littermates aftc.1 delivcrv) were made available through the departments of Anatom\and Animal Husbandry of the University of Wisconsin from experiments in which the animals were slaughtered and the embryos and fetuses removed immediately. Littermates were made available after birth. Comparison of the slaughterhouse material with that obtained from the living embryo or fetus showed no significant differences. The individual membranes (pig chorion, amnion, yolk sac, and allantois; rat yolk sac, amnion, and chorion) from both animals were also studied. All the tissues were homogenized in 10 volumes of 0.14 cetyltrimethylammonium bromide (CTB) for use in the reaction mixtures. Assay procedures for the different enqmes were those pxwiollsl~ described (Brown and Cohen, 1959). Per cent of gestation in all the figures refers to fetal weight as :I percentage of birth weight in the rat (Holtzman strain average hirtlj weight, 6 gm; gestation time, 22 davs) and crown-rump length as the percentage of birth length in the $g (average birth length, 27 cm. crown-rump; gestation time, 110-120 days ). The growth rates of thrscx two animals are graphically presented in Fig. 1. Specific activity for carbamyl phosphate synthetase and ornithillca transcarhamylase refers to micromoles of citrulline produced per milligram of protein per minute. Specific activity for arginine syntbetasc refers to micromoles of urea produced per milligram of protein per hour and for arginase refers to micromoles of luea per milligram of protein per minute. Specimens were obtained at random. A total of 38 individual stages were examined for the studies on the pig, and 50 pregnant rats were used. The data are significant as an expression of the trends in enzyme activity. Fetuses with identical weights ol lengths showed some differences in activit!,; not enough animals w(‘re obtained to permit statistical expression at anv given point during
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ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
20 30 40 50 60 70 60 90 100 110 120 GESTATION DAYS FIG. 1. Prenatal growth of rats and pigs. The data for fetal rats (Holtzman strain) are our own; those for fetal pigs were taken from the paper by Warwick ( 1928 )
gestation. With this in mind, averages for each 10% increase in gestation were calculated and plotted. When more than one determination is involved in any point on the figure, it is so indicated. A broken line has been used to indicate the trend in activity and has been used in describing the results of the study. The results are highly significant if the values observed with the youngest stages are compared with those obtained at term or in neonatal animals. This latter statement applies only when an apparent change was noted at birth. RESULTS
Carbamyl phosphate synthetase activity was absent from the liver of rate fetuses weighing approximately 0.5 gm but was found to be present in all embryos weighing a gram or more and increased to about one-fifth the adult level before birth. There was an exponential increase in enzyme activity in the neonatal period which approached the adult level in 4 days. Activity for this enzyme was found in the liver of all the pig embryos studied. The smallest embryo was approximately 28 days old. Technical difficulties precluded the examination of smaller specimens. The carbamyl phosphate synthetase activity in the pig fetus was found to increase during gestation up to the level
FETAL
517
UREA CYCLE ENZYMES
PIG
40
80
100 120
PERCENT
40
80
100 12c :
OF GESTATION
FK. 2. Carbamyl phosphate synthetase in fetal liver. Specific activity versus The reaction mixture contained 5 per cent gestation (see text for details). pmoles NH,HCO,, 5 pmoles ATT’, 5 amoles L-ornithine, 5 ~moles &‘-acetyf L-glutamate, 10 pmoles MgSO,, 2 units of highly purified beef liver ornithine transcarhamylase (Brown and Cohen, 1959), and enzyme preparation diluted to give 0.5 mg protein in this mixture with a final volume of 1 ml at pH 8.8. The tubes were incubated for 15 minutes at 37” C, deproteinized with 5 ml of 0.5 Al HClO,, and nliquots were then withdrawn for analysis for citrullinc. The adult Icvrls arc given as a reference.
of activity found in adults, and no change occurred thereafter. These results are shown graphically in Fig. 2. Carbamyl phosphate synthetase activity was not found in the fetal membranes of either animal. Ornithine transcarbamylase activity was found in the liver of both species in all specimens examined. The activity increased in a linear fashion in the rat before birth and exponentially the first 3 days after birth. The pig embryo behaved differently in that a linear increase was apparent up to a stage corresponding to approximately 50% of gestation. There was no significant change in the level of this enzyme activity following birth. No measurable activity was found in the fetal membranes of either species. These changes are shown graphicall) in Fig. 3. The arginine synthetase system, shown in reaction (3) (the two component enzymes, the condensing and cleavage enzvmes, were esti-
518
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
PIG
RAT
----ADULT --ADULT LEVEL.
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? .T’-p-;
*’ IO ,,A
LEVEL
’ k
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l: 2
l
7 3 ; r-H8
la I 40
I 80
PERCENT
I 100 120
*-a-f
3 L .,A -*- \ 2
“ ‘2 ‘2
3 I 40
I 80
I I 100 120
OF GESTATION
FIG. 3. Ornithine transcarbamylase in fetal liver. Specific activity versus per cent gestation (see text for details). The reaction mixture contained 20 &moles L-ornithine, 90 @moles glycylglycine, 5 @moles of dilithium or diammonium carbamyl phosphate, and the homogenate diluted to give 0.05 mg of protein in this mixture with a final volume of 2 ml at pH 8.1. The tubes were incubated for 15 minutes at 37” C and deproteinized and analyzed as described in the legend to Fig. 2. The adult levels are given as a reference. mated together) was not found to be present in significant quantities until shortly before birth in the rat liver, after which it increased exponentially in the neonatal period. Activity was found at all the
stages studied in the liver of the pig embryo. The graphic presentation in Fig. 4 suggests a linear increase of this enzyme with time. The difference between the earlier stages and those just before birth is significant. The data are not sufficient to establish linearity between these two points. The specific activity just before birth had a value about one-half that found in the adult and increased strikingly in the first 8 hours after birth. No activity was found in the membranes of either species. Arginase activity was present in the livers of all embryos studied. There was a linear increase of activity in the rat which became exponential after birth. The activity level was higher in the earliest pig embryo than in the youngest rat studied and increased in a linear fashion during gestation. A marked increase was apparent at birth in the pig.
FETAL
UREA
CYCLE
ENZYl4ES
RAT
PIG N 2 70 -100 X60 > k501 i-40
-80
, I.
,
Y
%
---ADULT
LEVEL
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.-
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.-
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OF GESTATION
FE. 4. The arginine synthetase system in fetal liver, Units versu:, per cent gestation (see text for details). The reaction mixture contained 5 pmoles MgSO:. 5 pmoles ATP, 5 pmoles L-citrulline, 5 pmoles L-aspartate, 50 pmoles potassimrI phosphate buffer, 20 units of arginase, and extract diluted to give 2-:3 mg oc protein in this mixture with a final volume of 1 ml at pH 7.0. The tubes were incubated for 1 hour at 37” C, deproteinized, and a suitable aliquot analvzed fol urea. The adult levels are given as a reference.
Arginase activity was found in the rat placenta with fetuses weighing less than 0.5 gm, but the urea blank values in these tissues were quite high; this made these assays of questionable significance. These chanses in arginase activity are shown in Fig. S. DISCUSSION
The fetal membranes of mammals have not been systematicall\ studied as to their role in synthetic and degradative processes invol&l in nitrogen metabolism. Their function prior to development of the functional fetal liver has been a matter of speculation for which there is little biochemical evidence available. A different developmental pattern for the urea cycle enzymes was found in the livers of the two species, but study of the fetal membranes has shown no activity in vitro. The difference between the two animals may be correlated with the maturity of the animal at birth, the development of the mesonephric kidney, and the thickness of the placental membrane.
520
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
PIG (u
RAT 1
i-----l7 586
,
40
80
PERCENT
100 120
---ADULT
LEVEL
f
40
80
100 120
OF GESTATION
FIG. 5. Arginase in fetal liver. Units versus per cent gestation. (see text for details). The reaction mixture contained 25 pmoles L-arginine, 0.5 pmoles MnCl,, 50 pmoles sodium glycinate buffer, and extract diluted to give 0.05 mg protein in this mixture with a final volume of 2 ml at pH 9.0. The tubes were incubated for 30 minutes at 37” C, deproteinized, and a suitable aliquot analyzed for urea. The adult levels are given as a reference.
The fetal membranes of the pig are apposed to the epithelial surface of the endometrium (placenta apposita). This simple apposition produces a fetal-maternal exchange membrane of six layers, which is the maximum possible. Because fetal-maternal exchange will be influenced by both the number of tissue layers in the placental membrane and the actual thickness of the membrane, it is to be expected that the pig would have a transplacental exchange rate among the lowest in mammals. This condition may be compared with the leathery eggshell of some reptiles which excrete urea by having it diffuse across this membrane (Needham, 1931; Baldwin, 1937). The pig fetus has the enzymes to synthesize urea very early in life at a time corresponding to the beginning function of the mesonephros and to the accumulation of urea in the allantoic vesicle (McCance and Dickerson, 1957). This receptacle appears to be necessary because of the relatively slow placental transfer of urea in the sheep and goat (Bella, 1958) and of NaZ4 in the pig and goat (Flexner and Gellhorn, 1942). These two animals have relatively very large allantoic vesicles. In
FETAL
UREA
CYCLE
ENZYMES
521
terms of over-all nitrogen excretion, the pig would appear to have a more critical need for detoxifying mechanisms because of its fetal membranes. The early appearance of the mesonephric kidney of the pig and the increasing amounts of urea found in the allantoic vesicle during the functioning period of this kidney (McCance and Dickerson, 1957) suggest that the mesonephros functions as an excretory organ at a time when the fetal membranes are least permeable (Flexner and Cellhorn, 1942). The newborn pig is relatively mature and though dependent upon the mother begins to forage almost immediately. The relatively early development of the urea cycle enzymes in the pig would seem to represent a means of protection against ammonia intoxication during development. In addition, the relatively high levels of these enzymes at term are a possible preparation for the dietary changes immediately after birth. The fetal rat has a membrane system more closely applied to the maternal blood stream (three layers). Here, through conjugation, the trophoblast is bathed in maternal blood. Because this membrane has fewer layers and is quantitatively thinner than the pig placental membrane, one might expect to find that transfer is much more rapid and efficient. Studies with Na?.’ (Flexner and Gellhorn, 1942) have shown that this is indeed the case. A similar situation obtains in human beings, where a significant difference has never been shown between fetal and maternal serum for nitrogenous end products. The fetal rat therefore would have less need of a urea-synthesizing system and could afford to develop the necessary enzymes relatively late, nearer term, when the need became more critical. Moreover, since the rat embryo has no functioning mesonephric kidney and no allantoic vesicle, it apparently depends upon the greater permeability and possible active transport mechanisms to transfer its nitrogenous end products to the mother. The activity of the rate-limiting enzymatic step in urea synthesis, the arginine synthetase system, does not become measurable until the eighteenth day of a 22-day pregnancy. It would thus appear that the fetal rat cannot synthesize urea until the period 34 days immediately before birth, corresponding to the functional stage of the metanephric kidney (Torrey, 1943; Wells, 1946). The rat is born relatively immature, with unopened eyes and no hair. It is completely dependent upon the mother for nourishment during neonatal maturation. ~vi-
522
ALFRED
L. KENNAN
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PHILIP
P. COHEN
dently, there is not the critical need in these relatively immaturely born animals for high levels of enzyme activity comparable to the newborn pig. This would seem to be correlated with the presence of a thin fetal membrane system during prenatal life and a relatively immature existence during the neonatal period. Upon considering the observations by Bremer (1916) on the interrelationships of the mesonephros and allantois of a series of mammals, one sees that the guinea pig, which has a thin fetal membrane system like that of the rat and human, has mesonephric glomeruli which appear at an intermediate stage in development. The guinea pig has no allantois and is practically mature at birth (gestation time, 68 days). Differences in enzymatic development between the rat and guinea pig have been shown for cytochrome oxidase and succinic dehydrogenase of brain (Flexner et al., 1941; Flexner and Flexner, 1946; Hamburgh and Flexner, 1957). The time of appearance of these enzymes in the fetal guinea pig brain is at 40-45 days; the enzymes appear in the rat brain at the sixth to tenth day postpartum. In a few isolated studies of our own, dealing with the urea cycle enzymes, it is clear that the fetal guinea pig can synthesize urea after the thirtieth day of gestation, but probably not before the twenty-sixth day. The guinea pig thus is in an intermediate stage with no real need in early embryonic life for urea formation because of its thin membranes. In the human being, mesonephric glomeruli were noted by Bremer (1916) at all stages of development, in contrast to the rat and guinea pig. In a limited number of observations by the authors (the smallest was 8 cm crown-heel length) enzyme activity for urea synthesis has been observed in all cases. Apparently urea crosses the thin fetal membrane system to the mother efficiently, for there is no allantoic vesicle in the human being. Passage of urea via the urethra during the last four months of pregnancy has been demonstrated with some accumulation in the amniotic fluid during this period (Friedberg, 1955). It is of some comparative interest to note that in marine teleost fishes, which do not synthesize urea, the functional adult kidney has been lost. Birds and saurian reptiles, which are uricotelic, have a marked reduction in the number of glomeruli and, of course, lack a functioning urea cycle. Thus there would appear to be some relationship between the ability to synthesize urea and the degree of kidney development. Collating our data with those of Bremer, we find that
FETAL
UREA
CYCLE
ENZYMES
523
the placental mammals apparently begin to synthesize urea when the embryonic glomerular kidney has developed. The time of appearance of the urea cycle enzymes in this group may, in turn, be correlated with the thickness of the fetal membrane system, the presence an d size of an allantoic vesicle, and the relative maturitv of the newborn animal. SUMMARY
Four enzymes of the urea cycle have been studied in the livers of fetal, neonatal, and adult rats and pigs. The development of the four enzymes for urea synthesis in the liver of fetal rats was found to be asynchronous, and urea did not appear to be synthesized at a significant rate until late fetal life. All four enzymes were found to be present at significant levels in the liver of the youngest pig embryo studied (28 days). The species difference, which is quite marked, has been discussed in relation to the fetal membranes of the two animals and the maturation of the fetus and newborn. In addition, the relation of these developmental patterns to the theory of recapitulation and the development of the excretory mechanism has also been discussed. REFERENCES BALDWIN, E. ( 1937). “An Introduction to Comparative Biochemistry.” 1st WI. Cambridge Univ. Press, London and New York. BALDWIN, E. (1952). “Dynamic Aspects of Biochemistry,” 2nd cd. Cambridgt Univ. Press, London and New York. BELLA DE GREGOHIO, (1958). A comparative studv of the concentrations of nonprotein, urea, and amino acid nitrogen in the maternal and fetal plasm;~s of sheep and goats. Yule J. Bid. and Med. 30, ~3-378. BOELL, E. 1. ( 1955). Energy exchange and enzyme development during cmhryogenesis. In “Analysis of Development” (B. H. Willicr, P. A. \Vt+ss, ant1 1.. Hamhurger, eds.) pp. 520-555. Saunders, New York. BHEMEH, J. L. ( 1916). The interrelations of the mesonephros, kidney and pl;lccxnt;l m different classes of animals. Am. J. Anut. 19, 179-209. Bnown, G. W., JR., and COHEN, P. P. (1958). B’losynthesis of urea in mvtamorphosing tadpoles, In “The Chemical Basis of Development” (w. I). McElroy and B. Glass, eds. ), pp. 495-51:3. John Hopkins Press, B:~ltilnorc~, Xlaryland. BHOWK, G. W., JR., and COHEN, P. P. ( 1959). Methods for the cluantit:~ti\y nss;,\’ of urea cycle enzymes in liver. J. Bid. Chem. 234, 1769-1774. BROWN, G. W., JR., BROWN, W. R., and COHEN, P. P. ( 1959). Lev~~ls ()f 1Ircii cycle enzymes in metamorphosing Rana wtesbeiana tadpoles. J. BinI. ~&rp,. 234, 1775-1780.
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evidence of the DALY, H., WELLS, L. J., and EVANS, G. ( 1947). Experimental secretion of urine by fetal kidney. PTOC. SOC. Exptl. Biol. Med. 64, 78-89. FLEXNER, L. B., and FLEXNER, J. B. (1946). Biochemical and physiological difVI. Succinic dehydrogenase and succinferentiation during morphogenesis. oxidase in the cerebral 27, 35-42. FLEXNER, L.
B.,
and
cortex of the fetal guinea pig. 1. Cellular GELLHORN, A.
( 1942).
The
comparative
Comp.
Physiol.
physiology
of
placental transfer. Am. J. Obstet. Gynecol. 43, 965-974. FLEXNER, J. B., FLEXNER, L. B., and STRAWS, W. L., JR. ( 1941). The oxygen cytochrome and cytochrome oxidase activity and histological consumption, structure of the developing cerebral cortex of the fetal guinea pig. J. CeIb_&r Comp. Physiol. 18, 355-368. FRIEDBERG, V. ( 1955). Untersuchungen
iiber
cologia, 140, 34-45. GREENSTEIN, J. P. ( 1954).
of Cancer,”
“Biochemistry
New York. HAMBURGH, M., and FLEXNER, L. B. differentiation during morphogenesis. hormone therapy on enzyme activities
die
fetale
Urinbildung.
2nd ed. Academic
GynaePress,
( 1957). Biochemical and physiological XXI. Eifect of hypothyroidism and of the developing cerebral cortex of the
rat. .I. Neurochem. 1, 279-288. HAMMETT, F. S. ( 1918a). Notes on the direct determination of urea and ammonia in placenta tissue. J. Bill. Chem. 33, 381385. HAMMETT, F. S. (1918b). The urea content of placentae from normal and toxemic pregnancies, J. Biol. Chem. 34, 515-520. LIGHTBODY, H. D. ( 1938). Variations associated with age in the concentration of arginase in the livers of white rats. J. Biol. Chem. 124, 169-178. LUSCHINSKY, H. L. ( 1951). The activity of ghrtaminase in the human placenta. Arch. Biochem. Biophys. 31, 132-140. MCCANCE, R. A., and DICKERSON, J. W. T. ( 1957). The composition and origin of the fetal fluids of the pig. J. Embryol. Exptl. Morphol. 5, 4350. MANDERSCHEID, H. (1933). Uber die Harnstoffbildung bei den Wirbeltieren. Biochem. Z. 263, 245-249. NATAF, B. (1953a). Progressive changes in alkaline phosphatase and arginase activities of the guinea pig placenta at different stages of gestation. Compt. rend. sot. biol. 147, 1564-1565. NATAF, B. (195315). Changes in the arginase activity of the guinea pig kidney during gestation and during treatment with certain sex hormones. Compt. rend. sot. biol. 147, 1701-1704. NATAF, B., and SFEZ, M. (1955). Evolution of arginase activity of the kidney during post-natal development of the guinea pig and albino rat. Compt. rend. sot. biol. 149, 81-84. NEEDHAM, J. ( 1931). “Chemical Embryology.” Macmillan, New York. NEEDHAM, J. ( 1942). “Biochemistry and Morphogenesis.” Cambridge Univ. Press, London and New York. PLENTL, A. A. ( 1957 ). The origin of amniotic fluid. In “Gestation” ( C. ViIIee, ea.), pp. 71-114. Josiah Macy, Jr. Foundation, New York.
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UREA
CYCLE
ENZYMES
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NADDE, I. C., and MCCANCE, Ft. A. ( 1959). Glutaminase activity of the fetal membranes and kidney of pigs. Nature 183, 115-116. SHhmmmcz, G., and MOYILEVSKAYA, F. G. ( 1954). Th e metabolism of glutamiuc and asparagine in the placentae and embryos of rats and rabbits. Riokhimiyc~ 19, 30-36. SLEILIONS, J. M., and MORRIS, W. H. ( 1916 ) . Th e non-protein nitrogen and urea in the maternal and the fetal blood at the time of birth. Bull. Johns Hopkim Hosp. 27, 343-350. SWINBACH, H. B., and MOOG, F. (1955). Cellular metabolism. In “Analysis of Development” (B. H. Willier, P. A. Weiss, and 1’. Hamburger, eds.), pp. 7090. Saunders, New York. TORREY, T. W. (1943). The development of the urinogcnital system of the albino rat. Am. J. Anat. 12, 113-147. WAHWICK, B. L. ( 1928). Prenatal growth of swine. J. Morphol. cmd Physiol. 46, 59-69. \L'ELLS, L. J. ( 1946). Observations on secretion of urine by kidneys of fetal rat\, Amt. Record 94. 504.
Studies Mammalian
of the Developing Fetus’
I. Urea Cycle Enzymes ALFRED
L. KENSAP?
:-\sD PHILIP
P. Corm
INTRODUCTION
The evolution of an adult animal from an embryo is accompanied by a complex series of biochemical changes including enzyme synthesis and activation to provide pathways for the specialized biochemical functions of the adult organism (Boell, 1955; Steinbach and Moog, 195s). During this developmental period there is a rapid exchange of water and metabolites between the mother and fetus. The fetal monkey (macaque) exchanges 1 liter of water per hour with the mother at term (Plentl, 1957). Following delivery, the oral intake of water will average less than 20 ml in a comparable period of time. This fiftyfold decrease in exchangeable water makes possible the elimination of nitrogen by different methods and appears to recapitulate the evolutionary historv of nitrogen cscretion as has~l on the availability of water. The evolutionary adaptations for nitrogen excretion as expressed bv Baldwin (1937, 1952) permit animals which live entirely in fresh water to excrete most of their nitrogen as ammonia since, with a plentiful water supply, ammonia can 1~ removed efficiently and rapidly and its toxicitv thus be minimized. Urea, whicll is less toxic than ammonia and is very sohtble in water, is the p’zcretion product under conditions of relative water shortage. Uric acid.
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ALFRED
L. KENNAN
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PHILIP
P, COHEN
which is almost insoluble in water and may be excreted in supersaturated solutions, would be expected to evolve as the nitrogen excretory end product under circumstances of limited water supplies or where the animal would have occasion to be away from the water for extended periods. In the passage from aquatic to terrestrial living, detoxication of ammonia requires not only a more efficient biosynthetic mechanism, but also a more efficient excretion system for the nitrogenous end products, Among land vertebrates, detoxication is usually accomplished by the predominant formation of either urea or uric acid. Ureotelism, a state in which the metabolic end product of protein nitrogen is predominantly urea, occurs in Amphibia and Mammalia; and uricotelism, a state in which the metabolic end product of protein nitrogen is predominantly uric acid, is found in the Sauropsida (snakes, lizards, and birds). Needham (1931, 1942) has suggested that the embryo excretes ammonia first and eventually develops a mechanism for nitrogen excretion characteristic of the species, recapitulating its evolution. The tadpole has been shown to change from ammonotelism to ureotelism during metamorphosis (Brown and Cohen, 1958; Brown et al., 1959). The placental mammals have not been systematically studied as to mode of nitrogen excretion during development nor as to the enzymes available for ammonia fixation. Since the mammal is ureotelic, it would be anticipated that during embryogenesis development of the enzymes for urea biosynthesis would take place in preparation for the nonaquatic environment after birth. A systematic study of nitrogen metabolism in fetal tissues was undertaken for the purpose of (1) investigating the activation of the enzymes necessary for ammonia fixation and urea synthesis; (2) assigning where possible these functions to the fetal liver and/or membranes; and (3) correlating the activity of the different enzymes with the stages of pregnancy and birth. Ammonia can be fixed as glutamate and glutamine, but the latter two compounds are not normally quantitatively important as urinary components of nitrogen excretion in mammals. Interest in this general problem dates back to the nineteenth century, but only in recent years could definitive enzyme studies be carried out (Brown and Cohen, 1959). There is extensive literature on studies carried out in lower forms to establish the mode of excretion (ammonia, urea, or uric acid) and to assign evolutionary
FETAL
UREA
CYCLE
ENZYMES
513
and taxonomic significance to these findings, but studies in the mammalian embryo and fetus have been limited for the most part to levels of ammonia and/or urea in their tissues, or in a few instances, to enzymes known to be involved in ammonia production or uptake. In the human species, observations have been made at different stages of pregnancy in an attempt to correlate the urea concentration of the amniotic fluid with that of the mother’s serum ( Friedburg, 1955). These studies revealed that there was some accumulation of urea in the amniotic fluid during the final four months of pregnancy. A thorough study of maternal and fetal serum concentrations of nonprotein nitrogen and urea revealed no differences in the human (Slemons and Morriss, 1916) and rat (Daly, et al., 1947) but suggested that the fetal serum had a higher concentration in animals with a thicker placental membrane (reviewed by Needham, 1931). Recently studies in the sheep and goat (Bella, 1958) have shown that the nonprotein nitrogen and urea levels were in general higher in fetal than in maternal plasma. In some instances the differences were quite marked. The ammonia and urea content of human placentas expelled at term has been determined (Hammett, 1918a, b). A wide, variation was found, but the placentas from pregnant patients with toxemia contained on the average more urea. Human fetal liver of three to four months’ gestation was studied with the tissue slice technique employed by Krebs and Henseleit ( Manderscheid, 1933). 1Jrtxa was formed under these conditions. Study of the human placenta (Luschinsky, 19.51) has shown that glutaminase activity decreased as pregnancy progressed. The fetal membranes of pigs at 46 and 90 days contain enzymes which are thought to be involved in regulation of the H+ concentration (carbonic anhydrase) and ammonia (glutaminase) in urine of adult animals (Radde and McCance, 1959). Studies of the formation of glntamint and asparagine in the placentas and fetal livers of rats and rabbits have shown that glutamine is synthesized in the fetal livers of both animals and in the placenta of the rabbit (Shmerling and Mo);ilevskaya, 1954). Arginase and the urea-synthesizing activity \vere low(lr in fetal liver when compared to adult tissue of the rat (Greenstein, 1954). Arginase has been studied in the livers of white rats from birth to maturity (Lightbody, 1938); changes in activity were noted at birth and were found to be affected bv diet and sexual maturity. The kidneys of the fetal rat and guinea pig were found to 1~~pra;:-
514
ALFRED
L. KENNAN
AND
PHILIP
P. CONES
tically devoid of arginase activity (Nataf and Sfez, 1955). In the guinea pig, this activity increased in the maternal kidney and decreased in the placenta (Nataf, 1953a, b). The availability of assay procedures for the urea cycle enzymes (shown in reactions 1-4) has made it possible to study this aspect of fetal development and further has permitted a comparison of the enzyme activities of maternal and fetal liver and fetal membranes. (1) Carbamyl phosphate synthetase 0 ACglut. II HCOI- + NHJ+ + 2 ATP ------) HzNCwP + 2 ADP + Pi ivrg++ (earbamyl phosphate) (2) Ornithine transcarbamylast: 0 HeNCmP + ornithinc + citrulline + P, (3) Arginine synthetase system (a) citrulline + aspartate + ,4TP -
-+ argininosuccinate + PPi + AMP
(b) argininosuccinate ---f arginine + fumarate (4) Arginase arginine + Hz0 -----f ornithine + urea
Reactions (3a) and (3b) were measured as a single over-all system. Animals of two placental types, the rat (hemochorial) and pig (epitheliochorial) were used in the present study. It is appreciated that these two animals are not comparable in the sense that maturation has become equivalent before birth, but rather, the data only show the time of acquisition of the urea-synthesizing enzymes in animals with these two placental types. PROCEDURES
Rats at different stages of pregnancy were stunned and decapitated. The fetuses with their membranes were removed and a portion of each was weighed and homogenized. In the earlier stages, the livers from several specimens were pooled to provide adequate material.
FETAL
UREA CYCLE ETZYMES
515
A portion of the maternal liver was taken in each experiment to provide a control, Rats were allowed to deliver and individual livers examined at selected intervals up to 21 days for enzymatic development postpartum, Several litters were studied in this way. Pig embryos and fetuses were available from a local abattoir and were obtained approximately 30 minutes after the sows were slaughtered. The crown-rump length of the animal was taken for age identification, and the livers were frozen with dry ice. A portion of the sow’s liver was taken concurrently for a control specimen. Selected embryos, fetuses, and piglets (21 days, 105 days, and littermates aftc.1 delivcrv) were made available through the departments of Anatom\and Animal Husbandry of the University of Wisconsin from experiments in which the animals were slaughtered and the embryos and fetuses removed immediately. Littermates were made available after birth. Comparison of the slaughterhouse material with that obtained from the living embryo or fetus showed no significant differences. The individual membranes (pig chorion, amnion, yolk sac, and allantois; rat yolk sac, amnion, and chorion) from both animals were also studied. All the tissues were homogenized in 10 volumes of 0.14 cetyltrimethylammonium bromide (CTB) for use in the reaction mixtures. Assay procedures for the different enqmes were those pxwiollsl~ described (Brown and Cohen, 1959). Per cent of gestation in all the figures refers to fetal weight as :I percentage of birth weight in the rat (Holtzman strain average hirtlj weight, 6 gm; gestation time, 22 davs) and crown-rump length as the percentage of birth length in the $g (average birth length, 27 cm. crown-rump; gestation time, 110-120 days ). The growth rates of thrscx two animals are graphically presented in Fig. 1. Specific activity for carbamyl phosphate synthetase and ornithillca transcarhamylase refers to micromoles of citrulline produced per milligram of protein per minute. Specific activity for arginine syntbetasc refers to micromoles of urea produced per milligram of protein per hour and for arginase refers to micromoles of luea per milligram of protein per minute. Specimens were obtained at random. A total of 38 individual stages were examined for the studies on the pig, and 50 pregnant rats were used. The data are significant as an expression of the trends in enzyme activity. Fetuses with identical weights ol lengths showed some differences in activit!,; not enough animals w(‘re obtained to permit statistical expression at anv given point during
516
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
20 30 40 50 60 70 60 90 100 110 120 GESTATION DAYS FIG. 1. Prenatal growth of rats and pigs. The data for fetal rats (Holtzman strain) are our own; those for fetal pigs were taken from the paper by Warwick ( 1928 )
gestation. With this in mind, averages for each 10% increase in gestation were calculated and plotted. When more than one determination is involved in any point on the figure, it is so indicated. A broken line has been used to indicate the trend in activity and has been used in describing the results of the study. The results are highly significant if the values observed with the youngest stages are compared with those obtained at term or in neonatal animals. This latter statement applies only when an apparent change was noted at birth. RESULTS
Carbamyl phosphate synthetase activity was absent from the liver of rate fetuses weighing approximately 0.5 gm but was found to be present in all embryos weighing a gram or more and increased to about one-fifth the adult level before birth. There was an exponential increase in enzyme activity in the neonatal period which approached the adult level in 4 days. Activity for this enzyme was found in the liver of all the pig embryos studied. The smallest embryo was approximately 28 days old. Technical difficulties precluded the examination of smaller specimens. The carbamyl phosphate synthetase activity in the pig fetus was found to increase during gestation up to the level
FETAL
517
UREA CYCLE ENZYMES
PIG
40
80
100 120
PERCENT
40
80
100 12c :
OF GESTATION
FK. 2. Carbamyl phosphate synthetase in fetal liver. Specific activity versus The reaction mixture contained 5 per cent gestation (see text for details). pmoles NH,HCO,, 5 pmoles ATT’, 5 amoles L-ornithine, 5 ~moles &‘-acetyf L-glutamate, 10 pmoles MgSO,, 2 units of highly purified beef liver ornithine transcarhamylase (Brown and Cohen, 1959), and enzyme preparation diluted to give 0.5 mg protein in this mixture with a final volume of 1 ml at pH 8.8. The tubes were incubated for 15 minutes at 37” C, deproteinized with 5 ml of 0.5 Al HClO,, and nliquots were then withdrawn for analysis for citrullinc. The adult Icvrls arc given as a reference.
of activity found in adults, and no change occurred thereafter. These results are shown graphically in Fig. 2. Carbamyl phosphate synthetase activity was not found in the fetal membranes of either animal. Ornithine transcarbamylase activity was found in the liver of both species in all specimens examined. The activity increased in a linear fashion in the rat before birth and exponentially the first 3 days after birth. The pig embryo behaved differently in that a linear increase was apparent up to a stage corresponding to approximately 50% of gestation. There was no significant change in the level of this enzyme activity following birth. No measurable activity was found in the fetal membranes of either species. These changes are shown graphicall) in Fig. 3. The arginine synthetase system, shown in reaction (3) (the two component enzymes, the condensing and cleavage enzvmes, were esti-
518
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
PIG
RAT
----ADULT --ADULT LEVEL.
? #/’
? .T’-p-;
*’ IO ,,A
LEVEL
’ k
‘I:
l: 2
l
7 3 ; r-H8
la I 40
I 80
PERCENT
I 100 120
*-a-f
3 L .,A -*- \ 2
“ ‘2 ‘2
3 I 40
I 80
I I 100 120
OF GESTATION
FIG. 3. Ornithine transcarbamylase in fetal liver. Specific activity versus per cent gestation (see text for details). The reaction mixture contained 20 &moles L-ornithine, 90 @moles glycylglycine, 5 @moles of dilithium or diammonium carbamyl phosphate, and the homogenate diluted to give 0.05 mg of protein in this mixture with a final volume of 2 ml at pH 8.1. The tubes were incubated for 15 minutes at 37” C and deproteinized and analyzed as described in the legend to Fig. 2. The adult levels are given as a reference. mated together) was not found to be present in significant quantities until shortly before birth in the rat liver, after which it increased exponentially in the neonatal period. Activity was found at all the
stages studied in the liver of the pig embryo. The graphic presentation in Fig. 4 suggests a linear increase of this enzyme with time. The difference between the earlier stages and those just before birth is significant. The data are not sufficient to establish linearity between these two points. The specific activity just before birth had a value about one-half that found in the adult and increased strikingly in the first 8 hours after birth. No activity was found in the membranes of either species. Arginase activity was present in the livers of all embryos studied. There was a linear increase of activity in the rat which became exponential after birth. The activity level was higher in the earliest pig embryo than in the youngest rat studied and increased in a linear fashion during gestation. A marked increase was apparent at birth in the pig.
FETAL
UREA
CYCLE
ENZYl4ES
RAT
PIG N 2 70 -100 X60 > k501 i-40
-80
, I.
,
Y
%
---ADULT
LEVEL
4-t l
:
l .-
420-
I30 E
t
cou
140
LEVEL
---ADULT
f
l/ y-*
.-
M.
.-
.I0
:
I’ 3
: :
l
l
e---h
IO *2
,.c1
L
40
00
PERCENT
t*
L 100
120
:
SC--
I
1
40
00
I 100
120
OF GESTATION
FE. 4. The arginine synthetase system in fetal liver, Units versu:, per cent gestation (see text for details). The reaction mixture contained 5 pmoles MgSO:. 5 pmoles ATP, 5 pmoles L-citrulline, 5 pmoles L-aspartate, 50 pmoles potassimrI phosphate buffer, 20 units of arginase, and extract diluted to give 2-:3 mg oc protein in this mixture with a final volume of 1 ml at pH 7.0. The tubes were incubated for 1 hour at 37” C, deproteinized, and a suitable aliquot analvzed fol urea. The adult levels are given as a reference.
Arginase activity was found in the rat placenta with fetuses weighing less than 0.5 gm, but the urea blank values in these tissues were quite high; this made these assays of questionable significance. These chanses in arginase activity are shown in Fig. S. DISCUSSION
The fetal membranes of mammals have not been systematicall\ studied as to their role in synthetic and degradative processes invol&l in nitrogen metabolism. Their function prior to development of the functional fetal liver has been a matter of speculation for which there is little biochemical evidence available. A different developmental pattern for the urea cycle enzymes was found in the livers of the two species, but study of the fetal membranes has shown no activity in vitro. The difference between the two animals may be correlated with the maturity of the animal at birth, the development of the mesonephric kidney, and the thickness of the placental membrane.
520
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
PIG (u
RAT 1
i-----l7 586
,
40
80
PERCENT
100 120
---ADULT
LEVEL
f
40
80
100 120
OF GESTATION
FIG. 5. Arginase in fetal liver. Units versus per cent gestation. (see text for details). The reaction mixture contained 25 pmoles L-arginine, 0.5 pmoles MnCl,, 50 pmoles sodium glycinate buffer, and extract diluted to give 0.05 mg protein in this mixture with a final volume of 2 ml at pH 9.0. The tubes were incubated for 30 minutes at 37” C, deproteinized, and a suitable aliquot analyzed for urea. The adult levels are given as a reference.
The fetal membranes of the pig are apposed to the epithelial surface of the endometrium (placenta apposita). This simple apposition produces a fetal-maternal exchange membrane of six layers, which is the maximum possible. Because fetal-maternal exchange will be influenced by both the number of tissue layers in the placental membrane and the actual thickness of the membrane, it is to be expected that the pig would have a transplacental exchange rate among the lowest in mammals. This condition may be compared with the leathery eggshell of some reptiles which excrete urea by having it diffuse across this membrane (Needham, 1931; Baldwin, 1937). The pig fetus has the enzymes to synthesize urea very early in life at a time corresponding to the beginning function of the mesonephros and to the accumulation of urea in the allantoic vesicle (McCance and Dickerson, 1957). This receptacle appears to be necessary because of the relatively slow placental transfer of urea in the sheep and goat (Bella, 1958) and of NaZ4 in the pig and goat (Flexner and Gellhorn, 1942). These two animals have relatively very large allantoic vesicles. In
FETAL
UREA
CYCLE
ENZYMES
521
terms of over-all nitrogen excretion, the pig would appear to have a more critical need for detoxifying mechanisms because of its fetal membranes. The early appearance of the mesonephric kidney of the pig and the increasing amounts of urea found in the allantoic vesicle during the functioning period of this kidney (McCance and Dickerson, 1957) suggest that the mesonephros functions as an excretory organ at a time when the fetal membranes are least permeable (Flexner and Cellhorn, 1942). The newborn pig is relatively mature and though dependent upon the mother begins to forage almost immediately. The relatively early development of the urea cycle enzymes in the pig would seem to represent a means of protection against ammonia intoxication during development. In addition, the relatively high levels of these enzymes at term are a possible preparation for the dietary changes immediately after birth. The fetal rat has a membrane system more closely applied to the maternal blood stream (three layers). Here, through conjugation, the trophoblast is bathed in maternal blood. Because this membrane has fewer layers and is quantitatively thinner than the pig placental membrane, one might expect to find that transfer is much more rapid and efficient. Studies with Na?.’ (Flexner and Gellhorn, 1942) have shown that this is indeed the case. A similar situation obtains in human beings, where a significant difference has never been shown between fetal and maternal serum for nitrogenous end products. The fetal rat therefore would have less need of a urea-synthesizing system and could afford to develop the necessary enzymes relatively late, nearer term, when the need became more critical. Moreover, since the rat embryo has no functioning mesonephric kidney and no allantoic vesicle, it apparently depends upon the greater permeability and possible active transport mechanisms to transfer its nitrogenous end products to the mother. The activity of the rate-limiting enzymatic step in urea synthesis, the arginine synthetase system, does not become measurable until the eighteenth day of a 22-day pregnancy. It would thus appear that the fetal rat cannot synthesize urea until the period 34 days immediately before birth, corresponding to the functional stage of the metanephric kidney (Torrey, 1943; Wells, 1946). The rat is born relatively immature, with unopened eyes and no hair. It is completely dependent upon the mother for nourishment during neonatal maturation. ~vi-
522
ALFRED
L. KENNAN
AND
PHILIP
P. COHEN
dently, there is not the critical need in these relatively immaturely born animals for high levels of enzyme activity comparable to the newborn pig. This would seem to be correlated with the presence of a thin fetal membrane system during prenatal life and a relatively immature existence during the neonatal period. Upon considering the observations by Bremer (1916) on the interrelationships of the mesonephros and allantois of a series of mammals, one sees that the guinea pig, which has a thin fetal membrane system like that of the rat and human, has mesonephric glomeruli which appear at an intermediate stage in development. The guinea pig has no allantois and is practically mature at birth (gestation time, 68 days). Differences in enzymatic development between the rat and guinea pig have been shown for cytochrome oxidase and succinic dehydrogenase of brain (Flexner et al., 1941; Flexner and Flexner, 1946; Hamburgh and Flexner, 1957). The time of appearance of these enzymes in the fetal guinea pig brain is at 40-45 days; the enzymes appear in the rat brain at the sixth to tenth day postpartum. In a few isolated studies of our own, dealing with the urea cycle enzymes, it is clear that the fetal guinea pig can synthesize urea after the thirtieth day of gestation, but probably not before the twenty-sixth day. The guinea pig thus is in an intermediate stage with no real need in early embryonic life for urea formation because of its thin membranes. In the human being, mesonephric glomeruli were noted by Bremer (1916) at all stages of development, in contrast to the rat and guinea pig. In a limited number of observations by the authors (the smallest was 8 cm crown-heel length) enzyme activity for urea synthesis has been observed in all cases. Apparently urea crosses the thin fetal membrane system to the mother efficiently, for there is no allantoic vesicle in the human being. Passage of urea via the urethra during the last four months of pregnancy has been demonstrated with some accumulation in the amniotic fluid during this period (Friedberg, 1955). It is of some comparative interest to note that in marine teleost fishes, which do not synthesize urea, the functional adult kidney has been lost. Birds and saurian reptiles, which are uricotelic, have a marked reduction in the number of glomeruli and, of course, lack a functioning urea cycle. Thus there would appear to be some relationship between the ability to synthesize urea and the degree of kidney development. Collating our data with those of Bremer, we find that
FETAL
UREA
CYCLE
ENZYMES
523
the placental mammals apparently begin to synthesize urea when the embryonic glomerular kidney has developed. The time of appearance of the urea cycle enzymes in this group may, in turn, be correlated with the thickness of the fetal membrane system, the presence an d size of an allantoic vesicle, and the relative maturitv of the newborn animal. SUMMARY
Four enzymes of the urea cycle have been studied in the livers of fetal, neonatal, and adult rats and pigs. The development of the four enzymes for urea synthesis in the liver of fetal rats was found to be asynchronous, and urea did not appear to be synthesized at a significant rate until late fetal life. All four enzymes were found to be present at significant levels in the liver of the youngest pig embryo studied (28 days). The species difference, which is quite marked, has been discussed in relation to the fetal membranes of the two animals and the maturation of the fetus and newborn. In addition, the relation of these developmental patterns to the theory of recapitulation and the development of the excretory mechanism has also been discussed. REFERENCES BALDWIN, E. ( 1937). “An Introduction to Comparative Biochemistry.” 1st WI. Cambridge Univ. Press, London and New York. BALDWIN, E. (1952). “Dynamic Aspects of Biochemistry,” 2nd cd. Cambridgt Univ. Press, London and New York. BELLA DE GREGOHIO, (1958). A comparative studv of the concentrations of nonprotein, urea, and amino acid nitrogen in the maternal and fetal plasm;~s of sheep and goats. Yule J. Bid. and Med. 30, ~3-378. BOELL, E. 1. ( 1955). Energy exchange and enzyme development during cmhryogenesis. In “Analysis of Development” (B. H. Willicr, P. A. \Vt+ss, ant1 1.. Hamhurger, eds.) pp. 520-555. Saunders, New York. BHEMEH, J. L. ( 1916). The interrelations of the mesonephros, kidney and pl;lccxnt;l m different classes of animals. Am. J. Anut. 19, 179-209. Bnown, G. W., JR., and COHEN, P. P. (1958). B’losynthesis of urea in mvtamorphosing tadpoles, In “The Chemical Basis of Development” (w. I). McElroy and B. Glass, eds. ), pp. 495-51:3. John Hopkins Press, B:~ltilnorc~, Xlaryland. BHOWK, G. W., JR., and COHEN, P. P. ( 1959). Methods for the cluantit:~ti\y nss;,\’ of urea cycle enzymes in liver. J. Bid. Chem. 234, 1769-1774. BROWN, G. W., JR., BROWN, W. R., and COHEN, P. P. ( 1959). Lev~~ls ()f 1Ircii cycle enzymes in metamorphosing Rana wtesbeiana tadpoles. J. BinI. ~&rp,. 234, 1775-1780.
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AND
PHILIP
P. COHEN
evidence of the DALY, H., WELLS, L. J., and EVANS, G. ( 1947). Experimental secretion of urine by fetal kidney. PTOC. SOC. Exptl. Biol. Med. 64, 78-89. FLEXNER, L. B., and FLEXNER, J. B. (1946). Biochemical and physiological difVI. Succinic dehydrogenase and succinferentiation during morphogenesis. oxidase in the cerebral 27, 35-42. FLEXNER, L.
B.,
and
cortex of the fetal guinea pig. 1. Cellular GELLHORN, A.
( 1942).
The
comparative
Comp.
Physiol.
physiology
of
placental transfer. Am. J. Obstet. Gynecol. 43, 965-974. FLEXNER, J. B., FLEXNER, L. B., and STRAWS, W. L., JR. ( 1941). The oxygen cytochrome and cytochrome oxidase activity and histological consumption, structure of the developing cerebral cortex of the fetal guinea pig. J. CeIb_&r Comp. Physiol. 18, 355-368. FRIEDBERG, V. ( 1955). Untersuchungen
iiber
cologia, 140, 34-45. GREENSTEIN, J. P. ( 1954).
of Cancer,”
“Biochemistry
New York. HAMBURGH, M., and FLEXNER, L. B. differentiation during morphogenesis. hormone therapy on enzyme activities
die
fetale
Urinbildung.
2nd ed. Academic
GynaePress,
( 1957). Biochemical and physiological XXI. Eifect of hypothyroidism and of the developing cerebral cortex of the
rat. .I. Neurochem. 1, 279-288. HAMMETT, F. S. ( 1918a). Notes on the direct determination of urea and ammonia in placenta tissue. J. Bill. Chem. 33, 381385. HAMMETT, F. S. (1918b). The urea content of placentae from normal and toxemic pregnancies, J. Biol. Chem. 34, 515-520. LIGHTBODY, H. D. ( 1938). Variations associated with age in the concentration of arginase in the livers of white rats. J. Biol. Chem. 124, 169-178. LUSCHINSKY, H. L. ( 1951). The activity of ghrtaminase in the human placenta. Arch. Biochem. Biophys. 31, 132-140. MCCANCE, R. A., and DICKERSON, J. W. T. ( 1957). The composition and origin of the fetal fluids of the pig. J. Embryol. Exptl. Morphol. 5, 4350. MANDERSCHEID, H. (1933). Uber die Harnstoffbildung bei den Wirbeltieren. Biochem. Z. 263, 245-249. NATAF, B. (1953a). Progressive changes in alkaline phosphatase and arginase activities of the guinea pig placenta at different stages of gestation. Compt. rend. sot. biol. 147, 1564-1565. NATAF, B. (195315). Changes in the arginase activity of the guinea pig kidney during gestation and during treatment with certain sex hormones. Compt. rend. sot. biol. 147, 1701-1704. NATAF, B., and SFEZ, M. (1955). Evolution of arginase activity of the kidney during post-natal development of the guinea pig and albino rat. Compt. rend. sot. biol. 149, 81-84. NEEDHAM, J. ( 1931). “Chemical Embryology.” Macmillan, New York. NEEDHAM, J. ( 1942). “Biochemistry and Morphogenesis.” Cambridge Univ. Press, London and New York. PLENTL, A. A. ( 1957 ). The origin of amniotic fluid. In “Gestation” ( C. ViIIee, ea.), pp. 71-114. Josiah Macy, Jr. Foundation, New York.
FETAL
UREA
CYCLE
ENZYMES
525
NADDE, I. C., and MCCANCE, Ft. A. ( 1959). Glutaminase activity of the fetal membranes and kidney of pigs. Nature 183, 115-116. SHhmmmcz, G., and MOYILEVSKAYA, F. G. ( 1954). Th e metabolism of glutamiuc and asparagine in the placentae and embryos of rats and rabbits. Riokhimiyc~ 19, 30-36. SLEILIONS, J. M., and MORRIS, W. H. ( 1916 ) . Th e non-protein nitrogen and urea in the maternal and the fetal blood at the time of birth. Bull. Johns Hopkim Hosp. 27, 343-350. SWINBACH, H. B., and MOOG, F. (1955). Cellular metabolism. In “Analysis of Development” (B. H. Willier, P. A. Weiss, and 1’. Hamburger, eds.), pp. 7090. Saunders, New York. TORREY, T. W. (1943). The development of the urinogcnital system of the albino rat. Am. J. Anat. 12, 113-147. WAHWICK, B. L. ( 1928). Prenatal growth of swine. J. Morphol. cmd Physiol. 46, 59-69. \L'ELLS, L. J. ( 1946). Observations on secretion of urine by kidneys of fetal rat\, Amt. Record 94. 504.