Beta Transforming Growth Factors (TGFß) at the Porcine Conceptus-Maternal Interface. Part I: Expression of TGFß1, TGFß2, and TGFß3 Messenger Ribonucleic Acids¹

^a Department of Veterinary Anatomy and Public Health, ^b Department of Animal Science, and ^c Center for Animal Biotechnology, Institute of Biosciences and Technology, Texas A&M University, College Station, Texas 77843–4458

	ABSTRACT

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Spatial and temporal mRNA expression of beta transforming growth factors (TGFß1, TGFß2, and TGFß3) in porcine uterus and conceptuses was determined during the peri-implantation period (Days 10–14 of gestation). Northern blotting identified a major 3.5-kilobase (kb) and a minor 2.5-kb transcript for TGFß1 mRNA. TGFß2 transcripts were 6.2 kb, 5.4 kb, and 2.7 kb, and a single 3.5-kb transcript was detected for TGFß3. With semiquantitative in situ hybridization analysis, progressive increases were detected in TGFßs 1, 2, and 3 mRNA expression in uterine luminal epithelium (ULE), uterine glands (UGs), and underlying stroma (stroma spongiosum, SS) from Days 10 through 14 of gestation (p < 0.05). In myometrium, TGFß mRNA expression did not differ between Days 10 through 14 of gestation.

In porcine conceptuses, TGFßs 1, 2, and 3 mRNA expression was detected in trophectoderm, endoderm, embryonic ectoderm, and mesoderm. For the three TGFß isoforms examined, mRNA expression increased 2- to 4-fold in trophectoderm and endoderm between Days 10 and 14 of gestation. TGFß1 mRNA levels increased significantly in embryonic ectoderm, but not mesoderm, between Days 12 and 14 of gestation; during that same time, TGFß2 mRNA levels increased, but no change was detected in TGFß3 mRNA levels, in embryonic ectoderm and mesoderm.

Progressive increases in TGFß mRNA expression in conceptus trophectoderm, endoderm, ULE, UGs, and SS suggest important roles for these growth factors in porcine conceptus development during the peri-implantation period.

	INTRODUCTION

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In pigs, marked uterine remodeling, conceptus differentiation, and implantation occur during the second week of gestation (peri-implantation period). Porcine conceptuses change from spherical to tubular to filamentous forms, undergo gastrulation, secrete estrogens as the signal for maternal recognition of pregnancy, and begin attachment to the endometrial epithelium during this period [1]. Implantation in pigs occurs between Days 13 and 18 of gestation; it is superficial and noninvasive and involves phases of trophectoderm-uterine epithelial cell apposition and adhesion. The factors that control these critical events in porcine conceptus development are not known.

Growth factors such as the beta transforming growth factors (TGFß) are thought to mediate many key events in conceptus development [2–4]. The three isoforms of TGFß (TGFß1, TGFß2, and TGFß3) exhibit differential expression during mammalian embryogenesis [5], are encoded for by distinct genes on separate chromosomes, and are thought to have diverse biological functions. The regulatory response elements in the promoters of the three different genes differ markedly and may explain their differential regulation [6].

TGFß mRNAs have been detected in mouse [7, 8] and ovine [9, 10] endometrial and placental tissues during the peri-implantation period. TGFß1 mRNA has been detected in equine endometrium, but not trophectoderm, during placentation [11]. The expression patterns of immunoreactive TGFßs and their receptors (type I and type II) in porcine peri-implantation conceptuses (Days 10–14 of gestation) suggest multiple roles for these growth factors in conceptus development and implantation [12].

TGFßs function in both autocrine and paracrine interactions; thus both the maternal uterus and the conceptuses represent potential sources for TGFß proteins. To better understand the mechanisms controlling conceptus differentiation during the peri-implantation period, expression of TGFßs 1, 2, and 3 mRNAs in porcine uterus and conceptuses was examined during the peri-implantation period.

	MATERIALS AND METHODS

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Sample Collection and Processing

Crossbred gilts of similar weight and genetic background were observed daily for estrous behavior and were mated at 12 and 24 h after the onset of estrus. Gilts were hysterectomized, using sterile techniques, at 10, 11, 12, 13, or 14 days after the onset of estrus, which was designated Day 0 (n = 3–5 gilts per day of gestation) [13]. Conceptuses were flushed from each uterine horn using sterile saline. Animal handling and surgical procedures were approved by the Institutional Animal Care and Use Committee at Texas A&M University.

Uterine samples (Days 10 through 14 of gestation, n = 3–5 gilts per day of gestation) were collected from approximately 10 cm below the tubo-uterine junction and fixed in 4% paraformaldehyde for 14–15 h, washed with 70% ethanol, and embedded in paraffin. Conceptus tissues from the same gilts, similarly processed, were the same samples as used for previous studies of TGFß protein expression [12].

Riboprobe Preparation

To generate a 600-base pair (bp) cRNA specific for TGFß2 mRNA, the HindIII-HindIII fragment was removed from the pig TGFß2 cDNA clone (gift from Dr. M. Murtaugh, University of Minnesota, St. Paul, MN; GenBank Accession #L08375, plasmid designation pig TGFß2). The remaining plasmid was religated and used to generate antisense (linearized with EcoRI, transcribed with SP6 RNA polymerase) and sense (linearized with HindIII, transcribed with T7 RNA polymerase) TGFß2 cRNAs. TGFß1 antisense (linearized with HindIII, transcribed with T7 RNA polymerase), TGFß1 sense (linearized with EcoRI, transcribed with SP6 RNA polymerase), TGFß3 antisense (linearized with BamHI, transcribed with T7 RNA polymerase), and TGFß3 sense (linearized with Xho I, transcribed with SP6 RNA polymerase) cRNAs were prepared from isoform-specific mouse cDNA sequences (GenBank Accession #M13177 and M32745 and plasmid designation pmTGFß1-a and pmTGFß3–11b, respectively) [14, 15]. These cRNAs were generated with high specific activity by omitting the addition of unlabeled dUTP and doubling the [-³²P]UTP (3000 Ci/mmol; NEN, Boston, MA) for Northern blotting or [-³⁵S]UTP (1250 Ci/mmol; NEN) for in situ hybridization, using the protocol described by Wilcox [16].

Northern Blotting

Total endometrial RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform extraction modification of the single-step method [17]. Poly(A)⁺ RNA was extracted from the endometrial tissues using the Fast Track 2.0 mRNA isolation kit (Invitrogen, San Diego, CA). Total endometrial RNA (40 µg) for TGFß1 Northern analysis, and poly(A)⁺ endometrial RNA (5 µg) for TGFß2 and TGFß3 Northern analysis, were electrophoresed through a 1.5% agarose-formaldehyde gel. RNA was then transferred overnight to a positively charged nylon membrane by capillary blotting with 10-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), and membranes were then baked at 80°C for 2 h. Antisense TGFßs 1, 2, and 3 cRNAs were labeled with [-³²P]UTP. Prehybridization, hybridization, and further washing steps were done using the buffers and conditions described by Ing et al. [18], including RNase A treatment (20 µg/ml) to verify the specificity of probe binding to the transcripts. Blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 1–3 days at -80°C.

The autoradiographs were then scanned (Art-Scan Pro, DPI Electronic Imaging Systems, Cincinnati, OH; w/ppc scanner) and images imported to NIH Image 1.60/ppc imaging software (W. Rasband; NIMH, Bethesda, MD). The software was used to generate optical density profiles of the complete blot. Peaks in the graphs represent specific transcripts detected within each lane.

In Situ Hybridization (ISH)

ISH was performed using the Wilcox protocol [16]. Briefly, paraffin-embedded tissue sections (uterus and conceptus) were cut (4 µm) and placed on Superfrost-Plus-coated slides (Statlab Medical Products, Lewisville, TX). After deparaffinization, prehybridization was performed at 42°C for 1–3 h. Hybridizations were performed with 5 x 10⁵ cpm [-³⁵S]UTP-labeled antisense TGFßs 1, 2, and 3 cRNAs at 55°C for 14–15 h. Nonspecific hybridization was determined using 5 x 10⁵ cpm [-³⁵S]UTP-labeled sense TGFß riboprobes on sections present on the same slide. After posthybridization washes, sections were treated with 20 µg/ml RNase A for 30 min. Slides were then dehydrated and air dried at 37°C for 1–3 h. The signal was visualized by exposing the sections to Kodak autoradiographic emulsion NTB-2 at 4°C for 2–12 wk and developing them in Kodak D-19. Sections were counterstained with hematoxylin. Several ISH experiments were conducted, and each run contained representative uterine and conceptus samples from each gestational day. Autoradiographic signals for all sections (Days 10, 11, 12, 13, and 14 of gestation) were visually evaluated and photographed by brightfield and darkfield microscopy (Zeiss Photomicroscope III; Oberkochen, Germany) to determine the general patterns of mRNA expression. A larger run was then performed with uterine (n = tissues from 3 gilts per day of gestation) and conceptus (n = 6–8 conceptuses obtained from 3 different litters per day of gestation) samples for semiquantitative analysis. Because of the limited number of slides on which ISH could be performed without artifact, representative uterine and conceptus tissues only from Days 10, 12, and 14 of gestation were included in this experiment.

Quantitation of ISH Results

To perform semiquantitative analysis of ISH data, sections from Days 10, 12, and 14 of gestation were hybridized and developed simultaneously. Sections were scanned into a computer using the Apple (Cupertino, CA) video player photoscanning system. The cell types were identified microscopically under brightfield conditions. Several non-overlapping areas for each cell type (from both sense and antisense sections) were separately scanned, and grains were counted. Fields were randomly selected by an individual who was blinded to the experiment and the identity of the slides.

Semiquantitative analysis was performed by counting the number of silver grains per area (15 x 40 µm²) per cell type per slide (6 areas of equal size were selected for antisense and 6 for control section) using the NIH Image 1.60/ppc imaging software. As the software could not differentiate between single grains and clusters of grains, preliminary experiments were performed to establish a correlation between grain count and grain area size. Single grains were estimated to have an area size of 0.5 µm² ± 0.08. Thus grains with area of 0.4–0.6 µm² were counted as single grains, those with area of 0.7–0.8 µm² as 1.5 grains, those with area of 0.9–1.2 µm² as two grains in a cluster, and so on. The number of specific grains on the antisense (positive) section was determined by subtracting the number of grains on the adjacent sense (control) section. Statistical analysis was performed using one-way ANOVA on ranks and Student-Neuman-Keuls method using Sigma Stat 2.0 statistical analysis software (Jandel Scientific, San Rafael, CA). Comparisons were made among each cell type in the various gestational days. Because of differences in the TGFßs 1, 2, and 3 cRNA specific activities, isoform specificity, and size, comparisons among different TGFß isoforms were not attempted.

	RESULTS

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Northern Analysis

To confirm the specificity of ISH results, Northern analysis was performed on representative uterine endometrial tissue RNA samples using TGFßs 1, 2, and 3 riboprobes. The various transcript sizes and their respective optical densities are shown in Figure 1. Northern analysis of uterine endometrial total RNA detected major 2.5-kilobase (kb) and minor 3.5-kb transcripts of TGFß1 mRNA. Another low-intensity band was observed at 28S size, but it was significantly diminished upon RNase A treatment; the other 2.5-kb and 3.5-kb intensities remained the same, indicative of some nonspecific binding of the TGFß1 cRNA to the 28S mRNA in our total endometrial RNA extracts (Fig. 1a). Poly(A)⁺ RNA Northern analysis of a uterine endometrial sample detected 2.7-kb, 5.2-kb, and 6.2-kb TGFß2 mRNA transcripts and a 3.5-kb TGFß3 mRNA transcript (Fig. 1, b and c).

View larger version (23K): [in this window] [in a new window]   FIG. 1. TGFß mRNA Northern analysis on representative endometrial RNA samples (Day 11 of gestation). a) TGFß1 mRNA (40 µg total RNA); b) TGFß2 mRNA (5 µg poly(A)+ RNA); c) TGFß3 mRNA (5 µg poly (A)+ RNA). Note transcript sizes in photographs. The corresponding graphs represent the optical densities (OD) of the scanned lane, showing specific transcript peaks and background levels of each blot along with length of each blot (mm).

TGFß mRNA Expression in Uterine Tissues

Messenger RNA expression of all three TGFß isoforms was detected in uterine luminal epithelium (ULE), uterine glands (UGs), stroma, and myometrium. TGFßs 1, 2, and 3 mRNA expression increased progressively in ULE and underlying stroma (stroma spongiosum, SS) from Day 10 to Day 12 and from Day 12 to Day 14 of gestation (Figs. 2 and 3), with resultant 2- to 4-fold (p < 0.05) increases in expression of each isoform from Day 10 through 14. Representative changes in TGFß2 mRNA expression, as visually analyzed by brightfield and darkfield photomicroscopy, are shown in Figure 2.

View larger version (101K): [in this window] [in a new window]   FIG. 2. TGFß2 mRNA expression in porcine endometrium (ISH; Days 10, 11, 12, 13, and 14 of gestation). Panels a, c, e, g, and i are Days 10, 11, 12, 13, and 14 endometrium antisense brightfield images with corresponding darkfield images in b, d, f, h, and j. Panels k (brightfield) and l (darkfield) are Day 14 endometrium sense controls; sUG, superficial uterine glands. Note progressive increase in TGFß2 mRNA expression from Day 10 to Day 14 of gestation. Similar results were obtained for TGFß1 and TGFß3 mRNA expression in endometrium (data not shown). Bar = 50 mm.

View larger version (26K): [in this window] [in a new window]   FIG. 3. Histograms representing TGFßs 1, 2, and 3 mRNA levels (grains per unit; 1 unit = 15 x 40 µm2) in porcine uterine cell types from Days 10, 12, and 14 of gestation (values are mean ± SD; n = 3 per day of gestation). sUG, superficial uterine glands; dUG, deep uterine glands; MY, myometrium; a,b,c indicate significant differences (p < 0.05) within a cell compartment.

The level of TGFß1 mRNA was higher (p < 0.05) in superficial UGs than in deep UGs at all days examined, and it tended to increase from Day 10 to 12 and Day 12 to 14 of gestation (p < 0.1); however, the differences were not statistically significant (Fig. 3). The temporal increase in TGFß2 mRNA expression in superficial UGs was significant (p < 0.05) only when Day 10 was compared to Day 14 of gestation; in deep UGs, TGFß2 mRNA expression increased significantly (p < 0.05) between Days 10 and 12. The increase in TGFß3 mRNA expression in superficial UGs and deep UGs was significant (p < 0.05) between Days 10 and 12 of gestation (Fig. 3).

No change was detected in expression of TGFßs 1, 2, and 3 mRNAs in myometrium between Days 10 and 14 of gestation (Fig. 3).

TGFß mRNA Expression in Conceptus Tissues

TGFß mRNA expression was detected in porcine conceptus trophectoderm (Te), endoderm, embryonic ectoderm (EE), and mesoderm on all days of gestation examined. TGFß1 and TGFß2 mRNAs increased progressively (2- to 4-fold; p < 0.05) in Te and endoderm, and differences were significant between Days 10 and 14 (Fig. 4). Significant increases in TGFß3 mRNA expression in Te were not detected until later, i.e., only between Days 12 and 14 of gestation. In sections in which embryonic disc was visible, expression of TGFßs 1, 2, and 3 was observed in both proximal and distal Te and endoderm cells. However, not all the sections contained embryonic discs; therefore, semiquantitative in situ analysis on Te and endoderm was performed only on Te in which a disc was not seen and in whose adjacent sections a disc was not seen. Although such Te is most likely to be distal to the disc, its precise location relative to the disc could not be definitively determined.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Histograms representing TGFßs 1, 2 and 3 mRNA levels (grains per unit; 1 unit = 15 x 40 µm2) in porcine conceptus cell types on Days 10, 12, and 14 of gestation (values are mean ± SD; n = 6–8 conceptuses per day of gestation). Endo, endoderm; Meso, mesoderm; a,b,c indicate significant differences (p < 0.05) within a cell compartment. * Insufficient for quantitation at Day 10; ** not present at Day 10.

TGFß1 mRNA expression in conceptus mesoderm at Day 14 of gestation tended to be higher than at Day 12 (p = 0.17), but the difference was not statistically significant; TGFß1 mRNA expression in EE was significantly (p < 0.05) higher at Day 14 compared to Day 12. TGFß2 mRNA expression in conceptus EE and mesoderm was significantly (p < 0.05) increased by Day 14, while TGFß3 mRNA levels remained unchanged (Fig. 4). Cell-specific TGFßs 1, 2, and 3 mRNA expression in porcine conceptuses is shown in representative brightfield and darkfield photomicrographs (Fig. 5).

View larger version (156K): [in this window] [in a new window]   FIG. 5. Cell type-specific expression of TGFß mRNAs in porcine conceptuses. Panels a, d, and g are brightfield images; b, e, and h are darkfield images of antisense hybridizations; c, f, and i are darkfield images of sense controls. TGFß1 mRNA expression in EE, mesoderm (Meso), and endoderm (Endo) of Day 14 conceptus (a, b); TGFß2 mRNA expression in Te and endoderm (Endo) of Day 12 conceptus (d, e); and TGFß3 mRNA expression in Te of Day 11 conceptus (g, h). Transcripts of all TGFß isoforms were detected in conceptuses on Days 10–14 of gestation. Bar = 50 mm.

	DISCUSSION

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Results of Northern analysis support the detection of isoform-specific TGFß transcripts by the cRNAs used in this study. Northern analysis identified 2.5-kb and 3.5-kb TGFß1 transcripts as has been reported in porcine tissues and platelets [19]. Our Northern analysis detected TGFß2 transcripts of 6.2, 5.2, and 2.7 kb. Multiple TGFß2 transcripts ranging from 6.2 to 2.7 kb have been reported; the sizes and number of TGFß2 transcripts vary among species. For example, in ovine tissues, five (6.2-, 5.2-, 4.8-, 4.0-, and 2.7-kb size) TGFß2 transcripts are reported [9], while in mouse tissues, four (6.0, 5.0, 4.0, and 3.5 kb) TGFß2 transcripts have been reported [20]. Our analysis does not exclude the possibility of other TGFß2 transcripts that may be present but below the limits of detection by Northern analysis. The single 3.5-kb transcript of TGFß3 mRNA concurs with previous findings on human and mouse TGFß3 transcript size [20–22].

The ISH studies indicated cell-specific TGFßs 1, 2, and 3 mRNA expression patterns in porcine uterus and conceptus tissues between Days 10 and 14 of gestation. TGFßs 1, 2, and 3 mRNA expression increased 2- to 4-fold in ULE cells in uterus and in conceptus Te from Day 10 to 14 of gestation. Immunohistochemically, increases in TGFßs 1, 2, and 3 protein expression in uterine luminal epithelium between Days 10 and 14 of gestation [23] and the presence of TGFßs in porcine conceptuses [12] during the same time period have also been reported. This apparent increase in expression of TGFßs in conceptus and maternal cell types actively involved in cell-to-cell contact during adhesion and implantation may represent an important step in conceptus-maternal interactions.

TGFßs can induce their own gene expression via autocrine or paracrine interactions [24, 25]. The presence of intense immunoreactive TGFßs in conceptus Te, endoderm, and EE as early as Day 10 of gestation [12], along with results of these present studies, suggests that conceptus TGFßs may, through paracrine mechanisms, induce TGFß mRNA expression in uterine cells.

The significant increase in uterine (in ULE and in SS) TGFß mRNA expression between Days 12 and 14 of gestation may also be due to effects of conceptus-secreted estrogens on uterine cells. TGFß gene expression can be regulated by steroid hormones [26–28], and studies of ovariectomized mice [20] and human endometrial stromal cells [22] have shown up-regulation (2- to 4-fold) of TGFßs 1, 2, and 3 mRNAs within 6 h of estrogen treatment. Porcine conceptuses secrete estrogens between Days 11 and 13 of gestation as the initial signal for pregnancy recognition [29], which may also increase expression of TGFßs as a part of maternal recognition of pregnancy.

Results of the present study, and results of a study on TGFß protein expression [12], indicate that porcine conceptuses can synthesize their own growth factors and that they are not completely dependent on a maternal source for TGFßs. The results also suggest differential regulation of TGFß expression in porcine conceptus EE, mesoderm, Te, and endoderm. TGFß1 mRNA expression in porcine EE increased between Days 12 and 14 of gestation and tended to increase in mesoderm, although the difference was not statistically significant. Similar, and statistically significant, increases in TGFß2 mRNA expression were detected in EE and mesoderm at this time. TGFß3 mRNA expression, however, was unchanged between Days 12 and 14 in EE and mesoderm. Interestingly, protein expression of all three TGFß isoforms (TGFß1, TGFß2, and TGFß3) tended to decrease during this time [12]. In porcine Te and endoderm, TGFß mRNA expression increased, and protein expression did not appear to change between Days 10 and 14 of gestation [12]. Discrepancies in TGFß protein and mRNA expression have been reported in various cell lines, indicating both transcriptional and posttranscriptional regulation in TGFß gene expression [30]. This posttranscriptional control could be due to the presence of "stem-loop forming regions" in the 5' flanking region of TGFßs 1, 2, and 3 genes [30–32]. The cytosolic factors controlling these inhibitory regions are not yet completely defined. Thus, although the mechanisms controlling the observed differences in porcine conceptus TGFß protein and mRNA expression are not clear, the data suggest cell type-dependent transcriptional and posttranscriptional regulatory pathways for TGFß expression in extraembryonic (Te and endoderm) and embryonic (EE and mesoderm) cells.

The results indicate that mRNA expression patterns for the three TGFß isoforms in porcine conceptus and endometrium are similar during the critical peri-implantation period of pregnancy in pigs. It remains to be determined whether these TGFßs have similar or diverse biological functions in conceptus development and implantation.

	ACKNOWLEDGMENTS

 
We thank Dr. H.L. Moses for providing TGFß1 and TGFß3 cDNAs and Dr. M. Murtaugh for providing TGFß2 cDNA. We thank Dr. T.L. Ott for expertise in pig management and surgery, and Dr. Bazer's laboratory for assistance with these procedures. The authors thank Sarah Christo and Barbara Merka for their histotechnology expertise. We also thank Dr. R.C. Burghardt for his expert assistance with photomicroscopy and photography and acknowledge the use of the College of Veterinary Medicine Image Analysis facilities. The use of Dr. L.C. Abbott's computer and video scanning system is gratefully acknowledged.

	FOOTNOTES

 
¹ Grant support: USDA grant #94–37203–1222.

² Correspondence. FAX: 409 847 8981; ljaeger{at}cvm.tamu.edu

Accepted: June 10, 1998.

Received: November 6, 1997.

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