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Biology of Reproduction 60, 305-311 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Nucleic Acid Sequence of Feline Preprorelaxin and Its Localization within the Feline Placenta1

Thomas Klonisch2,a, Sabine Hombach-Klonischa, Christine Froehlicha, Johannes Kauffoldb, Klaus Stegera, Berthold Huppertzc, and Bernd Fischera

a Department of Anatomy and Cell Biology, Faculty of Medicine, Martin Luther University of Halle-Wittenberg, D-06097 Halle (Saale), Germany b Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany c Institute of Anatomy, RWTH Aachen, D-52057 Aachen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cat placenta is known to secrete large amounts of relaxin. We employed uteroplacental tissue at approximately Day 35 of gestation to determine the nucleic acid sequence of feline preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Feline preprorelaxin cDNA was found to consist of 540 base pairs encoding a protein of 180 amino acids (aa). We identified a signal peptide of 25 aa, a B domain of 33 aa, a C domain of 98 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the B domain contained one substitution from the classical GRELVR motif (L->F). Feline preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a specific 1-kilobase transcript present in total RNA of feline uteroplacental tissue but not of liver tissue. Nonradioactive in situ hybridization was used to localize relaxin mRNA, and immunohistochemistry was used to localize the relaxin hormone and cytokeratin, in tissues of the feto-maternal interface recovered from two queens at Day 35 of gestation. Specific hybridization signals for relaxin mRNA were exclusively detected in cells located in the lamellar placental labyrinth but were absent from other placental and nonplacental uterine parts. The cells expressing relaxin mRNA also displayed immunoreactivity for cytokeratin and were, therefore, identified as trophoblast cells. Immunoreactive relaxin colocalized in those placental areas expressing relaxin mRNA. Trophoblast cells located at the villous chorioallantoic tips invading the endometrium and extravillous trophoblast cells in the junctional placental zone were devoid of relaxin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterodimeric polypeptide hormone relaxin is a member of the insulin-like superfamily, which also includes insulin, the insulin-like growth factors I and II, and the relaxin-like factor. All members of this family of growth factors appear to be synthesized as a preprohormone consisting of a signal peptide and B, C, and A domains, from the N to the C terminus, respectively. The relaxin molecules in different species display significant differences in their nucleic acid and amino acid sequences but always contain the conserved amino acid motif -R-X-X-X-R- close to the first cysteine of the B-peptide, which was shown to be important for binding to the relaxin receptor [1].

Relaxin attains the highest plasma levels during pregnancy and is known to have important functions during parturition [2, 3]. In the pregnant uterus, fetal trophoblast cells have been identified as the source of relaxin in the mare [4, 5], the rabbit [6], the golden hamster [7, 8], and the human [9]. In the guinea pig, relaxin has been localized in endometrial glands [10, 11]; and, in various species, endometrial granulocytes have also been found to contain relaxin [8]. Concentrations of relaxin detected in rabbit plasma during the periimplantation period seem to indicate a possible involvement of relaxin in implantation in that species [12].

Information is lacking on the sequence, expression, and possible functions of relaxin in feline species, including the domestic cat. Relaxin immunoreactivity in various feline tissues has revealed the feto-placental unit to be a major source of relaxin [13]. In the pregnant queen, relaxin serum immunoreactivity first becomes detectable at about Day 20 of gestation and reaches peak levels at about Day 35 of gestation. Thereafter, serum relaxin levels remain stable but decline rapidly shortly before parturition at around Day 60 of gestation [14]. In aborting pregnant queens suffering from dietary taurine deficiency, placental relaxin immunoreactivity started to deviate from normal by Day 25 of gestation [14].

In the study reported here, we employed uteroplacental tissue at approximately Day 35 of gestation to determine the cDNA sequence of feline preprorelaxin. Nonradioactive in situ hybridization was performed to localize relaxin mRNA transcripts, and immunohistochemistry was employed to detect relaxin and to characterize the placental cells expressing relaxin mRNA in the feto-placental unit of the pregnant queen.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Tissues and RNA Isolation

Uteroplacental tissues from two pregnant domestic cats (Felis catus) at approximately Day 35 of gestation, as determined by measurement of the crown-rump length of the fetus, were collected after hysterectomy. Feline liver was obtained from a euthanized nonpregnant animal and served as control tissue. All cat tissues used were obtained during the course of medically indicated procedures, and no animal was operated upon or killed specifically for this study. All tissues had been snap-frozen in liquid nitrogen and stored at -80°C until used. Total RNA was isolated with Trizol reagent (Life Technologies, Eggenstein, Germany). For the cloning of feline relaxin, mRNA was isolated from 75 µg of total uteroplacental RNA using oligo(dT)-coated magnetic beads according to the manufacturer's instructions (Dynal, Hamburg, Germany). The amount of mRNA isolated was determined by spectrophotometry at 260 and 280 nm [15].

Cloning of Feline Preprorelaxin by Reverse Transcription (RT)- and Rapid Amplification of 5'- and 3'-cDNA Ends (5'- and 3'-RACE)-Polymerase Chain Reaction (PCR)

We employed RT-PCR and 5'- and 3'-RACE-PCR to clone the cDNA for feline preprorelaxin from mRNA of uteroplacental origin. All PCR primers employed in this study (Table 1) flanked the putative single intron present at the N terminus of the C domain of relaxin to preclude any genomic DNA amplification (Fig. 1). Approximately 600 ng of mRNA was used for first-strand cDNA synthesis employing the Superscript reverse transcriptase kit and 500 ng/ml of oligo(dT) primer (both Life Technologies). PCR reactions were carried out in 50 µl of solution containing 1 µl of cDNA, 5 µl of 10-strength Advantage cDNA polymerase mix buffer, 100 µM of dNTP, 10 pmol of each primer (Table 1), and 2.5 U Advantage cDNA mix polymerase (Clontech, Heidelberg, Germany). The degenerate oligonucleotide primer pair 1 and 2 (Table 1), designed according to a relatively conserved amino acid region at the N terminus of the B domain and the C-terminus part of the A domain of porcine, human, and rat relaxin cDNA, was used for the initial amplification of a cDNA fragment of feline relaxin. The PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 56°C (both for 1 min each) and an elongation step for 2 min at 72°C, and a final extension cycle for 10 min at 72°C. The 3'- and 5'-RACE-PCR reactions were performed according to the manufacturer's instructions (Life Technologies) employing the gene-specific primers 5–8 and a universal primer (primer 9; Life Technologies) listed in Table 1. RACE-PCR reactions were run for 35 cycles at an annealing temperature of 68°C. Finally, the complete cDNA of feline preprorelaxin was amplified by RT-PCR for 40 cycles at an annealing temperature of 68°C using the specific primers 3 and 4 located at both ends of the coding sequence of feline preprorelaxin (Table 1). All PCR products were separated on a 1% low-melting point agarose gel, purified by Magic column extraction, and cloned into the pGEM-T vector (both Promega, Heidelberg, Germany). Sequence analysis of the PCR-amplified cDNA clones was performed employing the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer, Foster City, CA).


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  		TABLE 1. List of primers employed for RT-PCR and 5'- and 3'-RACE-PCR to clone the full-size cDNA of feline preprorelaxin.



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  		FIG. 1. Schematic representation of the results of the nucleic acid sequence analysis of feline preprorelaxin. Numbered arrows indicate the primers specified in Table 1 used for cDNA cloning by RT- and RACE-PCR techniques.

Northern Analysis

For Northern blot analysis, total RNA isolated from uteroplacental and liver tissues were run in a formaldehyde gel with both unlabeled RNA molecular weight markers (RNA ladder 0.44–1.77 kb; Life Technologies) and digoxigenin (DIG)-labeled RNA markers (Boehringer Mannheim, Mannheim, Germany). Gel sections for hybridization were blotted onto Hybond N+ nylon membranes (Amersham, Braunschweig, Germany) for 1.5 h by the downward alkaline blotting technique [16]. Membranes were UV-flashed, washed for 10 min in 4-strength SSC (4-strength SSC is 600 mM NaCl, 60 mM sodium-acetate, pH 7.0), dried for 2 h at 80°C, and then prehybridized for 30 min at 62°C in DIG-hybridization buffer (Boehringer Mannheim). The prehybridization solution was replaced with an equal volume of the same solution containing approximately 300 ng/ml of DIG-labeled cRNA probe, and hybridization continued overnight at 62°C. Membranes were washed twice for 15 min each in double-strength SSC/0.1% SDS at room temperature, and then twice for 20 min each in 0.5-strength SSC/0.1% SDS at 60°C before being processed for detection of chemiluminescence according to the manufacturer's instructions (Boehringer Mannheim).

DIG Labeling of cRNA and In Situ Hybridization

Synthesis of DIG-labeled cRNA has been described previously [5]. For nonradioactive in situ hybridization, feline uteroplacental cryocut sections (6 µm thick) attached to glass slides coated with 2% aminopropyltriethoxysilane (APTEX; Sigma, Deisenhofen, Germany) were processed according to the procedure described by Lewis and Wells [17] employing a 1:1000 dilution of an anti-DIG alkaline phosphatase-conjugated Fab antibody (Boehringer Mannheim) in 1% BSA. Specific signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (both Sigma). After counterstaining with hematoxylin, the slides were mounted in glycerol gel and examined under brightfield microscopy.

Immunohistochemistry

Immunohistochemical staining was performed on 6-µm-thick cryostat sections of OCT (Bayer Corp., Tarrytown, NY)-embedded feline uteroplacental tissue mounted on slides coated with 2% APTEX and allowed to air-dry overnight. The tissue sections were fixed in acetone for 10 min at 4°C, and endogenous alkaline phosphatase activity was inactivated by incubation in 20% acetic acid at 4°C for 15 sec. The tissue sections were blocked with 3% BSA in 0.5 M Tris-buffered saline (0.5 M TBS) for 30 min to saturate nonspecific binding sites. The primary mouse monoclonal antibody (Dako, Hamburg, Germany) to cytokeratin (MNF-116) at a concentration of 1:1000 or the rabbit polyclonal anti-relaxin antiserum R6 (generously provided by Prof. B.G. Steinetz, New York University Medical Center, NY) at 1:4000 were diluted in 0.5 M TBS containing a combination of 3% BSA plus 3% nonimmune goat serum. Sections were incubated overnight at 4°C in a moist chamber and then washed 3 times for 10 min each in 0.1 M TBS. Negative controls were performed in all cases by omitting the primary antibody. For relaxin immunohistochemistry, sections were also incubated with rabbit nonimmune serum as primary antibody instead of rabbit polyclonal anti-relaxin antiserum R6. The alkaline phosphatase anti-alkaline phosphatase (APAAP)-technique was employed for immunodetection of cytokeratin and feline relaxin. Sections were first incubated for 1 h at room temperature in 0.5 M TBS containing 1% BSA with an alkaline phosphatase-conjugated rabbit anti-mouse antibody (Z-0259; Sigma) at 1:50 or an alkaline phosphatase-conjugated goat anti-rabbit (Dianova, Hamburg, Germany) at 1:100. Sections were washed and incubated for 30 min at room temperature with a mouse APAAP (Dako) or rabbit APAAP (Sigma), respectively, both at 1:100 in 0.5 M TBS. The incubation with alkaline phosphatase-conjugated antibodies was repeated once, and specific immunoreactivity was visualized with the alkaline phosphatase substrate Histo Mark Red (KPL, Gaithersburg, MD). Finally, sections were counterstained with hematoxylin, mounted with glycerol gel, and examined under brightfield microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We employed RT- and RACE-PCR on mRNA isolated from uteroplacental tissue at Day 35 of gestation to determine the cDNA sequence of feline preprorelaxin. Control experiments using either mRNA or water as templates in the PCR reactions yielded no amplification products (data not shown). The degenerate primer pair used for the initial amplification of a cDNA fragment of the feline prorelaxin nucleic acid sequence resulted in the amplification of a 430-basepair (bp) fragment. The complete nucleic acid sequence of the feline preprorelaxin coding region was found to consist of 540 bp. According to the known cleavage sites of other preprorelaxin and relaxin-like molecules, feline preprorelaxin consisted of a signal peptide of 25 amino acid residues, a B domain of 33 residues, a C domain of 98 residues, and an A domain of 24 residues (Fig. 1). Northern analysis of total RNA from uteroplacental tissue at Day 35 of gestation revealed a single specific hybridization signal at approximately 1 kilobase (kb). No hybridization band was obtained with total RNA isolated from feline liver (Fig. 2).



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  		FIG. 2. Northern analysis was performed employing 20 µg of total RNA extracted from feline uteroplacental tissue at Day 35 of gestation (lane 1) and feline liver (lane 2). Equal amounts of total RNA were loaded on a 1% formaldehyde gel as illustrated by ethidium bromide staining and comparison of the 18S RNA bands (data not shown). Hybridization was performed at 62°C, and stringent washes were performed for 2 x 20 min in 0.5-strength SSC at 58°C. Upon chemiluminescent detection, a specific relaxin transcript of 1 kb was visualized in total RNA of uteroplacental tissue (lane 1). No hybridization signal for relaxin transcripts was obtained in total RNA extracted from liver tissue (lane 2). The blot was computer-scanned for presentation.

Nonradioactive in situ hybridization with a DIG-labeled feline relaxin antisense cRNA on cryosections of feline uteroplacental tissue at Day 35 of gestation revealed exclusive and specific hybridization signals in cells covering the lamellar part of the fetal chorioallantoic villi in the labyrinth of the feline zonary girdle placenta (Fig. 3, A, E, and G). Those cells lining the villous tips invading the maternal endometrium and found in close apposition to the transitional placental zone were devoid of relaxin mRNA (Fig. 3, A and G). Uterine tissue located in the paraplacental regions was devoid of relaxin mRNA, as were the cells of the placental transitional zone, the layer of uterine glands, and the maternal endo- and myometrium. Sections treated with the DIG-labeled sense cRNA were also devoid of hybridization signals (Fig. 3B).



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  		FIG. 3. Cryocut sections of the feto-maternal interface of the pregnant queen were subjected to either nonradioactive in situ hybridization for the detection of feline relaxin mRNA expression (A, B, E, and G) or rabbit APAAP immunohistochemistry with a rabbit polyclonal antibody specific for relaxin (R6) (C) and mouse APAAP immunohistochemistry with a mouse monoclonal antibody specific for cytokeratin (MNF-116; F and H). The feline trophoblast cells of the lamellar placental labyrinth (l) expressed relaxin mRNA (A, E, and G). Maternal endometrium of the para- and nonplacental areas were devoid of relaxin mRNA. Within the feline zonary placenta, transitional zone (t), uterine glands (u), myometrium (m), and tissue sections treated with the sense cRNA relaxin probe (B) were also devoid of hybridization signal. The trophoblast identity of the cells expressing relaxin mRNA was confirmed by their immunoreactivity for cytokeratin (F). Cytokeratin-positive feline trophoblast cells (arrows; H) located at the tip of the placental villous (v) invading the endometrium and extravillous trophoblast cells of the transitional zone (H) did not express relaxin mRNA (A and G). We detected specific relaxin immunoreactivity (C) only in the placental labyrinth (l) containing the trophoblast cells expressing relaxin mRNA. Negative controls for the rabbit and mouse APAAP techniques employed for relaxin and cytokeratin staining were performed omitting the primary antibodies (D) and (I). A, B) x29; E, F) x145; C, D, G–I) x72. Reproduced at 94%.

The cells of the lamellar placental labyrinth expressing relaxin mRNA stained immunopositive for cytokeratin and were, therefore, identified as feline trophoblast cells (Fig. 3F). We also detected cytokeratin immunoreactivity in those cells lining the tips of the chorioallantoic villi and in cells invading the transitional placental zone (Fig. 3H). However, these trophoblast cell populations did not express relaxin mRNA (Fig. 3, A and G). Employing the relaxin antiserum R6, relaxin immunoreactivity was exclusively localized in the placental labyrinth containing the villous trophoblast cells expressing relaxin mRNA (Fig. 3C). However, relaxin immunoreactivity was always localized in the maternal placental lamellae, indicating binding of the antibody to secreted relaxin, which is present in large amounts at this stage of feline pregnancy [14]. The trophoblast cells of the villous tips and the extravillous trophoblast cells in the junctional zone of the endometrium were devoid of relaxin immunoreactivity, as were the paraplacental tissues (Fig. 3C). Control experiments omitting the R6 relaxin antiserum (Fig. 3D) or the mouse monoclonal antibody to cytokeratin (Fig. 3I) consistently resulted in negative immunostaining. Similar negative results were obtained when the rabbit anti-relaxin R6 antiserum was replaced by rabbit nonimmune serum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The placenta is considered a major source of relaxin in the domestic cat although low levels of relaxin immunoreactivity have been detected in feline luteal, fetal, and uterine tissues [13]. Upon implantation at Days 13–14 [18], relaxin serum immunoreactivity is first detected at approximately Day 20 postovulation using a porcine relaxin RIA, and peak serum relaxin levels are detected between Days 30 and 35 of gestation [13]. Serum relaxin immunoreactivity decreases gradually 10–15 days prepartum and sharply declines shortly before parturition to be undetectable 24 h postpartum [13]. Similar profiles of relaxin immunoreactivity have been reported in placental tissue of queens throughout their approximately 65-day gestational period [13, 14]. Unlike previous researchers, who have found alternatively spliced human relaxin mRNA species [19], we detected only a single amplification product of 540 bp encoding for feline preprorelaxin. Within the B domain and close to the first cysteine residue, the amino acid sequence R-X-X-X-R (X representing any amino acid) is highly conserved among relaxin molecules. In feline preprorelaxin, this receptor recognition pentameric sequence (R-E-F-V-R) was found to be identical to that reported for the tammar wallaby relaxin (Fig. 4) [20] and, with the exception of a single substitution (Leu->Phe), was identical to the conserved classical receptor binding motif R-E-L-V-R found in pig, rhesus monkey, human, and various whale species [3]. The R6 relaxin antiserum we employed for immunolocalization of relaxin in the lamellar placental labyrinth has been reported to specifically interact with both arginine residues in the receptor binding site of the B domain [21,22]. These two basic amino acid residues are known to be essential for binding of relaxin to its receptor [1]. The C domain of feline prorelaxin was 98 amino acids long, slightly shorter than the C domain of mouse and guinea pig relaxin (102 residues) [23, 24]. Among the known prorelaxin molecules, the amino acid sequence R-X-K/R-R-{downarrow}X (X representing any amino acid, and the arrow indicating the cleavage site) at the C-domain/A-domain junction is a highly conserved consensus cleavage motif for furin [25,26]. The prorelaxin molecules of the pig [27], rat [28], hamster [29], tammar wallaby [20], and human H2 relaxin [30] contain the motif R-K-K-R, whereas the human H1 relaxin and the guinea pig relaxin have a single substitution in the first position (Gln and Pro for Arg). Feline prorelaxin contained a single substitution at the second position from a basic to a hydrophobic residue (Lys to Ile; Fig. 4). At the same position, the relaxin of both the rhesus and marmoset monkey contains a conservative substitution from Lys to Arg [31, 32]. In accordance with the amino acid sequence of all other relaxin molecules reported so far [3], the cysteine residues in the A and B domains of feline relaxin were highly conserved. Feline relaxin displayed the highest homology, of 56.6% identical amino acids, with porcine and equine preprorelaxin (Table 2) [27, 33], which is consistent with considerable variations in the amino acid sequences reported for relaxin molecules in different species [3]. Northern analysis revealed a single 1-kb relaxin gene transcript. Relaxin mRNA transcripts of similar size have been reported from the rat, [28], pig [34], horse [4], mouse [23], and guinea pig [24]. No larger relaxin transcripts were detected, as in the mouse [23], horse [33], and human [30]. This probably indicates that there is only a single gene for relaxin in the cat, as has been shown for the pig [27], rhesus monkey [31], horse [33], and tammar wallaby [20].



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  		FIG. 4. Nucleic acid sequence and deduced amino acid sequence of feline preprorelaxin cloned from uteroplacental tissue at Day 35 of gestation employing RT- and RACE-PCR. Two PCR amplicons from two amplifications were sequenced in both directions with the PRISM dye Deoxy Terminator cycle sequencing kit to exclude sequencing errors. Identical sequencing results were obtained in all cases.


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  		TABLE 2. Homology of feline preprorelaxin and single relaxin domains with relaxin molecules of other species.

Nonradioactive in situ hybridization and immunohistochemistry on the zonary placental girdle and paraplacental uterine tissues [35] at approximately Day 35 of gestation revealed the villous trophoblast cells of the lamellar placental labyrinth to be the sole source of relaxin mRNA and relaxin in the cat placenta. During Weeks 7–9 of gestation in the queen, the syncytiotrophoblast layer is reported to progressively replace the cytotrophoblast cells to become the predominant trophoblast cells in the feline term placenta [36]. The placentae used in this study were collected at approximately 4–5 wk of pregnancy, and cytotrophoblast cells should, therefore, be mainly responsible for relaxin expression at this stage. Relaxin has previously been localized in trophoblast cells of the developing equine placental microcotyledons [4, 5] and in the syncytiotrophoblast layer in the rabbit [6, 37] and human [9]. Feline relaxin serum immunoreactivity is undetectable during estrus cycle and pseudopregnancy [14]. Similar to data on the dog and horse [38, 39], and in agreement with declining relaxin serum levels determined in aborting cats suffering from taurine deficiency [13], our data seem to suggest that relaxin is a useful marker molecule to indicate and monitor placental sufficiency in feline species. Given the potential of relaxin to promote growth of rat uterine, cervical, and vaginal tissues [40, 41], the exclusive expression of relaxin by trophoblast cells of the microcotyledons in the pregnant mare has previously tempted us to suggest relaxin to be an important placental growth factor [5]. Despite the histological differences in placentation, those feline trophoblast cells expressing relaxin are also confined to the proliferative lamellar placental labyrinth of the cat. As in the horse and human, relaxin is already present during the early stages of placentation [5, 42].

We have previously reported the absence of relaxin mRNA transcripts in the invasive trophoblast cells of the equine chorionic girdle or endometrial cups, and the exclusive expression of relaxin in noninvasive trophoblast cells in the proliferating microcotyledons of the developing equine placenta [5]. In the feline placenta, both the trophoblast cells located at the tips of the chorioallantoic villi, which are in close contact with the endometrium, and the extravillous trophoblast cells invading the transitional placental zone are devoid of relaxin mRNA transcripts. In the human, the transition from a sessile villous trophoblast cell to an extravillous trophoblast cell with limited invasiveness marks a change in cellular differentiation patterns and involves activation and silencing of specific genes. Those genes known to be transcriptionally responsive during the invasive process include integrin subunits [43, 44], human leukocyte antigen G [45], and a number of matrix metalloproteinases and their inhibitors [46]. As in the temporally and spatially regulated switching of the integrin repertoire during human cytotrophoblast cell differentiation [47], it appears that, in the cat, differentiation of trophoblast cells involves down-regulation of relaxin transcriptional activity. Further studies are currently being conducted to determine those factors responsible for the regulation of relaxin gene transcription in trophoblast cells.


   ACKNOWLEDGMENTS
 
The authors would like to thank Prof. Bernhard G. Steinetz, Nelson Institute of Environmental Medicine, New York University Medical Center, NY, for generously providing the R6 relaxin antiserum. We would also like extend our gratitude to Dr. Wenkel at the Animal Clinic, Martin Luther University Halle-Wittenberg, for excellent cooperation.


   FOOTNOTES
 
1 S. Hombach-Klonisch was supported by the university program No. 3 "Wiedereinstiegsstipendium für Frauen" of the land Saxony-Anhalt. Back

2 Correspondence: T. Klonisch, Department of Anatomy and Cell Biology, Faculty of Medicine, Martin Luther University of Halle-Wittenberg, Grosse Steinstr. 52, D-06097 Halle (Saale), Germany. FAX: 49 345 557 1700; thomas.klonisch{at}medizin.uni-halle.de Back

Accepted: September 9, 1998.

Received: April 28, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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