Molecular Remodeling of Members of the Relaxin Family During Primate EvolutionGo

Thomas Klonisch2,, Christine Froehlich, Frank Tetens, Bernd Fischer and Sabine Hombach-Klonisch

Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Halle/Saale, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Employing comparative analysis of the cDNA-coding sequences of the unique preprorelaxin of the Afro-lorisiform Galago crassicaudatus and the Malagasy lemur Varecia variegata and the relaxin-like factor (RLF) of G. crassicaudatus, we demonstrated distinct differences in the dynamics of molecular remodeling of both hormones during primate evolution. The lorisiform and lemuriform preprorelaxin sequences encoded identical hormones, providing the first endocrinological evidence for the monophyletic origin of all Strepsirrhini. Structural analysis revealed the lemuriform members of the relaxin family to be potentially bioactive single-gene products. In contrast to the "two-prong" relaxin receptor-binding motif (RELVR) present within the B-domains of other primate relaxins, strepsirrhine relaxin contained a unique "three-prong" motif (RRLIR) with highest sequence homology to the receptor-binding motif of the evolutionarily much older skate relaxin. In contrast to relaxin, the RLF molecule was highly conserved during primate evolution and contained within its B-domain the putative relaxin receptor-binding motif and a pentameric sequence implicated in binding to specific RLF receptors. Mutually exclusive expression of strepsirrhine preprorelaxin and RLF were observed in the fetal villous trophoblast cells of the strepsirrhine placenta and postpubertal testicular Leydig cells, respectively, reflecting distinct functional roles for both hormones within the reproductive tract of Strepsirrhini.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Within the superfamily of insulin-like molecules which includes insulin, insulin-like growth factors I and II, placentin (Chassin et al. 1995Citation ), the recently discovered insulin-like factors 5 and 6 (INSL-5 and INSL-6) (Conklin et al. 1999Citation ), and relaxin/insulin-like factor 1 (RIF-1) (Hsu 1999Citation ), the heterodimeric polypeptide hormone relaxin and the structurally closely related relaxin-like factor (RLF), also known as INSL-3 (Adham et al. 1993Citation ; Burkhardt et al. 1994bCitation ), constitute the relaxin family. Relaxin and RLF are synthesized as preprohormones consisting of a signal peptide and B-, C-, and A-domains, from the N terminus to the C terminus, respectively. Despite significant differences in nucleic acid and amino acid sequence among species, all relaxins contain the conserved amino acid motif -R-X-X-X-R- close to the first cysteine of the B-peptide, which has been shown to be important for receptor binding (Büllesbach, Yang, and Schwabe 1992Citation ). Shifted four amino acids further C-terminal, a similar receptor-binding motif is present in the B-domain of RLF which facilitates the interaction with the relaxin receptor at a 100-fold lower binding affinity than relaxin (Büllesbach and Schwabe 1995Citation ). Located at the far C terminus within the RLF B-domain, an RLF receptor-binding motif is implicated in the interaction with specific RLF receptors (Büllesbach and Schwabe 1999aCitation ).

Attaining high relaxin plasma levels during pregnancy, relaxin is present during primate embryo implantation (Stewart et al. 1990, 1993, 1995Citation ; Einspanier et al. 1999Citation ). 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. 1989Citation ; Steinetz, Randolph, and Mahoney 1992, 1995Citation ). Functional roles for relaxin within human reproductive tissues have also been implicated during placentation (Sakbun et al. 1990Citation ; Bogic, Mandel, and Bryant-Greenwood 1995Citation ), parturition (Qin et al. 1997a, 1997bCitation ), and lactation (Tashima, Mazoujian, and Bryant-Greenwood 1994Citation ). In contrast to relaxin, large amounts of RLF are expressed by postpubertal testicular Leydig cells (Adham et al. 1993Citation ; Burkhardt et al. 1994bCitation ; Ivell et al. 1997Citation ), and the highest RLF serum concentrations are detected in postpubertal men (Büllesbach et al. 1999Citation ). In rodents, RLF expression is developmentally regulated (Zimmermann et al. 1997Citation ; Balvers et al. 1998Citation ) and induces gubernaculum testis formation to facilitate testicular descent (Nef and Parada 1999Citation ; Zimmermann et al. 1999Citation ). In the female reproductive tract, the ovary and the placenta have been identified as sources of RLF (Tashima et al. 1995Citation ; Bamberger et al. 1999Citation ).

Molecular evolution of the ancient hormone relaxin (Georges and Schwabe 1999Citation ) has raised concerns about the validity of the neo-Darwinian perception of molecular evolution as an orderly process over evolutionary time (Schwabe and Büllesbach 1994Citation , 1998, pp. 175–190). 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. 1989Citation ) and pigs (Haley et al. 1982Citation ), although fossil records separate both groups by around 58 Myr (Kumar and Hedges 1998Citation ). 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 1999Citation ), has revealed a unique strepsirrhine relaxin and distinct molecular evolutionary dynamics for both hormones during primate evolution.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Collection of Tissues and RNA Isolation
Postpartum placental tissue collected each from G. crassicaudatus and V. variegata rubra (both CITES number E-1874/96) and testicular tissue from one adult G. crassicaudatus (CITES number E1506/97) were snap-frozen in liquid nitrogen and stored at -80°C until they were used. Bouin-fixed testicular tissue was obtained from two captive postpubertal V. variegata variegata after medically indicated routine sterilization. Total RNA was isolated from cryopreserved reproductive tissues with Trizol reagent (Life Technologies, Eggenstein, Germany). For the isolation of mRNA, 75 µg of total uteroplacental and testicular RNA was incubated with oligo-d(T)-coated magnetic beads (Dynal, Hamburg, Germany) according to the manufacturer's instructions. The amount of mRNA isolated was determined by spectrophotometry at 260 and 280 nm (Sambrook, Fritsch, and Maniatis 1989Citation , p. E5).

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 transcriptase–polymerase 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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Table 1 List of primers Employed for RT-PRC and 5'/3'-RACE-PCR

 
Homology Analysis and Comparative Molecular Modeling
Protein alignments were performed with CLUSTAL W (Thompson, Higgins, and Gibson 1994Citation ) prior to maximum-likelihood calculations and phylogenetic tree reconstruction of prorelaxin and RLF by Puzzle, version 4.0.2 (Strimmer and von Haeseler 1996Citation ; Strimmer, Goldman, and von Haeseler 1997Citation ) and the TREEVIEW program (Page 1996Citation ). Comparative molecular modeling of the Galago-relaxin B-domain was performed with the SWISS-MODEL and the Swiss-Pdb Viewer (Guex and Peitsch 1997Citation ), according to the pdb-Brookhaven crystal structure coordinates of H2 relaxin (Eigenbrot et al. 1991Citation ). The POV-Ray computer program was used to produce high-quality molecular images.

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. 1995Citation ). 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. 1999Citation ). Testicular sections were incubated with polyclonal rabbit antisera against 17{alpha}-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 1993Citation ). The RLF antiserum had previously been characterized by both Western analysis and immunohistochemical staining (unpublished data).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Employing a combination of Smart-, RT- and RACE-PCR (table 1 ), we cloned from postpartum placental tissues the coding sequences of the preprorelaxins from the Afro-lorisiform G. crassicaudatus and the Malagasy lemur V. variegata rubra. Testicular tissue of adult G. crassicaudatus was devoid of relaxin amplification products (data not shown). Except for a conservative transversion (A->C) in position 507 of the lemur cDNA sequence, the 567-bp preprorelaxin cDNA-coding sequences expressed by G. crassicaudatus and V. variegata rubra encoded identical peptides of 188 amino acids (fig. 1 ). According to the known cleavage sites of preprorelaxin molecules from different species, preprorelaxin of Strepsirrhini consisted of a signal peptide of 21 amino acids (63 bp), a B-domain of 34 amino acids (102 bp), a C-domain of 109 amino acids (327 bp), and an A-domain of 24 amino acids (75 bp; fig. 1 ). The positions and numbers of cysteine residues were conserved. Analysis of the amino acid sequence alignment revealed a >70% difference in the amino acid sequence of the B-domain of lemuriform relaxin and the relaxin B-domains of Old and New World haplorrhine primates (fig. 2 and table 2 ). In contrast, the A-domain of strepsirrhine relaxin displayed degrees of amino acid homology similar among other primate species with highest amino acid homology to the relaxin 2 genes of hominoid and Old World primates (fig. 2 and table 2 ). With the exception of the likely nonfunctional gorilla and orangutan relaxin 1 genes (Evans, Fu, and Tregear 1994aCitation ), the classical receptor-binding motif (RELVR) located within the B-domain of all known primate relaxin molecules was significantly altered in strepsirrhine relaxin (RRLIR; fig. 2 ) displaying highest similarity with that of the phylogenetically >300-Myr-older skate relaxin (RDLIR; fig. 2 ). In addition to the N- and C-terminal arginine residues (Arg18 and Arg22) known to be important for relaxin's unique "two-prong" hormone-receptor interaction (Büllesbach, Yang, and Schwabe 1992Citation ), the strepsirrhine relaxin B-domain contained the guanidino group of a third arginine residue (Arg19) which is unique among relaxins and may extend the receptor interaction to a "three-prong" mode. Employing the crystal structure coordinates of H2 relaxin (Eigenbrot et al. 1991Citation ), comparative molecular modeling revealed a spindle-shaped B-domain of Galago-relaxin with a helix shortened by one turn. The three arginine residues (Arg18, Arg19, and Arg22) at the receptor-binding domain created an electrostatic potential that was significantly stronger than that in H2 relaxin or, indeed, any other known relaxin (fig. 3 ).



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Fig. 1.—Nucleic acid (lower numbers) and deduced amino acid sequence (upper numbers) of the coding sequence of the preprorelaxin molecules cloned from term placental tissues of the African lorisiform Galago crassicaudatus and the Malagasy lemur Varecia variegata rubra. Five independent PCR amplifications were sequenced in both directions with the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer) to exclude sequencing errors. Identical sequencing results were obtained in all experiments. The different domains of the lemuriform preprorelaxins are shown, and the putative position of the intron within the relaxin gene is indicated by an arrow. The putative receptor-binding motif is highlighted

 


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Fig. 2.—Alignment of the relaxin A- and B-domains of Galago crassicaudatus (Gcr) (AF317624) and the lemur (Varecia variegata rubra; Vva) with those of other species, including all known primate relaxin sequences. The unique putative receptor-binding motif within the B-domain of the lemuriform relaxin shares highest homology with the receptor-binding motif of skate relaxin. Differences in amino acid sequence are shown; hyphens identify missing amino acids, and dots indicate identical amino acids

 

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Table 2 Primate Relaxin Sequences (% difference)

 


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  Fig. 3.—Utilizing the Brookhaven crystal structure coordinates of H2 relaxin (Eigenbrot et al. 1991Citation ) as a structural reference and employing comparative molecular modeling with the SWISS-MODEL and the Swiss-Pdb Viewer, a proposed model structure of the relaxin B-domain of Galago crassicaudatus (Gcr) was generated. In contrast to the B-domain of human relaxin, the spindle-shaped relaxin B-domain of Gcr displayed a shorter helical section. Analysis of the molecular surface and electrostatic potential revealed an increase in positive potential (blue) over negative potential (red) in the B-domains of Gcr relaxin as compared with H2 relaxin. The three guanidino side groups of the arginine residues generated a strongly increased positive eletrostatic potential at the putative receptor-binding motif (RRLIR). The molecular images were produced with the POV-Ray computer program

 
RLF was cloned by RT- and RACE-PCR from postpubertal testicular tissue of G. crassicaudatus and consisted of a peptide of 131 amino acids (394 bp), with a signal peptide of 24 amino acids (72 bp), a B-domain of 31 amino acids (93 bp), a C-domain of 50 amino acids (150 bp), and an A-domain of 26 amino acids (78 bp; fig. 4 ). Within the B-domain of Galago-RLF, the cysteine residues, the putative relaxin receptor-binding motif (RALVR), and a pentameric sequence G23GPRW27, implicated in binding to specific RLF receptors (Büllesbach and Schwabe 1995, 1999bCitation ), were conserved (fig. 4 ). In contrast to relaxin, RLF displayed high amino acid homology among different species, with human (79%) and marmoset RLF (74%) showing the highest homology to Galago-RLF (table 3 ).



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Fig. 4.—Nucleic acid and deduced amino acid sequence of the coding sequence of the RLF cloned from postpubertal testicular tissue of the Afro-lorisiform Galago crassicaudatus (Gcr) (AF317625). Three independent PCR amplifications were sequenced in both directions to exclude sequencing errors, and identical sequencing results were obtained in all cases. Alignments of the different domains of Galago RLF with RLF sequences of other species, including the marmoset and the human, indicated high phylogenetic sequence conservation among RLF genes. The putative position of the intron within the RLF gene is indicated by an arrow, and the putative relaxin receptor (in bold) and RLF receptor-binding motif (in bold and italics) are highlighted

 

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Table 3 Homology in Amino Acid Sequence Between the Putative Relaxin-like Factor (RLF) Domains of {+}Galago crassicaudatus{-} and RLF Molecules of Other Species, Including the Two Known Primate RLF Sequences from the Human and the Marmoset

 
Puzzle, version 4.0.2, was employed for reconstruction of a phylogenetic tree based on the sequence relatedness of mammalian prorelaxin and RLF molecules by maximum-likelihood analysis implementing quartet puzzling as the tree search algorithm. The tree numbers indicated the estimates of the support values for each internal branch. The prorelaxins of the African lorisiform and Malagasy strepsirrhine lineage and the galagoid RLF nested in outgroups to corresponding prorelaxin and RLF sequences of other species, including primates (fig. 5 ).



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Fig. 5.—Phylogenetic relatedness based on the primary sequences of mammalian prorelaxin and RLF molecules presented as an unrooted quartet puzzling tree, with wallaby prorelaxin (Parry, Rust, and Ivell 1997Citation ) chosen as an outgroup. The tree represents a molecular phylogeny of relaxin and RLF proteins, not a phylogeny of the involved species. Strepsirrhine prorelaxin and RLF are nested in outgroups to corresponding primate sequences, suggesting independent molecular remodeling of both members of the relaxin family within the strepsirrhine clade. Numbers indicate estimates of the support values for each internal branch, and branch length corresponding to one amino acid substitution per 10 amino acids is indicated by the bar (bottom left). GenBank accession numbers are as follows: murine relaxin, 1350570; murine RLF, 1754739; human relaxin 1, 132280; human relaxin 2, 132281; human RLF, 3851207; chimpanzee relaxin 1, 1710080; chimpanzee relaxin 2, 1710081; marmoset RLF, 3850653; rhesus monkey relaxin, 132301; bovine RLF, 3719459; deer RLF, AF254740; caprine RLF, AF233686; porcine relaxin, 132309; porcine RLF, 1708498; equine relaxin, 2506784; feline relaxin, AF233688; canine relaxin, AF233687; camel relaxin, AF254739

 
In Strepsirrhini, both members of the relaxin family appear to be encoded by a single gene. Southern analysis of genomic DNA from G. crassicaudatus with a 32P-labeled Galago-preprorelaxin probe revealed hybridization signals at 9 kb for HindIII-restricted genomic DNA and 1.4 kb and 4 kb for EcoRI-restricted genomic DNA, whereas 32P-labeled Galago-RLF cDNA probe revealed single hybridization signals at 13 kb (HindIII digest) and 2 kb (EcoRI digest; data not shown).

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{alpha}-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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  Fig. 6.—Photomicrographs of cryostat sections of at-term placental tissue of Galago crassicaudatus subjected to nonradioactive in situ hybridization for the detection of relaxin mRNA expression (A, B) and rabbit or mouse alkaline phosphatase anti-alkaline phosphatase (APAAP) indirect immunohistochemistry with a rabbit polyclonal antibody specific for porcine relaxin (R6; C) and a mouse monoclonal antibody specific for cytokeratin (D). Specific hybridization signals for relaxin mRNA were observed in the fetal trophoblast cells covering the placental villi (v) (A). Placental stromal cells and tissue sections treated with the sense cRNA relaxin probe were devoid of hybridization signals (B). Immunoreactive relaxin was colocalized to cells (C) expressing relaxin mRNA and immunoreactive cytokeratin (D), confirming the trophoblast identity of these cells. Testicular cryosections of G. crassicaudatus resulted in poor histology. Therefore, paraffin-embedded testicular sections of the Malagasy lemur Varecia variegata variegata were employed for nonradioactive in situ hybridization under stringent washing conditions with a digoxigenin-labeled RLF cRNA probe of G. crassicaudatus, which revealed specific hybridization signals in Leydig cells (G), suggesting high homology between the galagoid and lemuroid RLFs. Testicular sections treated with the sense cRNA relaxin probe were devoid of hybridization signals (H). Testicular Leydig cells expressed immunoreactive RLF (I) and 17{alpha}-hydroxylase (F). Negative immunohistochemical control sections of placental (E) and testicular tissue sections (J) were obtained when the primary antibody was replaced with nonimmune serum of the same species. Magnifications: AHx216; I and Jx108

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
It is commonly assumed that primates diverged from other orders of placental mammals at the Cretaceous-Tertiary (Mesozoic-Cenozoic) boundary about 65 Myr, which coincides with a major reorganization of the terrestrial fauna and mammalian divergence, diversification, and radiation. The divergence between Strepsirrhini and Haplorrhini (Old and New World monkeys, apes, and humans) primates has been estimated at around 63 MYA, in the late Paleocene (Gingerich 1986Citation ; Shoshani et al. 1996Citation ). Likely initiated on the African continent some 62 MYA, the current ~20 living genera of strepsirrhine primates, including the African bush babies and other Afro-Asian lorises and the Malagasy lemurs, evolved by a combination of independent divergence and the eastward migration of lemurs across the Mozambique channel to the island of Madagascar during the early Eocene, signaling the beginning of their radiation by 54 MYA (Yoder et al. 1996Citation ). After the divergence of Cercopithecoidea from Hominoidea associated with the Oligocene-Miocene transition about 25 MYA, the relaxin gene, but not the RLF gene (Burkhardt et al. 1994aCitation ), appears to have undergone gene duplication (Crawford et al. 1984Citation ; Evans, Fu, and Tregear 1994bCitation ). Like those of other nonhominoid primates, the genome of G. crassicaudatus contained a single gene copy for relaxin and RLF (Crawford et al. 1989Citation ; Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999Citation ), and, in contrast to the human and the chimpanzee (Gunnersen et al. 1996Citation ), we detected only a single preprorelaxin amplification product in postpartum placental tissues. Despite an independent evolutionary history of lemurs and lorises of >60 Myr (Yoder et al. 1996Citation ) and despite reported differences in the nucleic acid and amino acid sequences for mitochondrial cytochrome b from the Afro-lorisiform Galago and the Malagasy lemuriform Varecia of 21.5% and 16.1%, respectively (Yoder, Vilgalys, and Ruvolo 1996Citation ), the preprorelaxin hormones of both species were identical. This provides the first endocrinological molecular evidence for an African monophyletic origin of all Strepsirrhini (Rumpler et al. 1986Citation ; Yoder 1994Citation ; Yoder et al. 1996Citation ; Yoder, Vilgalys, and Ruvolo 1996Citation ). DNA sequence analysis of three mitochondrial genes and one nuclear gene has recently established the genus Varecia to be phylogenetically placed basal to the other three genera of Lemuridae, grouped into two clades, Eulemur and Hapalemur/Lemur catta (Yoder and Irwine 1999Citation ). Varecia is, therefore, expected to display phenotypical and molecular characteristics plesiomorphic to all Malagasy lemurs. The identical preprorelaxin peptide sequences of Galago and Varecia would appear to reflect a mode of hormone-receptor interaction common to all Strepsirrhini but unique among primates and other mammals.

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 1997Citation ). 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. 1997Citation ; Safford et al. 1997Citation ; Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999Citation ). 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 1994Citation ) but displayed highest homology with the receptor-binding motif of the phylogenetically >300-Myr-older skate relaxin (RDLIR) (Büllesbach, Schwabe, and Callard 1987Citation ). 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 1988Citation ; Büllesbach, Yang, and Schwabe 1992Citation ), unique to the members of the relaxin family. In contrast to other relaxins (Sherwood 1994; Bryant-Greenwood and Schwabe 1994Citation ), 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 1994Citation ).

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 1995Citation ). 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 1995Citation ), whereas the interaction with specific RLF-receptors (Büllesbach and Schwabe 1999bCitation ) 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 1999aCitation ). Similar to human RLF, and with the exception of a single substitution (R26->L) in marmoset RLF (Zarreh-Hoshyari-Khah, Einspanier, and Ivell 1999Citation ), 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. 1999Citation ). In Strepsirrhini, like in other primates (Ivell 1997Citation ; Ivell et al. 1997Citation ), 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 1999Citation ). Similar to the epitheliochorial placenta of equids (Klonisch et al. 1997Citation ), exclusive expression of preprorelaxin was observed in fetal trophoblast cells of the epitheliochorial placenta of G. crassicaudatus (King 1993Citation ) at term. The human placental trophoblast expresses both RLF and H1 and H2 relaxin (Sakbun et al. 1990Citation ; Bogic, Mandel, and Bryant-Greenwood 1995Citation ; Tashima et al. 1995Citation ), 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, 1995Citation ; Einspanier et al. 1999Citation ). Given its conserved structure among Strepsirrhini, lemuriform relaxin may provide a useful tool for the monitoring of pregnancy in endangered strepsirrhine primates.


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
Molecular evolutionary remodeling and receptor-ligand interaction of the relaxin hormone within the strepsirrhine clade has generated a relaxin molecule that is likely common to all living lorises and lemurs but unique among primates. The structural changes mainly affected the B-domain encoded by the first of two exons of the strepsirrhine relaxin single gene copy, indicating distinct differences in the rates of nucleotide substitution over time between the two exons. In contrast to relaxin, the binding motifs for the interaction of RLF with the relaxin receptor and specific RLF receptors (Büllesbach and Schwabe 1995, 1999a, 1999bCitation ) were conserved in the strepsirrhine and haplorrhine lineage, reflecting a conservation of RLF structure and likely RLF function during primate evolution. Mutually exclusive expression of RLF and relaxin within different compartments of the male or female reproductive tract is a common phenomenon in both Strepsirrhini and Haplorrhini. Expression of two relaxin genes at the feto-maternal interface and coexpression of members of the relaxin family in the human placenta, which displays one of the most invasive trophoblast phenotypes of all species, is not observed in nonhominoid primates and must be regarded as a recent event in primate evolution.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 literature cited
 
We thank Mrs. Elke Bernhard and Sylke Vogt for excellent technical assistance. We also thank Mrs. Ruempler and her team at the lemur-breeding center, Cologne Zoo, Germany, for providing testicular tissues from V. variegata variegata. We are grateful to Professor J. I. Mason, University of Edinburgh, Scotland, for kindly providing the rabbit polyclonal antibodies to 3-ß-hydroxysteroid dehydrogenase and 17{alpha}-hydroxylase, and to Professor B. G. Steinetz, Nelson Institute of Environmental Medicine, New York University Medical Center, Old Forge Road, Tuxedo, N.Y., for providing the R6 rabbit anti-porcine relaxin antiserum. We also thank Dr. Luminita Göbbel, Department of Anatomy and Cell Biology, Martin Luther University Medical Faculty, for helpful discussion on the cladistics.


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Keywords: relaxin relaxin-like factor RLF INSL3 Strepsirrhini lemuriformes Back

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 Back


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Accepted for publication November 3, 2000.