Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Halle/Saale, Germany
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Abstract |
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Introduction |
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Attaining high relaxin plasma levels during pregnancy, relaxin is present during primate embryo implantation (Stewart et al. 1990, 1993, 1995
; Einspanier et al. 1999
). In hominoid primates, including humans, concentrations of circulating relaxin are maximal in the first trimester of pregnancy but barely exceed 2 ng/ml (Eddie et al. 1989
; Steinetz, Randolph, and Mahoney 1992, 1995
). Functional roles for relaxin within human reproductive tissues have also been implicated during placentation (Sakbun et al. 1990
; Bogic, Mandel, and Bryant-Greenwood 1995
), parturition (Qin et al. 1997a, 1997b
), and lactation (Tashima, Mazoujian, and Bryant-Greenwood 1994
). In contrast to relaxin, large amounts of RLF are expressed by postpubertal testicular Leydig cells (Adham et al. 1993
; Burkhardt et al. 1994b
; Ivell et al. 1997
), and the highest RLF serum concentrations are detected in postpubertal men (Büllesbach et al. 1999
). In rodents, RLF expression is developmentally regulated (Zimmermann et al. 1997
; Balvers et al. 1998
) and induces gubernaculum testis formation to facilitate testicular descent (Nef and Parada 1999
; Zimmermann et al. 1999
). In the female reproductive tract, the ovary and the placenta have been identified as sources of RLF (Tashima et al. 1995
; Bamberger et al. 1999
).
Molecular evolution of the ancient hormone relaxin (Georges and Schwabe 1999
) has raised concerns about the validity of the neo-Darwinian perception of molecular evolution as an orderly process over evolutionary time (Schwabe and Büllesbach 1994
, 1998, pp. 175190). The high sequence variability of relaxin within and between taxonomic groups among land mammals is contrasted by almost identical relaxins of sea mammals (Schwabe et al. 1989
) and pigs (Haley et al. 1982
), although fossil records separate both groups by around 58 Myr (Kumar and Hedges 1998
). In Strepsirrhini, which includes the Malagasy lemuriforms and the Afro-Asian lorisiforms, there is a complete lack of information on the molecular structure of relaxin and RLF. Determination of the cDNA-coding sequences of both preprorelaxin and RLF in the African lorisiform Galago crassicaudatus (greater bushbaby) and preprorelaxin in the Malagasy lemur Varecia variegata (ruffed lemur), regarded as phylogenetically plesiomorphic to all Malagasy lemurs (Yoder and Irwine 1999
), has revealed a unique strepsirrhine relaxin and distinct molecular evolutionary dynamics for both hormones during primate evolution.
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Materials and Methods |
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Cloning of Preprorelaxin and RLF
The preprorelaxin cDNA molecules from mRNA of uteroplacental tissue of G. crassicaudatus and V. variegata rubra and the RLF cDNA from mRNA of testicular tissue of G. crassicaudatus were cloned by reverse transcriptasepolymerase chain reaction (RT-PCR) and rapid amplification of the 5'- and 3'-cDNA ends (5'/3'-RACE-PCR). For cloning from the small amounts of uteroplacental mRNA available, Smart-PCR was used to amplify the cDNA pool according to the instructions of the manufacturer (Clontech, Heidelberg, Germany). All PCR primers employed flanked the putative single intron present at the N terminus of the C-domain of both relaxin and RLF to preclude genomic DNA amplification (table 1 ). For first-strand cDNA-synthesis, 500 ng of mRNA and 500 ng/ml of oligo d(T) primer were used with the Superscript reverse transcriptase kit (Life Technologies). PCR reactions were carried out in 50 µl of solution containing 1 µl of cDNA, 5 µl of 10 x 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). For the initial amplification of a cDNA fragment of the lemuriform prorelaxins, a forward oligonucleotide primer specific for H2-relaxin and a degenerate reverse primer designed according to a relatively conserved amino acid region at the C-terminal part of the A-domain of porcine, human, and rat relaxin cDNA were used (table 1
). Initial RT-PCR amplification of the Galago-RLF cDNA from testicular tissue was performed with an oligonucleotide primer pair specific for human RLF, followed by 3'/5'-RACE-PCR reactions employing gene-specific primers (table 1
). PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of 95°C and annealing at 60°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'/5'-RACE-PCR reactions were performed with universal and gene specific primers (table 1
) for 35 cycles at 68°C according to the manufacturer's instructions (Life Technologies). The complete cDNA-coding sequences of lemuriform preprorelaxin and RLF were amplified by RT-PCR for 40 cycles at an annealing temperature of 68°C using specific primers located at both ends of the coding sequences (table 1
). PCR products were purified by Magic column extraction, cloned into the pGEM-T vector (Promega, Heidelberg, Germany), and sequenced with the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer, Foster City, Calif.).
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Southern Analysis
Genomic DNA (20 µg) prepared from snap-frozen placental and testicular tissue of G. crassicaudatus employing the genomic tip 100 (Qiagen, Hilden, Germany) was digested with EcoRI or HindIII, separated on an 0.8 % agarose gel, and transferred to a Hybond N+ nylon membrane (Amersham Pharmacia, Freiburg, Germany). Filters were hybridized at 68°C overnight against the 32P-labeled cDNA of preprorelaxin and RLF of G. crassicaudatus. The next day, filters were washed twice at 68°C with 2 x SSPE/0.1% SDS (2 x SSPE is 0.34 M NaCl, 20 mM NaPO4, 2 mM EDTA, pH 7.7) and exposed to X-ray film for 5 days.
Digoxigenin-Labeling of cRNA and In Situ Hybridization
Synthesis of digoxigenin-labeled cRNA and nonradioactive in situ hybridization on uteroplacental cryocut sections of G. crassicaudatus and paraffin-embedded testicular tissue of V. variegata variegata (both 6 µm thick) have previously been described (Klonisch et al. 1995
). Specific signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma). After counterstaining with hematoxylin, the slides were mounted in glycerol gel and examined under bright-field microscopy.
Immunohistochemistry and Western Analysis
Acetone-fixed uteroplacental cryosections of G. crassicaudatus and dewaxed paraffin-embedded testicular tissue sections of V. variegata variegata (both 6 µm thick) were incubated in 20% acetic acid at 4°C for 15 s to inactivate endogenous alkaline phosphatase activity prior to blocking with 3% BSA in 0.5 M Tris-buffered saline (TBS) for 30 min to saturate nonspecific binding sites. For placental cryosections, the primary mouse monoclonal antibody (Dako, Hamburg, Germany) to cytokeratin (MNF-116) at a concentration of 1:1,000 or the rabbit polyclonal relaxin antiserum R6 at 1:4,000 (generously provided by Professor B. G. Steinetz, New York University Medical Center, N.Y.) was employed (Klonisch et al. 1999
). Testicular sections were incubated with polyclonal rabbit antisera against 17
-hydroxylase at a dilution of 1:1,000 (generously provided by Prof. Mason, University of Edinburgh, Scotland) and RLF at 1:200. The rabbit polyclonal serum against human RLF had been generated by immunizing rabbits with the specific peptide EKLCGHHFVRALVRV, located in the B-domain of RLF (BioGenes, Berlin, Germany) and predicted to be immunogenic using the computer program PREDITOP (Pellequer and Westhof 1993
). The RLF antiserum had previously been characterized by both Western analysis and immunohistochemical staining (unpublished data).
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Results |
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Mutually exclusive expression of relaxin and RLF was observed in the placenta and the testis of Strepsirrhini, respectively. Placental tissue of G. crassicaudatus revealed relaxin transcripts in cells covering the fetal villous with an antisense DIG-labeled preprorelaxin cRNA probe (fig. 6A
), but not with the corresponding sense cRNA (fig. 6B
). These cells also expressed immunoreactive relaxin (fig. 6C
) and cytokeratin (fig. 6D
) and were identified as fetal villous trophoblast cells. Fetal stromal cells or control sections treated with a rabbit nonimmune serum instead of the primary antiserum were devoid of immunostaining (fig. 6E
). In paraffin-embedded postpubertal testicular tissue sections of two V. variegata variegata lemurs, specific RLF hybridization signals were obtained exclusively in Leydig cells (fig. 6G
). Employing a rabbit antiserum generated against a specific RLF peptide sequence, Western analysis revealed a single immunoreactive 14.4-kDa band likely encoding for unprocessed RLF in postpubertal testicular extracts of G. crassicaudatus (data not shown). Employing this RLF-antiserum, immunoreactive RLF was detected in testicular Leydig cells (fig. 6I
) also expressing immunoreactive 17-hydroxylase, a marker enzyme for testosterone biosynthesis (fig. 6F
). Cells of the seminiferous epithelial cycle or testicular sections treated with the DIG-labeled sense RLF cRNA were devoid of hybridization signals (fig. 6H
), as were testicular sections with the primary antiserum replaced by a rabbit nonimmune serum (fig. 6J
).
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Discussion |
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Both members of the relaxin family appear to be under different evolutionary pressures. In contrast to relaxin, and with the exception of the RLF of rodents, the RLF hormone, including Galago RLF, is well conserved among species (Ivell 1997
). Expressing a single transcript encoding a pro-RLF peptide of 14.4 kDa, postpubertal testicular Leydig cells of G. crassicaudatus were devoid of splice variants previously described for the RLF of the marmoset, the human, and the mouse (Koskimies et al. 1997
; Safford et al. 1997
; Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999
). Employing a digoxigenin-labeled cRNA probe derived from Galago RLF, strong specific cross-hybridization with RLF transcripts in testicular Leydig cells of the ruffed lemur (V. variegata variegata) indicated a high degree of RLF sequence conservation among strepsirrhine primates. Both strepsirrhine members of the relaxin family displayed important structural features essential for bioactivity. The position and number of cysteine residues facilitating intra- and interchain cross-linking of properly folded A- and B-domains were conserved. Within the unique B-domain of strepsirrhine relaxin, a receptor-binding motif (RRLIR) was discovered that significantly differed from the RELVR pentameric sequence of all known primate relaxins (Bryant-Greenwood and Schwabe 1994
) but displayed highest homology with the receptor-binding motif of the phylogenetically >300-Myr-older skate relaxin (RDLIR) (Büllesbach, Schwabe, and Callard 1987
). The N- and C-terminal arginine residues within the receptor-binding motif are essential for binding relaxin to its uncloned receptor and provide the structural basis for a two-prong hormone-receptor interaction (Büllesbach and Schwabe 1988
; Büllesbach, Yang, and Schwabe 1992
), unique to the members of the relaxin family. In contrast to other relaxins (Sherwood 1994; Bryant-Greenwood and Schwabe 1994
), the second position within the receptor-binding motif of strepsirrhine relaxin contained a basic guanidino side chain (Arg18). Although the significance of the additional arginine residue within the receptor-binding motif of the relaxin for hormone-receptor interaction in loris and lemurs will have to be determined experimentally, it is tempting to speculate that in Strepsirrhini, the common two-prong relaxin receptor binding is extended to a unique three-prong interaction. Of the two relaxin domains, the A-domain of lemuriform relaxin was more conserved and contained the Gly14 residue, known to facilitate receptor binding by affecting the conformation of the relaxin heterodimer (Büllesbach and Schwabe 1994
).
Relaxin and RLF display weak cross-reactivity with their respective receptors, with the binding constant of RLF for the relaxin receptor being 100-fold lower than that of relaxin itself (Büllesbach and Schwabe 1995
). Like relaxin, cross-reactivity of RLF with the relaxin receptor appears to be mediated through the two arginines within the R16ALVR20 motif (Büllesbach and Schwabe 1995
), whereas the interaction with specific RLF-receptors (Büllesbach and Schwabe 1999b
) appears to require the pentameric motif G23GPRW27, located downstream of the putative relaxin receptor-binding motif within the B-domain of RLF (Büllesbach and Schwabe 1999a
). Similar to human RLF, and with the exception of a single substitution (R26
L) in marmoset RLF (Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999
), Galago-RLF also contained both conserved putative receptor-binding motifs within its B-domain and therefore acts as a bifunctional circulating hormone (Büllesbach et al. 1999
). In Strepsirrhini, like in other primates (Ivell 1997
; Ivell et al. 1997
), testicular postpubertal Leydig cells are an important source of RLF. Placental tissues of both lemuriform species studied were devoid of RLF. Northern analysis on marmoset placental RNA also had failed to provide evidence for RLF transcripts (Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999
). Similar to the epitheliochorial placenta of equids (Klonisch et al. 1997
), exclusive expression of preprorelaxin was observed in fetal trophoblast cells of the epitheliochorial placenta of G. crassicaudatus (King 1993
) at term. The human placental trophoblast expresses both RLF and H1 and H2 relaxin (Sakbun et al. 1990
; Bogic, Mandel, and Bryant-Greenwood 1995
; Tashima et al. 1995
), which makes it tempting to speculate that the duplication of the relaxin gene and the activation of the placental RLF gene may temporally and functionally coincide and reflect a rather recent development in primate evolution. Concentrations of relaxin in the plasma of the human, the rhesus monkey, and the marmoset during the peri-implantation period implicate relaxin as being involved in this critical period of feto-maternal interaction in these species (Stewart et al. 1990, 1993, 1995
; Einspanier et al. 1999
). Given its conserved structure among Strepsirrhini, lemuriform relaxin may provide a useful tool for the monitoring of pregnancy in endangered strepsirrhine primates.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Keywords: relaxin
relaxin-like factor
RLF
INSL3
Strepsirrhini
lemuriformes
2 Address for correspondence and reprints: Thomas Klonisch, Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Grosse Steinstrasse 52, D-06097 Halle/Saale, Germany. E-mail: thomas.klonisch{at}medizin.uni-halle.de
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