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Testis |
BIOQUAL, Inc., Rockville, Maryland 20850
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gonadotropin-releasing hormone, inhibin, male reproductive tract, spermatogenesis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In these previous studies, CDB-4022-induced infertility was not spontaneously reversible in male rats, which is consistent with the action of compounds that disrupt Sertoli cell structure and function [4]. However, reversibility of infertility is an important consideration for a male contraceptive. We showed previously that a series of four injections of the GnRH agonist, Lupron Depot, administered 3 wk apart beginning 1 wk before administration of a single oral dose of CDB-4022 (2.5 mg/kg) resulted in the return of spermatogenesis and fertility in the majority of treated rats [1]. Other investigators have shown that treatment with GnRH analogues, agonists or antagonists, prevents or reverses testicular damage induced by radiation [5, 6], chemotherapeutic agents [7, 8], an environmental reproductive toxicant (dibromochloropropane; [9]), or a Sertoli cell toxicant (2,5-hexanedione; [10]). Hence, one of the goals of the present study was to determine whether the GnRH antagonist, acyline, would provide similar protection or restimulation of spermatogenesis in CDB-4022-treated rats. The mechanism by which GnRH antagonists restore spermatogenesis following treatment with radiation or testicular toxicants may be through suppression of gonadotropins, testosterone, and/or spermatogenesis. To determine whether acyline-induced suppression of intratesticular testosterone (ITT) levels was critical for prevention of permanent CDB-4022-induced testicular damage, the ability of testosterone to reverse acyline's protective effect against CDB-4022-induced irreversible infertility and testicular damage in the rat was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprague-Dawley CD rats (Crl:CD(SD)IGS BR Stock) were purchased from Charles River Laboratories (Kingston, NY). All rats were housed in polycarbonate solid-floor cages with Bed-o-Cob or Beta-Chip bedding (Andersons Industrial Products Group, Maumee, OH) and received Purina laboratory rodent diet (#5001; Purina Mills, St. Louis, MO) and tap water ad libitum. The photoperiod was 14L:10D. All female rats were group-housed, whereas adult males were individually housed except during mating trials. Proven fertile male rats were used for experiments II and IV; male fertility was assessed by in-house mating trials before study assignment [1]. The environmental conditions of the animal rooms were maintained as recommended by the National Research Council in Guide for the Care and Use of Laboratory Animals [11] to the maximum extent possible. All study protocols were approved by BIOQUAL's institutional animal care and use committee.
Materials
The GnRH antagonist acyline (Ac-D-Nal-D-4-Cl-Phe-D-Pal-Ser-Aph(Ac)-D-Aph(Ac)-Leu-Lys(Ipr)-Pro-D-Ala-NH2) was synthesized by Multiple Peptide Systems (San Diego, CA) and was 98.4% pure based on HPLC analysis. CDB-4022 was synthesized by Research Triangle Institute (Research Triangle Park, NC) and used in experiment II. A second batch of CDB-4022, synthesized by Dr. P.N. Rao (Southwest Foundation for Biomedical Research, San Antonio, TX), was used in experiment IV. Both batches of CDB-4022 were considered >99% pure based on HPLC analysis and, as in previous reports [1, 2], were a racemic mixture of l- and d-enantiomers. [1, 2, 4, 5, 6, 7-3H]-5
-Dihydrotestosterone (5-DHT; 123 Ci/mmole) was purchased from Perkin Elmer Life Sciences (Boston, MA) and was used as the radioligand in the androgen-binding protein assays. Sterile Alzet osmotic minipumps (model 2002 or 2004) were purchased from Alza Corporation (Palo Alto, CA). Needles, syringes, anesthetics, and surgical supplies were purchased from NLS, Inc. (Baltimore, MD). Anesthetics included isoflurane, ketamine, and xylazine. Reagent-grade or molecular biology-grade chemicals and enzymes were purchased from Sigma (St. Louis, MO), Boehringer Mannheim (Indianapolis, IN), or Quantum Chemical Co. (Tuscola, IL). Food-grade sesame oil (Hain) was purchased from a local grocery store.
Immunoassays
Unextracted serum samples were assayed for testosterone using a commercially available coated-tube testosterone radioimmunoassay (RIA, Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA). The assay procedure followed the kit's directions except that the incubation period was increased from 3 h at room temperature to overnight at 2–6°C. This modification was made because it allowed more accurate measurement of a known quantity of testosterone in castrate rat serum. No testosterone was detected in a serum pool from castrated rats. RIA data were analyzed using a four-parameter sigmoidal curve fit (RiaSmart Data Reduction Program; Perkin Elmer Life Sciences, Meriden, CT). The limit of detection in this assay was 0.04 ng testosterone/ml, and the intra- and interassay variations were 7% (n = 46) and 15% (n = 46), respectively.
Inhibin B was determined in serum samples using an ultrasensitive Inhibin B ELISA assay kit (Serotec, Oxford, UK). Lyophilized human inhibin B extracted from follicular fluid (supplied with the kit) was reconstituted in castrate-adult-male-rat serum and a standard curve generated by serial dilution of the human inhibin B in the same serum [2]. The standard curve was calculated using a linear fit program (EIACalc Dynatech MR-500; Chantilly, VA). The limit of detection for the assay was 25 pg human inhibin B/ml and the intra- and interassay variations were 2% (n = 3) and 19% (n = 17), respectively.
Levels of the rat gonadotropins, rLH and rFSH, were determined in serum samples by RIA using reagents supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). RIA data were analyzed using a four-parameter sigmoidal curve fit as above. The limits of detection for these RIAs were 2.6 ng NIDDK-rFSH-RP-2/ml and 0.31 ng NIDDK-rLH-RP-3/ml. The intraassay variation was 5% (n = 10) and 9% (n = 4), and the interassay variation was 13% (n = 26) and 24% (n = 16) for the rLH and rFSH assays, respectively.
Androgen-Binding Protein Assay
Androgen-binding protein (ABP) content in the epididymal cytosol was determined by specific binding of [3H]-5DHT and was expressed as femtomol bound per milligram cytosolic protein [2, 12]. Briefly, each frozen epididymis was thawed on ice, minced, and homogenized in four volumes of TE buffer (50 mM Tris, 1.5 mM EDTA, pH 7.2–7.4) at room temperature. The homogenate was centrifuged at 110 000 x g for 30 min at 40°C. Endogenous steroids were removed from the resultant supernatant by dextran-coated charcoal precipitation (DCC: 0.05% dextran, 0.5% charcoal in TE buffer). Total protein was measured in an aliquot of charcoal-stripped cytosol using Pierce BCA protein assay (Rockford, IL). Charcoal-stripped epididymal cytosol (40–50 µl) was incubated with 1 nM [3H]-5DHT in the presence or absence of 500 nM unlabeled 5DHT for 2 h at 4°C. DCC was added to separate bound from free radioligand. Opti-Fluor (Perkin Elmer, Wellesley, MA) was added to the resultant supernatant, and the samples were counted in a Beckman LS 1800 liquid scintillation counter. The amount of rat ABP per mg of cytosolic protein was calculated using Excel (Excel version 5.0 spreadsheet; Microsoft Corp., Seattle, WA). This assay had a limit of detection of 10 fmol ABP/mg protein and intra- and interassay variations of 31% (n = 3) and 26% (n = 6), respectively.
Experiment I: Suppression of Testicular Function by Acyline
This preliminary study was undertaken to determine the daily dose of acyline and the treatment interval (2 or 4 wk) necessary for suppression of circulating testosterone and testicular spermatogenesis in adult male rats. The data from this study were used to design the subsequent experiment to assess the ability of this GnRH antagonist to prevent and/or reverse the effect(s) of CDB-4022 on the rat testis. Adult male rats (380– 450 g) were treated with acyline at 55, 105, or 210 µg rat–1 day–1 (3 rats/ group) or vehicle (2% Tween 80 in bacteriostatic water) for 2 (model 2002) or 4 (model 2004) wk via an Alzet osmotic minipump implanted into the subcutaneous space between the scapulae.
At the end of the treatment interval, the rats were anesthetized and exsanguinated. Serum was harvested by centrifugation and the levels of testosterone were determined by RIA. The ventral prostate, seminal vesicles, and testes were excised and weighed. Following removal of the tunica albuginea, the left testis was homogenized, spermatid head counts determined, and the data expressed as the number of spermatid heads per testis [13]. The right testis was preserved in Bouin fixative, washed, dehydrated, and embedded in glycol methacrylate medium (GMA). Hematoxylin-stained cross-sections (2 µm) of the testis were used to determine the percentage of seminiferous tubules containing later stage spermatids (elongated to condensed spermatids) [13]. A total of 200 tubules/testis were examined for each treatment group and 100 tubules/testis for the vehicle control group.
Experiment II: Ability of Acyline to Prevent CDB-4022-Induced Testicular Damage and Irreversible Infertility
The experimental design is depicted in Figure 1. Proven fertile male rats (400–470 g) were assigned to one of seven treatment groups (5/ group): 1) vehicle for Weeks –4–0 and vehicle orally on Week 0; 2) acyline for Weeks –4–0 and vehicle orally on Week 0; 3) vehicle for Weeks –4–0 and CDB-4022 orally on Week 0; 4) acyline for Weeks –4– 0 and CDB-4022 orally on Week 0; 5) acyline for Weeks 0–10 and CDB-4022 orally on Week 0; 6) CDB-4022 orally on Week 0 and acyline for Weeks 10–20; and 7) acyline for Weeks –4–10 and CDB-4022 orally on Week 0. CDB-4022 or 10% ethanol/sesame oil was administered as a single oral dose of 2.5 mg/kg or 5 ml/kg, respectively, on Week 0 (Week 0 = Day 0, the day of vehicle or CDB-4022 dosing). This dose of CDB-4022 had been determined previously to be the minimally effective or threshold dose for inducing infertility in 100% of adult male rats [1]. Infertility was not reversible in the majority of adult male rats at this dose. Two osmotic minipumps (Alzet model 2004) implanted subcutaneously between the scapulae were used to deliver 210 µg acyline rat–1 day–1 or 2% Tween 80/bacteriostatic water (12 µl rat–1 day–1) for 4 wk. These minipumps were surgically removed and replaced as required by the experimental design (see Fig. 1).
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Individual male rats were weighed at Weeks –4 and 0, every 3 wk after oral dosing, and at necropsy (Week 34). Mating trials were performed weekly during Weeks 1–8 and every 2 wk from Week 10 through Week 32 and the females examined for pregnancy status. At Week 34 of the study, the anesthetized male rats were exsanguinated and sera were harvested. Serum testosterone, rLH, rFSH, and inhibin B levels were determined. The ventral prostate, seminal vesicles, epididymides, and testes were excised and weighed. Spermatid head counts were determined for the left testis. The right testis was preserved for morphological assessment. The tubule differentiation index (TDI), defined as the percentage of seminiferous tubules undergoing spermatogenesis beyond formation of B spermatogonia, was determined in these sections [13]. The ABP content in the epididymal cytosol was measured. During this extended study, one male rat in group 1 and one male rat in group 3 died at Weeks 14 and 16 of the study, respectively, resulting in n = 4 for these groups.
Experiment III: Testosterone Replacement in Acyline-Treated Rats
To test the hypothesis that acyline protects the testes from irreversible damage via suppression of testosterone production, we wanted to verify that testosterone replacement via subcutaneous placement of a 6-cm Silastic implant would be sufficient to maintain spermatogenesis. Therefore, a study was undertaken to define testicular function in intact adult male rats (n = 5/group, 300–340 g) treated for 4 wk with vehicle (2% Tween 80/bacteriostatic water, 12 µl rat–1 day–1), acyline (210 µg acyline rat–1 day–1), or acyline and a subcutaneous 6-cm testosterone-filled Silastic implant (Silastic tubing of 0.078'' x 0.125; Speciality Manufacturing, Inc., Saginaw, MI). Testosterone-filled implants of this size were previously shown to raise intratesticular testosterone (ITT) and inhibit GnRH antagonist-induced recovery of spermatogenesis in irradiated rats [6]. Vehicle and acyline were administered using osmotic minipumps as described above. In this manner, all rats underwent a surgical procedure at the start of the study (Week 0). Rats were subjected to necropsy at Week 4. The ventral prostate, seminal vesicles, epididymides, and testes were excised and weighed. Spermatid head counts were determined from a portion of the homogenate prepared from the left testis. The remainder of the homogenate, prepared in TEGMD buffer (10 mM Tris [pH 7.2], 1.5 mM EDTA, 20 mM sodium molybdate dihydrate, 10% glycerol, 1 mM dithiothreitol), was centrifuged at 
109 000 x g for 1 h to obtain cytosol, which was assayed for ITT content. The right testis was preserved for morphological assessment. Sera harvested from collected blood were assayed for testosterone, inhibin B, rLH, and rFSH. Treatment-induced changes in serum hormone levels were reproduced in the subsequent experiment (experiment IV); therefore, the data from this specific experiment are not shown.
Experiment IV: Testosterone Replacement Blocks Acyline's Protective Effect Against CDB-4022-Induced Testicular Damage
The experimental design is depicted in Figure 1. Proven fertile male rats (400–470 g) were assigned to one of three treatment groups (7–8/ group): 1) vehicle (2% Tween 80/bacteriostatic water, 12 µl rat–1 day–1) for Weeks –4–0 and CDB-4022 at 2.5 mg/kg orally on Week 0; 2) acyline at 210 µg rat–1 day–1 for Weeks –4–0 and CDB-4022 at 2.5 mg/kg orally on Week 0; 3) acyline at 210 µg rat–1 day–1 and a subcutaneous 6-cm testosterone-filled Silastic implant for Weeks –4–0 and CDB-4022 orally at 2.5 mg/kg on Week 0.
Individual male rats were weighed at Weeks –4, –2, and 0, and every 3 wk after oral dosing and at necropsy (Week 20). Mating trials were performed weekly during Weeks 1 and 2 and then every 2 wk from Week 2 through Week 16 and the females examined for pregnancy status. Male rats were bled from the tail vein, before any treatment, at Weeks –4, –2, 0, 1, 3, 6, 9, and 15. At Week 20 of the study, the anesthetized male rats were exsanguinated and sera were harvested. Serum testosterone, rLH, rFSH, and inhibin B levels were determined from the samples collected throughout the study. The ventral prostate, seminal vesicles, epididymides, and testes were excised and weighed. Spermatid head counts were determined for the left testis. The right testis was preserved for determination of the TDI.
Statistical Analysis
Statistical analyses were performed using SigmaStat version 2.03 (SPSS Inc., Chicago, IL). All tests were two-tailed with significance set at = 0.05. Graphs were prepared using SigmaPlot 2001 (SPSS Inc.). The ratio of males rendered infertile and the ratio of males that recovered fertility were compared with a control using a z-test of proportions for each relevant experiment. Analysis of variance (ANOVA) was used to determine significant differences among treatment groups within an experiment. Whenever possible, a parametric ANOVA was used if the data met the test criteria of normality and homogeneity of variances. Data were transformed, as needed, to meet these assumptions. For data that did not met these criteria, a nonparametric Kruskal-Wallis ANOVA on ranks was performed. For measurements obtained from the same animal over time, an ANOVA for repeated measures was used. For a significant F-value, comparisons among treatment groups were determined by Bonferroni t-test for comparisons to control or Student-Newman-Keuls (SNK) multiple range test for all pairwise comparisons. Dunn method was used for multiple comparisons following the nonparametric Kruskal-Wallis ANOVA on ranks for experiments with an unequal number per group. Specific statistical tests are included below.
For experiment I, significant differences among the treatment groups in serum testosterone levels, the weights of paired testes and ventral prostate (log10 transformed), percentage of tubules containing elongated to condensed spermatids, and spermatid head counts were determined by ANOVA followed by Bonferroni t-test for multiple comparisons to the vehicle control group. The seminal vesicle weights were compared using a Kruskal-Wallis ANOVA on ranks followed by Dunn method for multiple comparisons to the control group.
For experiment II, body weights over time were compared using a two-way ANOVA for repeated measures. Comparisons among the treatment groups were assessed based on a parametric ANOVA followed by SNK test for a significant F-value for the following endpoints: paired testes, paired epididymal, and seminal vesicle weights; serum levels of testosterone, rLH, inhibin B, and epididymal levels of ABP. A Kruskal-Wallis ANOVA on ranks was performed for ventral prostate weights, spermatid head counts, TDI, number of normal conceptuses, and serum rFSH levels. SNK or Dunn method was used for multiple comparisons, as appropriate.
For experiment III, comparisons among the treatment groups were assessed based on a parametric ANOVA followed by SNK test for a significant F-value for the following parameters: paired testes, paired epididymal, ventral prostate, and seminal vesicle weights; ITT content; and serum levels of inhibin B and rFSH (log10 transformed). A nonparametric Kruskal-Wallis ANOVA on ranks was performed for spermatid head counts and serum levels of testosterone and rLH. A SNK multiple-range test was used to make comparisons across all groups for a significant F-value.
For experiment IV, body weights over time were compared using a two-way ANOVA for repeated measures. Comparisons among the three treatment groups were assessed based on a parametric ANOVA followed by SNK test for a significant F-value for the following parameters: paired testes, paired epididymal, ventral prostate, and seminal vesicle weights and spermatid head counts (log10 transformed). A Kruskal-Wallis ANOVA on ranks followed by Dunn method for multiple comparisons was used to compare TDI. Serum testosterone, rLH, rFSH, and inhibin B levels were compared over time using a one-way ANOVA for repeated measures followed by comparison to serum levels before initiation of any treatment (Week –4) using Bonferroni t-test for a significant F-value. For data that were not normally distributed, Friedman ANOVA on ranks for repeated measures was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum testosterone levels were significantly suppressed (P < 0.05) in acyline-treated rats as compared with vehicle-treated rats (3.47 ± 0.60 ng/ml, mean ± SEM), regardless of the dose or duration of treatment. Low, but detectable, serum testosterone levels were observed in rats treated with 55 µg rat–1 day–1 for 2 or 4 wk and 105 µg rat–1 day–1 for 4 wk (0.81 ± 0.59, 2.44 ± 1.47, and 0.14 ± 0.04 ng/ ml, respectively). However, serum testosterone levels were nondetectable in all rats treated with 210 µg rat–1 day–1 for 2 or 4 wk and in two of three rats receiving 105 µg rat–1 day–1 for 4 wk. One rat (#710) that was not suppressed at 105 µg rat–1 day–1 of acyline for 4 wk (serum testosterone levels of 6.71 ng/ml) was considered a statistical outlier and removed from all analyses. The weights of the androgen-dependent ventral prostate and seminal vesicles paralleled serum testosterone levels and were suppressed in acyline-treated rats in a dose- and time-dependent manner (data not shown), consistent with suppression of testosterone-mediated activity.
Testicular function, as assessed by testes weight, spermatid head counts, and the percentage of tubules containing mature spermatids, was suppressed by acyline treatment in a time- and dose-dependent fashion (Fig. 2). Significant suppression (P < 0.05) of all three endpoints was observed after 4 wk of acyline treatment at 105 and 210 µg rat–1 day–1. Although testosterone levels were suppressed within 2 wk of treatment at these doses, 4 wk of treatment was required to suppress testicular spermiogenesis. Because one rat (#710) was not suppressed at 105 µg rat–1 day–1 for 4 wk, the higher dose of 210 µg rat–1 day–1 for 4 wk was chosen to examine the potential of acyline treatment to prevent irreversible CDB-4022-induced testicular damage and infertility.
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Experiment II: Ability of Acyline to Prevent CDB-4022-Induced Testicular Damage and Irreversible Infertility
There were significant treatment- and time-related effects (P < 0.05) on the body weights of the male rats (data not shown). Suppression of body weight corresponded to the acyline treatment interval. For the duration of acyline-treatment (groups 2, 4, 5, 6, and 7), body weight gain was suppressed. However, this suppression was transient as male rats gained weight following cessation of acyline treatment. This transient decrease in body weight gain is consistent with suppression of testosterone production and, in turn, reduced anabolic activity.
The male rats treated with vehicle alone (group 1) maintained fertility throughout the study (up to Week 32; Table 1). Acyline-treatment during Weeks –4–0 resulted in infertility in all five male rats for study Weeks 1–6 (group 2; Table 1). Infertility was transitory, as all five male rats in this group recovered fertility within 16 wk after the removal of the acyline-containing osmotic pumps. In contrast, CDB-4022 alone rendered all four male rats infertile by Week 4, and none of these males regained fertility throughout the study (group 3; Table 1). Male rats treated with acyline before CDB-4022 administration (groups 4 and 7) were rendered infertile, but recovered fertility before study termination (Table 1). All five male rats treated with acyline on Weeks –4–0 followed by CDB-4022 treatment at Week 0 (group 4) recovered fertility by Week 10, whereas all five males treated with acyline on Weeks –4–9 followed by CDB-4022 treatment at Week 0 (group 7) recovered fertility by Week 20. When acyline treatment was initiated concurrently with CDB-4022 administration (Weeks 0–9, group 5) or after CDB-4022 treatment (Weeks 10–19, group 6), the male rats remained infertile for the duration of the study (up to Week 32; Table 1). Male rats that recovered fertility impregnated female rats yielding numbers of conceptuses that were not different from the vehicle-treated male rats (P = 0.49; Table 1).
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Male rats that recovered fertility (groups 2, 4, and 7) exhibited testes weights, spermatid head counts, and TDIs that were not different (P > 0.05) from those of vehicle-treated rats (group 1; Fig. 3) at necropsy on Week 34. In contrast, rats rendered infertile for the duration of the study (groups 3, 5, and 6) had suppressed testes weights as compared with those of vehicle-treated rats, no mature spermatids, and TDIs averaging less than 10% (Fig. 3). Likewise, epididymal weights were significantly suppressed (P < 0.05) in these rats (groups 3, 5, and 6) as compared with those in vehicle-treated rats, whereas rats in groups 2, 4, and 7 that had recovered fertility had epididymal weights that were not different (P > 0.05) from those of vehicle-treated rats (data not shown). Serum testosterone levels at Week 34 were significantly decreased (P < 0.05) in male rats treated with both acyline and CDB-4022 (groups 4, 5, 6, and 7), regardless of the interval of acyline treatment, as compared with those in vehicle-treated rats (Fig. 4A). However, serum testosterone levels were still sufficient to maintain fertility in treatment groups 4 and 7. Although serum testosterone levels appeared lower in rats treated with either acyline or CDB-4022 alone (groups 2 and 3, respectively), these were not significantly different (P > 0.05) from those in vehicle-treated animals. Serum testosterone levels were sufficient to sustain the weights of the sex accessory glands, as ventral prostate and seminal vesicles weights were not different (P = 0.17 and P = 0.13, respectively) across all treatment groups (data not shown). Despite the apparent decrease in serum testosterone levels, serum rLH levels were not different among the treatment groups, except for a significant increase (P < 0.05) in rLH levels in rats from groups 3 and 5 as compared with group 1 (Fig. 4A). Serum inhibin B levels were below the limit of detection in rats rendered irreversibly infertile (groups 3, 5, and 6), whereas rats that regained fertility had serum inhibin B levels that were similar to those of vehicle-treated rats (P > 0.05; Fig. 4B). Likewise, the levels of the Sertoli cell product ABP were nondetectable in the epididymides of rats in groups 3, 5, and 6, but ABP content in rats that recovered fertility (groups 2, 4, and 7; Fig. 5) was not different (P > 0.05) from that in vehicle-treated rats (group 1). Although inhibin B was suppressed in infertile rats, serum rFSH levels were significantly increased (P < 0.05) only in rats treated with CDB-4022 and acyline on Weeks 0 to 9 (group 5; Fig. 4B).
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Experiment III: Testosterone Replacement in Acyline-Treated Rats
Acyline treatment for 4 wk significantly suppressed (P < 0.05) testicular weight, spermatid head counts, and ITT content as compared with vehicle-treated rats (Fig. 6). ITT was significantly elevated (P < 0.05) in rats treated with both testosterone and acyline as compared with acyline alone, but ITT content was still lower (P < 0.05) than in vehicle-treated rats. However, testosterone replacement was sufficient to maintain spermatogenesis, as spermatid head counts in acyline plus testosterone treated rats were not different from those in vehicle-treated rats (P > 0.05). Spermatogenesis progressed only through the formation of round spermatids in acyline-treated rats, whereas mature spermatids (elongated and condensed) were present in the seminiferous tubules of acyline- and testosterone-treated rats (data not shown). Although testosterone replacement resulted in an increase in testes weight as compared with acyline treatment alone (P < 0.05), testes weights were still lower (P < 0.05) than in vehicle-treated rats (Fig. 6). The weights of the ventral prostate, seminal vesicles, and epididymides paralleled serum testosterone levels. Acyline treatment alone significantly suppressed (P < 0.05) serum testosterone levels to nondetectable and the weights of these androgen-dependent organs, whereas serum testosterone levels and the weights of these organs were not different (P > 0.05) from those in vehicle-treated rats when testosterone was coadministered with acyline (data not shown).
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Experiment IV: Testosterone Replacement Blocks Acyline's Protective Effect Against CDB-4022-Induced Testicular Damage
All CDB-4022-treated males in this experiment were rendered infertile (Table 2). As observed in experiment II, males pretreated with acyline recovered fertility by Week 10 following CDB-4022 treatment. However, the addition of testosterone to the acyline pretreatment completely reversed the protective effect(s) of the GnRH antagonist, as the male rats failed to recover fertility by Week 16. Testicular function, as assessed by testis weight, SHCs, and TDIs, in acyline plus CDB-4022-treated rats at necropsy (Fig. 7) was similar to those observed in vehicle-treated rats in the previous experiments (Figs. 3 and 6). In contrast, acyline plus testosterone treatment before administration of CDB-4022 resulted in suppression of testicular function that was not different from the suppression observed in the presence of CDB-4022 alone (P > 0.05). At study termination, epididymal weights were also significantly suppressed (P < 0.05) in males that were infertile, whereas the weights of the ventral prostate and seminal vesicles were not different (P > 0.21) among the treatment groups (data not shown).
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Serum hormone levels throughout the study are presented in Figures 8 and 9. Serum testosterone was suppressed by acyline treatment to nondetectable levels (ND, < 0.06 ng/ml), but returned to pretreatment levels after cessation of treatment (Week 3; Fig. 8A). The testosterone implant in combination with acyline resulted in elevated serum testosterone levels during treatment. After removal of the testosterone implant, serum testosterone decreased to ND at Week 1, and rats reestablished baseline testosterone levels by Week 3. In rats that received vehicle during the 4-wk pretreatment interval, serum testosterone levels before CDB-4022 treatment on Week 0 were significantly different from those at Week –4. This statistical difference may be due to the unusually high serum testosterone levels in three of the seven rats at Week –4. These high levels may be related to pulsatile release and/or the circadian rhythm of testosterone secretion. Subsequently, serum testosterone levels in this group of rats remained at a constant level from Week 0 through Week 20, and they were within the range observed in fertile males. Serum rLH levels were suppressed (P < 0.05) to ND (< 0.3 ng/ml) during the 4-wk acyline plus T treatment interval, and these returned to pretreatment levels by Week 3 (Fig. 8B). This trend was observed in rats pretreated with acyline alone, but the effect was not statistically different (P > 0.05). CDB-4022 treatment did not affect (P > 0.05) serum rLH levels. Serum inhibin B levels were suppressed to ND (<25 pg/ml) by Week 1 in rats treated with CDB-4022 alone or acyline + testosterone/CDB-4022 (Fig. 9A). These rats did not recover fertility (Table 2). In contrast, serum inhibin B levels were transiently elevated after CDB-4022 treatment in rats pretreated with acyline and returned to baseline by Week 6. These rats recovered fertility by Week 10 (Table 2). Serum rFSH levels were suppressed by acyline or acyline + testosterone pretreatment, but not to the same extent. Regardless, serum rFSH levels returned to pretreatment levels after cessation of treatment (Week 3, Fig. 9B). Serum rFSH levels were increased in rats treated with CDB-4022 alone. This increase corresponded to the decrease in serum inhibin B levels in these rats. Although suppression of serum inhibin B levels was also observed in acyline + testosterone/ CDB-4022-treated rats, serum rFSH levels were not increased above pretreatment levels in these rats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testosterone's permissive effects on testicular responses to drugs and radiation may be mediated through direct actions on the testis. The androgen receptor (AR) antagonist, flutamide, blocked testosterone-induced reversal of GnRH analogue-stimulated spermatogenic recovery in irradiated rats, implying a direct involvement of AR [6]. A subsequent study indicated that other potent androgens, the synthetic androgens R1881 and MENT and a 5-reduced androgen (5-DHT), had the same effect as testosterone, suggesting the effect was not specific for a particular androgen [15]. However, these actions were specific for androgens because 17ß-estradiol did not reverse the GnRH analogue-stimulated spermatogenic recovery [15]. These data further support an AR-mediated permissive effect on the testis because all of these androgens bind and activate AR ([16, 17], unpublished observations). The presence of AR in Sertoli cells ([18–20], our unpublished observations), the proposed primary target cell of CDB-4022 action [2], also fits with a direct androgen action.
However, possible paracrine factors produced by other testicular cells, especially differentiating germ cells and Leydig cells, may be involved in mediating testosterone's permissive effects on the testis. In particular, testosterone replacement in acyline-treated rats results in completion of spermiogenesis and the presence of mature spermatids in the seminiferous tubules. Hence, testosterone's block of acyline-stimulated spermatogenic recovery may be mediated, in part, by paracrine factor(s) produced by mature germ cells that render the Sertoli cell susceptible to the permanent adverse effects of CDB-4022. Although very limited correlative data exist, a role for germ cell-mediated regulation of inhibin B production and secretion from the Sertoli cell has been proposed [21–23]. Similar to azoospermic men, rats rendered irreversibly infertile by CDB-4022 have nondetectable serum inhibin B levels and tend to have seminiferous tubules lacking differentiating germ cells. In our previous studies, we showed that CDB-4022 affected inhibin B levels before an increase in germ cell apoptosis [2], suggesting that the inhibitory effects on inhibin B levels are due to direct actions of CDB-4022 on Sertoli cells rather than to the loss of germ cells. These direct CDB-4022 actions have also been shown to occur in cultured Sertoli cells [24]. Serum inhibin B levels remained nondetectable in CDB-4022-treated male rats rendered irreversibly infertile despite elevated serum rFSH levels. The elevation in rFSH from the pituitary is consistent with the release of negative feedback by inhibin B, and in this respect, the animal presented with a castrate phenotype [22]. Whether the lack of gonadotropin-stimulated inhibin B production and secretion in CDB-4022-treated infertile male rats is due to the absence of differentiating germ cells or irreversible damage to Sertoli cells has not yet been determined. CDB-4022-treated males that were pretreated with acyline and recovered fertility exhibited a transitory elevation of serum inhibin B levels followed by a reestablishment of both baseline serum inhibin B and rFSH levels. These data imply that this hormonal feedback axis is important in maintaining fertility in the rat. The possible role of mature germ cells in testosterone's block of acyline-stimulated spermatogenic recovery remains to be determined. GnRH analogs were also used to reverse the adverse testicular effects of 2,5-hexanedione (2,5-HD), a Sertoli cell toxicant, in adult male rats [10, 25]. In a subsequent study, administration of the Leydig cell toxicant, ethane dimethanesulphonate (EDS), resulted in suppression of ITT, but was not able to rescue the testes from 2,5-HD-induced damage [25]. In the same study, Lupron Depot treatment alone was able to prevent 2,5-HD-induced testicular damage, but the combination of Lupron Depot and EDS was not effective. The authors concluded that, in addition to the suppression of ITT, a paracrine factor(s) from the Leydig cell was required for rescue of testicular function. In a preliminary experiment in our laboratory, pretreatment with EDS alone prevented irreversible CDB-4022-induced testicular damage in all treated males, whereas only one of seven male rats recovered fertility when treated with vehicle before CDB-4022 (unpublished observations). The results suggest that, unlike 2,5-HD, the irreversible effects of CDB-4022 on the testes require testosterone but not the presence of Leydig cells. Collectively, these studies imply involvement of other testicular cell types and potential paracrine factors in the GnRH analogue-stimulated spermatogenic recovery, regardless of the agent used to induce infertility.
Although these studies indicate that testosterone most likely exerts its inhibitory effect directly on the testis, extratesticular sites of action may also contribute to testosterone's actions and cannot be conclusively ruled out. In particular, male rats treated with a combination of acyline + testosterone had elevated serum rFSH levels as compared with acyline-only-treated rats; however, serum rFSH levels were not so high as those observed in vehicle-treated males. In this experimental paradigm, testosterone reversed acyline-induced suppression of rFSH to some degree, and this was presumably through increased transcription of the FSH ß gene [26, 27]. To address the possible role of FSH in testosterone reversal of GnRH-analogue-stimulated spermatogenic recovery, Meistrich and Shetty [14, 28] treated irradiated adult male rats with exogenous FSH while suppressing endogenous hormones with a GnRH antagonist and flutamide. The addition of exogenous FSH inhibited the tubule differentiation observed in irradiated rats treated with GnRH antagonist and flutamide, but not to the same extent as that observed with androgens [6, 15]. These data support a role for the suppression of FSH as an important factor contributing to the protective effects of GnRH analogs on the testes.
The ability of GnRH-analogue treatment to reverse inhibition of spermatogenesis induced by radiation or multiple cytotoxic agents [5–10, 14, 28] implies that a common mechanism in spermatogonial differentiation is disrupted by these treatments and that suppression of testosterone and FSH either before or after the testicular injury is critical in stimulating spermatogonial differentiation. How these hormones, testosterone and FSH, are involved in the continued inhibition of spermatogonial differentiation in rats rendered infertile by radiation or cytotoxic treatment is not known. Of importance in elucidating the potential pathway(s) involved is determining the initial target cell of the toxic agent. Meistrich and Shetty [14] proposed four basic models for suppression of spermatogonial differentiation depending on the target cell, germ or somatic cell, and whether the pathological defect is a failure of spermatogonia to differentiate or is due to an increase in germ cell apoptosis. Because CDB-4022 appears to act on Sertoli cells, the proposed pathological defect would involve either the loss of a Sertoli cell-produced growth factor or stimulation of an apoptotic factor from the altered Sertoli cell. Thus, we are interested in determining effects of CDB-4022 on the expression of known Sertoli cell growth factors for germ cells (e.g., stem cell factor) and factors involved in the apoptotic pathway.
At present, there are few new promising candidates for male contraceptives. Steroid hormonal contraceptives are in the final stages of development but still have disadvantages, including the long delay between initiation of treatment and induction of infertility and the lack of a long-acting injectable or orally active androgen as part of the treatment regimen [29, 30]. Other proposed male contraceptives, including gossypol and lonidamine derivatives, have not moved forward due to undesirable side effects or toxicity issues [29, 30]. A recently discovered alkylated imino sugar, NB-DNJ, may be a promising candidate; however, further characterization of efficacy and safety are needed before development [30, 31]. Similar constraints also apply to CDB-4022. The lack of genetic or overt toxicity and other adverse side effects of the indenopyridines suggest that this family of compounds may have utility as male contraceptives [32–35]. Of particular concern, however, is CDB-4022's induction of irreversible infertility in adult male rats. In other species, including dogs and mice, reversible infertility was observed [33, 36]. The results of a preliminary study, performed in collaboration with the California Regional Primate Research Center (Davis, CA), indicated that the purified l-enantiomer of CDB-4022 induced spontaneously reversible suppression of sperm production as assessed in semen samples from adult cynomolgus monkeys following 1 wk of daily oral dosing (unpublished observations). Although these results in a nonhuman primate species are promising, this preliminary experiment needs to be confirmed and extended. Hence, the observation of irreversible infertility in CDB-4022-treated male rats may be specific to this species.
In conclusion, the GnRH antagonist, acyline, prevented CDB-4022-induced irreversible infertility in male rats when administered continuously over a 4-wk period before CDB-4022 treatment. Unlike results with radiation or other cytotoxic agents, concurrent or posttreatment with a GnRH analogue did not prevent irreversible infertility or restimulate spermatogenesis in CDB-4022-treated male rats. The protective effects of acyline appeared to be mediated by suppression of ITT, as they were reversed by exogenous testosterone. However, suppression of pituitary FSH may also be a contributing factor to acyline's protective action on the testes. Testosterone either renders the Sertoli cell responsive to CDB-4022's deleterious effects via a direct AR-mediated pathway or indirectly via suppression of the formation of mature spermatids and potential paracrine factors. Further research is required to elucidate the possible contributing factor(s) involved in GnRH analogue-stimulated spermatogenic recovery from testicular injury and the permissive role of androgens. Because this appears to be common to multiple modes of testicular injury, understanding the mechanism involved in the disruption of spermatogonial differentiation may have applications for men rendered infertile by reproductive toxicants. Continued development of CDB-4022 as a potential nonhormonal male contraceptive will depend on its safety profile in long-term studies and whether its antispermatogenic effect in primate species is reversible, either spontaneously or by additional treatments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Sheri Hild, BIOQUAL, Inc., 9600 Medical Center Dr., Rockville, MD 20850. FAX: 301/251-1260; shild{at}bioqual.com
Received: 23 December 2003.
First decision: 16 January 2004.
Accepted: 16 March 2004.
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-subunit messenger RNAs. Endocrinology 1992 130:726-734[Abstract]This article has been cited by other articles:
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, acyline/CDB-4022; 
, acyline + T/ CDB-4022