/. Embryol. exp. Morph., Vol. 16, 2, pp. 271-288, October 1966
With 2 plates
Printed in Great Britain
271
Enzymic differentiation of rat yolk-sac placenta
as affected by a teratogenic agent
By E.MARSHALL JOHNSON & RALPH SPINUZZ11
From the Department of Anatomy, College of Medicine,
University of Florida
INTRODUCTION
In 1927, Brunschwig presented experimental evidence which indicated that the
yolk-sac of the rat functioned as a site of transport between the mother and the
developing embryo. Everett (1935) was able to demonstrate that the maternal
blood within the central zone adjacent to the parietal wall of the yolk-sac was
circulating and that this area was indeed serving as a locale for exchange. He
further demonstrated that the allantoic lamellae and their circulation do not
become established in the rat until the end of day 11 or early on day 12; and
therefore, during days 10 and 11, circulating maternal blood in the central zone
serves as the only obvious source available for supply of substances to the embryo
as well as for removal of waste products from the embryo. For such transport
from the maternal to the embryonic organism, Reichert's membrane, which
appears to be the primary barrier of the parietal yolk-sac, was selectively permeable in that trypan blue crossed into the yolk-sac cavity but iron ammonium
citrate did not. Even after the chorioallantoic circulation had been established,
trypan blue injected into the maternal circulation readily passed through
Reichert's membrane and was quickly incorporated into the apical cytoplasm
of the proximal yolk-sac epithelium but did not appear in the chorionic lamellae
2 h after injection. In other words, during the very critical stages of early
differentiation and organogenesis in the rat, the yolk-sac epithelium would
appear to be the only available site for major transport. Even during somewhat
later embryonic stages the yolk-sac epithelium is able to incorporate macromolecules into itself more readily than are the chorioallantoic lamellae. There
does appear to be a change in the relative importance of the yolk-sac and the
chorioallantoic placenta as gestation progresses, however, because near term
the yolk-sac circulation could be interrupted and the fetuses survive; but if the
chorioallantoic circulation were interrupted late in gestation, the fetuses soon die
(Noer & Mossman, 1947). Furthermore, Brambell (1958) has reviewed papers
dealing with the acquisition of immunity prior to birth which appear to
1
Authors' address: Department of Anatomy, College of Medicine, University of Florida,
Gainesville, Florida, 32601, U.S.A.
272
E. M. JOHNSON & R. SPINUZZI
demonstrate temporal changes in transport of antibodies across the yolk-sac
in several species.
On a purely morphologic basis the visceral yolk-sac is not a static structure
either. This was made obvious by the observation of transformation of discontinuous blood islands into the vitelline circulatory bed and also was demonstrated by changes in the epithelial cytology as gestation progressed (Everett,
1935). At the ultrastructural level Dempsey (1953) showed that in the guinea-pig
the yolk-sac underwent progressive change during gestation. The bleb-like
evagination from the free surface membrane of the columnar epithelial cells
early in gestation became uniformly cylindrical microvilli associated with apical
granules and vacuoles as gestation advanced. Another indication of continued
morphologic development was the demonstration that the endoplasmic reticulum
became somewhat dilated toward term. Also, a continuous series of structures
from normal mitochondria to dense spherical bodies was described as being
degenerative changes indicative of over-all ageing of the yolk-sac as it became
relatively replaced in importance toward term by the chorioallantoic placenta.
Such changes were not observed in mitochondria of the rat's yolk-sac (Wislocki
& Dempsey, 1955), but on day 13 the microvilli were tall and branched and
associated with apparent pinocytotic vesicles and canaliculi (Padykula &
Wilson, 1960). By day 17 there was a marked regression indicated by microvilli
that were shorter and less branched and by canaliculi with fewer interconnexions.
In addition, the intercellular space between adjacent epithelial cells became
enlarged. Concomitant with these structural changes was a marked decrease in
the relative ability of the membrane to absorb vitamin B12 intrinsic factor
in vitro.
That the structural differentiation of the visceral yolk-sac is accompanied by
biochemical differentiation was demonstrated by Wislocki & Padykula (1953),
who found that PAS-positive material was present as early as day 10 and that
commencement of glycogen storage in the cytoplasm of the endoderm (Padykula
& Richardson, 1963) began on day 15. It reached a maximum concentration by
day 18 and decreased thereafter to term. Some glycogen was also encountered
in the mesenchymal cells between the yolk-sac epithelium and the vitelline blood
vessels. A similar pattern of differentiation was demonstrated for several
enzymes which had a relatively low level of activity on day 13, were more active
by day 16, and then declined until term (Padykula, 1958). Phosphatase activity
for glycerophosphate at alkaline pH and for adenosine triphosphate in tissue
sections was present in components of the apical cytoplasm and reached maximal
staining intensity by day 17. Through biochemical analysis, however, a higher
level of activity was demonstrated on day 19 which then declined markedly
through day 21. The adenosine triphosphatase activity appeared to be distributed
throughout the epithelial cytoplasm by day 19. Phosphatase activity at acid pH
was similarly localized to the apical cytoplasm at 15 days but had spread
throughout the cytoplasm by term. Acid phosphatase was the only exception to
Enzymic differentiation of yolk-sac
273
the general trend of decreasing activity toward term. Non-specific esterase
activity with a-naphthyl acetate also reached its maximum staining intensity
in the visceral yolk-sac epithelium between days 15 and 17, after which it declined. Succinic dehydrogenase activity studied with sodium succinate and
neotetrazolium compounds was difficult to localize but reached its greatest
level of activity by day 16 and declined, though not precipitously, until
term.
The present study was designed to determine whether the enzymic differentiation demonstrated by Padykula was associated with changes in specific isozymes
and multiple molecular forms of enzymically active proteins. The study also
included additional enzymes and encompassed the earlier days of gestation, and
lastly it included material treated with the potent teratogenic agent 9-methyl
pteroylglutamic acid. The teratogen was administered during the period of
differentiation and organogenesis of the embryo to determine whether this agent
could alter the normal molecular differentiation of the yolk-sac which appears
to be of considerable importance for exchange between mother and embryo.
The folic acid antagonist 9-methyl pteroylglutamic acid was chosen for study
because it is known to cause a variety of congenital defects in experimental
animals. Furthermore, this antagonist has previously been found to alter the
normal sequential appearance of electrophoretically mobile enzyme forms in
abnormally developing rat embryos (Johnson, 1965).
MATERIALS AND METHODS
Collection of tissues. Proestrus black-hooded rats, obtained from Research
Animals Inc., 80-120 days old and weighing between 180 and 240 g, were caged
overnight with black-hooded males. If sperm were found in a smear of vaginal
contents at 10.00 a.m. that moment was considered as day zero, i.e. the start
of the first day of gestation. The pregnant rats were maintained by a stock diet
and distilled water ad libitum which was supplemented twice weekly with lettuce.
For the first 7 days the diet was in the form of pellets, but on days 7-10 it was
powdered and fed in a diet jar which would be employed subsequently for
measurement of food intake because it reduced spillage and contamination with
excreta. This 3-day training resulted in normal food intake during days 10-13
when the diet was compounded of purified foodstuffs lacking pteroylglutamic
acid but containing 10 mg % of the potent antagonist 9-methyl pteroylglutamic
acid (9 m-equiv. PGA). Food intake ranged from 15 to 24 g/day and if any female
ate less than 15 g/day she was removed from the experiment. To reduce variability
in the time and rate at which a physiological deficiency was obtained, 1 mg of
9 m-equiv. PGA was administered in 1 ml of physiological saline solution by
stomach tube at the commencement of day 10. To terminate the deficiency the
rats were fed a synthetic diet fortified with a high level of the vitamin from days
13 through 15 and then were returned to the pelleted stock diet through day 20.
274
E. M. JOHNSON & R. SPINUZZI
Control rats were treated in a similar manner except that they received the
pelleted stock diet throughout gestation.
The PGA-deficient diet contained sucrose 63 %, alcohol-extracted casein
24 %, hydrogenated vegetable oil 8 %, salts no. 4 (Hegsted, Mills, Elvehjem &
Hart, 1941) 4 %, and PGA-deficient vitamin mixture 1 % to which was added
20 mg % 9-methyl pteroylglutamic acid. Per kilogram this diet contained 1-0 g
choline chloride, 400 mg inositol, 50 mg D-calcium pantothenate, 20 mg niacin,
10 mg j7-aminobenzoic acid, 10 mg riboflavin. 5 mg 2-methyl-l,4-naphthoquinone, 5 mg pyridoxine HC1, 5 mg thiamine HC1, and 300 fig D-biotin.
The synthetic fortified diet was identical to the PGA-deficient diet except that
9-methyl pteroylglutamic acid was omitted and 50 mg of crystalline pteroylglutamic acid was added per kilogram of diet.
Experimental and normal control rats were sacrificed on days 10, 11, 12, 13,
14, 16, 18 and 20 of pregnancy. The embryos were removed with the aid of a
dissecting microscope and the amnion and vitelline vessels, Reichert's membrane,
adherent decidua and chorioallantoic placenta were dissected free from the
visceral yolk-sac. The yolk-sacs were then pooled, homogenized in an equal
volume of triple-distilled water, stored at - 30 °C and subjected to zone electrophoresis in either starch or polyacrylamide gel within 21 days. No refrozen
homogenate was analysed for esterase as this resulted in altered patterns, probably indicating breakdown of larger proteins associated with the enzyme.
Utilization of distilled water rather than low molarity buffer, separating dehydrogenases in acrylamide and hydrolases in starch, permitted more enzyme systems
to be studied consistently and took cognizance of cautions such as were raised by
Markert (1963), Johnson (1964) and Vessell (1965). From six to twelve homogenates from different PGA-deficient pregnancies were electrophoretically
analysed for each day of gestation. Because the normal patterns of enzymic
differentiation were also unknown an equal number of homogenates from normal
pregnancies were similarly analysed. For histochemical localization of the
individual enzymes, yolk-sacs were also obtained intact and then either (1) fresh
frozen for cryostat sections or (2) vacuum embedded in low melting-point
paraffin.
Electrophoretic techniques. The equipment for separation in starch gel was
described previously (Johnson, 1964) and that for acrylamide gel is depicted in
Text-fig. 1. Separation was achieved at room temperature in 4 h with 6 V/cm
through the starch gel and in 20-30 min in acrylamide with 5 mA per gel.
Both the gel and tank buffers for esterase zymograms were 0 0 2 M tris/HCl,
pH 8-3, and staining was obtained with a-naphthyl acetate and Blue RR salt
(4', -amino-2', 5' dimethoxybenzanilide) buffered to pH 7-19 by 0-2 M tris/HCl.
For acid phosphatase the gel buffer was 0-02 M tris/HCl at pH 8-3 and the buffer
in the electrode tank was 0-03 Mborate (boric acid/sodium borate) at pH 8-4. Visualization of the bands of enzymic activity was by sodium a-naphthyl acid phosphate and the diazonium salt Blue RR in 0-02 M sodium acetate at pH 7-5, which
Enzymic differentiation of yolk-sac
275
12
A
B
C
Text-fig. 1. Equipment employed for electrophoretic separation in acrylamide gel.
A. For construction of the gel (A), a rubber stopper (1) from an injection bottle
was inverted and into this was placed a 6 cm long x 6 mm I.D. glass tube (2) which
had been capped with Saran Wrap (3). The tube was partially filled with hard gel (4)
which was overlain by the spacer gel (5) and capped by the gel bearing the sample
(6). The hard gel (4) was composed of 1 part 7-5 % acrylamide, 1 part accelerator,
1 part inhibitor and 1 part catalyst. The spacer gel was composed of 1 part 2 %
acrylamide, 1 part accelerator and 2 parts catalyst. The sample gel was composed of
2 parts 2-65 % acrylamide, 0-75 parts accelerator and 1 -5 parts catalyst. The buffer in
the electrode tanks was 0 0 8 M tris/glycine at pH 8-3.
B. For electrophoretic separation (B), the tube (2) containing the three gels was
inserted from below into a nylon sleeve (7) equipped with a rubber' O' ring (8) which
prevented buffer leaking from the upper buffer tank (9) into the lower tank (10).
Each buffer tank was equipped with a heavy-gauge platinum wire (11) and the entire apparatus was supported on rubber legs (12).
C. During staining (C), the hard gel (4) which had been removed from the glass tube
(2) by compressed air was placed in a 10 cm test tube (13) into which were placed
the appropriate substrate and co-factors (14) which were then incubated until the
bands of enzymic activity had developed (15). The reaction solution (14) was then
replaced by 5 % aqueous acetic acid and the tube was corked (16) for evaluation of
the banding, photographed and stored.
276
E. M. JOHNSON & R. SPINUZZI
changed to pH 6-1 when the sodium a-naphthyl acid phosphate and Blue RR
salt were added.
Visualization of malate and lactate dehydrogenase activity within the gels was
obtained within 30-60 min at 37 ° C with 0-2 M phosphate buffer (Na H2PO4/
Na2HPO4) at pH 7-4 in conjunction with 0-2 mg/ml phenezine methosulfate,
10 mg/ml diphosphopyridine nucleotide (DPN), 0-5 M hydrazine and 1 mg/ml
nitro-blue tetrazolium. The substrate for malate was 0-01 M malic acid and for
lactate 0-5 M sodium lactate.
Photographs of typically stained starch and acrylamide gels are shown in Plate 1,
figs. A and B. For simplification of analysis, a semiquantitative representation
of staining intensity and band width in the various zymograms (Hunter &
Markert, 1957) is depicted diagrammatically in Text-figs. 2, 3, 4 and 5.
Histochemical Techniques. For histochemistry of esterases, intact yolk-sacs
were taken at autopsy and immediately frozen with CO2 in triple-distilled water
for cryostat sectioning. These frozen tissues were stored at - 30 °C while wrapped
in Saran Wrap to avoid desiccation and were discarded after 3 weeks if not used.
Cryostat sections cut at 10 pi were stored at - 30 °C for no longer than 30 min
before they were fixed in cold neutral formalin for 1 min, rinsed in water and
placed into the substrate for esterase staining (Barka & Anderson, 1963). For
histochemical demonstration of acid phosphatases the fresh intact yolk-sacs were
fixed for 24 h in acetone at 4 °C, cleared for 15 min in benzene at 4 °C, infiltrated
and embedded in paraffin at 52 °C in vacuo within 45 min, sectioned at 10 /i,
deparaffinized in petroleum ether, hydrated in graded acetone and stained. Red
RC salt (diazotized 5-chloro-2-aminoanisole) and MgCl2 were employed for
24 h in 0-02 M sodium acetate buffer (sodium acetate/acetic acid) at pH 5-2 with
sodium alpha-naphthyl acid phosphate at 37 °C. The sections were counterstained with Mayer's haematoxylin and mounted in glycerin jelly.
For localization of lactate dehydrogenase (LDH) and malate dehydrogenase
(MDH) in tissue sections the yolk-sacs were prepared for sectioning as for
esterase but staining was obtained by the modified tetrazolium method of Barka
& Anderson (1963).
RESULTS
Teratogenic effects on the embryos. The timing and severity of the teratogenic
vitamin deficiency employed in this experiment were sufficient to cause approximately 10 % fetal death by day 21 and resulted in 95 % of the survivors having
club paws or shortened tails. In addition 80 % had ectopic kidneys or hyponephrosis (Johnson, manuscript in preparation).
Histological localization of enzymes. No differences were detected in the
localization of enzymic activity between yolk-sacs from normal control and
PGA-deficient mothers. There were observable differences, however, in both
the staining intensity and distribution of the enzyme reaction products on
successive days of gestation.
/. Embryol. exp. Morph., Vol. 16, Part 2
PLATE 1
+0
Fig. A. A typical starch gel of day 13 yolk-sac stained for esterase. The zones of enzymic
activity are designated in the same manner as in the zymogram in Text-fig. 2. (Actual size.)
Fig. B. A typical acrylamide gel of day 18 yolk-sac stained for MDH. The zones of enzymic
activity are designated in the same manner as in the zymogram in Text-fig. 4. x 3.
E. M. JOHNSON & R. SPINUZZI
facing p. 276
/. Embryol. exp. Morph., Vol. 16, Part 2
E. M. JOHNSON & R. SPINUZZI
PLATE 2
facing p. 277
Enzymic differentiation of yolk-sac
277
On day 10, staining for non-specific esterase was minimal and was located
almost entirely in the visceral yolk-sac endoderm though scattered particles were
present throughout the mesoderm. By day 14 the staining reaction was intense
(Plate 2, fig. C) and was distributed evenly within the epithelial cells. There was
moderate staining in the mesoderm which remained unchanged through day 20.
Acid phosphatase staining on day 10 was restricted to a weakly reactive zone of
cytoplasm distal to the characteristic apical vacuole (Everett, 1935) of the
epithelial cells (Plate 2, fig. D). By the 12th day the reaction was more intense
though still restricted to the apical cytoplasm of the epithelium. All cells did not
have equal levels of acid phosphatase, however, as strongly reactive cells sometimes had unreactive neighbours (Plate 2, fig. E). The level of enzymic activity
appeared to increase toward term but remained localized within the epithelium.
As at day 12, some cells continued to be stained less intensely than others
(Plate 2, fig. F). Granules of stained reaction-product for both malate and lactate
dehydrogenase were sparse at day 10 but were more numerous by day 13 and
remained essentially unchanged through term. At all stages of gestation the
reaction-product was located in the cytoplasm basal and lateral to the epithelial
cell nuclei. Moderate staining was observed in the yolk-sac mesoderm (Plate 2,
fig. G).
Electrophoretic analysis. Non-specific esterase. In addition to the positive
migration from the origin ( + 0) on days 13 through 16, there were nine distinct mobilities of enzymically active protein in the normal yolk-sac capable of
hydrolysing a-naphthyl acetate (Text-fig. 2). These bands of enzymic activity
PLATE 2
Fig. C. Fresh-frozen section of a day 14 yolk-sac stained with a-naphthyl acetate and Red RC
salt for esterase and counter-stained with Mayer's haemotoxylin. The staining reaction (SR)
is located primarily in the visceral endoderm (VE). Though photographically undetectable,
minute granules of the dye product were present in the visceral mesoderm (VM) but none
were seen in the embryo (is), x 250.
Fig. D. Fresh-frozen section of a day 10 yolk-sac stained with sodium a-naphthyl acid
phosphate and Red RC salt at pH 5-2 and counterstained with Mayer's haematoxylin. The
staining reaction was localized as a weakly reactive zone of the apical cytoplasm of the
endodermal cells. Note in Text-fig. 3 only origin staining was obtained by the electrophoretic
techniques. x450.
Fig. E. Section treated similarly to that in fig. D but from a day 12 yolk-sac. Note that some
cells had an intense deposit of the staining reaction (SR) but that other visceral endoderm
cells were relatively unstained (UC). x 350.
Fig. F. Section treated similarly to that in fig. E but from a day 16 yolk-sac. The staining
reaction was generally localized in the apical cytoplasm but relatively unstained cells (UC)
were also present, x 350.
Fig. G. A fresh-frozen but unfixed and non-counterstained section of a day 13 yolk-sac
stained for LDH. Distribution of the enzymic reaction was the same for both MDH and LDH
and was localized primarily in the visceral endoderm (SRE) but distinct granules were present
in the mesoderm (SRM) also, x 350.
11
12
13
14
X
16
18
Text-fig. 2. Diagrammatic representation of esterases from the visceral yolk-sac as seen in starch gels at days 10 through 20 of
gestation. The direction of migration was towards the top of the page. For each day studied, the zones of enzymic activity in normal
control yolk-sacs are placed on the left and the zones of activity from the PGA-deficient tissue are on the right, var = a zone of
enzymic activity not observed in every analysis, X = a zone of activity not present in the PGA-deficient tissue though it was
present in controls of the same gestational age.
10
+0
8
7
6
5
4
3A
3
3A
Enzymic differentiation of yolk-sac
279
were numbered in order, according to the distance migrated: that travelling the
greatest distance was designated as number 1 and the others were numbered in
series according to decreasing mobility. Seven forms were present on day 10 and
the greatest number was present one day later after the addition of no. 6 and a
trailing shelf (3 A) associated with one of the faster migrating proteins. Further
differentiation was evident on day 12 when either no. 7 was absent or perhaps
had become combined with no. 8 to form a larger zone which we have arbitrarily
called no. 8. The only further addition was the presence of a band near the anode
( + 0) on day 13 which persisted through day 16. The greatest staining intensity
for esterase was on days 14 and 16 after which the number of enzymic mobilities
as well as their intensities declined until term. No. 5 was absent on day 18 and
nos. 2 and 4 were not detected in all samples and invariably were absent on
day 20.
In yolk-sacs removed from pregnant rats treated with the folic acid antagonist
9 m-equiv. PGA from days 10 through 13, several alterations were produced
(Text-fig. 2) in the normal enzymic differentiation. Both the origin staining on
days 13 through 16 and the trailing shelf of no. 3 were absent on the basis of our
methods for detection. No. 6 was a day late in appearing and the combination
of the doublet 7-8 also was delayed by one day. Several bands were reduced in
width (e.g. no. 1 on day 16) and in staining intensity (e.g. no. 3 on day 14) for
various durations during development. Though band no. 5 was not detected on
day 18 in either experimental or normal yolk-sac, both 2 and 4 were still evident
by day 18 and no. 4 invariably was seen as late as day 20.
Acid phosphatase. A maximum of three forms of this enzyme of different
mobilities was present by day 16 in the normal yolk-sac (Text-fig. 3). In addition
to these, on day 10 a slight anodal migration ( + 0) was present which by day 11
had been joined by two mobile bands and by enzymic activity which had moved
negatively from the origin ( — 0). The third mobile form sometimes was detected
on day 14 and reached its maximum staining intensity by day 16 but was absent
2 days later. As can be seen in Text-fig. 3, no. 1 also reached its maximum staining intensity by day 16 after which it decreased somewhat in reactivity.
There was considerable variability among the enzymes as regards their
appearance in the PGA-deficient material. The negatively migrating originstaining normally appearing on day 11 was not detected until 24 h later whereas
mobility no. 3 was delayed approximately 48 h or until day 13, when it still was
not detected in all samples. Band no. 2 also was delayed in appearance until one
or two days later than normal and was detectable, even then, in only some of the
samples; however, we did not analyse yolk-sacs at days 17 or 19. In marked
contrast, howeyer, band no. 1 did appear on time and in apparently normal
concentration on day 11 in the experimental tissues as it did in the controls.
Malate dehydrogenase (MDH). Anodal origin staining and four migrating
forms of this enzyme were detected in polyacrylamide gel ( Text-fig. 4). Origin
staining and band no. 1 were present through the span of days studied. Both
l8
JEEM l6
10
Text-fig. 3. Diagrammatic representation of acid phosphatases from the visceral yolk-sac seen in starch gel.
(See legend for Text-fig. 2.)
to
oo
O
11
var
var
12
var
13
14
16
18
20
Text-fig. 4. Diagrammatic representation of malate dehydrogenases from the visceral yolk-sac seen in acrylamide gel. < = zone
of enzymic activity which will be detected in normal tissues at a later age but which has precociously appeared in PGAdeficient yolk-sacs. (See legend for Text-fig. 2.)
10
var
bo
oo
282
E. M. JOHNSON & R. SPINUZZI
nos. 2 and 3 appeared on day 11 and no. 2 persisted unaltered through day 20
while no. 3 was absent after day 13. A weakly reactive band present in varying
amounts (no. 4) was detected only on day 12.
By our methods we found two effects of the teratogenic treatment. The first
was the precocious disappearance of band 3 on day 13 just when this zone
attained maximum staining in the control. The second was the precocious
presence of minor band no. 4 on day 11 at which time it could not be detected
in the controls.
Lactate dehydrogenase {LDH). A maximum of six mobile forms of this
enzyme were detected during normal differentiation of the yolk-sac (Text-fig. 5).
By day 10 there were three major bands and one inconstantly detected band in
addition to the uniformly present forward migration from the origin ( + 0). Of
these, no. 3 persisted apparently unchanged to term whereas no. 2 was not
detectable by our method on day 11 though it was a rather intensely staining
band of variable width after day 12. Though isozyme no. 6 was the predominant
form present on day 10, it remained only as a faintly stained and inconstantly
detected band for the next 3 days after which it was not observed. Band no. 5
which was not always detected on day 10 increased in activity from day 11 through
13 and then remained unchanged through term. On day 12 the isozyme repertory
had increased to five by the addition of no. 4 which then did not change. The last
locale of enzymic activity (no. 1) appeared on days 14 and 16 as an inconstantly
present and rather weakly staining band.
Several changes were produced in this normal biochemical differentiation as a
result of the transitory maternal dietary deficiency of folic acid. LDH isozyme
3 was the only form apparently unaltered in yolk-sacs from PGA-deficient
mothers. The least altered was no. 1, which would normally have been encountered on day 16 as a variable minor band but which was absent from the
experimental material. In marked contrast was isozyme no. 6, which was a constantly major form on day 11 through 13 in the PGA-deficient yolk-sac but was
not detected in all normal control yolk-sacs of this age. Both nos. 2 and 4 were
altered more markedly in that no. 4 frequently appeared a day early and no. 2
was present on day 11 when it was transiently absent from the normals. Similarly
on day 14 both these bands showed changes in that no. 4 could not be resolved
and no. 2 was not detected in all homogenates of treated yolk-sac.
DISCUSSION
Normal enzymic differentiation. This investigation demonstrates that the yolksac placenta of the rat normally undergoes biochemical differentiation with
respect to several enzyme families. The maximum staining intensity by individual mobilities of acid phosphatase and non-specific esterase was present on
days 14 and 16. Though the maximum number of mobile esterases was present
on day 11 most of these bands were not strongly reactive. Biochemical involution
10
12
13
14
var
var
16
x
18
Text-fig. 5. Diagrammatic representation of lactate dehydrogenases from the visceral yolk-sac seen in acrylamide gel.
(See legends for Text-figs. 2, 4.)
11
.var
20
00
284
E. M. JOHNSON & R. SPINUZZI
was indicated for both of these families of enzymes at days 18 and 20. These
results are in agreement with those reported by Padykula (1958), who noted
a decline in histochemical reactivity by alkaline phosphatase, adenosine triphosphatase, succinic dehydrogenase and non-specific esterase toward term.
Electrophoretic analysis, in addition to confirming the rise in enzymic activity
during days 14-16 which was followed by a decline toward term, also demonstrated that individual esterases did not necessarily conform to this trend, as
esterase band no. 2 had its major reactivity on day 10 and no. 3 A was present
only on day 11. Similarly, two slow-moving bands (nos. 7 and 8) were present on
days 10 and 11 from normal yolk-sacs but these either had been combined into
one or were replaced by a new form migrating at the same speed on day 12. At
days 14 and 16 it might appear that nos. 7 and 8 could be due to diffusion from
areas of intense enzymic activity which gave the appearance of one band. Such
extensive diffusion is unlikely, however, because on day 12 only one band was
detected at this locale and its staining intensity was the same as that of the
doublet 7 and 8 on the preceding day.
Normal differentiation of acid phosphatase in the yolk-sac involved a repertory of three mobile forms invariably present by day 16 when the maximum
total staining was present. Biochemical evaluation of acid phosphatase activity
(Padykula, 1958) demonstrated no decline toward term though such a decline
appeared to be the case for two of the electrophoretic forms of the same enzyme
after day 16. No. 1 became less reactive, and though no. 3 appeared unchanged
no. 2 was lost. Evidently the loss of no. 2 and the decline of no. 1 was more than
compensated for by the increased origin staining ( + 0) on day 20. Thus, though
Padykula showed some increase in acid phosphatase staining through term, it
appears that this was achieved by the activity which remained at the origin and
in spite of both qualitative and quantitative decline in the electrophoretically
mobile repertory.
Though both esterase and acid phosphatase attained maximum staining at the
beginning of the third week both malate and lactate dehydrogenase activities
appeared to be greatest toward the end of the second week. MDH was quite consistent in the staining of two bands and at the origin but isozymes no. 3 and 4
were present only transiently. That both these forms were not present in all
homogenates of normal yolk-sac on days 11 and 12 probably was due to a level
of enzyme activity just at the maximum sensibility of the method. By day 13,
no. 3 appeared consistently and was then apparently well within the range of
sensitivity of the method, but by day 14 it was no longer detectable. Such early
differentiation and involution of an isozyme also was seen with LDH wherein
no. 6 was a consistent band only through day 13 and another minor band (no. 1)
was detected only on days 14 and 16 at a concentration apparently at the limit
of the method's detecting ability.
Abnormal enzymic differentiation. Though some enzymic forms appeared on
time and in apparently normal concentration in yolk-sacs from pregnant rats
Enzymic differentiation of yolk-sac
285
treated with a folic acid antagonist, several different classes of effects were produced in the differentiation of others. The least prominent of these changes was
reduced width of an enzyme band such as for esterases nos. 1 and 3 on day 16 or
LDH no. 2 on day 18. Such reduced width of the stained zone sometimes was
associated with reduced staining intensity such as esterase no. 3 on day 14 or
LDH no. 4 on day 12. A more obvious effect was delay in the normal appearance
of a form at a particular day. Such delay ranged from a 24 h period for the
negative migrating origin staining by acid phosphatase on day 12 to a 48-72 h
delay in appearance of acid phosphatase 3 on days 13 or 14. This type of effect
reached its maximum expression in the esterase origin staining normally
present on days 13, 14 and 16, which was not detected at any age in the experimental tissues though it may have been present on days 15 or 17 or at very low
concentration. Delay in disappearance of an enzymic form, or persistence of a
form beyond its normal time for involution, was observed both for esterases
2 and 4 and for acid phosphatase 2. A similar though quantitative persistence
was observed for LDH no. 6, which had the same level of activity as controls
on day 10 but was markedly greater in the experimental tissues on days 11,
12 and 13. Precocious absence of LDH no. 1 on day 16 and MDH no. 3
on day 13 was in contrast with the precocious presence of LDH no. 4 on
day 11.
General. The results of this electrophoretic evaluation of enzymic differentiation of the yolk-sac placenta have fundamental implications for several concepts.
With respect to electrophoretically mobile enzymes, this structure is a constantly
differentiating organ throughout gestation. Its physiologic characteristics at one
day must not be considered as indicative of those at another until demonstration of similarities actually have been shown. Such changing physiologic
properties have been demonstrated by Buxton (1952), who reported that antibodies were capable of crossing the yolk-sac epithelium most readily on day 17 in
the chick and by Halliday (1955) who reported an apparent increase in similar
transport from days 17 through 20 in the rat. Brambell, Hemmings & Rowlands (1948) found that in rabbits the blastocyst was permeable to both
homologous and heterologous proteins which Ferm (1956) suggested was a
result of histiotrophic nutrition characteristic of this stage. Anderson (1959)
termed the yolk-sac wall of the early blastocyst a 'leaky' membrane temporarily
freely permeable. Such a 'leaky' membrane concept is a useful one to convey
the idea of a state of differentiation at least with respect to antibody transport.
Passage of such large molecules is perhaps not directly due to the enzyme
repertory of the blastocyst, and similarly the apparent increase in permeability
of the yolk-sac late in gestation may not be due to the enzymes characteristic of
late gestation. However, it is more likely that increased permeability is the
result of altered morphology of the yolk-sac epithelium wherein spaces have
been demonstrated in the rat to form between adjacent cells towards term (Padykula & Wilson, 1960).
286
E. M. JOHNSON & R. SPINUZZI
Enzymic permutation of the yolk-sac as demonstrated in the current report
may, however, have effects on its permeability to smaller molecules of paramount
importance to the very labile stages of early embryonic development during which
the yolk-sac appears to be the most obvious site for exchange. Our current knowledge of placental transport is derived primarily from higher forms with a
rather inaccessible chorioallantoic placenta and presents data regarding exchange
in post-embryonic stages of development. Though considerable information has
been obtained from such material (Page, 1960), the yolk-sac is a more accessible
structure that can be handled in vitro for study of translocation of labelled
molecules which should give data concerning substances available to the early
embryo. How the abnormal enzymic repertory produced by a teratogenic
folic acid deficiency may affect transport and embryonic development for the
present remains a matter of conjecture but warrants investigation.
It is interesting to note that the adverse effects of folic acid deficiency on the
differentiation of phosphatase from the yolk-sac were quite different from those
observed in the embryo (Johnson, 1965). However, the study of abnormally
developing embryos involved a more severe teratogenic procedure in that
the deficient period was from days 8-10 which, though it resulted in
severe malformation of all embryos, also caused eventual death of the
embryos.
Abnormal organs and organ systems of the embryos and fetuses associated
with the yolk-sac analysed in the current report gave patterns of enzymic effects
dissimilar to those of the yolk-sac (Johnson, manuscript in preparation). This
may be an indication that the cellular environment in which the genome is
functioning has a role in influencing those portions of the DNA complement
susceptible to suppression of expression by this teratogen. It is not meant to be
implied that the results present in this communication necessarily are due to
direct action of the teratogen on the genome. On the contrary, an extranuclear effect may be indicated by the presence of LDH isozyme 4 in experimental
tissues prior to its normal time of appearance, which may be the result of a random association of enzyme monomers.
SUMMARY
1. Electrophoretic and biochemical techniques were applied to homogenates
of the rat's visceral yolk-sac.
2. Non-specific esterase, acid phosphatase, malate and lactate dehydrogenases
were shown, by electrophoretic techniques, to undergo sequential enzymic
differentiation from day 10 through day 20 of gestation. The enzyme repertory
on a particular day was achieved through both additions to and deletions from
the repertory of the preceding day. The greatest staining intensity by esterase and
acid phosphatase was late in gestation but for both malate and lactate dehydrogenase it was most intense during days 11-13.
Enzymic differentiation of yolk-sac
287
3. A teratogenic folic acid deficiency resulted in delayed appearance of
several enzyme forms but also resulted in some forms persisting beyond their
normal time for disappearance. The effects of the vitamin deficiency were quite
selective in that some enzyme forms appeared on time and in apparently normal
concentration in the PGA-deficient material.
RESUME
La differentiation des enzymes du sac vitellin de rat
sous Vinfluence d'un agent teratogene
1. Des techniques electrophoretiques et biochimiques ont ete appliquees a
des homogenats du sac vitellin visceral de rat.
2. II a ete possible de demontrer, par des techniques electrophoretiques, qu'il
existe entre le lOeme et le 20eme jour de gestation, une sequence dans la
differentiation d'une esterase non-specifique, de l'acide phosphatase, des malate
et lactate deshydrogenases. Chaque jour, le' repertoire' enzymatique se complete
par des additions et des deletions. Pour l'esterase et la phosphatase acide, la
coloration la plus intense apparait tard dans la gestation, alors que pour les
malate et lactate deshydrogenases, la coloration est particulierement intense
pendant la periode du lleme au 13eme jour.
3. Une carence teratogene en acide folique ralentit l'apparition de plusieurs
types d'enzymes, tandis que d'autres types enzymatiques persistent apres
l'epoque normale de leur disparition. Les effets de cette carence vitaminique se
sont reveles assez selectifs, puisque meme dans le materiel 'PGA'-deficient,
certains enzymes apparaissent au bon moment et a la concentration normale.
The authors wish to express their thanks to Mmes Hollis Hall and Rebecca Schwartz for
their technical assistance. We are also indebted to Professors James G. Wilson and Donald
C. Goodman for their constructive criticisms of the manuscript. This research was supported
by grant HD00109 from the National Institutes of Health, United States Public Health
Service.
REFERENCES
J. W. (1959). The placental barrier to gamma-globulins in the rat. Am. J. Anat.
104, 403-29.
BARKA, T. & ANDERSON, P. J. (1963). Histochemistry Theory, Practice and Bibliography.
New York: Hoeber Medical Division, Harper and Row, Publishers, Inc.
BRAMBELL, F. W. R. (1958). The passive immunity of the young mammal. Biol. Rev. 33,
488-531.
BRAMBELL, F. W. R., HEMMTNGS, W. A., & ROWLANDS, W. T. (1948). The passage of antibodies from the maternal circulation into the embryo in rabbits. Proc. R. Soc. Lond. B
135, 390-403.
BRUNSCHWIG, A. E. (1927). Notes on experiments in placental permeability. Anat. Rec. 34,
237-44.
BUXTON, A. (1952). On the transference of bacterial antibodies from the hen to the chick.
/. Gen. Microbiol. 7, 268-86.
DEMPSEY, E. W. (1953). Electron microscopy of the visceral yolk-sac epithelium of the
guinea pig. Am. J. Anat. 93. 331-63.
ANDERSON,
288
E. M. JOHNSON & R . S P I N U Z Z I
J. W. (1935). Morphological and physiological studies of the placenta in the albino
rat. /. exp. Zool. 70, 243-85.
FERM, V. H. (1956). Permeability of the rabbit blastocyst to trypan blue. Anat. Rec. 125,
745-59.
HALLEDAY, R. (1955). Prenatal and postnatal transmission of passive immunity to young rats.
Proc. R. Soc. Lond. 144, 427-30.
HEGSTED, D. M., MILLS, R. C , ELVEHJEM, C. A. & HART, E. B. (1941). Choline in the nutrition of chicks. J. biol. Chem. 138, 459-66.
HUNTER, R. L. & MARKERT, C. L. (1957). Histochemical demonstration of enzymes separated
by zone electrophoresis in starch gels. Science, N.Y. 125, 1294-5.
JOHNSON, E. M. (1964). Quantification of enzyme activity subsequent to zone electrophoresis
in starch gel. Anal. Biochem. 8, 210-16.
JOHNSON, E. M. (1965). Electrophoretic analysis of abnormal development. Proc. Soc. exp.
Biol. Med. 118, 9-11.
MARKERT, C. L. (1963). Lactate dehydrogenase isozymes: dissociation and recombination of
subunits. Science, N.Y. 140, 1329-30.
NOER, H. R. & MOSSMAN, H. W. (1947). Surgical investigation of the function of the inverted
yolk sac placenta in the rat. Anat. Rec. 98, 31-7.
PADYKULA, H. A. (1958). A histochemical and quantatitive study of enzymes of the rat's
placenta. /. Anat. 92, 118-29.
PADYKULA, H. A. & RICHARDSON, D. (1963). A correlated histochemical and biochemical
study of glycogen storage in the rat placenta. Am. J. Anat. 112, 215-41.
PADYKULA, H. A. & WILSON, T. H. (1960). Differentiation of absorptive capacity in the
visceral yolk sac of the rat. Anat. Rec. 136, 254 (abstr.).
PAGE, E. W. (1960). Physiology and biochemistry of the placenta. In J. P. Greenhill, Obstetrics,
chapter 3. Philadelphia: W. B. Saunders Co.
VESELL, E. S. (1965). Lactate dehydrogenase isozyme patterns of human platelets and bovine
lens fibres. Science, N.Y. 150, 1735-7.
WISLOCKI, G. B. & PADYKULA, H. A. (1953). Reichert's membrane and the yolk sac of the rat
investigated by histochemical means. Am. J. Anat. 92, 117-51.
WISLOCKI, G. B. & DEMPSEY, E. W. (1955). Electron microscopy of the placenta of the rat.
Anat. Rec. 123, 33-63.
EVERETT,
(Manuscript received 8 March 1966)