The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

There is evidence to suggest that an interaction occurs between the inner cell mass (ICM) and the trophectoderm of the mouse blastocyst. Microsurgically-isolated trophectodermal vesicles from 3 · 5-day mouse embryos not only fail to proliferate but also form giant cells when transferred to the uteri of pseudo-pregnant hosts (Gardner, 1972). If, however, ICMs are inserted into such vesicles prior to transfer, normal trophectodermal proliferation occurs (Gardner, Papaioannou & Barton, 1973). Subsequent analyses have shown that the rate of cellular proliferation within the trophectoderm overlying the ICM is higher than that found in the mural trophectoderm away from the inner cell mass (Copp, 1978). Together, these studies demonstrate that the mouse inner cell mass is not only able to promote trophectodermal proliferation but can also suppress the giant-cell transformation. Whether these findings are applicable to the early development of other mammals is not known. The object of the present study is to explore this problem in another species, namely the guinea pig. This particular species was chosen for two reasons. First, placentation in the guinea pig, in contrast to the mouse, occurs in a manner similar to that in man. Thus implantation is interstitial and trophoblastic growth is highly invasive and syncitial (Boyd & Hamilton, 1970; Amoroso, 1952). Second, observations suggest that the growth of guinea-pig trophectoderm may not be under ICM control because cells accumulate at the abembryonic pole both in vivo (Sansom & Hill, 1931 ; Blandau, 1959; Enders & Schlafke, 1965) and in vitro (Blandau & Rumery, 1957; Amoroso, 1959) thereby forming a multilayered, partially syncitial structure called the attachment cone. In addition, the proliferation of guinea-pig trophectoderm has been observed to continue in culture under conditions in which degeneration of the ICM was claimed to have occurred (Blandau, 1971). However, an accumulation of cells in the abembryonic part of the blastocyst does not prove that proliferation took place there especially since mouse mural trophectoderm has been shown to grow by cell recruitment rather than cellular division (Copp, 1979).

Furthermore, the observed ‘ICMdndependent’ proliferation of guinea-pig trophectoderm (Blandau, 1971) could have been due to the presence of unrecognized ICM derivatives which either continued to proliferate or, if quiescent, still retained the ability to promote trophectodermal growth. Therefore, the initial aim of the present study was to cut the guinea-pig blastocyst so as to produce two trophectodermal fragments of rougly equivalent size, one being composed of pure trophectoderm whilst the other contained ICM tissue as well. Thereafter, the subsequent growth of each fragment was followed in vitro. However, without cell markers it is not possible to discriminate the derivatives of polar trophectoderm from those of the inner cell mass making it somewhat difficult to interpret this type of study critically. Still, the guinea-pig egg cylinder is known to contain, by homology with the mouse, two presumptive polar trophectodermal derivatives, namely extraembryonic ectoderm and an ectoplacental giant-cell region (Sansom & Hill, 1931; Kaufman & Davidoff, 1977). Reconstitution (Gardner & Papaioannou, 1975) and injection (Rossant, Gardner & Alexandre, 1978) analyses of early mouse development show definitively that these tissues come from the polar trophectoderm. Unfortunately, in the guinea pig, such experimental studies are impracticable due to the lack of readily available enzyme polymorphisms, the low number of embryos per litter, and the non-expansive behaviour of trophectodermal vesicles. Nevertheless, the trophectodermal origin of extraembryonic ectoderm and the ectoplacental cone is also supported by the results of in vitro experiments in which tissues were isolated from the mouse egg cylinder (Rossant & Offer, 1977). Thus, it may be particularly instructive to isolate these same tissue layers from the guinea-pig egg cylinder and compare their nuclear DNA contents, mitotic indices, and cell numbers with those of the mouse both before and after growth in vitro. Since only blastomere-derived (Sherman, 1975) and thymidine-induced (Snow, 1973) trophectodermal vesicles have been studied previously in culture, the in vitro development of microsurgically sectioned mouse blastocysts has also been followed in the present study. The findings obtained suggest that guinea-pig, pre- and post-implantation, trophoblastic tissues grow in vitro in a manner similar to those of the mouse. However, until reconstitution experiments are carried out with the guinea-pig blastocyst in vivo these findings will remain suggestive.

(1) Animals

All animals were maintained on a regime of 12 h light: 12 h dark. For mice, ovulation was assumed to occur at the midpoint of the dark period (1.00 a.m.). The post-coital ages of mouse embryos were then estimated from the time of assumed ovulation (Time ‘0’ of pregnancy). For guinea pigs, ovulation was assumed to occur 10 h after the start of oestrous (Ediger, 1976), but the age of embryos was taken from the actual time of mating. In either species, oestrous females were placed with males and mating ascertained by the presence of a copulation plug.

(2) Recovery of embryos and dissection of tissues

All embryos were recovered from uteri dissected free of fat and debris in PB1 medium (Whittingham & Wales, 1969) plus 10% foetal calf serum. In all fifty-one 5 · 5-day blastocysts, twelve 10 · 5-day primitive-streak-stage egg cylinders, and four 17 · 5-day neural-tube-closure-stage embryos were recovered from the uteri of 59, random-bred, guinea pigs (Nuffield Inst. Med. Res., Oxford) killed with ether. Ninety-five 3 · 5-day blastocysts, 44 7 · 5-day primitive-streak-stage egg cylinders, and four neural-tube-closure-stage embryos were recovered from the uteri of 22 randombred, CFLP mice (Anglia Lab.) killed by cervical dislocation.

For the recovery of blastocysts, the methods described by Copp (1978) and Blandau (1971) were used. Immediately after recovery, blastocysts were transferred to pre-equilibrated tissue-culture medium and then microsurgically sectioned no more than 2 h later. Microsurgery was carried out using the methods of Gardner (1972). Thus, blastocysts were initially held at the abembryonic pole by means of a suction pipette. They were then cut equitorially with siliconized, glass microneedles parallel to the surface of the inner cell mass so that each ‘mural’ and ‘polar’ fragment contained roughly equal numbers of trophectodermal cells. Both fragments isolated from the same embryo were then grown as parallel cultures.

All egg cylinders and later-stage embryos were dissected in PB1 medium plus serum using glass microneedles made as described by Diacumakos (1973). Seven and one-half-day mouse extraembryonic ecoderm was isolated as described by Rossant & Offer (1977). Guinea-pig extraembryonic ectoderm was also removed from the embryo using ‘three cuts’ (see Fig. 1). Endoderm was then stripped away from the extraembryonic ectoderm using microneedle tips.

Fig. 1.

Primitive streak stage embryos of the guinea pig and the mouse. Schematic diagrams depict (a) 7 · 5-day mouse (modified from Snell & Stevens, 1966) and (b) 10 · 5-day guinea pig (compare with Fig. 1D, Ptyler & Strasser (1925) and Sansom & Hill (1931) Fig. 33) egg cylinders. Three-cut (‐ ‐ ‐) dissection method used to isolate trophectoderm-derived (◼) extraembryonic ectodermal, (Ex) primitive endodermal (▤), and embryonic ectodermal ▩ tissues. Primitive mesodermal (‘m’) cells partially line the guinea-pig exocoelomic cavity (ExC) whilst, in the mouse, mesoderm is more abundant forming a small allantoic bud (‘al’). Guinea pig does not have an ectoplacental cone (EPC) but only a rim of mesometrially-situated ectoplacental giant cells (EPGC).

Fig. 1.

Primitive streak stage embryos of the guinea pig and the mouse. Schematic diagrams depict (a) 7 · 5-day mouse (modified from Snell & Stevens, 1966) and (b) 10 · 5-day guinea pig (compare with Fig. 1D, Ptyler & Strasser (1925) and Sansom & Hill (1931) Fig. 33) egg cylinders. Three-cut (‐ ‐ ‐) dissection method used to isolate trophectoderm-derived (◼) extraembryonic ectodermal, (Ex) primitive endodermal (▤), and embryonic ectodermal ▩ tissues. Primitive mesodermal (‘m’) cells partially line the guinea-pig exocoelomic cavity (ExC) whilst, in the mouse, mesoderm is more abundant forming a small allantoic bud (‘al’). Guinea pig does not have an ectoplacental cone (EPC) but only a rim of mesometrially-situated ectoplacental giant cells (EPGC).

(3) Tissue culture

Following microdissection, all pre- and postimplantation tissues were grown in the alpha-modification of Eagle’s medium (Flow) with 10% foetal calf serum (Flow), nucleosides (3×10−5M), and antibiotics. Pre-implantation tissues were explanted in 30 mm plastic tissue-culture dishes (Sterilin) with 2·5 ml of medium and postimplantation tissues cultured in 50 mm Petri dishes (Sterilin) with 5-·0 ml of medium. Both were then incubated in 5% CO2 in air (37 °C). No more than one tissue fragment was explanted in a single Petri dish. Also, cultures were not disturbed for 18 h following explantation and the medium was not changed during the experimental period.

(4) Cytophotometry

(a) Recovery of tissues

All attached explants were mechanically removed from the surface of the Petri dish with the edge of a siliconized Pasteur pipette. For 5-day polar trophectoderm-ICM cultures, the attached trophectodermal base was initially separated from the organized ICM derivatives with a glass microneedle before being removed from the Petri dish.

(b) Dissociation of tissues

Preimplantation tissues, either freshly dissected or following culture, were dissociated using two different methods. The first was a modification of a technique developed by Evans, Burtenshaw & Ford (1972) whereby each tissue was initially transferred to hypotonic solution (1 % Na citrate; 15–60 sec; pH 8·45; 26 °C), fixed (three methanol/acetic acid), and then dissociated on acid-cleaned slides in several drops of 60 % acetic acid (30–60 sec; 26 °C). Comparable results were obtained when fresh tissues were placed directly into several drops of TVP (Bernstein, Hooper, Grandchamp & Ephrussi, 1973) enzyme solution on glass slides (30-60 sec; pH 7·5; 26 °C).

Postimplantation tissues, analysed immediately after dissection, were incubated in TVP (5–15 mm ; pH 7·5 ; 26 °C) and then transferred to slides in enzyme. Those analysed following culture were initially incubated in Ca2+- and Mg2+-free Hank’s balanced salt solution (HBSS, 30 min, 37 °C), placed into CTC (0-1 % collagenase, Worthington, N.J. ; 0·1% trypsin, Difco 1:250; and 10% chick serum (Flow), made up in deficient HBSS − 15 to 301 min, pH 7·6, gassed) solution, and then transferred on to slides in several drops of CTC. Afterwards, tissues in either CTC or TVP on glass slides were drawn through siliconized Pasteur pipettes of different bore sizes until a single cell suspension was produced. Slides were then dried beneath an air jet.

(c) Fixation and staining

The method used to fix and stain slides for cytophotometry is outlined in Table 1.

Table 1.

Procedure used to fix andfeulgen-strain tissues for cytophotometry

Procedure used to fix andfeulgen-strain tissues for cytophotometry
Procedure used to fix andfeulgen-strain tissues for cytophotometry
(d) Method of scanning

To reduce the risk of fading, slides were stored in the dark and not used for DNA measurements if more than 3 months had elapsed from the time of preparation. Nuclei selected for analysis were stained an intense magenta red and generally had well-delineated nuclear membranes. Although every nucleus in each chosen field was measured, usually not every field was scanned. Prior to staining, slides were smeared with fresh liver slices to provide known DNA contents. All measurements were carried out with a Vicker’s M-85 microdensitometer (λ = 585 nm).

(5) Histology

Pre- and postimplantation tissues were initially fixed in Bouin’s solution, dehydrated, cleared in cedar-wood oil, embedded in paraffin wax (56 °C), serially sectioned (5 μm), and then stained with haematoxylin and eosin.

(6) Determination of cell numbers and mitotic indices

Since it was usually not possible to see cell outlines, counts were frequently made by determining the number of nuclei on slides prepared for either cytophotometric or cytological study. Although it was assumed that changes in nuclear numbers reflected variations in the number of cells present, binucleates and multinucleates were occasionally seen in both guinea-pig and mouse tissues. However, the dissociation procedure used frequently disrupted cytoplasmic margins. Therefore, the precise number of uni, bi- and multinucleates in each preparation could not be determined. Approximate cell numbers were also estimated on serially-sectioned embryos. In the case of in vivo and in vitro, mouse and guinea-pig egg cylinders, all cells with clearly-defined nuclear membranes were counted in alternate serial sections. For the guinea pig, these were then assigned to the following anatomic regions: endoderm, see Ptyler & Strasser (1925); embryonic and extraembryonic ectoderm as well as the ectoplacental giant cell region, see Sansom & Hill (1931) and Mossman (1937) ; and extraembryonic mesoderm, see Kaufman & Davidoff (1976). The methods used to determine the mitotic indices (total number mitoses, total number cells scored × 100) of pre- and post-implantation guinea pig and mouse tissues are described beneath Tables 2 and 3.

Table 2.

Trophoblastic nature of the presumptive trophectodermal derivatives of the guinea pig

Trophoblastic nature of the presumptive trophectodermal derivatives of the guinea pig
Trophoblastic nature of the presumptive trophectodermal derivatives of the guinea pig
Table 3.

Development of isolated mural trophectoderm and polar trophectoderm-ICM fragments in vitro

Development of isolated mural trophectoderm and polar trophectoderm-ICM fragments in vitro
Development of isolated mural trophectoderm and polar trophectoderm-ICM fragments in vitro

(1) Trophoblastic nature of the presumptive trophectodermal derivatives of the guinea pig

At the primitive-streak stage, the 7 · 5-day mouse embryo is similar to the 10 · 5-day guinea-pig egg cylinder in several ways. Structurally, the presumptive embryonic and extraembryonic ectodermal regions of the 10 · 5-day guinea-pig embryo closely resemble those of the 7 · 5-day mouse egg cylinder (Fig. 1). Also, immediately after isolation from either the guinea pig or mouse embryo, these embryonic and extraembryonic tissues are totally diploid (2 − 4 ), contain considerable numbers of mitoses, and have approximately the same number of cells (Table 2 A). However, after 72 h in culture guinea-pig and mouse extraembryonic ectoderm not only stop dividing as indicated by a fall in mitotic index but also acquire a large number of nuclei with DNA contents greater than (Table 2B and Fig. 2). In addition, guinea-pig extraembryonic ectodermal cell numbers fall over the 72 h time course whereas those of the mouse increase (Table 2B). In contrast to extraembryonic ectoderm, mouse and guinea-pig embryonic ectodern not only remain diploid after 72 h in culture but also continue to divide (Table 2B).

Fig. 2.

Nuclear DNA contents of (a) 7·5-day mouse and (b) 10·5-day guinea pig extraembryonic ectoderm after 72 h in vitro.

Fig. 2.

Nuclear DNA contents of (a) 7·5-day mouse and (b) 10·5-day guinea pig extraembryonic ectoderm after 72 h in vitro.

At the neural-tube-closure stage, there are numerous trophoblastic giant cells in the trophoblastic tissues of the 10·5-day mouse embryo (Fig. 3a). At a comparable stage of development, there are also many giant cells in the placental disc of the 17·5-day guinea-pig embryo but their nuclear DNA contents are frequently less than those of the mouse (Fig. 3 b).

Fig. 3.

Nuclear DNA contents of (a) 10·5-day mouse secondary and (b) 17·5-day guinea-pig placental giant cells.

Fig. 3.

Nuclear DNA contents of (a) 10·5-day mouse secondary and (b) 17·5-day guinea-pig placental giant cells.

(2) Development of isolated mural trophectoderm and polar trophectoderm-ICM fragments in vitro

The 5·5-day guinea-pig blastocyst resembles the 3·5-day mouse embryo in several ways. Morphologically, the blastocysts of both species are quite similar except for a thickening of the guinea-pig’s abembryonic trophectoderm (Fig. 4). Also, shortly after microsurgical isolation, the trophectodermal constituents of both the guinea-pig and mouse blastocyst are almost totally diploid and contain approximately the same number of cells (Table 3 A). However, after 5 days growth in vitro, guinea-pig and mouse mural trophectoderm always acquire nuclei with DNA contents greater than , never have included mitotic figures, and do not increase in cell number (Table 3B and Fig. 5). In contrast to mural tissues, polar trophectoderm-ICM fragments form structures which resemble 10·5-day guinea-pig (14/17 cases) and 7·5-day mouse (4/30 cases) egg cylinders following five days in culture. These, in turn, contain two presumptive polar trophectodermal derivatives namely, the ectoplacental region and the extraembryonic ectodern. The latter is found to be, in both the mouse and the guinea pig, a highly cellular tissue which contains numerous mitoses and shows no evidence of giant-cell formation (Table 3B). If the in vitro-derived guinea-pig egg cylinder is dissected into its presumptive extraembryonic ectodermal, embryonic ectodermal, and ectoplacental components, isolated extraembryonic ectodern becomes giant, embryonic ectoderm remains diploid , and the ectoplacental region continues to endoreduplicate (Fig. 6).

Fig. 4.

5·5-day guinea-pig blastocyst (live) with abembryonic thickening (AbT) and Zona pellucida (ZP) penetrated by several pseudopodia. (‐ ‐ ‐ ) Represents plane of microsurgical section. Inverted phase. (× 1000).

Fig. 4.

5·5-day guinea-pig blastocyst (live) with abembryonic thickening (AbT) and Zona pellucida (ZP) penetrated by several pseudopodia. (‐ ‐ ‐ ) Represents plane of microsurgical section. Inverted phase. (× 1000).

Fig. 5.

Nuclear DNA contents of (a) 5 · 5-day guinea-pig and (b) 3 · 5-day mouse mural trophectodermal cells after 5 days in vitro.

Fig. 5.

Nuclear DNA contents of (a) 5 · 5-day guinea-pig and (b) 3 · 5-day mouse mural trophectodermal cells after 5 days in vitro.

Fig. 6.

Nuclear DNA contents of presumptive guinea-pig (a) embryonic ectoderm (b) extraembryonic ectoderm and (c) ectoplacental tissues after isolation from the in vitro-derived egg cylinder and an additional 72 h in culture.

Fig. 6.

Nuclear DNA contents of presumptive guinea-pig (a) embryonic ectoderm (b) extraembryonic ectoderm and (c) ectoplacental tissues after isolation from the in vitro-derived egg cylinder and an additional 72 h in culture.

In contrast to previous proposals (Blandau, 1971), the results of the present study suggest that the growth of guinea-pig trophectoderm in vitro is under the control of the inner cell mass. Thus, if guinea-pig trophectoderm is isolated from the ICM, it becomes giant. However, in the presence of the ICM, trophectodermal growth appears to continue thereby forming structures similar to the extraembryonic ectodermal and ectoplacental giant-cell regions of the 10 · 5-day guinea-pig embryo. If this in vitro-derived extraembryonic ectodermal tissue is subsequently isolated and grown in culture for an additional 72 h, it too becomes giant, a finding consistent with its proposed trophoblastic origin. Although these observations suggest that guinea-pig trophectodermal growth is under ICM control, it is still not possible to exclude an ICM contribution to presumptive guinea-pig trophoblast without performing reconstitution experiments. Nevertheless, the cytoarchitecture, nuclear DNA contents, mitotic indices, and cell numbers of the embryonic and extraembryonic ectodermal tissues of the guinea pig frequently resemble those of the mouse not only in vivo but also after isolation and growth in vitro. These observations therefore suggest that guineapig ICM cells do not make a substantial contribution to presumptive trophectodermal derivatives. In addition, they also suggest that the results of the present study are not artifacts of the tissue-culture environment.

The attachment cone of the guinea-pig blastocyst is probably not due to an ICM-independent proliferation of trophectoderm but is most likely derived from a transient accumulation of abembryonic trophectodermal cells. The fact that this is a temporary rather than a persistent trophectodermal growth is further supported by the observation that in vivo the attachment cone degenerates shortly after implantation (Sansom & Hill, 1931). Alternative explanations for the origin of this abembryonic thickening are possible but less likely. For example, the attachment cone could be, either partially or totally, composed of nontrophectodermal cells. Blandau & Rumery (1957) actually describe ‘macrophage-like’ cells in the abembryonic trophectoderm of the guinea pig. However, such presumptive mesodermal derivatives are generally confined to the exocoelomic cavity of the postimplantation, guinea-pig embryo (Ptyler & Strasser, 1925; Kaufman & Davidoff, 1977) or to the stroma of trophoblastic villi (Amoroso, 1952). Since primitive endoderm does not cover the abembryonic portion of the guinea-pig blastocoele cavity (Mossman, 1937; Amoroso, 1952), it would also not be expected to participate in the formation of the attachment cone. Therefore, the attachment cone is most likely to be totally trophectodermal in origin and, being located directly opposite the inner cell mass, probably destined to cease cell division (Copp, 1978) and become giant (Dickson, 1963, 1966) in a manner similar to mouse mural trophectoderm. However, definitive proof that guinea-pig trophectodermal growth depends on the ICM will only be obtained after blastocyst reconstitution experiments are performed.

I am very greatly indebted to Professor R. L. Gardner for his supervision of this work. I should also like to thank Dr A. J. Copp, Dr F. A. L. Clowes, Dr E. P. Evans, Dr C. F. Graham, Dr Anne McLaren, Dr P. W. Barlow, Professor A. C. Braun, and my laboratory colleagues for extremely helpful criticism, and Miss R. E. Woolston, Mr J. Haywood and Mr P. L. Small for excellent technical assistance. I am grateful to Professors F. R. Whatley (Botany School, Oxford) and H. Harris (Pathology School, Oxford) for providing the facilities to complete this work and to Professor J. W. Pringle and the Department of Zoology, Oxford, where this project was started. This work has been supported by fellowships to EBI from the International Agency for Research on Cancer, World Health Organization (IACR/R.882), the American Cancer Society (SPF 14), and the National Institutes of Health (1F32-H D05592-01). This study would not have been possible without the generous assistance of Mr H. Elvidge (Nuffield Institute for Medical Research, Oxford) who provided timed guinea-pig matings. Mr T. Smy (Zoology) and Mr C. Dear (Biochemistry, Oxford) have also kindly provided assistance with animals.

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