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Biology of Reproduction 59, 911-917 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Beta Transforming Growth Factors (TGFß) at the Porcine Conceptus-Maternal Interface. Part II: Uterine TGFß Bioactivity and Expression of Immunoreactive TGFßs (TGFß1, TGFß2, and TGFß3) and Their Receptors (Type I and Type II)1

Anupma Guptaa, Christopher M. Dekaneya, Fuller W. Bazera,b,c, Monique M. Madrigala, , and Laurie A. Jaegera,b,c

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&; University, College Station, Texas 77843–4458


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Porcine uterine tissues were collected from Days 10 to 14 of gestation (peri-implantation period) or corresponding days of the estrous cycle. Results indicated a marked increase in beta transforming growth factors (TGFß1, TGFß2, and TGFß3) and TGFß receptor (type I and type II) immunostaining in uterine luminal epithelium (ULE) between Days 10 and 14 of gestation, but there was no increase in ULE immunostaining on the corresponding days of the estrous cycle. Uterine glands and stroma were intensely immunopositive in pregnant gilts for TGFß isoforms and their receptors, but immunostaining was weak to undetectable in cycling gilts. No differences were detected in myometrium, in which immunostaining was moderate in both cycling and pregnant gilts. Additionally, TGFß2 and TGFß receptor (type I and type II) immunostaining was detected in uterine monocyte/macrophage-like cells.

Western blotting detected the presence of all three TGFß isoforms in uterine luminal flushings. The CCL64 cell TGFß bioassay detected bioactive TGFßs in uterine luminal flushings on Days 12, 13, and 14 of gestation. These results strongly indicate that uterine expression of TGFßs and their receptors is pregnancy specific and that bioactive TGFßs are present at the conceptus-maternal interface in the peri-implantation period in pigs. Thus TGFßs are likely to be involved in autocrine-paracrine interactions between the maternal uterus and the conceptus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Precisely regulated conceptus-maternal interactions and establishment of a uterine receptive state during the peri-implantation period are required for successful implantation. It is likely that uterine growth factors exert paracrine effects on the conceptuses that may act synergistically or additively with growth factors produced by the conceptuses. Transforming growth factor ßs (TGFßs) are a family of growth factors that have autocrine and paracrine effects and may play important roles in intercellular communications.

In most cases TGFßs are secreted in latent forms, bound to TGFß latency-associated peptide, and must be activated to elicit a biological response. The activation of latent TGFß could be by proteases, such as plasmin, or by other changes in the cellular microenvironment (reviewed in [1]). Active TGFßs 1, 2, and 3 are 25-kDa homodimers; they signal via cell surface serine/threonine kinase receptors (type I and type II) [2–4].

The isoform-specific roles of different TGFßs are not well defined, but TGFßs are involved in cellular proliferation, cellular differentiation, extracellular matrix protein and integrin modification, tissue repair, angiogenesis, and immunosuppression. All of these events occur during early pregnancy and/or the estrous cycle. Previous studies have identified TGFßs in endometrial and placental tissues of various species at various stages of gestation, and immunolocalization patterns vary among species [5–8]. TGFßs and TGFß receptors were also immunolocalized in porcine peri-implantation conceptuses (Days 10–14 of gestation) [9]. Results presented in a companion paper [10] indicate that TGFß mRNA up-regulation occurs in porcine conceptus and uterine tissues during the peri-implantation period, suggesting multiple sources of TGFß and important roles for TGFßs during this critical period of maternal recognition of pregnancy and establishment of the uterine receptive state in pigs.

The present study investigated 1) expression of TGFßs 1, 2, and 3 proteins and their receptors (type I and type II) in porcine uterine tissues collected on Days 10–14 of gestation (pregnant gilts) and the estrous cycle (cycling gilts) and 2) presence and bioactivity of TGFßs 1, 2, and 3 at the conceptus-maternal interface during the peri-implantation period.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Collection and Processing

Crossbred gilts of similar weight and genetic background were observed daily for estrous behavior and were either mated at 12 and 24 h after the onset of estrus or not mated and allowed to continue cycling. Gilts were hysterectomized using sterile techniques [11] on Days 10, 11, 12, 13, or 14 after onset of estrus, which was designated as Day 0 (n = 3–5 gilts per day of gestation). Animal handling and surgical procedures were approved by the Institutional Animal Care and Use Committee at Texas A&M University.

Uterine tissues were taken from approximately 10 cm below the tubo-uterine junction, fixed in 4% paraformaldehyde, and embedded in paraffin; these were the same samples as used for study of TGFß mRNA expression [10]. Uterine luminal proteins were collected by flushing the uterine lumen with sterile saline. Uterine luminal flushings were centrifuged at 1000 x g for 15 min to remove tissue particles and concentrated to a final volume of 5 ml by ultrafiltration (Centriprep concentrator 3, molecular weight cutoff, 3000 daltons; Amicon, Beverly, MA), and the total amount of protein was determined in each sample (Coomassie Plus protein assay; Pierce, Rockford, IL). Uterine luminal flushings were aliquoted and stored at -80°C.

Immunohistochemistry

Immunohistochemistry was performed using commercially available affinity-purified polyclonal rabbit antisera to TGFß1, TGFß2, TGFß3, and TGFß receptor types I and II (Santa Cruz Biotechnology, Santa Cruz, CA) and the same incubation conditions, dilutions, and control tissues as previously reported [9]. The peptide sequences used to generate these antisera are specific for TGFßs as shown in a search conducted using National Center for Biotechnology Information Basic Local Alignment Search Tool [12] and do not show any cross-reactivity among the TGFß isoforms, as indicated by the manufacturer.

For each antibody, representative uterine (n = tissues from 3–5 gilts per day) sections from each day of gestation were run together to allow for comparisons among them. Experiments were repeated such that each tissue was immunostained at least 2–3 times for each antibody. All slides were developed for 30–60 sec and counterstained with aqueous hematoxylin, and immunoreactivity was assessed using light microscopy. Results were obtained by visually comparing each positive section to its IgG control. All sections were evaluated by one investigator who had knowledge of the treatments; another, blinded investigator independently evaluated randomly selected sections.

Western Blotting

Samples of uterine luminal proteins (20 µg total uterine fluid protein; n = 3 gilts per day of gestation) and TGFß growth factor standards (human recombinant TGFß1; Gibco BRL, Gaithersburg, MD), porcine platelet TGFß2, and chicken recombinant TGFß3 (R&; Systems Inc., Minneapolis, MN)) were dissolved in reducing sample buffer (containing SDS and ß-mercaptoethanol) and boiled for 3 min; they were then cooled and analyzed by 7.5–20% gradient SDS-PAGE. After electrophoresis, the samples were transferred to nitrocellulose membranes using a Transblot apparatus (Bio-Rad Labs., Richmond, CA) at 100 V for 1 h at 4°C. Nonspecific binding was blocked in PBS with 3% BSA-0.2% Tween. The blots were incubated with primary antibodies to TGFß1, TGFß2, and TGFß3 at concentrations of 0.15 µg/ml. For controls, rabbit immunoglobulin (0.15 µg/ml) replaced the primary antibody, and a second control was performed in which primary antibody was immunoabsorbed with pure growth factor or control peptide (Santa Cruz Biotechnology) at 10 times the concentration of the primary antibody before addition to the blot. Horseradish-peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody. Immunoreactive proteins were detected using the Western blotting ECL system as directed by the manufacturer (cat. no. RP2209; Amersham Life Sciences, Buckinghamshire, England).

TGFß Bioassay

To determine whether TGFßs present at the conceptus-maternal interface are bioactive or latent, an in vitro TGFß bioassay was performed as described by Danielpour et al. [13]. Briefly, subconfluent CCL64 mink lung epithelial cells (Mv1Lu; American Type Culture Collection, Rockville, MD) were trypsinized, suspended in Dulbecco's Modified Eagle's medium-F12 medium containing 10% fetal bovine serum (FBS), pelleted at 500 x g for 3 min, and resuspended in assay medium (minimal essential medium, 0.2% FBS, 10 mM Hepes). Cells were seeded in wells of 24-well plates at 5 x 105 cells/500 µl.

After 1 h, 300 µl TGFß standards (5.0, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, and 0 ng/ml TGFß1 [porcine; R&; Systems]) (see Fig. 5a) or samples of uterine luminal flushings were added to each well in triplicate. Samples of uterine luminal flushings were either acid activated (addition of 0.5 µl of 0.5 N HCl/25 µl of sample at 4°C for 1 h) [14] to determine "acid activated" or "total" TGFß activity, or were left untreated to determine "native" TGFß activity at 4°C for 1 h. Acid-treated samples were neutralized by addition of 0.8 µl of 0.5 N NaOH/25 µl of sample. The same combination of HCl/NaOH was added to untreated samples. Next, each sample was diluted in assay medium to 0.025 µg protein/µl, sterile filtered, and kept on ice until assayed. A total of 7.5 µg protein was added to each well.

After 20 h, cells were pulsed with 0.25 µCi of [3H&; for 4 h at 37°C. Cells were then fixed in methanol-acetic acid for 1 h at room temperature, washed twice with 80% MeOH, and digested with 500 µl 0.05% trypsin for 30 min at 37°C. Next, 500 µl of 1% SDS was added to each well, and trays were left at room temperature overnight. Each well was harvested into scintillation fluid, and [3H&; uptake was counted using a Beckman LS 3801 liquid scintillation counter (Beckman Instruments, Palo Alto, CA). Data were expressed as percentage of control. Differences among days of gestation were compared using ANOVA and the Tukey-Kramer multiple comparisons test (Sigma Stat 2.0 statistical analysis software; Jandel Scientific, San Rafael, CA).

To verify identity of the growth-inhibitory activity observed in uterine luminal flushings, 10 µg/ml pan-specific TGFß-neutralizing antibody (cat. no. AB-100-NA; R&; Systems), 10 µg/ml rabbit IgG (cat. no. AB-105-C; R&; Systems), or 10 µl PBS was added to acid-treated and untreated samples after the final dilution with assay medium. Each treatment was assayed in triplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
No background staining was detected in control tissues used for TGFß1, TGFß2, TGFß3, or type I and type II receptors (see Fig. 2, d–f, and Fig. 3c). Marked differences in immunolocalization of TGFßs were detected in uterine tissues from cycling and pregnant pigs.

Immunohistochemical Localization of TGFßs 1, 2, and 3 in Uterine Tissues Collected from Pregnant and Cycling Gilts (Days 10–14)

Cycling gilts Light immunopositive TGFß1 reactions were detected on the apical membrane of ULE (Fig. 1, d and e) and uterine glands (UGs) (data not shown) on Days 10 through 14 of the estrous cycle; however, cytoplasmic reactions were not detected. A similar reaction pattern was detected for TGFß2 and TGFß3 proteins. The reaction pattern did not change, and intensity of staining between Days 10 and 14 of the estrous cycle was indistinguishable. Overall weak immunopositive TGFß reactions were detected in uterine stroma and myometrium on Days 10 through 14 of the estrous cycle (data not shown).



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  		FIG. 1. Immunohistochemical localization of TGFß1 in ULE (large arrowheads) and stromal fibroblasts (small arrowheads) of uterine tissues collected between Days 10 and 14 of gestation and the estrous cycle (n = 3 per day). Sections were counterstained with aqueous hematoxylin. a) Day 10 (pregnant); b) Day 12 (pregnant); c) Day 14 (pregnant); d) Day 10 (cycling); e) Day 14 (cycling); f) IgG control. Similar patterns of immunoreactivity were detected for TGFß2 and TGFß3 (data not shown). Note increase in TGFß expression from Day 10 through Day 14 of gestation in tissues collected from pregnant, but not cycling, gilts. Bar = 50 mm.

Pregnant gilts We detected a progressive increase in TGFß1, TGFß2, and TGFß3 immunostaining in ULE from Days 10 through 14 of gestation (Fig. 1, a–c). Apical immunostaining was detected on ULE cells from Days 10 to 14 of gestation. Cytoplasmic immunoreactions were detected in some epithelial cells on Day 11 of gestation (data not shown), and the number of immunopositive cells increased in subsequent days of gestation. TGFß protein expression was present in UGs on all the days of gestation examined (Fig. 2). In uterine stroma, moderate to intense immunopositive reactions were detected for all three TGFß isoforms in fibroblast-like cells and surrounding extracellular matrix (ECM) (Fig. 1, a–c). Myometrial immunostaining for TGFßs was weak, as described for tissues collected from cycling pigs (data not shown).



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  		FIG. 2. Immunolocalization of TGFßs in UGs (Day 13 of gestation). Sections were counterstained with aqueous hematoxylin. a) TGFß1; b) TGFß2; c) TGFß3. Panels d, e, and f are respective IgG controls. Note uniform TGFß1 and TGFß3 immunoreaction in glandular epithelium, and intense TGFß2-immunopositive macrophage/monocyte-like cells (arrowheads). Bar = 50 mm.

Some isoform-specific immunostaining differences were observed. TGFß1 immunoreactions were intense in superficial UGs and stratum spongiosum, but moderate to weak in deep UGs and stratum compactum between Days 10 and 14 of gestation (data not shown). In contrast, no regional differences in TGFß2 or TGFß3 immunoreactivity were observed in superficial and deep UGs and stromal cell layers. Within UGs, TGFß2 immunostaining was detected in cells previously identified as macrophage/monocyte cells (Fig. 2b) [15]. Similar TGFß2 immunostaining was also detected in other lymphocytic immune cells of stroma.

Immunohistochemical Localization of TGFß Receptors (Type I and Type II) in Uterine Tissues Collected from Pregnant and Cycling Gilts (Days 10–14)

Cycling gilts No changes in weak immunopositive staining for type I and type II receptors was detected from Days 10 through 14. Both receptor types were immunolocalized on the apical membrane of ULE (Fig. 3d) and UGs (data not shown), and cytoplasmic staining was observed in stromal and myometrial cells (data not shown).



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  		FIG. 3. Immunohistochemical localization of TGFß receptor type II in ULE (arrowheads) of uterine tissues collected between Days 10 and 14 of gestation and the estrous cycle. Sections were counterstained with aqueous hematoxylin. a) Day 10 (pregnant); b) Day 14 (pregnant); c) IgG control; d) Day 14 (cycling). Similar patterns of immunoreactivity were observed for TGFß receptor type I (data not shown). Note differences in TGFß receptor type II immunostaining between tissues collected from Day 14 pregnant (b) and Day 14 cycling (d) gilts. Bar = 50 mm.

Pregnant gilts Immunostaining for type I and type II receptors was weak and was localized in apical membranes of ULE (Fig. 3) and UG (data not shown) cells on Days 10 and 11 of gestation but was increased on Days 12, 13, and 14 of gestation, when immunostaining was present in both membranous and cytoplasmic components of the epithelial cells.

Fibroblast-like cells, macrophage/monocyte-like cells, and myometrial cells also stained positive for type I and type II receptors (data not shown) on all days of gestation examined.

TGFßs 1, 2, and 3 in Porcine Conceptus-Maternal Interface during the Peri-Implantation Period

Western blotting of uterine luminal flushings collected between Days 10 and 14 of gestation revealed specific immunoreactive protein bands for all three TGFß isoforms. Representative results for TGFß2 are shown in Figure 4. A 12.5-kDa protein and 25-kDa protein were detected for all three TGFß isoforms on Days 10 through 14 of gestation and TGFß standards. Additionally, 20- to 23-kDa bands were observed on Days 13 and 14 of gestation. Some higher molecular weight immunoreactive proteins in the stacking gel also reacted specifically with TGFß antibodies. The high molecular weight immunoreactive material in the stacking gel could not be resolved on the 7.5–20% gel, which was used to resolve the major (12.5 kDa and 25 kDa) forms of TGFßs. On a lower-percentage (5–12%) SDS-PAGE gel, in which all immunoreactive proteins entered the separating gel, a 65- to 67-kDa protein that was immunoreactive with TGFß1, TGFß2, and TGFß3 antibodies was detected. The immunoreactive 12.5-kDa, 20–23-kDa, 25-kDa, and high molecular weight stacking gel proteins were not detected in control blots in which primary antibody was replaced with immunoabsorbed primary antibody or an IgG (Fig. 4).



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  		FIG. 4. Western blot analysis of porcine uterine luminal flushings collected on Days 11 and 14 of gestation. Lanes 1, 3, 5, and 7 are uterine flushing proteins from Day 11; lanes 2, 4, 6, 8 are uterine flushings from Day 14. Lanes 1 and 2 are proteins incubated with TGFß2 primary antibody; lanes 3 and 4 are proteins incubated with control IgG; lanes 5 and 6 are proteins incubated with immunoabsorbed TGFß2 primary antibody; lanes 7 and 8 are proteins incubated with secondary antibody only. Note absence of specific immunoreactive bands in the blots incubated with various controls.

Measurement of TGFß Bioactivity in Uterine Luminal Flushings Collected between Days 10 and 14 of Gestation

Acid-activated uterine luminal flushings (collected on Days 10–14 of gestation) inhibited CCL64 cell [3H&; uptake. Results were expressed as percentage of control. This represents the activity of TGFßs that were in either latent or active form at the time of collection. Total (acid activated) TGFß activity on Days 11, 12, and 13 was greater than on Day 10 (p < 0.001) (Fig. 5b). Untreated uterine luminal protein (native TGFß activity) inhibited [3H&; uptake by CCL64 cells only on Days 12, 13, and 14 of gestation (Fig. 5b).



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  		FIG. 5. TGFß bioassay performed on uterine flushings collected from Days 10 to 14 of gestation. a) TGFß1 standard curve for the CCL64 (mink lung epithelial cell line). b) TGFß bioassay in uterine flushings. Equivalent amounts of protein were added to each well (n = 3–5 per day). Native activity represents active TGFßs in the unaltered fluids at the time of collection. Total activity represents bioactivity after acid activation of total TGFßs present in uterine flushings at the time of collection. Different letters (native) or symbols (total) indicate differences (p < 0.001–0.05) among different days of gestation. c) Representative pan-specific TGFß-neutralizing antibody results. Control represents equivalent amount of unaltered uterine flush proteins added to the cells. N.Ab represents uterine flush proteins preincubated with pan-specific TGFß-neutralizing antibody. IgG represents uterine flushing preincubated with IgG of the same species, class, and concentration as the N.Ab.

Addition of pan-specific TGFß-neutralizing antibody to acid-activated or native samples decreased (p < 0.01) total TGFß activity in all acid-activated samples and Day 12–14 native samples (Fig. 5c). No decrease in detectable active TGFßs was detected in samples incubated with rabbit IgG or PBS only.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results demonstrate that TGFß (TGFß1, TGFß2, and TGFß3) proteins increase in ULE between Days 10 and 14 of gestation, an increase that is parallel with the TGFß mRNA increases in the porcine uterus during the same time period [10]. These results, along with the reported increase in TGFß receptor (type I and type II) expression in pregnant uterine tissue, suggest that TGFßs modulate uterine biology and conceptus-maternal interactions during this critical time of maternal recognition of pregnancy and implantation in pigs.

We detected differential expression of TGFßs 1, 2, and 3 and their receptors (type I and type II) in porcine uterus collected between Days 10 and 14 of gestation and the estrous cycle. TGFßs have been detected in human endometrium throughout the menstrual cycle [16] and in first-trimester decidua [6]. Luminal and glandular immunostaining of TGFß1 and TGFß2 has been reported for peri-implantation mouse [5] and ovine [17] endometrium, but differences between cycling and pregnant uterine tissues as in the present studies have not been previously reported. The cause(s) for these differential TGFß expressions is not known. In other cell systems, TGFßs can up-regulate their gene expression [18]. Porcine conceptuses express immunoreactive TGFßs [9] during this time, suggesting up-regulation of endometrial TGFß expression via paracrine interactions. Alternatively, steroids may also modulate the expression of TGFßs in the porcine uterus. Estradiol has been shown to up-regulate TGFß1 and TGFß3 mRNA in human endometrial stromal cells [19]. Luminal estrogens secreted by peri-implantation conceptuses [20] may have similar up-regulatory effects on TGFßs in the porcine endometrium. Increase in TGFßs in stromal cells, induced by estradiol or other mechanisms, may then lead to an increase in TGFß protein in the uterus of the pregnant pigs through paracrine stromal-epithelial interactions.

The apical immunostaining at Day 10 of gestation with the absence of cytoplasmic immunostaining suggests the possibility that the observed TGFßs may have originated from the conceptus [9], but the presence of a similar reaction in the cycling gilts lessens this possibility. Likewise, the presence of low levels of TGFß mRNA expression [10] in the ULE at Day 10 of gestation suggests a basal level of TGFß protein expression that is below the levels of detection of the immunostaining technique used in the present study.

The increase in TGFß receptor type I and type II protein expression on Days 12–14 of gestation in pregnant but not cycling gilts strengthens the hypothesis that TGFßs are actively involved in cellular signaling between conceptus and maternal systems during the peri-implantation period. The mechanisms of TGFß signaling and receptor up-regulation are not completely understood. A recent report showed that decreases in TGFß receptor expression (types I, II, and III) in mouse implantation uterus cause alterations in TGFß signaling, resulting in delayed implantation [21], which suggests an important role for TGFß signaling in the process of implantation.

The present results are the first to show the presence of bioactive TGFßs at the conceptus-maternal interface during the peri-implantation period. The growth-inhibitory activity of TGFß appeared to be specific, since the bioactivity was lost on addition of pan-specific TGFß-neutralizing antibody. In some samples, CCL64 cell thymidine uptake was more than 100% of the control value, suggesting the presence of other mitogenic compounds in the uterine luminal flushings. Because of the complex nature of the uterine luminal flushings, absolute quantitation of TGFß activity could not be made. Bioactive TGFßs were detected on Days 12, 13, and 14 of gestation, but not on Days 10 and 11 of gestation. Porcine TGFß mRNAs have been detected at Days 10 through 14 of gestation in conceptus and ULE [10], suggesting that active TGFßs in uterine luminal fluids could have uterine or conceptus origin. Bioactive TGFßs at the conceptus-maternal interface may be relevant to maternal recognition of pregnancy and the onset of conceptus attachment to the ULE that occur during this time. The basis for this increased bioactivity is not known; however, proteases activate TGFßs in vitro [22], and one can speculate that TGFßs at the conceptus-maternal interface are activated by proteases secreted by the trophectoderm [23, 24]. Thus it is possible that conceptus controls TGFß activity and its influence on maternal epithelial cells.

As expected, the predominant TGFß-immunoreactive proteins in the uterine luminal flushings, as detected by Western blotting, were the 12.5-kDa (monomeric) and 25-kDa (dimeric) forms of the TGFß proteins. Additionally, high-molecular-weight immunoreactive protein was detected, and on Days 13 and 14 of gestation, 20- to 23-kDa protein bands were detected. Although the precise identity of these proteins remains to be determined, negative results of a search for homologous regions between the TGFß peptides and other known proteins (data not shown), and elimination of immunoreactive proteins by immunoadsorption with TGFß control peptides, support the identity of these proteins as TGFß, either in alternative forms or bound to other proteins from which they were incompletely dissociated during denaturing. TGFßs associate with numerous high-molecular-weight proteins, such as heparin [25], 60-kDa TGFß-binding protein that associates with heparin sulfate proteoglycans [26], human {alpha}2-HS glycoprotein, bovine fetulin [27], {alpha}2-macroglobulin [28], collagen type IV, fibronectin, decorin, and thrombospondin [29]; the standard SDS-PAGE denaturing conditions may have been inadequate to completely disrupt these associations.

The significance of the 20- to 23-kDa bands that, interestingly, were detected only on Days 13 and 14 uterine luminal flushings, remains unknown. TGFß1 and TGFß3 molecules of this apparent molecular weight have not been reported; however, Clark et al. [30] have reported a 20–23-kDa low-molecular-weight bioactive form of TGFß2 released from bone marrow-derived natural suppressor cells [30, 31]. The presence of a similar 20–23-kDa TGFß2-immunoreactive protein in the uterine luminal flushings at a time when increased TGFß bioactivity is present at the conceptus-maternal interface suggests the presence of a similar bioactive form of TGFß in the porcine uterus.

In the present study, intense TGFß2 immunoreaction in macrophage/monocyte-like cells and other lymphocytic immune cells in the porcine uterus was observed. Immunoreactive TGFß2 has been reported in macrophages and lymphocytes of ovine endometrium [17] and mouse decidua [31]. Hunt et al. [32] have also demonstrated macrophages in uterine glandular epithelium and demonstrated that TGFß from macrophages down-regulates expression of tumor necrosis factor {alpha}, interleukin-6, and interleukin-1 during the peri-implantation period [33]. These results suggest an immunosuppressive role for TGFß2. Immunosuppressive activity of TGFß2 and TGFß1 in the porcine uterus on Day 15 of gestation [34] has been reported, supporting immunological roles for TGFßs during pregnancy in swine.

Although the precise role(s) of TGFßs during the porcine peri-implantation period is not clear, they may mediate conceptus attachment to the uterine surface. TGFß strongly stimulates the expression of various ECM proteins like fibronectin, various collagens, proteoglycans, and their integrin receptor subunits (e.g., {alpha}1, {alpha}2, {alpha}3, {alpha}5, {alpha}v, ß1, ß3) in many cell types [35–37]. Oncofetal fibronectin is an ECM protein intensely expressed in porcine trophectoderm and endometrium during the implantation period [11] and is known to be up-regulated by TGFß1 in human trophectoderm [38]. Analysis of porcine implantation sites indicated that integrin subunits {alpha}4, {alpha}5, {alpha}v, ß1, and ß3 were expressed at sites of trophectoderm contact with the ULE [39]. Whether or not TGFßs have a role in modulating expression of ECMs and integrins in porcine trophectoderm remains to be determined.

In summary, the results of this study and the accompanying report [10] provide strong evidence for conceptus and maternal involvement in TGFß production, as well as pregnancy-specific TGFß protein up-regulation and activation at the conceptus-maternal interface, during the critical periods of maternal recognition of pregnancy and conceptus attachment (implantation) in pigs. Ongoing studies will determine factors controlling TGFß production by the peri-implantation uterus and conceptuses and will determine specific roles of the TGFßs in early embryonic development and implantation in swine.


   ACKNOWLEDGMENTS
 
The authors thank Lin Bustamante for excellent technical assistance, and Sarah Christo and Barbara Merka for their histotechnology expertise. We thank Dr. T.L. Ott for expertise in pig management and surgery, and Dr. Bazer's laboratory for assistance with these procedures. 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.


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

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

Accepted: June 1, 1998.

Received: November 6, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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