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a Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
b Howard Florey Institute, University of Melbourne, Parkville, Victoria 3052, Australia
ABSTRACT
The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.
follicular development, Leydig cells, relaxin, theca cells
INTRODUCTION
The relaxin-like factor (RLF) was first described as a testis-specific transcript from the porcine testis under the name Leydig insulin-like peptide (Ley-I-L) [1]. Since then, cDNA clones have been characterized from the testes and other tissues of a broad range of mammalian species (Fig. 1), including human [2–4], marmoset monkey [5], bovine [6], sheep [7], mouse [8], rat [9], dog [10], deer [11], goat [12], and a subprimate species [13]. In the primary structure of the encoded protein, RLF clearly belongs to the family of insulin- and relaxin-like molecules, which now includes nine members (Table 1), of which at least two are known to be duplicated in certain species, i.e., relaxin H1 and relaxin H2 in the human and anthropoid apes and insulin 1 and insulin 2 in rodents. The latest member of this family, relaxin 3, appears to be not a local duplication but to be present in several discrete groups of mammals [14]. In the meantime, genomic nomenclature has applied the name INSL3 (insulin-like factor 3) for the RLF gene. The term relaxin-like factor, used here for the encoded protein and its mRNA, derives both from the similarity of the primary sequence, particularly in the B domain, to that of relaxin and from the early observations that chemically synthesized RLF heterodimer was able to interact weakly with relaxin receptors but not with those for insulin [15]. Most recently, the fact that RLF and relaxin both probably use very similar receptors (see later), quite different from those used by insulin, IGF1, and IGF2, reinforces this viewpoint.
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The family appears to have evolved in part by local gene duplication. While some of the genes stand alone, others, like INSL4, INSL6, RLN1, and RLN2, obviously arose by local duplication (Fig. 2). Interestingly, both INSL6 and INSL3 are close to sequences for the Janus kinases 2 and 3, respectively, suggesting that, in this case, a relatively large locus may have been duplicated, possibly as part of a complete genome duplication. We have used the public and Celera Genomics databases to search for novel members of this peptide family [16]. No new members of the insulin/relaxin family were identified, although we were able to confirm that there are no mouse equivalents of human INSL4 or human gene H1 relaxin. Hence, as the two human relaxin genes (H1 and H2) are localized together with INSL6 and INSL4 on chromosome 19, it is probable that INSL4 and H1 relaxin are the result of a gene duplication that did not occur in nonprimates.
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While no RLF/INSL3 sequences are known from nonmammalian species, it is significant that a new relaxin-like species from a frog [17], as also the so-called relaxins from cartilaginous fish, are expressed to a high level in the testis as well as in female tissues [18], suggesting that RLF and relaxin may have functionally separated during the evolution of mammals. At around this time, RLF may have acquired its specific role as a prime factor in testicular descent (see later), a physiology unknown in nonmammalian vertebrates.
RLF PROTEIN
Like the other members of this peptide family, the cDNA predicts a three-domain structure (Fig. 1). Following a conventional signal peptide, which presumably permits access of the nascent protein to the endoplasmic reticulum, there are domains corresponding to the B-chain, C (connecting)-peptide, and A-chain of relaxin. Only very recently has the structure of an in vivo-synthesized protein been elucidated [19]. While the extraction procedure adopted probably prevented an analysis of larger forms, e.g., the prohormone, sufficient mature peptide could be extracted from bovine testis to show that, in this tissue at least, the pro-RLF precursor can be processed further in vivo to an A-B heterodimer.
Previously, based on the structure of relaxin and predicted protease cleavage sites, a synthetic sheep RLF peptide was produced [20], the structure of which is represented in Figure 3. Similar predictions have been used to synthesize RLF peptides from the rat [21], mouse [22], and human [15]. Synthetic human and mouse RLF labeled with 125I binds with high affinity to binding sites in the mouse uterus and brain [15, 22]. Furthermore, synthetic rat RLF has been shown to be bioactive because it is able to induce growth in whole-organ cultures of fetal rat gubernaculum [21]. Although an early report suggested that RLF may bind to relaxin receptors [15], recent data have shown that RLF peptides have only a very low affinity for these receptors [20, 21]. Furthermore, synthetic sheep RLF analogues with amino acid changes to insert a full relaxin-binding motif in the B chain result in minimal relaxin activity [23] (Fig. 3). Whereas in an earlier study, circular dichroism spectroscopy measurements indicated that RLF and relaxin had apparently similar secondary structures [15], more recent studies suggest that RLF is less helical [21, 23]. Therefore, it is likely that the RLF and relaxin molecules are sufficiently different in structure, thus influencing the conformation of the binding motif, which in turn contributes to the reduced activity of this RLF analogue with a relaxin receptor-binding motif. In addition, recent studies have shown that the residues that are important in the binding of RLF to its receptor are in the carboxy-terminal end of the B chain [24] (Fig. 3). Hence, relaxin and RLF evidently contain subtly different secondary structures and very different receptor-binding domains. Interestingly, the native RLF extracted from bovine testis showed slightly lower affinity for mouse uterine receptors than the synthetic peptide [19].
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Inspection of the pro-RLF sequence from all species implies that there should indeed be proteolytic processing in vivo since there is a conserved arginine residue close to the B-C domain transition that forms part of the corresponding cleavage signal for insulin and relaxin. However, while chemically synthesized A-B heterodimer based on such a predicted cleavage site is bioactive (see above), the recently characterized in vivo bovine peptide shows that cleavage of the B-C junction in vivo occurs nine residues further C-terminal to this conserved arginine (Fig. 3). It remains to be seen whether the native peptide is further cleaved by proteases in the blood or target tissues to produce the shorter form of the peptide that shows higher bioactivity. At the C-A transition, there is a highly unusual histidine-rich motif, which shares structural features with the furin protease motif possessed by relaxin. This prediction is indeed confirmed from the bovine peptide sequence extracted from testis tissue [19]. This important new report also indicated a variation in the cleavage site for the signal peptidase. While the majority of the signal peptide is evidently cleaved as predicted, approximately 20% of the extracted peptide shows evidence of an N-terminal extension of five amino acids, implying cleavage at an alternative residue [19].
Various polyclonal antibodies have been raised either against peptides or peptide fragments or against recombinant pro-RLF [4, 5, 10, 19, 25–27]. These antibodies appear to work very well in an immunohistochemical context and show that RLF is expressed as protein at a high level in the Leydig cells of the testis, in the follicular theca cells and luteal cells of the ovary, and in some other tissues (see later). The punctate pattern of Leydig cell staining [25] appears to localize the RLF epitopes to the perinuclear Golgi apparatus in these cells. While such antibodies work specifically in Western blots against recombinant protein (e.g., [5, 26]) and in some cases also recognize chemically synthesized A-B heterodimer, with one apparent exception [10], they so far have failed to recognize a protein from tissue extracts with this technique (unpublished data).
Antibodies raised against chemically synthesized heterodimer have been used successfully to establish immunoassays measuring human and rat RLF in peripheral blood [19, 22, 27]. It could be shown for the human that the highest concentrations (
2 ng/ml) are detected in the blood of adult men [27]. Prepubertal children and adult women have barely detectable levels. In rats, the highest peripheral RLF levels (2.4 ng/ml) are measured in perinatal blood of male animals [22], corresponding to high expression in the fetal Leydig cell population (see later). Following a prepubertal hiatus, levels increase again in male rats to reach moderate amounts (0.6 ng/ml) in adulthood. These findings are important in demonstrating that RLF is indeed a secreted hormone in vivo and is preferentially produced in male mammals. However, this does not exclude local paracrine functions in females or at other times in males.
RLF/INSL3 GENE AND ITS TRANSCRIPTION
Genomic sequences have been cloned for the human [28], pig [28], mouse [29, 30], rat [9], and bovine (unpublished data). The RLF/INSL3 gene comprises two exons, with an intron interrupting the C-peptide coding domain, just as for insulin and relaxin genes. Surprising was the discovery that the RLF/INSL3 gene is colinear within the 3' end of the gene encoding Janus kinase 3 (JAK3) [29]. This was subsequently confirmed also for the other species studied. Accordingly, the presumptive promoter region of the RLF/INSL3 gene is restricted within intron 23 of the JAK3 gene. Certainly, a short region of only about 500–700 base pairs from within this intron appears to be sufficient to govern specific RLF/INSL3 gene expression when transfected into mouse tumor Leydig cells [29, 30].
Preliminary promoter analyses using various mouse gene constructs have highlighted the importance of three putative binding motifs for the transcription factor steroidogenic factor-1 (SF-1) within the region immediately proximal to the transcription start site [29, 31]. Thus, the RLF/INSL3 gene is like many others expressed in Leydig and theca cells, which require SF-1 for expression.
Although there is only a single major transcript from the RLF/INSL3 gene of approximately 690 bases plus about 150 adenosines in the poly(A) tail, reverse transcription-polymerase chain reaction (RT-PCR) analysis has shown there to be several different minor transcripts resulting from alternative splicing [5, 32]. These all predict truncated proteins wherein a stop codon abbreviates the open reading frame following the B-peptide domain. There is as yet no evidence that any of these protein products are actually made in vivo.
REGULATION OF RLF/INSL3 GENE EXPRESSION
Transcripts of the RLF/INSL3 gene represent one of the most abundant mRNAs in the adult Leydig cell. The gene is completely down-regulated in the mesenchymal prepubertal Leydig cell precursors and is progressively up-regulated upon pubertal differentiation. This has been demonstrated particularly in mice and rats [8, 25] and was confirmed using the hypogonadal mutant mouse [25], where daily injections of hCG are required to induce differentiation of the adult Leydig cells. The RLF/INSL3 gene is also up-regulated in the fetal Leydig cell population, attaining maximal mRNA levels in the rat on about Day 19, shortly before birth, at a time when RLF is probably required for testicular descent (see later). Because the differentiation and androgen production of the fetal Leydig cell population is independent of gonadotropins, the fetal and neonatal testis of hypogonadal hpg mice express normal amounts of RLF. Although, there is a marked chronic gonadotropin-induced up-regulation of the RLF/INSL3 gene in Leydig cells during puberty, the mRNA appears to be constitutively expressed once full differentiation status has been reached. No classic effector of Leydig cell physiology was able to influence the levels of RLF mRNA in primary cell cultures [25] nor were significant differences in RLF mRNA or protein observed in human testicular biopsies from patients with various severe testicular pathologies [4]. The only situations where a marked change in RLF mRNA and protein concentrations in the testis are observed are in Leydig cell tumors or hyperplasias [33], where proliferation and dedifferentiation of the Leydig cells appear to contraindicate RLF expression. This finding is supported by the observation that Leydig tumor cell lines (e.g., MA10, R2C) exhibit much lower endogenous RLF mRNA levels than an equivalent number of primary Leydig cells ([25]; unpublished results). Also in the aging rat testis, when the steroidogenic function of the Leydig cells appears to deteriorate, there is a marked reduction of RLF mRNA expression [34]. Because of its constitutive but differentiation-dependent mode of expression, RLF is an ideal marker with which to follow changes in Leydig cell differentiation. As such, it has been used not only to follow the dynamics of puberty but also to monitor Leydig cell regeneration following treatment of rats with ethane dimethane sulfonate [9, 35] or the cyclic changes in the hamster testis following a shift from a long-day to a short-day paradigm (unpublished) or in the testes of PDGF-A knockout mice [36].
In the male, RLF appears to be made almost exclusively in the Leydig cells of the testis; only with the help of RT-PCR can transcripts be detected in other tissues, e.g., in the epididymis or in the prostate [5, 8, 30]. In the cow, RLF transcripts have also been detected by Northern analysis in the hypothalamus [6]. In the female, RLF appears to be somewhat more widely distributed. The ovary is the major source of RLF, with both mRNA and peptide being detectable in the follicular theca cells of antral follicles in ruminants [6, 7], humans [37], and the marmoset monkey [5] as well as in the corpora lutea and ovarian stroma of these species. RLF is also expressed, probably as a paracrine factor, in the uterine stroma of the marmoset monkey [5] and in placental tissues in a variety of different species [3, 11, 38]. In the marmoset uterus, an up-regulation of RLF expression in vivo also correlates with a shift from the proliferative to the more differentiated secretory phase of the cycle [5]. Finally, RLF has been detected in the thyroid and mammary glands [39, 40].
Most of these observations are the results of immunohistochemistry, in situ hybridization, or RT-PCR and generally offer only a static picture of RLF expression. Only for bovine theca cells has it been possible to undertake a dynamic study of RLF gene expression [6, 41]. In the ruminant ovary, RLF is expressed at much higher levels than in other species [6, 7], possibly linked to the evident natural loss of the gene for relaxin [42, 43]. In vivo RLF mRNA and protein appear to be maximally expressed in the theca cells of small to medium-sized antral follicles, with lower amounts in large follicles and almost undetectable levels in very early corpora lutea. With progression of the estrous cycle, RLF becomes readily detectable again in the large luteal cells of the mid- to late corpus luteum and increases to high levels in the corpus luteum of pregnancy until very shortly before parturition. Thus, also in the theca-lutein cell lineage, RLF appears to be expressed in a differentiation-dependent fashion. In primary theca cell cultures, RLF mRNA is initially down-regulated as the cells proliferate in the early phase of culture. A hiatus is reached at about 6 days, after which specific mRNA levels increase continuously for 2–3 wk. These cultures are serum-free and require only insulin or IGF1. LH added to the cultures inhibits RLF expression, as does serum [41].
RLF FUNCTIONS AND PHYSIOLOGY
Mice in which the INSL3 gene has been deleted have been produced by two independent groups [44, 45]. The major phenotype in both cases is cryptorchidism in the male, caused by a failure of the gubernacular ligament to shorten during the second phase of testicular descent. In later experiments [21, 22], it was shown that the rat prepartum gubernaculum possesses receptors for RLF and responds in organ culture by DNA synthesis and alteration of shape. The first phase of testicular descent is androgen dependent and involves the dissolution of the cranial suspensory ligament, which in the female holds the ovary in a perirenal position. In gain-of-function transgenic experiments, an RLF-expressing construct driven by a strong constitutive promoter was introduced into female mice [46, 47]. These females developed an ovary that was forced into the inguinal region by a developed gubernaculum. In addition, the transgenic animals showed a tendency to hernia of the abdominal muscles, possibly reflecting a role for RLF in helping the testes to pass into the scrotum. Other experiments have shown that estradiol negatively influences testicular descent, as in diethylstilbestrol-induced cryptorchidism, by suppressing RLF expression in the fetal Leydig cells [48, 49]. Several searches have been made for mutations in the human INSL3 gene in cases of spontaneous cryptorchidism [50–55]. While several polymorphisms have been detected, there appear, so far, to be no correlates with the pathological phenotype.
While the role of fetal RLF in testicular descent is well characterized, there is still no ascribed function for RLF in the adult male. Adult defects in spermatogenesis in the knockout mice appear to be solely due to a secondary effect of the cryptorchidism. Because there may be RLF receptors in the brain [15], RLF may be involved in some behavioral function.
Although the female INSL3 -/- mice are fertile, there is nevertheless a female phenotype. The American group [44], but not the German [45] group, observed that, in the homozygous knockouts, there was a considerable lengthening of the normal estrous cycle from an average of 6–7 days to 14–20 days and that the litter sizes were significantly smaller. The failure to note such changes in the German experiments was probably due to the genetic background of the mice used, which had substantially shorter normal estrous cycles. In a subsequent, more detailed analysis of the ovaries of the German knockout mice, it could be shown that these had a higher apparent rate of apoptosis in both follicles and corpora lutea, implying that RLF was in some way normally protecting these cells from entering the standard default pathway of apoptosis and atresia [56]. Recently, we have been able to provide reinforcement for this role in a semiquantitative study of RLF expression in bovine follicles in vivo [26]. During the process of follicle selection, when small antral follicles either become atretic (the default pathway) or enter a maturation pathway to become preovulatory follicles, specifically the basal granulosa cells either become apoptotic or not, respectively. Although all such secondary follicles expressed equally 3ß-HSD and P450scc, only those indicating basal granulosa cell apoptosis also indicated a significant loss of RLF expression in the adjacent theca interna layer. Thus, RLF expression in the theca layer of secondary follicles during the selection process is a clear indicator that these follicles will not enter the default atretic pathway, suggesting that RLF may be at least protective, if not determinant in follicle selection.
Because RLF is also expressed in a number of other tissues, including the placenta, it is likely to have several other, probably paracrine roles. Because, however, the INSL3 knockout mice do not appear to have any other obvious phenotypes, these paracrine roles are probably physiologically redundant with those of other factors.
RLF RECEPTOR
Until very recently, the receptors for relaxin and RLF were predicted to be like those for insulin and IGF1; now two seven-transmembrane-domain receptors have been identified, LGR7 and LGR8, which appear to fulfill the requirements of the receptors for relaxin and RLF, respectively [57]. While so far it has not been possible to characterize LGR8 at the pharmacological level as specifically binding RLF, the cloned receptor binds relaxin with a slightly lower affinity than LGR7. More significantly, however, mice and, more recently, humans with a deletion or a mutation, respectively, in the LGR8 (also known as Great) gene exhibit a cryptorchidism phenotypically identical to that caused by deletion of the INSL3 gene in mice [58, 59]. Expression of LGR8 mRNA broadly corresponds with those tissues where we might expect a function correlating with the expression of the peptide (brain, kidney, muscle, testis, thyroid, uterus, lymphocytes, bone marrow [57]). While it appears that the cloned LGR7 and LGR8 receptors respond to ligand by activating adenylate cyclase [57], at least for relaxin, other signal transduction pathways also seem to be activated [60]. An interesting observation, not yet confirmed for RLF and LGR8, is that relaxin is able to bind with full affinity to only the N-terminal ectodomain of the LGR7 receptor [57]. This ectodomain includes a cysteine-rich high-complexity region.
In a recent study looking at the effect of changes in the primary amino acid sequence of RLF on its ability to bind specific receptors, it was shown that particularly amino acids at the C-terminus of the B-chain, including the conserved tryptophan, were involved in ligand binding [24]. However, the recent observation that an in vivo extension of the B-chain by nine amino acids only marginally reduces ligand affinity [19] parallels the observation for relaxin binding that C-terminally extending the B-chain does not affect receptor activation [61]. The hormone-receptor interactions for LGR7 and LGR8 are thus clearly distinct from those of the insulin receptor, where the prohormone has substantially decreased activity. It is also important to note that neither the separate A- or B-chains for relaxin or RLF can interact with the receptor, confirming the importance of heterodimerization for maintaining a bioactive conformation of the hormones.
CONCLUSIONS
RLF is a new hormone and, thanks to modern techniques such as transgenesis, we have a hint at some of its possible functions. These are certainly only the tip of the iceberg, but already we have determined roles in testicular descent and possibly in follicle selection. More systematic research will only be possible with the development of bioactive agonists and antagonists, robust immunoassays, and in-depth knowledge of its receptor and intracellular signaling mechanisms. In the next few years, we should expect to see a major increase in our understanding of this new hormone accompanying the development of the appropriate methods and tools.
ACKNOWLEDGMENTS
We gratefully acknowledge the many friends and colleagues who have helped us in establishing RLF/INSL3 as an important target for research, specifically Professors Geoffrey Tregear and Freimut Leidenberger for their encouragement and support.
FOOTNOTES
First decision: 13 March 2002.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant Iv7-9) and the National Health and Medical Research Council of Australia (NHMRC; reg key 983001). R.B. is a recipient of an NHMRC RD Wright Fellowship. 
2 Correspondence: Richard Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 49 40 561908 64; ivell{at}ihf.de
Accepted: March 27, 2002.
Received: March 4, 2002.
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S. Sudo, J. Kumagai, S. Nishi, S. Layfield, T. Ferraro, R. A. D. Bathgate, and A. J. W. Hsueh H3 Relaxin Is a Specific Ligand for LGR7 and Activates the Receptor by Interacting with Both the Ectodomain and the Exoloop 2 J. Biol. Chem., February 28, 2003; 278(10): 7855 - 7862. [Abstract] [Full Text] |
