/. Embryo!, exp. Morph. Vol. 42, pp. 305-308, 1977
Printed in Great Britain © Company of Biologists Limited 1977
305
The onset of phosphatase activity
in early mammalian embryos
L. IZQUIERDO1
From the Faculty of Science, University of Chile
BYV. I S H I Y A M A A N D
SUMMARY
The onset of acid and alkaline phosphatase activity is determined by means of Burstone's
azo dye coupling methods in oocytes and embryos of the mouse, rat and hamster, and in
mouse embryos cultured in vitro. Acid phosphatase activity is detected in all cases while
alkaline phosphatase activity begins during the 4-cell stage and is always present thereafter.
The method is sensitive regarding detection of activity but does not permit the quantification
nor a precise localization of the enzymes.
INTRODUCTION
The activity and differential localization of acid and alkaline phosphatase in
mammalian embryos prior to implantation has been the subject of several
papers since the first studies by Mulnard (1955). Our interest has been centered
on alkaline phosphatase because the activity begins suddenly in early morulae
and its cytochemical demonstration suggests a spatial pattern (lzquierdo &
Marticorena, 1975; lzquierdo & Ortiz, 1975), thus providing some basis for a
model of cell differentiation during cleavage (lzquierdo, 1977). This report deals
with the onset of acid and alkaline phosphatase activity in mouse, rat and
hamster embryos, as recognized by Burstone's axo dye coupling methods.
MATERIALS AND METHODS
Albino Swiss CF1 mice provided oocytes and embryos. The oocytes, from
punctured follicles or from the oviduct, were derived from females induced to
superovulate, while the embryos were derived from untreated animals. Rat and
hamster embryos, were also obtained from untreated animals. The material was
recovered in Ringer solution and fixed. In other series, 2-cell mouse embryos
were cultured in vitro until the desired stage was reached, then rinsed in Ringer
solution and fixed. Cultures were performed according to Biggers, Whitten &
Whittingham (1971), but the albumin content of their standard medium was
doubled.
1
Author's address: Faculty of Science, University of Chile, Casilla 653, Santiago, Chile.
20-3
306
V. ISHIYAMA AND L. IZQU1ERDO
Acid phosphatase demonstration. The Naphtol AS phosphatase method
(Burstone, 1962) was adapted to our material by trying different fixatives,
fixation time, pH and incubation time. Best results were obtained fixing the
embryos for 1 h in cold 10% neutral formalin, rinsing 30min in H2O and
incubating for 5-6 h at 37 °C in freshly prepared medium; then the material was
rinsed in water for 20 min and whole-mounted in glycerol-gelatin for microscopic observation. The medium contained 1-5 mg of the substrate (Naphtol
AS-BI phosphate, Nutritional Biochemicals) dissolved in 0-1 ml of DMF
(AfTV-dimethylformamide, Merck), plus 10 ml of 0-1 M acetate buffer pH 5-4,
10 mg of the diazonium salt (fast violet B salt, Nutritional Biochemicals) and
2 drops of a 10 % MnCl2 solution. The mixture was stirred and filtered before use.
Alkaline phosphatase demonstration. The Naphtol AS phosphatase procedure
(Burstone, 1962) was carried out as previously reported (Izquierdo & Marticorena, 1975). After 1 h fixation in 10% cold neutral formalin and 30 min rinse
in H 2 O, the material was incubated during 30 min at 37 °C in a fresh medium
containing 1 mg of Naphtol AS-BI phosphate dissolved in 0 1 ml DMF, plus
10 ml of 0-1 M tris buffer pH 9-1 and 6 mg of fast violet; afterwards, the material
was rinsed in cold pH 7-4 buffer and whole-mounted.
Since the stain fades in about 1 week, microscopic observations and photographs were performed during the first 2 days. Different stages were routinely
processed simultaneously so as to attain valid comparisons. Enzyme inactivation
by heat and incubation without substrate were standard controls.
RESULTS
Acid phosphatase. The enzyme activity is detected at all times in mouse
oocytes and embryos developed in situ, mouse embryos developed in vitro, and
rat and hamster oocytes and embryos developed in situ (Table 1). The results
are decisive with regard to activity but the method does not allow us to quantify
this activity nor to describe the precise localization of the enzyme.
Alkaline phosphatase. Mouse embryos developing either in situ or in vitro, as
well as rat and hamster embryos, show a common pattern: all embryos with
less than 4 cells show a negative reaction, the reaction becomes positive during
the 4-cell stage and is always positive thereafter (Table 2). In the mouse, 4-cell
stages are considered early or late, depending on the stage of the accompanying
embryos.
An unexpected situation is revealed by mouse embryos cultured in vitro which
do not divide beyond 2, 3 or 4 cells: 23 out of 125 embryos arrested in 2-cell
stage and 87 out of 87 embryos arrested in 3- or 4-cell stages showed alkaline
phosphatase activity.
There is a clear-cut difference between a positive and a negative alkaline
phosphatase reaction, but the quantitative gradations and the cellular distribution of the enzyme were not detected with accuracy.
Onset of phosphatase activity in early embryos
307
Table 1. Acid phosphatase activity
Stage
Mouse,
developed
in situ
Mouse,
developed
in vitro
Rat,
developed
in situ
Hamster,
developed
in situ
Oocyte I
Oocyte II
1-cell
2-cell
3-cell
4-cell
5- to 6-cell
8- to 16-cell
Morulae
Blastocysts
Total
41/41
13/13
40/40
50/50
4/4
37/37
16/16
42/42
43/43
44/44
330
—
—
—
8/8
18/18
23/23
6/6
12/12
21/21
27/27
115
10/10
—
25/25
19/19
—
15/15
6/6
15/15
12/12
14/14
116
16/16
—
20/20
25/25
—
16/16
6/6
29/29
21/21
—
133
Table 2. Alkaline phosphatase activity
Stage
Mouse,
developed
in situ
Mouse,
developed
in vitro
Rat,
developed
in situ
Hamster,
developed
in situ
1-cell
0/49
—
0/20
0/18
2-cell
0/93
0/85
0/37
0/21
0/18
3-cell
—
—
—
4-cell
7/661
Early
37/37
1/40
11/20
11/20/
Late
—
6-cell
6/6
11/11
5/5
18/18
33/33
8-cell
49/49
38/38
20/20
• 33/33
17/17
16-cell
30/30
Morulae
43/43
19/19
20/20
25/25
62/62
28/28
—
Blastocysts
38/38
407
254
205
153
Total
Key to tables: results for each stage are expressed as number of embryos with positive
reaction over number of embryos analysed.
DISCUSSION
Using formalin fixation and Gomori's cytochemical method, Mulnard detected acid phosphatase activity beginning in the 1-cell rat embryo (1955) and
in the 8-cell mouse embryo (1965); however in the latter, formalin fixations
lasted more than 6 h and Mulnard mentions that Dalcq had obtained positive
reactions in uncleaved and early cleaving mouse eggs after shorter fixation.
Therefore, Mulnard's results may be consistent with those reported here and
with our electron microscope observations in the mouse egg (Izquierdo & Lopez,
unpublished results; Izquierdo, 1977). However, Solter, Damjanov & Skreb
(1973), using several cytochemical methods, detected acid phosphatase activity
308
V. ISHIYAMA AND L. IZQUIERDO
in the mouse beginning only at the 8-cell stage. We cannot resolve this discrepancy since no direct biochemical assay of this enzyme has been performed
on early mammalian embryos.
On the contrary, alkaline phosphatase in preimplantation mouse embryos
has been assayed and the results concerning the onset of activity coincide with
the observations made by means of Burnstone's cytochemical method (Izquierdo
& Marticorena, 1975). In this report we confirm and extend those findings to
mouse embryos cultured in vitro, and to rat and hamster embryos. Former
observations on differentiating mouse morulae with Gomori's procedure
(Izquierdo & Ortiz, 1975) do not contradict the above-mentioned results because we had modified the method so as to discriminate between inner cell mass
and trophoblast, disregarding specificity and sensitivity. Using a lead-citrate
adaptation of Gomori's method for the electron microscope, we also observed
that alkaline phosphatase activity begins during the 4-cell stage of the mouse
(Izquierdo & Marticorena, unpublished results; Izquierdo, 1977). Solter et al.
(1973) reported that the activity begins in the 8-cell mouse embryo, but since we
found that only some of the 4-cell stages show a positive reaction while all 8-cell
stages do, their results are probably not in contradiction with ours.
The activity of alkaline phosphatase detected on cultured mouse embryos
which had not developed beyond 4 cells suggest a disturbance of their developmental clock: they are older in time than in stage. Assuming that the cultured
4-cell embryos which we regarded as normal are also late developers, it is not
surprising that they all show alkaline phosphatase activity while less than 25 %
do when developed in situ (Table 2).
This work was supported by PNUD/UNESCO Regional Program RLA/006. V. Tshiyama
was in receipt of a research training grant from the same source. His present address is
Universidad de San Marcos, Laboratorio de Zoologia General, Ciudad Universitaria, Lima,
Peru.
REFERENCES
J. D., WHITTEN, W. K. & WHITTINGHAM, D. G. (1971). The culture of mouse
embryos in vitro. In Methods in Mammalian Embrology (ed. J. D. Daniel), pp. 86-115.
San Francisco: Freeman.
BURSTONE, M. S. (1962). Enzyme Histochemistry. New York: Academic Press.
IZQUIERDO, L. (1977). Cleavage and differentiation. In Development in Mammals, vol. 2 (ed.
M. H. Johnson), pp. 99-118. Amsterdam: North Holland Publishing Co.
IZQUIERDO, L. & MARTICORENA, P. (1975). Alkaline phosphatase in preimplantation mouse
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IZQUIERDO, L. & ORTIZ, M. E. (1975). Differentiation in the mouse morulae. Wilhelm Roux
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MULNARD, J. (1965). Studies of regulation of mouse ova in vitro. In Preimplantation Stages
of Pregnancy (ed. G. E. W. Wolstenholme & M. O'Connor), pp. 123-144. Boston: Little
Brown.
SOLTER, D., DAMJANOV, I. & SKREB, N. (1973). Distribution of hydrolytic enzymes in early
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BIGGERS,