The distribution of 5’-nucleotidase activity in pre-implantation mouse embryos is studied by means of a cytochemical method adapted from Uusitalo & Karnovsky (1977). The enzyme activity is detected, from the 4-cell stage up to the morula stage, on discrete patches of the cell membane between blastomeres. Appropriate controls show that this distribution is not a localization artifact due to selective retention of the enzyme reaction product in the narrow interblastomeric spaces. In early blastocysts, as the blastocoel expands the enzyme activity on its lining disappears. The external surface of the trophectoderm in early blastocysts lacks any enzyme activity, whereas in late blastocysts a strong enzyme activity is detected at the embryonic trophectoderm, decreasing in intensity towards the opposite pole of the embryo.

These results are compared to previous observations by other authors and the differences are mainly ascribed to differences in the cytochemical procedure employed.

We conclude that during cleavage a gradual cell membrane regionalization unfolds, revealing a pattern that may be related to morphogenesis; in particular, to the localization of zonular tight junctions around the peripheral blastomeres of the morula (Izquierdo, 1977; Izquierdo, López & Marticorena, 1980).

The inside-outside model originally proposed by Tarkowski & Wroblewska (1967) still offers the most coherent explanation for the differentiation of inner cell mass and trophectoderm during the transition from morula to blastocyst. The model aptly reduces the problem to a particular case of differential gene activity elicited by diverse cellular microenvironments. Nevertheless, positional information required for blastocyst morphogenesis (Izquierdo, 1977; Johnson, Pratt & Handyside, 1981) may depend, not on which genes are transcribed in different cells but rather on where a gene product localizes within a cell. A case in point is the proposed causal relationship between peripheral sealing of interblastomeric spaces by zonular tight junctions and the development of the blastocoel (Ducibella & Anderson, 1975; Izquierdo, Fernandez & López, 1976; Ducibella, 1977; Fernández & Izquierdo, 1980). If tight junctions were exclusively found around peripheral blastomeres, one might assume that their localization could be explained by differential gene activity leading to the synthesis of specific tight-junction components. However, incipient focal junctions, admittedly not easy to classify, can be seen between inner blastomeres of the morula (Ducibella, Albertini, Anderson & Biggers, 1975; Izquierdo et al. 1976). Furthermore, when the number of blastomeres at blastulation is diminished by different procedures, peripheral sealing is not prevented even though proper inside and outside cells may not exist (Smith & McLaren, 1977; Fernández & Izquierdo, 1980; Izquierdo & Becker, 1982); this situation would preclude the emergence of an inside-outside morphogenetic pattern, if based on the interaction of cells that express different genes. These and other observations (see Johnson, Pratt & Handyside, 1980) led us to assume that the search for positional information demands, not only an analysis of cellular regionalization within the embryo but also a description of the regionalization of cellular components within embryonic cells.

We described in a previous work the regionalization of the cell membrane in preimplantation mouse embryos as revealed by the distribution of alkaline phosphatase activity (Izquierdo et al, 1980). Results were interpreted according to a model which proposes that a morphogenetic pattern emerges during cleavage as a result of localized formation of discrete patches of ‘new’ Cell membrane (Izquierdo, 1977). In the present work we have recourse as a marker to the cytochemical demonstration of 5’-nucleotidase activity. Two reports have already dealt with the onset and distribution of 5’-nucleotidase activity in preimplantation embryos (Vorbrodt, Konwinski, Solter & Koprowski, 1977; Nizeyimana-Rugina & Mulnard, 1979); however, this matter requires further investigation for several reasons: (1) former observations were not intended to prove or disprove a morphogenetic model, (2) they disagree in several points and (3) cytochemical methods for the demonstration of 5’-nucleotidase have recently been revised and improved (Uusitalo & Karnovsky, 1977; Yamashina & Kawai, 1979; Klaushofer & von Mayersbach, 1979).

About 720 embryos from spontaneously ovulating Swiss-Rockefeller mice were used. The age of the embryos was reckoned starting at 0 h of the day in which the vaginal plug was detected. Oviducts were flushed with Biggers’ medium (Biggers, Whitten & Whittingham, 1971) and the embryos were processed for the cytochemical demonstration of 5’-nucleotidase activity according to a modified version of Uusitalo & Karnovsky’s method (1977). Modifications refer to the use of a different fixative and incubation medium. In preliminary trials we compared the effect of different fixatives containing tris-maleate or cacodylate buffers at pH values ranging from 6 to 7·5; we also compared the effect of different incubation media containing Pb(NO3)2 at concentrations ranging between 0·25 and 6 mM. Non-specific lead deposits were more frequent when high lead concentrations were used; however, results were not strictly proportionate, and even low lead concentrations occasionally produced unspecific deposits. In these preliminary trials 120 embryos were used.

Best results regarding sensitivity and specificity (see controls, below) were obtained with the following procedure. Embryos were fixed in 1 % (w/v) glutaraldehyde in 0·1 M-tris-maleate buffer pH 6 during 60 min at room temperature. After three rinses in 0·1 M-tris-maleate buffer pH 7·2, embryos were incubated during 50 min at 37 °C in a medium containing 0·1 M-tris-maleate buffer pH 7·2, 1·5 mM 5’AMP (Sigma), 1·5 mM Pb(NO3)2 and 10 mM-MgS04. The fixative, the buffers used for rinsing and the incubation medium contained 5% (w/v) glucose (Sundström & Mörnstad, 1975; E. Rodríguez, personal communication).

For light microscopy, after incubation the embryos were rinsed three times in tris-maleate buffer pH 7·2 and the enzyme reaction product was developed with 0·1 % (w/v) ammonium sulphide. The embryos were then whole-mounted in glycerol.

For electron microscopy, after incubation the embryos were rinsed three times in 0·1 M-cacodylate buffer pH 7·2 and then post-fixed in 3% (w/v) glutaraldehyde in cacodylate buffer during 60 min at room temperature. After rinsing in buffer, post-fixation continued for 60 min at room temperature With OSO4 (w/v) in 0-1 M-cacodylate buffer. The embryos were rinsed again in buffer, dehydrated in graded acetone and embedded in a low-viscosity resin (SpUrr, Poly science). Thin sections were obtained in a Porter-Blum ultramicrotome, the grids were stained during 2 min in 4 % (w/v) uranyl acetate in methanol and observed in a Philips EM 300 electron microscope.

Two kinds of control were used. In order to determine the specificity of the enzyme reaction we tested (a) incubation in substrate-free medium ; (b) incubation with sodium β-glycerophosphate or ATP as analogous substrates; (c) incubation without the activator; (d) incubation at 0 °C; (e) incubation after heating the embryos for 30 min at 95 °C; (/) incubation in a medium with 0·05 M-NaF. In order to discard a localization artifact that might be produced by retention of the enzyme reaction product in the interblastomeric spaces, we fused early morulae with 2-cell embryos, according to a method described elsewhere (Izquierdo et al. 1980). These chimaeras were cultured for 5 h before being processed for 5’nucleotidase demonstration.

In embryos with less than four blastomeres, 5’-nucleotidase activity is absent, but it is consistently present in embryos with more than four blastomeres. Embryos with 4 cells, recovered before 48 h of development, intermingled with 2- and 3-cell embryos, show a negative reaction, while the reaction is positive in 4-cell embryos recovered after 58 h of development, intermingled with early morulae. That is, the onset of enzyme activity, as recognized by this cytochemical method, coincides with the late 4-cell stage.

Light microscopy shows .that the enzyme reaction product appears between blastomeres as small patches that become larger as cleavage proceeds. In morulae and early blastocysts the external surface of the embryo lacks any enzyme activity. In late blastocysts, which have more than 106 h of development, enzyme activity can be detected on the external surface of the polar trophectoderm, gradually decreasing towards the opposite pole of the embryo (Figs. 1-5). Light microscope observations, excluding control series, were performed on 198 embryos.

Figs. 1.

Whole mounts of embryos in different stages of development. Bars represent 20μm.

Fig. 1. Late 4-cell embryo (58 h of development). A patch of 5’-nucleotidase activity is seen between the two blastomeres in focus.

Figs. 1.

Whole mounts of embryos in different stages of development. Bars represent 20μm.

Fig. 1. Late 4-cell embryo (58 h of development). A patch of 5’-nucleotidase activity is seen between the two blastomeres in focus.

Fig. 2.

Early morula (58 h of development). The enzyme activity is detected in interblastomeric spaces whereas no activity is detected on the surface of the embryo.

Fig. 2.

Early morula (58 h of development). The enzyme activity is detected in interblastomeric spaces whereas no activity is detected on the surface of the embryo.

Fig. 3.

Late morula (80 h of development). Observations similar to Fig. 2.

Fig. 3.

Late morula (80 h of development). Observations similar to Fig. 2.

Fig. 4.

Early blastocyst (84 h of development). The enzyme activity is detected between cells of the inner mass and in some places of the blastocoel (b) lining. No activity is detected on the outer aspect of the trophectoderm.

Fig. 4.

Early blastocyst (84 h of development). The enzyme activity is detected between cells of the inner mass and in some places of the blastocoel (b) lining. No activity is detected on the outer aspect of the trophectoderm.

Fig. 5.

Late blastocyst (104 h of development). Enzyme activity is detected between cells of the inner mass and on the outer surface of the trophectoderm at the embryonal pole.

Fig. 5.

Late blastocyst (104 h of development). Enzyme activity is detected between cells of the inner mass and on the outer surface of the trophectoderm at the embryonal pole.

The controls on the localization of the enzyme reaction product, using aggregated embryos, showed that each embryo presented the characteristic pattern of its stage and that no activity was found in the artificial cleft between them (Fig. 6). Twenty-three embryo pairs were observed. As to controls designed to test the specificity of the enzyme activity, all gave negative results (Fig. 7). One hundred and seventy-six embryos were observed with the light or the electron microscope during these tests.

Fig. 6.

A 2-cell embryo (later cleaved into 3 cells) paired with an early morula and cultured for 5 h. The enzyme activity is detected between blastomeres of the morula. No activity is detected in the 3-cell embryo nor on the surface between both embryos.

Fig. 6.

A 2-cell embryo (later cleaved into 3 cells) paired with an early morula and cultured for 5 h. The enzyme activity is detected between blastomeres of the morula. No activity is detected in the 3-cell embryo nor on the surface between both embryos.

Fig. 7.

Whole mounts of morulae. From left to right: normal control embryo; embryo incubated with 1·5 mM sodium β-glycerophosphate instead of 5’AMP; embryo incubated without Mg2+; embryo incubated with 0·05 M-NaF.

Fig. 7.

Whole mounts of morulae. From left to right: normal control embryo; embryo incubated with 1·5 mM sodium β-glycerophosphate instead of 5’AMP; embryo incubated without Mg2+; embryo incubated with 0·05 M-NaF.

Although there is evidence that the fixation procedure breaks down the permeability barrier to phosphatase substrates (Uusitalo & Karnovsky, 1977) electron microscopy shows that the enzyme reaction product is confined to the surface of the plasma membrane. At the 4-cell stage the enzyme reaction product appears in discrete regions of the cell surface of adjoining blastomeres. These regions grow as development continues, but always remain restricted to interblastomeric surfaces. Therefore a distinct regionalization of the plasma membrane can be recognized in peripheral blastomeres: the membrane facing the zona pellucida is devoid of enzyme reaction product, whereas the membrane facing the blastomeres is full of it. The precise boundary of these regions coincides with the site where zonular tight junctions become established (Figs. 8 and 9).

Figs. 8-11.

Electron micrographs of embryos in different stages of development. Bars represent 1 μm.

Fig. 8. Early morula (60 h of development). The enzyme reaction product is confined to the interblastomeric surface and does not extend beyond the site where tight junctions are localized (arrow). The cell surface facing the zona pellucida (zp) lacks reaction product. Unspecific lead deposits are observed in the perivitelline space.

Figs. 8-11.

Electron micrographs of embryos in different stages of development. Bars represent 1 μm.

Fig. 8. Early morula (60 h of development). The enzyme reaction product is confined to the interblastomeric surface and does not extend beyond the site where tight junctions are localized (arrow). The cell surface facing the zona pellucida (zp) lacks reaction product. Unspecific lead deposits are observed in the perivitelline space.

Fig. 9.

Late morula (80 h of development). The wide interblastomeric spaces are lined by the enzyme reaction product.

Fig. 9.

Late morula (80 h of development). The wide interblastomeric spaces are lined by the enzyme reaction product.

During blastulation, the cell membranes surrounding the nascent blastocoel reveal 5’-nucleotidase activity, but as the cavity expands its lining of enzyme reaction product becomes discontinuous and soon disappears. Electron micrographs suggest that the wall of the cytoplasmic vesicles which empty their contents into the blastocoel may contribute to its enlarging surface; however, a unit membrane encircling these vesicles is not clearly seen (Figs. 10 and 11).

Fig. 10.

Nascent blastocyst (80 h of development). A cytoplasmic ‘vesicle’ (v) emptying its contents into the growing blastocoel (b). The blastocoel is still lined by the enzyme reaction product whereas no reaction product is seen on the vesicle wall.

Fig. 10.

Nascent blastocyst (80 h of development). A cytoplasmic ‘vesicle’ (v) emptying its contents into the growing blastocoel (b). The blastocoel is still lined by the enzyme reaction product whereas no reaction product is seen on the vesicle wall.

Fig. 11.

Nascent blastocyst (80 h of development). The enzyme reaction product lining the expanding blastocoel (b) is discontinuous.

Fig. 11.

Nascent blastocyst (80 h of development). The enzyme reaction product lining the expanding blastocoel (b) is discontinuous.

Fourth-day blastocysts show enzyme reaction product between cells of the inner mass and in few patches around the blastocoel surface. Enzyme activity is rarely detected at the small contact surface between trophectoderm cells or at the external surface of the embryo. In fifth-day blastocysts, enzyme activity appears on the outer surface of the trophectoderm at the embryonal pole, decreasing in intensity towards the abembryonal pole (Figs. 12-19). Electron microscope observations, excluding control series, were performed on approximately 180 embryos. A summary of our results is presented in fig. 20.

Fig. 12-15.

Late blastocyst (104 h of development). Strong enzyme activity is detected on the external surface of the trophectoderm at the embryonal pole (Fig. 12), gradually decreasing towards the opposite pole (Figs. 13, 14) where no activity is detected (Fig. 15); Bar represents 1μm.

Fig. 12-15.

Late blastocyst (104 h of development). Strong enzyme activity is detected on the external surface of the trophectoderm at the embryonal pole (Fig. 12), gradually decreasing towards the opposite pole (Figs. 13, 14) where no activity is detected (Fig. 15); Bar represents 1μm.

Fig. 16-19.

Another late blastocyst (104 h of development) showing a similar distribution of the enzyme reaction product; this diminishes from the embryonal pole (Fig. 16) towards the abembryonal pole (Fig. 19). Bar represents 1 μm.

Fig. 16-19.

Another late blastocyst (104 h of development) showing a similar distribution of the enzyme reaction product; this diminishes from the embryonal pole (Fig. 16) towards the abembryonal pole (Fig. 19). Bar represents 1 μm.

Fig. 20.

Diagram representing 5’-nucleotidase activity in different developmental stages. The enzyme reaction product is indicated by bold lines.

Fig. 20.

Diagram representing 5’-nucleotidase activity in different developmental stages. The enzyme reaction product is indicated by bold lines.

Our observations are in keeping with those by Vorbrodt et al. (1977) except in that they report 5’-nucleotidase activity on the external surface of the embryo beginning at the advanced morula stage, while we do not observe it until the late blastocyst. The results reported by Nizeyimana-Rugina & Mulnard (1979) barely show any resemblance to ours, as these authors describe enzyme activity on the embryo surface at all stages and also within cells, in the form of cytoplasmic and nuclear clusters. The discrepancies, particularly those between our observations and those of Nizeyimana-Rugina & Mulnard, can be at least partly ascribed to differences in the method employed for demonstrating the enzyme activity; especially to differences in fixative composition and in lead content of the incubation medium.

Parallel biochemical and cytochemical experiments on mouse lymphocytes have shown that the standard fixation with glutaraldehyde in cacodylate buffer pH 7·2-7·4 causes an important inhibition of 5’-nucleotidase activity which can be avoided by using a slightly acidic fixative buffered with tris-malaete (Uusitalo & Karnovsky, 1977). These resits have been confirmed in acinar cells of rat salivary glands and in liver tissue (Yamashina & Kawai, 1979; Klaushofer & von Mayersbach, 1980). Since similar results were obtained in our preliminary trials, the composition of the fixative we used thereafter differs considerably from that used by Vorbrodt’s group or by Nizeyimana-Rugina & Mulnard.

Better preservation of the enzyme activity by proper fixation allows for a reduction of lead concentration in the incubation medium, thus avoiding the formation of non-specific lead deposits. Uusitalo & Karnovsky (1977) used 1-8 mM-Pb(NO3) instead of 3·6 mM, which is the concentration in the standard Wachstein-Meisel medium (Wachstein & Meisel, 1957). Klaushofer & von Mayersbach (1979) found that in liver rat tissue, prepared by n-butanol freeze substitution, best results were obtained with only 0·6 mM, and our preliminary trials also showed the advantage of low concentration. We used 1·5 mM-Pb(NO3)2 in the routine procedure, Vorbrodt et al. (1977) used 3·6 mM and Nizeyimana-Rugina & Mulnard (1979) used 60 mM.

Our controls support the specificity of the cytochemical method employed, and most probably discard a localization artifact caused by selective retention of the reaction product between blastomeres. The interblastomeric spaces experimentally produced by fusing embryos retain no reaction product and later, when enzyme activity appears on the external surface of advanced blastocyst, the reaction product showed no sign of diffusion. We then conclude that our procedure is a reliable method for the cytochemical demonstration of 5’-nucleotidase activity, and hence that the cell membrane regionalization it reveals deserves an interpretation.

Observations reported here fit in with a model which proposes that localized ‘new’ cell membrane is formed during cleavage, thus developing a pattern which might provide every blastomere with information about its age and position (Izquierdo, 1977). According to this model, we have formerly interpreted the cell membrane regionalization revealed by alkaline phosphatase activity (Izquierdo et al. 1980), and the same rationale would fit the observations reported here. Briefly: since no 5’-nucleotidase activity is detected prior to the 4-cell stage and a large quantity of cell membrane is required for cleavage, after the 4-cell stage one might recognize ‘old’ cell membrane, which is devoid of 5’-nucleotidase activity, from ‘new’ cell membrane, which presents activity. If ‘new’ cell membrane is formed at the cleavage furrows, leaving a patch between sister blastomeres, the outer surface of peripheral blastomeres would remain covered solely by ‘old’ cell membrane. This interpretation accounts quite well for our results, not so well for Vorbrodt’s results and not at all for results obtained by Nizeyimana-Rugina & Mulnard.

Our interpretation may also account for the appearance of 5’-nucleotidase activity on the outer surface of advanced blastocysts and for the stronger reaction on the polar trophectoderm as compared to the mural trophectoderm. It has been shown that trophectoderm cells close to the inner cell mass divide faster than those farther away (Copp, 1978); therefore, trophectoderm cells at the abembryonal pole, which have gone through fewer divisions, according to our model should have less ‘new ‘membrane, that is, less 5-’nucleotidase activity.

In its most extreme form, the interpretation we offer assumes that discrete patches of cell membrane are formed at the leading edge of cleavage furrows, and indeed, evidence has already been advanced in other materials which would support such an idea (review in Izquierdo, 1977). However, several objections can be raised to this scheme as applied to preimplantation embryos. First, both 5’-nucleotidase and alkaline phosphatase (Ishiyama & Izquierdo, 1977 ; Izquierdo et al. 1980) show a negative reaction in early 4-cell stage and a positive reaction in late 4-cell stage, without intervening cell divisions; secondly, the cytochemical demonstrations of 5’-nucleotidase and alkaline phosphatase have not revealed any cytoplasmic component that might be regarded as a membrane precursor, near to the place where the membrane is supposedly assembled. Even a less extreme interpretation, if based on the formation of patches of’new’ cell membrane, would imply that these enzymes are prevented from diffusing on the plane of the membrane; however, up to now we have seen no ultrastructural evidence of cytoplasmic attachments in appropriate positions (see Izquierdo et al. 1980) and our observations on the effect of cytoskeletal inhibitors are still indecisive.

In this and former reports we have considered 5’-nucleotidase and alkaline phosphatase activities exclusively as markers of cell membrane regionalization without any regard for their physiological role in preimplantation embryos. This remains to be elucidated. In other better known and comparable systems, such as lymphoblast differentiation and lymphocyte proliferation, the role of 5’-nucleotidase seems to be independent of cell membrane formation (Dornard, Bonnafous, Gavach & Mani, 1979; Edwards et al. 1979). It is worth mentioning though, that by analogy with the formation of cell wall in bacteria it has been suggested that phosphatases, such as 5’-nucleotidase and alkaline phosphatase, might be involved in the synthesis of cell membrane (Fishman, 1974).

We have as yet found no direct relationship between 5’-nucleotidase or alkaline phosphatase and the establishment of zonular tight junctions; however, the regionalization revealed by these enzymes offers a clue to the study of spatial signals that may be required, among other things, for the positioning of cell junctions and the morphogenesis of the blastocyst.

This research was supported by grants from the Ford Foundation, PNUD/UNESCO and the University of Chile.

Biggers
,
J. D.
,
Whitten
,
W. K.
&
Whittingham
,
D. G.
(
1971
).
The culture of mouse embryos in vitro
.
In Methods in Mammalian Embryology
(ed.
J. D.
Daniel
), pp.
86
115
.
Copp
,
A. J.
(
1979
).
Interaction between inner cell mass and trophectoderm of the mouse blastocyst. II. The fate of the polar trophectoderm
.
J. Embryol. exp. Morph
.
51
,
109
120
.
Dornard
,
J.
Bonnafous
,
J. C.
Gavach
,
C.
&
Mani
,
J. C.
(
1979
).
5znucleotidase-facilitfcted adenosine transport by mouse lymphocytes
.
Biochemie
61
,
973
977
.
Ducibella
,
T.
,
Albertini
,
D. F.
,
Anderson
,
E.
&
Biggers
,
J. D.
(
1975
).
The preimplantation mammalian embryo; characterization of intercellular junctions and their appearance during development
.
Devi Biol
.
45
,
231
250
.
Ducibella
,
T.
&
Anderson
,
E.
(
1975
).
Cell shape and membrane changes in the eighbcell mouse embryo: pre-requisites for morphogenesis of the blastocyst
.
Devi Biol
.
47
,
45
58
.
Ducibella
,
T.
(
1977
).
Surface changes of the developing trophoblast cell
.
In Development in Mammals
, vol.
1
, (ed.
M. H.
Johnson
), pp.
5
30
.
Edwards
,
L. N.
,
Gelfand
,
E. W.
,
Burk
,
L.
,
Dosch
,
H. M.
&
Fox
,
I. H.
(
1979
).
Distribution of 5’nucleotidase in human lymphoid tissues
.
Proc. natn. Acad. Sci. U.S.A
.
76
,
3474
3476
.
Fernández
,
M. S.
&
Izquierdo
,
L.
(
1980
).
Blastocoel formation in half and double mpuse embryos
.
Anat. Embryol
.
160
,
77
81
.
Fishman
,
W. H.
(
1974
).
Perspectives on alkaline phosphatase isoenzymes
.
Am. J. Med
.
56
,
617
646
.
Ishiyama
,
V.
&
Izquierdo
,
L.
(
1977
).
The onset of phosphatase activity in early mammalian embryos
.
J. Embryol. exp. Morph
.
42
,
305
308
Izquierdo
,
L.
,
Fernández
,
M. S.
&
Lopez
,
T.
(
1976
).
Cell membrane and cell junctions in differentiation of preimplanted embryos
.
Archs Biol. Med. exp
.
10
,
130
134
.
Izquierdo
,
L.
(
1977
).
Cleavage and differentiation
.
In Development in Mammals
, vol.
2
, (ed.
M. H.
Johnson
), pp.
99
118
.
Izquierdo
,
L.
,
López
,
T.
&
Martincorena
,
P.
(
1980
).
Cell membrane regions in preimplantation mouse embryos
.
J. Embryol. exp. Morph
.
59
,
89
102
.
Izquierdo
,
L.
&
Becker
,
M. I.
(
1982
).
Effect of Li+ on preimplantation mouse embryos
J. Embryol. exp. Morph
.
67
,
151
159
.
Johnson
,
M. H.
,
Pratt
,
H. P. M.
&
Handyside
,
A. H.
(
1981
).
The generation and recognition of positional information in the preimplantation mouse embryo
.
In Cellular and Molecular Aspects of Implantation
(ed.
S. R.
Glasser
&
D. W.
Bullock
), pp.
55
74
.
Klaushofer
,
S. M.
&
Von Mayersbach
,
H.
(
1979
).
Freeze substituted tissue in 5’nucleotidase histochemistry. Comparative histochemical and biochemical investigations
.
J. Histochem. Cytochem
.
27
,
1583
1587
.
Nizeyimana-Rugina
,
E.
&
Mulnard
,
J.
(
1979
).
Ultrastructural localization of 5’nucleotidase in preimplantation mouse embryos
.
Archs. Biol
.
90
,
131
140
.
Smith
,
R.
&
McLaren
,
A.
(
1977
).
Factors affecting the time of formation of the mouse blastocoele
.
J. Embryol. exp. Morph
.
41
,
79
92
.
Sundstrôm
,
B.
&
Mônrstad
,
H.
(
1975
).
Lead citrate-containing media for use at alkaline pH: their stabilization with glucose and increased buffer strength
.
Stain Technol
.
50
,
287
288
.
Tarkowski
,
A. K.
&
Wroblewska
,
J.
(
1967
).
Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage
.
J. Embryol. exp. Morph
.
18
,
155
180
.
Uusitalo
,
R. J.
&
Karnovsky
,
M. J.
(
1977
).
Surface localization of 5’nucleotidase on the mouse lymphocyte
.
J. Histochem. Cytochem
.
25
,
87
96
.
Vorbrodt
,
A.
,
Konwinski
,
M.
,
Solter
,
D.
&
Koprowski
,
H.
(
1977
).
Ultrastructural cyto-chemistry of membrane-bound phosphatases in preimplantation mouse embryos
.
Devi Biol
.
55
,
117
134
.
Wachstein
,
M.
&
Meisel
,
E.
(
1975
).
Histochemistry of hepatic phosphatases at a physiologic pH
.
Am. J. Clin. Pathol
.
27
,
13
23
.
Yamashina
,
S.
&
Kawai
,
K.
(
1979
).
Cytochemical studies on the localization of 5’nucleotidase in the acinar cells of the rat salivary glands
.
Histochem
.
60
,
255
263
.