Molecular Human Reproduction, Vol. 5, No. 2, 104-108,
February 1999
© 1999 European Society of Human Reproduction and Embryology
Expression of relaxin-like factor is down-regulated in human testicular Leydig cell neoplasia
1 Institute of Anatomy and Cell Biology, Martin Luther University Halle–Wittenberg, Grosse Steinstr. 52, D-06097 Halle (Saale), 2 Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, D-22529 Hamburg and 3 Department of Urology, Westfaelische–Wilhelms University of Muenster, Albert Schweizer Str. 33, D-48149 Muenster, Germany
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In addition to their role in steroidogenesis in the male, testicular Leydig cells constitutively express large amounts of the peptide relaxin-like factor (RLF), also known as Ley-IL. The Leydig cell-derived RLF belongs to the insulin-like superfamily, which also includes relaxin, insulin and the insulin-like growth factors, and within the testis is a specific marker of Leydig cells. Little information is available either on the regulation of gene expression or on the function of this Leydig cell-derived peptide. In the present study we have investigated the expression pattern of human RLF in patients with rare Leydig cell hyperplasia and adenoma. The expression of both mRNA and protein appear to be decreased in hyperplastic Leydig cells, whereas in the Leydig cell adenomas studied, large central areas of the adenoma were devoid of RLF mRNA and protein. Only Leydig cells located at the periphery of the adenoma displayed expression of RLF, with full agreement between in-situ hybridization and immunohistochemistry. It thus appears that the expression of the RLF gene and its products are down-regulated in Leydig cell hyperplasia and adenoma, consistent with a concomitant dedifferentiation of these cells.
adenoma/human testis/hyperplasia/Leydig cell/relaxin-like factor
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In recent years several new members of the insulin-like family, which includes relaxin, insulin and insulin-like growth factors, have been discovered in tissues of the female and male reproductive tracts of various species including human. These include the preprorelaxin-like molecule SQ10 in rabbit (Fields et al., 1995
), placentin (INSL4) expressed in human trophoblast cells (Chassin et al., 1995), and the Leydig cell-derived relaxin-like factor (RLF), also known as the Leydig-insulin-like peptide (Ley-IL) (Adham et al., 1993; Ivell, 1997). All members of the family appear to be synthesized as a preprohormone consisting of a signal peptide, and B-, C- and A-domains, from the N- to the C-terminus, respectively. Whether the C-domain is processed in the functional product, as in insulin and relaxin, is still not known for all species and all factors.
The RLF gene has been shown to be expressed in porcine, murine and human prenatal and postnatal testicular Leydig cells (Adham et al., 1993; Burkhardt et al., 1994; Pusch et al., 1996). In the human testis, RLF has been identified at the protein level as a specific marker for Leydig cells (Ivell et al., 1997). RLF has also been cloned from a bovine testis cDNA library and shown to be expressed in various bovine tissues of both the male and female reproductive tract as well as in the ovary (Bathgate et al., 1996). In addition, the human RLF is not restricted to the male reproductive tract but has also been detected in the corpus luteum and placental tissue in women by polymerase chain reaction (PCR) amplification and Northern analysis (Tashima et al., 1995). Expression of RLF in the corpus luteum is not unexpected since both theca and luteal cells as well as testicular Leydig cells derive from the mesonephric mesenchyme and, therefore, share embryological homology (Hunter, 1995).
Information on the physiological functions of the RLF in the testis is still lacking. Although preliminary reports for knock-out mice deficient in RLF indicate its possible role in spermatogenesis (Adham et al., 1997), the spatial distribution of RLF did not differ in patients suffering from severe oligozoospermia and azoospermia of different histopathological origin (Ivell et al., 1997). This would appear to exclude a direct involvement of RLF in these cases of disturbed spermatogenesis. As for the detection of relaxin in the rat hypothalamus (Gunnerson et al., 1995), the PCR amplification of message for RLF also in the bovine hypothalamus (Bathgate et al., 1996) has led to the hypothesis that this product of the testicular Leydig cells may be part of a neuroendocrinological feedback loop with the Leydig cell-derived RLF playing an important but as yet unidentified role in this hormonal circuit.
Leydig cell neoplasias are rare benign testicular tumours, whose appearances range from focal or diffuse hyperplasia to extensive Leydig cell adenoma, with displacement of adjacent seminiferous tubules. Neoplastic Leydig cells have been shown to have altered expression patterns for steroid receptors and intermediate filaments (Düe et al., 1989). In order to gain further information on a possible role for RLF in Leydig cell neoplasia, we have investigated the expression of RLF at both mRNA and protein levels in Leydig cell hyperplasia and adenoma in the human testis.
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Materials and methods |
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Human testicular tissues
Human testicular biopsy material from ten patients (aged 44–76 years; mean: 62.7) suffering from obstructive azoospermia with normal endocrine values and histologically normal spermatogenesis (score 9–10; Holstein and Schirren, 1983
) was obtained for histological diagnosis and used as control for immunohistochemistry and in-situ hybridization. In addition, testes of ten patients with Leydig cell hyperplasia (aged 29–46 years; mean: 36.1) and two patients with bilateral Leydig cell adenoma (aged 24 and 38 years) were selected for this study based on the histological diagnosis. Biopsy specimens were fixed in Bouin's solution for 6–12 h and temporarily stored in 70% ethanol at 4°C prior to embedding in paraffin wax according to standard procedures. Human testicular tissue revealing normal spermatogenesis (score 9–10) after histological examination was obtained after orchidectomy from patients with prostatic cancer (aged 52 and 76 years). The testicular tissue was snap frozen in liquid nitrogen and stored at –80°C until used for RNA extraction.
Cloning of human Leydig cell-derived RLF and digoxigenin labelling of cRNA
Total RNA was extracted from frozen normal testicular tissue using Trizol Reagent (Life Technologies, Eggenstein, Germany). Five µg of total RNA were used in reverse transcriptase PCR (RT–PCR) employing oligo-dT at a concentration of 500 ng/ml and superscript reverse transcriptase (Life Technologies) for first strand cDNA synthesis. Oligonucleotide primers specific for the nucleic acid sequence of human RLF (forward primer 5'-ATG GAC CCC CGT CTG CCC GCC-3' and reverse primer 5'-TCA GTA GGG ACA GAG GGT CAG CA-3') were used for PCR and flanked the single intron to preclude any genomic DNA amplification. PCR reactions were carried out in 50 µl solution containing 1 µl of cDNA, 100 µM of dNTP, 10 pmol of each primer, 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 2 mM MgCl2 and 2.5 U Taq polymerase. The PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 68°C, both for 1 min each, and an elongation step for 2 min at 72°C. After a final extension cycle for 10 min at 72°C, the 550 bp PCR fragment was purified on a 1% low melting point agarose gel, followed by Magic column extraction (Promega, Heidelberg, Germany) and cloned into the pGEM-T vector (Promega). Following verification of the clone by sequence analysis with the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer, Foster City, CA, USA), 5 µg of the plasmid containing the insert for Leydig cell-derived RLF were digested with the restriction enzymes Not I (antisense cRNA) and Sal I (sense cRNA; both New England Biolabs; Schwalbach/Taunus; Germany), phenol-extracted and precipitated for cRNA synthesis. One µg of digested and extracted plasmid was used for cRNA synthesis employing a cRNA synthesis kit (AMS Biotechnology; Wiesbaden, Germany) and a 10x digoxigenin (DIG)–RNA labelling mix (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. Digoxigenin-labelled cRNA was precipitated with 1/8 volume of 4 M LiCl and 3 volumes of 100% ethanol at –70°C for 1 h and washed once with 75% ethanol. The vacuum dried pellet was redissolved in 70 µl of diethylpyrocarbonate–water and the quantity of cRNA was determined spectrophotometrically at 260 and 280 nm and by dot blot analysis of serial dilutions of DIG-labelled cRNA spotted onto nylon membranes (Hybond N; Amersham, Braunschweig, Germany) according to the method of Jackson (1991).
Non-radioactive in-situ hybridization
Prior to non-radioactive in-situ hybridization, paraffin wax-embedded human testicular sections (6 µm thick) of normal and pathological human testicular tissues that had been attached to glass slides coated with 2% aminopropyltriethoxysilane (APTEX; Sigma, Deisenhofen, Germany) were dewaxed, rehydrated and permeabilized with proteinase K at 30 µg/ml for 30 min at 37°C. Further processing of the sections for in-situ hybridization and detection of the digoxigenin-labelled RNA was performed according to the procedure described by Lewis and Wells (1992) employing a 1:1000 dilution of an anti-digoxigenin alkaline phosphatase-conjugated Fab antibody (Boehringer Mannheim) in 1% bovine serum albumin. Specific signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. After counterstaining with haematoxylin, the slides were mounted in glycerol gel and examined under bright-field microscopy.
Immunohistochemistry
The polyclonal rat antiserum RA15 specific for human Leydig cell-derived RLF was employed for immunocytochemistry as described previously with slight modifications (Davidoff and Schulze, 1990; Ivell et al., 1997). Briefly, human testicular tissue sections were dewaxed, rehydrated and endogenous peroxidase activity was suppressed by treatment with 3% H2O2 for 45 min at room temperature. Sections were blocked for 60 min with 10% rabbit serum followed by an overnight incubation at 4°C with the specific anti-RLF primary rat antiserum diluted 1:2000 in 2% blocking solution (2% of rabbit normal serum diluted in Tris-buffered saline, TBS). Sections were washed six times for 5 min each in TBS followed by incubation with a 1:500 dilution in 2% blocking solution of biotinylated anti-rat immunoglobulin (Ig)G from rabbit (Dianova, Hamburg, Germany) for 1 h at room temperature. Sections were again washed six times for 5 min each in TBS, before adding rat peroxidase-conjugated peroxidase–antiperoxidase (PAP) complex (Dianova) diluted 1:500 in 2% blocking solution for 1 h at room temperature. After further washing, as above, the steps using biotinylated anti-IgG and peroxidase-conjugated PAP complex were repeated, each for 30 min. Finally sections were incubated with avidin–biotin–peroxidase (ABC) complex (Vector Laboratories, Burlingame, MA, USA) for 1 h at room temperature, washed and developed in diaminobenzidine substrate solution for 3–5 min. Thereafter, sections were mounted in Faramount medium (Dako, Hamburg, Germany). Primary sera were replaced by preimmune sera from the same animals as controls. Additional control experiments were also conducted to exclude non-specific binding of the rat antiserum to the glutathion-S-transferase tag used to purify the recombinant human RLF (Ivell et al., 1997).
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Using PCR primers that flanked the single intron in the N-terminal region of the C-domain of RLF, a specific PCR fragment of 550 bp was obtained, which could be confirmed by sequencing. Employing non-radioactive in-situ hybridization with digoxigenin-labelled cRNA derived from the cloned RLF-specific PCR fragment, an intense staining of Leydig cells from normal human testis was obtained with the antisense cRNA probe (Figure 1
) whereas the sense cRNA was negative in all tissues tested (not shown). In the Leydig cell adenoma tissue investigated, a pronounced loss of expression for Leydig cell-derived RLF was evident with large areas of the adenoma being devoid of the RLF-specific signals (Figure 3). In human testicular tissue with focal Leydig cell hyperplasia, the in-situ hybridization revealed a gradual depletion of specific mRNA from the periphery to the centre of the hyperplasia, whereas expression of the RLF gene in adjacent normal Leydig cells was unaffected (Figure 5).
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In agreement with the results obtained from the in-situ hybridization, immunohistochemical analysis of normal human testicular tissue revealed strong staining for RLF in normal Leydig cells (Figure 2). In Leydig cell adenoma, only the Leydig cells located at the circumference of the adenoma displayed substantial staining for RLF protein, while those towards the centre of the adenoma were negative (Figure 4). Focal Leydig cell hyperplasia demonstrated a marked decrease in the number of Leydig cells expressing RLF (Figure 6). Control experiments omitting the primary antiserum and testing for cross-reactivity to the GST-tag were negative (data not shown).
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Although RLF has been cloned in various species and mRNA for RLF has been detected in different tissues (Adham et al., 1993
; Burkhardt et al., 1994; Bathgate et al., 1996; Pusch et al., 1996), information on a possible function for RLF is still lacking. We have previously shown that RLF is a specific and constitutively expressed product of the adult human testicular Leydig cell, with the expression patterns of the RLF unchanged in testes of infertile men with a variety of oligo- and azoospermia of different aetiology as compared to healthy fertile individuals (Ivell et al., 1997). This implies that RLF is probably not directly involved in postpubertal germ cell function. Here we show that there is a substantial decrease and loss of gene expression for RLF in Leydig cells with benign neoplasia. The gradual loss of gene expression for RLF in Leydig cell hyperplasia and adenoma from the periphery to the centre of the neoplasia may have important implications for the study of RLF gene regulation and suggests that RLF may be a useful marker to discriminate between normal and neoplastic Leydig cells. Although no regulatory effectors have been identified that would affect the apparently constitutive expression of RLF in adult mouse Leydig cells, absolute RLF mRNA levels are much lower in Leydig cell lines derived from testicular tumours (Pusch et al., 1996). This would suggest that, since RLF expression is also very low in prepubertal rodent Leydig cells (R.Ivell, unpublished), Leydig cell neoplasia indeed involves a regression to a less differentiated phenotype.
Both hyperplastic and adenomatous testicular Leydig cells display altered gene expression for a variety of different molecules. In a subpopulation of tumour cells, transition from a normal to a hyperplastic Leydig cell in the human testis has been shown to result in increased expression for both oestrogen and progesterone receptors and altered expression of certain cytokeratins, such as the panel of cytokeratins 2–6 and 9–12 detected by the antibody KL-1 (Düe et al., 1989). Oestrogen agonists such as diethylstilboestrol have been shown to induce Leydig cell adenomata in mice via a paracrine mechanism that does not involve LH (Huseby and Samuels, 1977; Huseby, 1980; Navickis et al., 1981). The enhanced expression of oestrogen receptor and the ability of Leydig cells to secrete oestrogens (Castle and Richardson, 1986) may activate an autocrine self-enhancing process in which increased expression of oestrogen receptors, rather than an elevated secretion of oestrogen, stimulates the proliferation of neoplastic Leydig cells (Düe et al., 1989).
Leydig cells are also reported to contain high levels of insulin-like growth factor (IGF)-I and the corresponding IGF-I receptor (Lin et al., 1990). IGF-I and RLF belong to the insulin-like superfamily and IGF-I is known to stimulate steroidogenesis via specific receptors present on Leydig cells (Lin et al., 1986a,b). The possibility cannot be excluded that RLF may affect the expression of IGF-1 as has recently been shown in vitro and in vivo for relaxin in porcine granulosa cells (Oleth and Bagnell, 1995; Oleth et al., 1997). In fact both relaxin and the closely related RLF appear to be able to bind to the relaxin receptor and RLF enhances relaxin-mediated widening of the mouse symphysis pubis (Büllesbach and Schwabe, 1995; Parsell et al., 1996), indicating that although their physiological functions may differ, they may operate through similar tyrosine-linked receptors like insulin and IGF-I. For lack of a bioactive peptide it is not known whether also RLF may influence steroidogenesis.
It has long been known that hyperplasia of Leydig cells occurs in cryptorchidism, testicular feminization and in some conditions of impaired spermatogenesis including Sertoli cell-only syndrome (Gotoh et al., 1984; Gondos, 1987; Wong and Horvath, 1987). In addition, a considerable number of chemical substances, including well-known mutagens, have been identified that can cause testicular Leydig cell hyperplasia and adenoma in rats, mice and dogs. Of these agents, a number of non-DNA-reactive compounds either compete with testosterone and dihydrotestosterone for binding to the androgen receptor or block certain steps in the biosynthesis of both androgens (Clegg et al., 1997). As a consequence of prolonged exposure, low testosterone levels may, via pituitary feedback, result in elevated levels of luteinizing hormone (LH) which, by binding to LH receptors on Leydig cells, can induce them to proliferate. Preliminary studies have suggested that there may be specific receptors for RLF in the mouse uterus and brain (Büllesbach and Schwabe, 1995). However, detailed knowledge on the distribution and regulation of RLF receptors is still completely lacking. Such information could provide important clues as to a possible action of the Leydig cell-derived RLF, and it has been suggested that RLF might play a role in reproductive homeostasis (Ivell et al., 1997), possibly involving feedback to the brain and pituitary.
In summary, we have demonstrated that RLF expression is down-regulated in human Leydig cell hyperplasia and adenoma. Subsequent studies are in progress to extend the phenotypic characterization of those cells devoid of RLF and to try to analyse the regulatory processes that influence RLF expression in Leydig cell neoplasia.
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Acknowledgments |
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The authors would like to thank Mrs Christine Froehlich, Rosemarie Rappold and Heidrun Mrusek for their excellent technical assistance.
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4 To whom correspondence should be addressed
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