Control of trophoblastic growth

Placenta (:983), 4, 3o7-328

Review Article: Control of Trophoblastic Growth E. B. ILGREN Department of Zoology, University of OxJbrd, OxJbrd OX1 3PS, UK

ABSTRACT The investigations described herein have been aimed at elucidating the ways in which trophoblastic proliferation, endoreduplication, and muhinucleation are controlled. The early post-implantation, trophectodermal derivatives of the mouse remain diploid and continue to divide even after they move away from the inner cell mass. This is due, in part, to the maintenance of close cell contacts within these tissues which suppress the giant cell change and keep the early post-implantation trophoblast in a diploid, non-giant state. However, such close cell contacts are not able to promote continued trophoblastic cell division. This suggests that other factors, such as contact with inner cell mass (ICM) derivatives, are needed to sustain trophoblastic proliferation. Conversely, the loss of close cell contacts and an appropriate tissue shape as well as the absence o f l C M derivatives promote giant cell transformation. In the mouse, giant cell transformation may begin through a binucleate phase and subsequently proceed via endomitosis and/or endoreduplication. However, in species other than the mouse, trophoblastic giant cells may not arise through a binucleate phase but may, instead, become multinucleate and syncytial. Furthermore, in some species (e.g. ruminants), trophoblastic multinucleation may be consequential upon a multipolar mitosis without concomitant cytokinesis whilst in others (e.g., man) it may depend upon a cell fusion event with secondary syncytialization. The growth of guinea pig trophectoderm and its derivatives appears to be under ICM control. Thus the multilayered, abembryonic structure found within the guinea pig blastocyst, called the attachment cone, and the early post-implantation trophectodermal derivatives, presumed to be homologous to mouse extra-embryonic ectoderm, both stop dividing and become giant when isolated from the ICM and ICM-derived tissues. The development of primitive endoderm proceeds in a manner similar to the growth of trophectoderm. Thus the giant cell change appears to be a general feature of trophectodermal and primitive endodermal growth during mouse and guinea pig embryogenesis. Moreover, the giant cell change appears to b'egin in endoderm after trophectoderm has begun to degenerate. Endodermal polyploidization may occur during the growth of primitive, ICM-like, embryonal carcinoma cells as they form tissues with the biochemical and morphological features of normal primitive endoderm. Tumour-derived and normal primitive endoderm may also grow in a manner similar to trophectoderm since they both contain binucleate giant cells, possess polyploid mitotic figures, and display growth patterns potentially attributable to changes in homotypic and/or heterotypic cellular interactions.

307

308

E. B. llgren

PROPERTIES OF T R O P H O B L A S T I C TISSUES Trophectoderm is the first tissue to differentiate in the mammalian embryo (Amoroso, i952 ). It forms the outer layer of the btastocyst and displays a precocious form of differentiation which involves cellular vesiculation, fluid pumping, the formation ofzonula occludens and an inability to aggregate with like tissues, and the capacity to initiate a decidual response (discussed by Gardner 1972, 1974). The post-implantation derivatives of trophectoderm are called trophoblast. During development, tropboblast acquires distinct endocrinological (e.g., rodents: Billington, ~97r; Heap, Flint and Gadsby, 1979; man: Diczfalusy, 1974; Gordon and Chard, 1979; Villee, 1979) and immunological (e.g., rodents: Billington, 1971; Carter, 1976, i978; Billington, I965, i979; Chatterjee-Hasrouni and Lala, 1979; Sellens, 197z; Sellens, Jenkinson and Billington, 1978; man: Billington, 1979; Goodfellow et al, 1976; McIntyre and Faulk, 1979; Beer and Billington, 1978; Kawago, Kawana and Sakamoto, 198o) properties. It is also known to be invasive in utero (e.g., man: Boyd and Hamilton, 196o; mouse: Billington, 197 I; Cowell and Kirby, 1968; Cowell, 197z; Snow, 1973; rabbit: Glenister, 1961, 1965, 197o, vole: Copp, 198o; Ozdzenski and Mystowska, I976), in ectopic sites (e.g., mouse lung, kidney, spleen, testis, brain, liver and tumours: Carr, I979; Kirby, i96zb , I963a, I963b, I964, 1965; Kirby and Cowell, 1968; rat kidney: Kirby, i962a; pig ureter: Samuel, 197I; and hamster testis: Billington, 1966) and even when grafted xenogeneically (e.g., mouse trophoblast in rat: Kirby, I96za; and chick: Tickle, Crawley and Goodman, 1978; human trophoblast in rat: Obata et al, i976; hamster trophoblast in mouse: Billington 1966). In utero, trophoblast may invade vessels (e.g., hamster: Billington, 1966; Pijnenborg, Robertson and Brosens, 1975; man: Boyd and Hamilton, I96O; DeWolfet al, 198o; Hamilton and Boyd, 1966; Pijnenborg et al, 198o; guinea pig: Davies, Dempsey and Amoroso, I96Ia, 196Ib; rat: Bridgeman, 1948a, 1948b; Legrand, I974; Peel and Bulmer, 1977) and, at times, may be carried through the systemic circulation (e.g., man: Douglas et al, 1959; Noyes, 196o; chinchilla: Billington and Weir, 1967). Invasion is probably due in part to cellular migration (e.g., in vivo, hamster: Billington, 1971; Orsini, 1954; mouse: Amoroso, 1952; in vitro, vole: Copp, I98O; mouse: Sobel et al, i978 , 198o; Sherman and Wudl, i976), phagocytosis (mouse: Gardner, 1974; rat: Dickson and Bulmer, I96O; AI-Abass and Schulrz, I966; Enders and Schlafke, i969; man: Foldes, Schwartz and Keharty, i975) , and the release of cytolytic agents (e.g., guinea pig: Owers and Blandau, i971; and mouse: Strickland, Reich and Sherman, i976 ). Pre- (e.g., mouse: Copp, I978 ) and early post-implantation (e.g., extraembryonic ectoderm, mouse: Rossant and Offer, 1977) trophoblast is frequently found to be diploid (z-4~:) and dividing. However, after these tissues lose contact with the inner cell mass (e.g., mouse: Gardner, I97Z; Copp, I978) and its derivatives (e.g., mouse: Rossant and Offer, 1977), they also lose their ability to divide. Although many somatic cells also stop dividing after a limited number of cell divisions (Pardee, i974) , they usually come to rest in the G~ phase of the cell cycle and thereby remain diploid (z~.). In contrast, trophoblastic cells often stop dividing in G 2 (Brodskii and Uryvaeva, I977) and then become giant with nuclear DNA contents greater than 47:. Such giant cells, depending largely on species and location, may be either uni-, bi-, or multinucleate. Those with only a few nuclei are thought to grow via polyploidization and endoreduplication (Zybina, I963; Brodskii and Uryvaeva, i977; Nagl, I978), whilst others, with many nuclei, are thought to arise via cell fusion and syncytialization (tbr review see Billington, i97 I). The present aim is to review ways in which trophoblastic proliferation, endoreduplication, and syncytialization may be controlled. Tl~e control mechanisms thought to underly each of these cellular processes have thus been considered by examining the growth of trophoblast, as well as tissues that appear to grow in a manner similar to trophoblast, either in utero, in vitro, or in ectopic sites.

Control of Trophoblastic Growth

3o9

NORMAL DIFFERENTIATION OF MOUSE T R O P H E C T O D E R M AND T H E PRIMARY T R O P H O B L A S T I C GIANT CELL LAYER The processes leading to the differentiation oftrophectoderm probably begin as early as the twoto four-cell stage (Graham and Deussen, 1978) and depend largely on cell position (Kelly, i975). The differentiation process itself involves five cleavage divisions and is accompanied by blastomere compaction, tight junction formation, and cavitation (Smith and McLaren, I977). Compaction takes place at the eight- (e.g. mouse, rat, rabbit) to 16- (e.g., man, baboon: Ducibella, I977) cell stage and is associated with the onset of tight junction formation (Ducibella and Anderson, 1976; Ducibella, 1977) and junctional communication (Lo and Gilula, I979). During compaction, other changes occur in membrane surface charge, glycoprotein composition, and microvillous structure (review: Ducibella, I977; see also Brulet et al, i98o ). By the 3z-cell stage, the outer presumptive trophectodermal cells may be distinguished from the inside cell population destined to form the inner cell mass since the nuclei of the future ICM are more compact than those of the trophectoderm (Graham, 1973). Mature trophectoderm appears following the formation of the blastocoele cavity (Gardner, Papaioannou and Barton, 1973; Rossant and Lis, 1979; Rossant and Vijh, i98o), blastulation being accomplished by the release of fluid from cytoplasmic vesicles and its subsequent coalescence within intercellular spaces (Calarco and Brown, 1969). Apical tight junctions, responsible for the retention of blastocoele fluid (Nadjicka and Hillman, i974) , mature between trophectodermal cells (Ducibella, I977). Concurrently, maculae adherens fix the ICM to the polar trophectoderm (Ducibella, i977). The trophectoderm thus resembles a 'tight epithelium' with an Na +-dependent pump capable of maintaining a distinct ionic environment (Borland and Tasca, 1974; Nadjicka and Hillman, 1974; Borland, Biggers and Lechene, i975) and transferring certain amino acids (Nadjicka and Hillman, 1974). Blastocyst formation is also accompanied by an increase in oxygen consumption (McLaren, 1973), glucose utilization (Brinster, 1967) and CO_~production (Brinster, 1967) as well as a metabolic switch to the Embden-Meyerhoff and tricarboxylic acid pathways (McLaren, 1973; Benos and Balaban, 1983). At the late blastocyst stage, the polar trophectoderm continues to divide whilst cell division in mural regions declines (Copp, I978). As the abembryonic mural trophectodermal cells stop dividing, their nuclei enlarge (ca. i x7 h.s. p.c. Dickson, 1963; Barlow, Owen and Graham, i972), and presumably become giant (Barlow and Sherman, I972; Barlow, Owen and Graham, 1972.). The giant cell transformation proceeds from the abembryonic pole towards polar-ICM areas and is completed in about 15 hours (Dickson, 1966). During this interval, the blastocyst almost doubles in length (Dickson, 1966) and the trophectoderm becomes greatly attentuated (Barlow and Sherman, 1972). Tissue stretching, a concomitant reduction in the number of intercellular contacts, and the onset of cellular polarization (Johnson, Peatt and Handyside, I981 ) may secondarily serve to promote the giant cell change (Barlow and Sherman, 1972). Nevertheless, trophectodermal endoreduplication (e.g., Sherman and Atienza-Samols, I979) as well as the polyploidization (Surani, Barton and Burling, i98o), mult'inucleation (Sherman and Atienza-Samols, 1979), and even the biochemical differentiation oftrophectoderm (Chew and Sherman, 1975; Sellens and Sherman, 198o) can still apparently proceed in the absence of tissue interactions. During the periimplantation period, an amorphous material forms between the inner cell mass and the trophectoderm (Nadjicka and Hillman, 1974), whilst junctional communication between these two tissues appears to be altered (Lo and Gilula, i979). At implantation, mechanical constraints placed on the implanting blastocyst by the uterine wall may redirect polar to mural trophectodermal migration so that the polar trophectoderm secondarily accumulates over the ICM (Gardner and Papaioannou, 1975) and thus forms the ectoplacental cone and the extra-

3io

E. B. llgren

embryonic ectoderm (Gardner, Papaioannou and Barton, I973; Gardner and Papaioannou, 1975; Copp, I979). These structures may, in turn, push the embryonic ectoderm farther into the blastocoele cavity, thereby giving rise to the early egg cylinder (Copp, i978 ). The mural trophectoderm surrounding the mouse egg cylinder is then called the primary giant cell layer, a tissue composed of uninucleate and occasional binucleate giant cells (Amoroso, i95z).

C O N T R O L OF T R O P H E C T O D E R M A L P R O L I F E R A T I O N Contrary to earlier claims (Krebs, Krebs and Beard, i95o), trophoblast grows and spreads in a manner that is fundamentally different from the way tumours proliferate within host tissues (e.g., for tumours see Braun, i978 ). Thus, trophoblastic growth is not an uncontrolled cellular proliferation but rather a highly controlled, self-limited process. This idea is supported by the followingobservations. First, trophoblast does not.metastasize even when it extensively invades vessels (Kirby, I963). Secondly, it is not serially transplantable (Avery and Hunt, i969) and its life span appears to be limited in utero, in ectopic sites (Kirby, i965; Avery and Hunt, i972; Sherman and Wudl, I976), and in vitro (Dorgan and Schultze, i97i; Sherman and Wudl, I976 ). In utero, isolated trophoblastic giant cells may be found deep in the myometrium up to three . weeks beyond term (e.g., rat: Shintani, Glass and Page, 1966; man" Boyd and Hamilton, 196o; hamster: Orsino, I954) but they usually persist in these sites in a non-dividing state. Similarly, the giant cells which surround the rat (Amoroso, I952; Zybina, Kudryavtseva and Kudryavtseva, I979) and mouse (Muentener and Hsu, i977) embryo often degenerate by the end of pregnancy. In ectopic sites and in vitro, trophoblast also degenerates at a time that is roughly equivalent to the end of gestation in that species (Kirby, 1965; Billington, 197 i). Even if trophoblast is transplanted to irradiated hosts (Koren, Abrams and Behrman, i968; Zeilmaker, i971 ) or intraperitoneal diffusion chambers (Koren et al, I97o), its life span is still prolonged only slightly. Also claims that secondary trophoblastic giant cells can proliferate in vitro and thereby 'escape' the normal restraints placed upon their life span (Sherman, 1975b) still await confirmation since the giant nuclei found in these cultures may have been either nontrophoblastic (Ilgren, 198oa,b ) and/or neoplastic (Sherman, I975b ) in origin. Taken together these observations suggest that trophectodermal proliferation proceeds in a controlled manner. The existence of controls is indicated by two types of studies: namely (I) morphological investigations which show that there is regional differentiation in the trophoblastic tissues of normal and delayed embryos, tissue patterns established according to the distance between the trophectoderm and the inner cell mass, and (z) experimental analyses which demonstrate that trophectodermal proliferation is dependent on contact with the inner cell mass.

REGIONAL DIFFERENTIATION IN THE T R O P H O B L A S T I C TISSUES OF N O R M A L AND DELAYED EMBRYOS Tissues containing both diploid and giant nuclei often display remarkably constant developmental patterns that are, in many cases, both tissue- and species-specific (Nagl, I978). Regional differences in the 'karyological anatomy' (in sensu Tsermak-Woess, 1956) and cellular organization of these tissues have suggested that the switch from diploidy to polyploidy is a controlled process. Regional differentiation may be seen in trophoblastic tissues at three stages of development: namely, before implantation, in the early post-implantation period, and towards the end of pregnancy (Table i). Before implantation, there is regional differentiation in

311

Control of Trophoblastic Growth

Table tA. Regional differentiation in the trophectoderm

Species/tissue

Condition

Mouse blastocyst (3.5 day)

In utero

Mural trophectodermal nuclei larger than polar

(Gardner and Papaioannou, 1975)

Abembryonic pole--initial site of giant cell change Abembryonic pole--reduced mitotic index Abembryonic pole--most prominent phagocytic activity Abembryonic pole~ytoplasmic blebs and projections Abembryonic pole--maximal Con-A binding Abembryonic pole--maximal lysosome, acid phosphatase, lipid Abembryonic pole--initial site of giant cell change in blastocysts transferred to spleen and kidney Abembryonic pole--initial site of giant cell change and DNA synthesis Abembryonic pole--initial site of giant cell change in blastocysts grown on strips of immature mouse uteri Abembryonic pole--initial point of attachment to substrate

(Dickson, x963a)

In utero

Abembryonic pole--initial site of giant cell change

(Bridgeman, t948a, I948b )

In utero

Abembryonic pole--initial site of syncytial giant cell change Abembryonic pole--large numbers of microfilaments and vessels Abembryonic pole--initial site of syncytial giant cell change Abembryonic pole--initial site of proteolysis

(Blandau, 197x)

In ecotopic sites In activated delay In vitro

Rat blastocyst (4.5 day) Guinea pig blastocyst

In vitro

Rabbit blastocyst (8.o days)

Observations

In utero

Abembryonic pole--site of fusion with uterine epithelium Abembryonic pole--altered meta/anaphase ratio Equatorial to abembryonic syncytial knob formation

(Copp, ~979) (Gardner, 1974) (Nadjicka and Hillman, i974) (Fein and Toder, 1978) (Dickson, 1969) (Kirby, t963a, I965) (Holmes and Dickson, t975) (Dickson, i963b ) (Grant, I973)

(McLaren and Hensleigh, i975)

(Parr, i973) (llgren, 1979, unpub.) (Owers and Blandau, 1971) (Schlalke and Enders, i975) (Moog and Lutwak-Mann, x958) (Hay, i965)

Sheep blastocyst In utero (I6 days)

Abembryonic pole--large numbers of crystals and liposomes

(Winterberger-Torres and Flechon, 1974)

Ground squirrel blastocyst

In utero

Abembryonic pole--initial site syncytial giant cell change

(Boyd and Hamilton, 1952)

Primate blastocyst

In utero

Abembryonic pole--initial site 'sprout-like' outgrowths

(Boyd and Hamilton, 1952)

Lateral-abembryonic polc~syncytial plaques

(Schlafke and Enders, 1975)

Ferret blastocyst In utero

E. B. llgren

312 Table lB. Regional differentiation in early post-implantationand placental trophoblast

Species/tissue

Condition

Observations

Post-implantation trophoblast

Mouse (7.5 day) Extra--embryonic ectoderm ectoplacental cone

In utero

Secondary giant cells

Mouse (,o day) Primary giant cell layer

In Utero

Mouse (7.5 day) Ex. emb. ectoderm and ectoplacental cone

In ectopic sites

Ectopically transferred In vitro Mouse (7.5 day) Ex. emb. ectoderm and ectoplacental cone explanted

Rat (8.5 day) ectoplacental cone secondary giant cells

Mitotic index: (Ex Ect > EPC core > EPC periphery) (P < o.ooi) (%) G2 nuclei: (EPC core >Ex Ect) (P 4~z:(EPC periphery > EPC core) (%) Large sized nuclei: (EPC periphery > EPC core)

(llgren, 1981a) (llgren, 1981a) (llgren, 1981a) (Gardner, Papaioannou and Barton, 1973; Muentener & Hsu, 1977)

(%) Large-sized nuclei: 45% Abemb. Pole: 40% Equator: 3% cone

(Chew and Sherman,.*975)

Extent giant cell change--EPC more advanced than Ex-Emb. Ect. Abemb. half EPC less advanced than Emb. half EPC

(Gardner & Papaioannou, 1975)

Site giant nuclei and extent giant cell change--EPC core more advanced than Ex. Emb. Ect. cultures

(Ilgren, 1981a) (llgren, 1981c)

H3Thy. incorp: EPC core > EPC periphery Mitotic index: EPC core > EPC periphery

/ ~

Base villi~mall Langhan's cells and mitoses Tips villi--larger Langhan's cells 'Capillary free' interlobium-small nuclei 'Capillary invaded' interlobium--polyploid swellings

(Boyd and Hamilton, 196o)

(Grobstein, 195o)

In utero (Peel and Bulmer, 1977)

Placental trophoblast

Human (near term) Cytotrophoblast

Guinea pig Syncytiotrophoblast

In utero (Boyd and Hamilton, 196o)

t

(Kaufman and Davidoff, 1977)

the trophectoderm of a n u m b e r of species. T h u s , in the mouse, the largest nuclei are located at the abembryonic pole of the blastocyst (Table i). Similarly, the rate of cell division in the polar trophectoderm is higher than in mural regions (Copp, x978a; I978b ). Such regional changes are probably complete in less than 24 hours and are, in addition, associated with a drop in mitotic index, changes in cell surface properties, altered histochemical characteristics, and an increased ability to phagocytose particular matter (Table i). In the guinea pig and the ground squirrel, syncytial giant cells initially appear within the abembryonic mural trophectoderm (Table 0. Furthermore, 'zonal variations' in trophoblastic growth have also been seen in sheep trophectoderm and these have.been said to be directly correlated with the distance between the

Control of Trophoblastic Growth

313

trophoblastic tissues and the inner cell mass and/or its derivatives (Winterberger-Torres and Flechon, I974). Moreover, the timing of regional differentiation appears to be under maternal control. Thus, during lactational or ovariectomy delay (Dickson, i966; Sherman and Barlow, I97Z; McLaren, I973) the onset of these regional changes are postponed, but following the administration ofoestrogen and the cessation of delay such zonal differences appear (McLaren, i973; Holmes and Dickson, I975).

EXPERIMENTAL ANALYSES WHICH D E M O N S T R A T E T H A T T R O P H E C T O D E R M A L P R O L I F E R A T I O N IS D E P E N D E N T ON C O N T A C T WITH T H E INNER CELL MASS If an ICM is microsurgically removed from a mouse blastocyst (Gardner, i972) or is prevented from developing either chemically (e.g., colcemid, cytarabinoside, BuDR: Sherman, i975; U 3thymidine: Ansell, 1975; Ansell and Snow, i975) or with irradiation (Goldstein, Spindle and Pedersen, i975) , a trophectodermal vesicle is produced whose cells initially stop dividing and subsequently become giant ( > 4~:). However, if an ICM is inserted into a trophectodermal vesicle shortly after microsurgery, normal trophectodermal proliferation can be sustained (Gardner, Papaioannou and Barton, t973). Thus, the ICM appears to act as an 'inducing' tissue and the observed trophectodermal proliferation appears to be the result of an ICMtrophectoderm, inductive interaction (Gardner and Papaioannou, I975). Although the precise nature of the inductive stimulus (i.e., cell contact-, humoral-, and/or matrix-mediated) is not known, it is not species-specific (Gardner, 1975) and appears to act locally since the site of trophectodermal proliferation usually correlates with the position of the inner cell mass (Gardner and Papioannou, 1975). In addition there appears to be some correlation between the amount of ICM tissue present and the degree of trophectodermal proliferation observed (Ansell and Snow, x975). The aforementioned morphological and experimental studies clearly support the idea that the proliferation of trophectoderm is under the control of the inner cell mass. However, there are two observations that appear to challenge this hypothesis. First, during the early postimplantation development of the mouse, some trophectodermal derivatives (e.g., extraembryonic ectoderm: Rossant and Offer, I977) continue to divide even after they move away from the ICM. Second, during the pre-implantation development of the guinea pig, trophectodermal cells accumulate at the abembryonic pole of the blastocyst, i.e., at the point most distant from the inner cell mass (Blandau and Rumery, I957; Blandau, i971 ).

C O N T R O L OF T R O P H O B L A S T I C P R O L I F E R A T I O N DURING EARLY P O S T I M P L A N T A T I O N MOUSE DEVELOPMENT In utero some polar trophectoderm derivatives remain diploid and dividing even though the). have moved away from the inner cell mass (Rossant and Offer, i977). However, if these initially diploid, post-implantation trophectodermal derivatives are microsurgically isolated from the embryo and grown as cellular monolayers in vitro or as ectopic grafts in vivo, they fail to divide and subsequently become giant (Rossant and Offer, 1977). Such findings suggest that continued contact with ICM derivatives, e.g. extra-embryonic endoderm and/or mesoderm, may be needed to promote post-implantation trophoblastic proliferation in vivo. This is also supported by the fact that regional differentiation, similar to that Ibund within trophectoderm, is also seen

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E. B. llgren

within post-implantation trophoblast (Table i). Nevertheless, it is still not clear whether postimplantation trophoblastic cell division is sustained because of a specific ICM-inductive effect or because nearby ICM derivatives maintain these trophectoderm-derived tissues in a certain configuration which, in turn, serves to promote their continued proliferation (discussed by Rossant and Offer, i977) and their final contributions to the formation of the mature mouse placenta ( Johnson and Rossant, I98I). Recent studies have helped to determine which of these explanations is correct (Ilgren, I98Ic). Thus, initially diploid post-implantation mouse trophoblast was grown in vitro so that close cell contacts would be maintained within these tissues (Ilgren, ~98Ic). The maintenance of close cell contacts could, in turn, suppress the giant cell change but was not, in itself, able to promote trophoblastic proliferation (Ilgren, 198ic ). This suggests that additional factors, such as the presence of ICM derivatives; are also probably required to sustain trophoblastic cell division during early post-implantation mouse developments (see also Rossant and Lis, I98i ).

C O N T R O L OF T R O P H O B L A S T I C P R O L I F E R A T I O N IN T H E GUINEA PIG Several observations suggest that the growth of guinea pig trophectoderm may not be under ICM control. Thus, cells have been shown to accumulate at the abembryonic pole of the guinea pig blastocyst in vitro and in vivo (Blandau and Rumery, 1957; Blandau, 1971). Similarly, guinea pig trophectoderm has been said to proliferate in culture under conditions in which the degeneration of the ICM is claimed to have occurred (Blandau and Rumery, i957; Blandau, i971 ). It has been shown, however, that if abembryonic mural trophectoderm and extraembryonic ectoderm are isolated from the guinea pig embryo and grown in vitro, they not only stop dividing but also become giant (Ilgren, i98oa; also see Figure i). Such findings suggest that the growth of guinea pig trophectoderm and its derivatives may also be underICM--control in a manner similar to mouse trophoblast (Ilgren, i98oa; also see Ilgren, 198Ib,d).

THE MANNER IN WHICH DIPLOID T R O P H E C T O D E R M FIRST BECOMES GIANT: ORIGIN OF THE T R O P H O B L A S T I C GIANT CELL A high proportion oftrophectodermal cells lose their ability to divide and subsequently become giant during development (Amoroso, 1952; Copp, I978 ). In the mouse and the rat, these giant cells are uni- (Wislocki and Dempsey, i955; Bulmer and Dickson, I96o; Kirby, I964) and occasionally binucleate (Table 2) whilst in other species they are frequently multinucleate and syncytial (see below). Origin of the trophoblastic giant cell change in the mouse and the rat: binucleation Although post-implantation mouse trophoblast first becomes giant and thus contains nuclei with more than four times the haploid (E) amount of DNA immediately before implantation (Barlow, Owen and Graham, i972; Nagl, I978), the manner in which this occurs is not known. However, certain non-trophectodermal tissues (e.g., liver: Carriere, i969) initially become giant when nuclear division is not followed by cytokinesis, i.e., via a binucleate phase. Furthermore, the results of recent experimental studies suggest that mouse trophectoderm may also first become giant through a binucleate growth phase (Ilgren, i98ia; see also Table 2 and Figure 2) in a manner similar to these non-trophectodermal tissues (Carriere, I969).

315

Control of Trophoblastic Growth 5.5 d a y GUINEA PIG BLASTOCYST

MTB

~M : ~D

_

\ ...6.

GIANT CELL PLAQUE

/ ~

~

EMB ECT

YSE

3daysINVITRO EX E M B

EPGC ~ l h ~ : ~ J

parnd

EGG CYLINDER

Figure I. Schematic diagram depicting fate of abembryonic mural trophectoderm (MTB) and extra-embryonic ectoderm (EX EMB) after isolation from the guinea pig embryo and growth in vitro. From llgren 098xb), with kind permissionof the editor of Anatomy and Embryology. END = extra--embrionicendoderm; ICM = inner cell mass;PTB = potar trophectoderm; EPGC = ectoplacental giant cells; YSE = yolk sac endoderm.

M e c h a n i s m by which D N A a c c u m u l a t e s d u r i n g the t r o p h o b l a s t i c g i a n t cell t r a n s f o r m a t i o n o f the m o u s e a n d the rat: endocycles By the ninth day of pregnancy in the mouse and the rat, mitoses are found to be confined largely to the core of the ectoplacental cone (Amoroso, i952; Snell and Stevens, 1966 ). Shortly thereafter, mitotic activity is said to be particularly pronounced in the chorion, especially where it makes contact with allantoic mesoderm (Barlow and Sherman, t972; Gardner, Papaioannou and Barton, I973). After ten days p.c., trophoblastic cell division declines and the chorionic trophoblast as well as the secondary giant cell layer contain virtually no mitotic figures (Andreeva and Zavarsin, i964; Zybina, i97o; Zybina and Grischenko, i97o; Avery and Hunt, I972). By [2 days' gestation, cell division and D N A synthesis take place largely within the junctional zone of the placenta (Peel and Bulmer, i977). As cells leave this area and migrate into the uterine stroma, they stop dividing, undergo two endocycles, and thus form the tertiary giant cell layer (e.g., rat, 8-I6~: nuclei: Zybina, 197o; Zybina and Grischenko, I97O; see also Orsini, 1954 (hamster); Copp, I98O (volt)). In contrast to this tertiary giant cell movement, the cells that remain at the implantation site frequently (e.g., rat: 20 to 35 per cent) become binucleate, undergo more than four endocycles, and subsequently contribute to the formation of the secondary giant cell layer (e.g., rat, 64-io42~ nuclei: Zybina, I97O; Zybina and Grischenko, I97O ). Once maximal nuclear D N A content levels are reached (mouse, ca. z56-io24~: , i2 days p.c.: Muentener and Hsu, I977, and rat, t4 to ]7 days p.c." Jollie, i964; Dorgan and Schultze, I971 ) over one-half of all trophoblastic cells may be giant (e.g., mouse: Chapman, Ansell and McLaren, I972). Moreover, as the D N A content rises, the duration of the endocycle also tends to increase (Zybina, i963a ) even though the length of Endo-S remains relatively constant (ca. 6 to 7

316

E. B. llgren Table 2. Binucleation and the growth of trophectoderm and its derivatives

Species/tissues

Observations

Mouse

7.5--day primary giant cells lo.5-day secondary giant cells Mouse

3.5~ blastocyst transferred to kidney and brain

In utero: Binucleate giant cells Some incorporate H3-thymidine In ectopic sites: Binucleate giant cells

Kirby, 1965; Fawcett, i95o

In mutant embryos: tw3z embryos: 'periphery'--more binucleates as well as fewer tighter junctions os embryos: 'paired' nuclei with tetraploid metaphases ay embryos: occasional binucleates seen

Mouse

Blastocysts

Chatterjee-Hasrouni and Lala, 1979; Ilgren, t981a; Saccoman, Morgan and Wells, i965

Hillrnan and Hillman, i975; Hillman, Hillman and Wileman, I97o Patterson, t979 Calarco and Pederson, 1976

In vitro: (~-) and (~-) blastomeres form Sherman and Atienza-Samols, E979; binucleate giant cells in 7O0/o cases Sherman, 1975a Binucleate, polyploid metaphase Surani, Barton and Burling, 198o figures

Mouse

Trophectodermal vesicles Blastomere-derived Cytochalasin-induced Rat

7.5-day primary giant cells lo.5-day secondary giant cells 15-day secondary giant cells

In utero: Binucleate giant cells

Dickson and Bulmer, 196o; Alden, 1948. (%) Binucleates increase with time Zybina and Grischenko, 197o (day 16, 7%; day 15, 19% ) and vary with location (day 15, periphery, zO~ centre, 35%)

Sheep

22-day chorionic areolar tissue

(%) Binucleate giant cells: Wimsatt, 195o, 1951 (maximal, margin; moderate, crypts; absent, centre)

| Nuclearl(, ~ ("~Acytokineticp,/~-~ Nuclear_( ~ Acytokineti~'~--~ c Fusion ~e~

,oo Figure 2a

Nuclear

Control of Trophoblastic Growth

317

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24 T i m e in vitro ( H o u r s )

72

Figure2. (a) Schematic diagramdepicting the possible mechanismsof binucleationand polyploidizationin the cells of initially diploid, trophectodermalderivativesgrownin vitro. (b) Frequencydiagramdepictingthe changesin binucleate cell numbers and polyploidy in initially diploid, trophectodermalderivativosgrown in vitro. From llgren (198[a). hours; Zybina, i963b ). Several mechanisms have been proposed to account for the way in which the trophoblastic giant cells of the mouse and the rat accumulate these large amou'nts of nuclear DNA.

Non-replicative mechanisms: phagocytosis and cellfusion. It has been proposed that trophoblastic giant cells phagocytose maternal nuclei and subsequently reutilize the ingested nuclear D N A (for review see Graham, I973). Graham, however, has pointed out that nuclear engulfment may occur either as a rare event or one which is physiologically unimportant since the engulfed nuclei are probably genetically inactive. Thus, phagocytosis probably does not account for the large amounts of nuclear D N A seen within the trophoblastic giant cells of rats and mice. Although various workers have suggested that the trophoblastic giant cells of the mouse and the rat arise via cell fusion (for review, Graham, 1973), there is experimental evidence which suggests that the trophoblastic giant cell change does not occur in this manner (Chapman, Ansell and McLaren, 197z; Gearhart and Mintz, 1972 ). Rephcative mechanisms: endocycles. Endocycles are periodic rounds of D N A replication that take place within an intact nuclear membrane (Nagl, I978 ). They occur in the absence of nuclear and cytoplasmic division with or without chromatin condensation and chromosome formation. Endocycles may be endomitotic, polytenic, and/or endoreduplicative. Endomitotic cycles usually occur at relatively low ploidy levels (ca. 16-32~: , rat: Zybina, i97o; Zybina and Grischenko, [97o ) when chromatin condensation is followed by endochromosome formation. Thus, at endometaphase, a polyploid ( > zn) number of normal-sized endochromosomes are formed. Such endomitotic metaphase figures are found within post-implantation mouse

318

E. B.

llgren

trophoblast in vitro (Ilgren, 198ic) and trophoblastic giant cell layers in vivo (Zybina, I961 , I97O; Zybina et al, 1973, I975a , I975b; Zybina and Chervogryadskaya, i976 ). Polytenic endocycles take place when endochromosome formation is not followed by endochromosome separation. Thus, nuclei with a diploid number of giant polytene chromosomes are produced (Nagl, i978 ). The trophoblastic giant cells 0fthe mouse (Snow and Ansell, I974), the rat (Zybina, 197o), and the rabbit (Zybina, I97O) are said to be polytenic (see also Zybina, I972 ). Ho~vever, Barlow and Sherman (1974) were not able to find polytene chromosomes in the trophoblastic giant cells of either the rat or the mouse. This, in turn, suggests that trophoblastic polytenic cycles, if they do occur, produce giant polytene chromosomes which are structurally different from those found in other tissues (Nagl, I978 ). Endoreduplicative cycles take place when chromatin condensation arid chromosome formation fail to occur (Alden, i948; Saccoman, Morgan and Wells, 1965; Sherman, McLaren and Walker, 1972; Nagl, i978 ). Endoreduplication generally occurs at DNA content levels exceeding 32?: (Zybina and Grischenko, 197o; Zybina, I97o ). As the endoreduplicative process proceeds, polymerase levels tend to increase. The final polymerase levels attained, however, are not proportional to the large amounts of DNA found within the trophoblastic giant nuclei. Thus, the replication of trophoblastic DNA may require relatively small amounts of polymerase to continue (Sherman and Kang, i973; see also Kalf et al, i98o ). There is a proportionate replication of the entire trophoblastic nuclear genome, a proposal supported by at least two observations. The first is the finding that the DNA contents of the entire trophoblastic nucleus and its sex chromatin body display a parallel increase in value (Nagl, t972 ). The second observation is that the main band-to-satellite DNA ratio remains constant as trophoblastic nuclei increase in size (Sherman, McLaren and Walker, i972 ). ORIGIN OF T R O P H O B L A S T I C GIANT CELLS FOUND IN SPECIES O T H E R THAN THE MOUSE AND T H E RAT: M U L T I N U C L E A T I O N Normally, multinucleate cells are found only in certain tissues (e.g., bone: Chambers, 1978; skeletal muscle: Lambert, 1946; Chambers, I978; bone marrow: Japa, 1943; liver: Wilson and Leduc, i948; placenta: Amoroso, 1952; nerve: Penfield, 1932; and decidua: Ansell, Barlow and McLaren, i974) , and their occurrence elsewhere is usually pathological (Lewis, I927; Haythorn, i929; Chambers, 1978). It is generally possible to distinguish, on the basis of nuclear number and mitotic activity, two types oftrophoblastic multinucleate cells. The first are found most commonly within the trophoblastic tissues of man, the guinea pig, and the rabbit. They may have as many as 50 nuclei and include virtually no mitotic figures (see below). In this respect they resemble the multinucleates found in skeletal muscle and bone (Lambert, 1946; Young, I962; Chambers, i978), tissues thought to arise via the fusion of myoblastic (Kalderon, I98O) and osteoclastic (Chambers, i978 ) precursors respectively. The second type of trophoblastic multinucleate cell is seen in the placentae of ruminants (Wimsatt, 195o; 195 i). It is generally smaller than the first, usually containing fewer than ten nuclei and occasional included mitotic figures (see below). Cell fusion and the origin o f the trophoblastic multinucleate cell There is relatively little syncytiotrophoblast in mouse and rat placentae (Wislocki and Dempsey, i955; Bulmer and Dickson, i96o ). This may, in turn, account for the fact that a cell fusion event has not, as yet, been detected within mouse trophoblast (e.g., mouse: Chapman, Ansell and McLaren, I972; Gearhe:frt and Mintz, 1972). In other species, however, trophoblastic multinucleates are commonly seen either early in development (e.g., in guinea pig abembryonic

Control of Trophoblastic Growth

319

trophectoderm: Enders, I97 I, and human polar trophectoderm: Brewer, i937) , in the periimplantation period (e.g., in rabbit trophoblastic knob tissue: Enders and Schlafke, 1969) or near term (e.g., in rabbit: Enders and Schlafke, i97i; guinea pig: Kaufman and Davidoff, I977; and human: Boyd and Hamilton, I966 ) placentae. The syncytiotrophoblastic cells found within human and guinea pig placentae contain relatively large numbers of small nuclei, virtually no included mitotic figures, and little evidence of DNA synthesis (e.g., man: Amoroso, I952; Thiede, 196o; Galton, I962; Enders, 1965; Gerbie, Hathaway and Brewer, i968; Billington, I97I; guinea pig: Davies, Dempsey and Amoroso, I96Ia, I96Ib; Kaufman and Davidoff, i977). The DNA contents of most human syncytio- (96 per cent = 2~:; Galton, I962; Quinlivan, 1962) and cytotrophoblastic (82 per cent = 2E; Galton, I962) nuclei are diploid. The latter, however, continue to divide, whilst the former remain quiescent (Galton, 1962). Furthermore, autoradiographic (Midgely et al, i963) , electron microscopic (for review see Billington, i97i), and histochemical (for review see Billington, i97I ) observations strongly suggest that human s'yncytiotrophoblast is derived from cytotrophoblastic precursors and not from maternal decidual cells (Boyd and Hamilton, i96o ). Other studies suggest that the syncytiotrophoblastic tissues of the guinea pig (Enders, I97I), the rabbit (Enders and Schlafke, I97I ) and man (Galton, I962; Billington, i97i; Blandau, i972 ) arise through the fusion of cytotrophoblastic daughter cells (see also studies ofsyncytiotrophoblastic cells with fragments'ofdegenerating cell membranes within their cytoplasm, i.e., 'transitional cells'--rabbit: Glenister, i97o; man: Enders, i965; Billington, i971 ). There is little experimental evidence to support the hypothesis that trophoblastic tissues grow via cell fusion. In fact, skeletal muscle is the only tissue that has been shown by experimental means to undergo fusion during development (Mintz and Baker, i967). However, it is interesting to note that skeletal muscle and syncytiotrophoblast have at least one property in common, namely, the diploid (2E, G1) resting nature of their nuclei (Strehler, Konigsberg and Kelly, i963; Love, Stoddard and Grasso, I969). Myoblastic fusion does, in fact, appear to be consequential upon the G t diploid resting state (Konigsberg and Buckley, 1974), to be initiated in vitro by a reduction in essential metabolites (Konigsberg and Buckley, I974), and to be altered by changes in temperature and calcium (Kalderon, I98o). In contrast to myoblastic fusion, the factors able to influence trophoblastic syncytialization are.not known. Although some workers claim that mesenchyme can initiate and/or mediate the fusion of cytotrophoblastic precursors (e.g., rabbit, ruminants, shrew: see discussion, Gienister, i96I; mouse: Hernandez-Verdun, i975; hamster: Carpenter, i972), such claims are based almost exclusively upon comparative histological studies and are thus purely correlative. Moreover, the timing, position, and extent :of syncytialization are said to be highly variable in many species (Schlafke and Enders, 1975) and the trophoblast found immediately next to maternal vessels in rat and mouse placentae is thought to be cellular rather than syncytial (Schlafke and Enders, 1975). Furthermore, multinucleates may be found within pure trophoblastic tissues (e.g., blastomere-derived, trophectodermal vesicles: Sherman and Atienza-Samols, 1979, cultures of post-implantation mouse trophoblast: Ilgren, i98ic ). Together, these observations argue against the idea that mesenchyme mediates the syncytialization of trophoblast. There is also little experimental evidence to support claims that hypoxia can influence trophoblastic syncytialization (e.g., rabbit: Boving, i959; Glenister, t965) and/or cytotrophoblastic proliferation (e.g. man~ normal development: McKay et al, i958; Fox, 1979; pathological states: Fox, i964, 197o; Beischar et al, i97o ). Multipolar mitosis and the origin of the trophoblastic multinucleate cell The trophoblastic tissues of ruminants occasionally contain cells with three, four, or five nuclei and mitotic figures (Wimsatt, I95o, I95 i). Although it is not clear how these multinucleate cells

3zo

E. B. llgren

arise, certain observations offer a clue to their origin. Thus, the trophoblastic multinucleates of ruminant placentae resemble those seen within rodent liver (e.g., aged rodent hepatocytes with occasional polyploid, mitotic figures: Wilson and Leduc, i948), nerve and decidua with polyploid metaphase nuclei (e.g., neurones: Penfield, i932; Yarygin, Kenschor and Komarovich, i969; Willmer, i97o; Mann and Yates, i973; Brodskii and Uryvaeva, I977; and decidual cells: Sachs and Shelesnyak, 1955; Dupont, Dulac and Mayer, i97i ) and also bone marrow (e.g., megakaryocytes with several nuclei in metaphase: Japa, 1943). These findings suggest that the type of multinucleate cell found within ruminant trophoblast might arise through a multipolar mitosis without concomitant cytokinesis. In order to obtain further evidence in support of this idea, mouse decidua were analysed both cytologically and cytophotometrically (Ilgren, Evans and Burtenshaw, 1981). Decidua were chosen for ~rudy since their rate of cell division (Finn and Martin, i967) is generally much higher than that of mature liver (Carriere, i969) , brain (Jacobson, I978 ) and trophoblast (Wimsatt, i95o , I95I ). Thus it is potentially easier to find multipolar mitotic figures in decidua than in hepatic, neural, or trophoblastic tissues. In fact, such multipolar mitotic figures have been seen in decidual tissues and these were found to contain excessive amounts ofDNA ( > 4~:)and numerous sets of chromosomes (Ilgren, Evans and Burtenshaw, I98I ). Such findings suggest that multinucleation, as it occurs in mouse decidua and tissues somewhat similar to decidua, e.g., bovine trophoblast, may result from a multipolar nuclear division without concomitant cytokinesis (Ilgren, Evans and Burtenshaw, I981 ).

C O N T R O L OF T H E T R O P H O B L A S T I C GIANT CELL T R A N S F O R M A T I O N The post-implantation mouse embryo displays an exceedingly complex spatial distribution of rates of cellular growth and division. This complexity virtually precludes an experimental analysis of the control mechanisms which underly the growth of post-implantation trophoblastic tissues in vivo. Despite these difficulties, the problem of post-implantation trophoblastic growth control can still be approached by studying the behaviour of initially diploid, post-implantation, trophectodermal derivatives in vitro (Ilgren, I98Ia,c ) and the development of certain nontrophectodermal extra-embryonic tissues in vivo (Ilgren, i98ob; Ilgren and Littlefield, i98i; Ilgren, I981a ). Such in vitro analyses have been aimed at determining whether the trophoblastic giant cell change could be influenced by alterations in tissue shape and the degree of intercellular contact. These in vivo studies were designed to explore the possibility that the giant cell transformation might be a more generalized developmental phenomenon than was previously appreciated, occurring not only within trophectoderm- but also with extra-embryonic endoderm- and mesoderm-derived tissues (also see lineage studies, Gardner and Papaioannou, 1975). This possibility was initially suggested by studies of higher plant embryos (Nagl, I978 ). These demonstrated that the giant cell change is a general feature of the development of angiosperm extra-embryonic membranes, i.e., suspensor and endosperm (Nagl, i978 ). The suspensor in particular has been said to grow in a manner similar to trophoblast (Nagl, I978 ). Additional studies have shown that mouse endoderm and plant endosperm may also grow in similar ways. Thus, primitive endoderm normally becomes giant during development (Ilgren, i98ob ) and the endodermal giant cell change itself appears to be correlated with the growth of trophectoderm (Ilgren, I98id ). Finally, it has been demonstrated that tumour cells with the biochemical (Lo and Gilula, 198oa) and morphological (Lo and Gilula, i98ob ) features of primitive endoderm also become giant as they differentiate in vitro (Ilgren and Littlefield, 198 i). Moreover, if these giant (i.e. embryonal carcinoma cell-derived endoderm: Lo and Gilula, I98oa,

Controlof TrophoblasticGrowth

32t

198ob ) cells are treated with a mitogen (Ilgren and Littlefield, i98I), they are still able to undergo mitosis and thus behave in a manner similar to the endodermal giant cells normally found in the visceral yolk sac of the mouse (Ilgren, i98ob ).

CONCLUSIONS

9Control of trophoblastic proliferation in early post-implantation mouse development. Several conditions are needed to promote the growth of post-implantation mouse trophoblast. First, an appropriate tissue shape and close cell contacts are needed to suppress the trophoblastic giant cell transformation and to maintain these tissues in a largely diploid, non-giant state. Moreover, since an appropriate tissue shape cannot, on its own, promote trophoblastic cell division, ICMderivatives, e.g. extra-embryonic endoderm and/or mesoderm, are probably also needed to stimulate normal trophoblastic growth. Control of trophoblastic proliferation in the guinea pig. The growth of guinea pig trophectoderm and its early post-implantation derivatives appear to be under ICM control. Thus, the multilayered attachment cone of the guinea pig blastocyst is probably not due to an ICMindependent proliferation of trophectoderm but is most likely derived from a transient accumulation of abembryonic trophectodermal cells. Origin of the trophoblastic giant cell: binucleation. In the mouse, post-implantation trophoblast appears to become giant through a binucleate phase. Thus, the growth of mouse trophectoderm occurs, to a certain extent, in a manner similar to the polyploidization of mouse liver. Origin of the trophoblastic giant cell: multinucleation. The finding of multinucleates in pure, trophectoderm-derived tissues suggests that trophoblastic multinucleation, at least in the mouse,' does not depend upon prior contact with mesenchyme or mesenchymal tissues. Multipolar, polyploid mitotic figures were found in mouse decidua, tissues known to contain multinucleates similar to the ones seen in the trophoblastic derivatives of ruminants. This suggests that the multinucleate cells seen in some forms of trophoblast may arise in a manner similar to those found in rodent decidual tissues, i.e., via a multipolar mitosis without concomitant cytokinesis. Mechanism by which DNA accumulates during the trophoblastic giant cell transformation. Polyploid metaphases were found within post-implantation mouse trophoblast that had chromosomes arranged either randomly or in pairs. This suggests that at least some mouse trophoblastic cells increase their DNA via polyploid mitotic and/or endomitotic cycles. Control of the trophoblastic giant cell transformation. The degree to which trophectodermderived cells endoreduplicate their nuclear genome and thus become giant can be influenced by changes in tissue shape and the extent of intercellular contact. Thus, cell separation, spreading, and stretching appear to promote the giant cell change not only within trophectodermal derivatives but also within tissues known to behave in a manner similar to trophectoderm, namely extra-embryonic endoderm. Moreover, the giant cells found in normal and neoplastic extra-embryonic tissues are not necessarily terminal and incapable of cell division since they may be found in mitosis either before and/or following mitogenic stimulation. Comparative

3zz

E. B. llgren

s t u d i e s also s u g g e s t t h a t t h e o n s e t o f t h e g i a n t cell c h a n g e in o n e e x t r a - e m b r y o n i c m e m b r a n e m a y be t e m p o r a l l y c o r r e l a t e d w i t h t h e d e v e l o p m e n t a n d / o r d e g e n e r a t i o n o f a n e a r b y p l a c e n t a l tissue layer.

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Boyd, J. D. & Hamilton, W. I. (1966) Electronmicroscopic observations on the cytotrophoblastic contribution to the syncytium in the human placenta. Journal of Anatomy, loo, 535-548Braun, A. C. (1978) The Story of Cancer. London: Addison-Wesley Press. Brewer, J. J. (1937) Abnormal human ovum in a stage preceding the primitive streak (The Edwards-Jones-Brewer Ovum). American Journal of Anatomy, 61,429-453 . Bridgeman, J. (g948a) A morphological study of the development of the rat. I. An outline of the development of the placenta of the white rat. Journal of Morphology, 83, 61-85. Bridgeman, J. (1948b) A morphological study of the development of the rat. II. A histological and cytological study of the development of the chorioallantoic placenta of the white rat. Journal of Morphology, 83, 195-zz3. Brinster, R. L. (1967) Carbon dioxide production from glucose by pre-implantation mouse embryos. ExperimentalCell Research, 47, 271-277. Brodskii, V. Ya & Uryvaeva, 1. V. 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