BIOLOGY OF REPRODUCTION 71, 348±358 (2004) Published online before print 24 March 2004. DOI 10.1095/biolreprod.103.026989

The Ability of a -Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone1

Sheri Ann Hild,2 Barbara J. Attardi, and Jerry R. Reel BIOQUAL, Inc., Rockville, Maryland 20850

ABSTRACT returned to baseline in rats protected by acyline pretreatment. Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 These data indicate that pretreatment with acyline was able to Intratesticular (ITT) is known to play a critical prevent irreversible infertility in CDB-4022-treated rats, where- role in the maintenance of spermatogenesis. We have used acy- as posttreatment with acyline did not promote spermatogonial line, a GnRH antagonist, to suppress testosterone (T) produc- differentiation, as has been observed by others in rats that re- tion, and acyline and T implants to study the prevention of ir- ceived GnRH analogs and various other testicular toxicants. reversible infertility induced by CDB-4022. Vehicle or acyline Suppression of ITT and possibly rFSH by acyline appeared to be -group) at a crucial in preventing irreversible infertility induced by CDB/5 ؍ was administered to proven fertile male rats (n dose (210 ␮g/day) that completely suppressed (P Ͻ 0.05) T pro- 4022. In this regard, our results are similar to those of investi- duction, as measured by serum T, and testicular function, either gators who have studied other testicular toxicants. Continued before, concurrent with, or after vehicle or a single oral dose of development of CDB-4022 as a potential male contraceptive 2.5 mg CDB-4022/kg (Week 0). Vehicle-treated males remained will depend largely on its safety profile and whether its antisper- fertile, whereas acyline-treated males exhibited transitory infer- matogenic activity is reversible in primates. tility. CDB-4022 alone caused irreversible infertility in all males. Importantly, CDB-4022-treated males recovered fertility when gonadotropin-releasing hormone, inhibin, male reproductive acyline was started before CDB-4022 (Weeks Ϫ4 to 0; Weeks tract, spermatogenesis, testosterone Ϫ4–9), but not when acyline was administered concurrently with or after CDB-4022 (Weeks 0–9; Weeks 10–19). At the end INTRODUCTION of this study (Week 34), testes weights, spermatid head counts (SHC), and tubule differentiation indices (TDI) were suppressed CDB-4022, [4aRS,5SR,9bRS]-2-ethyl-2,3,4,4a,5,9b- (P Ͻ 0.05) in infertile CDB-4022-treated males, but in rats that hexahydro-8-iodo-7-methyl-5-[4-carbomethoxyphenyl]- recovered fertility, these parameters were similar (P Ͼ 0.05) to 1H-indeno[1, 2-c]pyridine-hydrochloride, was shown pre- those in vehicle-treated males. In addition, serum inhibin B and viously to induce infertility in the majority of adult male epididymal -binding protein levels were nondetectable rats treated with this compound [1, 2]. This antispermato- in infertile CDB-4022-treated rats. To test whether suppression genic effect of CDB-4022 appears to be mediated by dis- of ITT was critical for prevention of CDB-4022-induced infertil- ruption of Sertoli cell structure and function, resulting in group) received vehicle, acyline increased apoptosis of differentiating spermatogonia and/8–7 ؍ ity, proven fertile rats (n alone, or acyline and a T implant for 4 wk before CDB-4022 spermatocytes [2]. In contrast, CDB-4022 does not appear (Week 0). The T implant increased ITT in acyline-treated rats. Although ITT was lower (P Ͻ 0.05) in the T-implanted males to affect Leydig cells, as Leydig cell morphology, circulat- than in untreated rats, it was sufficient to sustain spermiogen- ing levels of testosterone, and libido were not affected [1± esis. Serum rFSH levels were also elevated in rats treated with 3]. However, the germ cell is still a possible site of direct .acyline ؉ T as compared with acyline alone during the treatment action for CDB-4022 induction of infertility interval, but rFSH was still lower than in vehicle-treated rats. In these previous studies, CDB-4022-induced infertility Rats in all treatment groups were rendered infertile initially, but was not spontaneously reversible in male rats, which is con- the acyline ؉ CDB-4022-treated rats recovered fertility by Week sistent with the action of compounds that disrupt Sertoli In contrast, rats treated with CDB-4022 alone or acyline ؉ cell structure and function [4]. However, reversibility of .10 -T ؉ CDB-4022 remained infertile until at least Week 16. Testes infertility is an important consideration for a male contra -weights, SHC, and TDI were within normal ranges for acyline ؉ ceptive. We showed previously that a series of four injec CDB-4022-treated rats, but were decreased (P Ͻ 0.05) in CDB- tions of the GnRH agonist, Lupron Depot, administered 3 or acyline ؉ T ؉ CDB-4022-treated rats. Serum inhibin wk apart beginning 1 wk before administration of a single 4022- B levels were nondetectable by Week 1 in males rendered irre- oral dose of CDB-4022 (2.5 mg/kg) resulted in the return versibly infertile by CDB-4022; levels increased transiently and of spermatogenesis and fertility in the majority of treated rats [1]. Other investigators have shown that treatment with 1Supported by National Institute of Child Health and Human Develop- GnRH analogues, agonists or antagonists, prevents or re- ment (NICHD) contract N01-HD-6-3259 awarded to BIOQUAL, Inc. A verses testicular damage induced by radiation [5, 6], che- portion of this work was presented at the 35th annual meeting of the motherapeutic agents [7, 8], an environmental reproductive Society for the Study of Reproduction (2002), Abstract 105. toxicant (dibromochloropropane; [9]), or a Sertoli cell tox- 2Correspondence: Sheri Hild, BIOQUAL, Inc., 9600 Medical Center Dr., Rockville, MD 20850. FAX: 301/251-1260; e-mail: [email protected] icant (2,5-hexanedione; [10]). Hence, one of the goals of the present study was to determine whether the GnRH an- tagonist, acyline, would provide similar protection or re- Received: 23 December 2003. stimulation of spermatogenesis in CDB-4022-treated rats. First decision: 16 January 2004. Accepted: 16 March 2004. The mechanism by which GnRH antagonists restore sper- ᮊ 2004 by the Society for the Study of Reproduction, Inc. matogenesis following treatment with radiation or testicular ISSN: 0006-3363. http://www.biolreprod.org toxicants may be through suppression of , 348 T REVERSES ACYLINE PROTECTION AGAINST CDB-4022 349 testosterone, and/or spermatogenesis. To determine whether analyzed using a four-parameter sigmoidal curve ®t as above. The limits acyline-induced suppression of intratesticular testosterone of detection for these RIAs were 2.6 ng NIDDK-rFSH-RP-2/ml and 0.31 (ITT) levels was critical for prevention of permanent CDB- ng NIDDK-rLH-RP-3/ml. The intraassay variation was 5% (n ϭ 10) and 9% (n ϭ 4), and the interassay variation was 13% (n ϭ 26) and 24% (n 4022-induced testicular damage, the ability of testosterone ϭ 16) for the rLH and rFSH assays, respectively. to reverse acyline's protective effect against CDB-4022-in- duced irreversible infertility and testicular damage in the rat was also investigated. Androgen-Binding Protein Assay Androgen-binding protein (ABP) content in the epididymal cytosol MATERIALS AND METHODS was determined by speci®c binding of [3H]-5␣DHT and was expressed as femtomol bound per milligram cytosolic protein [2, 12]. Brie¯y, each fro- Animals zen 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 Sprague-Dawley CD rats (Crl:CD(SD)IGS BR Stock) were purchased temperature. The homogenate was centrifuged at 110 000 ϫ g for 30 min from Charles River Laboratories (Kingston, NY). All rats were housed in at 40ЊC. Endogenous steroids were removed from the resultant supernatant polycarbonate solid-¯oor cages with Bed-o-Cob or Beta-Chip bedding by dextran-coated charcoal precipitation (DCC: 0.05% dextran, 0.5% char- Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 (Andersons Industrial Products Group, Maumee, OH) and received Purina coal in TE buffer). Total protein was measured in an aliquot of charcoal- laboratory rodent diet (#5001; Purina Mills, St. Louis, MO) and tap water stripped cytosol using Pierce BCA protein assay (Rockford, IL). Charcoal- ad libitum. The photoperiod was 14L:10D. All female rats were group- stripped epididymal cytosol (40±50 ␮l) was incubated with 1 nM [3H]- housed, whereas adult males were individually housed except during mat- 5␣DHT in the presence or absence of 500 nM unlabeled 5␣DHT for 2 h ing trials. Proven fertile male rats were used for experiments II and IV; at 4ЊC. DCC was added to separate bound from free radioligand. Opti- male fertility was assessed by in-house mating trials before study assign- Fluor (Perkin Elmer, Wellesley, MA) was added to the resultant superna- ment [1]. The environmental conditions of the animal rooms were main- tant, and the samples were counted in a Beckman LS 1800 liquid scintil- tained as recommended by the National Research Council in Guide for lation counter. The amount of rat ABP per mg of cytosolic protein was the Care and Use of Laboratory Animals [11] to the maximum extent calculated using Excel (Excel version 5.0 spreadsheet; Microsoft Corp., possible. All study protocols were approved by BIOQUAL's institutional Seattle, WA). This assay had a limit of detection of 10 fmol ABP/mg animal care and use committee. protein and intra- and interassay variations of 31% (n ϭ 3) and 26% (n ϭ 6), respectively. Materials The GnRH antagonist acyline (Ac-D-Nal-D-4-Cl-Phe-D-Pal-Ser- Experiment I: Suppression of Testicular Function Aph(Ac)-D-Aph(Ac)-Leu-Lys(Ipr)-Pro-D-Ala-NH2) was synthesized by by Acyline Multiple Systems (San Diego, CA) and was 98.4% pure based on HPLC analysis. CDB-4022 was synthesized by Research Triangle Institute This preliminary study was undertaken to determine the daily dose of (Research Triangle Park, NC) and used in experiment II. A second batch acyline and the treatment interval (2 or 4 wk) necessary for suppression of CDB-4022, synthesized by Dr. P.N. Rao (Southwest Foundation for of circulating testosterone and testicular spermatogenesis in adult male Biomedical Research, San Antonio, TX), was used in experiment IV. Both rats. The data from this study were used to design the subsequent exper- batches of CDB-4022 were considered Ͼ99% pure based on HPLC anal- iment to assess the ability of this GnRH antagonist to prevent and/or re- ysis and, as in previous reports [1, 2], were a racemic mixture of l- and verse the effect(s) of CDB-4022 on the rat testis. Adult male rats (380± Ϫ1 Ϫ1 d-enantiomers. [1, 2, 4, 5, 6, 7-3H]-5␣-Dihydrotestosterone (5␣-DHT; 123 450 g) were treated with acyline at 55, 105, or 210 ␮g rat day (3 rats/ Ci/mmole) was purchased from Perkin Elmer Life Sciences (Boston, MA) group) or vehicle (2% Tween 80 in bacteriostatic water) for 2 (model and was used as the radioligand in the androgen-binding protein assays. 2002) or 4 (model 2004) wk via an Alzet osmotic minipump implanted Sterile Alzet osmotic minipumps (model 2002 or 2004) were purchased into the subcutaneous space between the scapulae. from Alza Corporation (Palo Alto, CA). Needles, syringes, anesthetics, At the end of the treatment interval, the rats were anesthetized and and surgical supplies were purchased from NLS, Inc. (Baltimore, MD). exsanguinated. Serum was harvested by centrifugation and the levels of Anesthetics included iso¯urane, ketamine, and xylazine. Reagent-grade or testosterone were determined by RIA. The ventral prostate, seminal vesi- molecular biology-grade chemicals and enzymes were purchased from Sig- cles, and testes were excised and weighed. Following removal of the tunica ma (St. Louis, MO), Boehringer Mannheim (Indianapolis, IN), or Quan- albuginea, the left testis was homogenized, spermatid head counts deter- tum Chemical Co. (Tuscola, IL). Food-grade sesame oil (Hain) was pur- mined, and the data expressed as the number of spermatid heads per testis chased from a local grocery store. [13]. The right testis was preserved in Bouin ®xative, washed, dehydrated, and embedded in glycol methacrylate medium (GMA). Hematoxylin- stained cross-sections (2 ␮m) of the testis were used to determine the Immunoassays percentage of seminiferous tubules containing later stage spermatids (elon- gated to condensed spermatids) [13]. A total of 200 tubules/testis were Unextracted serum samples were assayed for testosterone using a com- examined for each treatment group and 100 tubules/testis for the vehicle mercially available coated-tube testosterone radioimmunoassay (RIA, control group. 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±6ЊC. This Experiment II: Ability of Acyline to Prevent CDB-4022- modi®cation was made because it allowed more accurate measurement of Induced Testicular Damage and Irreversible Infertility 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 The experimental design is depicted in Figure 1. Proven fertile male a four-parameter sigmoidal curve ®t (RiaSmart Data Reduction Program; rats (400±470 g) were assigned to one of seven treatment groups (5/ Perkin Elmer Life Sciences, Meriden, CT). The limit of detection in this group): 1) vehicle for Weeks Ϫ4±0 and vehicle orally on Week 0; 2) assay was 0.04 ng testosterone/ml, and the intra- and interassay variations acyline for Weeks Ϫ4±0 and vehicle orally on Week 0; 3) vehicle for were 7% (n ϭ 46) and 15% (n ϭ 46), respectively. Weeks Ϫ4±0 and CDB-4022 orally on Week 0; 4) acyline for Weeks Ϫ4± Inhibin B was determined in serum samples using an ultrasensitive 0 and CDB-4022 orally on Week 0; 5) acyline for Weeks 0±10 and CDB- Inhibin B ELISA assay kit (Serotec, Oxford, UK). Lyophilized human 4022 orally on Week 0; 6) CDB-4022 orally on Week 0 and acyline for inhibin B extracted from follicular ¯uid (supplied with the kit) was recon- Weeks 10±20; and 7) acyline for Weeks Ϫ4±10 and CDB-4022 orally on stituted in castrate-adult-male-rat serum and a standard curve generated by Week 0. CDB-4022 or 10% ethanol/sesame oil was administered as a serial dilution of the human inhibin B in the same serum [2]. The standard single oral dose of 2.5 mg/kg or 5 ml/kg, respectively, on Week 0 (Week curve was calculated using a linear ®t program (EIACalc Dynatech MR- 0 ϭ Day 0, the day of vehicle or CDB-4022 dosing). This dose of CDB- 500; Chantilly, VA). The limit of detection for the assay was 25 pg human 4022 had been determined previously to be the minimally effective or inhibin B/ml and the intra- and interassay variations were 2% (n ϭ 3) and threshold dose for inducing infertility in 100% of adult male rats [1]. 19% (n ϭ 17), respectively. Infertility was not reversible in the majority of adult male rats at this dose. Levels of the rat gonadotropins, rLH and rFSH, were determined in Two osmotic minipumps (Alzet model 2004) implanted subcutaneously serum samples by RIA using reagents supplied by the National Institute between the scapulae were used to deliver 210 ␮g acyline ratϪ1 dayϪ1 or of Diabetes and Digestive and Kidney Diseases (NIDDK). RIA data were 2% Tween 80/bacteriostatic water (12 ␮l ratϪ1 dayϪ1) for 4 wk. These 350 HILD ET AL.

FIG. 1. Design for experiments II and IV. Experiment II: Male rats were assigned to groups (5/group) and treated as described in Materials and Methods. Two osmotic minipumps (Alzet model 2004) implanted subcutaneously (sc) were used to deliver 210 ␮g acyline ratϪ1 dayϪ1 or vehicle, 2% Tween 80/bacteriostatic water (12 ␮lratϪ1 dayϪ1), for 4 wk. These minipumps were surgically removed and replaced as re- quired. CDB-4022 or vehicle (10% etha- nol/sesame oil) was given as a single oral dose of 2.5 mg/kg or 5 ml/kg, respectively, on Day 0 (Day 0 ϭ Week 0). Experiment IV: Male rats were assigned to groups (7–

8/group) and treated as indicated above. Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 Acyline was administered s.c. at 210 ␮g ratϪ1 dayϪ1; a 6-cm testosterone-filled Si- lastic implant was placed s.c.; CDB-4022 was administered orally at 2.5 mg/kg on Day 0.

minipumps were surgically removed and replaced as required by the ex- and acyline were administered using osmotic minipumps as described perimental design (see Fig. 1). above. In this manner, all rats underwent a surgical procedure at the start Individual male rats were weighed at Weeks Ϫ4 and 0, every 3 wk of the study (Week 0). Rats were subjected to necropsy at Week 4. The after oral dosing, and at necropsy (Week 34). Mating trials were performed ventral prostate, seminal vesicles, epididymides, and testes were excised weekly during Weeks 1±8 and every 2 wk from Week 10 through Week and weighed. Spermatid head counts were determined from a portion of 32 and the females examined for pregnancy status. At Week 34 of the the homogenate prepared from the left testis. The remainder of the ho- study, the anesthetized male rats were exsanguinated and sera were har- mogenate, prepared in TEGMD buffer (10 mM Tris [pH 7.2], 1.5 mM vested. Serum testosterone, rLH, rFSH, and inhibin B levels were deter- EDTA, 20 mM sodium molybdate dihydrate, 10% glycerol, 1 mM dithio- mined. The ventral prostate, seminal vesicles, epididymides, and testes threitol), was centrifuged at ϳ109 000 ϫ g for 1 h to obtain cytosol, which were excised and weighed. Spermatid head counts were determined for was assayed for ITT content. The right testis was preserved for morpho- the left testis. The right testis was preserved for morphological assessment. logical assessment. Sera harvested from collected blood were assayed for The tubule differentiation index (TDI), de®ned as the percentage of sem- testosterone, inhibin B, rLH, and rFSH. Treatment-induced changes in se- iniferous tubules undergoing spermatogenesis beyond formation of B sper- rum hormone levels were reproduced in the subsequent experiment (ex- matogonia, was determined in these sections [13]. The ABP content in the periment IV); therefore, the data from this speci®c experiment are not epididymal cytosol was measured. During this extended study, one male shown. rat in group 1 and one male rat in group 3 died at Weeks 14 and 16 of the study, respectively, resulting in n ϭ 4 for these groups. Experiment IV: Testosterone Replacement Blocks Acyline’s Protective Effect Against CDB-4022-Induced Testicular Experiment III: Testosterone Replacement in Acyline- Damage Treated Rats The experimental design is depicted in Figure 1. Proven fertile male To test the hypothesis that acyline protects the testes from irreversible rats (400±470 g) were assigned to one of three treatment groups (7±8/ damage via suppression of testosterone production, we wanted to verify group): 1) vehicle (2% Tween 80/bacteriostatic water, 12 ␮l ratϪ1 dayϪ1) that testosterone replacement via subcutaneous placement of a 6-cm Si- for Weeks Ϫ4±0 and CDB-4022 at 2.5 mg/kg orally on Week 0; 2) acyline lastic implant would be suf®cient to maintain spermatogenesis. Therefore, at 210 ␮g ratϪ1 dayϪ1 for Weeks Ϫ4±0 and CDB-4022 at 2.5 mg/kg orally a study was undertaken to de®ne testicular function in intact adult male on Week 0; 3) acyline at 210 ␮g ratϪ1 dayϪ1 and a subcutaneous 6-cm rats (n ϭ 5/group, 300±340 g) treated for 4 wk with vehicle (2% Tween testosterone-®lled Silastic implant for Weeks Ϫ4±0 and CDB-4022 orally 80/bacteriostatic water, 12 ␮l ratϪ1 dayϪ1), acyline (210 ␮g acyline ratϪ1 at 2.5 mg/kg on Week 0. dayϪ1), or acyline and a subcutaneous 6-cm testosterone-®lled Silastic im- Individual male rats were weighed at Weeks Ϫ4, Ϫ2, and 0, and every plant (Silastic tubing of 0.078Љϫ0.125; Speciality Manufacturing, Inc., 3 wk after oral dosing and at necropsy (Week 20). Mating trials were Saginaw, MI). Testosterone-®lled implants of this size were previously performed weekly during Weeks 1 and 2 and then every 2 wk from Week shown to raise intratesticular testosterone (ITT) and inhibit GnRH antag- 2 through Week 16 and the females examined for pregnancy status. Male onist-induced recovery of spermatogenesis in irradiated rats [6]. Vehicle rats were bled from the tail vein, before any treatment, at Weeks Ϫ4, Ϫ2, T REVERSES ACYLINE PROTECTION AGAINST CDB-4022 351

FIG. 2. Effects of continuous administra- tion of acyline on testicular function in ex- periment I. Asterisks (*) indicate significant differences (P Ͻ 0.05) from vehicle-treated rats. Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021

0, 1, 3, 6, 9, and 15. At Week 20 of the study, the anesthetized male rats levels of inhibin B and rFSH (log10 transformed). A nonparametric Krus- were exsanguinated and sera were harvested. Serum testosterone, rLH, kal-Wallis ANOVA on ranks was performed for spermatid head counts rFSH, and inhibin B levels were determined from the samples collected and serum levels of testosterone and rLH. A SNK multiple-range test was throughout the study. The ventral prostate, seminal vesicles, epididymides, used to make comparisons across all groups for a signi®cant F-value. and testes were excised and weighed. Spermatid head counts were deter- For experiment IV, body weights over time were compared using a mined for the left testis. The right testis was preserved for determination two-way ANOVA for repeated measures. Comparisons among the three of the TDI. treatment groups were assessed based on a parametric ANOVA followed by SNK test for a signi®cant F-value for the following parameters: paired Statistical Analysis testes, paired epididymal, ventral prostate, and seminal vesicle weights and spermatid head counts (log10 transformed). A Kruskal-Wallis ANOVA on Statistical analyses were performed using SigmaStat version 2.03 ranks followed by Dunn method for multiple comparisons was used to (SPSS Inc., Chicago, IL). All tests were two-tailed with signi®cance set compare TDI. Serum testosterone, rLH, rFSH, and inhibin B levels were at ␣ϭ0.05. Graphs were prepared using SigmaPlot 2001 (SPSS Inc.). compared over time using a one-way ANOVA for repeated measures fol- The ratio of males rendered infertile and the ratio of males that recovered lowed by comparison to serum levels before initiation of any treatment fertility were compared with a control using a z-test of proportions for (Week Ϫ4) using Bonferroni t-test for a signi®cant F-value. For data that each relevant experiment. Analysis of variance (ANOVA) was used to were not normally distributed, Friedman ANOVA on ranks for repeated determine signi®cant differences among treatment groups within an ex- measures was used. periment. Whenever possible, a parametric ANOVA was used if the data met the test criteria of normality and homogeneity of variances. Data were RESULTS transformed, as needed, to meet these assumptions. For data that did not met these criteria, a nonparametric Kruskal-Wallis ANOVA on ranks was Experiment I: Suppression of Testicular Function performed. For measurements obtained from the same animal over time, an ANOVA for repeated measures was used. For a signi®cant F-value, by Acyline comparisons among treatment groups were determined by Bonferroni t- Serum testosterone levels were signi®cantly suppressed test for comparisons to control or Student-Newman-Keuls (SNK) multiple range test for all pairwise comparisons. Dunn method was used for mul- (P Ͻ 0.05) in acyline-treated rats as compared with vehicle- tiple comparisons following the nonparametric Kruskal-Wallis ANOVA on treated rats (3.47 Ϯ 0.60 ng/ml, mean Ϯ SEM), regardless ranks for experiments with an unequal number per group. Speci®c statis- of the dose or duration of treatment. Low, but detectable, tical tests are included below. serum testosterone levels were observed in rats treated with For experiment I, signi®cant differences among the treatment groups 55 ␮g ratϪ1 dayϪ1 for 2 or 4 wk and 105 ␮g ratϪ1 dayϪ1 in serum testosterone levels, the weights of paired testes and ventral pros- for 4 wk (0.81 Ϯ 0.59, 2.44 Ϯ 1.47, and 0.14 Ϯ 0.04 ng/ tate (log10 transformed), percentage of tubules containing elongated to con- ml, respectively). However, serum testosterone levels were densed spermatids, and spermatid head counts were determined by Ϫ1 Ϫ1 ANOVA followed by Bonferroni t-test for multiple comparisons to the nondetectable in all rats treated with 210 ␮g rat day for Ϫ1 vehicle control group. The seminal vesicle weights were compared using 2 or 4 wk and in two of three rats receiving 105 ␮g rat a Kruskal-Wallis ANOVA on ranks followed by Dunn method for multiple dayϪ1 for 4 wk. One rat (#710) that was not suppressed at comparisons to the control group. 105 ␮g ratϪ1 dayϪ1 of acyline for 4 wk (serum testosterone For experiment II, body weights over time were compared using a two- levels of 6.71 ng/ml) was considered a statistical outlier and way ANOVA for repeated measures. Comparisons among the treatment groups were assessed based on a parametric ANOVA followed by SNK removed from all analyses. The weights of the androgen- test for a signi®cant F-value for the following endpoints: paired testes, dependent ventral prostate and seminal vesicles paralleled paired epididymal, and seminal vesicle weights; serum levels of testoster- serum testosterone levels and were suppressed in acyline- one, rLH, inhibin B, and epididymal levels of ABP. A Kruskal-Wallis treated rats in a dose- and time-dependent manner (data not ANOVA on ranks was performed for ventral prostate weights, spermatid shown), consistent with suppression of testosterone-medi- head counts, TDI, number of normal conceptuses, and serum rFSH levels. ated activity. SNK or Dunn method was used for multiple comparisons, as appropriate. For experiment III, comparisons among the treatment groups were as- Testicular function, as assessed by testes weight, sper- sessed based on a parametric ANOVA followed by SNK test for a signif- matid head counts, and the percentage of tubules containing icant F-value for the following parameters: paired testes, paired epididy- mature spermatids, was suppressed by acyline treatment in mal, ventral prostate, and seminal vesicle weights; ITT content; and serum a time- and dose-dependent fashion (Fig. 2). Signi®cant 352 HILD ET AL.

TABLE 1. Treatment-induced infertility and recovery of fertility in adult mination (Table 1). All ®ve male rats treated with acyline male rats (experiment II). on Weeks Ϫ4±0 followed by CDB-4022 treatment at Week No. male rats 0 (group 4) recovered fertility by Week 10, whereas all ®ve No. males recovering males treated with acyline on Weeks Ϫ4±9 followed by rendered fertility/no. male No. normal CDB-4022 treatment at Week 0 (group 7) recovered fertility infertile/no. rats rendered conceptuses/ by Week 20. When acyline treatment was initiated concur- Treatment group males treated infertile pregnant rata rently with CDB-4022 administration (Weeks 0±9, group Group 1: 5) or after CDB-4022 treatment (Weeks 10±19, group 6), Vehicle/vehicle 0/4 — 10 Ϯ 2 the male rats remained infertile for the duration of the study Group 2: (up to Week 32; Table 1). Male rats that recovered fertility vehicle/acyline impregnated female rats yielding numbers of conceptuses (weeks Ϫ4–0) 5/5b 5/5c 12 Ϯ 2 Group 3: that were not different from the vehicle-treated male rats 4022/vehicle 4/4b 0/4 — (P ϭ 0.49; Table 1).

Group 4: Male rats that recovered fertility (groups 2, 4, and 7) Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 4022/acyline exhibited testes weights, spermatid head counts, and TDIs (weeks Ϫ4–0) 5/5b 5/5c 13 Ϯ 1 that were not different (P Ͼ 0.05) from those of vehicle- Group 5: treated rats (group 1; Fig. 3) at necropsy on Week 34. In 4022/acyline (weeks 0–9) 5/5b 0/5 — contrast, rats rendered infertile for the duration of the study Group 6: (groups 3, 5, and 6) had suppressed testes weights as com- 4022/acyline pared with those of vehicle-treated rats, no mature sper- (weeks 10–19) 5/5b 0/5 — matids, and TDIs averaging less than 10% (Fig. 3). Like- Group 7: wise, epididymal weights were signi®cantly suppressed (P 4022/acyline Ͻ 0.05) in these rats (groups 3, 5, and 6) as compared with Ϫ b c Ϯ (weeks 4–9) 5/5 5/5 13 1 those in vehicle-treated rats, whereas rats in groups 2, 4, a Mean Ϯ SEM for Week 32 of the study. There were no significant dif- and 7 that had recovered fertility had epididymal weights ferences (P ϭ 0.49) in the number of normal conceptuses per pregnant that were not different (P Ͼ 0.05) from those of vehicle- female among the treatment groups. b Significantly different (P Ͻ 0.05) from vehicle control (group 1). treated rats (data not shown). Serum testosterone levels at c Significantly different (P Ͻ 0.05) from CDB 4022 treatment alone (group Week 34 were signi®cantly decreased (P Ͻ 0.05) in male 3). 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). How- suppression (P Ͻ 0.05) of all three endpoints was observed ever, serum testosterone levels were still suf®cient to main- Ϫ1 after 4 wk of acyline treatment at 105 and 210 ␮g rat tain fertility in treatment groups 4 and 7. Although serum Ϫ1 day . Although testosterone levels were suppressed within testosterone levels appeared lower in rats treated with either 2 wk of treatment at these doses, 4 wk of treatment was acyline or CDB-4022 alone (groups 2 and 3, respectively), required to suppress testicular spermiogenesis. Because one these were not signi®cantly different (P Ͼ 0.05) from those Ϫ1 Ϫ1 rat (#710) was not suppressed at 105 ␮g rat day for 4 in vehicle-treated animals. Serum testosterone levels were Ϫ1 Ϫ1 wk, the higher dose of 210 ␮g rat day for 4 wk was suf®cient to sustain the weights of the sex accessory glands, chosen to examine the potential of acyline treatment to pre- as ventral prostate and seminal vesicles weights were not vent irreversible CDB-4022-induced testicular damage and different (P ϭ 0.17 and P ϭ 0.13, respectively) across all infertility. treatment groups (data not shown). Despite the apparent decrease in serum testosterone levels, serum rLH levels Experiment II: Ability of Acyline to Prevent CDB-4022- were not different among the treatment groups, except for Induced Testicular Damage and Irreversible Infertility a signi®cant increase (P Ͻ 0.05) in rLH levels in rats from There were signi®cant treatment- and time-related ef- groups 3 and 5 as compared with group 1 (Fig. 4A). Serum fects (P Ͻ 0.05) on the body weights of the male rats (data inhibin B levels were below the limit of detection in rats not shown). Suppression of body weight corresponded to rendered irreversibly infertile (groups 3, 5, and 6), whereas the acyline treatment interval. For the duration of acyline- rats that regained fertility had serum inhibin B levels that treatment (groups 2, 4, 5, 6, and 7), body weight gain was were similar to those of vehicle-treated rats (P Ͼ 0.05; Fig. suppressed. However, this suppression was transient as 4B). Likewise, the levels of the Sertoli cell product ABP male rats gained weight following cessation of acyline were nondetectable in the epididymides of rats in groups 3, treatment. This transient decrease in body weight gain is 5, and 6, but ABP content in rats that recovered fertility consistent with suppression of testosterone production and, (groups 2, 4, and 7; Fig. 5) was not different (P Ͼ 0.05) in turn, reduced anabolic activity. from that in vehicle-treated rats (group 1). Although inhibin The male rats treated with vehicle alone (group 1) main- B was suppressed in infertile rats, serum rFSH levels were tained fertility throughout the study (up to Week 32; Table signi®cantly increased (P Ͻ 0.05) only in rats treated with 1). Acyline-treatment during Weeks Ϫ4±0 resulted in in- CDB-4022 and acyline on Weeks 0 to 9 (group 5; Fig. 4B). fertility in all ®ve male rats for study Weeks 1±6 (group 2; Table 1). Infertility was transitory, as all ®ve male rats in Experiment III: Testosterone Replacement in Acyline- this group recovered fertility within 16 wk after the removal Treated Rats of the acyline-containing osmotic pumps. In contrast, CDB- 4022 alone rendered all four male rats infertile by Week 4, Acyline treatment for 4 wk signi®cantly suppressed (P and none of these males regained fertility throughout the Ͻ 0.05) testicular weight, spermatid head counts, and ITT study (group 3; Table 1). Male rats treated with acyline content as compared with vehicle-treated rats (Fig. 6). ITT before CDB-4022 administration (groups 4 and 7) were was signi®cantly elevated (P Ͻ 0.05) in rats treated with rendered infertile, but recovered fertility before study ter- both testosterone and acyline as compared with acyline T REVERSES ACYLINE PROTECTION AGAINST CDB-4022 353

FIG. 3. Testicular function in rats treated with CDB-4022 alone or in combination with acyline in experiment II. Testis weights, SHCs, and TDIs were suppressed in rats that did not recover fertility (Table 1). Asterisks indicate significant differences (P Ͻ 0.05) from vehicle-treated rats (group 1). Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021

alone, but ITT content was still lower (P Ͻ 0.05) than in vehicle-treated rats. However, testosterone replacement was suf®cient to maintain spermatogenesis, as spermatid head counts in acyline plus testosterone treated rats were not different from those in vehicle-treated rats (P Ͼ 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 Ͻ 0.05), testes weights were still lower (P Ͻ 0.05) than in vehicle-treated rats (Fig. 6). The weights of the ventral prostate, seminal vesicles, and epi- didymides paralleled serum testosterone levels. Acyline treatment alone signi®cantly suppressed (P Ͻ 0.05) serum testosterone levels to nondetectable and the weights of these androgen-dependent organs, whereas serum testoster- one levels and the weights of these organs were not differ- ent (P Ͼ 0.05) from those in vehicle-treated rats when tes- tosterone was coadministered with acyline (data not shown).

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 re- versed the protective effect(s) of the GnRH antagonist, as the male rats failed to recover fertility by Week 16. Testic- ular 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 FIG. 4. Serum hormone levels at study termination (Week 34) in treated plus testosterone treatment before administration of CDB- rats from experiment II. A) Serum testosterone and rLH levels. B) Serum 4022 resulted in suppression of testicular function that was inhibin B and rFSH levels. Asterisks indicate significant differences (P Ͻ not different from the suppression observed in the presence 0.05) from vehicle-treated rats (group 1). ND ϭ nondetectable. 354 HILD ET AL.

TABLE 2. CDB-4022-induced infertility in male rats: testosterone reverses protective effect of acyline (experiment IV). No. male rats No. males recovering rendered fertility/no. male No. normal infertile/no. rats rendered conceptuses/ Treatment group males treated infertile pregnant rata Group 1: vehicle/4022 8/8 0/8 — Group 2: acyline/4022 7/7 7/7b 14 Ϯ 12 Group 3: acyline ϩ T/4022 8/8 0/8 — a Ϯ Mean SEM for Week 16 of the study. Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 b Significantly different (P Ͻ 0.05) from groups 1 and 3.

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 signi®cantly different from those at Week Ϫ4. This statistical difference may be due to the unusually high serum testosterone levels in three of the seven rats at Week Ϫ4. 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 sup- pressed (P Ͻ 0.05) to ND (Ͻ 0.3 ng/ml) during the 4-wk FIG. 5. Epididymal ABP levels in treated male rats from experiment II. acyline plus T treatment interval, and these returned to pre- Epididymal ABP content was nondetectable (ND, Ͻ10 fmol/mg protein) treatment levels by Week 3 (Fig. 8B). This trend was ob- in male rats that did not recover fertility (Table 1). served in rats pretreated with acyline alone, but the effect was not statistically different (P Ͼ 0.05). CDB-4022 treat- of CDB-4022 alone (P Ͼ 0.05). At study termination, ep- ment did not affect (P Ͼ 0.05) serum rLH levels. Serum ididymal weights were also signi®cantly suppressed (P Ͻ inhibin B levels were suppressed to ND (Ͻ25 pg/ml) by 0.05) in males that were infertile, whereas the weights of Week 1 in rats treated with CDB-4022 alone or acyline ϩ the ventral prostate and seminal vesicles were not different testosterone/CDB-4022 (Fig. 9A). These rats did not recov- (P Ͼ 0.21) among the treatment groups (data not shown). er fertility (Table 2). In contrast, serum inhibin B levels Serum hormone levels throughout the study are present- were transiently elevated after CDB-4022 treatment in rats ed in Figures 8 and 9. Serum testosterone was suppressed pretreated with acyline and returned to baseline by Week by acyline treatment to nondetectable levels (ND, Ͻ 0.06 6. These rats recovered fertility by Week 10 (Table 2). Se- ng/ml), but returned to pretreatment levels after cessation rum rFSH levels were suppressed by acyline or acyline ϩ of treatment (Week 3; Fig. 8A). The testosterone implant testosterone pretreatment, but not to the same extent. Re- in combination with acyline resulted in elevated serum tes- gardless, serum rFSH levels returned to pretreatment levels tosterone levels during treatment. After removal of the tes- after cessation of treatment (Week 3, Fig. 9B). Serum rFSH tosterone implant, serum testosterone decreased to ND at

FIG. 6. Testicular function following acyline or acyline plus testosterone FIG. 7. Testosterone reversal of the protective effect(s) of acyline against treatment for 4 wk in experiment III. Means with different superscript CDB-4022-induced damage of testicular function in experiment IV. letters for each endpoint were significantly different from one another (P Means with different superscript letters for each endpoint were signifi- Ͻ 0.05). cantly different from one another (P Ͻ 0.05). ND ϭ nondetectable. T REVERSES ACYLINE PROTECTION AGAINST CDB-4022 355 Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021

FIG. 8. Serum testosterone (A) and rLH (B) levels in treated rats through- out the study in experiment IV. Symbols indicate significant differences FIG. 9. Serum inhibin B (A) and rFSH (B) levels in treated rats throughout (P Ͻ 0.05) from pretreatment sample (Week Ϫ4) for each treatment group the study in experiment IV. Symbols indicate significant differences (P Ͻ as follows: *, Vehicle/CDB-4022; †, acyline/CDB-4022; ‡, acyline ϩ T/ 0.05) from pretreatment sample (Week Ϫ4) for each treatment group as CDB-4022. follows: *, vehicle/CDB-4022; †, acyline/CDB-4022; ‡, acyline ϩ T/CDB- 4022. 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 in- of mature differentiated germ cells. In contrast, a 2-wk acy- hibin B levels was also observed in acyline ϩ testosterone/ line exposure period did not completely block the formation CDB-4022-treated rats, serum rFSH levels were not in- of mature spermatids even though serum testosterone was creased above pretreatment levels in these rats. suppressed to nondetectable levels, nor was a 2-wk acyline pretreatment effective in preventing permanent CDB-4022- induced infertility (unpublished observations). Exogenous DISCUSSION testosterone treatment reversed the protective effect(s) of We showed previously that a single oral dose of CDB- acyline against permanent CDB-4022-induced infertility, 4022 induced irreversible infertility in the majority of male indicating a primary role for testosterone in rendering the rats [1, 2]. The Sertoli cell appeared to be the primary target Sertoli cell responsive to CDB-4022's deleterious effects, of CDB-4022 action [2], and CDB-4022-induced suppres- either directly or indirectly. Although ITT in acyline ϩ tes- sion of inhibin B and epididymal ABP, as observed in the tosterone-treated rats was not restored to levels observed in present study, are consistent with a Sertoli cell-mediated vehicle-treated males, ITT was suf®ciently elevated to effect. The GnRH agonist, Lupron Depot, provided partial maintain spermiogenesis as mature elongated, condensed protection from CDB-4022-induced testicular damage when spermatids were present. These results suggest that sup- treatment was initiated 1 wk before CDB-4022 [1]. In the pression or stimulation of other intratesticular and/or extra- present study, the GnRH antagonist, acyline, provided com- testicular factors may be required in addition to suppression plete protection from irreversible CDB-4022-induced infer- of testosterone for the protective effects of GnRH antago- tility when administered continuously over 4 wk before nist treatment to occur. CDB-4022. Indeed, unlike infertility induced by other cy- Testosterone's permissive effects on testicular responses totoxic agents or radiation (see [14] for review), concurrent to drugs and radiation may be mediated through direct ac- or posttreatment with the GnRH antagonist acyline did not tions on the testis. The androgen receptor (AR) antagonist, prevent irreversible infertility or restimulate spermatogen- ¯utamide, blocked testosterone-induced reversal of GnRH esis in CDB-4022-treated male rats. The 4-wk acyline-treat- analogue-stimulated spermatogenic recovery in irradiated ment interval not only suppressed serum testosterone and rats, implying a direct involvement of AR [6]. A subsequent ITT but also suppressed spermiogenesis, resulting in a loss study indicated that other potent , the synthetic 356 HILD ET AL. androgens R1881 and MENT and a 5␣-reduced androgen alone prevented irreversible CDB-4022-induced testicular (5␣-DHT), had the same effect as testosterone, suggesting damage in all treated males, whereas only one of seven the effect was not speci®c for a particular androgen [15]. male rats recovered fertility when treated with vehicle be- However, these actions were speci®c for androgens because fore CDB-4022 (unpublished observations). The results 17␤- did not reverse the GnRH analogue-stimulat- suggest that, unlike 2,5-HD, the irreversible effects of ed spermatogenic recovery [15]. These data further support CDB-4022 on the testes require testosterone but not the an AR-mediated permissive effect on the testis because all presence of Leydig cells. Collectively, these studies imply of these androgens bind and activate AR ([16, 17], unpub- involvement of other testicular cell types and potential lished observations). The presence of AR in Sertoli cells paracrine factors in the GnRH analogue-stimulated sper- ([18±20], our unpublished observations), the proposed pri- matogenic recovery, regardless of the agent used to induce mary target cell of CDB-4022 action [2], also ®ts with a infertility. direct androgen action. Although these studies indicate that testosterone most However, possible paracrine factors produced by other likely exerts its inhibitory effect directly on the testis, ex- testicular cells, especially differentiating germ cells and tratesticular sites of action may also contribute to testoster- Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 Leydig cells, may be involved in mediating testosterone's one's actions and cannot be conclusively ruled out. In par- permissive effects on the testis. In particular, testosterone ticular, male rats treated with a combination of acyline ϩ replacement in acyline-treated rats results in completion of testosterone had elevated serum rFSH levels as compared spermiogenesis and the presence of mature spermatids in with acyline-only-treated rats; however, serum rFSH levels the seminiferous tubules. Hence, testosterone's block of were not so high as those observed in vehicle-treated males. acyline-stimulated spermatogenic recovery may be medi- In this experimental paradigm, testosterone reversed acy- ated, in part, by paracrine factor(s) produced by mature line-induced suppression of rFSH to some degree, and this germ cells that render the Sertoli cell susceptible to the was presumably through increased transcription of the FSH permanent adverse effects of CDB-4022. Although very ␤ gene [26, 27]. To address the possible role of FSH in limited correlative data exist, a role for germ cell-mediated testosterone reversal of GnRH-analogue-stimulated sper- regulation of inhibin B production and secretion from the matogenic recovery, Meistrich and Shetty [14, 28] treated Sertoli cell has been proposed [21±23]. Similar to azoosper- irradiated adult male rats with exogenous FSH while sup- mic men, rats rendered irreversibly infertile by CDB-4022 pressing endogenous hormones with a GnRH antagonist have nondetectable serum inhibin B levels and tend to have and ¯utamide. The addition of exogenous FSH inhibited seminiferous tubules lacking differentiating germ cells. In the tubule differentiation observed in irradiated rats treated our previous studies, we showed that CDB-4022 affected with GnRH antagonist and ¯utamide, but not to the same inhibin B levels before an increase in germ cell apoptosis extent as that observed with androgens [6, 15]. These data [2], suggesting that the inhibitory effects on inhibin B lev- support a role for the suppression of FSH as an important els are due to direct actions of CDB-4022 on Sertoli cells factor contributing to the protective effects of GnRH ana- rather than to the loss of germ cells. These direct CDB- logs on the testes. 4022 actions have also been shown to occur in cultured The ability of GnRH-analogue treatment to reverse in- Sertoli cells [24]. Serum inhibin B levels remained non- hibition of spermatogenesis induced by radiation or multi- detectable in CDB-4022-treated male rats rendered irre- ple cytotoxic agents [5±10, 14, 28] implies that a common versibly infertile despite elevated serum rFSH levels. The mechanism in spermatogonial differentiation is disrupted elevation in rFSH from the pituitary is consistent with the by these treatments and that suppression of testosterone and release of negative feedback by inhibin B, and in this re- FSH either before or after the testicular injury is critical in spect, the animal presented with a castrate phenotype [22]. stimulating spermatogonial differentiation. How these hor- Whether the lack of gonadotropin-stimulated inhibin B pro- mones, testosterone and FSH, are involved in the continued duction and secretion in CDB-4022-treated infertile male inhibition of spermatogonial differentiation in rats rendered rats is due to the absence of differentiating germ cells or infertile by radiation or cytotoxic treatment is not known. irreversible damage to Sertoli cells has not yet been deter- Of importance in elucidating the potential pathway(s) in- mined. CDB-4022-treated males that were pretreated with volved is determining the initial target cell of the toxic acyline and recovered fertility exhibited a transitory ele- agent. Meistrich and Shetty [14] proposed four basic mod- vation of serum inhibin B levels followed by a reestablish- els for suppression of spermatogonial differentiation de- ment of both baseline serum inhibin B and rFSH levels. pending on the target cell, germ or somatic cell, and wheth- These data imply that this hormonal feedback axis is im- er the pathological defect is a failure of spermatogonia to portant in maintaining fertility in the rat. The possible role differentiate or is due to an increase in germ cell apoptosis. of mature germ cells in testosterone's block of acyline-stim- Because CDB-4022 appears to act on Sertoli cells, the pro- ulated spermatogenic recovery remains to be determined. posed pathological defect would involve either the loss of GnRH analogs were also used to reverse the adverse tes- a Sertoli cell-produced growth factor or stimulation of an ticular effects of 2,5-hexanedione (2,5-HD), a Sertoli cell apoptotic factor from the altered Sertoli cell. Thus, we are toxicant, in adult male rats [10, 25]. In a subsequent study, interested in determining effects of CDB-4022 on the ex- administration of the Leydig cell toxicant, ethane dimeth- pression of known Sertoli cell growth factors for germ cells anesulphonate (EDS), resulted in suppression of ITT, but (e.g., stem cell factor) and factors involved in the apoptotic was not able to rescue the testes from 2,5-HD-induced dam- pathway. age [25]. In the same study, Lupron Depot treatment alone At present, there are few new promising candidates for was able to prevent 2,5-HD-induced testicular damage, but male contraceptives. Steroid hormonal contraceptives are in the combination of Lupron Depot and EDS was not effec- the ®nal stages of development but still have disadvantages, tive. The authors concluded that, in addition to the sup- including the long delay between initiation of treatment and pression of ITT, a paracrine factor(s) from the Leydig cell induction of infertility and the lack of a long-acting inject- was required for rescue of testicular function. In a prelim- able or orally active androgen as part of the treatment reg- inary experiment in our laboratory, pretreatment with EDS imen [29, 30]. Other proposed male contraceptives, includ- T REVERSES ACYLINE PROTECTION AGAINST CDB-4022 357 ing gossypol and lonidamine derivatives, have not moved and Sertoli cell structure and function by the indenopyridine CDB- forward due to undesirable side effects or toxicity issues 4022 in rats. Biol Reprod 2001; 65:1771±1779. 3. Cook CE, Wani MC, Jump JM, Lee Y-W, Fail PA, Anderson SA, Gu [29, 30]. A recently discovered alkylated imino sugar, NB- Y-Q, Petrow V. Structure-activity studies of 2,3,4,4a,5,9b-hexahy- DNJ, may be a promising candidate; however, further char- droindeno[1,2-c]pyridines as antispermatogenic agents for male con- acterization of ef®cacy and safety are needed before de- traception. J Med Chem 1995; 38:753±763. velopment [30, 31]. Similar constraints also apply to CDB- 4. Boekelheide K. Sertoli cell toxicants. In: Russell LD, Griswold MD 4022. The lack of genetic or overt toxicity and other ad- (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:552± verse side effects of the indenopyridines suggest that this 575. 5. Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reissmann T, Russell family of compounds may have utility as male contracep- LD, Shetty G, Wilson G, Meistrich ML. Enhancement of a spermato- tives [32±35]. Of particular concern, however, is CDB- gonial proliferation and differentiation in irradiated rats by gonadotro- 4022's induction of irreversible infertility in adult male rats. pin-releasing hormone antagonist administration. Endocrinology In other species, including dogs and mice, reversible infer- 2000; 141:37±49. tility was observed [33, 36]. The results of a preliminary 6. Shetty G, Wilson G, Huhtaniemi I, Shuttlesworth GA, Reissmann T, study, performed in collaboration with the California Re- Meistrich ML. Gonadotropin-releasing hormone analogs stimulate and Downloaded from https://academic.oup.com/biolreprod/article/71/1/348/2667448 by guest on 29 September 2021 testosterone inhibits the recovery of spermatogenesis in irradiated rats. gional Primate Research Center (Davis, CA), indicated that Endocrinology 2000; 141:1735±1745. the puri®ed l-enantiomer of CDB-4022 induced spontane- 7. Kangasniemi M, Wilson G, Parchuri N, Huhtaniemi I, Meistrich ML. ously reversible suppression of sperm production as as- Rapid protection of rat spermatogenic stem cells against procarbazine sessed in semen samples from adult cynomolgus monkeys by treatment with a gonadotropin-releasing hormone antagonist (Nal- following 1 wk of daily oral dosing (unpublished obser- Glu) and an (¯utamide). Endocrinology 1995; 136: vations). Although these results in a nonhuman primate spe- 2881±2888. 8. Meistrich ML, Parchuri N, Wilson G, Kurdoglu B, Kangasniemi M. cies are promising, this preliminary experiment needs to be Hormonal protection from cyclophosphamide-induced inactivation of con®rmed and extended. Hence, the observation of irre- rat stem spermatogonia. J Androl 1995; 16:334±341. versible infertility in CDB-4022-treated male rats may be 9. Meistrich ML, Wilson G, Porter K, Huhtaniemi I, Shetty G, Shuttles- speci®c to this species. worth GA. Restoration of spermatogenesis in dibromochloropropane In conclusion, the GnRH antagonist, acyline, prevented (DBCP)-treated rats by hormone suppression. Toxicol Sci 2003; 76: CDB-4022-induced irreversible infertility in male rats when 418±426. 10. Blanchard KT, Lee J, Boekelheide K. Luprolide, a gonadotropin-re- administered continuously over a 4-wk period before CDB- leasing hormone agonist, reestablishes spermatogenesis after 2,5-hex- 4022 treatment. Unlike results with radiation or other cy- anedione-induced irreversible testicular injury in the rat, resulting in totoxic agents, concurrent or posttreatment with a GnRH a normalized stem cell factor expression. Endocrinology 1998; 139: analogue did not prevent irreversible infertility or restim- 236±244. ulate spermatogenesis in CDB-4022-treated male rats. The 11. National Research Council. Guide for the Care and Use of Laboratory protective effects of acyline appeared to be mediated by Animals. Washington, DC: Institute of Laboratory Animal Resources Commission on Life Sciences, National Academy Press; 1996. suppression of ITT, as they were reversed by exogenous 12. Danzo BJ, Eller BC. The ontogeny of biologically active androgen- testosterone. However, suppression of pituitary FSH may binding protein in rat plasma, testis, and epididymis. Endocrinology also be a contributing factor to acyline's protective action 1985; 117:1380±1388. on the testes. Testosterone either renders the Sertoli cell 13. Meistrich ML, van Beek EAB. Spermatogonial stem cells: Assessing responsive to CDB-4022's deleterious effects via a direct their survival and ability to produce differentiated cells. In: Chapin AR-mediated pathway or indirectly via suppression of the RE, Heindel JJ (eds.), Methods in Toxicology, vol. 3, Part A: Male Reproductive Toxicology. San Diego, CA: Academic Press; 1993: formation of mature spermatids and potential paracrine fac- 106±123. tors. Further research is required to elucidate the possible 14. Meistrich ML, Shetty G. Inhibition of spermatogonial differentiation contributing factor(s) involved in GnRH analogue-stimu- by testosterone. J Androl 2003; 24:135±148. lated spermatogenic recovery from testicular injury and the 15. Shetty G, Wilson G, Hardy MP, Niu E, Huhtaniemi I, Meistrich ML. permissive role of androgens. Because this appears to be Inhibition of recovery of spermatogenesis in irradiated rats by differ- common to multiple modes of testicular injury, understand- ent androgens. Endocrinology 2002; 143:3385±3396. ing the mechanism involved in the disruption of spermato- 16. Kemppainen JA, Langley E, Wong CI, Bobseine K, Kelce WR, Wil- son EM. Distinguishing androgen receptor agonists and antagonists: gonial differentiation may have applications for men ren- distinct mechanism of activation by medroxyprogesterone acetate and dered infertile by reproductive toxicants. Continued devel- dihydrotestosterone. Mol Endocrinol 1999; 13:440±454. opment of CDB-4022 as a potential nonhormonal male con- 17. Hartig PC, Bobseine KL, Britt BH, Cardon MC, Lambright CR, Wil- traceptive will depend on its safety pro®le in long-term son VS, Gray LE Jr. Development of two androgen receptor assays studies and whether its antispermatogenic effect in primate using adenoviral transduction of MMTV-luc reporter and/or hAR for species is reversible, either spontaneously or by additional endocrine screening. Toxicol Sci 2002; 66:82±90. 18. Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical treatments. localization of the androgen receptor in rat and human tissues. En- docrinology 1990; 127:3180±3186. ACKNOWLEDGMENTS 19. Takeda H, Chodak G, Mutchnik S, Nakamoto T, Chang C. Immuno- histochemical localization of androgen receptors with mono- and poly- We are grateful for the technical expertise of Janet Burgenson, Eileen clonal antibodies to androgen receptor. J Endocrinol 1990; 126:17± Curreri, Jennifer Lane, David Gropp, Trung Pham, Lisa Radler, Bruce Till, 25. and Devi Weier. 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