J. Anat. (1972), 111, 1, pp. 43-54 43With 11 figuresPrinted in Great Britain

Fine structure of human amniotic epithelium followingshort-term preservation in vitro

A. D. HOYESDepartment of Anatomy, St Mary's Hospital

Medical School, London W. 2

(Accepted 5 November 1971)

INTRODUCTION

In 1965 Thomas described two distinct types of cell in the human amnioticepithelium and more recently Armstrong, Wilt & Pritchard (1968) have reported thepresence ofup to four morphological variants in this epithelium at term. The materialused in both of these studies was taken from placentae which had been stored inHanks's solution for up to an hour before processing and, although Wilt, Miller &Ruiter (1964) found that preservation of the membranes in this solution produces nosignificant alteration in the light microscopical appearance of the epithelium, similarvariations in the morphology of the cells have not been reported in many of the otherultrastructural investigations of the mature amnion. While, therefore, the work ofThomas (1965) and Armstrong et al. (1968) indicates that the cells are capable of ahigh degree of structural lability, it appears that at least some of their findings mayhave been due to alterations in the structure of the cells during the period when theamnion was stored in Hanks's solution. The extent to which the fine structure of thecells may be modified by preservation of the amnion in this solution has now beensubjected to further investigation. In addition, since the occurrence of significantchanges in their structure under such conditions may be of relevance to the evaluationof the findings of in vitro studies on the transport of small ions across the epithelium,the changes produced by Hanks's solution have been compared with those seen inmaterial stored in some of the other media used in recent in vitro studies of fluidtransport across the epithelium, and also with those produced by normal saline andhuman Ringer-lactate.

MATERIALS AND METHODS

Specimens of reflected amnion were obtained from six placentae within 15minutes of delivery after a normal labour at term. The amnion was rapidly strippedfrom the underlying chorion and, after rinsing in Ringer-lactate, was cut into squaresapproximately 1 cm wide. Some of these were used as control specimens and wereimmediately fixed in glutaraldehyde. The remainder were immersed in either Hanks'ssolution TC (Difco), 09 % sodium chloride (Sterivac), compound sodium lactate(Ringer-lactate/Hartmann's solution; Evans Medical), Gey's solution (Gey & Gey,1936), or in the solution employed by Scoggin et al. (1964). The osmolarity of thesolutions was measured on an Advanced osmometer and their pH on an EIL meter.During preservation of the specimens, the solutions were maintained at room

temperature, and the exposure of the whole specimen to the solution was ensured byoccasional gentle agitation. After one hour the specimens were removed to 2 %glutaraldehyde in cacodylate buffer (Gordon, Miller & Bensch, 1963) and fixed for10-20 minutes before being post-fixed in buffered osmium tetroxide (Palade, 1952).Dehydration was accomplished in ethyl alcohol and the specimens were embedded inTAAB embedding resin, usingBDMA as accelerator. Thin sections were cut on eithera Porter-Blum or Huxley ultramicrotome and were mounted on uncoated grids andstained with lead citrate (Reynolds, 1963). The grids were then examined in a SiemensElmiskop 1 or Philips EM 300 electron microscope.

RESULTS

Control materialThere was no evidence in any of the control specimens of the kind of structural

diversity described in the epithelium by Thomas (1965) and Armstrong et al. (1968),or of the presence of the light and dark cells found by Wynn and his co-workers(Wynn & French, 1968; Wynn & Corbett, 1969) in the epithelium of other mammals.The surface microvilli were pleomorphic and were covered by an extraneous layer offilamentous material similar to that present on the microvilli of the periderm of thefetal epidermis and the superficial layer of the umbilical cord epithelium (Hoyes,1968, 1969) (Fig. 3). Numerous spaces were present between the adjacent cells (Fig. 1)and formed a complex system of intercellular channels which extended from the baseof the epithelium to the surface where, as noted by Bourne (1962) and Schmidt (1963),they were in open communication with the amniotic cavity. The intercellular channelswere rarely widely dilated and most were either partially or completely filled byvillous-like processes of the plasma membranes (Figs. 2, 4). In the intervals betweenthe spaces, similar folds formed interlocking processes with those of adjacent cellsand the plasma membranes were united by attachment plaques, most of which weretypical desmosomes (Fig. 1). Well developed hemidesmosomes were present on thebasal plasma membrane, and this was also often folded to form the basal or footprocesses described in all other investigations of the epithelium. The thick basementmembrane underlying the epithelium resembled that described by Thomas (1965)and portions of the membrane frequently extended into the intervals between the footprocesses and into the intercellular spaces (Fig. 5).The cytoplasm contained numerous bundles of filaments and these formed an

interlacing network which extended from just below the terminal web associated withthe surface membrane to the base of the cells (Fig. 5). The filaments were closelyrelated to the desmosomes on the lateral membranes, but were often separated fromthe hemidesmosomal plaques on the basal membrane by a significant interval.Pinocytotic activity was pronounced at the lateral cell membranes, especially in thelower half of the cells (Fig. 5), and was also visible at the basal part of the membrane.Clear vesicles of a similar size to the pinocytotic vesicles were present in all parts ofthe cytoplasm and were also numerous in the region of the Golgi apparatus (Fig. 4).This was almost invariably situated close to the upper surface of the nucleus and,in all but a few instances, consisted of a single group of cisternae. Larger vesicles,some of which contained filamentous material, were also commonly related to the

44 A. D. HOYES

Human amniotic epithelium 45apparatus (Fig. 4) and similar vesicles were distributed throughout the superficialparts of the cells. Although such vesicles were often situated close to the surface, theywere only occasionally seen to communicate with the plasma membrane (Fig. 3). Themitochondria were scattered, and the intramitochondrial matrix was rarely of morethan moderate electron density (Fig. 2). Dilated cisternae of granular endoplasmic

Fig. 1. Amniotic epithelium in a control specimen. mv, surface microvilli; ics, intercellularspaces; bm, basement membrane; d, desmosomes. x 9200.

reticulum were present in all areas of the cytoplasm (Fig. 2) and their membraneswere only incompletely covered by ribosomes. Numerous free ribosomes were alsopresent in the cytoplasm but showed no evidence of aggregation into chains orpolyribosomes. Microtubules, although reported as being numerous in the cells byWynn & French (1968), were only occasionally visible; centrioles were seen in onlytwo instances and lipid droplets (Fig. 4) were numerous in only two of the sixspecimens.

46 A. D. HOYES

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Human amniotic epithelium

Stored materialHanks's solutionThe quality of preservation of material stored in this solution was generally good,

and there was still no evidence of the development in the epithelium of the kind ofstructural diversity described by Thomas (1965) and Armstrong et al. (1968). Theintercellular spaces were generally wider than in the corresponding control material,and often they were extremely dilated. Pinocytotic activity was still evident at thelateral and basal membranes, but did not appear to have undergone any reduction orincrease compared with that seen in control material. There were, however, majorchanges in the degree of development of the Golgi apparatus. At least two groups ofcisternae were now usually visible and the cells commonly contained four or moregroups of cisternae (Fig. 6). The groups of Golgi cisternae were also often separatedfrom the nucleus and, although it generally remained supranuclear in position (Fig. 6),the apparatus occasionally extended into the basal part of the cells. The larger vesiclespresent in the supranuclear cytoplasm were also more numerous (Fig. 6) and thefilamentous material covering the microvilli often appeared to be increased in amounlt.In those specimens in which they were present, areas of reduced electron densitycould be seen in the lipid droplets (Fig. 6). The density of the mitochondrial matrixwas frequently increased, and the dilatation of the sacs ofendoplasmic reticulum wasless pronounced than in the control material. There was, however, no evidence ofany significant alteration in the amount of endoplasmic reticulum present in thecells, or in the number and arrangement of the ribosomes.

Gey's solution and Scoggin's solutionAlthough the density of the cytoplasm and of the-matrix of the mitochondria was

often greater in these specimens than in material stored in Hanks's solution, themorphology of the epithelial cells was otherwise closely similar. The intercellularspaces were similarly dilated and pinocytotic activity was still abundant at the lateralcell membranes. The Golgi apparatus was also extensive, large vesicles wereprominent in the supranuclear cytoplasm, and filamentous material was presentin increased amounts on the surface of the microvilli.

Normal salineThe appearance of the cells in specimens stored in this solution was also similar to

that in material stored in Hanks's solution. Compared with the control material, thewidth of the intercellular spaces was often markedly increased (Fig. 8), considerablepinocytotic activity was still evident at the lateral cell membranes, and the Golgi

Fig. 2. Villous folds (vf) in intercellular spaces near the surface of the epithelium. mv, microvilli;mt, mitochondria; er, granular endoplasmic reticulum. Control specimen. x 27500.Fig. 3. Filamentous material (fm) on the surface of the microvilli. va, supranuclear vesicle incommunication with the surface membrane. Control specimen. x 68000.Fig. 4. Small (v) and large vesicles (va) near the Golgi apparatus (g). vf, villous folds in the inter -cellular spaces; 1, lipid droplet. Control specimen. x 22600.Fig. 5. Basement membrane material (bm) in the intercellular spaces in a control specimen.f, cytoplasmic filaments; pcv, pinocytotic vesicles. x 24 500.

47

48 A. D. HOYES

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Human amniotic epitheliumapparatus was generally much more highly developed (Fig. 7). Large vesicles werenumerous in the supranuclear cytoplasm and were still often apparent both in theregion of the Golgi apparatus (Fig. 7) and close to the surface membrane. Densematerial could also often be seen in the lumen of the vesicles (Fig. 7) and, as inspecimens preserved in Hanks's solution, a thick layer of similar material was presenton the surface of the microvilli (Fig. 9).

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Ringer-lactateThe quality of preservation of material stored in this solution was often less satis-

factory than that of material stored in the other solutions and there was frequentlyevidence of the formation of clear areas in the cytoplasm, and of dilatation ofthe mitochondria and the sacs of endoplasmic reticulum. In the better preservedspecimens, however, the morphology of the cells was closely similar to that of the

Fig. 6. Enlarged Golgi apparatus in a specimen preserved in Hanks's solution. n, nucleus; va,supranuclear vesicles; 1, lipid droplet. x 24500.Fig. 7. Enlarged Golgi apparatus in a specimen preserved in normal saline. va, supranuclearvesicles. x 30000.

49

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Human amniotic epithelium

Table 1. Composition, osmolarity andpH ofpreservative solutions

AmnioticHanks Gey Scoggin Saline Ringer fluid

Na 141 149 157 154 131 126K 6 5 6 5 4Mg 2 3 2 - -Ca 3 3 4 4Cl 145 130 166 154 111 105HCO3 4 27 3 -HPO4 0 7 2 -H2PO4 0 4 0-2SO4 0-8 0-6Lactate - - - 29Glucose 6 3 6 -

Osmolarity 299 289 313 290 274 250-270pH 7-1 8-1 7 5 8-0 7-4

Ionic concentrations are expressed as m-equiv/l and osmolarity as m-osmol/l. The figures for theamniotic fluid are mean values computed from the Table given by Seeds (1968).

control material. The intercellular spaces were therefore usually small, and theinterlocking processes formed by the membranes of adjacent cells were often pro-nounced (Fig. 11). Pinocytotic activity was still evident at the lateral plasma mem-branes and small vesicles could be seen close to the Golgi apparatus. Only a singlegroup of Golgi cisternae was normally visible in the cells (Fig. 10), and there was noconsistent alteration in the number ofthe larger supranuclear vesicles or in the amountof filamentous material present on the surface of the microvilli.

DISCUSSION

Although the presence of such features as the invagination of the basement mem-brane into the intercellular spaces and the separation of the cytoplasmic filamentsfrom the basal hemidesmosomes is suggestive of considerable retraction of the cellsafter delivery, the fine structure of the amniotic epithelium in the control material em-ployed in the present investigation was similar to that described by such authors asSchmidt (1963) and Wynn & French (1968), and may be regarded as being reasonablyclose to that of the normal epithelium in vivo. The absence of significant differencesin the morphology of the individual epithelial cells in these specimens also suggeststhat the various types of cell described by Thomas (1965) and Armstrong et al. (1968)are not normally present in the epithelium. The appearance of considerable changesin the degree of development of the Golgi apparatus and in the number of supra-nuclear vesicles in material preserved in Hanks's solution indicates that the formation

Fig. 9. Increased filamentous material (fm) on the surface of the microvilli in a specimenpreserved in normal saline. x 44700.Fig. 10. Supranuclear cytoplasm in a specimen preserved in Ringer-lactate. g, Golgi apparatus.x 13600.Fig. 11. Small intercellular spaces (ics) and extensive interlocking of villous folds of adjacentcells (arrows) in a specimen stored in Ringer-lactate. x 21 800.

4-2

51

of the highly active 'Golgi' cells described by Thomas (1965) may have beenrelated to the storage of the membranes in this solution before fixation. Since,furthermore, similar changes were seen in material preserved in Gey's solution and inthat used by Scoggin et al. (1964), it may be assumed that the media employed inmany of the in vitro studies of fluid transport across the epithelium also producedconsiderable changes in the structure and function of the epithelium.The presence of sites of open communication between the intercellular spaces and

the amniotic cavity is now well established, and, although the structure of the epi-thelium is not unlike that of many of the other epithelia involved in the activetransport of fluid, it is probable that these form the major pathway for such transportacross the epithelium. The occurrence of considerable pinocytotic activity at thelateral membranes indicates, however, that the cells are capable of the active absorp-tion of materials from the spaces, and the presence of similar vesicles in the region ofthe Golgi apparatus suggests that these are subsequently transported to the apparatusand incorporated into any secretory product elaborated in this structure.The larger vesicles which were seen in the supranuclear cytoplasm were also often

related to the apparatus and their frequent occurrence close to, but only occasionallyin contact with, the surface membrane suggests that they are secretory rather thanabsorptive in nature, and form a further component of a mechanism for the activetransport of materials from the intercellular spaces to the amniotic cavity. Thefilamentous material present on the surface of the microvilli was similar to thatconsidered to form a mucous coat on the surface of the other epithelia lining theamniotic cavity (Hoyes, 1968, 1969). Filamentous material was also present in thelumen of the vesicles, and the marked increase in the amount of the surface materialseen, in association with the increase in the degree of development of the Golgiapparatus and in the number of supranuclear vesicles in specimens preserved inHanks's solution, may be regarded as evidence that it is formed in the apparatus andincluded in the secretory product released at the surface of the cells.The changes in the Golgi apparatus and in the number of supranuclear vesicles

seen in material preserved in Hanks's solution and normal saline, and in both Gey'sand Scoggin's solutions, could not be demonstrated in specimens stored in Ringer-lactate. Although a number of factors may have contributed to this result, comparisonof the osmolarity and composition of the various preservative solutions employedwith the values given by Seeds (1968) for the amniotic fluid (Table 1) suggests that itwas due to the close similarity of the osmolarity and sodium ion concentration of theRinger-lactate solution to that of the fluid. The appearance of the changes in thestructure of the cells in the other solutions may have been related to the fact that theirosmolarity and sodium content were invariably much higher than in the amnioticfluid. While, therefore, there was no evidence of any alteration in the amount ofpinocytosis at the lateral membranes in any of the stored material, it appears that thesecretory activity of the epithelium may be controlled by variations in the compositionof the amniotic fluid and that it reaches a maximum only under conditions in whichthe composition of the fluid approximates to that of the maternal plasma. The recentwork of such authors as Gillibrand (1969) has confirmed that, in later pregnancy, thetonicity and concentration of ions such as sodium in the amniotic fluid are almostinvariably lower than in the maternal serum. It is therefore probable that, under

52 A. D. HOYES

Human amniotic epitheliumnormal conditions, only small amounts of secretion are elaborated by the cells, andthat the secretory activity of the amnion is in some way directed towards maintainingthe level of the osmolar deficit between the amniotic fluid and the maternal plasma.

SUMMARY

After preservation of specimens of term amnion for an hour in Hanks's solution,there were marked changes in the fine structure of the amniotic epithelium. Theseincluded a considerable increase in the degree of development of the Golgi apparatusand were consistent with significant alterations in the functional activity of the cells.Similar changes were seen in material stored in a number of other solutions, but werenot apparent in specimens preserved in Ringer-lactate. Correlation of the osmolarityand composition of the various preservative solutions employed with values givenfor the amniotic fluid indicated that their appearance was related to exposure of theepithelium to media which were either hypertonic with respect to the fluid orcontained higher concentrations of ions such as sodium.

The author is indebted to Professor P. J. Huntingford and the other members ofthe staff of the Obstetric Department, St Mary's Hospital, W. 2, for making availablethe material used in this study and for the osmolarity measurements, and toProfessor K. A. Porter of the Department of Histopathology and ExperimentalPathology, St Mary's Hospital Medical School, for permission to use the Philipselectron microscope provided by the Wates Foundation. The technical assistance ofMr B. Martin is also gratefully acknowledged.

REFERENCES

ARMSTRONG, W. D., WILT, J. C. & PRITCHARD, E. T. (1968). Vacuolation in the human amnion cell studiedby time-lapse photography and electron microscopy. American Jouirnal of Obstetrics and Gynecology102, 932-948.

BOURNE, G. (1962). The Human Amnion and Chorion. London: Lloyd-Luke.GEY, G. 0. & GEY, M. K. (1936). The maintenance of human normal cells and tumor cells. I. Preliminary

report: Cultivation of mesoblastic tumors and normal tissue and notes on methods of cultivation.American Journal of Cancer 27, 45-76.

GILLIBRAND, P. N. (1969). Changes in the electrolytes, urea and osmolality of amniotic fluid with advanc-ing pregnancy. Journal of Obstetrics and Gynaecology of the British Commonwealth 76, 898-905.

GORDON, G. B., MILLER, L. R. & BENSCH, K. G. (1963). Fixation of tissue culture cells for ultrastructuralcytochemistry. Experimental Cell Research 31, 440-443.

HOYES, A. D. (1968). Electron microscopy of the surface layer (periderm) of human foetal skin. JournalofAnatomy 103, 321-336.

HOYES, A. D. (1969). Ultrastructure of the epithelium of the human umbilical cord. Journal of Anatomy105, 149-162.

PALADE, G. E. (1952). A study of fixation for electron microscopy. Journal of Experimental Medicine 95,285-298.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron micros-copy. Journal of Cell Biology 17, 208-213.

SCHMIDT, W. (1963). Struktur und Funktion des Amnioepithels von Mensch und Huhn. Zeitschrift fiurZellforschung und mikroskopische Anatomie 61, 642-660.

SCOGGIN, W. A., HARBERT, G. M., ANSLOW, W. P., VAN'T RIET, B. & MCGAUGHEY, H. S. JR. (1964).Fetomaternal exchange of water at term. American Journal of Obstetrics and Gynecology 90, 7-16.

SEEDS, A. E. (1968). Amniotic fluid and fetal water metabolism. In: Intrauterine Development (Ed. A. C.Barnes.) Philadelphia: Lea and Febiger.

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54 A. D. HOYES

THOMAS, C. E. (1965). The ultrastructure of human amnion epithelium. Journal of Ultrastructure Research13, 65-84.

WILT, J. C., MILLER, D. & RUITER, J. (1964). Human amnion for tissue culture. Canadian Journal ofMicrobiology 10, 169-174.

WYNN, R. M. & FRENCH, G. L. (1968). Comparative ultrastructure of the mammalian amnion. Obstetricsand Gynecology 31, 759-774.

WYNN, R. M. & CORBETT, J. R. (1969). Ultrastructure of the canine placenta and amnion. AmericanJournal of Obstetrics and Gynecology 103, 878-887.