Cells of both rat and mouse morulae can be stained vitally to reveal an asymmetry in the organization of their cytoplasm. In each cell of the rat 8-cell embryo a column of organelles develops between the nucleus and the embryo periphery as revealed by toluidine blue, acridine orange and horseradish peroxidase (HRP). Although cells of the mouse morula lack the blatant asymmetric distribution of organelles observed in rat cells, a long pulse (> 3 h) of HRP to compact 8-cell mouse embryos revealed a distinct restricted localization of the enzyme not evident at earlier pre-compaction stages. The cytoplasmic polarity generated in these embryos can be demonstrated in cells of intact embryos, and also in cells disaggregated from embryos before vital staining.

The polarization hypothesis (Johnson, Pratt & Handyside, 1981) proposed recently to explain the generation and maintenance of spatial differentiation in the early preimplantation mouse embryo postulates the establishment in the 8-cell embryo of a radial asymmetry which is expressed in individual cells in the form of an axial polarity. The generation of surface polarity in cells of the 8-cell mouse embryo has been shown already (Handyside, 1980; Ziomek & Johnson, 1980; Reeve & Ziomek, 1981). This study demonstrates that cells of 8-cell rat and mouse embryos also show a cytoplasmic polarity absent at earlier stages.

The existence of localized cytoplasmic material in the mammalian embryo has long been proposed (Dalcq, 1957). Vital staining with toluidine blue (Izquierdo, 1955) and acridine orange (Austin & Bishop, 1959), and studies by transmission electron microscopy (TEM) (Izquierdo & Vial, 1962; Schlafke & Enders, 1967) have shown a very clear reorganization of cytoplasmic components at the 8-cell-stage of the rat embryo. Thus, whereas organelles of earlier stages have a relatively uniform cytoplasmic distribution (Sotelo & Porter, 1959), those of the 8-cell embryo are restricted largely to a column extending from the nucleus of each cell to the embryo periphery (Izquierdo & Vial, 1962; Schlafke & Enders, 1967; Dvorak, 1978). In contrast, the mouse embryo has been reported to display no such overt cytoplasmic polarization (Calarco & Brown,1969), although microtubules may orientate parallel to areas of cell contact, and mitochondria occupy the cortical region (Ducibella, Ukena, Karnovsky & Anderson, 1977). This report confirms that in the mouse 8-cell embryo, unlike that of the rat, no blatant asymmetry is observed in the pattern of staining by toluidine blue and acridine orange. In contrast, long pulses of horseradish peroxidase (HRP) did result in a polarized distribution of ingested enzyme in the cytoplasm of cells of compact morulae of both rat and mouse.

Embryo collection

Female HC-CFLP mice (4–5 weeks; Hacking & Churchill) were super-ovulated with intraperitoneal injections of 5 i.u. of pregnant mare’s serum (PMS: Folligon, Intervet), followed after 44–48 h by 5 i.u. of human chorionic gonadotrophin (hCG: Chorulon, Intervet). Females were paired with HC-CFLP males, and vaginal plugs taken as an indication of mating. Mice were killed by cervical dislocation and embryos were flushed from the oviducts, at times between 49 and 68 h post-hCG, with phosphate-buffered medium 1 supplemented with 0·4% (w/v) bovine serum albumin (PB1 +0·4% BSA) (Whittingham & Wales, 1969), and were cultured at 37°C in medium 16 with 0·4% (w/v) BSA (Whittingham, 1971) in 5% CO2 in air.

Female Wistar rats (250 g body weight; Olac Limited) were paired with Wistar males, and examined the next morning when spermatozoa in the vaginal smear indicated day 1 of the pregnancy. Rats were killed by an ether overdose, and embryos flushed from the oviducts with PBl+0·4% (w/v) BSA or pregassed culture medium, kept below pH 7·4. Four-cell embryos were obtained late on day 3, and 8-cells during the afternoon of day 4. Embryos were cultured in either a modification of the standard mouse medium of Biggers, Whitten & Whittingham (1971) with 25% (v/v) foetal calf serum (Mayer & Fritz, 1974) or in T6’ modified Tyrode’s medium with 0·1% (w/v) BSA (Wood & Whittingham, 1980).

Zonae pellucidae of both rat and mouse embryos were removed by a 15- to 30-sec incubation in prewarmed (37°C) acid Tyrode’s solution (pH 2·5) + 0·4% (w/v) polyvinylpyrrolidone (Nicolson, Yanagimachi & Yanagimachi, 1975).

Disaggregation

Disaggregation into single cells was accomplished by pipetting embryos with a flame-polished micropipette after incubation in either trypsin/EDTA or calcium-free medium, both pre-equilibrated for at least 30 min at 37°C in 5% CO2 in air. After disaggregation, cells were restored immediately to the culture medium. The disaggregating media were as follows.

(i) Trypsin/EDTA

Rat embryos were incubated in 0·5% (w/v) trypsin + 0·2% (w/v) EDTA (Gibco) in calcium-free medium 16. After 5 min, when decompaction was complete, the medium was drawn off and replaced with a large volume of either PB1 +0·4% (w/v) BSA, or pregassed culture medium.

(ii) Calcium-free medium

Mouse embryos were incubated for 10–30 min in calcium-free medium 16 + 0·6% (w/v) BSA.

Toluidine blue

Cells or embryos were placed in a 1 /50000 (w/v) solution of toluidine blue in Tyrode’s medium for 20 min before fixation in a solution formed from water and saturated aqueous solutions of mercuric chloride, phosphotungstic acid and ammonium molybdate in the volumetric ratio of 6:3:1:2 (Izquierdo, 1955). After fixation for at least 1 h, embryos and cells were> washed in water, dehydrated through a graded alcohol series, cleared in toluene, and mounted in Depex (Gurr Limited).

Acridine orange

Embryos and cells were incubated in a range of concentrations of acridine orange (Gurr Limited) between 1/20000 (w/v) and 1 /250 (w/v) in PB1 + 0·4% (w/v) BSA. The fluorescent staining pattern was observed directly, and 1/1000 (w/v) acridine orange gave optimum differential fluorescence. The viability of compacted 8-cell embryos (67 h post-hCG) was examined by culture, either continuously or for 2 h followed by restoration to control medium, in 1/1000 (w/v) acridine orange in medium 16 + 0·4% (w/v) BSA at 37°C in 5% CO2 in air.

Horseradish peroxidase (HRP)

Embryos and cells were incubated in 2 mg/ml HRP (Sigma Type II) in medium 16 + 0·4% (w/v) BSA (mouse) or T6’ modified Tyrode’s medium + 0·1% (w/v) BSA (rat) at 37°C for 3-10 h in 5% CO2 in air. Cells were rinsed in PB1 +0·4% (w/v) BSA, and fixed in 4% (w/v) paraformaldehyde (Anderson & Co. Ltd) in phosphate-buffered saline (PBS) at 4°C for 1 h, before further washing and storage in PB1 +0·4% (w/v) BSA at 4°C. Cells were stained for HRP by the aminoethylcarbazole (AEC; Sigma) method (Pearse, 1968). Two mg AEC were dissolved in 10 ml 50 mM acetate buffer pH 5·0 to which a drop of 3% (v/v) hydrogen peroxide was added immediately before use. After staining for 10·60 min, rat embryos were mounted in glycerine jelly (Cavanaugh, 1964), while, for better resolution, mouse cells were examined in wells of a tissuetyping slide (Baird & Tatlock) in drops of PB1 +0·4% (w/v) BSA under oil.

Indirect immunofluorescence

HRP-treated embryos were incubated for 5 min in 25μl drops of a rabbit antiserum (RAMS) to mouse species antigens (Gardner & Johnson, 1975; Handyside, 1980) diluted 1 in 15 in PBl+0·4% (w/v) BSA + 0·02% (w/v) sodium azide, followed by thorough washing in PB 1 + BSA + azide, and a similar incubation in fluorescein-conjugated goat anti-rabbit IgG (FITC-GAR IgG; Miles Labs) diluted 1 in 15 in PB1 + BSA + azide. The embryos were washed again, and disaggregated into single cells which were fixed in 4% (w/v) paraformaldehyde in PBS at 4°C for 1 h, before being stained for HRP.

Immunosurgery

Inner cell masses (ICMs) were isolated by immunosurgery (Solter & Knowles, 1975). Blastocysts were incubated for 5 min in RAMS (diluted 1:10 with PB1) at 37°C, washed extensively in PB1 +0·4% (w/v) BSA, and incubated for 30 min in guinea-pig complement (Flow Labs.) (diluted 1:10 with PB1) at 37°C. After thorough washing, the inner cell masses were separated from the lysed trophectodermal cells by drawing the embryos through a finely pulled Pasteur pipette.

Cell counting

Embryos and inner cell masses were prepared and fixed according to Tarkowski (1966). Nuclei were stained with a millipored 1% (w/v) suspension of Giemsa (Raymond A. Lamb). Cell numbers were not recorded for the very small minority of spreads which showed either excessive clustering or dispersion of nuclei.

Light microscopy

A Zeiss Universal microscope, which was fitted with incident source HBO 50, III RS condenser and Zeiss filter set 487709, was used to examine cells for acridine orange staining and HRP-stained cells for FITC-labelling. Kodak Tri-X 35 mm film was used for both bright-field and fluorescence photography. Cells mounted in Depex or glycerine jelly were examined with a Zeiss Ultraphot II microscope, and photographed on Pan F film.

Transmission electron microscopy

Embryos were fixed for 1 h at room temperature in 2·5% (v/v) glutaraldehyde in 0·1 M sodium cacodylate buffer at pH 7·4. Embryos were washed with the buffer, and postfixed in 1% (w/v) osmium tetroxide in 0-1 M sodium cacodylate buffer. After dehydration through a graded alcohol series, embryos were infiltrated and embedded in Spurr resin (Spurr, 1969). Sections, 50 nm thick, were cut with a glass knife, and stained with uranyl acetate (Gibbons & Grimstone, 1960) followed by lead citrate (Reynolds, 1963). Sections were viewed in a Siemens Elmiskop I microscope.

1 Toluidine blue

Vital staining of rat embryos with toluidine blue revealed uniform cytoplasmic staining in 4-cell blastomeres (Fig. 1) and a polarized pattern in 8-cell blastomeres (Fig. 2). The metachromasia with toluidine blue iis considered by Izquierdo & Vial (1962) to be associated with vesicles which are rich in mucopolysac-charides and acid hydrolases (Stastna, 1974). At the 8-cell stage there is a segregation of organelles into a column extending from the nucleus of each cell to the embryo periphery (Fig. 3). Five different staining patterns were identified in single cells disaggregated from embryos and then stained (Fig. 4), When classified according to staining pattern, cells from 8-cell embryos showed a high incidence of cytoplasmic polarity compared with cells from 4-cell embryos which lacked cytoplasmic columns (Table 1). However, when all cells of individual embryos were examined, non-polarized cells were detected in many embryos in which the majority of cells were polarized (W. J. D. Reeve, un-published observations). Culture of both intact embryos and single cells in vitro before staining resulted in a slight decrease in the incidence of polarity (Table 1). In contrast to the rat embryo, cells from 8-cell mouse embryos stained with toluidine blue offered only a hint of polarity which was lost after fixation.

Table 1

Cytoplasmic staining patterns with toluidine blue of cells dissociated from rat embryos before staining

Cytoplasmic staining patterns with toluidine blue of cells dissociated from rat embryos before staining
Cytoplasmic staining patterns with toluidine blue of cells dissociated from rat embryos before staining
Fig. 1

The 4-cell rat embryo shows uniform cytoplasmic staining after immersion in toluidine blue.

Fig. 1

The 4-cell rat embryo shows uniform cytoplasmic staining after immersion in toluidine blue.

Fig. 2

An 8-cell rat embryo stained with toluidine blue reveals a column extending from the nucleus of each cell to the embryo periphery.

Fig. 2

An 8-cell rat embryo stained with toluidine blue reveals a column extending from the nucleus of each cell to the embryo periphery.

Fig. 3

The region of intense staining corresponds to columns of organelles seen in TEM (× 17000), N, nucleus; Z, zona pellucida; C, column of organelles.

Fig. 3

The region of intense staining corresponds to columns of organelles seen in TEM (× 17000), N, nucleus; Z, zona pellucida; C, column of organelles.

Fig. 4

Rat 8-ceii embryos were disaggregated to single cells and stained with toluidine blue. Five categories of staining pattern were recognized, (a) Tight column. Metachromasia extends in band (width less than nuclear diameter) from the nucleus to the periphery of the cell, (b) Loose column. The column is wider than the nucleus, (c) Nuclear cap. The metachromasia extends less than half-way from the nucleus to the cell periphery, (d) Nuclear ring. Metachromasia surrounds the nucleus, (e) Uniform stain. Stain occurs throughout the cytoplasm.

Fig. 4

Rat 8-ceii embryos were disaggregated to single cells and stained with toluidine blue. Five categories of staining pattern were recognized, (a) Tight column. Metachromasia extends in band (width less than nuclear diameter) from the nucleus to the periphery of the cell, (b) Loose column. The column is wider than the nucleus, (c) Nuclear cap. The metachromasia extends less than half-way from the nucleus to the cell periphery, (d) Nuclear ring. Metachromasia surrounds the nucleus, (e) Uniform stain. Stain occurs throughout the cytoplasm.

2 Acridine orange

Acridine orange stains the DNA of the nucleus green, and lysosomes and ribonucleic acids are considered to be associated with the orange cytoplasmic staining (Austin & Bishop, 1959; Allison & Young, 1969). Staining of intact 8-cell rat embryos and their dissociated cells revealed asymmetries of cytoplasm similar to those observed with toluidine blue. After being stained with acridine orange, dissociated rat cells were sorted on the basis of cytoplasmic polarization, and then further stained with toluidine blue. The two stains were localized in similar patterns. Thus, of 16 cells which stained with acridine orange to reveal a column stretching from the nucleus to the cell surface, all showed columns after staining with toluidine blue; seven cells had nuclear caps or rings when stained with both acridine orange and toluidine blue; and of four cells shown by acridine orange to have a scattered distribution of organelles, staining with toluidine blue showed two to have nuclear caps, one to have a uniform staining pattern and one to be unscorable. In contrast, only a minority of dissociated cells of both pre-compact and compact mouse embryos revealed an asymmetry of cytoplasmic staining with acridine orange over a range of concentrations. Just 16% of 53 cells from pre-compact 8-cell embryos and 9% of 64 cells from compact 8-cell embryos revealed a columnar cytoplasmic staining pattern. There was a similar low incidence of cytoplasmic stain concentrated in nuclear caps, nuclear rings and dispersed cytoplasmic aggregates. In contrast, 53% and 66% of the cells from pre-compact and compact 8-cell embryos, respectively, showed a uniform cytoplasmic pattern of staining.

3 Horseradish peroxidase

Incubation with HRP revealed a polarized HRP accumulation in compact rat embryos (Fig. 5) and a similar pattern was also found in cells from compact mouse morulae (Figs 8, 9). All cells in any one embryo showed very similar patterns of stain distribution. Thus the staining patterns of intact embryos were classified as either uniform, localized or aggregated. Embryos showing a localized pattern of staining appeared to have one restricted mass of stain near the nucleus of each cell, while embryos classed as aggregated appeared to have a more diffuse distribution of HRP-containing vesicles. Mouse embryos, which were either 4-cells (Fig. 6) or pre-compact 8-cells (Fig. 7) at the termination of pulses of HRP of up to 10 h duration, did not show a restricted localization of HRP-containing vesicles. The incidence of localized HRP-containing vesicles increased in the more overtly compact embryos (Table 2). Whereas 64% of precompact 8-cell embryos had cells which showed a uniform distribution of HRP-containing vesicles, only 14% of compact embryos had cells which did so.

Table 2

Incidence of HRP staining patterns in intact%-cell mouse embryos

Incidence of HRP staining patterns in intact%-cell mouse embryos
Incidence of HRP staining patterns in intact%-cell mouse embryos
Fig. 5

A rat morula stained with HRP to show cytoplasmic polarity. Individual cells which show a polarized distribution of HRP are arrowed. (The other cells also show a restricted localization of the enzyme, but are not in focus.)

Fig. 5

A rat morula stained with HRP to show cytoplasmic polarity. Individual cells which show a polarized distribution of HRP are arrowed. (The other cells also show a restricted localization of the enzyme, but are not in focus.)

Fig. 6

A mouse 4-cell embryo shows a uniform cytocortical distribution of HRP.

Fig. 6

A mouse 4-cell embryo shows a uniform cytocortical distribution of HRP.

Fig. 7

A pre-compact 8-cell mouse embryo shows widespread distribution of HRP.

Fig. 7

A pre-compact 8-cell mouse embryo shows widespread distribution of HRP.

Fig. 8

A compact 8-cell mouse embryo shows the restricted cytoplasmic localization of HRP peripheral to the nucleus of each cell. Two cells are arrowed.

Fig. 8

A compact 8-cell mouse embryo shows the restricted cytoplasmic localization of HRP peripheral to the nucleus of each cell. Two cells are arrowed.

Fig. 9

Cells of an 8-cell mouse embryo stained with (a) HRP and (6) fluorescent ligand before embryo disaggregation. Each pole of fluorescent ligand-binding overlies the restricted cytoplasmic localization.

Fig. 9

Cells of an 8-cell mouse embryo stained with (a) HRP and (6) fluorescent ligand before embryo disaggregation. Each pole of fluorescent ligand-binding overlies the restricted cytoplasmic localization.

Disaggregation of pre-labelled individual compact embryos showed that restricted localization can occur in all 8 cells of an embryo, although one or two cells in any one embryo may not appear polarized. Double staining with HRP and fluorescent antibody demonstrated that the localization of HRP lies under the fluorescent pole described previously (Fig. 9) (Handyside, 1980; Ziomek & Johnson, 1980; Reeve & Ziomek, 1981). The incidences of the different patterns of localization of HRP-containing vesicles (Fig. 10) were similar for cells labelled in situ before disaggregation, and for cells from embryos completely disaggregated before incubation with HRP (Table 3).

Table 3

Incidence of staining patterns in cells of compact 8-cell mouse embryos stained with HRP (68-72 h post-hCG) before or after disaggregation

Incidence of staining patterns in cells of compact 8-cell mouse embryos stained with HRP (68-72 h post-hCG) before or after disaggregation
Incidence of staining patterns in cells of compact 8-cell mouse embryos stained with HRP (68-72 h post-hCG) before or after disaggregation
Fig. 10

Dissociated cells of mouse 8-cell embryos showed several patterns of localization of HRP vesicles, (a) Tight localization. (6) Loose localization, (c) Nuclear ring, (d) Aggregates, (e) Uniform.

Fig. 10

Dissociated cells of mouse 8-cell embryos showed several patterns of localization of HRP vesicles, (a) Tight localization. (6) Loose localization, (c) Nuclear ring, (d) Aggregates, (e) Uniform.

4 Viability of embryos

Mouse embryos cultured in toluidine blue lysed within 1 h, and acridine orange also proved harmful to development. Compacted 8-cell embryos (67 h post-hCG), whether exposed to acridine orange for 2 h or continuously, retained a compact appearance at 100 h post-hCG when control embryos were expanded blastocysts. At 125 h post-hCG, all embryos pulsed earlier for 2 h with acridine orange had formed abnormal blastocysts with extruded cells, while morulae still in acridine orange had lysed. Prolonged culture in HRP did not affect development so adversely (Table 4).

Table 4

Toxicity of HRP for cultured 2-cell mouse embryos (49 h post-hCG)

Toxicity of HRP for cultured 2-cell mouse embryos (49 h post-hCG)
Toxicity of HRP for cultured 2-cell mouse embryos (49 h post-hCG)

Cells of rat and mouse 8-cell embryos show evidence of a cytoplasmic polarity absent at earlier stages of development. The results for the rat embryo confirm and extend previous observations, but hitherto no equivalent polarity in cells of the mouse embryo has been described.

There is abundant cytological evidence from light and electron microscopic studies on rat embryos to support the results obtained by staining with toluidine blue and acridine orange. In the rat 4-cell embryo, organelles lose the scattered distribution of earlier stages (Sotelo & Porter, 1959), and tend to localize at the periphery and around the nucleus (Mazanec & Dvorak, 1963; Schlafke & Enders, 1967; Stastna, 1974). At the 8-cell stage, each cell shows a definite cytoplasmic segregation involving the organization of almost all organelles into a column extending from the nucleus to the embryo periphery. The column contains most of the mitochondria, the small regions of the Golgi apparatus, the endoplasmic reticulum (mostly agranular) and a heterogeneous assortment of vesicles (Izquierdo & Vial, 1962; Schlafke & Enders, 1967; Stastna, 1974). These cytoplasmic columns are revealed by vital staining with both toluidine blue (Fig. 2) (Izquierdo, 1955) and acridine orange (Austin & Bishop, 1959). Outside the column, extensive areas of more dense homogeneous cytoplasm contain a few mitochondria and vesicles, and large amounts of proteinaceous lamellae considered to be storage material used in cleavage (Dvorak et al. 1975; Dvorak, Travnik & Stankova, 1977). The columns are stable, and persist even in isolated cells cultured for several hours (Table 1). There are, however, problems in the use of the rat embryo for a dynamic study of polarization. First, the supply of embryos is limited as superovulation in the rat is not an established technique. Second, published data on the culture of preimplantation rat embryos is scarce, and successful culture difficult. Although culture of 8-cell embryos to blastocysts has been reported as 80% or more successful (Folstad, Bennet & Dorfman, 1969; Mayer & Fritz, 1974), the media used by these authors and also the T6’ medium of Wood & Whittingham (1980) never gave a success rate above 60% (W. J. D. Reeve, unpublished results). Lastly, development in vitro of the rat embryo over the 4-cell stage is particularly difficult (Suzuki & lizuka, 1969; Mayer & Fritz, 1974), thus preventing culture over the period in which cytoplasmic polarity is generated.

The compact mouse embryo lacks the blatant cytoplasmic segregation described for the rat 8-cell embryo when examined by TEM (Calarco & Brown, 1969), although showing a surface polarization of microvilli (Ducibella et al. 1977; Reeve & Ziomek, 1981), numerous microtubules orientated parallel to the apposed membranes of blastomeres, and mitochondria localized to the cortex (Ducibella et al. 1977). Neither toluidine blue nor acridine orange staining patterns provided conclusive evidence of cytoplasmic polarity in blastomeres of compact 8-cell mouse embryos.

In contrast, when HRP was used as a vital stain, cells of both rat and mouse 8-cell embryos showed a pronounced cytoplasmic polarity. HRP differs from acridine orange and toluidine blue in its active uptake by cells, and its dependence on cellular mechanisms for transport, ultimate localization and metabolism. It has proved a useful tracer in studies of endocytosis (reviewed Silverstein, Steinman & Cohn, 1977) as it is non-toxic and its enzymic activity can be demonstrated histochemically (Graham & Karnovsky, 1966). HRP uptake is by fluid pinocytosis, and washing before fixation ensures that adsorbed enzyme is removed from the cell surface (Steinman & Cohn, 1972; Steinman, Silver & Cohn, 1974). In published data on preimplantation rabbit (Hastings & Enders, 1974) and rat (Schlafke & Enders, 1972) embryos, HRP pulses never exceeded 60 min, and provided mostly information on uptake patterns at the cell surface. Few vesicles were observed before the 8-cell stage, and endocytosis increased by the blastocyst stage, at which vesicles were restricted mainly to the supranuclear region.

Four-cell mouse embryos showed a uniform distribution of HRP reaction product in the cytocortex, but poor cytoplasmic staining, after prolonged pulses of HRP (Fig. 6). However, after pulses as short as 3 h, some 8-cell embryos were shown to have localization of reaction product between the nucleus of each cell and the embryo periphery (Figs. 8, 9). The increase in the incidence of dispersed aggregates and of restricted localization of HRP stain in cells was associated with compaction (Table 2). The restricted localization of HRP-containing vesicles shown by a minority of pre-compact 8-cell embryos is consistent with the generation of surface polarity before overt cell flattening (Ziomek & Johnson, 1980; Reeve & Ziomek, 1981). Interestingly, in the rat embryo, compaction as assessed by cell flattening occurs at the 4-cell stage, although the cytoplasmic organelles become organized into columns only at the 8-cell stage. The polarity of HRP localization in the intact 8-cell mouse embryo does not depend on differences in the area of exposed cell surface, since it is also shown by dissociated cells incubated in HRP (Table 3).

There is no obvious ultrastructural basis for the restricted localization of HRP stain. The HRP reaction product is thought to coincide with the Golgi apparatus (Steinman et al. 1974; Piasek & Thyberg, 1979), but information on the Golgi apparatus of the rodent embryo is confined almost entirely to the rat. The quantity of Golgi changes little during cleavage stages (Dvorak et al. 1977).

Although early cleavage stages were shown to have Golgi zones near both the cell membrane and nucleus, in the 8-cell embryo the membranous components are aggregated and small Golgi zones are present throughout the organelle-rich regions of cytoplasm (Schlafke & Enders, 1967). However, Stastna (1978) reported no obvious relationship between the Golgi apparatus and the nucleus or blastomere surface in cleavage-stage embryos. The Golgi complex of the pre-implantation mouse embryo is not prominent. The occasional small regions of stacked cisternae in early cleavage stages develop to larger regions of stacked cisternae in the morula (Calarco & Brown, 1969). The Golgi complex of the blastocyst of both the rat (Schlafke & Enders, 1963, 1967; Stastna, 1972, 1974, 1978) and the mouse (Calarco & Brown, 1969; Nadijcka & Hillman, 1974) embryo is located in a juxta-nuclear position.

The demonstration that mouse 8-cell blastomeres are cytoplasmically polarized on an identical axis to the surface polarity already described (Handyside, 1980; Ziomek & Johnson, 1980; Reeve & Ziomek, 1981) suggests a reorganization of cell structures and function at this developmental stage. Such a reorganization is consistent with the first postulate of the polarization hypothesis (Johnson, Pratt & Handyside, 1981) which suggests that the operation of axial polarity in 8-cell blastomeres involves the allocation of ICM- and trophectoderm-like properties to basal and apical portions, respectively, of the cell. Distinct cell lineages could then be generated by subsequent divisions of the polarized cells. An asymmetric cellular distribution at division has already been demonstrated for features of surface polarization (Johnson & Ziomek, 1980), and the stability and lack of toxicity of HRP may permit a similar analysis of the conservation of cytoplasmic polarity.

I am grateful to John Bashford and Ian Edgar for technical help, and to Drs M. H. Johnson, C. A. Ziomek and A. H. Handyside for valuable discussion. This work was supported by grants from the Medical Research Council and the Ford Foundation to Dr Johnson, and by an MRC research studentship.

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