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Articles |
a Department of Structural & Cellular Biology, University of South Alabama College of Medicine, Mobile, Alabama 36688
b Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
c Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, NIH, Research Triangle Park, North Carolina 27709
d Department of Animal Science, University of Florida, Gainesville, Florida 32611
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The placenta is a major source of relaxin in the rabbit. Placental extracts were shown to possess relaxin bioactivity [1, 5, 6], and relaxin immunostaining was demonstrated in the placental syncytiotrophoblast using antibodies to both a relaxin isolate of rabbit placenta and to porcine relaxin [5, 7]. In addition, cultured rabbit trophoblast cells were shown to undergo differentiation and transform into relaxin-containing syncytiotrophoblast that secrete relaxin [8]. However, relaxin immunostaining could not be detected in the pregnant rabbit ovary [5], and when pregnant rabbits were ovariectomized on Day 13, blood levels of relaxin were not different from those in sham controls [4]. Thus, although the ovary is the source of circulating relaxin in mammalian species such as the mouse, rat, pig, and human [9, 10], it has been ruled out as a major source of relaxin in the rabbit.
The nucleotide sequence of a preprorelaxin-like gene identified in the rabbit placenta [11] was identical to SQ10, a gene that is expressed during squamous differentiation of rabbit tracheal epithelial cells following injury [12]. Like relaxin [9, 10], SQ10 consists of an A and a B chain that are linked by two disulfide bonds [12]. The amino acid sequence of a portion of the B chain of relaxin has been conserved and represents the putative receptor-binding domain [13]. However, the amino acid sequence deduced from the nucleotide sequence of placental SQ10 [11] indicated a modification in this receptor-binding domain, and one would assume that the protein would not possess relaxin bioactivity. Since a biologically active rabbit relaxin has been isolated [1, 5, 6], the objective of this study was to determine the amino acid sequence of the B chain of a bioactive placental isolate of rabbit relaxin and to compare its sequence with that deduced from the nucleotide sequence of rabbit placental SQ10.
Relaxin has been immunohistochemically localized in rabbit placental syncytiotrophoblast [5, 7, 9]. However, these results could indicate synthesis or sequestering by receptor-mediated endocytosis. This question was addressed with in situ hybridization using a riboprobe to SQ10.
The results of these studies indicate that SQ10 is the rabbit relaxin and that the syncytiotrophoblast is a site of synthesis for this protein, suggesting a potential role for this hormone in implantation.
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MATERIALS AND METHODS |
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Rabbit placentas, obtained from Pel-Freeze, Inc. (Roger, AR), were extracted using the procedure of Griss et al. [14]. Partially frozen placentas were homogenized with a Polytron (model no. PT1035; Brinkmann Instruments, Westbury, NY) in ice-cold extraction medium (acetone:H2O:12 N HCl, 5:2.82:0.18, v:v:v) using 5 ml/g of placenta. The procedure was modified by the addition of protease inhibitors (1 mM 
-toluenesulfonyl fluoride and 10 mM EDTA, disodium salt) to the extraction medium. The homogenate was stirred for 24 h at 4°C and centrifuged at 30 000 x g for 30 min, and the precipitate was discarded. Five volumes of acetone were added to the supernatant, and this solution was stored at 4°C for 24 h. A precipitate formed and was collected by centrifugation (30 000 x g; 15 min). The precipitate was air dried and ground to a fine powder with a mortar and pestle.
Ion Exchange Chromatography
One gram of the powder was dissolved in 30 ml of column buffer (0.05 M ammonium acetate, pH 5.5) and applied to a 2.5 x 27-cm column of carboxymethyl cellulose (CM 52; Whatman, Clifton, NJ), and unbound proteins were eluted. A step-wise gradient of 0.05, 0.1, 0.3, and 0.5 M NaCl in column buffer was used to elute bound proteins. The flow rate was maintained at 1.0 ml/min. The protein eluted from each step was lyophilized, dissolved in H2O, dialyzed versus H2O, and lyophilized to a dry powder.
Western Blotting
A sample from each step of the column gradient was suspended in sample buffer (10 mM sodium phosphate [pH 7.2] and 1% SDS; with and without 5% mercaptoethanol), boiled for 1 min, and subjected to gel electrophoresis. The resolving gel consisted of 15% acrylamide (15% T, 3% Cbis) in 0.1 M sodium phosphate (pH 7.2) and 0.1% SDS. The running buffer was 0.1 M sodium phosphate (pH 7.2) and 0.1% SDS. The slab gel was run at 60 mA for 18 h and fixed in a solution of 10% acetic acid and 50% methanol. The gel was stained with 0.25% Coomassie blue in 7% acetic acid and 25% methanol and destained in 7% acetic acid and 25% methanol.
Matching sets of samples run on the same gel were not stained. Instead, these samples were electrophoretically transferred onto nitrocellulose membrane, and relaxin-immunoreactive protein bands were identified with peroxidase-antiperoxidase complex immunochemistry using antiporcine relaxin antiserum (1:250) as previously described [5].
Electroblotting and Amino Acid Sequencing
To isolate relaxin-immunoreactive protein for sequence analysis, a sample of 0.5 M NaCl eluate from the CMC (carboxymethyl cellulose) column was separated in multiple lanes on a mini-slab gel (8 x 7 x 0.075 cm) employing the Tris-tricine-SDS gel system [15]. The samples were dissolved in Laemmli's sample buffer [16] and loaded onto multiple lanes of the mini-gel (3% T, 3% Cbis stacking gel; 15% T, 3% Cbis resolving gel). The electrophoresis was carried out in Tris-tricine buffer (0.1 M Tris, 0.1 M tricine, and 0.1% SDS, pH 8.25) at 30 mA until the dye front was 1 cm from the bottom of the gel.
After separation by Tris-tricine-SDS gel, proteins were transferred electrophoretically at 70 V onto a layer of polyvinylidene difluoride (PVDF) membrane (Immobilon-PSQ; Millipore Corp., Bedford, MA) in a buffer containing 0.025 M Tris, 0.192 M glycine, 20% MeOH, pH ~8.3 [17], for 3 h at 4°C.
Proteins were visualized by staining with 0.1% Coomassie blue in 50% ethanol and 10% acetic acid for 5 min. After destaining and rinsing with distilled water, the membrane was air dried, and the protein bands of interest, i.e., bands corresponding to the reducible 7000 Mr relaxin-immunoreactive band, were excised and subjected to N-terminal amino acid sequence analysis (Protein Sequencer Model 470 A/B; Applied Biosystems, Foster City, CA) by the Protein Chemistry Core Laboratory of the interdisciplinary Center of Biotechnology Research at the University of Florida. Protein data banks were searched using the National Center for Biotechnology Information Database Search program [18].
In Situ Hybridization
Restriction enzymes, SP6 and T7 RNA polymerase, and RNase inhibitor were purchased from Promega (Madison, WI). [-35S]UTP and x-ray film were purchased from Amersham (Arlington Heights, IL). Proteinase K and RNase-free DNase I were purchased from Boehringer-Mannheim (Indianapolis, IN). NTB-2 was purchased from Eastman Kodak Co. (New Haven, CT). All general and molecular biology grade chemicals were purchased from Fisher (Pittsburgh, PA).
Implantation sites were collected from pregnant rabbits (Day 28 of gestation) and fixed in 4% paraformaldehyde overnight. Tissues were washed with 50% ethanol and 70% ethanol three times each for 30 min at 4°C, dehydrated, and embedded in Paraplast Plus (Sherwood Medical Labs, St. Louis, MO). Paraffin wax blocks were cut at 5-µm serial sections and placed on Plus slides (Fisher). The slides were dried overnight at 37°C, placed in desiccated boxes, and stored at 4°C until used in situ hybridization.
A 473-base pair SQ10 cDNA fragment located between the XhoI and BamHI restriction sites in pCR II (Invitrogen Corp., Carlsbad, CA) was used to generate riboprobes from linearized plasmids. SQ10 riboprobes synthesized with T7 RNA polymerase were antisense and transcripts synthesized with SP6 RNA polymerase were sense. Plasmid template (1 µg) was added to polymerase reaction mix containing 50 mM NaCl; 40 mM Tris-HCl (pH 7.5); 6 mM MgCl2; 10 mM dithiothreitol; 40 mM spermidine; 0.5 mM each of ATP, CTP, and GTP; 50 µCi of [-35S]UTP (> 1000 Ci/mmol); 60 units RNase inhibitor; and 20–70 units T7 or SP6 RNA polymerase. Reaction mix was incubated at 37°C for 1 h. DNase I (23 units) was added and incubation continued for 15 min at 37°C to digest DNA templates. [-35S]Riboprobes were separated from unincorporated [-35S]UTP on Bio-Rad (Richmond, CA) P30 spin columns, and probes were stored at -80°C.
Tissue sections were prepared for in situ hybridization as previously described by Lee et al. [19]. Briefly, slides were incubated with proteinase K for 30 min (37°C) and washed with PBS (4°C). Slides were incubated with 0.25% acetic anhydride in 0.1 M triethanolamine to reduce nonspecific binding, dehydrated in ethanol, and dried. 35S-Labeled riboprobes were placed on each slide (50 ng/ml) and incubated at 50°C overnight. After RNase treatment to remove unbound riboprobes, slides were dehydrated, dipped in emulsion NTB-2:distilled water (1:1) for 3 sec, and dried for 2 h. Slides were then placed in a light-tight box containing desiccant and exposed for 1–3 wk (4°C). Slides were developed with D19 (Kodak), counterstained with Mayer's hematoxylin (Sigma Chemical Co., St. Louis, MO), and coverslipped. Results were analyzed with brightfield and darkfield microscopy on an Olympus (Tokyo, Japan) BX50 microscope equipped with a 35-mm camera. Controls consisted of substitution of the sense riboprobe for the antisense riboprobe.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 7-kDa protein was electrophoretically transferred from an SDS gel to a PVDF membrane, excised, and submitted to amino acid sequencing. The sequence (Fig. 3) was identical to that deduced from the nucleotide sequence of the B chain of rabbit placental SQ10 [11]. Only 8 of 33 amino acids were identical to those in the B chain of porcine relaxin. The most significant difference between SQ10 and relaxin from 23 other species was observed in the receptor-binding domain in the B chain. In relaxin, this domain is GRXXXR, where X represents a variety of amino acids. In SQ10, this domain is RXXXXR.
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Placental tissue from a Day 28 pregnant rabbit was processed for in situ hybridization using a riboprobe to SQ10. Labeling was observed in the syncytiotrophoblast of the placental villi, but it was absent over the amnion, chorion, or decidua (Fig. 4, A–D). No labeling was observed with the sense riboprobe control (Fig. 4E). The pattern of in situ hybridization labeling over the syncytiotrophoblast was similar to the pattern of immunostaining using antiserum to porcine relaxin (Fig. 4F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A 7-amino acid sequence (B11 to B17) that represents the putative relaxin receptor-binding domain has been conserved in the B chain of relaxin from 23 species. This segment consists of glycine, arginine, three amino acids, and arginine. The arginines are the prongs for ligand binding [13]. The amino acid sequence of the B chain of purified rabbit placental SQ10 is arginine, four amino acids, and arginine. Although the flexibility of the receptor segment may be compromised by the loss of glycine, it may be compensated for by separating the arginines by four instead of three amino acids. Although this difference may, in part, account for the lower specific bioactivity of the rabbit relaxin as compared to porcine relaxin [5, 6], the potency of relaxin is different from species to species in which the 7-amino acid segment has been conserved. For example, rat relaxin has a lower specific activity in the mouse interpubic ligament relaxin bioassay than does porcine relaxin, and shark relaxin is inactive [9].
Our studies suggest that SQ10 is the rabbit relaxin. Polymerase chain reaction amplification of rabbit placental RNA encoding the product that hybridized with relaxin oligonucleotide probes revealed only SQ10 [11]. In this current study, the 7-kDa protein was the only immunoreactive protein band in the bioactive fraction, and there were no secondary amino acid sequences in the immunoreactive protein band used for sequencing.
The processing of SQ10 appears to be different in the trachea and in the placenta. Although there are amino acid motifs in SQ10 C peptide to A chain (arginine 151; lysine 152, 153; arginine 154) and C peptide to B chain (arginine 54) that could be cleavage sites for a trypsin-like endopeptidase, tracheal epithelial cells appear to lack a convertase necessary for processing the prohormone. Using our antibody with Western blotting, only those forms with a molecular weight similar to that of the prohormone were observed in tracheal extracts or culture medium from tracheal epithelial cells [12]. Although higher molecular weight forms of rabbit placental relaxin have been reported [20], in this current study and in previous studies [5, 6], the hormone form was observed in placental extracts.
Rabbit tracheal epithelial cells express SQ10 during the differentiation and transformation of injured pseudostratified columnar epithelial cells into stratified squamous epithelial cells and may be involved in the wound-healing process [12]. Although the role of rabbit placental SQ10 is unknown, our studies suggest a role in implantation. The in situ hybridization labeling pattern for SQ10 in syncytiotrophoblast cells, the differentiated and transformed trophoblast cells of the placental villus, was identical to the pattern observed with relaxin immunostaining using porcine relaxin antiserum. During early implantation, relaxin immunostaining has been reported also in differentiated trophoblast cells at the antimesometrial attachment sites (knob cells) and in differentiated trophoblast cells (symplasm) at the mesometrial implantation sites [21].
Relaxin promotes epithelial cell proliferation in the rat mammary gland lactiferous ducts and cervix, and it remodels connective tissue in the rat and pig uterus, mammary gland, and cervix [10]. Thus, as a product of differentiated trophoblast cells that are involved in blastocyst attachment, implantation, and placental expansion, SQ10 may act in a paracrine manner to remodel endometrial connective tissue in order to promote these processes. In addition, relaxin has been shown to promote increased vascularity in the rat cervix [22], mouse mammary gland [23], and human uteroplacenta [24]. Thus, another potential role for relaxin is to increase blood flow at the early implantation site that would support the developing fetus.
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FOOTNOTES |
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Accepted: March 25, 1999.
Received: January 6, 1999.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
