Differentiation of trophoblast endocrine cells

Placenta

(1996),

17,

CURRENT

277-289

TOPIC

Differentiation

M. J. Soaresa, Department Paper

of

of Trophoblast

B. M. Chapman,

Physiology,

accepted

12

University

December

Endocrine

C. A. Rasmussen, of Kansas

Medical

Center,

The placenta provides highly specialized functions during gestation that are critical for the normal development of the embryo/fetus. Acquisition of these important responsibilities hinges on the coordinated development and differentiation of the trophoblast cell lineage. In this review, we will focus on research related to the endocrine differentiation of the rat placenta. Although the rat has not been a frequent choice for placental investigation, it offers numerous experimental advantages that will be elaborated below.

OF THE RAT PLACENTA

Trophoblast cells originate via a multilineage differentiation pathway (Gardner, 1983; Gardner and Beddington, 1988; Rossant, 1995; Soares et al., 1995) and are the parenchymal cells of two placental structures, the choriovitelline and chorioallantoic placenta (Jollie, 1964, 1965; Enders, 1965; Davies and Glasser, 1968). Each placental structure is fundamental to the success of pregnancy. The choriovitelline placenta is physiologically significant between implantation and mid-gestation and is comprised of a single differentiated trophoblast cell type, the trophoblast giant cell. From mid-gestation onwards, the chorioallantoic placenta assumes the major responsibilities for the development of the fetus. Four differentiated trophoblast cell phenotypes comprising the rat chorioallantoic placenta can be readily identified: (1) trophoblast giant cells, (2) spongiotrophoblast cells, (3) glycogen cells, and (4) syncytial trophoblast cells (Table 1). Initial identification of differentiated cells was based on morphological and positional criteria. Trophoblast giant cells of the choriovitelline and chorioa To whom correspondence should be addressed. This review is based on a lecture given by the author on receipt of the Adriana & Luisa Castellucci Award on the occasion of the VIth meeting of the European Placenta Group in Spa, Belgium, September 1995. This work was supported by grants from the National Institute of Child Health and Human Development (HD 20676, HD 29036, HD 29797) 01431tOO4/96/050277

+ 13 $12.00/O

G. Dai, T. Kamei Kansas

City,

and K. E. Orwig

Kansas

66160,

USA

1995

INTRODUCTION

ORGANIZATION

Cells

allantoic placentae arise by a process called endoreduplication (i.e. continued DNA synthesis without cell division; Ilgren, 1983). This cell type can exhibit invasive characteristics and it possessessignificant endocrine activities. Spongiotrophoblast cells are morphologically distinct from trophoblast giant cells; however, they also exhibit endocrine activities. The glycogen cell accumulates glycogen and is a potential energy reserve (Davies and Glasser, 1968), whereas syncytial trophoblast cells are multinucleated cells that probably arise from the fusion of trophoblast progenitor cells, and have been implicated in bi-directional transport of nutrients and wastes (Sibley, 1994). As indicated, the location of these differentiated trophoblast cells is not random. Two morphologically and functionally distinct zones within the chorioallantoic placenta are evident (Soares, 1987): (1) the junctional zone and (2) the labyrinth zone (Figure 1). The junctional zone contains trophoblast giant cells positioned at the maternal interface and also contains spongiotrophoblast and glycogen cells. The labyrinth zone contains trophoblast giant cells, syncytial trophoblast cells and fetal mesenchyme and vasculature. This zone is located at the fetal interface, is the location of maternal-fetal exchange, and is defined by the extent of penetration of fetal vasculature. Factors controlling decisions for continued stem cell proliferation, differentiation toward one of the four trophoblast phenotypes, or the morphogenetic organization into a placental structure are just beginning to be appreciated.

PLACENTAL

ENDOCRINOLOGY

The rat placenta synthesizes and secretes a number of polypeptide and steroid hormones (see Sherman, 1983; Soares et al., 1991). We will focus our discussion on the PRL gene family and two steroid hydroxylases involved in progesterone and androgen biosynthesis (Table 2). A brief comment regarding two prominent human placental hormones not readily detectable in the rat or mouse placenta is merited. Trophoblast cells in the mature rat or mouse placenta do not appear capable of expressing c1or p subunits of the glycoprotein hormone gene family, including chorionic gonadotropin (Wurzel et al., 1983; 0

1996 W. B. Saunders

Company

Ltd

278

Placenta Table

1

Cell type Trophoblast

giant cell

Spongiotrophoblast

cell

Glycogen cell Syncytial trophoblast

a Components

cell

, Characteristics of differentiated

trophoblast

Differentiation

Proposed function

Choriovitelline placenta; junctional and labyrinth zonesa (maternal interface) Junctional zone (intraplacental) Junctional zone (intraplacental) Labyrinth zone (fetal interface)

Endoreduplication

Endocrine invasion

Unknown

Endocrine

Unknown

Energy reservoir

Fusion ?

Transport

and

placenta

+--F

.

-Figure 1. Cross-section giant cell

Table

2. Endocrine

of a mature

rat chorioallantoic

Vol. 17

cell types

Location

of the chorioallantoic

(1996),

placenta

and a schematic

markers of trophoblast cell differentiation

Rat placental prolactin family: Classical members: Placental lactogen-I subfamily: Placental lactogen-I (PL-I) Placental lactogen-I mosaic (PL-Im) Placental lactogen-I variant (PL-Iv) Placental lactogen-II (PL-II) Nonclassical members: Prolactin-like protein-A (PLP-A) Prolactin-like protein-B (PLP-B) Prolactin-like protein-C subfamily: Prolactin-like protein-C (PLP-C) Prolactin-like protein-C variant (PLP-Cv) Prolactin-related protein (d/tPRP) Steroid hydroxylases: Cytochrome P450 side chain cleavage enzyme (P45Oscc) Cytochrome P450 17~ hydroxylase (P45Oc17)

Tepper and Roberts, 1984; Carr and Chin, 1985) or the aromatase enzyme which is required for oestrogen biosynthesis (see Sherman, 1983 for a review). Failure to express these endocrine genes may be attributed to the absence of critical c&acting elements within the regulatory DNA of the rat/mouse genes (Nilson et al., 1991).

--u representation

Placental

\

of a mature

’

k?ASyncytial -Fetal

rat chorioallantoic

Spongiotrophoblast Glyeogen cells

TGC trophoblast mesenchyme and vasculature placenta.

TGC,

trophoblast

PRL family

The placental PRL family consists of a group of proteins structurally related to pituitary PRL. Inclusion in the family does not imply functional relatedness (Soares et al., 1991).

Historical aspects. A series of early reports suggested the existence of a luteotropin or mammotropin associated with the placenta (Pencharz and Long, 1931; Selye, Collip and Thomson, 1933; Astwood and Greep, 1938; Cerruti and Lyons, 1960). The pioneering efforts of Henry G. Friesen and Frank Talamantes provided the impetus for the discovery of what is now a relatively large family of proteins related to pituitary PRL. Although we presently acknowledge the existence of’at least nine members of the rat placental PRL family (see Table 2), at the beginning of the 1980s we were aware of only a single placental lactogen (PL) in the rat and mouse. This protein was characterized based on its PRL-like biological actions, and showed a pattern of expression during gestation similar to human PL (progressive increase as gestation advanced). As a result of the isolation and characterization of this initial PL, now referred to as PL-II (Robertson and Friesen, 1975; Colosi et al., 1982; Duckworth, Kirk and Friesen, 1986; Jackson et al., 1986), three additional members of the PRL family were identified: (1) PL-I (Robertson,

et al.: Trophoblast

279

Differentiation

Table Year

3. Discovery of members of the rat/mouse

placental prolactin family

Prolactin

Removal of the anterior pituitary is compatible with pregnancy (Pencharz and Long, 1931; Selye et al., 1933) 1938 Luteotropic activity identified in placental extracts (Astwood and Greep, 1938) Mammotropic activity identified in placental extracts (Cerruti and Lyons, 1960) 1960 1975 and 1981 Isolation of PL-II (Robertson and Friesen, 1975; Colosi et al., 1981) Identification of mouse and rat PL-I (Robertson et al., 1982; Soares et al., 1983) 1982-83 1984 Isolation of mouse proliferin cDNA (Linzer and Nathans, 1984) Isolation of mouse proliferin related protein cDNA (Linzer and Nathans, 1985) 1985 1986 Isolation of PL-II and PLP-A cDNAs (Jackson et al., 1986; Duckworth et al., 1986a,b) 1987 and 1990 Isolation and characterization of PL-I (Colosi et al., 1987a,b; Robertson et al., 1990) 1988 Isolation of rat PLP-B cDNA (Duckworth et al., 1988) 1990-1991 Identification and characterization of PLP-C (Ogilvie et al., 1990a; Deb et al., 1991b,c) 1991 Identification and characterization of PL-Iv (Deb et al., 1991a; Robertson et al., 1991) Identification and characterization of d/tPRP (Roby et al., 1993) 1993 1994 Isolation of PL-Im cDNA (Hirosawa et al., 1994) 1995 Identification and characterization of PLP-Cv (G. Dai and M. J. Soares, unpublished results)

1931-1933

Gillespie and Friesen, 1982; Robertson et al., 1990; Soares et al., 1983; Colosi et al., 1987; Colosi, Talamantes and Linzer, 1987); (2) PRL-like protein-A (PLP-A, Duckworth, Peden and Friesen 1986); (3) PLP-B (Duckworth, Peden and Friesen, 1988); and as these were characterized four other members were discovered: (1) PL-I variant (PL-Iv, Deb et al., 1991a; Robertson, Schroetter and Friesen, 1991); (2) PLP-C (Ogilvie et al., 1990a; Deb et al., 1991b,c); (3) PLP-C variant (PLP-Cv, G. Dai and M. J. Soares, unpublished results); (4) decidual/ trophoblast PRL-related protein (d/tPRP, Roby et al., 1993). An additional member closely related to PL-I, referred to as PL-I mosaic (FL-Im), has been reported (Hirosawa et al., 1994; G. Dai and _M.J. Soares, unpublished results). At present it is not clear whether PL-lm represents an allellic version of PL-I or is indeed a separate gene. Two members of the mouse placental PRL family without apparent rat homologues, termed proliferin and proliferin-related protein, have also been identified and characterized (Linzer and Nathans, 1984, 1985). A timeline of the discoveries of the placental PRL family is presented in Table 3. We have further classified members of the PRL family into subgroups based on function (classical versus non-classical) and structural relatedness (e.g. PL-I and PLP-C subfamilies; Table 2). The nomenclature for the placental PRL family is not ideal; however, it is probably inappropriate to change until we have a better idea of the functions of each of the members. General features.The placental PRL family represents the major secretory proteins of the rat placenta. All receive some type of carbohydrate modification except PL-II (Deb et al., 1989a,b, 199la,b; Deb and Soares, 1990; Roby et al., 1993). The nature of the carbohydrate modifications is very much dependent upon the cell type and protein involved (S. Manzella, M. J. Soares, and J. Baenziger, unpublished results).

Each of the genes has been localized to chromosome 17 of the rat genome (Deb et al., 1991~; Duckworth et al., 1993; Roby et al., 1993; Cohick et al., 1996). To date, the published gene structures for a few members of the placental PRL gene family have been consistent with that reported for pituitary PRL. This is a well-characterized S-exon/4-intron structure. Pituitary PRL genes from several species (Nicoll, Mayo and Russell, 1986), mouse PL-II (Shida, Ross and Linzer, 1992), and two bovine placental PRL (Ebbitt et al., 1989; Kessler and Schuler, 1991) genes all possess this structure. In contrast, at least two members of the rat placental PRL family which are part of the PLP-C subfamily have an additional exon. PLP-Cv and d/tPRP have a single additional exon located between exons 2 and 3 of the prototypical PRL exon/intron arrangement (K. E. Orwig, G. Dai and M. J. Soares, unpublished results). This unique exon may encode for domains that influence biological activity. Biological activities. The actions of members of the placental PRL family can be divided into two groups based on their activation of PRL receptor signalling pathways. PL-UPL-Im, PL-Iv, and PL-II can activate PRL receptor signalling pathways and are considered classical members (Robertson, Gillespie and Friesen, 1982; Robertson, et al., 1994; Cohick et al., 1996; G. Dai and M. J. Soares, unpublished results), whereas the PLPs and d/tPRP are not effective PRL agonists and are considered non-classical members (Deb et al., 1993; Conliffe et al., 1994; Rasmussen et al., 1994; C. B. Cohick and M. J. Soares, unpublished results). This categorization is not meant to be entirely restrictive or an absolute but to provide an initial framework for future investigations. It is evident that we have much to learn about the biological activities of the placental PRL family.

Placenta

280

(1) Classical actions. We have insight into the temporal and spatial pattern of PL-I/PL-Im, PL-II, and PL-Iv expression, and we know that they use the PRL receptor signal transduction system. These ligands are critical to the success of pregnancy in that circulating pituitary PRL levels are minimal from mid-gestation until just before to parturition (Neill, 1980; Soares et al., 1991). However, the specific involvement of these trophoblast hormones in the regulation of pregnancy-specific processes is rudimentary. PL-I, PL-II, and PL-Iv are present in maternal circulation (Robertson, Gillespie and Friesen, 1982; Soares et al., 1983; Ogren et al., 1989; Robertson, Cosby and Shiu, 1995), and thus, are likely to affect an array of different known PRL targets throughout mother (liver, immune system, reproductive tract, ovaries, etc.; Soares et al., 1991). More specifically, PL-I and PL-II have been shown to bind ovarian PRL receptors and specifically stimulate luteal cell progesterone production (MacLeod et al., 1989; Galosy and Talamantes, 1995). PL-Iv activates the PRL receptor signalling pathway but appears to do so less efficiently than PRL, PL-I, or PL-II (Robertson, Cosby and Shiu, 199:; Cohick et al., 1996). The physiological significance of the production of PRL agonists of different potencies is not known. The fetus is also a potential target for the actions of classical members of the placental PRL family. PRL receptors are widely distributed in the fetus (Royster et al., 1995) and targeted mutation of the mouse PRL receptor results in prenatal lethality (Ormandy et al., 1995) further demonstrating the importance of the PRL receptor ligands of trophoblast origin. Considerable attention has recently focused on signalling pathways downstream of the PRL receptor. The non-receptor tyrosine kinase Jak2 is activated when PRL binds to its receptor (Campbell et al., 1994; Dusanter-Fourt et al., 1994; Lebrun et al., 1994; Rui, Kirken and Farrar, 1994). PL-Iv can also effectively interact with PRL receptors resulting in the activation of Jak2 (Cohick et al., 1996). Jak2 then conveys signals to the nucleus via the STAT family of transcriptional regulators (Gouilleux et al., 1994). Other kinases (Fyn and Raf-1) have also recently been identified that appear to participate in PRL signal transduction pathways (Clevenger and Medaglia, 1994; Clevenger, Torigoe and Reed, 1994). Much remains to be accomplished in investigating the mechanism of action of classical members of the placental PRL family. Is the activation of the PRL receptor signalling cascade similar for each classicalmember of the PRL family? Are there other receptor signalling systems or modes of action used by the trophoblast PRL receptor ligands? The placental PRL family represents a group of natural variants of pituitary PRL and thus valuable tools for dissecting the PRL receptor signalling pathway. (2) Non-classical actions. Our understanding of the biological roles of members of the PRL family not using the PRL receptor signalling system has been modest. Nonetheless, we are beginning to obtain some insights. Recently, two nonclassical members of the mouse placental PRL family (proliferin and proliferin-related protein) have been demonstrated to influence blood vessel development (Jackson et al., 1994;

(1996),

Vol. 17

Linzer, 1995). Through a series of in vitro and in vivo assays Linzer and his colleagues have shown that proliferin is angiogenic and proliferin-related protein is anti-angiogenic (Jackson et al., 1994). A 16 kDa proteolytic fragment of pituitary PRL also antagonizes blood vessel development and may share receptors with proliferin and proliferin-related protein (Clapp and Weiner, 1992; Clapp et al., 1993). Most interestingly, proliferin-related protein possessesa region of homology with the unique exon 3 that is found in the PLP-C subfamily. Whether members of the PLP-C subfamily similarly affect endothelial cell function is yet to be resolved. Proliferin has also been shown to specifically bind to uterine membrane preparations and to stimulate DNA synthesis in cell cultures containing a mixture of uterine cell types (Nelson et al., 1995). Thus, at least some of the non-classical members of the PRL family may participate in the establishment of the uteroplacental environment required for a successful pregnancy. Another unique feature of the rat placental PRL family is that its members are expressed in: (1) cell, (2) temporal, and (3) positional patterns that are very useful for assessingtrophoblast cell differentiation (Figure 2).

Expression patterns.

(1) Cell-specific patterns. In this section, we focus on two trophoblast cell types (trophoblast giant cells and spongiotrophoblast cells) and curiously decidual cells. Trophoblast giant cells are the exclusive sources of PL-I and PL-II, and during the latter part of gestation are minor but significant contributors to the production of PLP-A, PLP-C, PLP-Cv, d/tPRP, and PL-Iv (Soares, Julian and Glasser, 1985; Campbell et al., 1989; Duckworth, Schroedter and Friesen, 1990; Faria et al., 1990a, 1991; Deb et al., 1991a,b,c; Rasmussen, Orwig and Soares, 1995). Spongiotrophoblast cells are the major sources of the PLPs, d/tPRP, and PL-Iv and the exclusive trophoblast source of PLP-B (Campbell et al., 1989; Duckworth, Schroedter and Friesen, 1990; Ogilvie et al., 1990b; Deb et al., 1991a,b,c; Rasmussen, Orwig and Soares, 1995). d/tPRP and PLP-B are also expressed in antimesometrial decidua cells, which differentiate from uterine stromal cells (Croze et al., 1990; Gu et al., 1994; Rasmussen, Orwig and Soares, 1995). (2) Temporal-specific patterns. There are four distinct temporal patterns of expression for members of the PRL gene family during the 21-day-gestation period of the rat (Figure 3). The transient expression of PL-I between days 6-11 of gestation represents the first type of pattern (Faria et al., 1990a; Robertson et al., 1990). A second pattern is typified by the PL-II profile, which is initiated just before the termination of PL-I expression and continues until term (Campbell et al., 1989; Faria et al., 1990a). The third pattern is represented by PLP-A, PLP-C, PLP-Cv, and PL-Iv. These hormones are first expressed between days 13 and 15 of gestation and continue until term (Campbell et al., 1989; Deb et al., 1991a,b,c; Robertson, Schroedter and Friesen, 1991; Roby and Soares, 1993). The final pattern exemplifies a coordinated

281

Soares et al.: Trophoblast Differentiation

PL-I /

I

PL-II

Trophoblast

giant cell

Trophoblast

giant cell

Spongiotrophoblast/ trophoblast giant cell

PL-A, PLP-C, PL-Iv

Decidua/spongiotrophoblast/ trophoblast giant cell*

dkPRP, PLP-B ;

14

7

2’1

Day of gestation Figure 2. Cell- and temporal specific characteristics of PRL family gene expression in the developing rat placenta and decidua. The scale on the bottom of the diagram refers to the duration of gestation in the rat. Positions of the horizontal bars represent the duration of expression. (0) Trophoblast giant cell source. (m) dual spongiotrophoblast cell and trophoblast giant cell source. (a) Decidual expression. The prominent diagonal line within the PL-II horizontal bar denotes a shift from junctional zone to labyrinth zone expression. ( ’ ) Denotes a distinction between the expression of d/tPRP and PLP-B. Please note that PLP-B is not expressed by trophoblast giant cells.

FBS (10-20s)

0

12

3

Figure 3. Schematic representation of serum (FBS) containing culture medium. containing culture medium results in the the enrichment of trophoblast giant cells.

the experimental manipulation of Rcho-1 trophoblast cell differentiation. The cells are routinely grown in fetal bovine At confluence, the cells are shifted to horse serum (HS) containing medium. Continued maintenance of the cells in FBS generation of a mixed population of stem and differentiated cells. Shifting the Rcho-1 trophoblast cells to HS results in The scale on the bottom of the diagram refers to the duration of culture.

effort between decidual and trophoblast cells. PLP-B and d/tPRP are first expressed in uterine stromal cells at the time of decidualization,

-

Day of culture

continue

until

mid-gestation,

then

their

expression shifts to trophoblast cells within the junctional zone of the chorioallantoic placenta resembling the PLP/PL-Iv pattern (Croze et al., 1990; Duckworth, Schroedter and Friesen, 1990; Rasmussen, Orwig and Soares, 1995). (3) Positional-specific patterns. The behaviour of trophoblast cells can be influenced by their location within the

developing rat placenta. This is best illustrated by observing the endocrine behaviour of trophoblast giant cells within the junctional and labyrinth zones of the chorioallantoic placenta. Within the junctional zone, trophoblast giant cells have the capacity to express PL-I, and subsequently, PL-II, PLPs, and PL-Iv; however, within the labyrinth zone, trophoblast giant cells are limited to the expression of PL-II (Campbell et al., 1989; Duckworth, Schroedter and Friesen, 1990; Deb et al., 1991a,b,c; Robertson, Schroedter and Friesen, 1991).

Placenta

282

Divergence in the behaviours of trophoblast giant cells from the choriovitelline and chorioallantoic placenta are evident also (Soares, Julian and Glasser, 1985). These positional-specific phenotypes may be related to inherent differences in the trophoblast giant cells from the two locations or their intraplacental environments. (4) Overview. Thus, it is evident that there is interesting phenomenology regarding the patterns of expression of members of the rat placental PRL gene family during pregnancy. A formidable task lies before us in understanding the orchestration of events that control cell-, temporal- and positional-specific aspects of placental PRL family gene expression. Elucidation of the molecular mechanisms directing these events will likely also provide us with considerable information on the regulation of differentiation of trophoblast endocrine cells.

Steroid

hydroxylases

Trophoblast cells of the mouse and rat prominently express two steroid hydroxylases essential for the biosynthesis of steroid hormones: cytochrome P450 side-chain cleavage enzyme (P45Oscc) and cytochrome P450 17a hydroxylase (P45Oc17). P45Oscc is the rate-limiting enzyme for steroidogenesis, catalyzing the conversion of cholesterol to pregnenolone; whereas, P45Oc17 is central to the biosynthesis of androgens (Waterman and Simpson, 1989; Hum and Miller, 1993). Another steroid metabolizing enzyme, 3p hydroxysteroid dehydrogenase (3PHSD), responsible for the conversion of pregnenolone to progesterone, is also required for steroidogenesis (Sherman, 1983). Activities of each of these enzymes in trophoblast cells is not discernibly different from those associated with other steroidogenic tissues. Detection of 3J3HSD activities provided the initial evidence for a steroidogenic role of the rat placenta, and more specifically, for a steroid metabolizing capability of trophoblast giant cells (Samuels et al., 1951; Deane et al., 1962; Sherman, 1983). Although additional characterization of rat placental 3PHSD has not been reported, placental P45Oscc and P45Oc17 enzymatic activities, protein, and mRNAs have been demonstrated (Aringer, Eneroth and Nordstrom, 1979; Johnson and Sen, 1990; Durkee et al., 1992). Placental expression of P45Osccand P45Oc17 is restricted to trophoblast giant cells of the choriovitelline placenta and the junctional zone of the chorioallantoic placenta (Johnson, 1992; Schiff et al., 1993; Yamamoto et al., 1994, 1995b). Expression is intitiated post-implantation and continues throughout the remainder of gestation (Durkee et al., 1992). Mouse decidual cells have also been reported to express P45Oscc(Schiff et al., 1993). Trophoblast giant cell progesterone production has been implicated as a regulator of uteroplacental function (Soares and Talamantes, 1985; Soares and Glasser, 1987) directly and indirectly via its conversion to androgens (Gibori et al., 1988). The principal androgen secreted by the rat and mouse placenta is androstenedione, an aromatizable androgen (Soares and

(1996),

Vol. 17

Talamantes, 1983; Jackson and Albrecht, 1985; Warshaw et al., 1986). Consequently, androgens of trophoblast origin have been viewed principally as substrates for oestrogen biosynthesis. This may be especially relevant in the ovary where estrogens are involved in the maintenance of the corpus luteum, and thus, crucial to the successof pregnancy (Jackson and Albrecht, 1985; Gibori et al., 1988). However, it is also possible that placental androgens may directly contribute to the physiology of pregnancy, independent of their contribution to the biosynthesis of oestrogens. Placentae from most species express P45Oscc; whereas, P45Oc17 expression is conspicuously absent from the human placenta (Sherman, 1983; Waterman and Simpson, 1989; Hum and Miller, 1993). Mechanisms governing this species difference in P45Oc17 expression have not been determined. ANALYSIS OF TROPHOBLAST ENDOCRINE DIFFERENTIATION

CELL

Even though the trophoblast lineage emanates from the first differentiation step during development, our understanding of the cellular and molecular mechanisms controlling trophoblast cell differentiation is nominal. Some insight into regulatory pathways controlling trophoblast cell endocrine differentiation has been obtained through the development and use of in vitro models for studying trophoblast cell differentiation and the identification and characterization of trophoblast cell differentiation-dependent genes. We will address the differentiation of the two principal endocrine cells of the rodent placenta: (1) trophoblast giant cells and (2) spongiotrophoblast cells. Trophoblast

giant

cell

Fundamental to the control of trophoblast giant cell differentiation is the process of endoreduplication and the acquisition of endocrine activities (Soares et al., 1993, 1995). These represent two differentiated endpoints that have been used extensively to monitor trophoblast giant cell development. giant cell dzferentiation. Dissection of any cell differentiation pathway is facilitated by the availability of tissue culture systems. Ideally, a culture system would permit controlled investigation of the transition from the proliferative state to the differentiated state. Rodent trophoblast cells have been inherently difficult to study. Primary cultures are difficult to isolate to homogeneity and do not proliferate; most cell lines readily proliferate, but show limited abilities to differentiate (Soares et al., 1993). In contradistinction, the Rcho-1 trophoblast cell line has provided a useful in vitro model for studying regulatory pathways controlling trophoblast giant cell differentiation (Faria and Soares, 1991).

An in vitro model of trophoblast

Rcho-1 trophoblast cells were derived from a rat choriocarcinoma (Teshima et al., 1983; Faria and Soares, 1991). Trophoblast tumors are an unusual phenomenon in rodents but can be

Soares et al.: Trophoblast

Differentiation

experimentally induced in the pregnant rat by removal of the fetus and extrauterine displacement of the placenta (Shintani, Glass and Page, 1966; Teshima et al., 1983). A rat choriocarcinoma generated by Teshima and colleagues was shown to be transplantable, exhibit giant cells, and when transplanted to female rats stimulated mammary gland development (Teshima et al., 1983; Verstuyf, Sobis and Vandeputte, 1989). The transplantable choriocarcinomas express significant amounts of PL-I which is specifically localized to giant cells within the tumors (Faria et al., 1990b). Cell lines were established from the transplantable rat choriocarcinoma and referred to as RCHO (Verstuyf et al., 1990) and Rcho-1 (Faria and Soares, 1991). The cell lines appear to share many attributes but may also differ in a few characteristics (Faria and Soares, 1991; Duckworth et al., 1993). The following comments refer primarily to Rcho-1 trophoblast cells. In vitro, Rcho-1 trophoblast cells recapitulate trophoblast giant cell development, including endoreduplication and the expression of trophoblast giant cell-specific members of the placental PRL family and steroid hydroxylases (Faria and Soares, 1991; Hamlin et al., 1994; Hamlin and Soares, 1995; Yamamoto et al., 1994, 1995a,b). Rcho-1 trophoblast cells appear to be restricted to the trophoblast giant cell lineage (Faria and Soares, 1991). Growth and differentiation of the Rcho-1 trophoblast cells can be experimentally manipulated (Figure 3; Faria and Soares, 1991; Hamlin et al., 1994). Routinely, Rcho-1 trophoblast cells are maintained in a proliferative state by exposing the cells to fetal bovine serum (FBS) and minimizing cell-cell contact. Differentiation is induced by growing the cells to confluence and substitution of horse serum for FBS (i.e. removal of the FBS-derived mitogens). If the cells are left in FBS, some differentiation towards giant cells occurs; however, the proliferative population is also maintained resulting in a mixed culture (Figure 3). Culturing in the presence of horse serum selects for differentiated giant cells. The adhesive characteristics of the Rcho-1 trophoblast cells can be used also to discriminate relevant cell populations. Differentiated giant cells are more adherent than proliferative stem cells. The ability to induce differentiation is stable for at least 30 passages. Expression of differentiation-specific markers is restricted to morphologically distinct giant cells and temporally reproduces the in situ acquisition of giant-cell specific endocrine markers (Faria and Soares, 1991; Hamlin et al., 1994; Yamamoto et al., 1994, 1995b). PL-I and steroid hydroxylase expression is initiated during early stages of differentiation followed by PL-II, and then PLP-A and PLP-C expression. The only apparent difference that we have observed is the failure of PL-I expression to terminate as it does in vivo which might suggest the existence of extratrophoblast signals regulating PL-I expression. Given the limitations of any in vitro model, Rcho-1 trophoblast cells offer considerable promise as a means for elucidating regulatory mechanisms controlling trophoblast giant cell differentiation. Rcho-1 trophoblast cells have been used for the identification of intracellular signalling pathways controlling

283

trophoblast giant cell development and trophoblast cell-specific gene expression.

giant

Putative molecular

giant

mechanisms

controlling

trophoblast

Discovery of trophoblast giant cell differentiation-dependent regulatory proteins situated from the surface to the nucleus of the trophoblast cell is advancing along two fronts: (1) dissection of intracellular signalling cascades and (2) elucidation of the transcriptional mechanisms controlling trophoblast giant cell-specific gene expression. cell

dtferentiation.

(1) Intracellular signalling cascades. Trophoblast giant cell differentiation is initiated through the development of cell-cell interactions and is facilitated by the removal of mitogenic stimuli (Faria and Soares, 1991; Hamlin et al., 1994; Hamlin and Soares, 1995). Progression along the trophoblast giant cell differentiation pathway and maintenance of the differentiated trophoblast giant cell phenotype are under intrinsic control mechanisms, involving, at least in part, tyrosine kinase signalling pathways (Hamlin and Soares, 1995; B. M. Chapman and M. J. Soares, unpublished results). Three members of the Src family of non-receptor tyrosine kinases (SYC, yes, lyn) are the most prominent tyrosine kinases activated in proliferating and differentiating trophoblast cells (Hamlin and Soares, 1995; T. Kamei, G. P. Hamlin and M. J. Soares, unpublished results). Activation of lyn tyrosine kinase is directly associated with the differentiation of trophoblast giant cells (T. Kamei, G. P. Hamlin and M. J. Soares, unpublished results). Identification of mechanisms responsible for the activation of lyn tyrosine kinase and the nature of its substrates are currently being pursued. We propose that lyn tyrosine kinase participates in the initiation, progression and/or maintenance of trophoblast giant cell differentiation. Src has also been implicated in the regulation of the endocrine differentiation of human trophoblast cells (Rebut-Bonneton, Boutemy-Roulier and Evain-Brion, 1993). In a search for putative trophoblast regulatory genes, three research groups independently identified the same basic helixloop-helix transcription factor, referring to it as Thing-l (Hollenberg et al., 1995), Hxt (Cross et al., 1995), or e-HAND (Cserjesi et al., 1995). They showed that Thing-I/Hxt /e-HAND exhibited a pattern of tissue-specific expression that was consistent with a trophoblast regulatory factor. Furthermore, Cross and coworkers showed that overexpression of Thing-l/Hxt /e-HAND promoted various aspects of trophoblast differentiation, including stimulation of the PL-I gene promoter in cells committed to the trophoblast giant cell lineage (Cross et al., 1995). Current data are compatible with Thing-l/Hxt /e-HAND serving as an important regulator of the trophoblast giant cell lineage. (2) Trophoblast giant cell-specific gene expression. Experimentation has been reported on the transcriptional regulation of four genes specific to trophoblast giant cells: PL-I, PL-II, PLP-A, and P45Oscc. The Rcho-1 trophoblast cell line has proven an effective tool in analysing regulatory regions controlling PL-I gene

284

expression. A region 274 bp upstream of the PL-I transcriptional start site has been shown to direct trophoblast giant cell-specific gene expression in vitro (Shida et al., 1993). Linzer and his colleagues have further demonstrated the involvement of AP-1 and GATA elements within this regulatory segment and the participation of fus and jun proteins and GATA-2 and GATA3 transcription factors (Shida, Ng and Linzer, 1993; Shida et al., 1993; Ng et al., 1994). GATA factors have also been implicated in the transcriptional control of the u- and P-subunit genes of CG in human trophoblast cells (Steger et al., 1994). Targeted disruption of either the GATA-2 or GATA3 genes is associated with mid-gestation lethality (Tsai et al., 1994; Pandolfi et al., 1995), consistent with the participation of GATA factors in the regulation of trophoblast giant cell function. Mid-gestation GATA3 null placentae are morphologically abnormal and express reduced levels of PL-I (Ma et al., 1995). Differentiation dependent changes in GATA factor binding activity have not been reported. Some evidence for an increase in AP-1 binding activity associated with trophoblast giant cell differentiation has been demonstrated (Shida, Ng and Linzer, 1993). Upstream elements of the signalling cascade culminating in an increase in AP-1 and/or GATA factor activity remain to be determined. The ras/mitogen-activated protein kinase pathway can potentially influence the activity of both AP-1 and GATA factors (Karin, 1995; Towatari et al., 1995) and thus may be a reasonable candidate pathway for upstream regulation of the differentiation-dependent activation of PL-I. The PL-II promoter has proven to be somewhat more difficult to study. Approximately 2.1 kb of 5’ flanking DNA associated with the PL-II gene has been shown to contain elements that specifically direct expression of a reporter gene to labyrinthine trophoblast giant cells in transgenic mice (Shida, Ross and Linzer, 1992). This regulatory region is located between 2.7 kb and 569 bp upstream of the PL-II transcription start site. A 2.7 kb PL-II promoter-reporter construct was also effective in primary cultures established from late gestation placentae (Shida, Ng and Linzer, 1993a). Curiously, this 2.7 kb mouse PL-II promoter-reporter construct and an 800 bp rat PL-II promoter-reporter construct were ineffective in the Rcho-1 trophoblast cell line (Shida, Ng and Linzer, 1993; Vuille et al., 1993). Please recall that differentiated Rcho-1 trophoblast cells express PL-II mRNA and protein (Faria and Soares, 1991). Additionally, the Rcho-1 PL-II transcript sequence is identical to that previously reported for the PL-II transcript isolated from the rat placenta (Duckworth, Kirk and Friesen, 1986; G. Dai and M. J. Soares, unpublished results). Why are these PL-II promoter-reporter constructs ineffective in the Rcho-1 trophoblast cells? A couple of possibilities are worthy of consideration. Please remember that trophoblast giant cell behaviour differs depending upon its location. Giant cells within the junctional zone express an array of endocrine genes, including PL-II, whereas giant cells located within the labyrinth zone are more selective and express only PL-II. Differentiated Rcho-1 trophoblast cells possess the junctional zone phenotype. The transgenic and in vitro primary culture

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transfection analyses performed by Linzer and co-workers were compatible with a labyrinthine pattern of expression (Shida, Ross and Linzer, 1992, Shida, Ng and Linzer, 1993). Eliciting a junctional zone/Rcho-1 trophoblast cell PL-II pattern of expression may require additional elements associated with the PL-II gene. Alternatively, could there be another PL-II gene containing an identical coding region but possessing distinct regulatory regions? PLP-A promoter-reporter constructs containing either 4.6 kb or 975 bp of 5’ flanking DNA are active in RCHO cells but not in rat pituitary cells (Vuille et al., 1993). There are several putative cis-acting elements within the -975 bp PLP-A promoter construct; however, their involvement in directing trophoblast giant cell specific expression of PLP-A has not been reported. The ontogeny of steroidogenesis in trophoblast giant cells is associated with transcriptional activation of the P45Osccgene (Yamamoto et al., 1994, 1995a). An 83 bp region located between -5 11 and -428 of the P45Osccpromoter is required for the differentiation-dependent activation of P45Osccin Rcho-I trophoblast cells (Yamamoto et al., 1995a). Interestingly, this represents an entirely different region than involved in regulating P45Osccin other steroidogenic tissues (Yamamoto et al., 1995a). In addition, two prominent activators of P45Osccgene expression in the gonads and adrenal, cyclic AMP/protein kinase A and steroidogenic factor-l, do not participate in activation of the P45Osccgene in trophoblast giant cells (Yamamoto et al., 1995a). At present, we are attempting to further narrow the trophoblast giant cell-specific P45Oscc gene regulatory region and identify proteins interacting with the region.

Spongiotrophoblast

cell

Research directed toward understanding the regulatory factors governing spongiotrophoblast cell differentiation has been affected by the paucity of in vitro models. Rcho-1 trophoblast cells do not express genes restricted to spongiotrophoblast cells (PLP-B) and thus, would not appear to be a suitable model for studying spongiotrophoblast cell differentiation (Faria and Soares, 1991). A spongiotrophoblast primary culture system has been recently described (Lu, Deb and Soares, 1994). The primary spongiotrophoblast cells spontaneously differentiate attaining the capacity to express spongiotrophoblast cell-specific genes, including PLP-B, and can be modulated by exposure to retinoic acid (Lu, Deb and Soares, 1994). These cells may prove useful for some studies on spongiotrophoblast cell differentiation or may represent a suitable starting cell population for establishing an immortalized spongiotrophoblast cell line. Most of the progress in elucidating possible mechanisms controlling spongiotrophoblast cell development have been garnered from gene targeting and transgenic strategies. Mash-2 is a helix-loop-helix transcription factor specifically expressed in developing trophoblast cells (Guillemot et al., 1994, 1995). Targeted disruption of the Mash-2 gene results in mid-gestation lethality associated with the failure of spongiotrophoblast cell

Soares et al.: Trophoblast

Differentiation

285 Differentiated

cell

PL-I, PL-II, PLP-C, PLP-Cv, d/tPRP, PL-Iv, P45Osq P45Oc17

PLP-A, Tyrosine protein GATA AP-1 Thing-l/Hxtie-HAND

kinases Giant

cell

PLPs, a

Mash-2 EGF-R CIEBPP?

d/tPRP, (PLP-B)

PL-Iv,

cell

Figure 4. Overview of the control of trophoblast endocrine cell differentiation. A putative stem cell gives rise to two trophoblast cells capable of endocrine activities: (1) trophoblast giant cells and (2) spongiotrophoblast cells. Trophoblast giant cells are characterized by their expression of placental lactogen-I (PL-I), placental lactogen-II (PL-II), placental lactogen-I variant (PL-Iv), prolactin-like protein-A (PLP-A), prolactin-like protein-C (PLP-C), PLP-C variant (PLP-Cv), decidual/trophoblast prolactin-related protein (d/tPRP), cytochrome P450 side chain cleavage (P45Oscc), and cytochrome P450 17a hydroxylase (P45Oc17). Spongiotrophoblast cells are characterized by their expression of the PLPs, PL-Iv, and d/tPRP. Trophoblast giant cells are the sole source of PL-I and PL-II, whereas spongiotrophoblast cells are the sole placental source of PLP-B. Non receptor tyrosine protein kinases and GATA, AP-1, and Thing-I/Hxt/e-HAND transcription factors are implicated in the development of the trophoblast giant cell lineage, whereas the epidermal growth factor receptor (EGF-R) and the transcriptional regulator, Mash-2 have been implicated in the differentiation of spongiotrophoblast cells. There is also some evidence for a possible involvement of C/EBPP with the regulation of spongiotrophoblast cell-specific gene expression.

development (Guillemot et al., 1994). Mash-2 interacts with DNA elements containing a conserved motif referred to as an E-Box (Johnson et al., 1992). 5’ Flanking DNA for at least three genes expressed in spongiotrophoblast cells (PLP-A, PLP-Cv, and the 43 11 gene) contain consensus E-Box sequences (Vuille et al., 1993; Calzonetti, Stevenson and Rossant, 1995; G. Dai and M. J. Soares, unpublished results). The 4311 gene encodes for a putative secretory protein of unknown function (Lescisin, Varmuza and Rossant, 1988). A 340 bp region located 3.7 kb upstream of the 4311 gene transcription start site contains two E-Box elements and is capable of targeting fi-galactosidase to spongiotrophoblast cells in transgenic placentae (Calzonetti, Stevenson and Rossant, 1995). Consequently, Mash-2 may be a pivotal regulatory gene for both the development of spongiotrophoblast cells and the expression of spongiotrophoblast-specific endocrine genes. Epidermal growth factor receptor (EGFR) null mutant mice on a 129/Sv genetic background die at mid-gestation with a spongiotrophoblast cell defect that resembles the placental defect in Mash-2 null mutants (Sibilia and Wagner, 199.5; Threadgill et al., 1995). Interestingly, EGFR null mutants on other genetic backgrounds have entirely different phenotypes (Sibilia and Wagner, 1995; Threadgill et al., 1995; Miettinen et al., 1995). The data suggest a potentially complex involvement of the EGFR in controlling spongiotrophoblast cell development, possibly via interactions with Mash-2. A family of pregnancy-specific glycoprotein genes (rnCGM) are expressed in the rat placenta and at least one member, rnCGM-1, apparently expressed in spongiotrophoblast

cells (Rebstock et al., 1993; Chen, Chen and Chou, 1994). RnCGM-3, another member whose intraplacental localization has not been reported, is regulated by C/EBPP, a member of the bZIP classof transcription factors (Chen et al., 1995). The rnCGM-3 C/EBPP DNA binding element does not appear in 5’ flanking DNA of other spongiotrophoblast cell specific genes. Thus, at this juncture the significance of C/EBPP in regulating the endocrine differentiation of spongiotrophoblast cells is uncertain.

Overview

The regulatory pathways directing differentiation along the trophoblast giant cell and spongiotrophoblast lineages are clearly distinct (see Figure 4). Based on the existing evidence, trophoblast giant cell differentiation appears to be influenced by non-receptor tyrosine protein kinase pathways and the activities of GATA, AP-1, and Thing-1/Hxt/e-HAND transcription factors. In contrast, spongiotrophoblast cell development appears to be regulated by an EGF receptor signalling pathway and the transcriptional regulators, Mash-2 and possibly C/EBPP. The specific responsibility of each component in the regulatory network controlling trophoblast cell differentiation requires further investigation. Nonetheless, we now have the beginnings of a framework for elucidating the regulatory pathways controlling differentiation of the endocrine cells of the rat placenta.

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FINAL

COMMENTS

There are several keys to expanding our understanding of the differentiation of trophoblast endocrine cells. First of all, an appreciation of the biological roles of the endocrine products of the cells is essential. Trophoblast endocrine cells do not operate in a vacuum but instead are likely impacted directly or indirectly by other cell populations that are targets for their endocrine products. Second, we need additional in vitro models, especially representing the spongiotrophoblast cell lineage. This is not a simple task but may benefit from advances in targeting gene expression with spongiotrophoblast cellspecific promoters (Calzonetti et al., 1995). These cell populations are needed for investigating intracellular signalling pathways and cell-specific gene expression. Third, we need to identify &-elements and trans-acting factors responsible for several trophoblast giant cell- and spongiotrophoblast cellspecific genes using both in vitro and transgenic paradigms. Patterns should emerge for composite cell, temporal, and positional specific regulatory elements and factors. These mechanisms for controlling gene activation are likely to provide insight into the regulation of trophoblast cell differentiation as has been shown for muscle, pituitary, adipose, and haematopoietic cell development (Karin, Castrillo and Theill, 1990; Orkin, 1992; Olson, 1993; Smyth, Sparks and Wharton, 1993). A fourth objective should be a thorough investigation of the relationship between genes pivotal to cell cycle control and trophoblast cell endoreduplication. Information forthcoming from such studies will not only provide insight into the differentiation of trophoblast giant cells but also facilitate the identification of novel cell cycle regulatory genes. Finally, we can derive benefit

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from understanding trophoblast regulatory pathways in other species. Differences in the regulation of primate and rodent trophoblast cells exist. The cyclic AMP/protein kinase A pathway plays a prominent role in the control of gene expression in differentiated human trophoblast cells (Strauss et al., 1992) but not in rodent trophoblast cells (Yamamoto et al., 1995a, b). It is not unreasonable to predict that the regulatory mechanisms governing trophoblast cell development will show at least some aspects of conservation acrossspecies. In support of this notion, promoters for human trophoblast-specific genes have been demonstrated to specifically target the activation of reporter genes to the mouse placenta (Bokar et al., 1989; Strauss et al., 1994). Such data suggest that at least some of the transcriptional machinery in primate and rodent trophoblast cells must be shared. Putative trophoblast regulatory genes identified from non-rodent speciesare likely to be evaluated in the mouse or rat. Thus, we need to obtain a better understanding of the trophoblast lineage in other species. Are there functional equivalents of trophoblast giant cells or spongiotrophoblast cells in primates, ruminants, or other species?Morphological criteria for identifying cellular homologues may not be sufficient. Conservation may be evident at the level of the intracellular signalling pathways, including the transcriptional machinery controlling trophoblast cell-specific gene expression. This review was based on a lecture presented on the occasion of being recognized for our placental research with the 1995 Adriana & Luisa Castellucci Award. We would like to thank the Castellucci family very much for their interest in placental research and in establishing and supporting this special award.

ACKNOWLEDGEMENTS We would like to acknowledge current colleagues in our laboratory, Christopher B. Cohick, Thomas J. Peters, Bing Liu, and Dr Heiner Mueller, and also some previous trainees and research associates that made valuable contributions: graduate students: Teresa Faria, Gary Hamlin, and Xing-jian Lu; and postdoctoral fellows: Santanu Deb, Kazuyoshi Hashizume, Katherine F. Roby, and Toshiya Yamamoto; and research associates: Douglas Larsen and Lei Xu. It is important also that we acknowledge a number of collaborators and contributors of valuable reagents: Drs JoAnne Richards, -Michel Vandeputte, Frank Talamantes, Daniel Linzer, Henry Friesen, Jacques Baenziger, Joan Hunt, Shinichi Teshima, Kunio Shiota, Claude Szpirer, Simon CM. Kwok, James L. Voogt, and Elke W-interhager.

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