/. Embryol. exp. Morph. Vol. 51, pp. 109-120, 1979 109 Printed in Great Britain © Company of Biologists Limited 1979 Interaction between inner cell mass and trophectoderm of the mouse blastocyst II. The fate of the polar trophectoderm By A. J. COPP1 From the Department of Zoology, Oxford SUMMARY Selective labelling of polar trophectoderm cells in early mouse blastocysts has allowed the fate of polar cells to be followed during in vitro and in vivo blastocyst development. Results show that there is cell movement from polar to mural regions as blastocysts grow. This indicates that trophectoderm cells directly opposite the inner cell mass are the oldest mural cells. However, after implantation polar cells invaginate into the blastocoelic cavity and contribute to the extra-embryonic ectoderm. It is suggested that the morphogenetic changes occurring in the mouse embryo at implantation result from the maintenance of a balance between (a) regional differences in rates of cellular proliferation, and (b) mechanical constraints on the direction in which growth can occur. INTRODUCTION It was predicted in a previous paper, on the basis of cellular proliferation rates in the various blastocyst regions, that there is cell movement from polar to mural trophectoderm as blastocysts develop (Copp, 1978; see also Gardner & Papaioannou, 1975). This paper reports experiments in which the fate of polar trophectoderm cells was followed, both before and after implantation, using melanin granules as a marker. Trophectoderm cells readily phagocytose melanin granules, and retain them at least until implantation has occurred, apparently with no subsequent transfer of granules between cells (Gardner, 1975). Further- more, melanin granules are easily visualized in standard histological prepara- tions. Therefore, apart from the problem of dilution as labelled cells continue to divide, melanin granule labelling fulfills most of the requirements for a useful biological marker (Weston, 1967). MATERIALS AND METHODS Blastocysts were flushed from the uteri of pregnant CFLP females between 3 days 14 h and 3 days 16 h after the estimated time of ovulation (see Copp, 1978), and were stored in PB1-medium (PB1, Whittingham & Wales, 1969) 1 Author's address: Department of Zoology, South Parks Road, Oxford 0X1 3PS, U.K. 8 EMB 51 110 A. J. COPP 100 Fig. 1. Partially polar-herniated blastocyst before labelling. Fig. 2. Completely polar-herniated blastocyst after labelling. plus 10 % heat-inactivated foetal calf serum (FCS). Any blastocysts which had collapsed were allowed to re-expand in PB1 + FCS at 37 °C. Each expanded blastocyst was held by suction and a slit was made in the zona pellucida over the polar trophectoderm using a pair of straight needles controlled by a Leitz micromanipulator assembly (Gardner, 1978). Operated blastocysts were cultured at 37 °C in PB1 + FCS under paraffin oil (Boots, U.K. Ltd) until blastocoelic expansion caused herniation of the inner cell mass (ICM) plus covering polar trophectoderm through the slit in the zona pellucida. When part of the polar region had emerged, herniation was arrested by removing blasto- cysts to PB1 + FCS at room temperature. A suspension of melanin granules was prepared by teasing apart retinae from pigmented mice of various strains in alpha modification of Eagle's medium supplemented with 30 fim adenosine, guanosine, cytidine and uridine, and 10 /an thymidine (a, Flow Labs) plus 10 % foetal calf serum. Partially herniated blastocysts (Fig. 1) were cultured in hanging drops of this suspension for 1 h at 37 °C, in an atmosphere of 5 % CO2 in air, after which most embryos showed complete polar-herniation (Fig. 2). Any blastocysts which had collapsed or showed mural herniation were rejected. All others were washed thoroughly in a + FCS to remove loosely adhering granules and their zonae were removed in acid Tyrode's solution, pH 2-5 (Nicolson, Yanagimachi & Yanagimachi, 1975). In order to study the fate of polar trophectoderm cells before implantation, labelled blastocysts were cultured in a + FCS, in bacteriological plastic dishes (Sterilin) under 5 % CO2 in air. After either 1 or 24 h of culture, blastocysts were fixed and prepared for analysis by serial reconstruction as described previously (Copp, 1978). Cell numbers were determined by counting nuclei in both the polar and ICM regions and in the mural subregions. Since cell outlines were not always easy to see, the number of labelled cells was estimated Fate of mouse polar trophectoderm 111 indirectly. The distribution of melanin granules was usually clumped, and it was considered that each 'clump' of melanin granules corresponded to a labelled cell. A 'clump' was defined as one or more granules occurring within a volume of cytoplasm which extended around each nucleus in any direction for a distance equal to the average length of a nucleus in that particular region. However, it was decided that 'clumps' should be confined within regional boundaries and therefore they were usually more extensive in certain directions than others (e.g. elongated, narrow 'clumps' in the polar region). In this way, 'clumps' were intended to resemble cells in both size and shape (Fig. 3). The 'clump':cell number ratio gave an estimated labelling index for each blastocyst region. In order to control for a possible time-dependent spreading of melanin granules in all directions throughout the blastocyst, mural-herniated blastocysts were also labelled and analysed in the same way (Figs. 4 and 5). It was not possible to control precisely whether the proximal or distal mural subregion herniated, so all mural-herniated blastocysts were pooled for analysis. The fate of polar trophectoderm cells after implantation was followed by transferring polar-labelled blastocysts to the uteri of pseudopregnant recipients 3 days 17 h after the estimated time of mating to sterile males. Labelled blasto- cysts were transferred to one uterine horn of each recipient female, and the contralateral horn received an equal number of unlabelled polar-herniated blastocysts. Recipients were killed 38 h later and uteri containing implantation sites were prepared for histological examination as described previously (Copp, 1978). Embryos had reached a very early egg-cylinder stage in which the polar trophectoderm was multilayered (Fig. 6). Extra-embryonic ectoderm was clearly forming in all embryos but none showed an accumulation of polar cells above the level of origin of the primary giant cells, indicating that the ecto- placental cone had not yet developed. Extra-embryonic ectodermal cells could not be reliably disinguished from more superficial polar cells, and so the whole polar region was analysed together. It could be distinguished from the embryonic ectoderm on the basis of: (a) its greater intensity of cytoplasmic and nuclear staining; (b) the shape and orientation of its nuclei, and (c) a space between the two regions. In addition, primary trophoblastic giant cells, visceral and parietal endoderm were recognizable. Serial sections were scored for the presence or absence of each tissue and for the presence of one or more labelled cells within them. RESULTS P re-imp Ian tat ion developmen t Twenty-four polar-labelled and 19 mural-labelled blastocysts were analysed. The number of melanin granules per blastocyst ranged from 28 to 648, except for a single polar-labelled blastocyst which contained four granules. This was felt to be an abnormally low level of labelling and the blastocyst was excluded from the granule-distribution analysis. The average number of granules per 8-2 112 A. J. COPP A Fig. 3. Drawings of serial sections of polar-labelled blastocysts fixed after (A) 1 h, and (B) 24 h of culture. Nuclei are drawn in outline. Dashed outlines indicate nuclei which were only faintly visible. Dots represent melanin granules, rectangles enclose 'clumps'. Note that 'clumps' usually extend to adjacent sections. Fate of mouse polar trophectoderm 113 100 Him Fig. 4. Partially mural-herniated blastocyst before labelling. Fig. 5. Completely mural-herniated blastocyst after labelling. Polar region Primary giant cells Embryonic ectoderm Visceral endoderm Parietal endoderm Fig. 6. (A) Section and (B) drawing of an early egg-cylinder developed from a polar-labelled blastocyst transferred to the uterus of a pseudopregnant recipient. Arrows indicate melanin granules. embryo did not differ significantly between polar-labelled blastocysts cultured for 1 and 24 h and the same was true for mural-labelled blastocysts (Table 1). This indicates that there is no substantial loss of melanin granules from blasto- cysts during 24 h of culture. Since there appears to be no transfer of granules between cells (Gardner, 1975), any alteration in the relative numbers of granules in different blastocyst regions must therefore indicate a redistribution of granule- containing cells, or their progeny, as development proceeds. Table 2 shows the distribution of melanin granule ' clumps' in blastocysts after 1 and 24 h of culture. These results indicate that, for polar-labelled 114 A. J. COPP Table 1. Numbers of melanin granules in polar- and mural-labelled blastocysts after 1 and 24 h of culture Average number of Region Hours of Number of melanin granules labelled culture blastocysts per blastocyst P Polar 1, 1..1. 112-..^v6, I, 1?4 > 0-()5 24 13 171-3 Mural x1 s9 ji.^.322- 3J \'1-25 > 01 24 10 216-6 * Student's ^-values from 'comparison of two means' tests. blastocysts: (1) there is an increase in the proportion of labelled cells in the proximal mural subregion after 24 h of development; (2) there is no comparable increase in distal and mural labelling; (3) there is no fall in the proportion of labelled polar cells, showing that all polar daughter cells inherit granules at least during the next 24 h of development. However, there is some dilution of label during this period since the average number of melanin granules per polar 'clump' fell from 8-6 after 1 h, to 5-9 after 24 h of culture; and (4) ICM labelling is negligible.