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Amniotic fluid proteins: Evidence for the presence of fetal plasma glycoproteins in mouse amniotic fluid
Amniotic fluid proteins: Evidence for the presence of fetal plasma glycoproteins in mouse amniotic fluid DAVID
L.
ERNEST
F.
Cincinnati,
PH.D.*
GUSTINE,
PH.D.
ZIMMERMAN,
Ohio
The origin of amniotic fluid firoteins in the mouse was investigated. Analysis of amniotic fluid proteins by fiolyacrylamide disk gel electrofihoresis reueaZed 3 major groups of firoteins: 3 transferrins (Trl to 3), 5 fetoproteins designated Bl to 5, and albumin. During Days 13.5, 14.5, and 15.5 of gestation, T73 and B4 and 5 increased in concentration in fetal filasma: although these proteins also increased in the amniotic fluid, they showed a 24 to 48 hour delay. Evidence was #resented which established that T72 was synthesized by the fetus, and T73, by both the fetus and mother; however, B4 and 5 were synthesized exclusively by the fetus. Thus, @@roteins and some transferrins in amniotic fluid are poteins derived from the fetus. The evidence also suggested albumin was $rimarily synthesized in the mother and trans$orted to the fetus and amniotic fluid. It is suggested that the amniotic &id acts as a storehouse for secreted proteins. The existence of fetus-sjecific proteins in amniotic fluid of other species is also discussed.
ALT H o u G H a number of investigators have estabhshed that human amniotic fluid at term contains principally serum proteins,ll 2 no one has yet established whether these proteins originate from the fetus or the mother or both. Results from electrophoretic and immunologic studies of human amniotic
fluid and fetal and maternal serum suggest that amniotic proteins resemble fetal serum proteins3-5 ; in contrast, other studies indicate a similarity to maternal serum proteins.lp 2v 6 7 Therefore, it is uncertain whether the amniotic fluid proteins originate from the fetus or mother or both. It has been established that, as early as 3 months, the human fetus is capable of synthesizing all of its serum proteins, except immunoglobulin G,g-ll but none has been shown to be transported to the amniotic fluid. Behrman, Parer, and deLannoy* have studied the formation of amniotic fluid in monkeys from which the fetus had been removed. They concluded from their data that amniotic fluid is initialIy formed by a transudate of maternal plasma but begins to resemble a transudate of the fetal body fluids as the fetus contribtues an increasing amount of urine and other body secretions. In our report, we present evidence which clearly demonstrates the presence of fetus-specific proteins in the amniotic fluid of mice.
From the Children’s Hosfiital Research Foundation, Defiartments of Pediatrics and Pharmacology, University of Cincinnati. Su#ported by grants from The American Cancer Society (T39), The Pharmaceutical Manufacturers Association, and a Center Grant in Mental Retardation (HD-05221). F;ec;ived
for
jublication
September
Accepted
for
jublication
Afiril
I,
17, 1972.
Refirint requests: Dr, Ernest F. Zimmerman, Children’s Hosfiital Research Foundation, Elland and Bethesda Avenues, Cincinnati, Ohio 45229. *Present address: U. S. Regional Research Laboratory, University Pennsylvania 168OI.
Pasture Park,
553
554
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M,aterial
and
and
October
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Am. J. Obstet.
methods
Inbred Strain C3H/An mice (Cumberland View Farms, Clinton, Tennessee) were used in all experiments. Evidence of a vaginal plug the morning after mating was the criterion for conception,1z and this time was designated Day 0.5 of gestation. Embryos at Days 13.5, 14.5, and 15.5 were isolated with the amniotic sac still intact. Amniotic fluid was removed carefully with a syringe. Samples contaminated with blood were discarded. Blood was collected with heparinized capillary tubes from an incision in the fetal thoracic region and from an inincision in the adult heart. After the capillary tubes were sealed and centrifuged, the clear plasma was collected. Individual amniotic fluid and fetal plasma samples were pooled for each litter. Proteins were incubated with neuraminidase as previously described.13 Production of antibody (goat) to purified bl-3 proteins from Day 12.5 amniotic fluid and purification of ,61-3 proteins have been previously described. I6 Purification of calf fetuin has been detailedI while that of ,&!I to 5 (Day 15.5 amniotic fluid) and /34 and 5 (Day 18.5 fetal plasma) was similar to ,f31 to 3 . A double-diffusion test for immunoprecipitates was performed by adding 20 ~1 of sera or antigen to the appropriate well of a Cordis IDF Cell II. The immunodiffusion cell was incubated overnight at room temperature. Incorporation of 3H-leucine for 24 hours in vivo was carried out from Day 14.5 to Day 15.5. Each of 2 pregnant dams was injected intraperitoneally with 50 ,UC (0.10 ml.) of 3H-r,-leucine,* 54 c. per millimole every 2 hours, a total of 4 times. Amniotic fluid, fetal plasma, and adult plasma were collected 24 hours after the first injection, as described above. Incorporation of 3H-glucosamine into cultured embryos was achieved as follows. After uterine walls were carefully removed, Day 14.5 embryos (12 from 2 litters), with all extraembryonic membranes and placentas intact, were placed in culture dishes contain*Schwm/Mann,
Orangeburg,
New
York.
15, 1972
Gynecd.
ing culture medium.13 An injection of 10 ~1 of 3H-n-glucosamine (1 .l PC, 1.3 c. per millimole) was made into the amniotic fluid with a 30 gauge needle, with little or no leakage of fluid occurring. The embryos were incubated at 37O C. for 24 hours in an atmosphere of 5 per cent carbon dioxide, 35 per cent nitrogen, and 60 per cent oxygen. Embryonic sacs were carefully blotted, then disrupted to obtain the amniotic fluid. Analytic separation of proteins was performed by polyacrylamide disk gel electrophoresis according to the method of Ornsteinl* and Davis.15 It was not necessary to concentrate the amniotic fluid samples (protein concentration: 1.5 to 2.5 mg. per milliliter). Absorbance patterns of stained gels were recorded with a Model 2400 spectrophotometer* at 600 nm. ; radioactive proteins in gels were analyzed as described previously.*s Results
Fig. I illustrates the characteristic electrophoretic separation in polyacrylamide gels of proteins from Day 15.5 amniotic fluid, fetal plasma, and adult plasma (solid lines). The mouse transferrins (Trl, 2, and 3) at a relative mobility of 0.4 and albumin at a relative mobility of 1.0 have been identified previously in this electrophoresis system.16 The group of 5 fetoproteins designated ,6-l to 5 (apparently analogous to calf fetuinf (relative mobility 0.8) were present only in amniotic fluid and fetal plasma and were not detected in adult plasma. The p-proteins and transferrins have been characterized as glycoproteins, each group showing microheterogeneity since each contains varying amounts of sialic acid13p I6 (Zimmerman and Madappally, unpublished observations). Treatment of these proteins with neurami*Gilford
Instrument
Labs.,
Inc.,
Oberlin,
Ohio.
tThat @-proteins are analogous to calf fetuin is based on the following similarities: (I) They are present only in the fetus and not in the mother; (2) they are phana proteins; (3) they are glycoproteins, as shown by a positive periodic acid-Schiff reaction; (4) they have similar electrophmetic mobilities in polyacrylamide gels; (5) they both have sialic acid, except that calf fetuin contains ahout twentyfold more than &proteins: (6) they demonstrate decreased electrophoretie mobilities after incubation with neuraminidase; (7) they both are purified by carrying out the same fractionation procedure.‘P
voblme Number
114 4
nidase should result in decreased mobilities due to loss of negatively charged sialic acid. This prediction was confirmed as shown in Fig. 1 (dashed line). With amniotic fluid and fetal plasma, this treatment resulted in the appearance of Trl and 2, while, with adult plasma, only Tr2 was present; in each case, Tr3 was no longer present. In both amniotic fluid and fetal plasma, p4 and 5 were missing or greatly reduced and pl to 3 were increased in amount, but in adult plasma the small protein peak present where p-protein would be (relative mobility 0.75) did not decrease in mobility. Thus, the effect of neuraminidase on the electrophoretic mobilities of amniotic fluid proteins more closely resembled the effect on fetal plasma proteins than on adult plasma proteins. Note also that before neuraminidase treatment the concentration of Tr3 in ad& plasma was much greater than that in either amniotic fluid or fetal plasma. We have shown elsewhere that Tr3 and ~5 in fetal plasma increase dramatically in concentration between Day 14.5 and 15.5.13 In order to examine the relationship between amniotic fluid and fetal plasma proteins, we compared the relative concentration of the transfer&s and &proteins from both sources at 3 different times of development. Gel absorbance patterns from Days 13.5, 14.5, and 15.5 are shown in Fig. 2. In amniotic fluid, Tr3 increased slightly at each time, but Trl and 2 remained essentially constant; /35 was unchanged from Day 13.5 to 14.5 but increased in amount equal to /32, 3, and 4 by Day 15.5; albumin was constant to Day 14.5 but increased by Day 15.5. In fetal plasma, Tr3 was essentially constant for all 3 days,13 while both Trl and 2 decreased at each time, /34 and 5 increased and /31, 2, and 3 decreased at each time, and albumin was essentially constant. Thus, changes with development observed for Trl to 3 and p I to 5 in fetal plasma were also observed in amniotic fluid, although in amniotic fluid they appeared to be delayed 24 to 48 hours with respect to fetal plasma. To substantiate that the p-proteins of fetal plasma and amniotic fluid (which show sim-
Amniotic
0
0.2
0.4
RELATIVE
0.6
fluid
0.8
protems
I.0
555
I.2
MOBILITY
Fig. 1. Effect
of neuraminidase on the mobilities of amniotic fluid, fetal plasma, and adult plasma proteins in polyacrylamide gels. The sample gels contained 155, 95, and 140 pg of protein, respectively. The gels were scanned at a full-scale absorbance of 3.0. Note that the base lines are at different levels.
ilar electrophoretic mobilities) are the same an immunologic experiment was proteins, performed. It has been observed that at Day 18.5 of gestation the developmental changes of p-proteins in fetal plasma have progressed to the point where only p-4 and 5 are present (Zimmerman and Bowen, unpublished observations). In addition, only /31 to 3 are found in the amniotic fluid at Day 12.5. Therefore, an antiserum to purified /31 to 3 proteins (amniotic fluid) was preparedt6 and its cross-reactivity to p4 and 5 proteins of fetal plasma was tested. With the use of the double-immunodiffusion method (Fig. 3), pl to 3 (amniotic fluid) (Well I ) cross-reacted with antisera (center well), yielding a single precipitin line. When /34 and 5 proteins purified from fetal plasma (Day 18.5) were tested, a single precipitin line was also observed (Well 2). Since this line fused with the precipitin line of pl to 3 and showed no spurs, it is concluded that the /?-proteins from the two fetal tissues are
5.56
Gustine
and
October 15, 1972 Am. J. Obstet. Gymcal.
Zimmerman
0
.2
.4
.6
.8
I.0
RELATIVE Fig. 2. Changes in concentrations of amniotic ment. Sample gels for Days 13.5, 14.5, and pg of protein, respectively; fetal plasma-68,
0
.2
.4
.6
I.0
MOBILITY fluid and fetal plasma proteins during 15.5 contained: amniotic fluid-102, 90, 96, and 102 pg of protein, respectively.
developand 104
similar. Further controls were utilized: Amniotic fluid from Day 12.5 also gave a single precipitin line as expected (Well 3) as did purified ,Gl to 5 (amniotic fluid, Day 15.5, Well 6). Control sera (Well 4) did not cross-react with the proteins in Well 3, and purified calf fetuin (Well 5) did not cross-react with anti-/31 to 3 in the center well. Further evidence that pl to 3 antibody (amniotic fluid) cross-reacts with ,84 and 5 antigen (fetal plasma) comes from the observation that when the immunoprecipitate of this reaction was subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate the /34 and 5 protein band was found (Zimmerman and Bowen, unpublished observations). TO determine the site of synthesis (maternal versus fetal) of the amniotic fluid proteins, we subjected Day 14.5 dams (2) to a 24 hour incorporation of 3H-leucine. The radioactive proteins were analyzed by gel electrophoresis; the separation of these proteins is shown in Fig. 4. No radioactive
immunologically
Fig. 3. Double-immunodiffusion test for immunoprecipitability of p-proteins and calf fetuin by antisera prepared against /31 to 3 proteins (Day 12.5 amniotic fluid). Center well, antisera; Well 1, purified /31 to 3 from Day 12.5 amniotic fluid; Well 2, purified p4 and 5 from Day 18.5 fetal plasma; Well 3, Day 12.5 amniotic fluid; Well 4, control sera; Well 5, purified calf fetuin; Well 6, purified /31 to 5 from Day 15.5 amniotic fluid. All antigens were present in a concentration of 100 /Lg per milliliter except Day 12.5 amniotic fluid which was 300 Pg per milliliter.
.8
Volune Number
114 4
,,I
1
17,
1
Amniotic
fluid
!
1
1
,
proteins
557
- ADULT PLASMA
FETAL PLASMA
300 - . Alb
f,5
250-
Alb
Tr3
”
0
20
40
0 GEL
40 20 SLICE NO
0
20
i
40
Fig. 4. Incorporation of sH-leucine in viva into proteins of Day 15.5 amniotic fluid, Day fetal nlasma. and adult Dlasma. The amount of nrotein for each ~1 was: amniotic fluid pg, f;tal p&ma 160 pg: and adult plasma 223. pg. Location of-transferrin, P-protein, albumin was determined from duplicate stained gels.
,&protein was present in the adult, while only radioactive /34 and 5 were present in amniotic fluid and fetal plasma. Radioactive transferrin (Tr3) and albumin were present in all three fluids. The identity of the radioactive peak from amniotic fluid at Gel Slice 52 has not been investigated. These results suggested, but did not prove, that p-protein was synthesized by the fetus and subsequently appeared in the amniotic fluid. To determine if these proteins were synthesized by the fetus rather than by the mother and transported to the fetus, Day 14.5 fetuses were isolated with the amniotic sacs and placentas intact and incubated for 24 hours in the presence of 3H-glucosamine. Then the amniotic fluid was isolated by breaking the membranes, and the proteins were separated by gel electrophoresis. Results in the upper panel of Fig. 5 show that a major peak of radioactivity corresponded to b4 and 5. In addition, some radioactivity was present at positions corresponding to Trl-3 and albumin. Finally, a major radioactive peak was found at Gel Slice 52 which could be free 3H-glucosamine or a radioactive protein. Evidence that the radioactivity which migrated with the absorbance peak of ,&protein was incorporated into the
15.5 100 and
protein and not nonspecifically absorbed to it was the following: 3H-Glucosamine was mixed with unlabeled amniotic fluid and subjected to electrophoresis. No radioactivity was associated with the p-protein. To establish that the radioactive proteins were /34 and 5, an aliquot of the amniotic fluid was incubated with neuraminidase (Fig. 5, middle panel). Both the radioactivity and absorbance peaks of p-protein were decreased in mobility, indicating conversion to /31 to 3.13 In addition, radioactive Tr3 was converted to Trl and 2. To show that the shift in mobilities was real, amniotic fluid treated with neuraminidase was mixed with untreated material and electrophoresed. The split peak of radioactivity (bottom panel) confirmed that /34 and 5 were present in the untreated portion and that pl to 3 were present in the neuraminidase-treated portion. We could react the 3H-p-proteins with /31, 2, and 3 antibody and show that the immunoprecipitated p-proteins were radioactive. Although in this experiment the fetuses were incubated for 24 hours, and thus possibly dead by this time, there was no obvious tissue degeneration when they were removed from the incubator. In later experiments, similar isotope incorporation into
558
Gustine
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October 15, 1972 Am. J. Obstet. Gynecol.
Zimmerman
GEL
SLICE
NO
Fig. 5. Incorporation of sH-glucosamine in vitro into Day 14.5 to 15.5 amniotic fluid proteins. Embryos were incubated as described in Material and methods; neuraminidase treatment (upper and middle panels) was carried out as described previously.13 Before the +neuraminidase sample was mixed with the -neuraminidase sample (lower panel), the pH was adjusted to 7.0 to 7.5 in order to inactivate the enzyme.
,&proteins was obtained from incubation periods of 1, 3, and 8 hours. Under those conditions, the fetuses were probably still alive. In any case, under the conditions of incubation with 60 per cent oxygen, incorporation of 3H-leucine (I, 3, 20 hours) into t0ta.l embryonic protein and /3-amniotic protein was nearly linear, indicating that these culture condiGons mimic the physiologic synthesis of p-protein. In summary, these data establish that p-protein was synthesized by the fetus. Comment As stated in the introduction, human amniotic fluid proteins consist primarily of serum proteins, but their origin is uncertain; they may come from the mother or the fetus. Our experiments were carried out with the mouse to study this problem. The data presented in Figs. 1 and 2 established that pl to 5 were present in both fetal plasma and amniotic fluid but not in maternal plasma. Moreover, ,01 to 5 from both sources
showed the same sensitivity to neuraminidase, and both showed developmental changes immunochemical experi(Fig. 2). Finally, ments indicated that p-proteins from fetal plasma and amniotic fluid were antigenically similar (Fig. 3). The results from incorporation of 3H-leucine in vivo (Fig. 4) also indicated that f3-protein was present only in fetus-associated fluids and further suggested that &protein was synthesized by fetal Gssues. These results, however, did not rule out the possibility that p-protein was synthesized by the mother and transported to the fetus. That /?-protein was, in fact, not synthesized by the mother was conclusively demonstrated by incorporation of 3H-glucosamine into p4 and 5 by embryos in vitro (Fig. 5, top panel), by the sensitivity of radioactive p-protein to neuraminidase (Fig. 5, middle of the radiopanel 1, and by precipitation active proteins by amniotic anti-PI, 2 and 3. Thus, since pl to 5 proteins were present in fetal plasma and amniotic fluid but not maternal plasma and since /?4 and 5 were synthesized by isolated fetuses, the ,&proteins present in amniotic fluid must be fetal proteins. The terms “fetal origin” and “fetal synthesis” mean that the proteins were synthesized in fetus-associated tissues and cellsthat is, the fetus itself, the placenta, the amnion, or cells in the amniotic fluid. Regardless of the specific fetal origin, the proteins were not synthesized by the mother. It is also likely that at least some of the transferrins in amniotic fluid are fetal proteins, since very little of Trl and 2 are seen in the adult and Tr3 was synthesized in vitro (Fig. 5). However, in these experiments, the incorporation of 3H-glucosamine into Tr3 (and possibly Trl and 2) was close to the background range and much less than that of the p-proteins. This is not surprising since the proportion of carbohydrate in human transferrin (6 per cent) I7 is much lower than in calf fetuin (26 per cent) .I8 By incorporation of 14C-leucine and 3H-glucosamine into Day 12.5 and Day 14.5 mouse embryos in vitro, we have shown significant amounts
of
3, respectively.
14C-radioacGvity
I3 Thus,
in
Trl
Trl,
2,
and
to 3 are synthe-
Vdume Number
114 4
sized in the fetus. However, since Tr3 is also found in adult plasma in high concentrations (Fig. 1) and is actively synthesized by the mother (Fig. 4), it is also likely that some Tr3 in the amniotic fluid comes from the mother, That such a possibility could exist is supported by the fact that maternal serum transferrins of the rhesus monkey are found in the amniotic fluid.19 It seems probable that most albumin in the amniotic fluid was derived from the mother. The results from incorporation of sH-leucine in vivo (Fig. 4) showed active synthesis of albumin, which was present as radioactive albumin in amniotic fluid, fetal plasma, and adult plasma. By incorporation of 14C-leucine into mouse embryos in vitro,ls we have also found a minimal amount of radioactive albumin in amniotic fluid as compared to p-protein. Therefore, most of the albumin present in amniotic fluid must be synthesized by the mother and transported to the amniotic fluid, which is in accord with the result found in the rhesus monkey.2o It has been suggested by Bangham*” that the histologic features of the amnion and syncytial trophoblast of the placenta of the fetal monkey are similar to those of the entodermal lining of the yolk sac of the rabbit and the entoderm of the rat and that these membranes are likely to be responsible for the selective transmission of albumin. Whether these different cellular layers have the same absorptive capacity for proteins still remains to be determined. In any case, the selective transmission of maternal serum proteins across fetal membranes into amniotic fluid has also been shown in the human being.z1 A radioactive peak was found in the amniotic fluid which migrated faster than albumin (Figs. 4 and 5). Although the identity of this protein is not known, it could correspond to the amniotic fluid protein described in the monkey,19 which has a molecular weight of 35,000 and thus would be expected to migrate faster than albumin. Our results are consistent with the concept that the amniotic fluid acts as a reservoir for secreted proteins. The delay in developmental changes of plasma proteins in the
Amniotic
fluid proteins
559
amniotic fluid (Fig. 2) could reflect a much slower turnover of these proteins as compared to the fetal plasma proteins. These proteins may be transported by transudation to the amniotic fluid from the fetuP> M and through the amnion and placenta from the mother.z0 Some investigators have concluded that all human amniotic proteins are derived from the mother.‘, Z* G This may be due to the high concentration of albumin (60 per cent) which has been used as a marker, as well as the absence of other unique markers such as the 5 p-proteins and the 3 transferrins found in mice. Another difficulty in interpretation of both the human and monkey studies may have arisen from the lack of precise time-course analysis carried out during development. Thus, in the primates, with their attendant longer fetal periods,* the fetoprotein (e.g., c+fetoprotein analogous to mouse ,&protein and calf fetuin) may have already decreased to a very low concentration in the fetal serum”, *’ and thus in the amniotic fluid by the time of analysis. Second, earlier studies may not have appreciated the microheterogeneity that exists in these fetoproteins, e.g., calf fetuinZG; and the developmental changes occurring in the fetoproteins, e.g., mouse ,8-protein.13 Thus, primate fetoproteins in fetal plasma could have a different electrophoretic charge, due to a different quantity of sialic acid present, than the same type of protein that was synthesized earlier in development and is residing in the amniotic fluid (e.g., Fig. 2).lZ Although we have established that ,81 to 5 are fetal proteins, it is not possible to determine from our experiments their site of synthesis. Dancis, Braverman, and LindlO have shown that except for the y-globulins, human fetal serum proteins are synthesized by the fetal liver; however, synthesis by the placenta of plasma proteins, although low, was significant except for albumin. However, the site of synthesis of fetal plasma proteins in the mouse has not yet been established, and also *Day 15 of gestation in the mouse is comparable to about 8 weeks in the w~rnan,~~ while birth of the mouse takes place at about Day 20.
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the mechanism of regulation of their synthesis remains to be answered. Although it is difficult to extrapolate data from the mouse to the human being, nevertheless, an extraordinary similarity exists for the two fetoproteins: Both have a molecular weight of 70,000, and human fetoprotein contains two sialic residues per polypeptide chain while mouse p-proteins contain 0 to 2 sialic acid residues according to their degree of “biologic maturity” (Zimmerman, Bowen, and Madappally, unpublished observations) . Finally, the mouse fetoprotein immunologically cross-reacts with the human
one.z8 Thus, the results from this present work in mice have produced additional data concerning the origin of amniotic fluid proteins, the similarity of which could be further tested in human beings. If specific proteins in human amniotic fluid arc fetal in origin, the value of amniocentesis in the diagnosis of genetically inherited disorders could possibly be enhanced. For example, changes in the carbohydrate or polypeptide structures of the fetus-specific proteins in the amniotic fluid could have occurred in the diseased state, and these proteins found in the amniotic fluid could be analyzed for such changes.
REFERENCES
1.
Abbas, 1: 476,
2.
Derrin@on,
7. 8. 9. 10. Il. 12. 13.
T. M., 1960.
and
Tovey,
J. E.:
Br.
Med.
J.
14.
M. M., and Soothill, J. F.: J. Obstet. Gynaecol. Br. Commonw. 68: 755, 1961. Brzezinski, A., Sadovsky, E., and Shafrir, E.: AM. J. OBSTET. GYNECOL, 82: 800, 1961. Mendelbaum, B., and Evans, T. N.: AM. J. OBSTET.GYNECOL. 104:365.1969. Gitlin, D., and Boesman, M:: J. Clin. Invest. 45: 1826, 1966. Stander, .R. W., McNutt, C. C., Barton, D. M.. and Werts. C. E.: Am. ”T. Clin. Pathol. 42:’ 125, 1964. ’ Fischbacher, P. H., and Quinlivan, W. L. G.: AM. J. OBSTET. GYNECOL. 108: 1051, 1970. Behrman, R. E., Parer, J. T., and deLannoy, C. W.: Nature 214: 678, 1967. Rausen, A. R., Gerald, P. S., and Diamond, L. K.: Nature 192: 182, 1961. Dancis, J., Braverman, N., and Lind, J.: J. Clin. Invest, 36: 398. 1957. Hirschfeld, J., and Lunell, N. 0.: Nature 196: 1220, 1962. Walker, B. E., and Fraser, F. C.: J. Embryol. Exp. Morphol. 4: 176, 1956. Gustine, D. L., and Zimmerman, E. F.: Proc. Natl. Acad. Sci. U. S, A.: Submitted for publication,
15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
2.5.
Ornstein, L.: Ann. N, Y. Acad. Sci. 121: 321, 1964. Davis, B. J.: Ann. N. Y. Acad. Sci. 121: 404, 1964. Gustine, D. L., and Zimmerman, E. F.: Teratology. In press. Schultze$ H. E., Schmidtberger, R., and Haupt, H.: Biochem. Z, 329: 490, 1958. Spiro, R. G.: J. Biol. Chem. 235: 2860, 1960. Bangham, D. R., Hobbs, K. R,, and Tee, D. E. H.: J. Physiol. 158: 207, 1961. Bangham, D. R.: J. Physiol. 153: 265, 1960. Usategui-Gomez, M., and Morgan, D. F.: Proc. Sot. Exp. Biol. Med. 125: 819, 1967. Setnika, I., Agostoni, E., and Taglietti, A.: Proc. Sot. EXD. Biol. Med. 101: 842. 1959. Shaw, R. E., and Marriot, H. J.: J. ‘Obstet. Gynaecol. Br. Emp. 56: 1004, 1949. Rugh, R.: The Mouse-Its Reproduction and Development, Minneapolis, 1968, Burgess Publishing Company, py 297. Bergstrand. C. G.. and Czar. B.: Stand. T. Clin. Lab. Invest. 9: 277, 1957. Page, M.: Biochim. Biophys. Acta 236: 571, 1971. Ruoslahti, E., and Ruoslahti, M,: Int. J, Cancer 7: 218, 1971. Abelev, G. I.: Cancer Res. 28: 1344, 1968. ”
26. 27. 28.
L.
ERNEST
F.
Cincinnati,
PH.D.*
GUSTINE,
PH.D.
ZIMMERMAN,
Ohio
The origin of amniotic fluid firoteins in the mouse was investigated. Analysis of amniotic fluid proteins by fiolyacrylamide disk gel electrofihoresis reueaZed 3 major groups of firoteins: 3 transferrins (Trl to 3), 5 fetoproteins designated Bl to 5, and albumin. During Days 13.5, 14.5, and 15.5 of gestation, T73 and B4 and 5 increased in concentration in fetal filasma: although these proteins also increased in the amniotic fluid, they showed a 24 to 48 hour delay. Evidence was #resented which established that T72 was synthesized by the fetus, and T73, by both the fetus and mother; however, B4 and 5 were synthesized exclusively by the fetus. Thus, @@roteins and some transferrins in amniotic fluid are poteins derived from the fetus. The evidence also suggested albumin was $rimarily synthesized in the mother and trans$orted to the fetus and amniotic fluid. It is suggested that the amniotic &id acts as a storehouse for secreted proteins. The existence of fetus-sjecific proteins in amniotic fluid of other species is also discussed.
ALT H o u G H a number of investigators have estabhshed that human amniotic fluid at term contains principally serum proteins,ll 2 no one has yet established whether these proteins originate from the fetus or the mother or both. Results from electrophoretic and immunologic studies of human amniotic
fluid and fetal and maternal serum suggest that amniotic proteins resemble fetal serum proteins3-5 ; in contrast, other studies indicate a similarity to maternal serum proteins.lp 2v 6 7 Therefore, it is uncertain whether the amniotic fluid proteins originate from the fetus or mother or both. It has been established that, as early as 3 months, the human fetus is capable of synthesizing all of its serum proteins, except immunoglobulin G,g-ll but none has been shown to be transported to the amniotic fluid. Behrman, Parer, and deLannoy* have studied the formation of amniotic fluid in monkeys from which the fetus had been removed. They concluded from their data that amniotic fluid is initialIy formed by a transudate of maternal plasma but begins to resemble a transudate of the fetal body fluids as the fetus contribtues an increasing amount of urine and other body secretions. In our report, we present evidence which clearly demonstrates the presence of fetus-specific proteins in the amniotic fluid of mice.
From the Children’s Hosfiital Research Foundation, Defiartments of Pediatrics and Pharmacology, University of Cincinnati. Su#ported by grants from The American Cancer Society (T39), The Pharmaceutical Manufacturers Association, and a Center Grant in Mental Retardation (HD-05221). F;ec;ived
for
jublication
September
Accepted
for
jublication
Afiril
I,
17, 1972.
Refirint requests: Dr, Ernest F. Zimmerman, Children’s Hosfiital Research Foundation, Elland and Bethesda Avenues, Cincinnati, Ohio 45229. *Present address: U. S. Regional Research Laboratory, University Pennsylvania 168OI.
Pasture Park,
553
554
Gustine
M,aterial
and
and
October
Zimmerman
Am. J. Obstet.
methods
Inbred Strain C3H/An mice (Cumberland View Farms, Clinton, Tennessee) were used in all experiments. Evidence of a vaginal plug the morning after mating was the criterion for conception,1z and this time was designated Day 0.5 of gestation. Embryos at Days 13.5, 14.5, and 15.5 were isolated with the amniotic sac still intact. Amniotic fluid was removed carefully with a syringe. Samples contaminated with blood were discarded. Blood was collected with heparinized capillary tubes from an incision in the fetal thoracic region and from an inincision in the adult heart. After the capillary tubes were sealed and centrifuged, the clear plasma was collected. Individual amniotic fluid and fetal plasma samples were pooled for each litter. Proteins were incubated with neuraminidase as previously described.13 Production of antibody (goat) to purified bl-3 proteins from Day 12.5 amniotic fluid and purification of ,61-3 proteins have been previously described. I6 Purification of calf fetuin has been detailedI while that of ,&!I to 5 (Day 15.5 amniotic fluid) and /34 and 5 (Day 18.5 fetal plasma) was similar to ,f31 to 3 . A double-diffusion test for immunoprecipitates was performed by adding 20 ~1 of sera or antigen to the appropriate well of a Cordis IDF Cell II. The immunodiffusion cell was incubated overnight at room temperature. Incorporation of 3H-leucine for 24 hours in vivo was carried out from Day 14.5 to Day 15.5. Each of 2 pregnant dams was injected intraperitoneally with 50 ,UC (0.10 ml.) of 3H-r,-leucine,* 54 c. per millimole every 2 hours, a total of 4 times. Amniotic fluid, fetal plasma, and adult plasma were collected 24 hours after the first injection, as described above. Incorporation of 3H-glucosamine into cultured embryos was achieved as follows. After uterine walls were carefully removed, Day 14.5 embryos (12 from 2 litters), with all extraembryonic membranes and placentas intact, were placed in culture dishes contain*Schwm/Mann,
Orangeburg,
New
York.
15, 1972
Gynecd.
ing culture medium.13 An injection of 10 ~1 of 3H-n-glucosamine (1 .l PC, 1.3 c. per millimole) was made into the amniotic fluid with a 30 gauge needle, with little or no leakage of fluid occurring. The embryos were incubated at 37O C. for 24 hours in an atmosphere of 5 per cent carbon dioxide, 35 per cent nitrogen, and 60 per cent oxygen. Embryonic sacs were carefully blotted, then disrupted to obtain the amniotic fluid. Analytic separation of proteins was performed by polyacrylamide disk gel electrophoresis according to the method of Ornsteinl* and Davis.15 It was not necessary to concentrate the amniotic fluid samples (protein concentration: 1.5 to 2.5 mg. per milliliter). Absorbance patterns of stained gels were recorded with a Model 2400 spectrophotometer* at 600 nm. ; radioactive proteins in gels were analyzed as described previously.*s Results
Fig. I illustrates the characteristic electrophoretic separation in polyacrylamide gels of proteins from Day 15.5 amniotic fluid, fetal plasma, and adult plasma (solid lines). The mouse transferrins (Trl, 2, and 3) at a relative mobility of 0.4 and albumin at a relative mobility of 1.0 have been identified previously in this electrophoresis system.16 The group of 5 fetoproteins designated ,6-l to 5 (apparently analogous to calf fetuinf (relative mobility 0.8) were present only in amniotic fluid and fetal plasma and were not detected in adult plasma. The p-proteins and transferrins have been characterized as glycoproteins, each group showing microheterogeneity since each contains varying amounts of sialic acid13p I6 (Zimmerman and Madappally, unpublished observations). Treatment of these proteins with neurami*Gilford
Instrument
Labs.,
Inc.,
Oberlin,
Ohio.
tThat @-proteins are analogous to calf fetuin is based on the following similarities: (I) They are present only in the fetus and not in the mother; (2) they are phana proteins; (3) they are glycoproteins, as shown by a positive periodic acid-Schiff reaction; (4) they have similar electrophmetic mobilities in polyacrylamide gels; (5) they both have sialic acid, except that calf fetuin contains ahout twentyfold more than &proteins: (6) they demonstrate decreased electrophoretie mobilities after incubation with neuraminidase; (7) they both are purified by carrying out the same fractionation procedure.‘P
voblme Number
114 4
nidase should result in decreased mobilities due to loss of negatively charged sialic acid. This prediction was confirmed as shown in Fig. 1 (dashed line). With amniotic fluid and fetal plasma, this treatment resulted in the appearance of Trl and 2, while, with adult plasma, only Tr2 was present; in each case, Tr3 was no longer present. In both amniotic fluid and fetal plasma, p4 and 5 were missing or greatly reduced and pl to 3 were increased in amount, but in adult plasma the small protein peak present where p-protein would be (relative mobility 0.75) did not decrease in mobility. Thus, the effect of neuraminidase on the electrophoretic mobilities of amniotic fluid proteins more closely resembled the effect on fetal plasma proteins than on adult plasma proteins. Note also that before neuraminidase treatment the concentration of Tr3 in ad& plasma was much greater than that in either amniotic fluid or fetal plasma. We have shown elsewhere that Tr3 and ~5 in fetal plasma increase dramatically in concentration between Day 14.5 and 15.5.13 In order to examine the relationship between amniotic fluid and fetal plasma proteins, we compared the relative concentration of the transfer&s and &proteins from both sources at 3 different times of development. Gel absorbance patterns from Days 13.5, 14.5, and 15.5 are shown in Fig. 2. In amniotic fluid, Tr3 increased slightly at each time, but Trl and 2 remained essentially constant; /35 was unchanged from Day 13.5 to 14.5 but increased in amount equal to /32, 3, and 4 by Day 15.5; albumin was constant to Day 14.5 but increased by Day 15.5. In fetal plasma, Tr3 was essentially constant for all 3 days,13 while both Trl and 2 decreased at each time, /34 and 5 increased and /31, 2, and 3 decreased at each time, and albumin was essentially constant. Thus, changes with development observed for Trl to 3 and p I to 5 in fetal plasma were also observed in amniotic fluid, although in amniotic fluid they appeared to be delayed 24 to 48 hours with respect to fetal plasma. To substantiate that the p-proteins of fetal plasma and amniotic fluid (which show sim-
Amniotic
0
0.2
0.4
RELATIVE
0.6
fluid
0.8
protems
I.0
555
I.2
MOBILITY
Fig. 1. Effect
of neuraminidase on the mobilities of amniotic fluid, fetal plasma, and adult plasma proteins in polyacrylamide gels. The sample gels contained 155, 95, and 140 pg of protein, respectively. The gels were scanned at a full-scale absorbance of 3.0. Note that the base lines are at different levels.
ilar electrophoretic mobilities) are the same an immunologic experiment was proteins, performed. It has been observed that at Day 18.5 of gestation the developmental changes of p-proteins in fetal plasma have progressed to the point where only p-4 and 5 are present (Zimmerman and Bowen, unpublished observations). In addition, only /31 to 3 are found in the amniotic fluid at Day 12.5. Therefore, an antiserum to purified /31 to 3 proteins (amniotic fluid) was preparedt6 and its cross-reactivity to p4 and 5 proteins of fetal plasma was tested. With the use of the double-immunodiffusion method (Fig. 3), pl to 3 (amniotic fluid) (Well I ) cross-reacted with antisera (center well), yielding a single precipitin line. When /34 and 5 proteins purified from fetal plasma (Day 18.5) were tested, a single precipitin line was also observed (Well 2). Since this line fused with the precipitin line of pl to 3 and showed no spurs, it is concluded that the /?-proteins from the two fetal tissues are
5.56
Gustine
and
October 15, 1972 Am. J. Obstet. Gymcal.
Zimmerman
0
.2
.4
.6
.8
I.0
RELATIVE Fig. 2. Changes in concentrations of amniotic ment. Sample gels for Days 13.5, 14.5, and pg of protein, respectively; fetal plasma-68,
0
.2
.4
.6
I.0
MOBILITY fluid and fetal plasma proteins during 15.5 contained: amniotic fluid-102, 90, 96, and 102 pg of protein, respectively.
developand 104
similar. Further controls were utilized: Amniotic fluid from Day 12.5 also gave a single precipitin line as expected (Well 3) as did purified ,Gl to 5 (amniotic fluid, Day 15.5, Well 6). Control sera (Well 4) did not cross-react with the proteins in Well 3, and purified calf fetuin (Well 5) did not cross-react with anti-/31 to 3 in the center well. Further evidence that pl to 3 antibody (amniotic fluid) cross-reacts with ,84 and 5 antigen (fetal plasma) comes from the observation that when the immunoprecipitate of this reaction was subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate the /34 and 5 protein band was found (Zimmerman and Bowen, unpublished observations). TO determine the site of synthesis (maternal versus fetal) of the amniotic fluid proteins, we subjected Day 14.5 dams (2) to a 24 hour incorporation of 3H-leucine. The radioactive proteins were analyzed by gel electrophoresis; the separation of these proteins is shown in Fig. 4. No radioactive
immunologically
Fig. 3. Double-immunodiffusion test for immunoprecipitability of p-proteins and calf fetuin by antisera prepared against /31 to 3 proteins (Day 12.5 amniotic fluid). Center well, antisera; Well 1, purified /31 to 3 from Day 12.5 amniotic fluid; Well 2, purified p4 and 5 from Day 18.5 fetal plasma; Well 3, Day 12.5 amniotic fluid; Well 4, control sera; Well 5, purified calf fetuin; Well 6, purified /31 to 5 from Day 15.5 amniotic fluid. All antigens were present in a concentration of 100 /Lg per milliliter except Day 12.5 amniotic fluid which was 300 Pg per milliliter.
.8
Volune Number
114 4
,,I
1
17,
1
Amniotic
fluid
!
1
1
,
proteins
557
- ADULT PLASMA
FETAL PLASMA
300 - . Alb
f,5
250-
Alb
Tr3
”
0
20
40
0 GEL
40 20 SLICE NO
0
20
i
40
Fig. 4. Incorporation of sH-leucine in viva into proteins of Day 15.5 amniotic fluid, Day fetal nlasma. and adult Dlasma. The amount of nrotein for each ~1 was: amniotic fluid pg, f;tal p&ma 160 pg: and adult plasma 223. pg. Location of-transferrin, P-protein, albumin was determined from duplicate stained gels.
,&protein was present in the adult, while only radioactive /34 and 5 were present in amniotic fluid and fetal plasma. Radioactive transferrin (Tr3) and albumin were present in all three fluids. The identity of the radioactive peak from amniotic fluid at Gel Slice 52 has not been investigated. These results suggested, but did not prove, that p-protein was synthesized by the fetus and subsequently appeared in the amniotic fluid. To determine if these proteins were synthesized by the fetus rather than by the mother and transported to the fetus, Day 14.5 fetuses were isolated with the amniotic sacs and placentas intact and incubated for 24 hours in the presence of 3H-glucosamine. Then the amniotic fluid was isolated by breaking the membranes, and the proteins were separated by gel electrophoresis. Results in the upper panel of Fig. 5 show that a major peak of radioactivity corresponded to b4 and 5. In addition, some radioactivity was present at positions corresponding to Trl-3 and albumin. Finally, a major radioactive peak was found at Gel Slice 52 which could be free 3H-glucosamine or a radioactive protein. Evidence that the radioactivity which migrated with the absorbance peak of ,&protein was incorporated into the
15.5 100 and
protein and not nonspecifically absorbed to it was the following: 3H-Glucosamine was mixed with unlabeled amniotic fluid and subjected to electrophoresis. No radioactivity was associated with the p-protein. To establish that the radioactive proteins were /34 and 5, an aliquot of the amniotic fluid was incubated with neuraminidase (Fig. 5, middle panel). Both the radioactivity and absorbance peaks of p-protein were decreased in mobility, indicating conversion to /31 to 3.13 In addition, radioactive Tr3 was converted to Trl and 2. To show that the shift in mobilities was real, amniotic fluid treated with neuraminidase was mixed with untreated material and electrophoresed. The split peak of radioactivity (bottom panel) confirmed that /34 and 5 were present in the untreated portion and that pl to 3 were present in the neuraminidase-treated portion. We could react the 3H-p-proteins with /31, 2, and 3 antibody and show that the immunoprecipitated p-proteins were radioactive. Although in this experiment the fetuses were incubated for 24 hours, and thus possibly dead by this time, there was no obvious tissue degeneration when they were removed from the incubator. In later experiments, similar isotope incorporation into
558
Gustine
and
October 15, 1972 Am. J. Obstet. Gynecol.
Zimmerman
GEL
SLICE
NO
Fig. 5. Incorporation of sH-glucosamine in vitro into Day 14.5 to 15.5 amniotic fluid proteins. Embryos were incubated as described in Material and methods; neuraminidase treatment (upper and middle panels) was carried out as described previously.13 Before the +neuraminidase sample was mixed with the -neuraminidase sample (lower panel), the pH was adjusted to 7.0 to 7.5 in order to inactivate the enzyme.
,&proteins was obtained from incubation periods of 1, 3, and 8 hours. Under those conditions, the fetuses were probably still alive. In any case, under the conditions of incubation with 60 per cent oxygen, incorporation of 3H-leucine (I, 3, 20 hours) into t0ta.l embryonic protein and /3-amniotic protein was nearly linear, indicating that these culture condiGons mimic the physiologic synthesis of p-protein. In summary, these data establish that p-protein was synthesized by the fetus. Comment As stated in the introduction, human amniotic fluid proteins consist primarily of serum proteins, but their origin is uncertain; they may come from the mother or the fetus. Our experiments were carried out with the mouse to study this problem. The data presented in Figs. 1 and 2 established that pl to 5 were present in both fetal plasma and amniotic fluid but not in maternal plasma. Moreover, ,01 to 5 from both sources
showed the same sensitivity to neuraminidase, and both showed developmental changes immunochemical experi(Fig. 2). Finally, ments indicated that p-proteins from fetal plasma and amniotic fluid were antigenically similar (Fig. 3). The results from incorporation of 3H-leucine in vivo (Fig. 4) also indicated that f3-protein was present only in fetus-associated fluids and further suggested that &protein was synthesized by fetal Gssues. These results, however, did not rule out the possibility that p-protein was synthesized by the mother and transported to the fetus. That /?-protein was, in fact, not synthesized by the mother was conclusively demonstrated by incorporation of 3H-glucosamine into p4 and 5 by embryos in vitro (Fig. 5, top panel), by the sensitivity of radioactive p-protein to neuraminidase (Fig. 5, middle of the radiopanel 1, and by precipitation active proteins by amniotic anti-PI, 2 and 3. Thus, since pl to 5 proteins were present in fetal plasma and amniotic fluid but not maternal plasma and since /?4 and 5 were synthesized by isolated fetuses, the ,&proteins present in amniotic fluid must be fetal proteins. The terms “fetal origin” and “fetal synthesis” mean that the proteins were synthesized in fetus-associated tissues and cellsthat is, the fetus itself, the placenta, the amnion, or cells in the amniotic fluid. Regardless of the specific fetal origin, the proteins were not synthesized by the mother. It is also likely that at least some of the transferrins in amniotic fluid are fetal proteins, since very little of Trl and 2 are seen in the adult and Tr3 was synthesized in vitro (Fig. 5). However, in these experiments, the incorporation of 3H-glucosamine into Tr3 (and possibly Trl and 2) was close to the background range and much less than that of the p-proteins. This is not surprising since the proportion of carbohydrate in human transferrin (6 per cent) I7 is much lower than in calf fetuin (26 per cent) .I8 By incorporation of 14C-leucine and 3H-glucosamine into Day 12.5 and Day 14.5 mouse embryos in vitro, we have shown significant amounts
of
3, respectively.
14C-radioacGvity
I3 Thus,
in
Trl
Trl,
2,
and
to 3 are synthe-
Vdume Number
114 4
sized in the fetus. However, since Tr3 is also found in adult plasma in high concentrations (Fig. 1) and is actively synthesized by the mother (Fig. 4), it is also likely that some Tr3 in the amniotic fluid comes from the mother, That such a possibility could exist is supported by the fact that maternal serum transferrins of the rhesus monkey are found in the amniotic fluid.19 It seems probable that most albumin in the amniotic fluid was derived from the mother. The results from incorporation of sH-leucine in vivo (Fig. 4) showed active synthesis of albumin, which was present as radioactive albumin in amniotic fluid, fetal plasma, and adult plasma. By incorporation of 14C-leucine into mouse embryos in vitro,ls we have also found a minimal amount of radioactive albumin in amniotic fluid as compared to p-protein. Therefore, most of the albumin present in amniotic fluid must be synthesized by the mother and transported to the amniotic fluid, which is in accord with the result found in the rhesus monkey.2o It has been suggested by Bangham*” that the histologic features of the amnion and syncytial trophoblast of the placenta of the fetal monkey are similar to those of the entodermal lining of the yolk sac of the rabbit and the entoderm of the rat and that these membranes are likely to be responsible for the selective transmission of albumin. Whether these different cellular layers have the same absorptive capacity for proteins still remains to be determined. In any case, the selective transmission of maternal serum proteins across fetal membranes into amniotic fluid has also been shown in the human being.z1 A radioactive peak was found in the amniotic fluid which migrated faster than albumin (Figs. 4 and 5). Although the identity of this protein is not known, it could correspond to the amniotic fluid protein described in the monkey,19 which has a molecular weight of 35,000 and thus would be expected to migrate faster than albumin. Our results are consistent with the concept that the amniotic fluid acts as a reservoir for secreted proteins. The delay in developmental changes of plasma proteins in the
Amniotic
fluid proteins
559
amniotic fluid (Fig. 2) could reflect a much slower turnover of these proteins as compared to the fetal plasma proteins. These proteins may be transported by transudation to the amniotic fluid from the fetuP> M and through the amnion and placenta from the mother.z0 Some investigators have concluded that all human amniotic proteins are derived from the mother.‘, Z* G This may be due to the high concentration of albumin (60 per cent) which has been used as a marker, as well as the absence of other unique markers such as the 5 p-proteins and the 3 transferrins found in mice. Another difficulty in interpretation of both the human and monkey studies may have arisen from the lack of precise time-course analysis carried out during development. Thus, in the primates, with their attendant longer fetal periods,* the fetoprotein (e.g., c+fetoprotein analogous to mouse ,&protein and calf fetuin) may have already decreased to a very low concentration in the fetal serum”, *’ and thus in the amniotic fluid by the time of analysis. Second, earlier studies may not have appreciated the microheterogeneity that exists in these fetoproteins, e.g., calf fetuinZG; and the developmental changes occurring in the fetoproteins, e.g., mouse ,8-protein.13 Thus, primate fetoproteins in fetal plasma could have a different electrophoretic charge, due to a different quantity of sialic acid present, than the same type of protein that was synthesized earlier in development and is residing in the amniotic fluid (e.g., Fig. 2).lZ Although we have established that ,81 to 5 are fetal proteins, it is not possible to determine from our experiments their site of synthesis. Dancis, Braverman, and LindlO have shown that except for the y-globulins, human fetal serum proteins are synthesized by the fetal liver; however, synthesis by the placenta of plasma proteins, although low, was significant except for albumin. However, the site of synthesis of fetal plasma proteins in the mouse has not yet been established, and also *Day 15 of gestation in the mouse is comparable to about 8 weeks in the w~rnan,~~ while birth of the mouse takes place at about Day 20.
560
Gustine
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October 15, 19X Am. J. Obstet. Gynecol.
Zimmerman
the mechanism of regulation of their synthesis remains to be answered. Although it is difficult to extrapolate data from the mouse to the human being, nevertheless, an extraordinary similarity exists for the two fetoproteins: Both have a molecular weight of 70,000, and human fetoprotein contains two sialic residues per polypeptide chain while mouse p-proteins contain 0 to 2 sialic acid residues according to their degree of “biologic maturity” (Zimmerman, Bowen, and Madappally, unpublished observations) . Finally, the mouse fetoprotein immunologically cross-reacts with the human
one.z8 Thus, the results from this present work in mice have produced additional data concerning the origin of amniotic fluid proteins, the similarity of which could be further tested in human beings. If specific proteins in human amniotic fluid arc fetal in origin, the value of amniocentesis in the diagnosis of genetically inherited disorders could possibly be enhanced. For example, changes in the carbohydrate or polypeptide structures of the fetus-specific proteins in the amniotic fluid could have occurred in the diseased state, and these proteins found in the amniotic fluid could be analyzed for such changes.
REFERENCES
1.
Abbas, 1: 476,
2.
Derrin@on,
7. 8. 9. 10. Il. 12. 13.
T. M., 1960.
and
Tovey,
J. E.:
Br.
Med.
J.
14.
M. M., and Soothill, J. F.: J. Obstet. Gynaecol. Br. Commonw. 68: 755, 1961. Brzezinski, A., Sadovsky, E., and Shafrir, E.: AM. J. OBSTET. GYNECOL, 82: 800, 1961. Mendelbaum, B., and Evans, T. N.: AM. J. OBSTET.GYNECOL. 104:365.1969. Gitlin, D., and Boesman, M:: J. Clin. Invest. 45: 1826, 1966. Stander, .R. W., McNutt, C. C., Barton, D. M.. and Werts. C. E.: Am. ”T. Clin. Pathol. 42:’ 125, 1964. ’ Fischbacher, P. H., and Quinlivan, W. L. G.: AM. J. OBSTET. GYNECOL. 108: 1051, 1970. Behrman, R. E., Parer, J. T., and deLannoy, C. W.: Nature 214: 678, 1967. Rausen, A. R., Gerald, P. S., and Diamond, L. K.: Nature 192: 182, 1961. Dancis, J., Braverman, N., and Lind, J.: J. Clin. Invest, 36: 398. 1957. Hirschfeld, J., and Lunell, N. 0.: Nature 196: 1220, 1962. Walker, B. E., and Fraser, F. C.: J. Embryol. Exp. Morphol. 4: 176, 1956. Gustine, D. L., and Zimmerman, E. F.: Proc. Natl. Acad. Sci. U. S, A.: Submitted for publication,
15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
2.5.
Ornstein, L.: Ann. N, Y. Acad. Sci. 121: 321, 1964. Davis, B. J.: Ann. N. Y. Acad. Sci. 121: 404, 1964. Gustine, D. L., and Zimmerman, E. F.: Teratology. In press. Schultze$ H. E., Schmidtberger, R., and Haupt, H.: Biochem. Z, 329: 490, 1958. Spiro, R. G.: J. Biol. Chem. 235: 2860, 1960. Bangham, D. R., Hobbs, K. R,, and Tee, D. E. H.: J. Physiol. 158: 207, 1961. Bangham, D. R.: J. Physiol. 153: 265, 1960. Usategui-Gomez, M., and Morgan, D. F.: Proc. Sot. Exp. Biol. Med. 125: 819, 1967. Setnika, I., Agostoni, E., and Taglietti, A.: Proc. Sot. EXD. Biol. Med. 101: 842. 1959. Shaw, R. E., and Marriot, H. J.: J. ‘Obstet. Gynaecol. Br. Emp. 56: 1004, 1949. Rugh, R.: The Mouse-Its Reproduction and Development, Minneapolis, 1968, Burgess Publishing Company, py 297. Bergstrand. C. G.. and Czar. B.: Stand. T. Clin. Lab. Invest. 9: 277, 1957. Page, M.: Biochim. Biophys. Acta 236: 571, 1971. Ruoslahti, E., and Ruoslahti, M,: Int. J, Cancer 7: 218, 1971. Abelev, G. I.: Cancer Res. 28: 1344, 1968. ”
26. 27. 28.