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Regular Article |
a Center for Animal Biotechnology and Genomics, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
ABSTRACT
Epithelial-mesenchymal interactions (EMI) are necessary for epithelial cell proliferation, differentiation, and function in the uterus and are mediated, in part, by paracrine growth factors of stromal origin. The objective of this study was to determine if hepatocyte growth factor (HGF, scatter factor) and its receptor c-met were present in the ovine uterus and to characterize their temporal and spatial expression during the estrous cycle and pregnancy. Reverse transcription-polymerase chain reaction was used to clone partial cDNAs for ovine HGF and c-met from endometrial total RNA. Northern blot analysis of endometrial RNA revealed expression of a 6-kb mRNA for HGF and an 8-kb mRNA for c-met in ovine endometrium. In situ hybridization demonstrated that HGF mRNA was expressed by stromal cells of the endometrium, whereas c-met mRNA was localized exclusively to luminal and glandular epithelial cells. In the early conceptus, HGF mRNA was expressed by chorioallantoic mesenchyme, and c-met was expressed by trophectoderm. Steady-state levels of endometrial c-met mRNA increased after Day 9 in both cyclic and pregnant ewes. The HGF mRNA was expressed during both the estrous cycle and early pregnancy. Results indicate that HGF is a stromal-derived paracrine growth factor in the ovine uterus and placenta that is potentially involved in endometrial epithelial-stromal interactions and chorioallantoic stromal-trophectodermal interactions. In the ovine uterus, HGF may stimulate epithelial morphogenesis and differentiated function required for establishment and maintenance of pregnancy, conceptus implantation, and placentation.
Epithelial-mesenchymal interactions (EMI) have been implicated in morphogenesis and differentiated function of the uterus, a steroid hormone-responsive organ [1, 2]. Several ruminant endometrial cell types, including luminal (LE) and glandular epithelium (GE), stromal (ST) fibroblasts, myometrium, endothelium, and immune cells, are involved in paracrine interactions with one another. Stromal cells regulate proliferation, differentiation, and function of the epithelium. Reciprocal EMI are required to ensure the correct organization and function of the stroma as well as myometrium [3].
Coordinated actions of ovarian steroids, progesterone and estrogen, are essential to stimulate endometrial function via their respective nuclear receptors, progesterone receptor (PR) and estrogen receptor alpha (ER
). In the ovine uterus, PR and ER are expressed in all cell types and undergo temporal and spatial changes during the estrous cycle and pregnancy [4, 5]. Progesterone, the hormone of pregnancy, negatively autoregulates expression of its own receptor in the uterine LE and GE [5]. The absence of PR in uterine LE and superficial GE during the peri-implantation period has been observed in a number of mammalian species including sheep [5–7]. In the ovine uterus, endometrial PR gene expression can be detected in LE and GE on Days 9 and 11 of pregnancy and then declines to undetectable levels after Day 13 during the peri-implantation period [5]. From Day 25 of pregnancy to term, PR and ER expression is not detectable in uterine epithelia (unpublished observations). However, PR and ER expression is detectable in ST and myometrium throughout pregnancy [5] (unpublished observations). These observations strongly suggest that PR- and ER-positive stromal and myometrial cells mediate effects of progesterone and estrogen on ovine uterine epithelial cell function through production of paracrine growth factors.
Hepatocyte growth factor (HGF or scatter factor) is a pleiotropic mesenchymal growth factor that has potent mitogenic, morphogenic, and motogenic activities on cells of epithelial origin [8–10]. It is also an angiogenic factor [11] and an inhibitor of tumor growth and invasion [12]. The effects of HGF are mediated by c-met, a transmembrane type I tyrosine kinase receptor expressed in a variety of |epithelial tissues [13, 14]. Expression of HGF and c-met has been observed in many fetal and adult tissues including the uterus and placenta [15]. Recent evidence indicates that HGF regulates human endometrial epithelial cell proliferation and motility [16] and is a candidate mediator of estrogen actions (i.e., estromedin) in the primate uterus [17].
To date, HGF expression has not been described in any ovine tissues and organs. In order to determine the presence of HGF in sheep and investigate its potential role as a mediator of uterine epithelial–mesenchymal interactions, partial ovine HGF, and c-met cDNAs were cloned from ovine endometrial total RNA and used to examine temporal and spatial alterations in their expression in the ovine uterus during the estrous cycle and early pregnancy.
MATERIALS AND METHODS
Animals and Tissue Collection
All experimental and surgical procedures involving animals were approved by the Institutional Agricultural Animal Care and Use Committee, Texas A&M University (Animal Use Protocols 7–286 and AG-239). Mature ewes of primarily Rambouillet breeding were observed daily for estrus (Day 0) using vasectomized rams. After exhibiting at least two estrous cycles of normal duration (16–18 days), ewes were assigned on Day 0 to either cyclic or pregnant status. Ewes assigned to pregnant status were mated to intact rams at estrus and 12 h and 24 h later. Ewes were hysterectomized (n = 4 ewes/day) on Days 1, 3, 5, 7, 9, 11, 13, or 15 of the estrous cycle and Days 11, 13, 15, 17, 19, or 25 of pregnancy as described previously [18]. At hysterectomy, sections of the uterine wall from the midportion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). The remaining endometrium was physically separated by dissection from myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA isolation and analyses.
Reverse Transcription-Polymerase Chain Reaction Cloning of Partial cDNA for Ovine HGF and c-met
Total cellular RNA was isolated from Day 1 cyclic endometrial tissue using the Trizol reagent (Gibco-BRL, Grand Island, NY). Partial cDNAs for ovine HGF (328 bp) and c-met (450 bp) were amplified by reverse transcription–polymerase chain reaction (RT-PCR) from endometrial total RNA using primers based on human HGF (M60718) and rat c-met (X96786) sequences (HGF forward, 5'-ATTTGGCCATGAATTTGACC; reverse, 5'-TCGATAACTCTCCCCATTGC; c-met forward, 5'-CGGTCTTCAAGTAGCCAAGG; reverse, 5'-ACCAGTTCAGAAAACGGATGG). The PCR conditions were 1) 94°C for 6 min; 2) 1 min at 94°C, 2 min at 55°C, and 1 min at 72°C; for 34 cycles; and 3) 72°C for 10 min. The PCR products of the appropriate size were cloned using a T/A Cloning Kit (Invitrogen, Carlsbad, CA) and sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems, Foster City, CA) and an ABI PRISM automated DNA sequencer (P-E Applied Biosystems).
Northern Blot Analysis
Total cellular RNA from cyclic and pregnant endometrial tissues was isolated using Trizol (Gibco-BRL), and polyadenylated mRNA was purified from endometrial tissues using a Poly(A)+ Pure kit (Ambion, Austin, TX). In order to determine the size(s) of transcripts for HGF and c-met, endometrial polyadenylated mRNA for HGF (4 µg) or total RNA for c-met (20 µg) was separated by electrophoresis through a denaturing agarose gel, transferred to Nytran nylon membrane (Schleicher and Schuell, Keene, NH) by capillary blotting, and hybridized with a 32P-labeled antisense ovine HGF or c-met cRNA probe as previously described [19]. Autoradiographs were prepared using Kodak X-OMAT x-ray film.
Semiquantitative RT-PCR
Semiquantitative RT-PCR analysis was used to determine steady-state levels of HGF mRNA in endometrial total RNA from cyclic and pregnant ewes. First-strand cDNA was synthesized from total endometrial RNA isolated from each cyclic and pregnant ewe as described previously [20]. The cDNA was diluted with water (1:10 and 1:100) for use as template in PCR reactions. The PCR reactions contained 100 ng of each primer, 1 U AmpliTaq DNA polymerase (Perkin Elmer), 1 mM dNTPs, 5 µl RT cDNA (1:10 for HGF and 1:100 for ß-actin) in optimized buffer (Invitrogen, Carlsbad, CA; buffer F for HGF and buffer D for ß-actin). The dilution and volume of cDNAs used in PCR reactions were determined to be in the linear range to ensure that the amount of PCR product derived from a given amount of total RNA in a sample was directly proportional to the concentration of target mRNA in the sample. The same primers indicated above were used to amplify HGF (328 bp). Amplification of ß-actin (420 bp) was used as an internal control to correct for differences in the amount of RT cDNA used in each reaction. Primer sequences for ß-actin were: forward, 5'-CATCCTGACCCTCAAGTACCC; reverse, 5'-GTGGTGGTGAAGCTGTAGCC. The HGF and ß-actin PCR conditions were 30 cycles at 95°C for 30 sec, 55°C for 1 min, and 72°C for 1 min. The PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The DNA fluorescence in correctly sized bands was densitometrically quantitated using a video documentation system and the image analysis software (Alpha Innotech Corporation, San Leandro, CA). The ß-actin values were used as a covariate in statistical analyses to correct for differences in amounts of cDNA used for each PCR reaction and are expressed as relative light units.
Slot-Blot Hybridization Analysis
Steady-state levels of c-met mRNA were measured in endometrial samples from each cyclic and pregnant ewe using slot-blot analysis as described previously [19]. Total cellular RNA (20 µg) from each ewe was hybridized with radiolabeled antisense ovine c-met or 18S rRNA (pT718S; Ambion, Austin, TX) cRNA probes generated by in vitro transcription as previously described [19]. The radioactivity in each slot was quantitated by electronic autoradiography using an Instant Imager (Packard, Meriden, CT).
In Situ Hybridization Analysis
In situ hybridization was performed as previously described [21]. Uterine tissue sections (4–5 µm) from each ewe were hybridized with antisense or sense 35S-riboprobes for ovine HGF or c-met. After hybridization and washing procedures, slides were dipped in NBT-2 liquid photographic emulsion (Kodak, Rochester, NY) and stored at 4°C for 4 wk for HGF and 2 wk for c-met, respectively. Slides were then developed in Kodak D-19 developer and counterstained with hematoxylin.
Photomicroscopy
Digital photomicrographs of in situ hybridization slides were captured using a Zeiss Axioplan2 photomicroscope (Carl Zeiss, Inc., Thornwood, NY) fitted with a Hamamatsu chilled 3CCD color digital camera (Hamamatsu Corporation, Bridgewater, NJ). Digital images were assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA). Black and white photomicrographs were electronically printed using a Kodak DS8650 color printer.
Statistical Analysis
All quantitative data were subjected to least-squares ANOVA using the general linear models procedures of the Statistical Analysis System [22]. Analyses of the RT-PCR data included the ß-actin data as a covariate. Similarly, total counts were adjusted for differences in sample loading using 18S rRNA data as a covariate in slot-blot analysis. All tests of statistical significance were performed using the appropriate error terms according to the expectation of mean squares of error. Data are presented as least-square means with standard errors.
RESULTS
Expression of HGF and c-met mRNA was detected in ovine endometrium using RT-PCR. Partial cDNAs for ovine HGF and c-met were cloned and sequenced. The 328-base ovine HGF mRNA (GenBank accession number AF213396) shared high nucleotide sequence homology with that of human (96%), mouse (95%), and rat (94%). The 450-base sequence of ovine c-met mRNA (GenBank accession number AF213397) was 93%, 87%, and 86% identical to the sequences of the same respective species.
Northern blot analysis revealed a 6-kb transcript for HGF and an 8-kb transcript for c-met in RNA from the ovine endometrium (Fig. 1, A and B). The sizes of ovine HGF and c-met mRNA were similar to those of human [8, 23] and rat [24, 25].
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As illustrated in Figure 2, semiquantitative RT-PCR analysis indicated that steady-state levels of HGF mRNA expression (Fig. 3A) in the endometrium of cyclic ewes was highest on Days 1 and 5, decreased from Days 7 to 13, and then increased on Day 15 (P < 0.02, cubic). In pregnant ewes, endometrial HGF mRNA expression decreased between Days 11 and 13, increased from Day 13 to maximal levels on Days 15 and 17, and then decreased on Day 19 (P < 0.06, cubic). On Days 11, 13, and 15, HGF mRNA expression was not affected by pregnancy status (day x status, P > 0.10).
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As illustrated in Figure 3B, steady-state levels of endometrial c-met mRNA expression in cyclic ewes was low between Days 1 and 7 and then increased to maximal levels on Day 13 of the estrous cycle (P < 0.10, cubic). In pregnant ewes, c-met mRNA increased between Days 11 and 15, remained high through Day 17, and then decreased by Day 19 (P < 0.10, quadratic). On Days 11, 13, and 15, c-met mRNA levels were not affected by pregnancy status (day x status, P > 0.10).
In situ hybridization analyses revealed that HGF mRNA was expressed exclusively by the endometrial stroma, because hybridization signals above the sense control were not detected in the endometrial epithelium (Fig. 4) or myometrium (data not shown). Interestingly, the HGF mRNA was predominantly expressed in the stroma cells of the stratum compactum of the intercaruncular endometrium. Lower levels of HGF mRNA expression were detected in the stratum spongiosum stroma of the intercaruncular endometrium as well as in the aglandular caruncular areas. The expression of HGF mRNA in myometrium was either very low or at background levels. Low levels of HGF mRNA expression were detected in the chorioallantoic mesenchyme of the placenta on Day 25 of pregnancy.
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In situ hybridization analyses revealed that c-met mRNA was expressed exclusively in uterine LE and GE (Fig. 5). As compared to the sense control, c-met mRNA expression was not detected in stroma, myometrium, or endothelia of blood vessels. Although c-met mRNA expression did not differ in the caruncular and intercaruncular endometrial LE, differential expression of c-met mRNA was detected in the GE of the upper stratum compactum as compared to the lower stratum spongiosum. Expression of c-met mRNA in the upper GE was similar to that in the LE but was much lower in the deeper GE. In particular, levels of c-met mRNA levels in deep GE were lower than in shallow GE. In pregnant ewes, c-met mRNA expression was also detected in the trophectoderm of the chorioallantoic placenta, but overall levels appeared lower than in the adjacent uterine LE.
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DISCUSSION
This is the first report of HGF and c-met mRNA expression in the ovine uterus. Northern blot analysis revealed that a 6-kb mRNA was the primary transcript for ovine HGF in the ovine endometrium, although HGF may have alternative transcripts and protein isoforms [26]. Due to the low expression of HGF mRNA, semiquantitative RT-PCR, a more sensitive method in evaluating mRNA, was used to examine steady-state levels of HGF mRNA in ovine endometrium during the estrous cycle and early pregnancy. In situ hybridization analysis indicated that c-met mRNA was expressed exclusively by LE and GE, whereas HGF mRNA localization was restricted to stromal cells. This in vivo mRNA expression pattern strongly suggests that HGF is a paracrine growth factor candidate for a stromal mediator of ovine uterine epithelial cell functions. Hepatocyte growth factor has been reported to stimulate proliferation, migration, and lumen formation of human endometrial epithelial cells in culture [16]. Because HGF is a potential stromal-derived growth factor with receptors specific to uterine epithelial cells in sheep, we infer that HGF may have a role in proliferation, differentiation, and function of the uterine epithelia that occurs during pregnancy [27]. Based on the differential levels of c-met expression by the GE and HGF in the intercaruncular endometrium, the upper GE and LE may be more primary targets for the paracrine actions of HGF from the stratum compactum. These observations, together with the proliferative and morphogenic effects of HGF on mesenchymal-epithelial tissues such as mammary gland and lung [28, 29] suggest that HGF may be important in uterine gland proliferation, tubulogenesis, and branching morphogenesis that occurs during early pregnancy.
The HGF gene knockout experiments have demonstrated that HGF is an essential mediator of chorioallantoic mesenchymal-trophoblastic interactions required for placental organogenesis in the mouse [30]. In the present study, the c-met mRNA expression in trophectoderm and HGF mRNA expression in allantoic mesenchyme suggests a similar role for HGF in placental development and embryogenesis in the sheep. Further, the loss of the uterine LE during synepitheliochorial placentation during early pregnancy suggests that there may also be cross-talk between HGF from endometrial stroma and c-met in trophectoderm to direct conceptus development. The hormonal regulation of HGF in ovine endometrium is unknown. However, estrogen response elements have been identified in mouse HGF promoter, and HGF is transcriptionally up-regulated by estrogen in mouse ovaries and primate endometrium [17, 31]. Based on temporal aspects of ovine endometrial HGF mRNA expression, HGF mRNA increased on Day 15 of the cycle when PR are absent in the uterine epithelia and ER are present [4, 5]. However, HGF mRNA remains high during early pregnancy when PR remain high and ER are low to undetectable in stromal cells. Therefore, progesterone, the hormone of pregnancy, may play a role in regulation of HGF expression by uterine stromal cells that express PR. Similarly, steady-state levels of c-met mRNA increase in endometrial epithelia as circulating levels of progesterone increase and epithelial cell PR decrease during the estrous cycle and early pregnancy, implicating a role for progesterone in c-met mRNA regulation perhaps through progesterone-induced down regulation of PR. It has been demonstrated that inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor alpha can up-regulate mRNAs for both HGF and c-met [32]. Therefore, the expression of HGF and c-met may be coordinated by the actions of ovarian steroids and cytokines in a complex network. In vivo and in vitro experiments need to be conducted to elucidate the regulation of HGF and c-met expression in the ovine uterus by sex steroids and/or cytocines.
Hepatocyte growth factor is a heparin-binding glycoprotein secreted as an inactive single-chain precursor that is present in the extracellular matrix (ECM) [33]. Formation of physiologically active HGF heterodimers requires proteolytic cleavage of this precursor by various HGF convertases, including HGF activator and other serine proteases [34, 35]. The activity of HGF protein is, in part, regulated by this localized proteolytic activation. At present, HGF protein has not been localized in the ovine uterus. Whether it is secreted, accumulates in the ECM, or is activated and released by proteolytic processing remains to be determined. In addition, alterations in the endometrial ECM of the ovine uterus during the estrous cycle and early pregnancy are virtually unknown but represent potential mechanisms regulating actions of this paracrine growth factor network.
In conclusion, our results are the first to demonstrate that HGF and c-met mRNAs are expressed in the ovine uterus. Hepatocyte growth factor is a potential stromal mediator of uterine epithelial cell functions. Recently, fibroblast growth factor-10 (FGF-10) was also identified as a stromal-derived paracrine growth factor that may regulate ovine endometrial epithelial cell function, and FGF-7 (KGF) may also function in a similar manner (unpublished observations). Epithelial–mesenchymal interactions are important for maintaining the integrity of uterine function. Therefore, unique stromal-derived growth factors such as HGF, FGF-10, and KGF may play important roles in these interactions and may be critical for uterine epithelial cell functions and trophectoderm/chorion function necessary for the establishment and maintenance of pregnancy.
ACKNOWLEDGMENTS
We thank Margaret Joyce for technical assistance and Dr. Greg Johnson, Dr. JoAnn Fleming, and Hakhyun Ka for advice and comments. The authors thank Dr. Shawn W. Ramsey and Mr. Todd Taylor of the Texas A&M University Sheep and Goat Center for care and management of ewes.
FOOTNOTES
First decision: 10 January 2000.
1 Photomicrographs and digital images were prepared using facilities in the Texas A&M University College of Veterinary Medicine Image Analysis Laboratory that is supported, in part, by NIH grant P30 ES09106. 
2 Correspondence: Fuller W. Bazer, Center for Animal Biotechnology and Genomics, 442D Kleberg Center, Texas A&M University, College Station, TX 77843-2471. FAX: 409 862 2662; fbazer{at}cvm.tamu.edu
Accepted: January 28, 2000.
Received: December 15, 1999.
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