BIOLOGY OF REPRODUCTION 57, 119-127 (1997)
Marsupial Relaxin: Complementary Deoxyribonucleic Acid Sequence and Gene Expression in the Female and Male Tammar Wallaby, Macropus eugenil' Laura J. Parry, 2 Werner Rust, and Richard Ivell Division of Reproductive Sciences, IHF Institute for Hormone & Fertility Research, University of Hamburg, 22529 Hamburg, Germany ABSTRACT The nucleotide and derived amino acid sequences of tammar preprorelaxin were established by combined reverse transcriptase polymerase chain reaction and 3'- and 5'-rapid amplification of cDNA ends methods, using RNA from the corpus luteum of late pregnancy as template. Relaxin gene expression was then investigated in tissues at various stages of the 26-day pregnancy and in adult males. The full-length tammar relaxin preprohormone is 579 base pairs. The derived amino acid sequence contains a probable signal peptide of 26 amino acids, a B-domain of 31 amino acids, a C-domain of 111 amino acids, and an A-domain of 24 amino acids, with sequence homologies of 49%, 38%, 47%, and 47%, respectively, to dogfish, pig, and both human relaxins, for the combined A- and B-domains of the functional peptides. The conserved amino acid residues in the B-domain confirm a region shown to be essential for binding of the peptide to its receptor. A relaxin gene is expressed in several other tissues of pregnant tammars including the placenta, follicle, and hypothalamus. Northern analysis showed a 1-kilobase relaxin transcript in the corpus luteum and placenta. Using RNase protection assays, relaxin gene expression in the corpus luteum was greater in early and mid pregnancy, reduced at term, and absent postpartum. These data demonstrate relaxin biosynthesis in both the corpus luteum and placenta in this marsupial and suggest that a relaxin physiology has been conserved during mammalian evolution. INTRODUCTION The peptide hormone relaxin, commonly associated with connective tissue modifications of the reproductive tract before birth, has been isolated from many eutherian mammals as well as from nonmammalian species such as the shark and skate (reviewed in [1]). The ovaries, placenta, endometrium, and prostate gland are all tissue sources of the hormone, although highest circulating concentrations are measured during pregnancy in females [1-3]. The relaxin gene directs the synthesis of a preprohormone as the primary translation product. In most species, subsequent intracellular processing involves enzymatic cleavage of the signal peptide, a connecting peptide, and formation of interchain disulfide bonds to produce the bioactive relaxin peptide [4-7]. Recent molecular evidence points to the divergence of marsupials, together with monotremes, from the mammalian stock approximately 130 million years ago [8, 9]. A comparison of the primary structure of hormone molecules from such diverging groups of animals is useful to highlight
conserved motifs that may be important for the functioning of the molecule, e.g., receptor-binding sites and enzyme cleavage sites. Although marsupial relaxin has not been extracted and purified from any tissue, early studies demonstrated that crude extracts of corpora lutea from the pregnant tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula) contain bioactive relaxin [10]. Heterologous antisera to porcine relaxin have been used to show immunoreactive relaxin in the corpus luteum of both species [11, 12], which in the tammar is localized to membrane-bound, electron-dense granules in the luteal cell cytoplasm [13]. This finding would imply that marsupial and porcine relaxin share certain similarities in structure at the epitope level and that tammar preprorelaxin is likely to be transported in a typical secretory pathway within cytoplasmic granules. Analysis of the tammar relaxin gene, by highlighting conserved motifs, could therefore provide insight into general relaxin structure-function relationships. The first objective of this study was to determine the nucleotide and derived amino acid sequences of tammar preprorelaxin and compare them with known relaxin sequences of other species to establish regions of homology. The tissue and temporal specificity of relaxin gene expression was then investigated in reproductive and other tissues of male and female tammar wallabies for a comparison with the known eutherian physiology. MATERIALS AND METHODS Animals and Tissues Pregnant tammars of known gestational age (Day 0, 6, 14, 17, 22, 23, 24, 25, and 26 of pregnancy: n = 2 or 3 at each stage) were anesthetized with sodium pentobarbitone. Under sterile surgical conditions, the uteri and ovaries were exposed by laparotomy and the reproductive status of the animal was established. If pregnant, the animals were killed by overdose of anesthetic. The corpus luteum and follicle were dissected from the ovaries, uterine and placental tissues were dissected from the reproductive tract, and the brain was removed for recovery of neocortex and the hypothalamic region. Ovarian tissues were also collected from tammars at 6 and 9 days postpartum (n = 2 at each stage). Prostate and testis tissues were collected from 3 adult males killed by overdose with sodium pentobarbitone. All tissues were rapidly frozen in liquid nitrogen, transported to Hamburg, Germany, on dry ice, and stored at -80°C until used. Total RNA was isolated from 20-50 mg tissue by modification of the Chomczynski and Sacchi method [14] using RNase Clean (AGS GmbH, Heidelberg, Germany). Quantity and quality of the RNA were assessed by spectrophotometry at 260 and 280 nm and ethidium bromide staining after agarose gel electrophoresis.
Accepted February 24, 1997. Received December 30, 1996. 'Financial support provided by Grant v7/1-3 from the Deutsche Forschungsgemeinschaft. L.J.P. was in receipt of an Alexander von Humboldt Research Fellowship. 2Correspondence: L.J. Parry, Department of Zoology, University of Melbourne, Parkville, Victoria, 3052, Australia. FAX: +61 3 9344 7909; e-mail:
[email protected]
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120 FIG. 1. Diagram showing the compilation ot the full-length tammar relaxin cDNA sequence from independent RT-PCR subclones. The first two clones were isolated with the heterologous primers #578 and #mll Tammar-specific primers were then used to obtain multiple (n = 15) independent subclones from different tissues; most primer combinations were selected to span the intron/exon junction to ensure that the PCR products could not have arisen from genomic DNA. Primers are denoted by short, horizontal arrows with their numbers. The position of the putative intron is indicated with an arrow.
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