J. Embryol. exp. Morph. Vol. 41, pp. 289-294, 1977
Printed in Great Britain © Company of Biologists Limited 1977
289
SHORT PAPERS
Effects of the teratogenic folic acid
antagonist, 9-methyl pteroylglutamic acid, on
hydroxyproline levels in fetal rat limbs
By R. R. SCHMIDT, 1 K. P. CHEPENIK
AND B. V. PAYNTON
From the Daniel Baugh Institute of Anatomy, Jefferson Medical
College of Thomas Jefferson University, Philadelphia
SUMMARY
Pregnant rats were subjected to either a folic-acid-deficient regimen that produces multiple
congenital skeletal malformations, or a control folic-acid-supplemented regimen. Fetal
limbs were extirpated on days 16 and 18 of gestation, pooled from each litter, homogenized,
and aliquots set aside for hydroxyproline, protein and DNA determinations. We found that
(1) the amount of protein recovered per treated limb was approximately half that of controls
on both days, (2) the amount of protein recovered per treated or control day-18 limb was
twice that of a day-16 limb, (3) treated limbs constituted the same percentage of total body
weight as in controls on day 16, but a smaller percentage than in controls on day 18, and (4)
the concentration of hydroxyproline Og/mg protein) was significantly less for treated limbs
than for controls on day 18 of gestation. We noted also that: (1) lowest hydroxyproline
concentrations were found in limbs from treated fetuses with gross limb malformations, (2)
intermediate concentrations were found in limbs of treated fetuses not exhibiting gross limb
malformations, and (3) highest concentrations were found in control limbs. We suggest that
the treatment resulted in (1) a decreased rate of accumulation of protein in limbs prior to day
16, but not from day 16 to day 18, (2) a decreased rate of accumulation of some non-protein
component(s) in treated limbs from day 16 to day 18, and (3) an altered collagen metabolism.
INTRODUCTION
Multiple congenital skeletal malformations in fetal rats are produced by
initiating a transient maternal pteroylglutamic (folic, PGA) acid deficiency on
day 11 of gestation (Nelson, Asling & Evans, 1952; Asling, Nelson, Wright &
Evans, 1955; Johnson, Nelson & Monie, 1963). The folic acid antagonist,
9-methyl pteroylglutamic acid, is administered via stomach tube on day 11 of
gestation and then by diet until day 14 of gestation, in addition to dietary
withdrawal of folic acid for the same period. The regimen results in a high
malformation rate and a low fetal mortality rate. Regions most frequently
1
Author's address: Daniel Baugh Institute of Anatomy, Jefferson Medical College of
Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania 19107, U.S.A.
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R. R. SCHMIDT, K. P. CHEPEN1K AND B. V. PAYNTON
exhibiting malformations are the sternebrae, ribs and limbs. Various degrees
of skeletal dwarfism are reported also. Histologic examination of fetal rat
limbs obtained from PGA-deftcient mothers (Asling et al. 1955; Jaffe & Johnson, 1973) showed that condensation of limb mesenchyme occurs on schedule
on day 13 of gestation, but that, although cartilaginous models are present on
day 16, as in normal fetuses, they are deformed and retardation of ossification
centres is apparent by day 17. Localized regions of increased peri osteal ossification were observed, while endochondral ossification appeared markedly decreased. Morphologically, therefore, early stages of mesenchymal condensation
and chondrogenesis appeared to be normal, while later stages of chondrogenesis
and cartilage resorption preceding endochondral ossification appeared adversely
affected by the PGA deficiency. These observations prompted us to suggest
that the deformities and retarded ossification of the cartilaginous models might
be the result of the deposition of a quantitatively and/or qualitatively abnormal
cartilage matrix in the affected fetuses. We have therefore evaluated the accumulation of one intercellular matrix component, collagen, by measuring the
concentration of hydroxyproline in limbs obtained from malformed fetuses.
MATERIALS AND METHODS
Proestrus black hooded Long-Evans rats were caged overnight with males
of the same stock. The presence of sperm in a vaginal smear prepared at 10.00
a.m. the following morning indicated day 1 of gestation. Pregnant rats were
maintained on a stock diet of Purina laboratory chow and water ad libitum
until day 11 of gestation. Experimental females were then intubated with 1-0 ml
of a 1-Omg/ml 9-methyl pteroylglutamic acid2 (folic acid antagonist) solution
and fed a synthetic folic-acid-deficient diet containing 100 mg/kg of 9-methyl
pteroylglutamic acid until day 14 of gestation (Johnson et al. 1963). Control
females were intubated with 1-0 ml of a 1-0 mg/ml folic acid (Nutritional Biochemicals Corp.) solution on day 11 and maintained on a synthetic diet containing folic acid. All pregnant rats were intubated on day 14 with 1-0 ml of a
1-0 mg/ml solution of folic acid and fed the folic-acid-supplemented diet.
Animals consuming less than 20 gm/day of the synthetic diets were excluded
from the study. The stock laboratory diet was reinstituted on day 16 of gestation.
Pregnant rats were killed by decapitation on either day 16 or 18 of gestation,
and their uteri removed and placed in ice-cold, 0-05 M Trizma (Sigma) buffer
(pH 8-7) made 0-115 M in CaCl2. Fetuses were dissected free of their surrounding
membranes, blotted with filter paper and their wet weights determined. Fetal
limbs and livers were extirpated and their wet weights similarly obtained. Fetal
livers, serving as internal control tissue, would enable us to distinguish between
local versus generalized effects of the teratogenic regimen.
Forelimbs and hind limbs were pooled from each litter, placed in ice-cold
2
We wish to thank Dr E. W. Cantrall, Director, Preclinical Development, Lederle Laboratories, Pearl River, New York, for his generous donation of 9-methyl pteroylglutamic acid.
Hydroxyproline levels in fetal limbs
291
Table 1. Effect of maternal PGA deficiency on protein and DNA
content offetal limbs and livers
Gestational
day
16
18
Protein*
Group
DNA*
A
,
mg/limb
mg/liver
v
Control
0-334 ±003 (6) 1-697 ±0-15 (6)
Experimental 0-198±0-02 (5) 0-705 + 0-11 (5)
P < 0005
P < 0001
0-795 ±005 (7) 7050 ±0-2 (6)
Control
Experimental 0-435±001 (7) 4010±0-1 (6)
P < 0001
P < 0001
i
A
*
mg/limb
mg/liver
0028 ± 0003 (6)
0-016±0-001 (6)
P < 0005
0044 ±0-005 (6)
0-029±0-002 (5)
P < 005
0085 ± 0004 (6)
0052±0002 (5)
P < 0001
0-645 ±0066 (6)
0-389±0014(5)
P < 001
* Mean±s.E. followed by the number of observations in parentheses; each observation is
the mean of replicate determinations.
Table 2. Effect of maternal PGA deficiency on percentage of total
body weight accounted for by fetal limbs and livers
Total body weight (%)*
Gestational
day
A
,
1
Group
Limbs
Livers
16
Control
Experimental
3-79 ±0-33 (5)
411 ±0-35 (5)
7-47 ±0-16 (5)
7-11 ±0-13 (5)
N.S.D.f
N.S.D.
18
Control
Experimental
2-68 ±0-16 (5)
204±010 (5)
P < 0-01
9-74±0-25(5)
9-21 ±0-28 (5)
N.S.D.
* Mean ± S.E. followed by the number of observations in parthentheses.
t Not significantly different (P > 0-05).
0-05 M Trizma buffer (pH 8-7) made 0-115 M in CaCl2 and homogenized in an
ice-cold, glass homogenization tube by 9 passes of a teflon pestle rotating at
2000 rev./min. Aliquots (1-0 ml) of the homogenate saved for analysis of
hydroxyproline content were precipitated with an equal volume of ice-cold
20 % trichloroacetic acid (TCA). The acidified homogenate was centrifuged at
600 g for 5 min to produce a supernatant which was discarded and a sediment
which was resuspended in 4-0 ml of 6N-HC1 and hydrolyzed for 18-24 h at
120 °C. The hydrolysates were then evaporated to dryness and redissolved in
2-3 ml of deionized water. Hydroxyproline determinations were conducted
spectrophotometrically, according to the p-dimethylaminobenzaldehyde method
of Switzer & Summer (1971) and using 4-hydroxy-L-proline (Sigma) as the standard. Protein and DNA determinations were made using modifications of the
Lowry (Hartree, 1972) and Burton (Giles & Myers, 1965) procedures, respectively. Crystallized and lyophilized bovine serum albumin (Sigma) was used as
292
R. R. SCHMIDT, K. P. CHEPENIK AND B. V. PAYNTON
Table 3. Effect of maternal PGA deficiency on hydroxyproline
concentration in fetal limbs and livers
Hydroxyproline
Gestational
day
Group
Limbs*
Livers*
K
A
,
* ,
/*g/mg protein /*g/mg DNA /*g/mg protein /*g/mg DNA
16
Control
Experimental
1-23 ±0-08 (5)
1-15 ±0-29 (5)
18
Control
Experimental
5-15 + 0-61(5)
2-68±0-48(5)
P < 002
N.S.D.f
1609 ±1-23 (5) 0-425 ± 0 0 2 (5) 5-90 ± 0 3 5 (5)
16-39 + 0-71 (5) 0-415 ± 0 0 2 (5) 5-61 ±0-41 (5)
N.S.D.
N.S.D.
N.S.D.
78-32 ±7-27 (5) 0-428 ± 0 0 5 (6) 4-75 ±0-82 (5)
40-18±3-24 (5) 0-454 ± 004 (5) 4-66±0-32(5)
P < 0001
N.S.D.
N.S.D.
* Mean + S.E. followed by the number of observations in parentheses; each observation is
the mean of replicate determinations.
t Not significantly different (P > 0-05).
the standard in protein determinations; calf thymus deoxyribonucleic acid
(Aldrich Chemical Co.) was used as the standard for DNA quantitation.
RESULTS AND DISCUSSION
Maternal PGA deficiency resulted in a substantial decrease (approximately
50 %) in the amount of protein per treated fetal limb or liver when compared
to their respective controls on days 16 and 18 of gestation (Table 1). By day
18 the treated and control limbs both contained approximately twice as much
protein as did their day-16 counterparts (Table 1). Since the proportionate
increase in DNA in both treated and control limbs and livers from day 16 to
day 18 was similar, treated and control tissues were presumably growing at the
same rate (Table 1). It seems that the teratogenic regimen resulted in a significant decrease in the rate of protein accumulation in both the fetal limbs and
livers prior to day 16, but that by day 16 both tissues had essentially recovered.
On day 16 treated and control limbs made up a similar proportion of total
body weight, but by day 18 treated limbs constituted a markedly smaller proportion of total body weight than did control limbs (Table 2). This was not true
for fetal livers on day 16 or 18 (Table 2). Since (1) the rate of accumulation of
protein and DNA from day 16 to day 18 was similar in treated and control
limbs, (2) the proportion of total body weight accounted for by treated limbs
was less than that accounted for by control limbs on day 18, but not on day 16,
of gestation, and (3) the proportion of total body weight accounted for by
treated and control livers was similar, we suggest that there must have been a
substantial reduction in the net accumulation of some non-protein compound(s)
in the treated fetal limbs (skeletal tissue), but not in the livers (soft tissue),
during the period studied.
Hydroxyproline levels in fetal limbs
293
Hydroxyproline concentration, expressed as /tg per mg protein or /tg per mg
DNA, was found to be the same for both treated and control limbs and livers
on day 16 of gestation (Table 3), and for treated and control livers on day 18.
However, fetal limbs from treated mothers on day 18 had only half (52 %) the
hydroxyproline content of controls (Table 3). The percentage increase of hydroxyproline in fetal limbs from day 16 to day 18 of gestation was approximately twice as great in control as in treated tissues (Table 3). This suggests
that from day 16 to day 18 a selective decrease occurs in the accumulation of
hydroxyproline in fetal limbs from mothers treated with the teratogen, perhaps
reflecting an altered collagen metabolism.
A correlation was seen between the hydroxyproline concentration of fetal
limbs and the degree of structural malformation. The lowest hydroxyproline
concentrations were in limbs of treated fetuses that possessed obvious gross
malformations, intermediate concentrations were found in limbs of treated
fetuses not appearing malformed (less than 5 % of the total number of fetuses
examined), and the highest concentrations were obtained in control limbs.
The relationship between the quality and quantity of intercellular matrix
and fetal chondrogenesis has recently been studied in fetal and newborn
cartilage and/or chondrocytes obtained from genetic mutants possessing characteristic skeletal defects. Rhodes & Elmer (1975) demonstrated that fibular
chondrocytes from brachypod mice produced normal cartilage-type collagen,
but at a significantly slower rate than normal. They also reported an increased
accumulation of acid mucopolysaccharides in mutant cartilage and attributed
this to a failure in degradation. Similarly, Orkin, Pratt & Martin (1976) showed
that epiphyseal cartilages from brachymorphic mice synthesize undersulfated
glycosaminoglycans. Finally, Pennypacker & Goetinck (1976) demonstrated
that the composition and rate of synthesis of collagen in cartilage obtained from
day-14 nanomelic chick embryos was normal, but proteochondroitin sulfate
synthesis was defective.
The authors express their gratitude to Drs J. M. East and G. M. Rajala for their critical
review of the manuscript. We also wish to thank Ms Debra Tees for typing the manuscript.
This study was supported in part by a grant from the Human Growth Foundation.
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{Received 1 February, 1977, revised 29 April 1977)