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Human term placental villi in explant tissue culture
Human term placental villi in explant tissue culture I. Behavior
MARTIN Brooklyn,
C. New
CARR.
M.D.
York
D u R I N G studies on human term placental villi in conventional explant tissue culture, it was noted that the syncytial trophoblast layer deteriorated within a few days while adjacent tissue seemed to survive and gr0w.l This rather selective breakdown of the multinucleated layer paralleled the observation that the syncytial cell was exquisitely and especially sensitive to trypsin, chymotrypsin, and plasmin. When certain proteolytic inhibitors appeared in tissue culture to slow down or prevent this syncytial lysis, it was felt that this dissipation was yet another reflection of the selective sensitivity of this cell to certain proteolytic substances; and that such a substance(s) was present in these cu1tures.l Our own interest in proteolytic materials stemmed from the demonstration that beginning early in a normal gestation exceedingly large placental multinucleated trophoblasts seemed to be deposited into the maternal bloodstream in inordinate amounts, theoretically demanding a clearing agent to avoid their acting as multiple pulmonary emboli.” Such a clearing agent or system would have to possess proteolytic properties
to break down these cells and might be located either in the plasma or trophoblastic cell or both. Possibly the active system in the explant cultures was related to this hypothesized maternal clearing agent. Present studies were undertaken to confirm and enlarge upon these observations and eventually to characterize the enzyme system(s) involved in this lysis in tissue culture. Materials
From Department of Obstetrics and Gynecology, Jewish Hospital of Brooklyn. Supported by Research Grant U1313 of the Health Research Council of New York City. Presented at the Brooklyn Society, May 15, 1963.
and
methods
Placentas were obtained as sterile as possible at uncomplicated vaginal deliveries from patients with unremarkable antepartum and intrapartum courses. Immediately they were brought to the laboratory where, if not used then, they were placed in a refrigerator at 4’ C. Work was initiated on all placentas within 45 minutes of delivery. Initially a 2 by 2 cm. portion of tissue was removed with scalpels from an area midway between the center and the margin of the placenta, avoiding the site of the origin of the cord. This chunk of tissue was placed in a Petri dish where the maternal and fetal sides were trimmed away. Further slicing was done until a piece of tissue 0.5 by 0.5 by 0.5 cm. remained. This was transferred to another Petri dish, soaked, and rinsed in phosphate buffered saline and minced until bits of tissue 0.5 by 1.0 by 0.5-1.5 mm. remained. Further rinsing removed much of the maternal blood. The remaining fragments of placental tissue were then soaked in chicken
Gynecological
584
Volume Number
88 5
plasma (Baltimore Biological Laboratory) and transferred to coverslips where the excess chicken plasma was removed. In early preparations chicken embryo extract (Baltimore Biological Laboratory) was added to aid clotting, but, when it was noted that the chicken plasma clotted by itself on addition of the villous fragment, this step was eliminated. The coverslips were then placed inside Leighton tubes with Morton closures (Bellco Glass) and incubated at 37’ C. Small amounts of MEM Eagles with Hanks’ base and 10 per cent calf serum (Baltimore Biological Laboratory), were added to prevent drying and the chicken plasma was allowed to clot. Once the clot was formed additional medium was added. The tubes were once again put into an incubator and 95 per cent compressed air-5 per cent carbon dioxide were bubbled in slowly ( < 0.1 liter per minute). This was found to maintain the pH at 7.0 to 7.2. At predetermined intervals the tubes were removed; the medium pipetted out, cultured in blood agar and thioglycollate broth, and Bouin’s solution added as a fixative. The latter was removed after 24 hours and 80 per cent ethanol added and replaced periodically. After 3 to 4 weeks the cover-slips were removed from the tubes, passed through a conventional hematoxylin and eosin stain, mounted on slides, and numbered. These identifying numbers were covered and the slides were placed in random order from which they were examined, rated, and renumbered. After all the slides had been seen, they were then identified. In this way it was hoped bias could be minimized in the interpretation of the slides. The slides were rated according to our own system which depended on the degree and the amount of syncytial cell cytoplasmic and nuclear destruction. It was subjective but fairly reproducible and thus far workable. Three grades were employed (Fig. 1) . Stage A included intact normal-appearing syncytium; Stage B was for decreased to absent cytoplasmic staining associated with vacuolization and/or mild ballooning of the cell wall. Stage C encompassed the syn-
Term
placental
villi
in explant
tissue
cultwre
585
cytium which had ballooned to over twice its formerly estimated diameter and invariably had little cytoplasmic staining. Each slide was reviewed by inspecting the syncytial lining on all the explants on that slide and arbitrarily deciding the amount of intactness or dissipation expressed as a percentage in each grade. Results Usually within the initial 24 hours the entire explant retained good cytoplasmic nuclear staining capabilities and little change was noted (Figs. 2 and 3). However, in some cases fine granular basophilic material was noted contiguous to the syncytial cell wall (Fig. 4) ; and at infrequent intervals in this lining there was decreased to absent cytoplasmic staining basally with a slight to marked ballooning of the outer cell wall (Fig. 5). This cytoplasmic dissolution was not necessarily preceded or accompanied by the bordering basophilic material. In three out of fourteen cultures in chick plasmachick embryo extract clots at least 10 per cent of the syncytial lining showed these changes by this time. A similar percentage
----Core
tissue
c
Fig. blast
1. Staging of breakdown layer in villous explant
of syncytial culture.
trophe
586
March 1, 1964 Am. J. Obst. & Gym.
Carr
of dissolution was noted where chick plasma clot was employed (Table I) _ After 48 hours the syncytium had larger areas of absent cytoplasmic staining with marked ballooning and occasional disrup-
Fig. 2. Twenty-four plasma clot--Stage (x100.)
A,
hour intact
tion of the cell wall. In these areas there was decreased to absent nuclear staining (Fig. 6). Indeed one saw faint nuclear outlines, nuclear skeletons, which suggested that the nuclei had swollen slightly prior to their
chick villus.
Fig. 3. Twenty-four plasma clot-Stage (~400.)
A,
hour intact
chick villus.
Volume
88
Number
5
dissipation (Fig. 7). Other areas with intact syncytial staining also exhibited absent nuclei, and about these and other areas concentrations of basophilic granular material were noted. This material was also noted in
Term
placental
villi
in explant
tissue
culture
587
the core tissue of the villus but usually did not parallel the extent of that seen about the villus. Yet, many nuclei of the fibrous and endothelial core seemed pyknotic. In any event, at this time in the chick plasma-
Fig. 4. Twenty-four hour chick plasma clot--Stage A, intact villus with basophilic granular material in vicinity of lining cell. (x400.)
Fig.
5. Twenty-four hour chick plasma-chick embryo extract clotStage B, intact villus with diminished syncytial cytoplasmic staining near base of cell with ballooning of cell wall and decreased nuclear staining.
(x400.)
588
Carr
March 1, 1964 Am. J. Obst. &Gym.
Fig. 6. Forty-eight hour chick plasma-chick embryo extract clot-Stage B, ballooning and decreased syncytial cytoplasmic staining with decreased number of nuclei and survival of core tissue stainability. (x400.)
Fig. 7. Forty-eight hour chick plasma-chick embryo extract clot-swelling and slight decreased staining in the syncytial nuclei while core nuclei are either vesicular or shrunken and hyperchromatic. (Approx. x800.)
Volume Number
88 5
Term
placental
villi
in explant
tissue
culture
589
590
March 1, 1964 Am. J. Obst. & Gynec.
Carr
Table I. Status of the syncytial in term culture
human
Syncytiotsophoblast remaining as Stage (%) Chicken
plasma-chick
Above 90 75 to 90 Below 75 Chick
plasma
trophoblast villous explant
placental
1
I
I
A embryo
extract
clot
11 2 1
8 4 2
5 5 3
I3 I! 0
7 3 ?
5 6 3
clot
Above 90 75 to 90 Below 75
chick embryo extract clot, 6 of 14 cultures were observed to have at least 10 per cent level of syncytial breakdown while 2 of these were more extensive. Again the chick plasma clot cultures demonstrated a similar ratio of breakdown (Table I). At 72 hours, the syncytiolysis was similar to that seen earlier although a bit more extensive and more advanced, and here occasionally an explant was noted where the entire syncytial lining was destroyed with the remains lying a distance from the surviving villous core tissue (Fig. 8). Again here the chick plasma chick embryo extract clot and the chick plasma clot culture are comparable (Table I) It should be mentioned that by the third day the endothelial and fibrous tissue still retained their eosinophilia while their nuclei had become pyknotic or more vesicular than originally. If this tissue had been continued in culture fibroplastic growth would have resulted. In addition, if minimal syncytiolysis had taken place by the third day usually the syncytium was still present by the fifth day. Present studies did not go beyond this time, because they were primarily concerned with the syncytial dissolution which was more reliably and easily evaluated with 3 day cultures. On adding the same chick plasma to the medium (0.1 c.c./l.O C.C. of medium) that was used in producing the explant clot, it was found that syncytiolysis could be accelerated, so that by 48 hours 50 per cent
of the syncytium showed signs of dissipation as compared to 5 per cent in the control, and by 72 hours no intact syncytium was found. Decreasing concentrations of the chick plasma resulted in slowed rate of breakdown although still two to five times the rate of the controls. Greater amounts of chick embryo extract were needed (0.5 c.c.jl.0 C.C. of medium) to increase the rate and extent of lysis and this also diminished with lowered concentrations. Comment Explant culture syncytial lysis is an interesting phenomenon; and it should be understood that it is quite variable, that is, the events described can occur within one day or not. at all. Indeed, while one portion of the syncytium underwent violent dissolution, a neighboring villous of the same explant did have a well-preserved syncytial layer (Fig. 9). Small differences were not considered significant but marked changes were noted. Moreover, several well-prepared explants were necessary per slide to correctly evaluate over-all behavior. Within these bounds syncytial behavior was fairly reproducible providing duplicate conditions were met. The steps of cytoplasmic breakdown were fairly clear while those of nuclear death deserve some comment. The basophilic granularity was most likely extrusion of nuclear material from the syncytial layer, and probably represented an early stage of syncytial nuclear degeneration, although the core cells might yet have been the principal source. Nevertheless, the syncytial nuclear deterioration appeared to differ from that of the villous core nucleus, the latter became pyknotic, while the lining cell nuclei seemed to swell slightly prior to gradual loss of their staining ability. The dissipation of the nucleus and the cytoplasm did not parallel each other, for one saw the cytoplasm eosinophific with absent nuclei while the reverse was witnessed also. This suggested that different processes might be concerned in the breakdown of these cellular constituents, although nuclear death might he a
Volume Number
88 5
product of some previous cytoplasmic breakdown. The events of the syncytial cytoplasmic dissolution were very much like those produced using certain proteolytic enzymes, and suggested strongly the presence here of a proteolytic enzyme. Changes in the rate of lysis by variations in chick plasma or chick embryo extract concentrations suggested that these materials contained at least a portion of this system. While major variations in the response of different placental syncytia to the same clot type were observed, it would be premature to offer this as proof that a syncytial factor was involved. Numerous possibilities exist which involve enzyme, activator, and inhibitor substances in multiple combinations. These include syncytiolysis by a chick plasma and/or chick embryo extract factor, or by chick plasma and/or chick embryo extract activating a syncytial cytoplasmic factor; or the breakdown might have involved a combination of chick plasma and/or chick embryo extract factors and cytoplasmic factors working independently of each other to produce the same end. Moreover, the fetal blood, core tissue, or maternal blood might have played a role. The usual day or 2 delay before breakdown occurred may be the reflection of a slowed activation process or inhibitory degeneration time. In any event, the syncytial breakdown seen in these cultures with survival of adjacent endothelial and mesothelial tissue is
Term
placental
villi
in explant
tissue
culture
591
intriguing and once again reflects the unique and exquisite sensitivity of these cells to certain proteolytic materials. This type of enzyme (s) is located somewhere within a viiloussplasma clot, and it is not difficult to envision a similar, if not the same, system in the living maternal organism. This would be of great importance, for if one believes that the placenta throughout most of the human gestation normally deposits substantial numbers of syncytial cells into the maternal bloodstream, and these cells being quite large must be cleared and destroyed lest they clog the mother’s pulmonary vascular bed, then a proteolytic enzyme system would be needed?; and it might very well be that responsible for the syncytial lysis here. Efforts are now concerned with identifying the system present in these cultures. Summary
The syncytial trophoblastic lining of human term placental villi was observed to have undergone breakdown within a few days after this tissue was placed in explant culture in a chick plasma clot. This behavior is attributed to the presence of a proteolytic enzyme(s) . The possible role of this enzyme(s) in a normal pregnancy is discussed. The author wishes to thank Dr. Edwin M. Gold for his encouragement and to acknowledge the excellent technical assistance of Mrs. Marcela Preininger.
REFERENCES
1. Brunner, K. T., Carr, M. C., and Thomas, L.: Unpublished data.
2. Thomas, L., Douglas, G. W., and Carr, M. C.: Tr. A. Am. Phys. 72: 140, 1959.
MARTIN Brooklyn,
C. New
CARR.
M.D.
York
D u R I N G studies on human term placental villi in conventional explant tissue culture, it was noted that the syncytial trophoblast layer deteriorated within a few days while adjacent tissue seemed to survive and gr0w.l This rather selective breakdown of the multinucleated layer paralleled the observation that the syncytial cell was exquisitely and especially sensitive to trypsin, chymotrypsin, and plasmin. When certain proteolytic inhibitors appeared in tissue culture to slow down or prevent this syncytial lysis, it was felt that this dissipation was yet another reflection of the selective sensitivity of this cell to certain proteolytic substances; and that such a substance(s) was present in these cu1tures.l Our own interest in proteolytic materials stemmed from the demonstration that beginning early in a normal gestation exceedingly large placental multinucleated trophoblasts seemed to be deposited into the maternal bloodstream in inordinate amounts, theoretically demanding a clearing agent to avoid their acting as multiple pulmonary emboli.” Such a clearing agent or system would have to possess proteolytic properties
to break down these cells and might be located either in the plasma or trophoblastic cell or both. Possibly the active system in the explant cultures was related to this hypothesized maternal clearing agent. Present studies were undertaken to confirm and enlarge upon these observations and eventually to characterize the enzyme system(s) involved in this lysis in tissue culture. Materials
From Department of Obstetrics and Gynecology, Jewish Hospital of Brooklyn. Supported by Research Grant U1313 of the Health Research Council of New York City. Presented at the Brooklyn Society, May 15, 1963.
and
methods
Placentas were obtained as sterile as possible at uncomplicated vaginal deliveries from patients with unremarkable antepartum and intrapartum courses. Immediately they were brought to the laboratory where, if not used then, they were placed in a refrigerator at 4’ C. Work was initiated on all placentas within 45 minutes of delivery. Initially a 2 by 2 cm. portion of tissue was removed with scalpels from an area midway between the center and the margin of the placenta, avoiding the site of the origin of the cord. This chunk of tissue was placed in a Petri dish where the maternal and fetal sides were trimmed away. Further slicing was done until a piece of tissue 0.5 by 0.5 by 0.5 cm. remained. This was transferred to another Petri dish, soaked, and rinsed in phosphate buffered saline and minced until bits of tissue 0.5 by 1.0 by 0.5-1.5 mm. remained. Further rinsing removed much of the maternal blood. The remaining fragments of placental tissue were then soaked in chicken
Gynecological
584
Volume Number
88 5
plasma (Baltimore Biological Laboratory) and transferred to coverslips where the excess chicken plasma was removed. In early preparations chicken embryo extract (Baltimore Biological Laboratory) was added to aid clotting, but, when it was noted that the chicken plasma clotted by itself on addition of the villous fragment, this step was eliminated. The coverslips were then placed inside Leighton tubes with Morton closures (Bellco Glass) and incubated at 37’ C. Small amounts of MEM Eagles with Hanks’ base and 10 per cent calf serum (Baltimore Biological Laboratory), were added to prevent drying and the chicken plasma was allowed to clot. Once the clot was formed additional medium was added. The tubes were once again put into an incubator and 95 per cent compressed air-5 per cent carbon dioxide were bubbled in slowly ( < 0.1 liter per minute). This was found to maintain the pH at 7.0 to 7.2. At predetermined intervals the tubes were removed; the medium pipetted out, cultured in blood agar and thioglycollate broth, and Bouin’s solution added as a fixative. The latter was removed after 24 hours and 80 per cent ethanol added and replaced periodically. After 3 to 4 weeks the cover-slips were removed from the tubes, passed through a conventional hematoxylin and eosin stain, mounted on slides, and numbered. These identifying numbers were covered and the slides were placed in random order from which they were examined, rated, and renumbered. After all the slides had been seen, they were then identified. In this way it was hoped bias could be minimized in the interpretation of the slides. The slides were rated according to our own system which depended on the degree and the amount of syncytial cell cytoplasmic and nuclear destruction. It was subjective but fairly reproducible and thus far workable. Three grades were employed (Fig. 1) . Stage A included intact normal-appearing syncytium; Stage B was for decreased to absent cytoplasmic staining associated with vacuolization and/or mild ballooning of the cell wall. Stage C encompassed the syn-
Term
placental
villi
in explant
tissue
cultwre
585
cytium which had ballooned to over twice its formerly estimated diameter and invariably had little cytoplasmic staining. Each slide was reviewed by inspecting the syncytial lining on all the explants on that slide and arbitrarily deciding the amount of intactness or dissipation expressed as a percentage in each grade. Results Usually within the initial 24 hours the entire explant retained good cytoplasmic nuclear staining capabilities and little change was noted (Figs. 2 and 3). However, in some cases fine granular basophilic material was noted contiguous to the syncytial cell wall (Fig. 4) ; and at infrequent intervals in this lining there was decreased to absent cytoplasmic staining basally with a slight to marked ballooning of the outer cell wall (Fig. 5). This cytoplasmic dissolution was not necessarily preceded or accompanied by the bordering basophilic material. In three out of fourteen cultures in chick plasmachick embryo extract clots at least 10 per cent of the syncytial lining showed these changes by this time. A similar percentage
----Core
tissue
c
Fig. blast
1. Staging of breakdown layer in villous explant
of syncytial culture.
trophe
586
March 1, 1964 Am. J. Obst. & Gym.
Carr
of dissolution was noted where chick plasma clot was employed (Table I) _ After 48 hours the syncytium had larger areas of absent cytoplasmic staining with marked ballooning and occasional disrup-
Fig. 2. Twenty-four plasma clot--Stage (x100.)
A,
hour intact
tion of the cell wall. In these areas there was decreased to absent nuclear staining (Fig. 6). Indeed one saw faint nuclear outlines, nuclear skeletons, which suggested that the nuclei had swollen slightly prior to their
chick villus.
Fig. 3. Twenty-four plasma clot-Stage (~400.)
A,
hour intact
chick villus.
Volume
88
Number
5
dissipation (Fig. 7). Other areas with intact syncytial staining also exhibited absent nuclei, and about these and other areas concentrations of basophilic granular material were noted. This material was also noted in
Term
placental
villi
in explant
tissue
culture
587
the core tissue of the villus but usually did not parallel the extent of that seen about the villus. Yet, many nuclei of the fibrous and endothelial core seemed pyknotic. In any event, at this time in the chick plasma-
Fig. 4. Twenty-four hour chick plasma clot--Stage A, intact villus with basophilic granular material in vicinity of lining cell. (x400.)
Fig.
5. Twenty-four hour chick plasma-chick embryo extract clotStage B, intact villus with diminished syncytial cytoplasmic staining near base of cell with ballooning of cell wall and decreased nuclear staining.
(x400.)
588
Carr
March 1, 1964 Am. J. Obst. &Gym.
Fig. 6. Forty-eight hour chick plasma-chick embryo extract clot-Stage B, ballooning and decreased syncytial cytoplasmic staining with decreased number of nuclei and survival of core tissue stainability. (x400.)
Fig. 7. Forty-eight hour chick plasma-chick embryo extract clot-swelling and slight decreased staining in the syncytial nuclei while core nuclei are either vesicular or shrunken and hyperchromatic. (Approx. x800.)
Volume Number
88 5
Term
placental
villi
in explant
tissue
culture
589
590
March 1, 1964 Am. J. Obst. & Gynec.
Carr
Table I. Status of the syncytial in term culture
human
Syncytiotsophoblast remaining as Stage (%) Chicken
plasma-chick
Above 90 75 to 90 Below 75 Chick
plasma
trophoblast villous explant
placental
1
I
I
A embryo
extract
clot
11 2 1
8 4 2
5 5 3
I3 I! 0
7 3 ?
5 6 3
clot
Above 90 75 to 90 Below 75
chick embryo extract clot, 6 of 14 cultures were observed to have at least 10 per cent level of syncytial breakdown while 2 of these were more extensive. Again the chick plasma clot cultures demonstrated a similar ratio of breakdown (Table I). At 72 hours, the syncytiolysis was similar to that seen earlier although a bit more extensive and more advanced, and here occasionally an explant was noted where the entire syncytial lining was destroyed with the remains lying a distance from the surviving villous core tissue (Fig. 8). Again here the chick plasma chick embryo extract clot and the chick plasma clot culture are comparable (Table I) It should be mentioned that by the third day the endothelial and fibrous tissue still retained their eosinophilia while their nuclei had become pyknotic or more vesicular than originally. If this tissue had been continued in culture fibroplastic growth would have resulted. In addition, if minimal syncytiolysis had taken place by the third day usually the syncytium was still present by the fifth day. Present studies did not go beyond this time, because they were primarily concerned with the syncytial dissolution which was more reliably and easily evaluated with 3 day cultures. On adding the same chick plasma to the medium (0.1 c.c./l.O C.C. of medium) that was used in producing the explant clot, it was found that syncytiolysis could be accelerated, so that by 48 hours 50 per cent
of the syncytium showed signs of dissipation as compared to 5 per cent in the control, and by 72 hours no intact syncytium was found. Decreasing concentrations of the chick plasma resulted in slowed rate of breakdown although still two to five times the rate of the controls. Greater amounts of chick embryo extract were needed (0.5 c.c.jl.0 C.C. of medium) to increase the rate and extent of lysis and this also diminished with lowered concentrations. Comment Explant culture syncytial lysis is an interesting phenomenon; and it should be understood that it is quite variable, that is, the events described can occur within one day or not. at all. Indeed, while one portion of the syncytium underwent violent dissolution, a neighboring villous of the same explant did have a well-preserved syncytial layer (Fig. 9). Small differences were not considered significant but marked changes were noted. Moreover, several well-prepared explants were necessary per slide to correctly evaluate over-all behavior. Within these bounds syncytial behavior was fairly reproducible providing duplicate conditions were met. The steps of cytoplasmic breakdown were fairly clear while those of nuclear death deserve some comment. The basophilic granularity was most likely extrusion of nuclear material from the syncytial layer, and probably represented an early stage of syncytial nuclear degeneration, although the core cells might yet have been the principal source. Nevertheless, the syncytial nuclear deterioration appeared to differ from that of the villous core nucleus, the latter became pyknotic, while the lining cell nuclei seemed to swell slightly prior to gradual loss of their staining ability. The dissipation of the nucleus and the cytoplasm did not parallel each other, for one saw the cytoplasm eosinophific with absent nuclei while the reverse was witnessed also. This suggested that different processes might be concerned in the breakdown of these cellular constituents, although nuclear death might he a
Volume Number
88 5
product of some previous cytoplasmic breakdown. The events of the syncytial cytoplasmic dissolution were very much like those produced using certain proteolytic enzymes, and suggested strongly the presence here of a proteolytic enzyme. Changes in the rate of lysis by variations in chick plasma or chick embryo extract concentrations suggested that these materials contained at least a portion of this system. While major variations in the response of different placental syncytia to the same clot type were observed, it would be premature to offer this as proof that a syncytial factor was involved. Numerous possibilities exist which involve enzyme, activator, and inhibitor substances in multiple combinations. These include syncytiolysis by a chick plasma and/or chick embryo extract factor, or by chick plasma and/or chick embryo extract activating a syncytial cytoplasmic factor; or the breakdown might have involved a combination of chick plasma and/or chick embryo extract factors and cytoplasmic factors working independently of each other to produce the same end. Moreover, the fetal blood, core tissue, or maternal blood might have played a role. The usual day or 2 delay before breakdown occurred may be the reflection of a slowed activation process or inhibitory degeneration time. In any event, the syncytial breakdown seen in these cultures with survival of adjacent endothelial and mesothelial tissue is
Term
placental
villi
in explant
tissue
culture
591
intriguing and once again reflects the unique and exquisite sensitivity of these cells to certain proteolytic materials. This type of enzyme (s) is located somewhere within a viiloussplasma clot, and it is not difficult to envision a similar, if not the same, system in the living maternal organism. This would be of great importance, for if one believes that the placenta throughout most of the human gestation normally deposits substantial numbers of syncytial cells into the maternal bloodstream, and these cells being quite large must be cleared and destroyed lest they clog the mother’s pulmonary vascular bed, then a proteolytic enzyme system would be needed?; and it might very well be that responsible for the syncytial lysis here. Efforts are now concerned with identifying the system present in these cultures. Summary
The syncytial trophoblastic lining of human term placental villi was observed to have undergone breakdown within a few days after this tissue was placed in explant culture in a chick plasma clot. This behavior is attributed to the presence of a proteolytic enzyme(s) . The possible role of this enzyme(s) in a normal pregnancy is discussed. The author wishes to thank Dr. Edwin M. Gold for his encouragement and to acknowledge the excellent technical assistance of Mrs. Marcela Preininger.
REFERENCES
1. Brunner, K. T., Carr, M. C., and Thomas, L.: Unpublished data.
2. Thomas, L., Douglas, G. W., and Carr, M. C.: Tr. A. Am. Phys. 72: 140, 1959.