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Reproduction (2008) 135 351-355
DOI: 10.1530/REP-07-0330
Copyright © 2008 Society for Reproduction and Fertility
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RESEARCH

Cryptorchidism induced in normal rats by the relaxin-like factor inhibitor

Erika E Büllesbach1, Fredric R Boockfor2, George Fullbright1 and Christian Schwabe1

Departments of1 , Biochemistry and Molecular Biology and2 Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425, USA

Correspondence should be addressed to C Schwabe; Email: schwabec{at}musc.edu


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Cryptorchidism is a serious problem, which affects 2–5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The relaxin-like factor (RLF, also known as insulin-like-3 (INSL3) peptide, or Leydig insulin-like peptide) and its receptor, the leucine-rich repeat G protein-coupled receptor 8 (LGR8, alternatively called RXFP2), are largely responsible for the physiological migration of the testicles from their original high abdominal position into the scrotum (Kumagai et al. 2002, Adham & Agoulnik 2004, Hutson & Hasthorpe 2005). Knockout of either the hormone (Nef & Parada 1999, Zimmermann et al. 1999) or the receptor gene (Overbeek et al. 2001) resulted in high abdominal retention of the testis and infertility in homozygous male mice. RLF, produced by the Leydig cells (Adham et al. 1993), activates the LGR8 receptors (Gorlov et al. 2002) in the gubernaculum, resulting in growth changes that facilitate intra-abdominal migration of the testes to the inguinal canal (Hutson & Hasthorpe 2005, Amann & Veeramachaneni 2007).

During recent studies, we have discovered that both the signaling and binding sites of RLF are located on different non-overlapping segments of the molecule (Büllesbach & Schwabe 2005), and that signaling requires both of the sites to work in concert to initiate cAMP production (Büllesbach & Schwabe 2007). Deletion of the A-chain signaling site produced a competitive inhibitor of RLF (RLFi) that retained all of the binding avidity of native RLF but failed to signal cAMP production. RLFi caused 50% inhibition at equimolar RLF concentrations in binding assays on LGR8 bearing 293T cells (Büllesbach & Schwabe 2005). Presently, we are reporting the induction of cryptorchidism in normal rats by injections of RLFi.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
RLFi, an RLF derivative devoid of the signal initiation region of the A-chain (Fig. 1), can inhibit the effect of endogenous RLF by displacing the active hormone from its receptor in whole cell assays (Büllesbach & Schwabe 2005). Presently, we are evaluating the application of RLFi in vivo to establish a rat model of cryptorchidism. Based on the timing of the first Leydig cell appearance in rodents (12.5 days post coitum (dpc); O'Shaughnessy et al. 2006) and the observation that RLF mRNA is first detected in the mouse at 13.5 dpc by RT-PCR (Zimmermann et al. 1997), we administered RLFi to inhibit the physiological action of endogenous RLF throughout prenatal development. Therefore, RLFi was administered to the mother starting at day 12 of pregnancy via an osmotic pump. This treatment did not cause adverse effects even at the highest concentration. At the time of delivery, the litter size of the control and experimental rats was normal with equal distribution of males and females. The number of males investigated in this study is listed in Table 1. The experiment was terminated at day 23 post partum (pp), the time when testicular descent in control rats is almost completed. At that time, blood was collected from the mother to determine the inhibitor concentration by a competitive radio-ligand LGR8 binding assay. By this assay, one obtains a sum of circulating inhibitor and endogenous hormone not including degradation products. Animals treated with the highest dose (20 mg/ml) of RLFi showed 8 ng/ml (±1 ng/ml) in the mother's serum, while no LGR8-specific binding was detected in the sera of the offspring or in control rats. Considering that the circulating endogenous RLF in females is negligible these experiments do suggest that from days 12 pc to 23 pp a constant level of ~8 ng/ml of the inhibitor was present in the mother's serum. Endogenous rat RLF for comparison peaks at a concentration of 2–3 ng/ml in male rat serum at day 19 pc (Boockfor et al. 2001). At day 23 pp, endogenous RLF in the male averages 0.37±0.1 ng/ml (Boockfor et al. 2001), which is below the detection level of the receptor binding assay.


Figure 1
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  		Figure 1 The primary structure of RLFi (black). Eight residues (grey) have been deleted from the N-terminal end of the A-chain of RLF to produce the competitive inhibitor.

 

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  		Table 1 Number of male rats used in this study and the position of the testes at day 23 pp.

 
The position of the testes in RLFi-treated animals was evaluated at day 23 pp. Animals showing bilaterally descended testes, the unilaterally cryptorchid, and bilaterally cryptorchid were listed separately. The location of the testes in bilaterally cryptorchid rats treated with 5 mg/ml was at the entrance to the inguinal canal whereas at the dose of 10 mg/ml, 2 out of 45 rats, and at the dose of 20 mg/ml, 5 out of 22 rats showed both testes in a position midway between the kidney and the beginning of the inguinal canal (Table 1). This suggests that we can modulate the severity of the syndrome by manipulating the concentration of RLFi.

As outlined in Table 1, bilateral cryptorchidism increases with the concentration of RLFi. The difference is significant (P<0.05) at doses of ≥10 mg/ml and did not appear to be more pronounced at 20 mg/ml. An example of the position of the gonads in treated (10 mg/ml) and untreated pups at day 23 pp is shown in Fig. 2A and B. The excised tissues show the testis attached to the gubernaculum and the cranial suspensory ligament (CSL). Comparison of the corresponding tissues from control and experimental rats revealed a more translucent gubernaculum in the experimental rat while the CSL regressed more in the control experiment (Fig. 2C).


Figure 2
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  		Figure 2 Photograph of the position of testes in day 23 post partum rats: (A) a control animal that shows the scrotal position of gonads and (B) an experimental animal at a dose of 10 mg/ml with inguinal retention. Frame (C) shows the dissected male reproductive tract of a control (left) and an experimental animal at a dose 10 mg/ml (right). The gubernaculum (G) of the experimental animal appears larger and more transparent than that of the control, while the CSL of the control animal regresses. T, testis; G, gubernaculum; CSL, cranial suspensory ligament.

 
Induction of cryptorchidism is dependent upon successful delivery of the agent to the pups, whereas rescue of a cryptorchid animal would require the transport of RLF from the pregnant mother across the placenta to the fetus. Accessibility of this pathway was confirmed when timed-pregnant rats were injected (i.p.) with 50 µg human RLF (hRLF) in PBS 2 days prior to parturition. After 30 min, hRLF is detected at about the same concentration in the experimental groups and is absent in the controls (Fig. 3). Since the antibody is hRLF specific, the natural spike of rat RLF occurring at that time of development (Boockfor et al. 2001) is not detected.


Figure 3
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  		Figure 3 Concentration of human RLF in rats at 19 dpc. Timed-pregnant rats received i.p. injections of either 50 µg human RLF in PBS or PBS control. The rats were killed after 30 min, sera collected and human RLF determined by RIA, using a polyclonal rabbit anti-human RLF antiserum (one representative experiment is shown). M, male fetus; F, female fetus. A serial dilution curve (1:1 v/v) of human RLF at a starting concentration of 100 ng/ml runs parallel to the rat serum samples.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Competitive inhibition is potentially the most informative way to perturb biological processes. In this article, we report the discovery of a competitive inhibitor of a hormone that is crucial for a physiological function, namely, the proper positioning of the gonads in males. Cryptorchidism is a serious human disorder that has remained refractory to preventive treatment for the lack of information that may become available if a good animal model can be developed. The identification of RLF as a necessary component of the physiological mechanism of testicular descent by gene deletion (Nef & Parada 1999, Zimmermann et al. 1999) and the discovery of RLFi (Büllesbach & Schwabe 2005, 2007) have created the potential for a significant break in a vexing problem.

Our investigations were performed with a recently discovered competitive inhibitor, RLFi, which displaced 50% of RLF from the receptor (LGR8) at equimolar concentration (Büllesbach & Schwabe 2005). Present experiments involving rats were designed to provide a constant level of RLFi to the fetuses such as to inhibit the prenatal spike of RLF, which occurs about 2 days before parturition (Boockfor et al. 2001, McKinnell et al. 2005). Between the time of birth and sexual maturity low levels of RLF are produced first by fetal Leydig cells and thereafter by adult Leydig cells (McKinnell et al. 2005, Mendis-Handagama et al. 2007). The physiological effect of postnatal RLF, however, is unknown and in our model it would be unaffected unless the pups take up RLFi during suckling. It has been shown in beagles that the hormone relaxin reaches the pups through the milk (Goldsmith et al. 1994).

Although RLFi effectively inhibits intra-abdominal migration of the testis in a dose-dependent manner, complete inhibition as described for the RLF (Insl3; Nef & Parada 1999, Zimmermann et al. 1999) or GREAT (Lgr8; Overbeek et al. 2001) knockout mouse has not been observed. From the mass action law, we know that saturation of a receptor is approached asymptotically and that the highest concentration may not achieve total displacement of RLF. This may be especially true since compensatory mechanisms of the endogenous hormones are not suppressed.

Gene deletion, which established a role for RLF in testicular retention, is a global procedure that deletes the RLF function from every tissue including those that are behind biological barriers. It is known that peptide hormones in different compartments may play different roles, which may precondition the organism for the proper function of a hormone in a target tissue. The RLF receptors noted in the gubernaculum (Boockfor et al. 2001, Overbeek et al. 2001), for example, are also observed in the uterus and the brain (Büllesbach & Schwabe 1995), and LGR8 mRNA was detected in other tissues as well (Overbeek et al. 2001, Hsu et al. 2003). Presently, we are unaware of a specific function of RLF in these tissues. Relaxin in the central nervous system (CNS), for example, is independent of the peripheral relaxin system. While CNS relaxin affects the timing of pregnancy it has no influence on the course of labor once initiated (Summerlee et al. 1998). As concerns the RLF system such possibilities must be considered.

Receptor-level inhibition methods are limited to cases where the signaling and the binding sites of a hormone are separate and independent. This represents a problem considering that the first signaling site has only recently been clearly identified in a peptide hormone (Büllesbach & Schwabe 2005). The experimental results presented here confirm the causal connection between RLF and cryptorchidism and are in harmony with the results obtained with Insl3–/– mice. The graded and time-dependent inhibition of testes descent by RLFi offers an opportunity to study multiple aspects of this complex process and to discover the time at which preventive application of RLF will be most effective. Human neonates normally would show descended testicles so that failure or retardation of the physiological process is obvious at birth.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents
RLF and RLFi were chemically synthesized as described in detail elsewhere (Büllesbach & Schwabe 2005). Briefly, each chain was synthesized by Fmoc chemistry using differentially protected cysteine side chains. Stepwise removal of these protecting groups allowed the direct synthesis of the three disulphide bonds. The synthetic products were characterized by reversed phase HPLC, matrix-assisted laser desorption/ionization mass spectrometry, competitive binding to LGR8, and their ability to stimulate cAMP production.

Animals
All procedures were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. Timed-pregnant (day 10) Sprague–Dawley rats (weight 250–270 g) were purchased from Harlan (Indianapolis, IN, USA) and maintained on a 12 h light:12 h darkness cycle with water and laboratory chow ad libitum. The rats were acclimated to the environmental conditions for 2 days before the treatment.

Effect of RLFi on male pups
At day 12 dpc, a mini-osmotic pump (model 2004D; Alza, Cupertino, CA, USA) was implanted in the peritoneal cavity. Anesthesia was induced with a premixed cocktail of xylazine (10 mg/kg) and ketamine (90 mg/kg) administered intra-peritoneally. A small cut was made in the lower abdomen under the rib cage to insert the mini-osmotic pump loaded with either 50 mM acetate buffer, pH 5.0 (vehicle), or with RLFi (5 mg/ml=0.9 mM, 10 mg/ml=1.8 mM, or 20 mg/ml=3.6 mM in acetate buffer). The pumps delivered 0.25 µl/h, which corresponds to 1.23 µg/h (0.225 nmol/h), 2.47 µg/h (0.45 nmol/h), and 4.95 µg/h (0.9 nmol/h) RLFi respectively. At this rate, the pump has the capacity to operate for 33 days. Once the mini-osmotic pumps were inserted, the incision was sutured. The animals recovered fully within 15 min. The pups were left with their mother until the experiment was terminated at day 23 pp.

Evaluation of testes migration
At 23 days of age, male pups were killed by CO2 anoxia and the abdominal cavity exposed. Testes were identified as abdominal or scrotal and the retained testes were classified as inguinal or, if located midway between the kidney and the beginning of the inguinal canal, as mid-abdominal.

Determination of RLFi in rat serum
At day 23 pp, blood was collected from the dams and the male and female pups. Sera of the pups belonging to the same litter and the same sex were pooled and serial diluted with binding buffer (20 mM HEPES, pH 7.5, 1% BSA, 0.1 mg/ml lysine, 1.5 mM CaCl2, 50 mM NaCl, 0.01% NaN3). Receptor binding assays were conducted using 293T/17 cells stably transfected with LGR8 as previously described (Büllesbach & Schwabe 2006). Serum concentrations of RLFi were obtained by comparison with a hRLF standard dose–response curve run in parallel.

Placental passage of hRLF
At 19 dpc, timed-pregnant rats were injected (i.p.) with 50 µg hRLF dissolved in 50 mM PBS (concentration: 0.5 µg/µl). After 30 min, the pregnant rats were killed by CO2 anoxia, decapitated, and trunk blood collected. The blood of pups of the same litter and the same sex were pooled, the sera collected, and serial diluted (1:1 v/v or 1:3 v/v) with RIA buffer (50 mM phosphate buffer, pH 7.4, supplemented with 0.15 M NaCl, 1% BSA, and 0.01% sodium azide). Three independent experiments were performed using a total number of three control rats and five experimental rats.

RIA
Rabbit anti-hRLF antibody (AB-9956) was produced against synthetic hRLF at the Monoclonal and Polyclonal Antibody Facility at the Medical University of South Carolina. The anti-serum was used for RIAs in combination with 125I-Tyr(A9)hRLF as tracer (Büllesbach & Schwabe 1999). Immobilized goat anti-rabbit IgG antibody was used to separate bound and free tracer for {gamma}-counting. All samples were compared with a hRLF standard curve.

Statistical analysis
Values are expressed as means±S.E.M. and data were analyzed using Student's paired t-test or one-way ANOVA. Statistical significance (P<0.05) is indicated.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
This work was supported by Grant 1-R01-HD40406 from the National Institutes of Health and by the Medical University of South Carolina, Research Committee. We thank Robert Bracey for technical assistance and Dr Bernard G Steinetz for constructive comments. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Received 18 July 2007
First decision 7 September 2007
Revised manuscript received 27 November 2007
Accepted 4 December 2007

   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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