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Endocrinology Vol. 141, No. 1 430-437
Copyright © 2000 by The Endocrine Society

ARTICLES

Cellular Localization of the Human Chorionic Gonadotropin ß-Subunit in Transgenic Mouse Placenta

Brian L. Strauss and Irving Boime

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Irving Boime, Department of Molecular Biology and Pharmacology, Washington, University School of Medicine, St. Louis, Missouri 63110. E-mail: iboime{at}pcg.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CG is a human placental glycoprotein expressed in first-trimester trophoblasts. To examine the regulation of the CGß-subunit in an in vivo model, we previously constructed transgenic mice containing the CGß gene cluster and demonstrated its expression in the placenta. Here, we determine the cell type responsible for CGß synthesis in the mouse by immunohistochemical and in situ hybridization analyses. Unexpectedly, the protein and messenger RNA were not detected in trophoblast or elsewhere in the chorioallantoic placenta but in the parietal endoderm, a separate extraembryonic component of the placenta. The identity of this CGß-producing layer was confirmed by the presence of laminin A, a known protein of the parietal endoderm extracellular matrix. However, we observed heterogeneity, with respect to synthesis of laminin A and CGß; parietal endoderm cells expressing CGß at high levels synthesized less laminin A, and vice versa. The absence of CGß production in trophoblasts of the transgenic mouse demonstrates a lack of transcriptional equivalence between rodent and human trophoblasts. The data are consistent with the hypothesis that, in human placenta, one or more transcriptional factors coevolved as members of the CGß gene cluster underwent duplication.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CG IS THE PLACENTAL member of the family of glycoprotein hormones, which includes the pituitary hormones lutropin, follitropin, and TSH, all heterodimeric proteins with an identical {alpha}-subunit and a unique ß-subunit. CG is produced by trophoblast cells at maximal levels in the first trimester and declines thereafter to barely detectable levels at term. Trophoblast differentiation is associated with activation of the CGß and {alpha}-subunit genes; cytotrophoblasts expressing the {alpha}-subunit gene differentiate to multinucleated intermediates, which also express CGß, then further differentiate into a syncytium (1, 2, 3, 4, 5, 6, 7).

CGß is encoded by a cluster of six genes, presumably duplicated from an ancestral LHß-subunit gene (8, 9, 10). Little information is known regarding the specific proteins or DNA elements involved with its transcriptional activation, although general regulatory regions have been identified through use of human choriocarcinoma cell lines (11, 12, 13). Because CG is present only in equids and primates, the lack of a suitable animal model has hindered the study of gestational changes in CGß expression and the role of trophoblast differentiation in regulating CGß synthesis in vivo. To address this issue, we constructed transgenic mice containing the CGß gene cluster (Fig. 1Go). Previously, we reported that these mice express CGß messenger RNA (mRNA) in placenta with the same relative abundance of the transcripts as in the human (ß5 > ß3,ß8 >> ß7,ß1,ß2) and the apparent molecular weight equivalent to that seen in human trophoblasts (14). In contrast to the human placenta (where CGß synthesis is high early in gestation), in the transgenic mouse, it is synthesized only in the last third of pregnancy, from day 14 until term (day 19).



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  		Figure 1. Map of the CGß gene cluster and ßcos cosmid boundaries. Number assignments are those of Boorstein et al. (44 ).

 
Like their human counterparts, rodent trophoblasts can form syncytium, but they also differentiate to mononucleated spongiotrophoblast, or into large polyploid cells known as trophoblast giant cells (Fig. 2Go). To determine the cell type(s) responsible for CGß synthesis in the mouse and to compare the differentiation events associated with mouse and human trophoblast development, we performed imunohistochemistry and in situ hybridization on sections of mouse placentas. We anticipated that one or more of these trophoblast types would likely express the transgene. However, we find that CGß is not detected in trophoblasts but rather in another extraembryonic tissue, the parietal endoderm. Our results identify a distinct population of parietal endoderm adjacent to the chorioallantoic placenta, which may have an alternative role from the parietal endoderm in the yolk sac placenta. The data demonstrate that, with respect to supporting CGß synthesis, rodent and human trophoblasts are not the same.



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  		Figure 2. Diagrams of mouse extraembryonic membranes. A, Cross-section of term mouse extraembryonic tissues. By term, the parietal endoderm has been resorbed on the side opposite the embryo and the visceral yolk sac is exposed to the uterine lumen. Parietal endoderm remains covering the placenta and as a remnant at the placental edge. B, Schematic of the chorioallantoic placenta in A. The two layers of the placenta are illustrated: the junctional zone containing trophoblast giant cells (larger cells) and cytotrophoblast (smaller); and the labyrinthine zone with an inset showing the trilaminar barrier between maternal and fetal blood, containing layers of trophoblast giant cells, syncytiotrophoblast, and cytotrophoblast.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation
Construction of mice transgenic for the human CGß gene cluster has previously been described (14). Hemizygous transgenic ICR male mice were mated with nontransgenic ICR females, which were then killed by cervical dislocation on day 18 of pregnancy (the appearance of a vaginal plug marking day 0). The uterus was removed, and placentae were separated from embryos. Uterine tissue covering the placenta was included to ensure all of the junctional layer of the placenta was retained. DNA was extracted from embryos to determine which animals contained the transgenes, as previously described (14). Placentae designated for immunohistochemistry were fixed in Bouin’s solution (0.9% picric acid, 9% formaldehyde, 5% acetic acid) for 6 h, followed by dehydration in 50% ethanol and storage in 70% ethanol until paraffin embedding. Placentae retained for in situ hybridization were placed in OCT embedding medium (Tissue-Tek, Torrance, CA) in molds and frozen over liquid nitrogen vapor. Experimental protocols for mouse experiments were approved by the Washington University Animal Studies Committee. Human placental tissue was obtained from archival surgical pathology clinical specimens, in accordance with Washington University’s Human Studies Committee guidelines.

Immunohistochemistry
Tissue was embedded in paraffin and sectioned on a microtome at a thickness of 7 µm. The slides were prepared for immunofluorescence as previously described (15). Briefly, slides were deparaffinized with xylene, rehydrated in ethanol, and transferred to water. Sample slides were stained with hematoxylin and eosin (H&E) to examine morphology. The remaining slides were used for immunofluorescence and were successively soaked 5 min each in water, PBS (8.0 g/liter sodium chloride, 0.2 g/liter potassium chloride, 1.14 g/liter sodium monophosphate, 0.2 g/liter potassium diphosphate, pH 7.2), and blocking buffer [PBS containing 1% BSA, 0.2% nonfat powered milk, 0.3% Triton X-100 (Sigma, St. Louis, MO)]. Slides were incubated overnight at 4 C, with primary antiserum diluted 1:1200 in blocking buffer. The sera used were normal rabbit serum, and antisera raised in rabbits against CGß (see Ref. 18), mouse placental lactogen 2 (PL2; see Ref. 20), and the human common glycoprotein {alpha}-subunit . The slides were washed three times in PBS 5 min each and incubated for 1 h at room temperature with Cy3-conjugated affinity-purified donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:400 in blocking buffer. The slides were washed three times in PBS 5 min each and mounted with 1:1 PBS/glycerol. The specimens were examined under brightfield (H&E-stained sections) or fluorescence (immunofluorescent sections) and photographed with Fujichrome 1600D film (Fuji Film Co., Paramus, NJ).

In situ hybridization
Tissue was sectioned on a cryostat at a thickness of 7 µm and fixed in 4% paraformaldehyde as described (16), except that protease K treatment was omitted. Hybridization was performed with [35S]-labeled probes including sense and antisense RNAs from pGEM vectors containing complementary DNA (cDNA) encoding CGß (14), mouse PL2 (see Ref. 21), or laminin A (17). The labeled probes were first resolved on a 5% polyacrylamide gel to ensure they were of correct size, and 500,000 cpm of probe was added to each slide. Hybridization and washing conditions were as described (8, 16). Air-dried slides were coated with Eastman Kodak Co. NTB-2 emulsion, exposed at 4 C for 3–8 days, developed, and stained with H&E. The slides were examined under the microscope with brightfield and darkfield condensers and photographed with Fujichrome 1600D film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the location of the CGß-subunit , we performed fluorescent immunohistochemistry on sections of mouse transgenic placentae obtained on day 18 of gestation, 1 day before term. CGß antiserum (18), applied to first-trimester human placenta, stained the outer syncytiotrophoblast (in Fig. 3Go, A and B, are H&E staining and fluoresence, respectively). No labeling was seen in the interior layer of cytotrophoblasts or fibrovascular connective tissue cores. In the mouse, CGß antiserum detected a discrete set of strongly stained cells (Fig. 4Go). Panel A shows the section stained by H&E, with the edge of the chorioallantoic placenta covered by the single cell layer of parietal endoderm (p), an extraembryonic tissue that forms part of the yolk sac and is distinct from trophoblast (Fig. 2Go). In B, only this layer of parietal endoderm was immunoreactive, bending around the edge of the placenta and continuing in the remnant of periplacental yolk sac. C shows this relationship more clearly by superimposing the immunofluorescence data in red, over the H&E brightfield image in grayscale. No other cells were immunoreactive, including the spongiotrophoblasts and trophoblast giant cells (t) of the chorioallantoic placenta. The visceral endoderm (v), morphologically distinct but closely related embryologically to the parietal endoderm, also lacked a detectable signal (B). Not all parietal endoderm cells visible on routine sections were immunofluorescent; some morphologically identical parietal endoderm cells were unreactive. As expected, no signals were seen in placenta of nontransgenic mice (Fig. 4Go, D and E) or when either nonimmune rabbit serum or antiserum to the human glycoprotein common {alpha}-subunit were substituted for CGß antiserum (data not shown). This analysis was repeated for two other independent lines of mice expressing the CGß transgenes, and we obtained the same results (data not shown), demonstrating that the CGß expression pattern is not the result of transcriptional activation at a particular chromosomal insertion site.



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  		Figure 3. Immunohistochemical distribution of the CGß-subunit in first-trimester human placenta. The regions corresponding to syncytiotrophoblasts (s) and maternal lumen (m) are indicated. A and B correspond to brightfield and fluorescence photomicrographs, respectively, of sections treated with CGß antiserum.

 


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  		Figure 4. Cellular localization of the CGß-subunit in transgenic placenta. Day-18 placentae were analyzed immunohistochemically using polyclonal antiserum to the CGß-subunit (A–E) or to mPL2 (F and G). The sections were examined by brightfield (A, D, and F) and immunofluorescence (B, E, and G). A and B, Transgenic placenta; D and E, nontransgenic placenta; F and G, transgenic placenta with antiserum to mPL2. C is a composite of A and B, and it displays the immunofluorescence in red over the brightfield image in grayscale. Parietal endoderm (p), visceral endoderm (v), and trophoblast giant cells (t) are shown (magnification 125x).

 
To address whether the lack of CGß immunostaining in trophoblasts was attributable to protein degradation caused by autolysis, placental sections were stained for mouse PL2 (Fig. 4Go, F and G). The placental lactogens are a family of well-characterized protein hormones similar to GH, which are secreted from rodent trophoblast at different times in pregnancy (19). PL2 is expressed at high levels from the trophoblast giant cells beginning at midgestation (20, 21). Antiserum to PL2 gave the expected pattern of immunostaining of trophoblast giant cells (Fig. 4Go, F and G), with no staining of the parietal endoderm. The data demonstrate that the endogenously-synthesized PL2 can be visualized in these trophoblast sections, and absence of CGß immunostaining is not caused by tissue degradation.

Our immunofluorescence experiments do not exclude movement of already-synthesized protein from other cells to those of the parietal endoderm. For example, CGß might originate in the trophoblast giant cells but be transported to the parietal endoderm. To identify the cells in which the CGß genes were transcriptionally active, we used in situ hybridization to visualize the CGß mRNA (Fig. 5Go). In agreement with the immunofluorescence experiments, only parietal endoderm hybridized with the antisense RNA probe to CGß (Fig. 5Go, A and B). As expected, hybridization was not seen in sections of transgenic placenta hybridized with a sense CGß probe (Fig. 5Go, C and D) or in nontransgenic placenta hybridized with this CGß antisense probe (Fig. 5Go, E and F). Only a subset of parietal endoderm cells hybridized with the CGß probe, consistent with the immunohistochemical data. The population of CGß- and non-CGß-producing parietal endoderm are morphologically identical. That the trophoblasts are intact is shown by the hybridization observed with the PL2 antisense probe (22) in spongiotrophoblasts and the trophoblast giant cells (Fig. 5Go, G and H). The pattern was identical to that seen with immunohistochemical analysis, and no PL2 signal was detected in parietal endoderm cells.



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  		Figure 5. In situ hybridization of CGß and PL2 mRNAs in sections of mouse placenta. Sections of transgenic placentae were hybridized with an antisense cDNA probe to CGß mRNA (A and B), sense cDNA of CGß (C and D), or an antisense probe to mPL2 mRNA (G and H). E and F show hybridization of antisense CGß probe to a nontransgenic placental section. A, C, E, and G; and B, D, F, and H are paired brightfield and darkfield photomicrographs, respectively, of the same regions.

 
The parietal endoderm secretes several extracellular matrix proteins that comprise Reichert’s membrane, a structure forming a semipermeable barrier between the parietal endoderm and a more distal layer of trophoblast. One of the proteins in Reichert’s membrane is the heterotrimeric complex laminin. The laminin A subunit is specific for parietal endoderm, and is present at much higher levels than in other extraembryonic tissues (23). To obtain further evidence that CGß is expressed in parietal endoderm, sequential sections were hybridized with an antisense probe to laminin A (Fig. 6Go). The H&E brightfield (A) shows a layer of parietal endoderm stretching from the lower left to the upper right, as it overlies the placental trophoblast in the upper left. Visceral endoderm is present in the lower right. The in situ hybridization CGß and laminin A mRNAs are highlighted in the darkfield micrographs (B and C) and show expression in the same single layer of cells. No detectable signal was seen using a sense probe to laminin A (data not shown).



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  		Figure 6. Localization of laminin mRNA in parietal endoderm. Two separate experiments are shown in A–C and D–G. The first experiment depicts a single area of a transgenic placenta probed with antisense cDNAs to laminin A [lam A; brightfield (A) and darkfield (B)] and to CGß cDNA [darkfield (C); 125x]. The second experiment, shown under lower magnification, compares regions of parietal endoderm that display complimentary signals of CGß and laminin mRNAs. D and E, CGß; F and G, laminin. Bars indicate the regions of high CGß/low laminin A expression (bent bar) and low CGß/high lamanin A expression (straight bar) (62x).

 
Although laminin A was found in the same cell layer as CGß, our experiments identified an unexpected relationship between their expression: The cells which hybridized most strongly to the CGß probe hybridized weakly with laminin A, and vice versa (Fig. 6Go, D–G). This is demonstrated under lower magnification showing approximately half the placenta and its endodermal covering. E shows regions of parietal endoderm that hybridize to the CGß probe (bent bar); note that, as the parietal endoderm continues along the edge of the placenta, no signal is detected for CGß (straight bar). G shows the same placenta hybridized to the laminin A antisense probe. The less intense laminin A hybridization corresponds to the CGß-positive region (bent bar), whereas there is more intense hybridization in the zone lacking CGß mRNA (straight bar). Morphologically, the two sets of parietal endoderm cells are indistinguishable. This relationship between CGß and laminin A was seen in the other two independent lines of transgenic mice in all experiments. The hybridization pattern of laminin A is not the result of the transgenic construction; a similar laminin A hybridization profile, i.e. regions of greater and lesser signal, was observed in nontransgenic mice (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A key finding in this study is the unexpected expression of the human CGß gene cluster in rodent parietal endoderm but not in trophoblast. The transcriptional pattern of the CGß genes is comparable with that seen in human trophoblasts. Moreover, the expression occurred in a subset of parietal endoderm cells. These observations raise two issues: 1) the functional comparison between rodent and primate placenta; and 2) within the rodent conceptus, the relationship between parietal endoderm and trophoblast. Murine and primate placenta share several features, including the contact of maternal blood with fetal trophoblast. There is also a significant difference between the rodent and primate systems. The primate placenta is referred to as chorioallantoic; chorio refers to the interaction of trophoblast with maternal endometrium, and allantoic derives from the allantois, a structure which provides the blood vessels for the placenta. Rodents also have a chorioallantoic placenta but, in addition, possess a yolk sac placenta (Fig. 2Go) (24), which serves as the major fetal-maternal route before the formation of the chorioallantoic placenta (25, 26). It consists of visceral and parietal yolk sacs, the latter of which is composed of the parietal endoderm layer (source for a thick extracellular matrix, Reichert’s membrane) and a sheet of trophoblast giant cells. By term, the parietal endoderm has been resorbed, except for a portion covering the chorioallantoic placenta (27, 28). Parietal endoderm and trophoblasts have a separate embryological derivation. Trophoblastic cells arise from trophectoderm, whereas both parietal and visceral endoderm are derived from primitive endoderm (24, 29) which, in turn, is derived from the inner cell mass. While the trophectoderm and parietal endoderm arise from distinct lineages, studies in rodents and primates indicate that trophectoderm and parietal endoderm cells interact. Parietal endoderm cells differentiate while they are in contact with trophectoderm (30, 31). In the rodent, there is a paracrine relationship between parietal endoderm and trophoblasts, e.g. PTH-related peptide is elaborated by the trophectoderm and induces differentiation of parietal endoderm (32, 33). Summers et al. (34) have observed that initiation of CG synthesis in the marmoset monkey coincides with the appearance of parietal endoderm and suggest that the inner cell mass/parietal endoderm is critical for marmoset CG synthesis. In this regard, it has also been proposed that human CG has a trophic action on differentiating trophoblasts in human placenta (35, 36, 37). An additional developmental link between the two tissues is that both trophoblast and parietal endoderm undergo nonrandom X chromosome inactivation, implying that similar mechanisms govern transcriptional regulation in these tissues (38, 39). Taken together, these experiments suggest that there exists a developmental interaction between inner cell mass and trophoblast derivatives and that the parietal endoderm and trophoblast share a cooperative functional association.

Several observations imply that the expression of the CGß genes in parietal endoderm and lack thereof in trophoblasts is not related to the transgenic DNA used. First, the six CGß genes were transcribed at the same ratios in the transgenic mouse, as seen in the human placenta in vivo (8). Second, synthesis is restricted to a particular placental cell type and gestational period (14); and third, expression was seen in three different transgenic lines, indicating that transcription of the CGß cluster in parietal endoderm did not result from a particular transgenic integration site. Previously, we reported that two members of the CGß gene cluster that were silent in mouse and human placenta were ectopically expressed in transgenic mouse brain (14). Thus, only the genes transcribed in human placenta are expressed in rodent parietal endoderm. We cannot exclude the possibility that the 36-kb construct used here lacks necessary transcriptional elements required for trophoblast-specific expression, e.g. a sequence analogous to the locus control region of the globin multigene cluster (40), which is located many kilobases from the structural genes themselves. However, much smaller subclones of the CGß construct are sufficient for correct expression in human choriocarcinoma trophoblast cell lines, in which the gene is transcriptionally activated as the cells differentiate (7, 41). Moreover, the common glycoprotein {alpha}-subunit of the mouse is expressed only in the pituitary and not in the placenta. However, a reporter gene driven by the human {alpha}-promoter was active in the placenta of transgene mice (42), but the cell type was not identified. Taken together, these data show that synthesis of the CG subunits in transgenic mice is tissue specific.

Although gross morphology differs between human and rodent placenta, there is no reason a priori to assume that the corresponding trophoblasts would differ functionally, because such cells perform similar transport and endocrine functions. Consistent with this hypothesis, the human aromatase P450 gene, normally synthesized in several tissues including syncytiotrophoblast (unlike the mouse gene, which is not expressed in placenta), was produced in the labyrinthine trophoblast in transgenic mice (43). However, the absence of a transcriptional system that supports CGß synthesis in rodent trophoblasts indicates a lack of equivalence among the species for transcribing this gene. The data are consistent with the hypothesis that, in human placenta, one or more transcriptional factors coevolved as the distinct members of the CGß gene cluster underwent duplication (44).

Our observation that CGß and laminin are expressed in subsets of parietal endoderm cells provides further evidence for heterogeneity of parietal endoderm cells (31). The cell populations show reciprocal expression, i.e. cells expressing more CGß synthesize less laminin, and vice versa. Intriguingly, it is the region of parietal endoderm remaining at term, after the bulk has been resorbed, that produces CGß. We suggest that two populations of PE can be distinguished: one that includes the yolk sac and lays down Reichert’s membrane, and another associated with the expression of the CGß transgene.

In summary, we find that mouse and human trophoblasts are not equivalent in transcriptional regulation, despite having extensive functional similarities, and that analogous trophoblast functions may be performed by parietal endoderm cells in the mouse. The results also reveal a strategy for investigating the functions of parietal endoderm, about which little is known other than as a source of Reicherts membrane. By expressing marker proteins or toxins specifically in that portion of parietal endoderm expressing CGß, it should be possible to identify and follow the fate of these cells in vivo.


   Acknowledgments
 
The authors thank Dr. Kevin Roth, Elvie Taylor, and Bill Coleman of the Washington University Histology Core Facility for their advice and services. The authors also thank Drs. Frank Talamantes for providing antiserum to PL2, Daniel Linzer for PL2 cDNA, and Yoshihiko Yamada for laminin A cDNA. We are grateful to Drs. John Nilson and Robin Pittman for their helpful comments and discussion throughout the course of this investigation, and to Drs. Jeffrey Ross and Mark Boothby for their critical comments regarding the manuscript. We thank Ms. Mary Wingate for her excellent assistance in preparing the manuscript.

Received August 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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