/ . Embryol. exp. Morph. Vol. 35, 3, pp. 545-559, 1976
Printed in Great Britain
545
Foetal and placental ornithine
decarboxylase activity in the rat - effect of maternal
undernutrition
By JOHN P. G. WILLIAMS 1 AND PETER A. McANULTY 2
From the Department of Growth and Development,
Institute of Child Health, London
SUMMARY
The activity of the enzyme ornithine decarboxylase and the accumulation of nucleic acids
were examined in the rat foetus and placenta during normal development and during
maternal undernutrition.
Maternal undernutrition resulted in a reduced rate of increase in weight of both the foetus
and placenta towards the end of gestation. In the placenta the failure to increase in weight
was accompanied by a failure of DNA to increase. In the foetus the amount of DNA failed
to increase over a short period and then made a compensatory response to return to a normal
amount by the end of gestation. Undernutrition failed to affect RNA in either the foetus or
placenta. These results differ from those obtained during maternal protein deficiency.
The activity of foetal and placental ornithine decarboxylase was normal during much of
gestation in the undernourished group. However, at the same time as the compensatory
increase in foetal DNA, there was a marked increase in enzyme activity in both tissues. It is
suggested that a single compensatory stimulus is responsible for the changes in both ornithine
decarboxylase and DNA.
INTRODUCTION
High polyamine concentrations and polyamine-synthesizing enzyme activities
have been reported in embryos and larvae of the toad Xenopus laevis (Russell,
1971), in the chick embryo (Raina, 1963; Caldarera, Barbiroli & Moruzzi,
1965; Russell & Lombardini, 1971), in the rat foetus (Russell & McVicker,
1972) and in the human foetus (Sturman & Gaull, 1974). High rates of polyamine synthesis and high polyamine concentrations have also been reported in
the rat (Maudsley & Kobayashi, 1971; Gunaga, Sheth, Gunaga & Rao, 1973)
and human placenta (Gunaga et al. 1972).
The polyamines spermidine and spermine are associated with nucleic acid and
protein synthesis. Heby & Agrell (1971) have reported that the polyamines bind
to both DNA and RNA and this can account for many of their effects on nucleic
1
Author's address: Department of Biological Sciences, City of London Polytechnic, Old
Castle Street, London, El 7NT.
2
Author's address: Strangeways Research Laboratory, Wort's Causeway, Cambridge
CB1 4RN.
546
J. P. G. WILLIAMS AND P. A. McANULTY
acid and protein synthesis. The polyamines have been reported to enhance
RNA polymerase activity (MacGregor & Mahler, 1967; Raina & Janne, 1970),
to stabilize newly formed RNA (Raina & Janne, 1970), to stabilize and aggregate ribosomal units (Siekevitz & Palade, 1962), to play a role in ribosomal
attachment to membranes (Khawaja, 1971), and to inhibit ribonuclease
activity (Brewer, 1972). The polyamines have also been shown to stimulate
DNA polymerase B of rat brain (Chiu & Sung, 1972) and it has been suggested
that they may affect chromatin condensation (Anderson & Norris, 1960). Since
the polyamines are of importance in nucleic acid synthesis it is not surprising
to find that they occur in high concentrations in rapidly growing tissues (Snyder
& Russell, 1970; Russell, 1973).
The first step in the biosynthesis of the polyamines is the decarboxylation of
ornithine by ornithine decarboxylase (EC4.1.1.17) to form the diamine
putrescine (Janne & Raina, 1968; Pegg & Williams-Ashman, 1968; Russell &
Snyder, 1968). Spermidine is synthesized from putrescine by the addition of a
propylamine group from S-adenosyl (5')-3-methylthiopropylamine (Janne,
Schenone & Williams-Ashman, 1971) and spermine is synthesized from spermidine by the addition of a further propylamine group (Raina & Hannonen, 1971).
The rate-limiting step in the biosynthesis of the polyamines is the formation of
putrescine by ornithine decarboxylase, and the importance of this regulatory
role of ornithine decarboxylase is reflected by its short half-life, which has been
reported to be as little as 10 min (Russell & Snyder, 1969).
During intrauterine growth retardation the rate of nucleic acid and protein
synthesis is reduced in the foetus (Zeman & Stanbrough, 1969; Oh & Guy,
1971; Hill et al. 1971). This can result in reduced amounts of nucleic acids and
proteins in the newborn, and these deficits may persist during postnatal development (Roux, 1971a, b; McLeod, Goldrick & Whyte, 1972). In the case of intrauterine growth retardation caused by maternal malnutrition, nucleic acid and
protein synthesis are also reduced in the placenta (Brasel & Winick, 1972;
Rosado et al 1972).
The purpose of the present investigation was to examine whether the changes
that occur in nucleic acid accumulation in the foetus and placenta during
maternal malnutrition were related to changes in the activity of ornithine
decarboxylase, the regulator of polyamine biosynthesis.
MATERIALS AND METHODS
Animals
Female black-hooded rats that had previously reared at least one litter
successfully, were used throughout the study.
Foetal and placental ornithine decarboxylase
547
Experimental procedure
The rats were caged individually following the identification of spermatozoa
in vaginal smears, and allotted to one of two nutritional regimes. The first set of
animals were allowed unrestricted access to the normal diet throughout gestation ('control' animals). The second set were fed normally for the first 5 days of
gestation, but for the remainder of the gestation period they were given half the
mean daily food intake of the first 5 days (Dickerson & McAnulty, 1972). All
animals were allowed free access to water.
Animals were killed by cervical dislocation at 10.00 h daily between days 13
and 21 of gestation. The uterine horns were exposed and animals with less than
eight conceptuses were rejected to minimize variations. The foetuses and
placentas were excised and weighed. At each gestational age six control and six
undernourished rats were used.
Preparation of tissue extracts
From each pregnant female two tissue pools were prepared, one of all the
foetuses and one of all the placentas. Immediately after weighing the tissues
were minced, and aliquots were homogenized in a MSE homogenizer with a
25 ml micromasticator assembly. The vessel containing the tissue was surrounded with ice during homogenization. Aliquots of the homogenate for
enzyme measurement were analysed immediately and the remaining homogenate
was stored at - 15 °C to be examined later for nucleic acids and protein.
For enzyme determinations, aliquots of the homogenate were further
homogenized in 5 vol. of 50 mM-sodium/potassium phosphate buffer (pH 7-0),
containing 5 mM-pyridoxal-5'-phosphate, 5 mM-dithiothreitol (both obtained
from Koch-Light Laboratories Ltd, Colnbrook, Bucks.) and 2 mM-EDTA in a
Thomas homogenizer. The homogenates were centrifuged for 20 min at 20000 g
at 4 °C in a MSE High Speed 25 centrifuge. The resulting supernatants were
used for enzyme determinations and soluble protein estimations.
Nucleic acids were extracted from aliquots of the tissue homogenate by the
method of Munro & Fleck (1966).
Ornithine decarboxylase assay
The method used was essentially that of Russell & Snyder (1968) but the
incubation vessels were a modification of those described by Jones, Hampton
& Preslock (1972). The incubation medium consisted of 1-6 ml of the buffer
used in the enzyme preparation, and the incubation vessel was a plastic scintillation vial (Packard Instrument International S.A., Breda, Holland) with a hole
bored near its top covered with Sellotape®; 0-4 ml of the enzyme preparation
was added to the incubation medium and preincubated at 37 °C for 10 min
in a shaking water bath; 0-5/*Ci of DL-ornithine-l-(carboxy-14C)-monohydrochloride (29 mCi/mmol), obtained from the Radiochemical Centre Ltd,
548
J. P. G. WILLIAMS AND P. A. McANULTY
Amersham, Bucks., was diluted with unlabelled L-ornithine monohydrochloride
(Koch-Light Laboratories Ltd, Colnbrook, Bucks.), and added to each incubation vial to give a final concentration of 0-5 mM ornithine monohydrochloride
and a final specific activity of 0-25 mCi/mmol. The vial was sealed with its
plastic cap, and fixed in the lid of the cap with three spots of Copydex® was
a 1-5 cm 3MM grade filter paper circle (Reeve Angel Scientific Ltd, London),
on which three drops of a 1-0 M solution of Hyamine® hydroxide in methanol
(Koch-Light Laboratories Ltd, Colnbrook, Bucks.) had been placed. The vials
were incubated for 30 min at 37 °C in the shaking water bath. The reaction was
stopped by the injection of 1 ml of 2 M-citric acid through the Sellotape®covered hole in the vial, and the hole was resealed with a further piece of
Sellotape®. Incubation was continued, for another 30 min to ensure that all
released 14CO2 was absorbed by the filter paper disc. The filter paper disc was
then freed from the vial cap and placed in another plastic scintillation vial
containing 10 ml of toluene with 0-8 % 2,5-diphenyloxazole and 0-01 % 1,4-di
(2-(5-phenyloxazolyl)) benzene (both from Hopkin and Williams Ltd, Chad well
Heath, Essex). Radioactivity was measured in a Philips Automatic Liquid
Scintillation Analyser, and quenching was corrected by the use of an external
standard. The activity of ornithine decarboxylase was expressed as pmol 14CO2
produced in 30 min by 1 mg of soluble protein.
Nucleic acid determination
DNA was determined by the diphenylamine method of Burton (1956) as
modified by Giles & Myers (1965), and RNA by the ultra-violet absorption
method of Munro & Fleck (1966). Highly polymerized DNA from calf thymus
and highly polymerized RNA from yeast (both from BDH Chemicals Ltd,
Poole, Dorset) were used as standards.
Protein determination
Protein was measured in the enzyme preparations by the method of Lowry,
Rosebrough, Farr & Randall (1951) using bovine serum albumin (BDH
Chemicals Ltd, Poole, Dorset) as standard.
Water content determination
To determine water content, samples of the original homogenates were
heated at 97 °C in tared containers until they reached constant weight.
RESULTS
Foetal and placental weights
The weight of both the foetus and the placenta was lower in the experimental
group than in the control group towards the end of gestation (Fig. 1). On the
21st day of gestation this deficit amounted to 25 % in the foetus and 35 % in the
Foetal and placental ornithine decarboxylase
549
5
.-»-?*?
13
14
15
16
17
18 19
Age (postconceptual clays)
20
21
Fig. 1. The weight of the rat foetus and placenta during normal gestation and
during maternal undemutrition. Each point is the mean of the pooled tissues from
six females, and the S.E.M. is indicated. Where no S.E.M. is shown the limits of
variability fall within the area of the symbol: # , normal foetuses; O, foetuses from
undernourished mothers; A, normal placentas; A» placentas from undernourished
mothers.
placenta. The difference in weight did not become apparent in the foetus
until the 17th day of gestation, but thereafter there was a significant difference between control and experimental groups (P < 0-001). In the placenta a
difference in weight could be detected earlier, on the 15th day of gestation
(P < 0-01).
Foetal and placental water content
In neither tissue was there any significant difference between experimental and
control groups in water content throughout the period of gestation investigated.
Foetal and placental nucleic acids
The concentrations of DNA and RNA showed few differences between the
experimental and control groups in both the foetuses and placentas (Table 1).
Throughout gestation the concentration of RNA was slightly higher in the
undernourished foetuses and placentas than in the normal tissues, but on only
one occasion did this difference become significant. On the 16th day of gestation the concentration of DNA was elevated in both undernourished foetuses
and placentas.
35
EMB 35
15
16
17
18
19
20
21
300±0-61 2-31±0-20
3-52±0-25 2-64±011*
3-41 ±0-39
3-50±0-50
3-48±0-56 3-59±O18 3-76±0-32 2-96±0-31
4-25 ±0-79 4-70±0-39* 4-23 ±0-52 3-49 ±0-36
y
z
o
5-63 ±0-45 3-82 ±0-33 >
6-09±0-61 4-87 ±0-38
o
2-87 ±0-51 2-00±0-31
3-58±0-79 2-64±0-30 Z
1-44 ±0-39 1-39 ±007
l-60±0-19 l-52±018
Statistical significance of difference between mean values for normal and control groups: * P < 005; ** P < 001; all other differences are
insignificant.
Values are expressed as mg/g and are means ±S.E.M. for six tissue pools.
5-39±0-55
710±0-59
5-62±0-54
7-25±0-63
1-82 + 011
1-62 ±0-09
6-43 ±0-45 6-66±0-28
6-54±0-86 7-10±0-60
6-30±0-34
6-92±0-65
1-96±0-02 l-85±O-13 2-56±0-29 202±018
2-15±018 2-40±015* 1-87 ±0-22 l-92±0-37
3-63±0-74 2-47±0-19
3-91±0-28 3-27±0-23 319±017 3-07±0-21 2-40±0-20
2-28 ±0-20 1-69 ±011
4-54±0-27 4-33 ±0-39** 3-45±0-21 416±019* 2-76±003 2-51 ±016* 3-48±0-18** 2-81 ±0-26 204±008
14
RNA
Normal foetuses 10-71 ± 0-68 6-89 ± 1 08
Undernourished
9-95 ±0-46 7-22 ±0-93
foetuses
Normal placentas 703±1-76 3-55±0-60
Undernourished
7-27 ±1-44 5-31 ± 0-73
placentas
DNA
Normal foetuses
Undernourished
foetuses
Normal placentas
Undernourished
placentas
13
Gestational age (postconceptual days)
Table 1. Nucleic acid concentrations in the rat foetus and placenta during normal development and during
intrauterine growth retardation caused by maternal undernutrition
Foetal and placental ornithine decarboxylase
551
9 i-
< 5
Z
a
2 4
13
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 2. The DNA content of rat foetuses during normal gestation and during
maternal undernutrition. Each point is the mean of all of the foetuses from six
females, and the S.E.M. is indicated; # , normal foetuses; O, foetuses from undernourished mothers.
10
r
I I
0-9
0-8
0-7
06
05
04
0-3
02
0-1
13
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 3. The DNA content of rat placentas during normal gestation and during
maternal undernutrition. Each point is the mean of all of the placentas from six
females, and the S.E.M. is indicated; # , normal placentas; O, placentas from undernourished mothers.
35-2
552
J. P. G. WILLIAMS AND P. A. McANULTY
20
18
16
^
14
E
l
10
c
8
6
13
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 4. The RNA content of rat foetuses during normal gestation and during
maternal undernutrition. Each point is the mean of all of the foetuses from six
females, and the S.E.M. is indicated; # , normal foetuses; O, foetuses from undernourished mothers.
The absolute amount of DNA in the experimental foetuses increased normally
until the 16th day of gestation (Fig. 2), but between days 16 and 17 it failed to
show the normal increase and fell below the control values (P < 0-001). The
amount of DNA began to increase again after day 17, but was still below normal
values on day 18 (P < 0-001). Between days 18 and 19 the amount of DNA in
the experimental foetuses increased rapidly and was higher than in the controls
on day 19 (P < 0-05). Thereafter there was no difference in the amount of foetal
DNA in the control and experimental groups. The amount of DNA in the
placentas of the experimental group also increased normally until the 16th day
of gestation (Fig. 3), but then failed to increase at the normal rate. Unlike the
foetuses, the deficit was not made up later, and there was a permanent deficit in
placental DNA. In neither the foetus nor the placenta were there any differences
in the absolute amounts of RNA between the control and experimental groups
(Figs. 4 and 5).
The weight: DNA ratios were also calculated, and on the 21st day of gestation
the ratio in the undernourished foetuses was significantly below that in the
control foetuses (0-46 ±0-02 and 0-59 ±0-04, respectively; P < 0-01). In the
placentas there was no significant difference in the ratios of the control and
undernourished groups (0-77 ± 0-07 and 0-74 ± 0-07, respectively).
Foetal and placental ornithine decarboxylase
553
20
18
1-6
To |-4
OS
1 1-0
<u
o
E
08
0-6
0-4
0-2
13
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 5. The RNA content of rat placentas during normal gestation and during
maternal undernutrition. Each point is the mean of all of the placentas from six
females, and the S.E.M. is indicated; • , normal placentas; O, placentas from undernourished mothers.
Foetal and placental ornithine decarboxylase activity
During normal development of the foetal rat, the activity of ornithine decarboxylase decreased from day 13 onwards (Fig. 6). In the undernourished
foetuses, however, the normal curve was followed until the 15th day of gestation,
but between days 15 and 16 the activity increased and was significantly greater
than in normal foetuses on days 16 (P < 0-02) and 17 (P < 0-01). Thereafter the
activity decreased at a similar rate to that of the normal foetuses.
In the normal placentas a peak occurred in ornithine decarboxylase activity
on the 14th day of gestation (Fig. 7). The activity decreased between days 14
and 15, and then plateaued until the 18th day of gestation, after which the
activity decreased. As in the foetuses, the activity of ornithine decarboxylase in
the experimental placentas followed the normal curve up to the 15th day of
gestation, but then increased above normal activities between days 15 and 16.
On days 16 and 17 the enzyme activity was significantly greater in the retarded
placentas than in the normal placentas (P < 0-01 and P < 0-05, respectively).
Again, as in the foetus, the enzyme activity curve was similar in the placentas of
both groups from the 18th day of gestation onwards.
554
J. P. G. WILLIAMS AND P. A. McANULTY
600
=
r
500
400
•e s
300
200
^
O
100
13
I
1
I
I
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 6. The activity of ornithine decarboxylase of rat foetuses during normal
gestation and during maternal undernutrition. Each point is the mean of all of
the foetuses from six females, and the S.E.M. is indicated; • , normal foetuses;
O, foetuses from undernourished mothers.
DISCUSSION
It has long been believed that the foetus is spared some of the effects of
maternal malnutrition. In the study reported here, deficiency in foetal weight
did not occur until some twelve days after the initiation of diet restriction,
although the effects were apparent in the placenta a little earlier. The time of the
retardation in weight gain of these two tissues was however later than that
reported by Brasel & Winick (1972) for maternal protein deficiency.
At 21 days the amount of DNA in the foetuses from undernourished mothers
was the same as in the control foetuses. This is in contrast with the reports of
Zeman & Stanbrough (1969) and of Brasel & Winick (1972) who found a
reduction in the DNA content of newborn rats from protein-deficient mothers.
The pattern of DNA accumulation however was not the same in the normal and
undernourished animals. Between 16 and 17 days of gestation DNA content
failed to increase in the undernourished group, but the deficit was made up by
an increased rate of accumulation between days 17 and 19. In the undernourished
placentas the rate of accumulation of DNA fell below normal, but did not show
Foetal and placental ornithine decarboxylase
555
2500 r
2000
1500
looo
500
13
14
15
16
17
18
19
20
21
Age (postconceptual days)
Fig. 7. The activity of ornithine decarboxylase of rat placentas during normal
gestation and during maternal undernutrition. Each point is the mean of all of
the placentas from six females, and the S.E.M. is indicated; • , normal placentas;
O, placentas from undernourished mothers.
a compensatory increase. Since DNA can be used as an index of cell number
these results suggest that foetal cell number can be maintained despite undernutrition, but placental cell number is reduced. In the undernourished foetuses
the weight:DNA ratio was reduced, indicating a reduced mean cell size. In the
studies on maternal protein deficiency it was found that both foetal and placental
cell size remained normal (Zeman & Stanbrough, 1969; Brasel & Winick,
1972).
The total RNA in the foetus and placenta was not affected by undernutrition.
This result is again at variance with the effects of protein deficiency, during which
foetal RNA decreases and placental RNA increases (Zeman & Stanbrough,
1969; Brasel & Winick, 1972).
Ornithine decarboxylase activity in the normal foetuses decreased throughout
the period of gestation examined. In the placenta the activity increased initially
but then fell. The activity in the placenta is the highest so far reported for a
normal rat tissue, and this probably reflects the great metabolic activity of the
placenta. The foetal ornithine decarboxylase activities reported in this study are
much higher than those reported by Russell & McVicker (1972), and there are
also differences in the developmental profile of the enzyme's activity. These
differences are probably due to the use of dithiothreitol in the present study,
and to the use of different strains of rat.
556
J. P. G. WILLIAMS AND P. A. McANULTY
In the foetuses and placentas from undernourished mothers, changes are seen
in ornithine decarboxylase activity before any other response. In both tissues the
enzyme activity increased between days 15 and 16, and remained elevated until
day 17. The timing of this increase occurs just before the compensatory increase
in foetal DNA, so these responses may be related. Ornithine decarboxylase
characteristically responds to anabolic stimuli (Morris & Fillingame, 1974), and
thus there may be a compensatory stimulus during undernutrition of the foetus
which maintains normal cell number. The identity of a possible stimulus
remains a matter of conjecture, especially as the production of the majority of
hormones in the rat foetus does not begin until after the time that the stimulus
occurs. One possibility is that the ornithine decarboxylase-stimulating factor
that occurs in foetal calf serum (Morley, 1972, 1974) may also occur in the rat
foetus, and may be involved in the responses observed in this study. It is also
possible that chorionic somatomammotrophin (placental lactogen) plays a role.
This hormone is secreted by the foetal placenta, passes into the maternal circulation, and may modify maternal glucose metabolism to maintain the glucose
needs of the foetus (Josimovich, Kosor & Mintz, 1969). Serum somatomammotrophin increases in pregnant humans in response to a prolonged fast
(Tyson, Austin & Farinholt, 1971), so its possible role cannot be dismissed.
Thus, maternal undernutrition from the 5th day of gestation results in
reduced weight gain of the rat foetus and placenta. In the foetus a compensatory
response occurs, manifested by a maintenance of cell number and an increase in
ornithine decarboxylase activity, although mean cell size is reduced. Changes in
ornithine decarboxylase activity may be of use in studying compensatory
responses by the foetus.
This research was supported by the Medical Research Council through a long-term grant
to Professor J. M. Tanner. We would like to thank Professor Tanner for his suggestions
during the preparation of this paper, Miss J. A. Lewis for technical assistance, and Mrs
C. A. McAnulty for typing the manuscript.
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(Received 7 November 1975, revised 14 January 1976)