Reviews of Reproduction (1997) 2, 133–138

Biology of the relaxin-like factor (RLF)

Richard Ivell

Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany

The relaxin-like factor (RLF) is a novel member of the insulin–IGF–relaxin family of hormones and growth factors. Also known as the Leydig cell insulin-like factor (Ley-I-L), this peptide and the mRNA encoding it appear to be expressed in very large quantities in the Leydig cells of the testis. However, it is also produced in the ovary of a number of species in both follicular theca cells and in the corpus luteum of the cycle and pregnancy. RLF gene transcripts have been identified at a much lower level of expression throughout the bovine female reproductive tract and also in the hypothalamus. Although data are limited, it would appear that RLF represents a new differentiation-related factor with specific functions linked to reproductive physiology in male and female mammals.

Independently in two different laboratories a gene was ident- 1994b). Although studies on the regulation of the RLF gene are ified because of its very high level of expression and tissue still very preliminary, the promoter region of the mouse gene specificity in differential cDNA cloning projects involving does not appear to contain marked homology to other known either porcine (Adham et al., 1993) or mouse (Pusch et al., 1996) genes. Computer analysis has identified motifs for binding the testicular tissue. These cDNA clones were shown to encode a constitutive transcription factor SP1 and possibly also for ster- novel peptide factor with structural homology to the insulin– oidogenic factor 1 (Koskimies et al., 1997; Zimmermann et al., IGF–relaxin family of hormonal factors. Originally named 1997), although experimental work is necessary to examine the Leydig cell insulin-like factor (Ley-I-L) gene and later the these more fully. However, constructs comprising about 0.7 kb relaxin-like factor (RLF) gene, it is expressed at very high levels of the region upstream of the transcription start site appear to in the Leydig cells of all mammalian species so far investigated. be sufficient to permit expression specifically in Leydig cells Estimates from the bovine testis imply that RLF transcripts (Koskimies et al., 1997). account for approximately 5% of all transcripts expressed by The primary structure of the mRNA transcript produced adult Leydig cells (Bathgate et al., 1996). However, contrary to from this gene has been analysed in a number of species earlier notions, the gene is not only expressed in the testis; (porcine, Adham et al., 1993; human, Burghardt et al., 1994a; RLF transcripts have been identified in the human ovary and Ivell et al., 1997; mouse, Pusch et al., 1996; rat, W. Pusch and trophoblast (Tashima et al., 1995), as well as in ruminant ovary, R. Ivell, unpublished; bovine, Bathgate et al., 1996; sheep, Roche uterus and placental tissues (Bathgate et al., 1996; Roche et al., et al., 1996; and marmoset monkey, R. Zarreh-Hoshyari-Khah, 1996), and subsequently in the mouse (Zimmermann et al., 1997; A. Einspanier and R. Ivell, unpublished). Although there is M. Balvers and R. Ivell, unpublished) and marmoset ovary variability between species such that, for example, the mouse (R. Zarreh-Hoshyari-Khah, A. Einspanier and R. Ivell, unpub- cDNA sequence shares only 51% homology with the human lished). In addition, a chemically synthesized human RLF mol- sequence – a fact that prevents some probes from hybridizing ecule has been shown to interact specifically with relaxin across species – all encode polypeptides of about 14 kDa with receptors, albeit at low affinity, but not with insulin or IGF re- very similar structural organization (Fig. 1). This is the struc- ceptors (Büllesbach and Schwabe, 1995). Because it appears ture shared by all members of the insulin–IGF–relaxin family. neither to show insulin specificity, nor to be expressed only in First, there is a classic signal peptide domain at the N-terminus, Leydig cells, it is more appropriate to refer to this hormone as which is most likely used to traffic the molecule into the endo- the relaxin-like factor (Büllesbach and Schwabe, 1995). plasmic reticulum and hence into the secretory pathway. This is followed by a polypeptide which, by homology of cysteine residues and potential endopeptidase cleavage signals, is sep- Gene and peptide structure arable into B, C (connecting) and A domains, using the nomen- Subsequent cloning of the human, porcine (Burghardt et al., clature defined for the proinsulin molecule. 1994a) and mouse (Koskimies et al., 1997; Zimmermann et al., There is hardly any information on the RLF/Ley-I-L mol- 1997) genomic sequences showed that RLF/Ley-I-L is encoded ecule at the peptide level. Antibodies have only recently become by a single-copy gene, comprising only two exons with the available (for example, Ivell et al., 1997), and these have not yet single intron interposing within the open-reading frame en- provided information as to whether the RLF molecule is indeed coding the C (connecting) peptide domain (see below). This secreted, or if it is processed into its component moieties (as exonic organization is similar to that for the relaxin and insulin are insulin or relaxin in most species), or produced as an un- genes. In humans, the RLF/Ley-I-L gene has been localized to processed molecule (as are IGF-I and IGF-II). That RLF is most the short arm of chromosome 19 (p13.2–p12; Burghardt et al., probably secreted from the cells where it is made is shown © 1997 Journals of Reproduction and Fertility 1359-6004/97 $10.00 Downloaded from Bioscientifica.com at 10/01/2021 05:14:48AM via free access 134 R. Ivell

Signal peptide Human RLF MDPRLPAWALVLLGPALV Porcine RLF MDPHPLTWALVLLGPALA Bovine RLF MDRRPLTWALVLLGPALA Mouse RLF M-RAPL--LLMLLALGSA * * ** B-domain FALGPAPTPEMREKLCGHHFVRALVR LSRAPAPAQEAPEKLCGHHFVRALVR IALGPAAAQEAPEKLCGHHFVRALVR LR-SPQP-PEARAKLCGHHLVRTLVR * * ****** ** ***

VCGGPRWSTEARRP-AAGGDRELLQW LCGGPRWSPEDGRAV-AGGDRELLQW LCGGPRWSSEEDGRPVAGGDRELLRW VCGGPRWSPEATQPV-ETRDRELLQW ******* * ***** * C-domain LERRHLLHGLVADSNLTLGPGLQPLP LEGQHLFHGLMASGDPMLVLAPQPPP LEGQHLLHGLMASGDPVLVLAPQPLP LEQRHLLHALVADVDPAL--DPQLPR ** ** * * * * * A-domain QTSHHHRHHRAAATNPARYCCLSGCT QASGHHHHRRAAATNPARHCCLSGCT QASRHHHHRRATAINPARHCCLSGCT QASQRQRRSAAT--NAVHRCCLTGCT * * * * *** ***

QQDLLTLCPY RQDLLTLCPH RQDLLTLCPH QQDLLGLCPH **** ***

Fig. 1. Comparative structure of the relaxin-like factor (RLF) precursor polypeptide from several eutherian species. Asterisks indicate absolutely conserved amino acids. The putative receptor binding motif in the B-domain is boxed. indirectly by the structure of the N-terminal signal peptide moi- little or no local RLF production, would lead one to assume ety, and by the fact that secretion of marmoset RLF has been that RLF is indeed a secreted peptide factor like all other mem- demonstrated when the protein is produced biotechnologically bers of the insulin–IGF–relaxin family, and presumably able in baculovirus-infected insect cells (R. Zarreh-Hoshyari-Khah to enter the circulation or otherwise reach receptors in target and R. Ivell, unpublished). Finally, the demonstration that high tissues. affinity RLF-specific receptors appear to be present in the Cleavage of the full-length polypeptide can only be inferred mouse uterus (Büllesbach and Schwabe, 1995), a tissue with from the homology of the endopeptidase cleavage signals to

Downloaded from Bioscientifica.com at 10/01/2021 05:14:48AM via free access Biology of the relaxin-like factor (RLF) 135 those found also in the relaxin molecules from various species, the observation that in MA10 cells and primary mouse Leydig and which are thought to be substrates for enzymes such as furin cells in culture, none of the classic Leydig cell effectors and (Renegar et al., 1996). Although it seems likely that these are secretogogues (hCG, phorbol esters, atrionatriuretic peptide) cleaved in vivo – the human molecule, chemically synthesized influence the level of RLF mRNA in these cells (Pusch et al., like relaxin as an A–B heterodimer (Büllesbach and Schwabe, 1996). Furthermore, in a clinical study evaluating immuno- 1995) and shown to interact with specific receptors would sup- reactive RLF in the testes of patients with a variety of severe port this viewpoint – it should be noted that both IGFs are and less severe disorders, there appeared to be no significant bioactive without cleavage of the C-domain, and that porcine fluctuation in staining intensity, as was observed with the prorelaxin is just as bioactive as its cleaved product (Layden Leydig cell marker, 3β-hydroxysteroid dehydrogenase (Ivell and Tregear, 1996). et al., 1997). Thus, irrespective of the physiological environ- One of the most interesting features of the RLF peptide ment, the RLF gene appears to be expressed constitutively in structure is the conservation of a peptide epitope in the middle the adult Leydig cell and may leave the cell via the unregulated of the B-domain, comprising the motif X-R-X-X-X-R-X, which is pathway. very similar to the epitope identified as the receptor binding However, the RLF gene does appear to be developmentally site of relaxin (Büllesbach et al., 1992). The only difference is regulated. Analysis of rat and mouse testes through develop- that in all RLF molecules this feature is shifted four amino acids ment show that although RLF mRNA can be detected by PCR further C-terminal to the conserved cysteine residue. The avail- in the mouse embryo 13 days post coitum (Zimmermann et al., ability of this motif in approximately the same position on a 1997), the principal upregulation occurs during puberty (Pusch molecule whose three-dimensional structure is likely to be very et al., 1996; Zimmermann et al., 1997). In agreement with this similar to that of relaxin probably accounts for the moderate observation is the finding that in the adult hypogonadal (hpg) but specific affinity of RLF for relaxin receptors, but not for mouse, whose gonads remain effectively in a prepubertal state insulin or IGF receptors (Büllesbach and Schwabe, 1995). because of a deletion in the GnRH gene, RLF mRNA and pep- tide are undetectable. Only after several days treatment with hCG, which forces the testes to differentiate, does RLF become Leydig cell expression detectable (R. Ivell, M. Balvers, H. Charlton and A. N. Spiess, The RLF/Ley-I-L gene is expressed as a common, approxi- unpublished). mately 0.9 kb transcript in all testicular tissues so far examined. It was originally identified by differential cloning in the porcine Ovarian expression testis in a study looking for testis-specific transcripts (Adham et al., 1993), and independently in the mouse testis (Pusch et al., In the original studies that analysed RLF gene expression by 1996) in a study looking for clones upregulated preferentially northern hybridization, RLF was considered to be exclusively in the azoospermic mutant w/wv mouse compared with wild a testis-specific gene (Adham et al., 1993; Pusch et al., 1996). type mice. In situ hybridization in the porcine (Adham et al., Tashima et al. (1995), however, were able to show a weak 1993), mouse (Pusch et al., 1996), bovine (Bathgate et al., 1996), northern signal in human ovary and trophoblast, and sheep (Roche et al., 1996), and human testis (Ivell et al., 1997) Zimmermann et al. (1997) detected a similar signal in the mouse confirm that, within the testis, RLF is expressed exclusively in ovary. However, neither of these signals were comparable in the interstitial Leydig cells (Fig. 2). This has also been con- magnitude to those identified in the ruminant ovary (Bathgate firmed in studies using purified Leydig cells from mouse testis et al., 1996; Roche et al., 1996). Here, northern hybridization in which expression of RLF was greatly enriched compared with indicated that RLF mRNA was present in the ovary at levels as whole testis (Pusch et al., 1996; Zimmermann et al., 1997). Also, great or greater than those in the testis. In situ hybridization mouse Leydig tumour cells (MA10) show expression of the RLF showed that the strongest signals were not only in the theca transcript (Pusch et al., 1996; Zimmermann et al., 1997). Most re- cells of preovulatory and atretic follicles (Bathgate et al., 1996), cently, immunohistochemistry in the human (Fig.2; Ivell et al., but also in the corpus luteum of the mid–late cycle and in preg- 1997), mouse and marmoset (M. Balvers, A. Einspanier and nancy. Interestingly, the RLF gene was expressed only weakly R. Ivell, unpublished) confirm that, within the testis, only the in the corpus luteum of the early cycle, and was completely Leydig cells express the RLF peptide. downregulated in corpora lutea undergoing luteolysis. We The RLF transcript is exceedingly common. Calculations for have recently been able to confirm a comparable pattern of ex- the bovine testis suggest that some 5% of all transcription in pression in marmoset and mouse ovaries using immunohisto- Leydig cells goes to making this peptide. However, the adult chemistry with RLF specific antibodies (R. Ivell, A. Einspanier Leydig cell has characteristically little rough endoplasmic reti- and M. Balvers, unpublished). culum and is virtually devoid of classic 100–160 nm dense-core The very high expression of the RLF gene in the ruminant secretory granules, features which should be prominent in a ovary, by comparison with other species, is interesting since it cell secreting a large amount of a secretory polypeptide. suggests that only ruminants lack a gene for the related hor- Assuming that RLF is indeed secreted, the only explanation mone, relaxin (Hartung et al., 1995), which in most other mam- is that RLF is made not via the regulated granule-dependent mals is also expressed in ovarian theca and luteal cells and secretory pathway, but via the constitutive pathway, much as plays an important role in female reproduction. Yet ruminants hCG is produced in the placenta. This implies that RLF is do appear to have specific relaxin receptors, and exhibit a trafficked via the Golgi directly in small vesicles to the plasma characteristic relaxin-dependent physiology (Anderson et al., membrane, where it is released in a relatively unregulated 1995). In view of the ability of human RLF to interact with fashion. In support of an unregulated mode of production is relaxin receptors (see above), it is attractive to speculate that

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RLF may be functionally substituting for relaxin in ruminants factor can be acutely regulated through the agency of specific such as the sheep and cow. binding proteins, as in the case of the IGFs. Supporting a role It was pointed out in the context of the Leydig cell that RLF in differentiation is the finding that RLF is a marker for a gene expression appears to be constitutive, once differentiation specific stage of differentiation in the Leydig cell, and possibly of the cell had occurred. This appears also to be true for the also for theca/luteal cells. Adham and coworkers have recently bovine ovarian theca cell, in which in situ hybridization shows succeeded in producing knock-out mice whose RLF/Ley-I-L no change in the level of RLF mRNA expression, irrespective gene has been ablated. Preliminary reports suggest that only of whether the theca cells are parts of preovulatory or atretic the males are impaired and show a marked disturbance in follicles (Bathgate et al., 1996). spermatogenesis (Adham et al., 1997). A full study of these mice, including possible subtle effects in development, is eagerly awaited. Other tissues Very little is known about RLF expression in other tissues. Conclusion Except in the human where a very weak signal is detectable in trophoblast (Tashima et al., 1995), northern hybridization has The relaxin-like factor, RLF, is a highly expressed novel mem- consistently failed to show any specific RLF transcripts in any ber of the insulin–IGF–relaxin hormone family that is specifi- tissues other than testis or ovary. In a more extensive study of cally expressed in reproductive tissues with a common pattern bovine tissues using RT–PCR, Bathgate et al. (1996) were able to in males and females of all mammals so far investigated. Like detect RLF specific signals in endometrium and myometrium the related hormone, relaxin, it is to be expected that besides of the uterus both in the cycle and in pregnancy, and in car- certain principal functions, possibly in the differentiation of uncle and chorion, as well as in epididymis. Very weak signals reproductive tissues, it may have other local or more subtle were also detectable in heart and lung, and, suprisingly, there functions, for example within the brain. At the current stage was a moderate signal in the hypothalamus. Considering that of research, the tools available to investigate these functions specific RLF receptors were detected in the mouse brain in a are extremely limited. It is therefore of paramount importance heterologous assay using a human RLF peptide (Büllesbach to develop bioactive peptides, agonists and antagonists, and to and Schwabe, 1995), this last observation may point to a role establish sensitive immunoassays and receptor assays, as well for RLF also in the central nervous system. as appropriate in vivo and in vitro test systems in which to analyse this new factor. Only then will we be able to allocate it an appropriate place in the menagerie of reproductive Receptors and function endocrinology. All the evidence at present points to RLF being a novel secreted factor with a role to play in the testis and in the ovary. The author thanks colleagues and coworkers for helpful advice and Certainly for the testis and possibly also for the ovary, based permission to report their unpublished results, especially Ross Bathgate, on the level of gene transcription and the relative intensity of Wolfgang Pusch, Almuth Einspanier, Ilpo Huhtaniemi, Pasi Koskimies, immunostaining, RLF should be secreted at a level that is Rasoul Zarreh-Hoshyari-Khah, Marga Balvers, Werner Rust, and Ergin Kilic. In addition, the author thanks the Deutsche Forschungsgemein- likely to have not only local effects but also systemic effects. So schaft for financial support (grants Iv7/4-1-7 and Iv7/1-3). far, relevant receptors have only been sought for and found in mouse uterus and brain (Büllesbach and Schwabe, 1995) using a heterologous assay based on a synthetic A–B heterodimeric References human peptide. Additionally, there is evidence for local sys- Key references are identified by asterisks. tems in the uterus, possibly also involving the placenta during *Adham IM, Burkhardt E, Benahmed M and Engel W (1993) Cloning of a pregnancy, and in the brain. Altogether, this is a pattern of ex- cDNA for a novel insulin-like peptide of the testicular Leydig cells Journal pression very reminiscent of what is known for the related hor- of Biological Chemistry 268 26 668–26 672 mone, relaxin. Adham IM, Zimmermann S and Engel W (1997) Leydig insulin-like As with relaxin and the other members of this family, in- hormone-deficient mice show premeiotic arrest and germ cell death Reproduction in Domestic Animals 32 73 cluding the other newly described member, placentin (Koman Anderson LL, Gazal OS, Dlamini B and Li Y (1995) The role of relaxin in et al., 1996), we might expect that RLF is involved in growth ruminants. In Progress in Relaxin Research pp 428–438 Eds AH McLennan, and differentiation, particularly of reproductive tissues. The G Tregear and G Bryant-Greenwood. World Scientific Publishing, observation that its expression appears to be constitutive Singapore *Bathgate RAD, Balvers M, Hunt N and Ivell R (1996) Relaxin-like factor rather than regulated may preclude it from acute feedback gene is highly expressed in the bovine ovary of the cycle and pregnancy: roles, such as one finds for insulin or for inhibin. However, sequence and messenger ribonucleic acid analysis Biology of Reproduction one should not ignore the possibility that a constitutive 55 1452–1457

Fig. 2. Localization of relaxin-like factor (RLF) mRNA and peptide in the human testis by in situ hybridization and immunohistochemistry. (a) In situ hybridization using an antisense 35S-labelled cRNA probe photographed using direct illumination. (b) In situ hybridization using a sense cRNA probe as control. (c) Immunohistochemistry using an antibody raised against a recombinant human RLF peptide. (d) Control for the immunohistochemistry where the primary antiserum was replaced by an equivalent preimmune serum. (For methodological details, see Ivell et al., 1997).

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*Büllesbach E and Schwabe C (1995) A novel Leydig cell cDNA-derived pro- Koskimies P, Ivell R, and Huhtaniemi I (1997) Genomic structure and pro- tein is a relaxin-like factor Journal of Biological Chemistry 270 16 011–16 015 moter analysis of the mouse testis-specific relaxin-like factor gene Abstract Büllesbach E, Yang S and Schwabe C (1992) The receptor-binding site of VIth International Congress of Andrology Salzburg, May 1997 human relaxin II Journal of Biological Chemistry 267 22 957–22 960 Layden SS and Tregear GW (1996) Purification and characterization Burghardt E, Adham IM, Hobohm U, Murphy D, Sander C and Engel W of porcine prorelaxin Journal of Biochemical and Biophysical Methods 31 (1994a) A human cDNA coding for the Leydig insulin-like peptide (Ley-I-L) 69–80 Human Genetics 94 91–94 Pusch W, Balvers and Ivell R (1996) Molecular cloning and expression *Burghardt E, Adham IM, Brosig B, Gastmann A, Mattei MG and Engel W of the relaxin-like factor from the mouse testis Endocrinology 137 (1994b) Structural organization of the porcine and human genes coding 3009–3013 for a Leydig cell-specific insulin-like peptide (Ley-I-L) and chromosomal Renegar RH, Kwok SCM and McMurty JP (1996) Prohormone convertases localization of the human gene (INSL3) Genomics 20 13–19 in the porcine ovary and hamster placenta Biology of Reproduction 51 Hartung S, Kondo S, Abend N, Hunt N, Rust W, Balvers M, Bryant- (Supplement) 241 Greenwood G and Ivell R (1995) The search for ruminant relaxin. Roche PJ, Butkus E, Wintour EM and Tregear G (1996) Structure and ex- In Progress in Relaxin Research pp 439–456 Eds AH McLennan, G Tregear pression of Leydig insulin-like peptide mRNA in the sheep Molecular and and G Bryant-Greenwood. World Scientific Publishing, Singapore Cellular Endocrinology 121 171–178 *Ivell R, Balvers M, Domagalski R, Ungefroren H, Hunt N and Schulze W Tashima L, Hieber D, Greenwood F and Bryant-Greenwood G (1995) The (1997) Relaxin-like factor: a highly specific and constitutive new marker for human Leydig insulin-like (Ley-I-L) gene is expressed in the corpus Leydig cells in the human testis Molecular Human Reproduction 3 101–108 luteum and trophoblast Journal of Clinical Endocrinology and Metabolism Koman A, Cazaubon S, Vouraud PO, Ullrich A and Strosberg AD (1996) 80 707–710 Molecular characterization and in vitro biological activity of placentin, a Zimmermann S, Schöttler P, Engel W and Adham IM (1997) Mouse Leydig new member of the insulin gene family Journal of Biological Chemistry 271 insulin-like (Ley-I-L) gene: structure and expression during testis and 20 238–20 241 ovary development Molecular Reproduction and Development 47 30–38

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