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Development of structure and function in the mammalian yolk sac
I)EVELOPI\fENTAL
BIOLOGY
Development
II. Vitamin JULIUS
13, 349-369
of Structure and Function Mammalian Yolk Sac BE Uptake
J. DEREN,?, R HELEN
Depurtment Department
of Physiology, of Biological
( 1966)
by Rabbit
A. PADYKULA,
Haroard Sciences, Accepted
AND
Yolk
in the Sad
T. HASTINGS
WILSON~
Medical School, Boston, IMassachusetts, and Wellesley College, Wellesley, Massuchusetts December
13, 1965
INTRODUCTION
In the preceding paper (Padykula et al., 1966) it was shown that the visceral yolk sac of the rat incubated in vitro with radioactive vitamin B,, was capable of accumulating this large molecule. The uptake was stimulated two- to fivefold by rat gastric intrinsic factor (IF), the mucoprotein essential for B,, absorption by the small intestine. B,, uptake per gram tissue, both in the presence and absence of IF, decreased with increasing age of the fetus, being highest at 13 days of gestation, the earliest stage tested, and lowest at term. Membrane invagination and vacuole formation which are characteristic of cellular uptake of macromolecules were evident in the endodermal cells of the yolk sac. The demonstration by Brambell and his collaborators (Brambell et al., 1948, 1949, 1951b; Brambell, 1958) that the rabbit yolk sac is the major route for the transfer of antibodies from mother to fetus suggested to us the possibility that the transfer of vitamin B,, is mediated via a similar route. The visceral yolk sac accumulated considerable vitamin B,, in the absence of added IF by an energydependent mechanism. Intrinsic factor enhanced uptake with a more ‘Supported in part by research grants from the U.S. Public Health Service (AM-05736 and HD-01026). ‘Postdoctoral Fellow of the U.S. Public Health Service (5-FZ-GM-15, 144). ” Present address: Department of Medicine, Maimonides Hospital, Brooklyn, New York. ’ Supported by a Research Career Development Award of the U.S. Public Health Service ( 5-K3-GM-15, 306). 349
350
DEREN,
PADYKULA,
AND
WILSON
pronounced effect as term approached. In viva experiments were performed in an attempt to evaluate the physiological role of the yolk sac in the transfer of vitamin B,, from mother to fetus. MATERIALS
AND METHODS
Pregnant white New Zealand rabbits and Sprague-Dawley rats were obtained from a breeding farm which carefully recorded the time of coitus and segregated the females. Guinea pigs were obtained from a commercial source which did not segregate the sexes. The approximate length of gestation in the guinea pig was estimated from the number and weight of the fetuses, according to the tables prepared by Draper (1920). The gestation period for the rabbit is approximately 32 days; the guinea pig 65 days; and the rat 22-23 days. Cytological
Techniques
Yolk sacs were isolated at 16 and 28 days, rinsed briefly in isotonic saline, and fixed in a buffered osmium tetroxide solution (Palade, 1952). After standard embedding in Epon, plastic sections were cut at l-2 p of a Porter-Blum ultramicrotome, stained with toluidine blue, and examined with the light microscope, Ultrathin plastic sections were stained with uranyl acetate and Karnovsky’s lead stain (1961). They were examined with a Siemens Elmiskop I. In Vitro Experiments Pregnant rabbits, guinea pigs, or rats were stunned by a blow on the head and exsanguinated by cutting the carotid vessels. The visceral portion of the yolk sac was carefully excised from the chorion and placed into cold saline until the beginning of the incubation. Yolk sacs were incubated intact, in halves, or quarters depending upon the size of the tissue and the experimental protocol. Incubation was carried out in a 25-ml Erlenmeyer flask in an oscillating water bath at 37°C. Each flask contained 2 ml of bicarbonate-saline (Krebs and In some flasks 0.1 ml of the Henseleit, 1932) with 1 mpg Co”‘B,,/ml. appropriate IF preparation was added. At the end of an hour the tissue was removed, rinsed once in cold saline, blotted, weighed, and counted in a well-type scintillation counter. In Vivo Experiments 1. Parenteral injection injected subcutaneously
of pregnant animals. Pregnant animals were with Co57B12 and sacrificed after different
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
351
time intervals. The radioactivity in the maternal and fetal organs as well as the placental membranes was determined. 2. Intrauterine injection. Pregnant rabbits 25-28 days were premedicated with atropine and anesthetized with Nembutal. A midline lower abdominal incision was made, then a ligature was placed around the junction of one uterine horn with the cervix without interfering with the vascular supply. A small incision was then made in the tubal end of the uterus, and a thin polyethylene catheter attached to a syringe was introduced and advanced to the opposite end of the uterine horn. One milliliter of CO”~B~~ (containing 1.04 mpg Co”‘B,,/ ml) per conceptus was injected into the uterine cavity during withdrawal of the polyethylene catheter. This procedure ensured exposure of each yolk sac to the radioactive vitamin. The perforation in the uterus was ligated, and the abdomen was closed. Twenty-four or 48 hours after the intrauterine injection the rabbits were killed by a blow on the head, and the radioactivity in the maternal and fetal organs and placental membranes was determined. 3. Ligation of vitelline vessels. Pregnant rabbits (25-28 days) were premeditated with atropine and anesthetized with Nembutal. The abdomen was opened, and a small incision was made through the uterine wall overlying the main vitelline vessels, according to the method of Brambell et al. (1949). Slight pressure was exerted on the conceptus, forcing the visceral yolk sac to protrude through the uterine incision. A ligature was placed over the protruding yolk sac, which included the vitelline stalk. The tissue was then pressed back into the uterine cavity, and the uterine wall was sutured. The abdominal incision was repaired, and the rabbit was injected subcutaneously with Co”‘B,,. Twenty-four hours later the rabbit was killed, and the radioactivity in the appropriate organs was determined. Materials CO”‘B~~ was purchased from Merck and Company. Hog intrinsic factor was kindly provided by Dr. C. W. Pettinga, Eli Lilly Company (lots Nos. 258-458-B-241 and 258-417-B-98-5). It was diluted in saline to contain 1 mg/ml, and 0.1 ml was added to the appropriate flask. The rat, rabbit, and hamster IF were prepared by homogenizing the glandular mucosa in ten times its weight of saline and centrifuging at 1000 g for 10 minutes. A 0.1 ml sample of the supernatant was added to the appropriate flask. 2,4-Dinitrophenol was purchased from Mann
352
DEREN,
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AND
WILSON
FIG. 1. Visceral yolk sac, rabbit, 16 days’ gestation. The absorptive epithelium (AE) stains darkly and is thrown into folds around a central core of mesenchyme carrying the vitelline vessels (VU). The vitelline vessels immediately below the absorptive epithelium are thin-walled endothelial tubes that have large are evident within the vitelline vessels. lumens. Various stages of erythropoiesis The exocoelom is lined by a flattened mesothelium (m). The visceral basement membrane is inconspicuous at this time. The absorptive cells are further magnified in Fig. 3. 2 p plastic section; toluidine blue. Magnification: X 300.
DEVELOPMENT
Research Laboratories Organic Chemical.
OF
THE
MAMMALIAN
and sodium iodoacetate
YOLK
SAC
353
was from Eastman
RESULTS
Morphology Light microscopy. The rabbit, like the rat, has two placentas, a discoidal labyrinthine chorioallantois and an inverted yolk sac. The chorioallantoic placenta is fully developed on about the 12th day of gestation, and the yolk sac placenta is established even earlier (Amoroso, 1952). The parietal wall of the yolk sac (bilaminar omphalopleure) has broken down by the 15th day, and thereafter the visceral wall (visceral splanchnopleure) is directly exposed to the uterine contents. All our studies were performed after the loss of the parietal wall. It is important to point out one major difference between placentation in the rabbit and rat. In the rabbit, a wide annular chorion extends from the margin of the placental disc and connects with the yolk sac membrane (Mossman, 1937; Brambell et al., 195Ib). This chorionic margin is for the most part nonvascular, but its outer, trophoblastic surface is freely exposed to the uterine contents. In the present experiments, the chorion was dissected away from the yolk sac. At 16 days, the visceral yolk sac of the rabbit resembled that of the rat at 13 days (Figs. 1 and 3). The absorptive epithelium was composed of tall columnar cells that rested on a highly vascular mesenchymal layer in which hematopoiesis was evident. A mesothelium lined the exocoelomic surface. The free surface of the absorptive endodermal cells was modified into a brush border. The apica cytoplasm of the absorptive cells was packed with droplets of various sizes, many of which stained heavily with basic dye (Fig. 3). Large empty vacuoles were seen in the infranuclear cytoplasm; these most likely represented sites of stored lipid which was dissolved out by the preparative procedures. FIG. 2. Visceral yolk sac, rabbit, 28 days’ gestation. The three tissue layers of this membrane are evident here. The thick, darkly stained absorptive epithelium (AE) rests upon the loose mesenchymal layer containing vitelline vessels (vu). The mesothelium (m) demarcates the exocoelomic surface of the membrane. The absorptive cells are further enlarged in Fig. 4. 2 p plastic section; toluidine blue. Magnification: X 300.
354
DEREN,
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AND
WILSON
FIG. 3. Absorptive epithelium, rabbit yolk sac, 16 days’ gestation. free surface of the closely apposed absorptive cells, the brush border evident. The supranuclear cytoplasm contains numerous droplets and that stain lightly or heavily with basic dye. The oval nuclei possess two nucleoli. The lumen of the vitelline vessel ( UV) is in close proximity absorptive epithelium. 2 p plastic section; toluidine blue. Magnification:
At the is barely vacuoles or more to the x 1300.
DEVELOPMENT
OF
THE
MAMMALIAX
YOLK
SAC
355
of the rabbit yolk sac At 28 days, the absorptive epithelium resembled that of the rat near term quite closely, except that the epithelial ridges were barely penetrated by mesenchyme and blood vessels (Fig. 2). At this time there was no sign of hematopoiesis. A brush border capped the free surface of the endodermal cells. The supranuclear cytoplasm was highly vacuolated, although fewer basophilie droplets were evident. Large intercellular spaces were now present between the absorptive cells (Fig. 4). The visceral basement membrane was considerably thicker than that present at 16 days. The appearance of intercellular spaces and the thickening of the visceral basement membrane also occurred during the development of the rat yolk sac (Padykula et al., 1966). Electron microscopy. The free surface of the absorptive cells resembled that of the rat yolk sac closely (Figs. 5 and 6). At 16 days and 28 days, the microvilli were well developed, and invaginations of the plasma membrane occurred at the bases of the microvilli (Fig. 5). A thick-walled canalicular system filled the cytoplasm immediately below the microvillous border. The invaginations and canaliculi were lined by a filamentous coat that may represent a special surface for protein attachment. In close proximity to the canaliculi, absorptive vacuoles occurred which contained a loose precipitate. As in the rat may placenta, the microvilli, invaginations, canaliculi, and vacuoles represent an intracellular pathway for transporting macromolecules (Padykula et al., 1966). At 16 days, the lateral cell membranes of the absorptive cells were closely and uniformly apposed (Fig. 5), whereas near term, large intercelluIar spaces appeared intermittently between the lateral ceII membranes (Fig. 6). These spaces contained a delicate precipitate, and this suggested the possibility of a pathway for intercellular transport. Near term, a similar developmental change in the lateral associations of absorptive cells occurred in the rat yolk sac (Padykula et al., 1966). FIG. 4. Absorptive epithelium, rabbit yolk sac, 28 days’ gestation. The brush border at the free surface of the absorptive cells is indistinct. The supranuclear cytoplasm is fiIled primarily with pale-staining droplets and vacuoles. The nuclei are somewhat more basal in position and more irregular in form than at 16: days. Intercellular spaces have appeared between absorptive cells. A conspicuous difference between this and the earlier stage is the thickening of the visceral basement membrane (vm) which intervenes between the absorptive cells and the vitelline vessels. 2 JJ plastic section; toluidine blue. Magnification: x 1300.
356
DEREN,
PADYKULA,
AND
WILSON
In Vitro Studies When rabbit yolk sac segments were incubated in the presence of CoS7-labeled vitamin B,,, there was an accumulation of the isotope with little variation from day 15 to term (Fig. 7). This level of uptake of B,, by rabbit yolk sacs was greater than that observed with the distal intestine from the same litter in the absence of added intrinsic factor (Table 1). When hog IF was added to the incubation TABLE 1 Blz UPTAKE BY FETAL RABBIT ILEUM AND YOLK SAC OF A SIMILAR AGES COS~BIZ accumulated Sample
27-31 day yolk sacs 27-31 day distal half of intestine
a,
(m&l00
alone
0.15 f 0.01” (19) 0.06 f 0.01 (15)
mg tissue
wet weight) ~__ HIS + hog IT’
0.93 * O.l@ (19) 0.32 + 0.03c (15)
a Pregnant rabbits were sacrificed, and t,he visceral yolk sac and the distal half of the fetal intestine were removed. The yolk sacs were divided in half and incubated for 1 hour at 37°C in a shaking water bath in 2 ml bicarbonate-saline containing 1 mpg Co57B12 per milliliter, 0.1 of hog IF (Eli Lilly, 1 me/ml) was added to one of the flasks. The distal intestine was cut into segments and incubated as above. Result,s are expressed as mean values h SE. The number of experiments is given in parentheses. b P < 0.01 when compared to Ble upt)ake by intestine. c P < 0.01 when compared to uptake in the absence of IF.
medium, a twofold stimulation was observed at day 15 with a more pronounced effect as term approached. As seen from Table 2, rabbit and rat IF, in addition to hog IF, enhanced uptake in both rabbit yolk sacs and fetal intestine, whereas hamster IF failed to stimulate uptake. Rat IF stimulated B 12 uptake by both rat yolk sacs and adult rat intestine, whereas rabbit, hog, and hamster IF were ineffective. Thus the rabbit and rat yolk sac displayed the same species specificity FIG. 5. Absorptive cell, visceral yolk sac, rabbit, 16 days’ gestation. The microvillous border (mu) is cjuite irregular in form. Invaginations of the apical The most superficial cytoplasm is filled plasma membrane are evident ( arrow). with various profiles through the canaliculi system (c). The single large absorptive vacuole (AV) contains a fine, loose precipitate. The invaginations, canaliculi, and vacuoles have dense walls which are composed of a smooth limiting membrane and a thick inner filamentous lining. The lateral cell surfaces are closely apposed (arrows). D, desmosome; M, mitochondria. Electron micrograph. Magnification: x 21,300.
DEVELOPfitEST
OF
THE
MAhfhfAI,IAN
YOLK
SAC
357
358
DEREN,
PADYKULA,
AND
TABLE SPECIES
WILSON
2
SPECIFICITY OF INTRINSIC FACTOR STIMULATION YOLK SAC AND INTESTINE PHOM THE RAT Ratio
SalIlplZ2
Rabbit yolk sac Rabbit fetal ileum Rat yolk sac Adult rat gut?
Rabbit
IP
4.9 (4)
18
(2)
1.1 (3) 0.9
BD uptake Bn uptake Rat
IF
2.5 (3) 14 (2) 9.9 (2) 10
OF BU UPTAKE AND RABBITS
in the presence in the absence Hog
IF
6.0 (19) 5.5 (15) 0.88 (3) 1.0
IN THE
of IF of IF
Hamster
IF
1.0 (3) 0.95 (3) 0.41 (2, 0.50
4 Incubations were carried out in a shaking water bath for 1 hour at 37°C in a 25 ml flask containing 2 ml bicarbonate-saline with 1 mpg Co6’B12 per milliliter. Hog IF, kindly provided by Eli Lilly and Company, was diluted to contain 0.1-1.0 mg/ml. Rat, rabbit, and hamster IF were prepared by homogenizing the gastric glandular mucosa in ten times its weight of saline and centrifuging at 1000 g for 10 minutes. One-tenth milliliter of the diluted hog IF or the supernatant was added to the appropriate flask. The number of experiments is given in parentheses. * Data taken from Wilson and Strauss (ISFig).
for IF as the fetal and adult ileum of the corresponding species (Wilson and Strauss, 1959). To determine whether the uptake of B,, by rabbit yolk sacs in the absence of added IF represented an active process of accumulation or nonspecific tissue binding, uptake was measured in the presence of various inhibitors. As seen from Table 3, the addition of 1 mM 2,4-dinitrophenol (DNP) to the incubation medium significantly lowered the B,, uptake. Incubation in the presence of 10 mM glucose, completely reversed the DNP inhibition. The IF-stimulated B,, uptake was similarly inhibited by 1 mM DNP, and the inhibition was abolished by 10 mM glucose. The IF-stimulated B,, was also inFIG. 6. Absorptive cell, visceral yolk sac, rabbit, 28 days’ gestation. The free surface of the absorptive cell near term resembles that of the earlier stage (Fig. 6) in the presence of a microvillus (mu), canaliculi (c), and absorptive however, in the associations of the vacuoles (AV). There is a difference, lateral cell surfaces. Intermittent enlargements of the intercellular space (IC) have appeared, and they contain some precipitate. At intervals, the lateral plasma membranes are held in close association by desmosomes (D). Also, near term, cytoplasmic fibrils (f) appear to be more numerous. ER, cisternae of the M, mitochondria; G, Golgi membranes. Elecgranular endoplasmic reticulum; tron micrograph. Magnification: X 23,000.
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
359
360
DEREN,
EFFECT OF INHIBITORS ON
PADYKULA,
B1?
AND
TABLE
3
TJPTAKE
BY
WILSON
RABBIT
YOLK
SAC
in
Vitw
Number of yolk sacs
~~ ~~~ I3
0.17 * 0.02
.~____
0.068 + 0.006 I’ < O.OOlb
En + IF
Biz + IF
i
DNP
0.14 + 0.07 P > 0.3” P < O.OlC Bn + IF
0.073 * 0.002 P < O.OOlb
74
HI? + IF
Bn
+ IF
+ iodoacetate
+ DTiP
+ glucose
0.47 * 0.1 P > 0.6b P < 0,005’ Bn
+ IF
+ iodoacetate
+
glWXl3
7
0.53 k 0.089
0.1 f 0.01 P < 0.005”
0.071 * 0.017 1’ < 0.01b 1’ > 0.025~
n Pregnant rabbits (28-30 days) were sacrificed by a blow on the head, and the uterus and contained concepti were removed. The visceral yolk sacs were carefully excised from the chorion and divided into four quarters. Two quarters were used in measuring the baseline Biz uptake, and the other quarters were incubat,ed with inhibitor or inhibitor plus glucose. Incubations were carried out in a 25-ml flask with 2 ml bicarbonate-saline containing 1 rnlg CoSrBiz/ml. Dinitrophenol (DXP) and iodoacetate were added to a final concent,rat,ion in the incubation medium of 1 mM and glucose to a final concentration of 10 mM. Hog 1F (0.1 ml of 1 mg/ml, Eli Lilly Co.) was added to the appropriate flasks. Results are presented as mpg CO~~B&OO mg tissue wet weight and are expressed as mean values + SE. * Level of significance as compared to uptake in t’he absence of inhibitor. c Level of significance as compared to uptake in the presence of inhibitor.
hibited by 1 mM iodoacetate, an effect not reversed by glucose. In other experiments these inhibitory effects were observed at iodoacetate concentrations of 0.1 mM. In Vivo Studies In order to investigate the role of the yolk sac in the transfer of B,, from mother to fetus, a series of in viva experiments were performed. First, an attempt was made to determine the rate of B,, uptake by the yolk sac, as compared with the other fetal organs, following injection of radioactive B,, into the maternal circulation, A series of rabbits near term (25th~28th day of gestation) were in-
DE\‘ELOPMENT
Ok’ THE
I.40 . 1 Y
;
YOLK
WM
l
&
(lcuX
a/one
T (11
x with B,g f Hog /f
1.20
361
SAC
(3)
UPrAKE ,’
I
<
MAMMALIAN
f 22 1.00 i= I
1 (9) :
3 0.60 L t
(2)
(4)
1 1
x
IIO,T
15
20 FETAL
25
-
AGE (DAYS)
FIG. 7. Developmental differences in vitamin B1? and Blz-IF uptake by the rabbit yolk sac measured in vitro. Yolk sacs were carefully excised from the surrounding chorion. Incubations were carried out for 1 hour at 37°C in 2 ml of bicarbonate-saline containing 1 mpg Co6iB1?/ml. One-tenth ml of hog IF (1 mg/ml) was added to some flasks. The tissue was rinsed once in cold saline and counted in a well-type scintillation counter. Results are expressed as mean values f SE.
I~STRIRUTIOK
OF CoS7-&
TABLX 4 1~ FETAL ORGANS AND ~IEMBRANES
ISJECTION Time interval between injection and sacrifice (hours)
Liver Xiumber of
pregnallt rabbit
N~rrlher of f&Uses
I‘ Pregnant rabbits (Z-28 CO~‘B~?. At timed intervals membranes
OF PREGKANT
were
weighed
Total (p&q)
Kidney
I~~nav~~ous
PlWXlt~l disc
Yolk ____
sac
Total
Total
(WA )
hg)
COKLC. b-w,’ Total sm) (!a)
._,~~.~
COIlC. ‘FU{
AFTER
RABBITW
COIlC.
days) were injected the animals mere and counted. Results
COIX
ha/ am)
via the ear vein with 100-200 mpg sacrificed and the fet,al organs are expressed as meitn values.
and
362
DEREN,
PADYKULA,
AND
WILSON
jetted with 100-200 mpg Cos7B1? and then sacrificed at various time intervals. Table 4 shows that the rabbit yolk sac contained large amounts of B,, at all time intervals after injection. At 48 hours the B,? content of the yolk sac on a weight basis was more than 10 times higher than any other tissue examined. Thus, the rabbit yolk sac has a remarkable avidity for the vitamin. Similar injection experiments were performed with guinea pigs (Table 5). Although appreciable B,, was taken up by the yolk sac, the quantities were consistently less than in the fetal liver and kidneys.
DISTRIBUTION
or‘
TABLE 5 CoS7B12 IN FETAL ORGANS INJECTION
Duratiw of gestation (dws)
52 54 60 63
(1) (1) (1) (1)
Interval between irljectio~k and sacrifice (hours)
24 48 24 24
Liver
OF PREGNANT
AND MEMBRANES GUINEA PIGS”
Kidneys
Yolk sac
AFTER
THE
Placental disc
EL:;
ChC. (wgl .Xm)
Total h+R 1
Fz? Km)
Total G/d
COllC. hwK/W)
Total (w!z)
cont. (wd ml)
71 89 73 26
22 25 17 6.1
110 160 140 57
200 320 210 82
20 1s 15 5.6
27 23 16 6.1
280 160 280 71
59 35 45 15
6 Guinea pigs were injected subcutaneously with 5 mpg Co5rB12. Twenty-four or 48 hours later, the animal was sacrificed by a blow on the head and Co”‘B12 content of fetal organs and membranes was determined. Results are expressed as mean values + SE. The number of experiments is given in parentheses.
To determine whether the vitamin B1, that reached the uterine lumen can be transferred directly to the fetus via the yolk sac, CO”~B~?was injected into one horn of an anesthetized pregnant rabbit. The abdominal incision was closed and the animal sacrificed 24-48 hours later. In each instance the concentration of B,, was greater in the fetuses of the injected horn than in those of the uninjected horn (Table 6). An additional observation that may be related to the mode of transfer of B,, from the yolk sac to the fetus was the finding of significant B,, in the amniotic fluid and fetal gastric juice (Table 7). Moreover, the concentration of the label in the aspirated gastric contents was greater than that found in the amniotic fluid at both 24 and 48 hours. In an attempt to assess the physiological role of the yolk sac in B,, transport, the vitelline vessels of several fetuses in a litter were
DEVELOPMEIL’T
OF THE
MAMMALIAN
T,iBLE
YOLK
363
SAC
6
IETRAUTERINE INJECTION" Interval between injection and sacrifice (hours)
Yolk SLC
24
Rabbit no. 1 Illjected horn Korrinjected horn 11abbit no. 2 Injected horn Soninject,ed horn Rabbit no. 3 Injected horn Noninjected horn I:abbit, no. 4 Injected horn Koninjected horn Rabbit no. <5 Inject,ed horn Xoninjcrted horn
4.81 (4) 0.67 (4)
1.31 (2) 0.30 (2)
4700 (‘2) 0.57 (2)
3.4 (2, 0.41 (4)
0.53 (2) 0.19 (2)
310 (2,
0.82 (2) 0.044 (4)
0.15 (2) 0.025 (4)
170 (2) 0.73 (4)
4.2 (2) 0 18 (3)
2.9 (2) 0.01 (3)
380 (2) 4.5 (3)
11 (3) 0.10 (5)
10 (3) 0.05 (5)
310 (3) 2.0 (5)
24 0.41 (4)
24
48
4s
a Pregnant rabbits (“5-27 days) were anesthetized with &her after atropinization. The abdomen was opened and a ligature was placed around the junction of one llt,erinc horn and the cervix. Approximately 1 ml (1 mpg Co6’B,2, per milliliter) per conceptus was injected via the tubal end into the uterine lumen. The abdomen was closed. After 24-48 hours the animal was sacrificed and the radioactivitJy in the fetal organs and membranes was counted. Results are presented as mean values, and 1he number of fetuses is given in parentheses. TABLE 7 COSCENTRATION OF CoS7Blz IS AMNIOTIC FLTJID ASU GASTRIC JUICE OF THE FETAL NABBIT AFTER ~JXILATERAL INTRAUTERINE INJECTION"
Uterine horn Injected Noninjected n’oninjected Noninjected Injected Injected Injected Injected ‘1The experimental
protocol
Itlterval between injection -and sacrifice (hours) 48 48 48 48 48 24 24 24 is described in Table 6.
Gastric juice
:Ymniotic fluid
120 1.2 5 5 5.4 59 33 91 9.2
16 0.9 2 0 1 3 10 18
53 4.9
364
DEREN,
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AND
WILSON
ligated, employing the technique described by Brambell et al. ( 1949 ). Following closure of the abdominal incision, CoS7B12 was administered to the mother intravenously. The rabbits were sacrificed 2P 48 hours later, and the fetal organs were assayed for radioactive B,,. No significant differences were detected between those fetuses whose vitelline circulation was interrupted and control fetuses from the same uterine horn. The experiments, however, were found to be technically difficult and, although a number of experiments appeared satisfactory, further studies are needed. DISCUSSION
The high molecular weight, the chemical complexity, and the binding of vitamin B,, to serum proteins would lead one to expect a special placental transfer mechanism to provide the fetus with an adequate amount of this coenzyme. This transfer mechanism must be extremely efficient, as the serum B,, level in the fetus exceeds that in the maternal host (Killander and Vahlquist, 1954; Okuda et al., 1956; Boger et al., 1957). As a result of this fetal accumulation, the maternal serum B,, level falls during pregnancy in spite of enhanced intestinal absorption of the vitamin (Boger et al., 1956; Hellegers et al., 1957). However, the mechanism and route of transport from the maternal to the fetal circulations have not been extensively studied. Two observations suggest that the yolk sac may be involved in . . the transfer of vitamin B,, from mother to fetus. Brambell and associates (Brambell et al., 1948, 1949, 1951a-c; Brambell, 1958) have clearly demonstrated that the rabbit chorioallantoic placenta is impermeable to protein and that passive transfer of immunity is mediated via the yolk sac and the vitelline circulation. The pronounced protein binding of serum B,, (Rosenthal and Saratt, 1952; Rosenthal and Austin, 1962; Rosenthal et al., 1962; Wolff et al., 1952; Bertcher and Meyer, 1957; Gregory and Holdsworth, 1959) would suggest that the chorioallantoic placental route may be quantitatively less important than the yolk sac route in vitamin B,, transfer. Furthermore, the progestational rabbit uterus secretes vitamin B,, into the lumen at concentrations greatly exceeding that of the maternal serum (Jacobson and Lutwak-Mann, 1956). The detection of labeled B,, in fetal organs following the parenteral injection of a pregnant rat had been described previously (Chow
IlEVELOPMENT
OF THE
hIAhfhIALIAN
YOLK
SAC
365
et al., 1951; Rice et al., 1958). Our results confirm this phenomenon in rats as well as in rabbits and guinea pigs. Moreover, there was considerable accumulation of the label in the visceral yolk sac. This was especially prominent in the yolk sac of the rabbit where the concentration exceeded by more than tenfold the concentration in other fetal organs. Although Co”‘B12 was detected in the rat and guinea pig yolk sacs, its concentration did not exceed that found in the fetal liver and kidney. The rat yolk sac, in spite of a demonstrated greater avidity for the vitamin in vitro, accumulated less B,, than the rabbit yolk sac following the parenteral administration to the maternal host. The demonstration of vitamin B,, uptake in oitro lends further support to the possible physiological role of the yolk sac in B,, transport in uiuo. The inhibition of B,, uptake by 1 mhl DNP is evidence that B,, uptake, both in the presence and the absence of IF, is an energy-dependent process, The addition of glucose to the incubation medium enabled this glycogen-free tissue (Davies, 1956) to overcome the DNP inhibition. Presumably the quantities of ATP generated by glycolysis were adequate to provide the energy necessary for B,, uptake. The inhibition of uptake by l-0.1 miLl iodoacetate and the failure of glucose to restore uptake to the noninhibited level are compatible with this hypothesis. Iodoacetate probably does not interfere with the interaction of B,, with IF, as --SH groups are not involved in binding (Grasbeck, 1958). Injecting vitamin B,, directly into one horn of the uterine lumen resulted in a marked accumulation of the isotope by the yolk sac of the injected horn and a higher concentration of the label in the organs of the respective fetuses. When similar experiments were performed in an attempt to evaluate the role of the yolk sac in iron transfer, no significant differences were observed between the fetuses of the injected and noninjected horns (Davies et nl., 1959). Thus, the B,, that appears in the uterine lumen is taken up, presumably, by the yolk sac, and transferred to the fetus. Although this is relatively clear evidence that the yolk sac possesses the capacity for such transfer, it does not provide information on the quantitative importance of this route under physiological conditions. Once the vitamin has accumulated in the absorptive cells, it is presumably transferred to the fetus via the vitelline vessels. Another possibility is diffusion across the exocoelom into the amniotic fluid,
366
DEREN,
PADYKULA,
AND
WILSON
Fetal swallowing of amniotic fluid has been shown to occur ( Wislocki, 1920; Windle, 1940), and thus vitamin B,, might conceivably be absorbed via the intestinal tract. Although the label was found in both the amniotic fluid and gastric juice, the concentration in the stomach exceeded that in the amniotic fluid. This indicates that the rate of water exit from the stomach exceeded the rate of vitamin B,? removal. These findings are similar to those made on gamma globulin concentration in gastric juice in the rabbit, an animal in which proteins are not transferred in significant quantities to the fetal circulation following per oral intubation of the fetus (Brambell et al., 195Ia,c). In the fetal rat, whose intestine is capable of absorbing gamma globulin, a concentration difference between gastric contents and amniotic fluid was not observed ( Halliday, 1955). These observations, together with the demonstration of absent IF from fetal rabbit intestine and the inability of fetal ileum to accumulate significant B12 in the absence of IF (Deren and Wilson, unpublished observations), are evidence that amniotic fluid swallowing is not an important mechanism for the transfer of B,, from the yolk sac to the fetal circulation. The effects of interrupting the vitelline circulation followed by injecting B,, into the mother were pursued in an attempt to estimate the roles of these two placental membranes. The lack of significant interference in BXz transfer following ligation of the vitelline vessel might lead one to conclude that the yolk sac does not contribute significantly to B,, transport in the rabbit. The conclusions to be drawn from this experiment, however, are limited, because the adequacy of the vitelline vessel interruption was not assessed by simultaneously injecting a specific gamma globulin known to traverse this route. Moreover, the parenteral administration of vitamin B1, may have led to an elevated free B,, level in the serum or to abnormal protein binding. The free B,, may be transported across the chorioallantoic placenta, whereas under more physiological circumstances, the extensive protein binding of B 12 may exclude it from this transfer mechanism. These in viva experiments, therefore, are open to many alternative explanations and are not considered decisive evidence against the yolk sac route of transfer. Thus, we have demonstrated that the rabbit yolk sac possesses an energy-dependent mechanism for vitamin B,, uptake in vitro and that this mechanism may be responsible for the net transfer to the
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
367
fetus of Blz injected into the lumen of the uterus in viuo. The presence of the vitamin in the placental disc of all animals studied suggest that B,, is also accumulated by chorioallantoic placenta. From our experiments, however, it is difficult to assess the quantitative importance of the yolk sac route on the one hand and the chorioallantoic placenta on the other in the transfer of vitamin B,, from the maternal to the fetal circulation. SUMMARY
When rabbit yolk sacs were incubated in vitro with Co5’B12, there was a greater accumulation of the vitamin than that observed with fetal ileum of a similar age. Intrinsic factor, with a species specificity similar to that observed in fetal and adult intestine, stimulated B,, uptake severalfold. The uptake of B12, both in the presence and absence of IF, was depressed when DNP or iodoacetate was added to the incubation medium. The addition of 10 mM glucose overcame the DNP inhibition but did not reverse the iodoacetate effect. Following the parenteral administration of CO”‘B~~ to pregnant rabbits, the label appeared in the fetal organs and extrafetal membranes. The accumulation in the rabbit yolk sacs was particularly striking after 24-48 hours. Unilateral intrauterine injection of B,, led to more of the label appearing in the fetal organs of the injected horn than in the noninjected control horn. Ligation of the vitelline vessels did not appear to effect the transfer of parenterally administered CO”~B,~ to the fetal organs. The contribution of the chorioallantoic placenta and the yolk sac placenta of the rabbit in the transfer of B12 from mother to fetus is discussed. The developmental morphology of the visceral yolk sac of the rabbit was compared with that of the rat. The absorptive cells of the visceral endoderm possess surface specializations related to the uptake of macromolecules. The technical is most gratefully
assistance of Miss Ann G. Campbell acknowledged.
and Miss Angela
De Carlo
REFERENCES AMOHOSO, E. C. (1952). Placentation. In Marshall’s “Physiology of Reproduction” (A. S. Parkes, ed.), Vol. II. Longmans, Green, New York. BEHTCHER, R. \V., and MEYER, L. M. (1957). CO” vitamin Blz binding capacity of normal human serum. Proc. SW. Exptl. Biol. Med. 94, 169-171.
368
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PADYKULA,
AND
WILSON
BOGER, W. P., WRIGHT, L. D., BECK, G. D., and BAYNE, G. M. (1956). Vitamin B,x: correlation of serum concentrations and pregnancy. Proc. Sot. Exptl. Biol. Med. 92, 140-143. L. D., and BECK, G. D. (1957). DifBOGER, W. P., BAYNE, G. M., WRIGHT, ferential serum vitamin B,? concentration in mothers and infants. New EngZ. J. Med. 256, 1085-1087. BRAMBELL, F. W. R. ( 1958). The passive immunity of the young mammal. Biol. Reo. Cambridge Phil. Sot. 33, 488531. BRAMBELL, F. W. R., HEMMINGS, W. A., and ROWLANDS, W. T. (1948). The passage of antibodies from the maternal circulation into the embryo in rabbits. Proc. Roy. Sot. B135, 390-403. BRAMBELL, F. W. R., HEMMINGS, W. A., HENDERSON, M., PARRY, H. J., and ROWLANDS, W. T. ( 1949). The route of antibodies passing from the maternal to the foetal circulation in rabbits. Proc. Roy. Sot. B136, 131-144. BRAMBELL, F. W. R., HEMMINGS, G. P., HEMMINGS, W. A., HENDERSON, M., and ROWLANDS, W. T. (1951a). The route by which antibodies enter the circulation after infection of immune serum into the exocoel in foetal rabbits. Proc. Roy. Sot. B138, 188-195. BBAMBELL, F. W. R., HEMMINGS, W. A., and HENDERSON, M. (1951b). “Antibodies and Embryos.” Oxford Univ. Press (Athlone), London and New York. BRAMBELL, F. W. R., HE~IMINGS, W. A., HENDERSON, M., OAKLEY, C. L., and ROWLANDS, W. T. (1951c). The accumulation of antibodies in the stomach contents of foetal rabbits. Proc. Roy. Sot. B138, 195-204. CHOW, B. F., BARROWS, L., and LING, C. T. ( 1951). The distribution of radioactivity in the organs of the fetus or of young rats borne by mothers injected with vitamin B,? containing Cow. Arch. Biochem. Biophys. 34, 151-157. DAVIES, J. ( 1956). Histochemistry of the rabbit placenta. .l. Anat. 90, 135-142. DAVIES, J., BROWN, E. B., STEWART. D., TERRY, C. W., and SISSON, J. (1959). Transfer of radioactive iron via the placenta and accessory fetal membranes in the rabbit. Am. J. Physiol. 197, 87-92. DRAPER, R. L. (1920). Prenatal growth of guinea pig. Anat. Record 18, 369-392. GRXSBECK, R. ( 1958 ) Action of group blocking agents and enzymes on intrinsic factor preparations. Acta Chem. Stand. 12, 142-144. GREGORY, R/I. E., and HOLDSWORTH, E. S. (1959). A study of protein-binding in the metabolism of vitamin B,?. Biochem. J. 72, 549-556. HALLIDAY, R. (1955). Prenatal and postnatal transmission of passive immunity to young rats. Proc. Roy. Sot. B144, 427-430. HELLEGERS, A., OKUDA, K., NESBITT, R. E. L., SMITH, D. W., and CHOW, B. F. ( 1957). Vitamin BIz absorption in pregnancy and in the newborn. Am. J. CZin. Nutr. 5, 327-331. JACOBSON, W., and LUTWAK-MANN, C. (1956). The vitamin B,s content of the early rabbit embryo. J. Endocrinol. 14, XIX-XX. KARNOVSKY, M. J. ( 1961). Simple methods for “staining with lead” at high pH in electron microscopy. J. Biophys. Biochem. Cytol. 11, 729-732. BX concentration in KILLANDER, A., and VAHLQUIST, B. ( 1954). The vitamin serum from term and premature infants. Nerd. Med. 51, 777-779.
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OF THE
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KREBS, H. A., and HENSELEIT, K. ( 1932). Untersuchungen iiber die Harnstoffbildung im Tierkorper. Z. Physiol. Chem. 210, 33-66. MOSSMAN, H. W. ( 1937). Comparative morphogenesis of the foetal membranes and accessory uterine structures. Contrib. Embryol. Carnegie Inst. Wush. 26, 129-246. OKUIZIA, K., HELLEGEHS, A., and CHOW, B. F. (1956). Vitamin BIr serum level and pregnancy. Am. J. Clin. Nutr. 4, 440-443. PADYKULA, H. A., DEREN, J. J., and WILSON, T. H. (1966). Development of structure and function in the mammalian yolk sac. I. Developmental morphology and vitamin B,, uptake of the rat yolk sac. Dewlop. Bid. 3, 311-348. PALADE, G. E. ( 1952). A study of fixation for electron microscopy. J. Erptl. Med. 95, 285-298. RICE, E. G., HEHNDON, J. F., VAN LOON, E. J., and GREENBERG, S. M. (1958). Enhancement of vitamin B1z absorption by n-sorbital as measured by maternal and fetal tissue levels in pregnant rats. Am. J. Physiol. 193, 513-515. ROSENTHAL, H. L., and SAHATT, H. P. (1952). The determination of vitamin B,? activity in human serum. J. Biol. Chem. 199, 433-442. ROSENTHAL, H. L., and AUSTEN, S. (1962a). Vitamin B,, unsaturated binding capacity of sera from various animals. Proc. Sot. Exptl. Med. 109, 179-181. ROSENTHAL, H. L., O’BHIEN, G., and AUSTIN, S. (1962b). Factors affecting the binding of cyanocobalamine to serum proteins from various animals. Arch. Rio&em. Biophys. 99, 319-325. WILSON, T. H., and STHAUSS, E. W. (1959). Some species differences in the intrinsic factor stimulation of Blz uptake by small intestine in z;itro. Am. J. Physiol. 197, 926-928. WINDLE, W. F. (1940). “Physiology of the Fetus: Origin and Extent of Function in Prenatal Life.” Saunders, Philadelphia, Pennsylvania. WISLOCKI, G. B. (1920). Experimental studies on fetal absorption. Contrib. Embryol. (Carnegie Inst. Wash. Publ. 274) 11, 47-60. WOLFF, R., KAHLIN, R., and ROYER, P. (1952). Sur l’existence dun pouvoir de combinaison du serum pour la vitamine B,?. Compt. Rend. Sot. Bid. 146, 1008-1010.
BIOLOGY
Development
II. Vitamin JULIUS
13, 349-369
of Structure and Function Mammalian Yolk Sac BE Uptake
J. DEREN,?, R HELEN
Depurtment Department
of Physiology, of Biological
( 1966)
by Rabbit
A. PADYKULA,
Haroard Sciences, Accepted
AND
Yolk
in the Sad
T. HASTINGS
WILSON~
Medical School, Boston, IMassachusetts, and Wellesley College, Wellesley, Massuchusetts December
13, 1965
INTRODUCTION
In the preceding paper (Padykula et al., 1966) it was shown that the visceral yolk sac of the rat incubated in vitro with radioactive vitamin B,, was capable of accumulating this large molecule. The uptake was stimulated two- to fivefold by rat gastric intrinsic factor (IF), the mucoprotein essential for B,, absorption by the small intestine. B,, uptake per gram tissue, both in the presence and absence of IF, decreased with increasing age of the fetus, being highest at 13 days of gestation, the earliest stage tested, and lowest at term. Membrane invagination and vacuole formation which are characteristic of cellular uptake of macromolecules were evident in the endodermal cells of the yolk sac. The demonstration by Brambell and his collaborators (Brambell et al., 1948, 1949, 1951b; Brambell, 1958) that the rabbit yolk sac is the major route for the transfer of antibodies from mother to fetus suggested to us the possibility that the transfer of vitamin B,, is mediated via a similar route. The visceral yolk sac accumulated considerable vitamin B,, in the absence of added IF by an energydependent mechanism. Intrinsic factor enhanced uptake with a more ‘Supported in part by research grants from the U.S. Public Health Service (AM-05736 and HD-01026). ‘Postdoctoral Fellow of the U.S. Public Health Service (5-FZ-GM-15, 144). ” Present address: Department of Medicine, Maimonides Hospital, Brooklyn, New York. ’ Supported by a Research Career Development Award of the U.S. Public Health Service ( 5-K3-GM-15, 306). 349
350
DEREN,
PADYKULA,
AND
WILSON
pronounced effect as term approached. In viva experiments were performed in an attempt to evaluate the physiological role of the yolk sac in the transfer of vitamin B,, from mother to fetus. MATERIALS
AND METHODS
Pregnant white New Zealand rabbits and Sprague-Dawley rats were obtained from a breeding farm which carefully recorded the time of coitus and segregated the females. Guinea pigs were obtained from a commercial source which did not segregate the sexes. The approximate length of gestation in the guinea pig was estimated from the number and weight of the fetuses, according to the tables prepared by Draper (1920). The gestation period for the rabbit is approximately 32 days; the guinea pig 65 days; and the rat 22-23 days. Cytological
Techniques
Yolk sacs were isolated at 16 and 28 days, rinsed briefly in isotonic saline, and fixed in a buffered osmium tetroxide solution (Palade, 1952). After standard embedding in Epon, plastic sections were cut at l-2 p of a Porter-Blum ultramicrotome, stained with toluidine blue, and examined with the light microscope, Ultrathin plastic sections were stained with uranyl acetate and Karnovsky’s lead stain (1961). They were examined with a Siemens Elmiskop I. In Vitro Experiments Pregnant rabbits, guinea pigs, or rats were stunned by a blow on the head and exsanguinated by cutting the carotid vessels. The visceral portion of the yolk sac was carefully excised from the chorion and placed into cold saline until the beginning of the incubation. Yolk sacs were incubated intact, in halves, or quarters depending upon the size of the tissue and the experimental protocol. Incubation was carried out in a 25-ml Erlenmeyer flask in an oscillating water bath at 37°C. Each flask contained 2 ml of bicarbonate-saline (Krebs and In some flasks 0.1 ml of the Henseleit, 1932) with 1 mpg Co”‘B,,/ml. appropriate IF preparation was added. At the end of an hour the tissue was removed, rinsed once in cold saline, blotted, weighed, and counted in a well-type scintillation counter. In Vivo Experiments 1. Parenteral injection injected subcutaneously
of pregnant animals. Pregnant animals were with Co57B12 and sacrificed after different
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
351
time intervals. The radioactivity in the maternal and fetal organs as well as the placental membranes was determined. 2. Intrauterine injection. Pregnant rabbits 25-28 days were premedicated with atropine and anesthetized with Nembutal. A midline lower abdominal incision was made, then a ligature was placed around the junction of one uterine horn with the cervix without interfering with the vascular supply. A small incision was then made in the tubal end of the uterus, and a thin polyethylene catheter attached to a syringe was introduced and advanced to the opposite end of the uterine horn. One milliliter of CO”~B~~ (containing 1.04 mpg Co”‘B,,/ ml) per conceptus was injected into the uterine cavity during withdrawal of the polyethylene catheter. This procedure ensured exposure of each yolk sac to the radioactive vitamin. The perforation in the uterus was ligated, and the abdomen was closed. Twenty-four or 48 hours after the intrauterine injection the rabbits were killed by a blow on the head, and the radioactivity in the maternal and fetal organs and placental membranes was determined. 3. Ligation of vitelline vessels. Pregnant rabbits (25-28 days) were premeditated with atropine and anesthetized with Nembutal. The abdomen was opened, and a small incision was made through the uterine wall overlying the main vitelline vessels, according to the method of Brambell et al. (1949). Slight pressure was exerted on the conceptus, forcing the visceral yolk sac to protrude through the uterine incision. A ligature was placed over the protruding yolk sac, which included the vitelline stalk. The tissue was then pressed back into the uterine cavity, and the uterine wall was sutured. The abdominal incision was repaired, and the rabbit was injected subcutaneously with Co”‘B,,. Twenty-four hours later the rabbit was killed, and the radioactivity in the appropriate organs was determined. Materials CO”‘B~~ was purchased from Merck and Company. Hog intrinsic factor was kindly provided by Dr. C. W. Pettinga, Eli Lilly Company (lots Nos. 258-458-B-241 and 258-417-B-98-5). It was diluted in saline to contain 1 mg/ml, and 0.1 ml was added to the appropriate flask. The rat, rabbit, and hamster IF were prepared by homogenizing the glandular mucosa in ten times its weight of saline and centrifuging at 1000 g for 10 minutes. A 0.1 ml sample of the supernatant was added to the appropriate flask. 2,4-Dinitrophenol was purchased from Mann
352
DEREN,
PADYKULA,
AND
WILSON
FIG. 1. Visceral yolk sac, rabbit, 16 days’ gestation. The absorptive epithelium (AE) stains darkly and is thrown into folds around a central core of mesenchyme carrying the vitelline vessels (VU). The vitelline vessels immediately below the absorptive epithelium are thin-walled endothelial tubes that have large are evident within the vitelline vessels. lumens. Various stages of erythropoiesis The exocoelom is lined by a flattened mesothelium (m). The visceral basement membrane is inconspicuous at this time. The absorptive cells are further magnified in Fig. 3. 2 p plastic section; toluidine blue. Magnification: X 300.
DEVELOPMENT
Research Laboratories Organic Chemical.
OF
THE
MAMMALIAN
and sodium iodoacetate
YOLK
SAC
353
was from Eastman
RESULTS
Morphology Light microscopy. The rabbit, like the rat, has two placentas, a discoidal labyrinthine chorioallantois and an inverted yolk sac. The chorioallantoic placenta is fully developed on about the 12th day of gestation, and the yolk sac placenta is established even earlier (Amoroso, 1952). The parietal wall of the yolk sac (bilaminar omphalopleure) has broken down by the 15th day, and thereafter the visceral wall (visceral splanchnopleure) is directly exposed to the uterine contents. All our studies were performed after the loss of the parietal wall. It is important to point out one major difference between placentation in the rabbit and rat. In the rabbit, a wide annular chorion extends from the margin of the placental disc and connects with the yolk sac membrane (Mossman, 1937; Brambell et al., 195Ib). This chorionic margin is for the most part nonvascular, but its outer, trophoblastic surface is freely exposed to the uterine contents. In the present experiments, the chorion was dissected away from the yolk sac. At 16 days, the visceral yolk sac of the rabbit resembled that of the rat at 13 days (Figs. 1 and 3). The absorptive epithelium was composed of tall columnar cells that rested on a highly vascular mesenchymal layer in which hematopoiesis was evident. A mesothelium lined the exocoelomic surface. The free surface of the absorptive endodermal cells was modified into a brush border. The apica cytoplasm of the absorptive cells was packed with droplets of various sizes, many of which stained heavily with basic dye (Fig. 3). Large empty vacuoles were seen in the infranuclear cytoplasm; these most likely represented sites of stored lipid which was dissolved out by the preparative procedures. FIG. 2. Visceral yolk sac, rabbit, 28 days’ gestation. The three tissue layers of this membrane are evident here. The thick, darkly stained absorptive epithelium (AE) rests upon the loose mesenchymal layer containing vitelline vessels (vu). The mesothelium (m) demarcates the exocoelomic surface of the membrane. The absorptive cells are further enlarged in Fig. 4. 2 p plastic section; toluidine blue. Magnification: X 300.
354
DEREN,
PADYKULA,
AND
WILSON
FIG. 3. Absorptive epithelium, rabbit yolk sac, 16 days’ gestation. free surface of the closely apposed absorptive cells, the brush border evident. The supranuclear cytoplasm contains numerous droplets and that stain lightly or heavily with basic dye. The oval nuclei possess two nucleoli. The lumen of the vitelline vessel ( UV) is in close proximity absorptive epithelium. 2 p plastic section; toluidine blue. Magnification:
At the is barely vacuoles or more to the x 1300.
DEVELOPMENT
OF
THE
MAMMALIAX
YOLK
SAC
355
of the rabbit yolk sac At 28 days, the absorptive epithelium resembled that of the rat near term quite closely, except that the epithelial ridges were barely penetrated by mesenchyme and blood vessels (Fig. 2). At this time there was no sign of hematopoiesis. A brush border capped the free surface of the endodermal cells. The supranuclear cytoplasm was highly vacuolated, although fewer basophilie droplets were evident. Large intercellular spaces were now present between the absorptive cells (Fig. 4). The visceral basement membrane was considerably thicker than that present at 16 days. The appearance of intercellular spaces and the thickening of the visceral basement membrane also occurred during the development of the rat yolk sac (Padykula et al., 1966). Electron microscopy. The free surface of the absorptive cells resembled that of the rat yolk sac closely (Figs. 5 and 6). At 16 days and 28 days, the microvilli were well developed, and invaginations of the plasma membrane occurred at the bases of the microvilli (Fig. 5). A thick-walled canalicular system filled the cytoplasm immediately below the microvillous border. The invaginations and canaliculi were lined by a filamentous coat that may represent a special surface for protein attachment. In close proximity to the canaliculi, absorptive vacuoles occurred which contained a loose precipitate. As in the rat may placenta, the microvilli, invaginations, canaliculi, and vacuoles represent an intracellular pathway for transporting macromolecules (Padykula et al., 1966). At 16 days, the lateral cell membranes of the absorptive cells were closely and uniformly apposed (Fig. 5), whereas near term, large intercelluIar spaces appeared intermittently between the lateral ceII membranes (Fig. 6). These spaces contained a delicate precipitate, and this suggested the possibility of a pathway for intercellular transport. Near term, a similar developmental change in the lateral associations of absorptive cells occurred in the rat yolk sac (Padykula et al., 1966). FIG. 4. Absorptive epithelium, rabbit yolk sac, 28 days’ gestation. The brush border at the free surface of the absorptive cells is indistinct. The supranuclear cytoplasm is fiIled primarily with pale-staining droplets and vacuoles. The nuclei are somewhat more basal in position and more irregular in form than at 16: days. Intercellular spaces have appeared between absorptive cells. A conspicuous difference between this and the earlier stage is the thickening of the visceral basement membrane (vm) which intervenes between the absorptive cells and the vitelline vessels. 2 JJ plastic section; toluidine blue. Magnification: x 1300.
356
DEREN,
PADYKULA,
AND
WILSON
In Vitro Studies When rabbit yolk sac segments were incubated in the presence of CoS7-labeled vitamin B,,, there was an accumulation of the isotope with little variation from day 15 to term (Fig. 7). This level of uptake of B,, by rabbit yolk sacs was greater than that observed with the distal intestine from the same litter in the absence of added intrinsic factor (Table 1). When hog IF was added to the incubation TABLE 1 Blz UPTAKE BY FETAL RABBIT ILEUM AND YOLK SAC OF A SIMILAR AGES COS~BIZ accumulated Sample
27-31 day yolk sacs 27-31 day distal half of intestine
a,
(m&l00
alone
0.15 f 0.01” (19) 0.06 f 0.01 (15)
mg tissue
wet weight) ~__ HIS + hog IT’
0.93 * O.l@ (19) 0.32 + 0.03c (15)
a Pregnant rabbits were sacrificed, and t,he visceral yolk sac and the distal half of the fetal intestine were removed. The yolk sacs were divided in half and incubated for 1 hour at 37°C in a shaking water bath in 2 ml bicarbonate-saline containing 1 mpg Co57B12 per milliliter, 0.1 of hog IF (Eli Lilly, 1 me/ml) was added to one of the flasks. The distal intestine was cut into segments and incubated as above. Result,s are expressed as mean values h SE. The number of experiments is given in parentheses. b P < 0.01 when compared to Ble upt)ake by intestine. c P < 0.01 when compared to uptake in the absence of IF.
medium, a twofold stimulation was observed at day 15 with a more pronounced effect as term approached. As seen from Table 2, rabbit and rat IF, in addition to hog IF, enhanced uptake in both rabbit yolk sacs and fetal intestine, whereas hamster IF failed to stimulate uptake. Rat IF stimulated B 12 uptake by both rat yolk sacs and adult rat intestine, whereas rabbit, hog, and hamster IF were ineffective. Thus the rabbit and rat yolk sac displayed the same species specificity FIG. 5. Absorptive cell, visceral yolk sac, rabbit, 16 days’ gestation. The microvillous border (mu) is cjuite irregular in form. Invaginations of the apical The most superficial cytoplasm is filled plasma membrane are evident ( arrow). with various profiles through the canaliculi system (c). The single large absorptive vacuole (AV) contains a fine, loose precipitate. The invaginations, canaliculi, and vacuoles have dense walls which are composed of a smooth limiting membrane and a thick inner filamentous lining. The lateral cell surfaces are closely apposed (arrows). D, desmosome; M, mitochondria. Electron micrograph. Magnification: x 21,300.
DEVELOPfitEST
OF
THE
MAhfhfAI,IAN
YOLK
SAC
357
358
DEREN,
PADYKULA,
AND
TABLE SPECIES
WILSON
2
SPECIFICITY OF INTRINSIC FACTOR STIMULATION YOLK SAC AND INTESTINE PHOM THE RAT Ratio
SalIlplZ2
Rabbit yolk sac Rabbit fetal ileum Rat yolk sac Adult rat gut?
Rabbit
IP
4.9 (4)
18
(2)
1.1 (3) 0.9
BD uptake Bn uptake Rat
IF
2.5 (3) 14 (2) 9.9 (2) 10
OF BU UPTAKE AND RABBITS
in the presence in the absence Hog
IF
6.0 (19) 5.5 (15) 0.88 (3) 1.0
IN THE
of IF of IF
Hamster
IF
1.0 (3) 0.95 (3) 0.41 (2, 0.50
4 Incubations were carried out in a shaking water bath for 1 hour at 37°C in a 25 ml flask containing 2 ml bicarbonate-saline with 1 mpg Co6’B12 per milliliter. Hog IF, kindly provided by Eli Lilly and Company, was diluted to contain 0.1-1.0 mg/ml. Rat, rabbit, and hamster IF were prepared by homogenizing the gastric glandular mucosa in ten times its weight of saline and centrifuging at 1000 g for 10 minutes. One-tenth milliliter of the diluted hog IF or the supernatant was added to the appropriate flask. The number of experiments is given in parentheses. * Data taken from Wilson and Strauss (ISFig).
for IF as the fetal and adult ileum of the corresponding species (Wilson and Strauss, 1959). To determine whether the uptake of B,, by rabbit yolk sacs in the absence of added IF represented an active process of accumulation or nonspecific tissue binding, uptake was measured in the presence of various inhibitors. As seen from Table 3, the addition of 1 mM 2,4-dinitrophenol (DNP) to the incubation medium significantly lowered the B,, uptake. Incubation in the presence of 10 mM glucose, completely reversed the DNP inhibition. The IF-stimulated B,, uptake was similarly inhibited by 1 mM DNP, and the inhibition was abolished by 10 mM glucose. The IF-stimulated B,, was also inFIG. 6. Absorptive cell, visceral yolk sac, rabbit, 28 days’ gestation. The free surface of the absorptive cell near term resembles that of the earlier stage (Fig. 6) in the presence of a microvillus (mu), canaliculi (c), and absorptive however, in the associations of the vacuoles (AV). There is a difference, lateral cell surfaces. Intermittent enlargements of the intercellular space (IC) have appeared, and they contain some precipitate. At intervals, the lateral plasma membranes are held in close association by desmosomes (D). Also, near term, cytoplasmic fibrils (f) appear to be more numerous. ER, cisternae of the M, mitochondria; G, Golgi membranes. Elecgranular endoplasmic reticulum; tron micrograph. Magnification: X 23,000.
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
359
360
DEREN,
EFFECT OF INHIBITORS ON
PADYKULA,
B1?
AND
TABLE
3
TJPTAKE
BY
WILSON
RABBIT
YOLK
SAC
in
Vitw
Number of yolk sacs
~~ ~~~ I3
0.17 * 0.02
.~____
0.068 + 0.006 I’ < O.OOlb
En + IF
Biz + IF
i
DNP
0.14 + 0.07 P > 0.3” P < O.OlC Bn + IF
0.073 * 0.002 P < O.OOlb
74
HI? + IF
Bn
+ IF
+ iodoacetate
+ DTiP
+ glucose
0.47 * 0.1 P > 0.6b P < 0,005’ Bn
+ IF
+ iodoacetate
+
glWXl3
7
0.53 k 0.089
0.1 f 0.01 P < 0.005”
0.071 * 0.017 1’ < 0.01b 1’ > 0.025~
n Pregnant rabbits (28-30 days) were sacrificed by a blow on the head, and the uterus and contained concepti were removed. The visceral yolk sacs were carefully excised from the chorion and divided into four quarters. Two quarters were used in measuring the baseline Biz uptake, and the other quarters were incubat,ed with inhibitor or inhibitor plus glucose. Incubations were carried out in a 25-ml flask with 2 ml bicarbonate-saline containing 1 rnlg CoSrBiz/ml. Dinitrophenol (DXP) and iodoacetate were added to a final concent,rat,ion in the incubation medium of 1 mM and glucose to a final concentration of 10 mM. Hog 1F (0.1 ml of 1 mg/ml, Eli Lilly Co.) was added to the appropriate flasks. Results are presented as mpg CO~~B&OO mg tissue wet weight and are expressed as mean values + SE. * Level of significance as compared to uptake in t’he absence of inhibitor. c Level of significance as compared to uptake in the presence of inhibitor.
hibited by 1 mM iodoacetate, an effect not reversed by glucose. In other experiments these inhibitory effects were observed at iodoacetate concentrations of 0.1 mM. In Vivo Studies In order to investigate the role of the yolk sac in the transfer of B,, from mother to fetus, a series of in viva experiments were performed. First, an attempt was made to determine the rate of B,, uptake by the yolk sac, as compared with the other fetal organs, following injection of radioactive B,, into the maternal circulation, A series of rabbits near term (25th~28th day of gestation) were in-
DE\‘ELOPMENT
Ok’ THE
I.40 . 1 Y
;
YOLK
WM
l
&
(lcuX
a/one
T (11
x with B,g f Hog /f
1.20
361
SAC
(3)
UPrAKE ,’
I
<
MAMMALIAN
f 22 1.00 i= I
1 (9) :
3 0.60 L t
(2)
(4)
1 1
x
IIO,T
15
20 FETAL
25
-
AGE (DAYS)
FIG. 7. Developmental differences in vitamin B1? and Blz-IF uptake by the rabbit yolk sac measured in vitro. Yolk sacs were carefully excised from the surrounding chorion. Incubations were carried out for 1 hour at 37°C in 2 ml of bicarbonate-saline containing 1 mpg Co6iB1?/ml. One-tenth ml of hog IF (1 mg/ml) was added to some flasks. The tissue was rinsed once in cold saline and counted in a well-type scintillation counter. Results are expressed as mean values f SE.
I~STRIRUTIOK
OF CoS7-&
TABLX 4 1~ FETAL ORGANS AND ~IEMBRANES
ISJECTION Time interval between injection and sacrifice (hours)
Liver Xiumber of
pregnallt rabbit
N~rrlher of f&Uses
I‘ Pregnant rabbits (Z-28 CO~‘B~?. At timed intervals membranes
OF PREGKANT
were
weighed
Total (p&q)
Kidney
I~~nav~~ous
PlWXlt~l disc
Yolk ____
sac
Total
Total
(WA )
hg)
COKLC. b-w,’ Total sm) (!a)
._,~~.~
COIlC. ‘FU{
AFTER
RABBITW
COIlC.
days) were injected the animals mere and counted. Results
COIX
ha/ am)
via the ear vein with 100-200 mpg sacrificed and the fet,al organs are expressed as meitn values.
and
362
DEREN,
PADYKULA,
AND
WILSON
jetted with 100-200 mpg Cos7B1? and then sacrificed at various time intervals. Table 4 shows that the rabbit yolk sac contained large amounts of B,, at all time intervals after injection. At 48 hours the B,? content of the yolk sac on a weight basis was more than 10 times higher than any other tissue examined. Thus, the rabbit yolk sac has a remarkable avidity for the vitamin. Similar injection experiments were performed with guinea pigs (Table 5). Although appreciable B,, was taken up by the yolk sac, the quantities were consistently less than in the fetal liver and kidneys.
DISTRIBUTION
or‘
TABLE 5 CoS7B12 IN FETAL ORGANS INJECTION
Duratiw of gestation (dws)
52 54 60 63
(1) (1) (1) (1)
Interval between irljectio~k and sacrifice (hours)
24 48 24 24
Liver
OF PREGNANT
AND MEMBRANES GUINEA PIGS”
Kidneys
Yolk sac
AFTER
THE
Placental disc
EL:;
ChC. (wgl .Xm)
Total h+R 1
Fz? Km)
Total G/d
COllC. hwK/W)
Total (w!z)
cont. (wd ml)
71 89 73 26
22 25 17 6.1
110 160 140 57
200 320 210 82
20 1s 15 5.6
27 23 16 6.1
280 160 280 71
59 35 45 15
6 Guinea pigs were injected subcutaneously with 5 mpg Co5rB12. Twenty-four or 48 hours later, the animal was sacrificed by a blow on the head and Co”‘B12 content of fetal organs and membranes was determined. Results are expressed as mean values + SE. The number of experiments is given in parentheses.
To determine whether the vitamin B1, that reached the uterine lumen can be transferred directly to the fetus via the yolk sac, CO”~B~?was injected into one horn of an anesthetized pregnant rabbit. The abdominal incision was closed and the animal sacrificed 24-48 hours later. In each instance the concentration of B,, was greater in the fetuses of the injected horn than in those of the uninjected horn (Table 6). An additional observation that may be related to the mode of transfer of B,, from the yolk sac to the fetus was the finding of significant B,, in the amniotic fluid and fetal gastric juice (Table 7). Moreover, the concentration of the label in the aspirated gastric contents was greater than that found in the amniotic fluid at both 24 and 48 hours. In an attempt to assess the physiological role of the yolk sac in B,, transport, the vitelline vessels of several fetuses in a litter were
DEVELOPMEIL’T
OF THE
MAMMALIAN
T,iBLE
YOLK
363
SAC
6
IETRAUTERINE INJECTION" Interval between injection and sacrifice (hours)
Yolk SLC
24
Rabbit no. 1 Illjected horn Korrinjected horn 11abbit no. 2 Injected horn Soninject,ed horn Rabbit no. 3 Injected horn Noninjected horn I:abbit, no. 4 Injected horn Koninjected horn Rabbit no. <5 Inject,ed horn Xoninjcrted horn
4.81 (4) 0.67 (4)
1.31 (2) 0.30 (2)
4700 (‘2) 0.57 (2)
3.4 (2, 0.41 (4)
0.53 (2) 0.19 (2)
310 (2,
0.82 (2) 0.044 (4)
0.15 (2) 0.025 (4)
170 (2) 0.73 (4)
4.2 (2) 0 18 (3)
2.9 (2) 0.01 (3)
380 (2) 4.5 (3)
11 (3) 0.10 (5)
10 (3) 0.05 (5)
310 (3) 2.0 (5)
24 0.41 (4)
24
48
4s
a Pregnant rabbits (“5-27 days) were anesthetized with &her after atropinization. The abdomen was opened and a ligature was placed around the junction of one llt,erinc horn and the cervix. Approximately 1 ml (1 mpg Co6’B,2, per milliliter) per conceptus was injected via the tubal end into the uterine lumen. The abdomen was closed. After 24-48 hours the animal was sacrificed and the radioactivitJy in the fetal organs and membranes was counted. Results are presented as mean values, and 1he number of fetuses is given in parentheses. TABLE 7 COSCENTRATION OF CoS7Blz IS AMNIOTIC FLTJID ASU GASTRIC JUICE OF THE FETAL NABBIT AFTER ~JXILATERAL INTRAUTERINE INJECTION"
Uterine horn Injected Noninjected n’oninjected Noninjected Injected Injected Injected Injected ‘1The experimental
protocol
Itlterval between injection -and sacrifice (hours) 48 48 48 48 48 24 24 24 is described in Table 6.
Gastric juice
:Ymniotic fluid
120 1.2 5 5 5.4 59 33 91 9.2
16 0.9 2 0 1 3 10 18
53 4.9
364
DEREN,
PADYKULA,
AND
WILSON
ligated, employing the technique described by Brambell et al. ( 1949 ). Following closure of the abdominal incision, CoS7B12 was administered to the mother intravenously. The rabbits were sacrificed 2P 48 hours later, and the fetal organs were assayed for radioactive B,,. No significant differences were detected between those fetuses whose vitelline circulation was interrupted and control fetuses from the same uterine horn. The experiments, however, were found to be technically difficult and, although a number of experiments appeared satisfactory, further studies are needed. DISCUSSION
The high molecular weight, the chemical complexity, and the binding of vitamin B,, to serum proteins would lead one to expect a special placental transfer mechanism to provide the fetus with an adequate amount of this coenzyme. This transfer mechanism must be extremely efficient, as the serum B,, level in the fetus exceeds that in the maternal host (Killander and Vahlquist, 1954; Okuda et al., 1956; Boger et al., 1957). As a result of this fetal accumulation, the maternal serum B,, level falls during pregnancy in spite of enhanced intestinal absorption of the vitamin (Boger et al., 1956; Hellegers et al., 1957). However, the mechanism and route of transport from the maternal to the fetal circulations have not been extensively studied. Two observations suggest that the yolk sac may be involved in . . the transfer of vitamin B,, from mother to fetus. Brambell and associates (Brambell et al., 1948, 1949, 1951a-c; Brambell, 1958) have clearly demonstrated that the rabbit chorioallantoic placenta is impermeable to protein and that passive transfer of immunity is mediated via the yolk sac and the vitelline circulation. The pronounced protein binding of serum B,, (Rosenthal and Saratt, 1952; Rosenthal and Austin, 1962; Rosenthal et al., 1962; Wolff et al., 1952; Bertcher and Meyer, 1957; Gregory and Holdsworth, 1959) would suggest that the chorioallantoic placental route may be quantitatively less important than the yolk sac route in vitamin B,, transfer. Furthermore, the progestational rabbit uterus secretes vitamin B,, into the lumen at concentrations greatly exceeding that of the maternal serum (Jacobson and Lutwak-Mann, 1956). The detection of labeled B,, in fetal organs following the parenteral injection of a pregnant rat had been described previously (Chow
IlEVELOPMENT
OF THE
hIAhfhIALIAN
YOLK
SAC
365
et al., 1951; Rice et al., 1958). Our results confirm this phenomenon in rats as well as in rabbits and guinea pigs. Moreover, there was considerable accumulation of the label in the visceral yolk sac. This was especially prominent in the yolk sac of the rabbit where the concentration exceeded by more than tenfold the concentration in other fetal organs. Although Co”‘B12 was detected in the rat and guinea pig yolk sacs, its concentration did not exceed that found in the fetal liver and kidney. The rat yolk sac, in spite of a demonstrated greater avidity for the vitamin in vitro, accumulated less B,, than the rabbit yolk sac following the parenteral administration to the maternal host. The demonstration of vitamin B,, uptake in oitro lends further support to the possible physiological role of the yolk sac in B,, transport in uiuo. The inhibition of B,, uptake by 1 mhl DNP is evidence that B,, uptake, both in the presence and the absence of IF, is an energy-dependent process, The addition of glucose to the incubation medium enabled this glycogen-free tissue (Davies, 1956) to overcome the DNP inhibition. Presumably the quantities of ATP generated by glycolysis were adequate to provide the energy necessary for B,, uptake. The inhibition of uptake by l-0.1 miLl iodoacetate and the failure of glucose to restore uptake to the noninhibited level are compatible with this hypothesis. Iodoacetate probably does not interfere with the interaction of B,, with IF, as --SH groups are not involved in binding (Grasbeck, 1958). Injecting vitamin B,, directly into one horn of the uterine lumen resulted in a marked accumulation of the isotope by the yolk sac of the injected horn and a higher concentration of the label in the organs of the respective fetuses. When similar experiments were performed in an attempt to evaluate the role of the yolk sac in iron transfer, no significant differences were observed between the fetuses of the injected and noninjected horns (Davies et nl., 1959). Thus, the B,, that appears in the uterine lumen is taken up, presumably, by the yolk sac, and transferred to the fetus. Although this is relatively clear evidence that the yolk sac possesses the capacity for such transfer, it does not provide information on the quantitative importance of this route under physiological conditions. Once the vitamin has accumulated in the absorptive cells, it is presumably transferred to the fetus via the vitelline vessels. Another possibility is diffusion across the exocoelom into the amniotic fluid,
366
DEREN,
PADYKULA,
AND
WILSON
Fetal swallowing of amniotic fluid has been shown to occur ( Wislocki, 1920; Windle, 1940), and thus vitamin B,, might conceivably be absorbed via the intestinal tract. Although the label was found in both the amniotic fluid and gastric juice, the concentration in the stomach exceeded that in the amniotic fluid. This indicates that the rate of water exit from the stomach exceeded the rate of vitamin B,? removal. These findings are similar to those made on gamma globulin concentration in gastric juice in the rabbit, an animal in which proteins are not transferred in significant quantities to the fetal circulation following per oral intubation of the fetus (Brambell et al., 195Ia,c). In the fetal rat, whose intestine is capable of absorbing gamma globulin, a concentration difference between gastric contents and amniotic fluid was not observed ( Halliday, 1955). These observations, together with the demonstration of absent IF from fetal rabbit intestine and the inability of fetal ileum to accumulate significant B12 in the absence of IF (Deren and Wilson, unpublished observations), are evidence that amniotic fluid swallowing is not an important mechanism for the transfer of B,, from the yolk sac to the fetal circulation. The effects of interrupting the vitelline circulation followed by injecting B,, into the mother were pursued in an attempt to estimate the roles of these two placental membranes. The lack of significant interference in BXz transfer following ligation of the vitelline vessel might lead one to conclude that the yolk sac does not contribute significantly to B,, transport in the rabbit. The conclusions to be drawn from this experiment, however, are limited, because the adequacy of the vitelline vessel interruption was not assessed by simultaneously injecting a specific gamma globulin known to traverse this route. Moreover, the parenteral administration of vitamin B1, may have led to an elevated free B,, level in the serum or to abnormal protein binding. The free B,, may be transported across the chorioallantoic placenta, whereas under more physiological circumstances, the extensive protein binding of B 12 may exclude it from this transfer mechanism. These in viva experiments, therefore, are open to many alternative explanations and are not considered decisive evidence against the yolk sac route of transfer. Thus, we have demonstrated that the rabbit yolk sac possesses an energy-dependent mechanism for vitamin B,, uptake in vitro and that this mechanism may be responsible for the net transfer to the
DEVELOPMENT
OF THE
MAMMALIAN
YOLK
SAC
367
fetus of Blz injected into the lumen of the uterus in viuo. The presence of the vitamin in the placental disc of all animals studied suggest that B,, is also accumulated by chorioallantoic placenta. From our experiments, however, it is difficult to assess the quantitative importance of the yolk sac route on the one hand and the chorioallantoic placenta on the other in the transfer of vitamin B,, from the maternal to the fetal circulation. SUMMARY
When rabbit yolk sacs were incubated in vitro with Co5’B12, there was a greater accumulation of the vitamin than that observed with fetal ileum of a similar age. Intrinsic factor, with a species specificity similar to that observed in fetal and adult intestine, stimulated B,, uptake severalfold. The uptake of B12, both in the presence and absence of IF, was depressed when DNP or iodoacetate was added to the incubation medium. The addition of 10 mM glucose overcame the DNP inhibition but did not reverse the iodoacetate effect. Following the parenteral administration of CO”‘B~~ to pregnant rabbits, the label appeared in the fetal organs and extrafetal membranes. The accumulation in the rabbit yolk sacs was particularly striking after 24-48 hours. Unilateral intrauterine injection of B,, led to more of the label appearing in the fetal organs of the injected horn than in the noninjected control horn. Ligation of the vitelline vessels did not appear to effect the transfer of parenterally administered CO”~B,~ to the fetal organs. The contribution of the chorioallantoic placenta and the yolk sac placenta of the rabbit in the transfer of B12 from mother to fetus is discussed. The developmental morphology of the visceral yolk sac of the rabbit was compared with that of the rat. The absorptive cells of the visceral endoderm possess surface specializations related to the uptake of macromolecules. The technical is most gratefully
assistance of Miss Ann G. Campbell acknowledged.
and Miss Angela
De Carlo
REFERENCES AMOHOSO, E. C. (1952). Placentation. In Marshall’s “Physiology of Reproduction” (A. S. Parkes, ed.), Vol. II. Longmans, Green, New York. BEHTCHER, R. \V., and MEYER, L. M. (1957). CO” vitamin Blz binding capacity of normal human serum. Proc. SW. Exptl. Biol. Med. 94, 169-171.
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AND
WILSON
BOGER, W. P., WRIGHT, L. D., BECK, G. D., and BAYNE, G. M. (1956). Vitamin B,x: correlation of serum concentrations and pregnancy. Proc. Sot. Exptl. Biol. Med. 92, 140-143. L. D., and BECK, G. D. (1957). DifBOGER, W. P., BAYNE, G. M., WRIGHT, ferential serum vitamin B,? concentration in mothers and infants. New EngZ. J. Med. 256, 1085-1087. BRAMBELL, F. W. R. ( 1958). The passive immunity of the young mammal. Biol. Reo. Cambridge Phil. Sot. 33, 488531. BRAMBELL, F. W. R., HEMMINGS, W. A., and ROWLANDS, W. T. (1948). The passage of antibodies from the maternal circulation into the embryo in rabbits. Proc. Roy. Sot. B135, 390-403. BRAMBELL, F. W. R., HEMMINGS, W. A., HENDERSON, M., PARRY, H. J., and ROWLANDS, W. T. ( 1949). The route of antibodies passing from the maternal to the foetal circulation in rabbits. Proc. Roy. Sot. B136, 131-144. BRAMBELL, F. W. R., HEMMINGS, G. P., HEMMINGS, W. A., HENDERSON, M., and ROWLANDS, W. T. (1951a). The route by which antibodies enter the circulation after infection of immune serum into the exocoel in foetal rabbits. Proc. Roy. Sot. B138, 188-195. BBAMBELL, F. W. R., HEMMINGS, W. A., and HENDERSON, M. (1951b). “Antibodies and Embryos.” Oxford Univ. Press (Athlone), London and New York. BRAMBELL, F. W. R., HE~IMINGS, W. A., HENDERSON, M., OAKLEY, C. L., and ROWLANDS, W. T. (1951c). The accumulation of antibodies in the stomach contents of foetal rabbits. Proc. Roy. Sot. B138, 195-204. CHOW, B. F., BARROWS, L., and LING, C. T. ( 1951). The distribution of radioactivity in the organs of the fetus or of young rats borne by mothers injected with vitamin B,? containing Cow. Arch. Biochem. Biophys. 34, 151-157. DAVIES, J. ( 1956). Histochemistry of the rabbit placenta. .l. Anat. 90, 135-142. DAVIES, J., BROWN, E. B., STEWART. D., TERRY, C. W., and SISSON, J. (1959). Transfer of radioactive iron via the placenta and accessory fetal membranes in the rabbit. Am. J. Physiol. 197, 87-92. DRAPER, R. L. (1920). Prenatal growth of guinea pig. Anat. Record 18, 369-392. GRXSBECK, R. ( 1958 ) Action of group blocking agents and enzymes on intrinsic factor preparations. Acta Chem. Stand. 12, 142-144. GREGORY, R/I. E., and HOLDSWORTH, E. S. (1959). A study of protein-binding in the metabolism of vitamin B,?. Biochem. J. 72, 549-556. HALLIDAY, R. (1955). Prenatal and postnatal transmission of passive immunity to young rats. Proc. Roy. Sot. B144, 427-430. HELLEGERS, A., OKUDA, K., NESBITT, R. E. L., SMITH, D. W., and CHOW, B. F. ( 1957). Vitamin BIz absorption in pregnancy and in the newborn. Am. J. CZin. Nutr. 5, 327-331. JACOBSON, W., and LUTWAK-MANN, C. (1956). The vitamin B,s content of the early rabbit embryo. J. Endocrinol. 14, XIX-XX. KARNOVSKY, M. J. ( 1961). Simple methods for “staining with lead” at high pH in electron microscopy. J. Biophys. Biochem. Cytol. 11, 729-732. BX concentration in KILLANDER, A., and VAHLQUIST, B. ( 1954). The vitamin serum from term and premature infants. Nerd. Med. 51, 777-779.
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KREBS, H. A., and HENSELEIT, K. ( 1932). Untersuchungen iiber die Harnstoffbildung im Tierkorper. Z. Physiol. Chem. 210, 33-66. MOSSMAN, H. W. ( 1937). Comparative morphogenesis of the foetal membranes and accessory uterine structures. Contrib. Embryol. Carnegie Inst. Wush. 26, 129-246. OKUIZIA, K., HELLEGEHS, A., and CHOW, B. F. (1956). Vitamin BIr serum level and pregnancy. Am. J. Clin. Nutr. 4, 440-443. PADYKULA, H. A., DEREN, J. J., and WILSON, T. H. (1966). Development of structure and function in the mammalian yolk sac. I. Developmental morphology and vitamin B,, uptake of the rat yolk sac. Dewlop. Bid. 3, 311-348. PALADE, G. E. ( 1952). A study of fixation for electron microscopy. J. Erptl. Med. 95, 285-298. RICE, E. G., HEHNDON, J. F., VAN LOON, E. J., and GREENBERG, S. M. (1958). Enhancement of vitamin B1z absorption by n-sorbital as measured by maternal and fetal tissue levels in pregnant rats. Am. J. Physiol. 193, 513-515. ROSENTHAL, H. L., and SAHATT, H. P. (1952). The determination of vitamin B,? activity in human serum. J. Biol. Chem. 199, 433-442. ROSENTHAL, H. L., and AUSTEN, S. (1962a). Vitamin B,, unsaturated binding capacity of sera from various animals. Proc. Sot. Exptl. Med. 109, 179-181. ROSENTHAL, H. L., O’BHIEN, G., and AUSTIN, S. (1962b). Factors affecting the binding of cyanocobalamine to serum proteins from various animals. Arch. Rio&em. Biophys. 99, 319-325. WILSON, T. H., and STHAUSS, E. W. (1959). Some species differences in the intrinsic factor stimulation of Blz uptake by small intestine in z;itro. Am. J. Physiol. 197, 926-928. WINDLE, W. F. (1940). “Physiology of the Fetus: Origin and Extent of Function in Prenatal Life.” Saunders, Philadelphia, Pennsylvania. WISLOCKI, G. B. (1920). Experimental studies on fetal absorption. Contrib. Embryol. (Carnegie Inst. Wash. Publ. 274) 11, 47-60. WOLFF, R., KAHLIN, R., and ROYER, P. (1952). Sur l’existence dun pouvoir de combinaison du serum pour la vitamine B,?. Compt. Rend. Sot. Bid. 146, 1008-1010.