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BOR - Papers in Press, published online ahead of print September 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061382
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BIOLOGY OF REPRODUCTION 77, 923–933 (2007)
DOI: 10.1095/biolreprod.107.061382
© 2007 by the Society for the Study of Reproduction, Inc.

Developmental Changes in Extracellular Matrix Messenger RNAs in the Mouse Placenta During the Second Half of Pregnancy: Possible Factors Involved in the Regulation of Placental Extracellular Matrix Expression1

Koji Y Arai  2 and Toshio Nishiyama 

Division of Matrix Biology, Scleroprotein Research Institute, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

ABSTRACT

Expression of procollagens (Col1a1/2, Col3a1, Col4a1/2, Col5a1/2) and fibronectin 1 (Fn1) in the mouse fetal placental tissue was examined during the second half of pregnancy. Ribonuclease protection assays (RPAs) revealed that levels of these mRNAs noticeably increased between Days 10 and 14 of pregnancy, and they remained at relatively constant levels thereafter. In situ hyridization showed that Col1a1 and Col4a1 mainly localized in the labyrinth, whereas Fn1 was expressed mainly in the spongiotrophoblast. Since members of the transforming growth factor-beta (TGFB) superfamily are involved in the regulation of extracellular matrix (ECM) expression in various tissues, mRNA levels of TGFB family members and their binding proteins were also examined by RPAs. Transforming growth factor-beta1–3 (Tgfb1–3), activin subunits (Inhba, Inhbb), follistatin (Fst), and follistatin-like 3 (Fstl3) were expressed in the placenta, whereas significant expression of myostatin (Mstn) was not detected. Although the expression patterns of Tgfb1–3 and Inhba in the placenta suggest possible involvement of TGFBs and activin A in the regulation of placental ECM expression, neither TGFBs nor activin A affected ECM mRNA levels in vitro. On the other hand, hypoxia significantly decreased Col1a1/2 and Col4a1/2 mRNAs in cultured placental cells, and a high-glucose condition significantly increased Col1a1 and Col3a1 mRNAs. Fn1 expression was increased under the high-glucose condition, although hypoxia also increased Fn1 expression to a lesser degree. These data suggest that an increase in oxygen tension and nutrient supply during placentation rather than TGFB family members may be responsible for the increase in the placental ECM mRNA expression.

deciduas, extracellular matrix, glucose, oxygen, placenta, pregnancy, TGFB family

INTRODUCTION

The placenta is an important mammalian reproductive organ responsible for fetal nutrition, respiration, and waste removal. During pregnancy, an increase in the functional capacity of the placenta (i.e., the ability to exchange materials between mother and fetus) is needed, because the supply of nutrients and oxygen across the placenta must meet the increasing requirement of the developing fetus.

The placenta is abundant in diverse extracellular matrices, such as fibril collagens [1, 2], type IV collagen [3], and fibronectin [4, 5]. An early study demonstrated that collagen concentrations (hydroxyproline concentrations) in the rat placenta increased during the last one third of pregnancy [1]. A recent study revealed that the overall organization of the placenta was retained in mouse embryos lacking type IV collagen genes (Col4a1/2), but an impaired development of the labyrinth layer was observed at Embryonic Day 11.5 (E11.5) [6]. These observations suggest that placental development is closely associated with extracellular matrix (ECM) production in addition to proliferation of placental cells. However, little is known about the developmental changes in placental ECM mRNA expression. In the present study, expression patterns of fibronectin, fibril collagens, and type IV collagen during the second half of mouse pregnancy were examined. Fibronectin is an important molecule for cell adhesion and migration. Previous studies have indicated that fibronectin is involved in the regulation of implantation and placental organization [7–10]. Fibril collagens are thought to be responsible for the mechanical strength of the tissue. Major fibril collagens contained in the placenta are type I, type III, and type V collagens [2]. The components of these fibril collagens are two type I alpha1 and one type I alpha2 chains for type I collagen, three type III alpha1 chains for type III collagen, two type V alpha1 and one type V alpha2 chains or each one of type V alpha1, type V alpha2, and type V alpha3 chains for type V collagen [11]. Type IV collagen forms a mesh and is a very important component of the basement membrane [11], which exists between the endothelial cells of fetal capillaries and a trilaminar layer of trophoblast cells in the labyrinthine zone [12–14]. Placental type IV collagen is mainly composed of type IV alpha1 and type IV alpha2 chains [3].

Expression of some ECM proteins, such as type I procollagens and fibronectin, is stimulated by transforming growth factor-betas (TGFBs) in many cell types and tissues [15]. The effects of TGFBs are, at least in part, mediated through Smad2, Smad3, and Smad4 [15, 16], and some members of the TGFB family, such as activins and myostatin (MSTN), also activate intracellular signaling pathways mediated by these Smads [17], suggesting that activins and MSTN are able to stimulate ECM expression. Previous studies revealed that TGFBs, activins, and MSTN are expressed in the placenta [18, 19]. These accumulating evidences suggest that the members of the TGFB family expressed in the placenta may be involved in the regulation of placental ECM production. In addition to the TGFB family, TGFB family-binding proteins follistatin (Fst) and follistatin-like3 (Fstl3, also known as follistatin-related gene [FLRG]) are expressed in the placenta and the decidua [20–22]. Activins and MSTN are identified as ligands of FST and FSTL3 [23]. Therefore, expression patterns of members of the TGFB family and their binding proteins, as well as the in vitro effects of TGFBs and activin A on ECM expression were also examined to elucidate roles of TGFB family members in the regulation of placental ECM production.

It is well known that placental gene expression is regulated by oxygen tension [24, 25]. Before establishing the maternal-fetal interface, the placenta is under a hypoxic environment. This hypoxic condition is required for normal placental gene expression [24, 25]. In the mouse placenta, the development of the labyrinth begins at 8.75 days postcoitum [26]. A previous study reported a striking increase in the diameter of the spiral arteries and the thickness of the labyrinth in the mouse placenta between E11.5 and E14.5 [27], indicating that a considerable increase in placental oxygen concentrations occurs between these periods. Studies on the human placenta demonstrated that ECM expression in placental fibroblasts is regulated by oxygen concentrations in vitro [28, 29], suggesting that oxygen tension is involved in the regulation of placental ECM expression in vivo. In addition to oxygen tension, nutrient supply probably increases as the labyrinthine layer develops. To elucidate possible involvement of oxygen tension and nutrient supply in the regulation of placental ECM expression, the in vitro effects of oxygen tension and glucose concentrations on placental cells also were examined.

MATERIALS AND METHODS

Animals

Virgin female mice of the C57BL/6J strain (7–9 wk old) were used. They were kept under a 12L:12D photoperiod (lights on at 0800 h) at 22°C–26°C. Food and water were available ad libitum. Each female was transferred to the cage of a single mature male and left until a vaginal plug was found. The day on which a vaginal plug was detected in the morning was designated as Day 0 of pregnancy. The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals prepared at the Tokyo University of Agriculture and Technology.

Tissue Samples

Mice were killed by cervical dislocation under ether anesthesia on Days 10, 12, 14, 16, and 18 of pregnancy. Placentae were collected, and the decidua basalis was separated from the fetal placental tissue with forceps. Because complete separation of the decidua basalis from the fetal placental tissue was difficult after Day 14, the fetal placental tissues obtained after Day 14 were probably contaminated with some decidual tissues. At each point, two or three placentae were collected from one mother, and the fetal placental tissues obtained from four pregnant mice were weighed after washing with PBS. For isolation of total RNA, the fetal placental tissues and the decidual tissues were collected from three pregnant mice at each point and quickly frozen in liquid nitrogen. The decidual tissues obtained from two or three implantation sites were pooled before freezing. One or two fetal placental tissues and one or two pools of the decidual tissues were collected from one pregnant mouse. The tissues were stored at –80°C until isolation of total RNA. Total RNA was prepared with TRIzol reagent (Invitrogen Co., Carlsbad, CA) according to the manufacturer's protocol.

Culture of Dispersed Placental Cells

Pregnant mice were killed by cervical dislocation under ether anesthesia on Day 12 of pregnancy. The fetal placental tissues were collected, washed twice with PBS, and minced with a sterilized razor on a Petri dish. The minced placental tissues were collected in a disposable plastic centrifuge tube with PBS. The tube was left until the placental tissues settled to the bottom of the tube. After careful removal of the supernatant with a pipette, a cell-dispersing solution (0.1% calcium chloride, 0.1% magnesium chloride, 0.1% BSA, 14 mg/ml collagenase [Type IA; Sigma-Aldrich Japan K.K., Tokyo, Japan], and 10 µg/ml DNase I [Sigma-Aldrich] in PBS) was added. The placental tissues were incubated in the solution with shaking at 37°C for 30 min. The cells were further dispersed by pipetting after the incubation and were collected by centrifugation at 500 x g for 10 min. After removing the supernatant, the cells were suspended in Dulbecco modified Eagle medium (DMEM)-high glucose (4500 mg/l; Sigma-Aldrich) containing 10% fetal calf serum (FCS). When the effects of glucose concentrations were examined, DMEM-low glucose (1000 mg/l; Sigma-Aldrich) was used instead of the DMEM-high glucose. The placental cells obtained from two or three placentae (3–4 x 104 viable cells) were plated on a 6-cm diameter culture dish and incubated overnight at 37°C under a humidified condition of 95% air and 5% CO2 to let the cells attach to the dish.

Effects of TGFBs and Activin A on ECM Expression in Cultured Placental Cells

After the cells had attached to the dish, medium was removed and replaced with DMEM-high glucose containing 0.1% FCS after washing twice with PBS. The placental cells then were incubated for 24 h in either the presence or absence of growth factors. Human recombinant TGFB1 and TGFB3 were purchased from Sigma-Aldrich. Recombinant activin A was prepared as previously described [30]. Final concentrations of the growth factors were 10 ng/ml for TGFBs and 20 ng/ml for activin A. After the incubation, total RNA was isolated from the cells with TRIzol reagent.

Effects of Hypoxia on ECM Expression in Cultured Placental Cells

After the cells had attached to the dish, medium was removed and replaced with DMEM-high glucose containing 10% FCS after washing twice with PBS. The culture dishes were placed in a 2-liter volume airtight jar (Mitsubishi Gas Chemical Co., Tokyo, Japan) in the presence of Anaeropack for Cell (Mitsubishi Gas Chemical) at 37°C. This device achieves a severe hypoxic condition (<1% O2) within 1 h and maintains about 5% CO2 [31]. Control dishes were incubated at 37°C under a humidified condition of 95% air and 5% CO2. After 24 h of incubation, total RNA was isolated from the cells with TRIzol reagent.

Effects of Glucose Concentrations on ECM Expression in Cultured Placental Cells

After the cells had attached to the dish, medium was removed and replaced with DMEM-low glucose containing 10% FCS after washing twice with PBS. The cells were incubated under either 1000 mg/l glucose or 4500 mg/l glucose conditions for 48 h. After the incubation, total RNA was isolated from the cells with TRIzol reagent.

Ribonuclease Protection Assays

The templates of the complementary RNA probes were prepared by RT-PCR. Complementary DNAs for procollagens (type I alpha1 [Col1a1], type I alpha2 [Col1a2], type III alpha1 [Col3a1], type IV alpha1 [Col4a1], type IV alpha2 [Col4a2], type V alpha1 [Col5a1], and type V alpha2 [Col5a2]), fibronectin 1 (Fn1), Tgfb1, Tgfb2, Tgfb3, Inhba, Inhbb, Mstn, Fst, and Fstl3 were prepared. The primers used are listed in Table 1. The PCR products were subcloned into pGEM-T easy vector (Promega K.K. Japan, Tokyo, Japan). The cDNAs for Col1a2, Col4a2, Fn1, Tgfb2, and Tgfb3 were cut out from the pGEM-T easy vector with EcoRI and subcloned into the EcoRI site of pBluescript II SK(+) vector. The Col5a2 cDNA was cut out from the pGEM-T easy vector with EcoRI and AatI, and the 236-base pair fragment was subcloned between the restriction sites EcoRI and EcoRV of pBluescript II SK(+) vector. Virus promoters of the plasmids were used for synthesizing the RNA probes. The plasmids containing the cDNA fragments were linearized by cutting appropriate restriction sites located in the multiple cloning sites of the plasmids, except for the Inhba and Inhbb cDNAs. The plasmids containing the Inhba and Inhbb cDNAs were linearized with BamHI and PvuII, respectively. These restriction enzymes cut inside the cDNAs and generate a 300-bp cDNA for Inhba and a 240-bp cDNA for Inhbb. As the internal standard control, beta-actin (Actb) mRNA was used. The template for the Actb probe containing a 250-bp mouse Actb cDNA was purchased from Applied Biosystems (Foster City, CA). The digoxigenin (DIG)-labeled complementary RNA probes were synthesized with SP6, T3, or T7 RNA polymerase in the presence of DIG-RNA labeling mix (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's protocol. Ribonuclease protection assays using the DIG-labeled RNA probes were performed as previously described [32]. Briefly, the DIG-labeled probes (1 ng each) were ethanol precipitated with the samples, hybridized overnight at 45°C with an exception for Tgfb1 at 50°C, and digested with an RNase A and T1 mixture. After inactivation of the RNases by proteinase K treatment, the protected probes were separated with an 8M-urea denaturating polyacrylamide gel and electrotransferred to a nylon membrane (nylon membrane, positive charge; Roche Diagnostics). The signals were detected with an alkaline phosphatase-labeled anti-DIG antibody (Roche Diagnostics), disodium 3-(4-methoxyspiro {I,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate (Roche Diagnostics), and an X-ray film (RX-U; Fujifilm, Tokyo, Japan), and they were analyzed with image-analyzing software Image J. Amounts of total RNA used for the assays were 2.5 µg for Actb and Fn1, 5 µg for Col4a1 and Col4a2, 10 µg for Col1a1, Col1a2, and Col3a1, and 15 µg for all the others.


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  		TABLE 1 PCR primers used to obtain cDNAs for mouse extracellular matrices, TGFB family members, and FST family members.

In Situ Hybridization for ECM mRNAs in the Placenta

The placentae obtained from Days 12, 14, and 16 were fixed in freshly prepared 4% (w/v) paraformaldehyde (PFA; Wako) in PBS overnight at 4°C and embedded in paraffin. The paraffin-embedded placentae were sectioned at 6-µm thickness and placed on glass slides coated with 3-aminopropyltriethoxysilane. The sections were deparaffined with xylene, rehydrated with a graded series of ethanol, and washed twice with PBS for 5 min each. Thereafter, the sections were treated with 2.5 µg/ml proteinase K in PBS for 10 min and 0.2% (w/v) glycine in PBS for 10 min, washed twice with PBS for 5 min each, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 20 min. After washing with 4x saline sodium citrate (SSC) twice for 10 min each, the sections were prehybridized in prehybridization buffer (50% deionized formamide, 2x SSC, 10 mM Tris–HCl [pH 7.5], 1x Denhardt solution, and 2 mg/ml yeast RNA) for 30 min at 42°C. The DIG-labeled complementary RNA probes (200 ng/ml) diluted in hybridization buffer (50% deionized formamide, 2x SSC, 10 mM Tris-HCl [pH 7.5], 1x Denhardt solution, 2 mg/ml yeast RNA, and 10% dextran sulfate) were denatured for 5 min at 80°C and hybridized overnight at 42°C under coverslips in a humidified box. After hybridization, the sections were washed in 2x SSC at 60°C three times for 20 min each, treated with RNase solution (10 mM Tris-HCl [pH 8.0], 0.5 M NaCl, and 20 µg/ml RNaseA) for 30 min at 37°C, and washed again with 0.1x SSC at 50°C three times for 20 min each. Before and after the treatment with RNaseA, the sections were washed with RNase diluent for 5 min. Thereafter, the cRNA probes were visualized with an alkaline phosphatase-conjugated anti-DIG antibody (Roche), nitroblue tetrazolium salt (Roche), and 5-bromo-4-chloro-3-indolyl phosphate (Roche) according to the procedure recommended by Roche Diagnostics. For negative controls, sense strand RNA probes were used instead of the cRNA probes.

Immunohistochemistry for Cytokeratin and In Situ Hybridization for S100a4 in Cultured Placental Cells

To examine the proportion of trophoblasts and other cells in the primary placental cell culture, the cells were stained with an anti-pancytokeratin rabbit polyclonal antibody (Histofine SAB-PO(R) kit, keratin/cytokeratin; Nichirei Bioscience, Tokyo, Japan) and an anti-rabbit IgG antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The dispersed placental cells were seeded on a cover glass (24 x 24 mm) in a six-well culture dish. The treatments described above, except for growth factor treatments, were carried out. After the treatments, the cells were washed twice with PBS and fixed in ice-cold acetone for 5 min. Thereafter, the fixed cells were treated with 0.3% H2O2 in methanol. After washing with PBS twice, the cover glass was blocked with 1% BSA-PBS for 20 min, then incubated with the prediluted primary antibody for 2 h and the secondary antibody (1:500 diluted) for 30 min. The cover glass was washed twice with PBS after the each antibody incubation. The signals were visualized with Vector NovaRED substrate kit (Vector Laboratories Inc., Burlingame. CA). The cells were counterstained with hematoxylin. The numbers of cytokeratin-positive and cytokeratin-negative cells were counted using a light microscope. Several fields were randomly examined until a total number of 500–1000 cells were counted for the each cover glass. Three cover glasses were examined for each treatment. To identify fibroblasts in the cultured cells, the cells on the cover glasses were fixed in 4% PFA-PBS for 15 min on ice, and in situ hybridization for the fibroblast marker S100a4 (also known as fibroblast-specific protein-1) was performed as described in the previous section. The primers used to obtain the template S100a4 cDNA for the cRNA probe were 5'-GGCAAAGAGGGTGACAAG-3' and 5'-TGTGCGAAGAAGCCAGAG-3'.

Statistical Analysis

Values are represented as means ± SEM. To compare more than three mean values, results were subjected to one-way analysis of variance, followed by Duncan multiple-range test. When comparing two mean values, either the Student t-test or the Mann-Whitney test was used. A value of P < 0.05 was considered to be significant. The correlation between parameters was determined using simple regression analysis.

RESULTS

Changes in Fetal Placental Tissue Weights and ECM Expression in the Fetal Placental Tissue During the Second Half of Mouse Pregnancy

The weights of the fetal placental tissue noticeably increased between Days 12 and 14 of pregnancy, and they remained constant thereafter (Fig. 1). Figure 2 shows changes in ECM mRNA expression in the fetal placental tissue. The levels of Col1a1 mRNA were higher than those of other fibril collagen mRNAs throughout the second half of pregnancy. The levels of Col1a1, Col1a2, and Col1a3 mRNAs noticeably increased between Days 10 and 14 of pregnancy. Thereafter, the expression of these mRNAs remained at relatively constant levels or slightly decreased until Day 18. As observed in Col1a1, Col1a2, and Col3a1 mRNAs, Col5a2 mRNA noticeably increased between Days 10 and 14, and it remained at constant levels thereafter. The signals for Col5a1 mRNA showed the lowest intensity among the fibril collagen mRNAs. The expression of Col5a1 mRNA continuously increased during the second half of pregnancy, although the change was not statistically significant. Type IV collagen showed higher expression than fibril collagens. Both Col4a1 and Col4a2 mRNAs continuously increased between Days 10 and 16 of pregnancy and remained at high on Day 18. The fetal placental tissue expressed very high levels of Fn1 mRNA. The changing pattern of Fn1 mRNA was similar to those of procollagen mRNAs. Regression analysis revealed that the fetal placental tissue weights were highly correlated with the ECM mRNA levels. The correlation coefficients were 0.85 for Col1a1, 0.63 for Col1a2, 0.91 for Col3a1, 0.95 for Col4a1, 0.96 for Col4a2, 0.90 for Col5a1, and 0.95 for Col5a2.


Figure 01
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  		FIG. 1 Developmental changes in the weights of the fetal placental tissue during the second half of mouse pregnancy. Values represent means ± SEM for 8–12 placentae obtained from four pregnant mice. Values with the same letters represent the minimum difference between values required for the points to be significantly different (P < 0.05).


Figure 02
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  		FIG. 2 A) Representative images of ribonuclease protection assays detecting Col1a1, Col1a2, Col3a1, Col4a1, Col4a2, Col5a1, Col5a2, and Fn1 mRNAs in the fetal placental tissue during the second half of mouse pregnancy. Actb mRNA was detected for the internal standard control. B) Developmental changes in levels of extracellular matrix mRNAs in the fetal placental tissue during the second half of mouse pregnancy. The signals obtained by the ribonuclease protection assays were analyzed with ImageJ, and the results are expressed as the intensity relative to Actb mRNA. Values represent means ± SEM for five placentae obtained from three pregnant mice (one or two placenta(e) per mother), except for Col4a1 and Col4a2 on Day 16 (three placentae from three mothers). Values with the same letters represent the minimum difference between values required for the points to be significantly different (P < 0.05).

Localization of Fn1, Col1a1, and Col4a1 mRNAs in the Placenta

Between Days 12 and 16, the localization patterns of Fn1, Col1a1, and Col4a1 in the placenta did not show significant changes. The representative sections from Day 12 are shown in Figure 3. Fn1 was localized mainly in the spongiotrophoblast. This distribution pattern of Fn1 is consistent with that of FN1 protein [33]. Some trophoblast cells in the labyrinthine layer also showed positive staining for Fn1 mRNA. Col1a1, a representative of fibril collagen genes, was mainly expressed in the labyrinthine zone. Weak signals were also observed in the spongiotrophoblast zone. The distribution pattern of Col4a1, a representative of basement membrane collagen genes, was similar to that of Col1a1. The distribution patterns are generally consistent with previous studies examining localization of ECM mRNAs [34] and proteins [35] in the rat, although the spongiotrophoblast showed stronger signals for Col4a1 mRNA than the labyrinth in the rat [34]. The labyrinth is the zone where fetal blood and maternal blood circulate within fetal capillaries and maternal blood spaces, respectively [12–14]. These labyrinthine interfaces consist of the endothelial cells of fetal capillaries, their associated basement membrane, and a trilaminar layer of trophoblast cells that directly lines the maternal blood spaces [12–14]. In the labyrinth, both Col1a1 and Col4a1 mRNAs were expressed in trophoblast cells. However, it was difficult to determine from the sections whether the mRNAs expressed in the endothelial cells.


Figure 03
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  		FIG. 3 Localization of Fn1 (upper), Col1a1 (middle), and Col4a1 (lower) mRNAs in the mouse placenta. Low-power (left two columns) and high-power (right two columns) views are shown. Placental sections from Day 12 of pregnancy were hybridized with DIG-labeled antisense (the first and the third columns) or sense (the second and the fourth columns) probes. The distribution patterns of the mRNAs on Days 14 and 16 were similar to those on Day 12. L, labyrinthine layer; Sp, spongiotrophoblast; Db, decidua basalis. Bars = 100 µm.

Changes in Levels of mRNAs Encoding TGFB Family Members and Their Binding Proteins in the Fetal Placental Tissue and Decidua During the Second Half of Mouse Pregnancy

The expression of Inhba mRNA in the fetal placental tissue increased as pregnancy progressed, and it was noticeably higher than that of Inhbb mRNA during late pregnancy (Fig. 4). Fstl3 mRNA expression in the mouse fetal placental tissue was relatively high on Days 10 and 12 of pregnancy, decreased between Days 12 and 16, and remained relatively low thereafter (Fig. 4). The expression of Fst mRNA in the mouse fetal placental tissue increased between Days 10 and 14 and remained at constant levels thereafter (Fig. 4). Interestingly, there were two protected bands when Tgfb1 mRNA was detected (Fig. 4A). The size of the long protected fragment (Tgfb1 L) was consistent with the expected size of the protected band (340 bases), whereas the short protected fragment (Tgfb1 S) was approximately 270 bases in length. In the fetal placental tissue, both the long and the short protected fragments for Tgfb1 mRNA increased between Days 10 and 16 and remained at relatively high levels during late pregnancy (Fig. 4). Tgfb2 mRNA remained at low levels throughout the second half of pregnancy compared with the other two isoforms (Fig. 4). Tgfb3 mRNA increased between Days 10 and 12 and gradually decreased thereafter (Fig. 4).


Figure 04
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  		FIG. 4 A) Representative images of ribonuclease protection assays detecting Inhba, Inhbb, Fstl3, Fst, Tgfb1, Tgfb2, and Tgfb3 mRNAs in fetal placental tissues during the second half of mouse pregnancy. Actb mRNA was detected for the internal standard control. In the ribonuclease assay for Tgfb1, two protected bands were detected. The long (Tgfb1 L) and the short (Tgfb1 S) fragments were approximately 340 and 270 bases in length, respectively. B) Developmental changes in levels of mRNAs encoding activin subunits, FST family proteins, and TGFBs in the fetal placental tissue during the second half of mouse pregnancy. The signals obtained by the ribonuclease protection assays were analyzed with ImageJ, and the results are expressed as the intensity relative to Actb. Values represent means ± SEM for five placentae obtained from three pregnant mice (one or two placenta(e) per mother), except for Tgfb1 (three placentae from three mothers). Values with the same letters represent the minimum difference between values required for the points to be significantly different (P < 0.05).

Decidual Inhba mRNA temporarily increased on Day 12, decreased to relatively low levels by Day 14, and remained at constant levels thereafter (Fig. 5). The expression of Inhbb mRNA in the decidua remained low on Days 10, 12, and 14. Thereafter, Inhbb mRNA increased until Day 18 (Fig. 5). During late pregnancy, there was no difference between the levels of Inhba and Inhbb mRNAs in the decidua. Fstl3 mRNA expression in the decidua was low compared with the placenta. Decidual Fstl3 mRNA decreased between Days 10 and 14 and remained at low levels until Day 18 (Fig. 5). The Fst mRNA levels decreased between Days 10 and 14. After the nadir on Day 14, Fst mRNA increased until Day 18 (Fig. 5). However, these changes in Fstl3 and Fst mRNAs in the decidua were not statistically significant. In the decidua, the intensity of the short protected fragment of Tgfb1 mRNA increased between Days 10 and 14 and remained at constant levels thereafter (Fig. 5). The expression of both Tgfb2 and Tgfb3 increased as pregnancy progressed (Fig. 5). The intensity of the long protected fragment for Tgfb1 mRNA was very faint compared with the short fragment during late pregnancy. In contrast to the short protected fragment of Tgfb1 mRNA and the other two isoforms, the intensity of the long protected fragment of Tgfb1 mRNA decreased as pregnancy progressed (Fig. 5).


Figure 05
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  		FIG. 5 A) Representative images of ribonuclease protection assays detecting Inhba, Inhbb, Fstl3, Fst, Tgfb1, Tgfb2, and Tgfb3 mRNAs in the mouse decidual tissue during the second half of pregnancy. Actb mRNA was detected for the internal standard control. In the ribonuclease assay for Tgfb1, two protected bands were detected. The long (Tgfb1 L) and the short (Tgfb1 S) fragments were approximately 340 and 270 bases in length, respectively. B) Developmental changes in levels of mRNAs encoding activin subunits, FST family proteins, and TGFBs. The signals obtained by the ribonuclease protection assays were analyzed with ImageJ, and the results are expressed as the intensity relative to Actb mRNA. Values represent means ± SEM for three decidual tissue pools obtained from three pregnant mice. Values with the same letters represent the minimum difference between values required for the points to be significantly different (P < 0.05). *Significant compared with other stages (P < 0.05). L, long; S, short.

The ribonuclease protection assay for Mstn detected a solid signal in the skeletal muscle (see supplemental Fig. 1, available at www.biolreprod.org ). However, a significant expression of Mstn was not detected in the fetal placental tissue and decidua throughout the period studied (supplemental Fig. 1).

Effects of Culture Conditions on Cell Population of the Primary Placental Cell Culture

To characterize the cultured placental cells, the cells were stained with anti-cytokeratin antibody or hybridized with S100a4 cRNA probe. Cytokeratin-positive cells (trophoblast cells) were predominant in the culture (Fig. 6A). After the overnight incubation for cell attachment, 88.7% ± 0.6% cells were cytokeratin positive (n = 3). Neither oxygen tension nor glucose concentrations changed the proportion of the cytokeratin-positive cells. Percentages of the cytokeratin-positive cells after the incubation under the several conditions were 88.6% ± 0.7%, 87.8% ± 1.6%, 86.0% ± 2.8%, and 88.2 ± 0.13% for normoxia, hypoxia, low-glucose, and high-glucose conditions, respectively (n = 3). The cytokeratin-negative cells were probably composed of fetal capillary endothelial cells, immune cells, and fibroblasts. We also carried out in situ hybridization for S100a4, a fibroblast marker known as fibroblast-specific protein-1 (Fig. 6, B and C). Under any conditions examined, only a few or less cells out of more than 100 cells were S100a4 positive. These results in combination with the results in Figure 3 suggest that the ECM mRNAs detected in this culture system are predominantly expressed in the trophoblast.


Figure 06
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  		FIG. 6 A) Immunohistochemistry of cytokeratin in cultured placental cells. Cells were counterstained with hematoxylin. The cell culture was mainly composed of cytokeratin-positive cells (trophoblast cells). There are seven cytokeratin-negative cells in this field (arrows), and 60 nuclei are present in the cytokeratin-positive cell masses. B, C) In situ hybridization for a fibroblast marker S100a4 in cultured placental cells. Images before (B) and after (C) the counterstaining with hematoxylin are shown. Arrowheads indicate S100a4-positive cells. The cells were obtained from fetal placental tissues from Day 12 of pregnancy. Bars = 100 µm.

Effects of Activin A, TGFBs, Hypoxia, and Glucose Concentrations on Placental ECM Expression

Because the expression of TGFB1, TGFB3, and activin A in the fetal placental tissue was relatively high, effects of these growth factors on placental ECM expression were examined in vitro. Unexpectedly, none of these growth factors could stimulate the expression of placental ECM mRNAs in vitro (Fig. 7), although the same batches of the growth factors could stimulate the TGFB/activin-responsive reporter construct p3TP-lux in hamster and mouse cell lines at the same concentrations used in this study (data not shown).


Figure 07
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  		FIG. 7 In vitro effects of activin A (20 ng/ml), TGFB1 (10 ng/ml), and TGFB3 (10 ng/ml) on levels of Col1a1, Col1a2, Col3a1, Col4a1, Col4a2, and Fn1 mRNAs in dispersed primary mouse placental cells. The cells were obtained from fetal placental tissues from Day 12 of pregnancy. The cells were stimulated with the growth factors for 24 h. Messenger RNAs were detected by the ribonuclease protection assays, and the signals were analyzed with ImageJ. The results were normalized with the levels of Actb mRNA and expressed as relative levels to control. Values represent means ± SEM for three independent experiments.

The hypoxic condition significantly decreased Col1a1, Col1a2, Col4a1, and Col4a2 mRNAs (Fig. 8A). Col3a1 mRNA also was decreased by hypoxia, although the difference was not statistically significant (Fig. 8A). Both hypoxia and the high-glucose condition significantly increased Fn1 mRNA, with a greater effect from the high-glucose concentration (Fig. 8). Col1a1 and Col3a1 mRNAs also increased significantly under the high-glucose condition.


Figure 08
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  		FIG. 8 In vitro effects of hypoxia (A) or glucose concentrations (B) on levels of Col1a1, Col1a2, Col3a1, Col4a1, Col4a2, and Fn1 mRNAs in dispersed primary mouse placental cells. The cells were obtained from fetal placental tissues from Day 12 of pregnancy. A) The cells were cultured under either a normoxic or a hypoxic condition for 24 h. B) The cells were cultured under either a low-glucose (1000 mg/l) or a high-glucose (4500 mg/l) condition for 48 h. Messenger RNAs were detected by the ribonuclease protection assays, and the signals were analyzed with ImageJ. The results were normalized with the levels of Actb mRNA and expressed as relative levels to normoxia (A) or low glucose (B). Values represent means ± SEM for three or four independent experiments. *Significant compared with normoxia or low-glucose control (P < 0.05).

DISCUSSION

An increase in the ability of the placenta to exchange materials between mother and fetus is necessary for supporting fetal growth. The size of the placenta is one of the crucial factors determining this functional capacity. In the present study, the weights of the fetal placental tissue increased noticeably between Days 10 and 14 of pregnancy (Fig. 1). The changing pattern of the fetal placental tissue weights is generally consistent with previous observations [36, 37] in which placentae with the decidua basalis were weighed. An early study revealed that collagen contents in the rat placenta increased during the last one third of pregnancy, and the increase in collagen contents was greater than the increase in placental weight [1]. The correlation between the weights of the fetal placental tissue and the placental ECM expression in the present study is consistent with the increase in placental collagen concentrations during placental development [1]. After Day 16, the placental weights did not change significantly, whereas previous studies demonstrated continuous increases in the thickness of the labyrinth until term [27, 37]. Furthermore the trophoblast-lined sinusoids became smaller and more intricate as pregnancy progressed [27]. These morphologic changes may enable the placenta to increase its functional capacity without increasing in size.

The distribution pattern of Col4a1 mRNA (Fig. 3), the impaired labyrinth formation in Col4a1/2 null mice [6], and the increase in the thickness of the labyrinthine layer during placental development [27, 37] suggest that the increase in Col4a1/2 mRNAs in the placenta during the second half of pregnancy (Fig. 2) is required for constructing normal feto-maternal interfaces. In the human placenta, collagen is deposited in the stroma of chorionic villi [38]. Although the precise structure of the labyrinth is different from that of the human corresponding tissue [14], the distribution pattern of Col1a1 mRNA (Fig. 3) suggests that the increases in fibril collagen mRNAs (Fig. 2) are also responsible for normal development of the labyrinthine layer. In contrast, Fn1 mRNA was localized mainly in the spongiotrophoblast, as observed in Fn1 protein [33]. The spongiotrophoblast is sandwiched by the labyrinthine layer and the decidua basalis. Therefore, the spongiotrophoblast may have a role in stabilizing the feto-maternal junction by producing Fn1, a gluelike protein. The distribution pattern of Fn1 mRNA may be responsible for the distinct response of Fn1 mRNA to hypoxia (Fig. 8A).

The higher expression of Inhba than Inhbb in the mouse placenta is consistent with the higher levels of inhibin A than inhibin B in maternal circulation during human pregnancy [39] and the immunohistologic localization of activin subunits in the equine placenta [40]. The changing pattern of Inhba mRNA in the fetal placental tissue is very similar to the changes in maternal circulating activin A in humans [39, 41], nonhuman primates [42], and hamsters [43], but is quite different from the decreasing activin A expression in the equine placenta during the latter two thirds of pregnancy [40]. The expression patterns of activin A probably depend on types of placenta (hemochorial/discoid or epitheliochorial/diffuse). The expression patterns of activin-binding proteins Fst and Fstl3 in the fetal placental tissue and the decidua suggest that Fstl3 in the fetal placental tissue is a major activin-binding protein in the mouse placenta. The changing patterns of activin-binding protein mRNAs in mice are the opposite of those in rats [21]. Although mRNA levels do not always reflect protein expression, the decrease in Fstl3 expression and the increase in Inhba expression in the fetal placental tissue suggest that activin bioactivity in the placenta strikingly increases from Day 12 to term.

With respect to Tgfb1–3, all three isoforms, especially Tgfb2, were expressed at relatively high levels in the decidua, whereas the fetal placental tissue expressed Tgfb1 and Tgfb3 as predominant forms. This expression pattern is quite different from that in the human chorionic villi, in which Tgfb2 and Tgfb3 are predominantly expressed [44]. It is not known whether the different expression patterns of Tgfb1–3 between the fetal and maternal tissues and between the species have any physiologic importance. The three isoforms of TGFBs bind to the same combination of type I and type II receptors TGFBR1 and TGFBR2 [45]. However, the TGFB2 signaling largely depends on the presence of betaglycan. Betaglycan is a membrane-anchored proteoglycan whose core protein binds with high-affinity TGFBs and facilitates TGFB binding to the signaling receptors [45]. This function is most apparent with TGFB2. TGFB2 on its own has low affinity for the type I and type II signaling receptors compared with TGFB1 and TGFB3, and cells that express these receptors but lack betaglycan are poorly responsive to TGFB2 [45]. The different expression patterns of Tgfb1–3 may have some physiologic significance through such a difference in the signaling mechanism.

In this study, two protected bands for Tgfb1 mRNA were detected (Figs. 4A and 5A). The size of the long protected fragment was consistent with the expected size of the protected band (340 bases), whereas the short protected fragment was approximately 270 bases in length. A previous study has shown that the mouse Tgfb1 gene uses two different promoters [46]. The first and second transcription start sites of the mouse Tgfb1 gene are located at 866 and 576 bp upstream of the translation start site, respectively [46]. The antisense probe for Tgfb1 used in this study corresponds to downstream of the second transcription start site, but the probe contains a 161-base 5' untranslated region. Therefore, the short protected fragment probably represents a third unknown Tgfb1 mRNA transcription start site, which would be located approximately 90 bp upstream of the translation start site. Although we could not find any putative promoter site around the position, the first and second promoters also are atypical [46], and our efforts have found that no promoter-finding program could detect the first and second promoters. Interestingly, the long and the short protected bands for Tgfb1 showed different changing patterns in the decidua. We do not know whether the different changing patterns of these two protected bands have any physiologic significance. Characterization of the promoter and transcript responsible for the short protected fragment remains to be performed.

A previous study has indicated that MSTN stimulates glucose uptake in the human placenta and is expressed in this tissue [19]. However, significant expression of Mstn was not detected in either the mouse fetal placental tissue or the decidua (supplemental Fig. 1). Although MSTN may regulate glucose uptake in the human placenta in an autocrine or paracrine fashion, in the mouse placenta, autocrine or paracrine regulation of placental functions by this growth factor does not likely exist. A previous study has shown that most circulating MSTN binds to its propepitide or FSTL3 in humans [47]. Acid activation of mouse serum revealed MSTN activity in a reporter gene assay, providing an estimate of the MSTN concentration at 80 ng/ml [48]. In contrast, Hills et al. described that identically treated human serum did not have detectable MSTN activity in this assay [47]. These results indicate the presence of relatively high concentrations of free MSTN in mouse plasma compared with human plasma. Therefore, it is possible that the circulating free MSTN may affect placental functions in the mouse.

TGFBs are well known to stimulate ECM expression in many cell types and tissues [15], including the human placenta [29, 49–51]. Activin also stimulates collagen production in renal fibroblasts [52] and fibronectin secretion from human chorionic villi [53]. Therefore, TGFBs and activins are the most probable factors involved in the regulation of ECM secretion in the mouse placenta. Most of the TGFBs are secreted as latent forms, and several factors are involved in the activation of latent TGFBs, such as the plasminogen activation cascade and thrombospondin 1 [45]. In addition, mRNA levels do not always reflect protein levels. Therefore, changes in Inhba and Tfgb1–3 mRNAs do not necessarily reflect changes in activities of activin A and TGFBs. Despite these facts, the increase in Inhba and Tgfb1 mRNAs in the fetal placental tissue during the second half of pregnancy, the increase in Tgfb1, Tgfb2, and Tgfb3 mRNAs in the decidua between Days 10 and 18, a temporary increase in fetal placental tissue Tgfb3 mRNA, and a temporary increase in decidual Inhba mRNA lead us to hypothesize that activin A and TGFBs may be responsible for the increase in placental ECM expression during the second half of mouse pregnancy. However, activin A and TGFBs failed to stimulate the placental ECM expression in vitro after the 24-h incubation (Fig. 7), although the same batches of the growth factors could stimulate the TGFB/activin-responsive reporter construct p3TP-lux within 24 h in hamster and mouse cell lines (data not shown). These results suggest that placental and decidual TGFBs and activins are not likely principal factors regulating placental ECM expression.

Although low oxygen tension increased expression of type I and type IV procollagens in human placental fibroblasts [28, 29], the procollagen mRNAs in the mouse placental cells were decreased by hypoxia in this study. The different responses may be due to differences in species, cell populations, culture periods, or oxygen concentrations. With the exception of Fn1, the in vitro effects of hypoxia on placental cells suggest that an increase in oxygen tension may be partly responsible for the increase in the placental ECM expression during the second half of mouse pregnancy. With respect to Fn1, its expression increased under the hypoxic condition in the mouse placental cells, as observed in human placental fibroblasts [28, 29]. However, high glucose concentration also increased Fn1 expression in the mouse placental cells, with greater effects than hypoxia. An increase in fibronectin expression in response to a high glucose concentration has been reported in bovine trabecular meshwork cells [54]. Col1a1 and Col3a1 mRNAs also increased in response to the high glucose condition. While the high-glucose concentration (4500 mg/l) corresponds to a hyperglycemic condition, the present results suggest that an increase in nutrient supply may be another factor responsible for the increase in the placental ECM expression. It is hypothesized that increasing oxygen tension and nutrient supply in the midgestational mouse placenta stimulate ECM production by trophoblast cells. Subsequently, this increase in extracelluar matrices facilitates construction of maternal-fetal interfaces, which in turn induces further increase in placental oxygen tension and nutrient supply.

In summary, the present study demonstrated increasing expression of the ECM mRNAs during the second half of pregnancy. Although expression patterns of Tgfb1–3 and Inhba in the fetal placental tissue and the decidua suggest involvement of TGFBs and activin A in the regulation of placental ECM expression, in vitro data suggest that during placentation, an increase in oxygen tension and nutrient supply rather than TGFB family members may be responsible for the increase in the placental ECM mRNA expression.

FOOTNOTES

1Supported in part by the Ito Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19580336). Back

Correspondence: 2Koji Y. Arai, Division of Matrix Biology, Scleroprotein Research Institute, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan. FAX: 81 42 367 5787; e-mail: kojiarai{at}cc.tuat.ac.jp

Received: 9 March 2007.

First decision: 4 April 2007.

Accepted: 4 September 2007.

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