The localization of non-specific alkaline phosphatase activity during cleavage and blastocyst formation has been investigated in the mouse by electron microscopy. The activity is detectable for the first time at the two-cell stage and is localized on the surface of the interblastomeric plasma membranes and on small cytoplasmic inclusions. It increases in the following stages, predominantly on the interblastomeric membranes, the outside membranes remaining devoid of reaction. From the four-cell stage on, small reactive grains are also observed in the crystalloid plates of the cytoplasm. At the morula stage, the plasma membranes of the inner mass cells are entirely marked by the reaction whereas the trophoblastic ceils are polarized, with their inner surfaces positive and outside surfaces negative. At the blastocyst stage the enzyme is gradually eliminated from the membranes bordering the blastocoel and from the interblastomeric furrows of the trophoblast and primary endoderm. The significance of the differential localization of the enzyme is discussed, especially in relation to the differentiation mechanisms of the trophoblast and inner cell mass.

In a previous paper (Mulnard, 1974) the localization of non-specific alkaline phosphatase in the early development of the rat and mouse were comparatively reevaluated by means of an improved technique involving fixation by glutaraldehyde. The original method of Gomori-Takamatsu, applied to whole mounted embryos, gave the following results in the case of the mouse: (1) the enzymic activity appears at the four-cell stage and perhaps, but not conclusively, at the two-cell stage, (2) at all cleavage stages it is mainly localized on the interblastomeric membranes and absent from the outside surfaces of the blastomeres. Activity was also found on small cytoplasmic inclusions, (3) at the blastocyst stage, the reaction fades rapidly in the trophoblastic and primary endodermal cells where it disappears from the interblastomeric membranes and from the surface bordering the blastocoel; hence in the egg-cylinder-stage embryo of the early 6th day the reaction appears to be restricted to the primary ectoderm, (4) the extra-embryonic ectoderm is devoid of reaction from the beginning of its formation.

Such a gross analysis was only a prelude to an ultrastructural study, the results of which are presented in this paper.

The animals were of the white (NMRL) or black (C57B1) randomly bred strains. Ovulation was always spontaneous and the day of discovery of the copulation plug was counted as the first of development. The embryos were prefixed for 1 h at 2 °C in a 2% solution of glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 8 % sucrose. They were then washed at 2 °C for 2·3 h in the buffer and incubated for 15 min at 37 °C. The incubation medium was that of Mayahara, Hirano, Saito & Ogawa (1967) with Reynold’s solution as a source of Pb ions (Tris-HCl buffer 0.2 M, pH 8.5, 1.4 ml ; Na β glycerophosphate 0.1 M, 2 ml; Reynold’s solution, 4 ml; 0.15% MgSO4, 2-6 ml). Sucrose was added at the final concentration of 8 % and pH was adjusted to 9.2·9.4 by 0.1 N-HCI. Control embryos were incubated in the same mixture in which glycerophosphate was replaced by the corresponding amount of buffer. After incubation, the embryos were rinsed for a few minutes in cacodylate buffer and immediately post-fixed for 45 min in a 2 % solution of OsO4in 0.1 M cacodylate buffer (pH 7.4) at 2 °C in the dark. After a short rinsing in the buffer, they were dehydrated and embedded in Epon, without propylene oxide treatment.

Thin (1-2 μm) or ultrathin (0·03 μm) sections were cut on a LKB microtome. The thin sections were mounted without staining for examination by phasecontrast. The ultrathin sections were directly examined with a M III Philips electron microscope. In order to verify the good preservation of the structures, some ultrathin sections were stained with uranyl acetate-lead citrate.

For each stage some embryos were treated after incubation and rinsing by diluted (NH4)2S and mounted for in toto examination.

No reaction could be detected in the uncleaved egg, whether fertilized or not. The first evidence of alkaline phosphatase activity was found at the two-cell stage (Fig. 1 A) : very small grains of lead precipitate are scattered on the adjacent membranes of the two blastomeres and mainly on the surface of the microvilli and on the walls of the thin cisternae which are already commonly present in the interblastomeric space. A few grains of the same size are found in the cytoplasm, either isolated or in small clusters, in the vicinity of the membrane.

Fig. 1.

Electron micrographs of the interblastomeric regions at the two-cell (A) and four-cell (B) stages. Mayahara’s method, uncontrasted. Small grains of lead phosphate precipitate on the plasma membranes, on the surface of the microvilli and on the wall of the few intercellular cisternae. Intracytoplasmic grains of the same size, isolated or in clusters, some of them in small crystalloid plates (CR). Obvious increase of the positive structures from stage II to stage IV.

Fig. 1.

Electron micrographs of the interblastomeric regions at the two-cell (A) and four-cell (B) stages. Mayahara’s method, uncontrasted. Small grains of lead phosphate precipitate on the plasma membranes, on the surface of the microvilli and on the wall of the few intercellular cisternae. Intracytoplasmic grains of the same size, isolated or in clusters, some of them in small crystalloid plates (CR). Obvious increase of the positive structures from stage II to stage IV.

At the four-cell stage (Fig. 1B) the localization is identical but the grains of precipitate are more numerous and thicker than at stage II. Also some of the intracytoplasmic clusters are larger and exhibit a crystalloid structure. At the two-cell as well as the four-cell stage the outside membrane of the blastomeres is devoid of reaction. The nuclei are also entirely negative.

At the eight-cell stage (Fig. 2) the situation is the same as at stage IV, with a continuous increase of the number and size of the reactive grains on the surface of the inside membranes of the cells and in the cytoplasm where large clusters of crystalloid appearance are found in increasing number (Fig. 2C). Higher magnification (Fig. 2D) indicates that the large clusters correspond to the well-known ‘crystalloid plates’ (see Calarco & Brown, 1969; Gianguzza & Mulnard, in which isolated or aggregated particles of lead phosphate precipitate are scattered. Again at stage VIII the outside membranes and nuclei are devoid of reaction.

FIGURE 2.

Distribution of the alkaline phosphatase activity at the eight-cell stage. Mayahara’s method, uncontrasted.

(A) An embryo treated by NH42S after incubation and photographed in toto : absence of reaction on the outside membranes facing the zona pellucida. Heavy reaction on the interblastomeric regions.

(B) Thin section examined under phase-contrast: positive reaction in the interblastomeric furrows and on a few cytoplasmic inclusions. No reaction on the outside membranes. The nucleoli have the same contrast in control sections of embryos incubated without substrate.

(C) Uncontrasted electron micrograph of the central region of an eight-cell embryo : increased number and size of the grains of lead precipitate on the membranes of three adjacent blastomeres. In the cytoplasm, numerous grains of precipitate, either isolated or aggregated in small clusters. Positive granular reaction on large crystalloid plates (CR).

(D) Higher magnification of the same preparation to demonstrate two crystalloid plates containing numerous positive inclusions. Fibrous material (F) is also visible but shows no reaction.

FIGURE 2.

Distribution of the alkaline phosphatase activity at the eight-cell stage. Mayahara’s method, uncontrasted.

(A) An embryo treated by NH42S after incubation and photographed in toto : absence of reaction on the outside membranes facing the zona pellucida. Heavy reaction on the interblastomeric regions.

(B) Thin section examined under phase-contrast: positive reaction in the interblastomeric furrows and on a few cytoplasmic inclusions. No reaction on the outside membranes. The nucleoli have the same contrast in control sections of embryos incubated without substrate.

(C) Uncontrasted electron micrograph of the central region of an eight-cell embryo : increased number and size of the grains of lead precipitate on the membranes of three adjacent blastomeres. In the cytoplasm, numerous grains of precipitate, either isolated or aggregated in small clusters. Positive granular reaction on large crystalloid plates (CR).

(D) Higher magnification of the same preparation to demonstrate two crystalloid plates containing numerous positive inclusions. Fibrous material (F) is also visible but shows no reaction.

The overall distribution of the membranar enzymic activity in the morula and young blastocyst of the 4th day (16—32 cells) is clearly demonstrated by phasecontrast examination of thin sections (Fig. 3A): the reaction is positive along the interblastomeric furrows and absent from the outside membranes of the trophoblast which now forms a continuous envelope around the inner cell mass. At the electron-microscopical level (Fig. 3B) the interblastomeric plasma membranes of trophoblastic as well as inner mass cells are seen to be coated with a more or less regular row of large and dense grains of lead phosphate precipitated on the outer surface of each membrane, as is clearly shown where the two membranes are locally separated to form intercellular cisternae. One can also see how abruptly the reaction stops at the junction between a peripheral interblastomeric furrow and the outside membrane of the two adjacent trophoblastic cells (Fig. 3B, arrow). The membranar reaction is interrupted along the few tight junctions which have developed between the blastomeres (Fig. 3C, arrow). In contrast with the increased membranar activity, the number of positive intracytoplasmic structures - isolated or aggregated granulations - has somewhat decreased.

FIGURE 3.

Morula stage (16-32 cells). Mayahara’s method, uncontrasted.

(A) Thin section photographed under phase-contrast: positive reaction on the inside membranes of all blastomeres. No reaction on the outside membranes of the trophoblast. The nucleoli have the same contrast as in control embryos.

(B) Low magnification electron micrograph of the peripheral region of a 16-cell morula: positive reaction on the adjacent plasma membranes of inner mass (IC) and trophoblastic (T) cells. Clear-cut limit between the positive interblastomeric and the negative outside membranes of two adjacent trophoblastic cells (arrow). The isolated or aggregated reactive grains and the crystalloid plates are less numerous than at stage VIII.

(C) Higher magnification of the central region of a similar morula showing the more or less regular row of grains of lead phosphate precipitate and their situation on the external aspect of the adjacent plasma membranes. Note the absence of reaction along the tight junction uniting two of the blastomeres (arrow).

FIGURE 3.

Morula stage (16-32 cells). Mayahara’s method, uncontrasted.

(A) Thin section photographed under phase-contrast: positive reaction on the inside membranes of all blastomeres. No reaction on the outside membranes of the trophoblast. The nucleoli have the same contrast as in control embryos.

(B) Low magnification electron micrograph of the peripheral region of a 16-cell morula: positive reaction on the adjacent plasma membranes of inner mass (IC) and trophoblastic (T) cells. Clear-cut limit between the positive interblastomeric and the negative outside membranes of two adjacent trophoblastic cells (arrow). The isolated or aggregated reactive grains and the crystalloid plates are less numerous than at stage VIII.

(C) Higher magnification of the central region of a similar morula showing the more or less regular row of grains of lead phosphate precipitate and their situation on the external aspect of the adjacent plasma membranes. Note the absence of reaction along the tight junction uniting two of the blastomeres (arrow).

In the early 5th day blastocyst a rapid change occurs in the distribution of the membranar alkaline phosphatase activity (Fig. 4): the reaction remains unchanged on the intercellular membranes of the primary ectoderm cells and practically also on the inside membranes of the cells which immediately surround them. The outside membranes of the trophoblastic cells remain negative. But the reaction has considerably faded in the intercellular furrows of the trophoblast and of the primary endoderm, where only a few very small grains can still be observed, and on the membranes, either trophoblastic or endodermal, which border the blastocoel. In the vicinity of the cell surfaces and especially along the endodermal wall, small cytoplasmic fragments, either free or still attached to the blastomeres and covered with tiny grains of lead precipitate, are seen floating in the blastocoelic fluid, giving the impression of a ‘desquamation’ of the cell surface. As in the morula isolated or aggregated positive granules and crystalloid plates are less frequently observed than at the eight-cell stage.

FIGURE 4.

Blastocyst of the late 4th day having spontaneously shed its zona pellucida. Maya-hara’s method, uncontrasted.

(A) Thin section examined under phase-contrast: heavy positive reaction in the interblastomeric furrows of the primary ectoderm (P.EC). No reaction detectable with that technique in the trophoblast (T) or the primary endoderm (P. EN).

(B) Low magnification electron micrograph of a similar blastocyst showing the intense positive reaction on the adjacent membranes of the primary ectoderm cells (P. EC). Absence of reaction on the outside membranes of the trophoblastic cells (T). Decreasing number and size of the positive grains in the interblastomeric furrows and on the blastocoelic surface of the trophoblast and primary endoderm (P. EN). Presence in the blastocoelic fluid of membranar debris covered with small positive grains, some still attached to the inside surface of the primary endoderm cells.

FIGURE 4.

Blastocyst of the late 4th day having spontaneously shed its zona pellucida. Maya-hara’s method, uncontrasted.

(A) Thin section examined under phase-contrast: heavy positive reaction in the interblastomeric furrows of the primary ectoderm (P.EC). No reaction detectable with that technique in the trophoblast (T) or the primary endoderm (P. EN).

(B) Low magnification electron micrograph of a similar blastocyst showing the intense positive reaction on the adjacent membranes of the primary ectoderm cells (P. EC). Absence of reaction on the outside membranes of the trophoblastic cells (T). Decreasing number and size of the positive grains in the interblastomeric furrows and on the blastocoelic surface of the trophoblast and primary endoderm (P. EN). Presence in the blastocoelic fluid of membranar debris covered with small positive grains, some still attached to the inside surface of the primary endoderm cells.

The electron-microscopical analysis has confirmed and given finer detail to the main results obtained by the method of Gomori-Takamatsu applied to whole embryos (Mulnard, 1974). Up to the morula stage non-specific alkaline phosphatase activity is observed on the inside membranes of all blastomeres and absent from their outside membranes. In the young blastocyst it disappears gradually from the trophoblast and from the primary endoderm except where these groups are in contact with the primary ectoderm cells.

Before any conclusion can be drawn from these observations, it is necessary to consider the possibility of an artifact due to selective inactivation or elimination of the superficial enzyme by the fixative or to removal of its reaction products. Selective and irreversible inactivation by glutaraldehyde is highly improbable: considering the easy penetration of the fixative, any irreversible action should affect the deep regions of the embryo as well as its surface, or at the least result in an outside-inside gradient of inactivation. The clear-cut limit between the positive interblastomeric reaction and the negative outside surfaces of adjacent trophoblastic cells of the morula (see Fig. 3B) is not explicable by an inactivation effect of the fixative. The hypothesis of a selective removal of the reaction product by the post-fixation and embedding procedures must also be discarded since control treatment of whole embryos by ammonium sulfide immediately after incubation gives the same negative result on the outside membranes (see Fig. 2A). Perhaps more difficult to eliminate is the possibility of a ‘scouring’ action of the fixative on the free membranes with subsequent removal of the membranar debris by washing, an action which would not affect the better protected interblastomeric membranes and, to a lesser degree, the membranes bordering the blastocoel where the debris would not be entirely washed away (Fig. 4). Such an artifact is also improbable when one considers (1) that at the blastocyst stage (Fig. 4) the same residual activity is observed not only along the membranes bordering the blastocoel but also in the interblastomeric furrows of the trophoblast and of the primary endoderm where the membranes should be protected against any removal effect, whereas the reaction remains unchanged between the cells of the primary ectoderm, (2) that after identical fixation procedure the reaction is positive on the outside membranes of the trophoblast for other membrane-bound phosphatases such as 5′-nucleotidase (unpublished observations).

However, the differential localization of the alkaline phosphatase in the preimplantation mammalian embryo remains a subject of controversy, in spite of the increasing precision of the cyto-enzymological methods. In a recent publication on membrane-bound phosphatases in the early development of the mouse, Vorbrodt, Konwinski, Solter & Koprowski (1977) describe an entirely different distribution of the alkaline phosphatase activity which is claimed to be predominantly localized on the outside and adjacent trophoblastic membranes of the blastocyst and only occasionally detected in the inner cell mass. Such a radical discrepancy can perhaps be explained by an unusually long incubation (60-120 min) at a relatively low pH (8-6 instead of 9-2-9-4) at which other hydrolytic enzymes present on the outside membranes - such as 5′-nucleotidase - are capable of cleaving glycerophosphate or CMP (see Goff & Milaire, 1974). On the other hand, our results are in good agreement with the conclusions of Izquierdo and his co-workers, who found the alkaline phosphatase activity of the mouse morula and blastocyst is definitely higher in the inner cell mass than in the trophoblast (Izquierdo & Marticorena, 1975; Izquierdo & Ortiz, 1975; Izquierdo 1977).

The accumulating evidence of a differential, temporal and spatial localization of non-specific alkaline phosphatase points to the possibility of a relation of the enzyme to the mechanisms which regulate the differentiation of the two fundamental groups of cells in the preimplantation embryo, inner cell mass and trophoblast. Up to the young blastocyst stage the membrane phosphatase activity is localized along the interblastomeric furrows, the free surfaces of the blastomeres being entirely negative. The result is that as soon as a cell becomes completely surrounded by others the enzymic activity extends to the entire surface of its plasma membrane whereas the peripheral blastomeres remain polarized, with their inside membranes positive and outside membranes devoid of activity. It is thus conceivable that the alkaline phosphatase is implicated in general surface modifications responsible for the differentiation of the two groups of blastomeres. Such a mechanism would be in accordance with Tarkowski & Wrobleska’s conception (1967) of a differentiation depending on the central or peripheral position of the blastomeres in the morula.

Also of special interest is the disappearance of the alkaline phosphatase from the cell groups of the blastocyst which undergo early extra-embryonic differentiation : trophoblast and primary endoderm. The ultrastructural analysis has confirmed this finding of a previous in toto study (Mulnard, 1974) : it shows a considerable reduction of the size and number of the grains of lead phosphate precipitated along the blastocoelic wall and in the interblastomeric furrows. It also suggests that the enzyme is possibly eliminated by a sort of desquamation of the cell membranes bordering the blastocoel and finally released with the membrane debris in the blastocoelic fluid. This modification could explain the difference observed by Calarco & Epstein (1973) in scanning electron microscopy between the cell surfaces bordering the blastocoel and the outer cell surface of the mouse blastocyst. However, it is not clear whether the cytoplasmic debris covered with positive grains and floating in the blastocoelic fluid represent a process of membrane elimination or the remnants of trophoblastic fragments isolated at the wall of the blastocoel during its formation (unpublished personal observations).

The functional significance of the non-specific alkaline phosphatase in the early mammalian development has not been elucidated so far. In the mouse, the predominant membranar localizations are remarkable because the final product of the reaction is situated on the external aspect of the interblastomeric plasma membranes (see Fig. 3), suggesting that the enzymic molecules are inserted in the membrane with their active group turned outside. This is consistent with the hypothesis that the alkaline phosphatase could be implicated, here as in various adult tissues (small intestine, kidney tubules… see Fernley, 1971), in the active transport of small molecules across the plasma membrane. In addition to the membrane reaction, more discrete sites of enzymic activity were observed in the depth of the cytoplasm. They show a maximum development between stages II and VIII and tend to be less frequent in the morula and blastocyst. The granules found in the vicinity of the membranes, either isolated or in small clusters, could be the cytochemical visualization of small Golgi vesicles representing the transportation sites of the enzyme to its functional membranar localization. Of particular interest is the granular reaction found in the crystalloid plates. It was previously demonstrated that these structures are closely associated with small elements of rough endoplasmic reticulum (Gianguzza & Mulnard, 1972). The positive granules observed in the plates (see Fig. 2D) would possibly correspond to sites of enzymic synthesis. Those interpretations are however conjectural in the present state of our knowledge.

We are indebted to W. Zwijsen for technical assistance.

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