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Regular Article |
a BIOQUAL, Inc., Rockville, Maryland 20850
b Department of Experimental Radiation Oncology, University of Texas, MD Anderson Cancer Center, Houston, Texas 77030
c Contraception and Reproductive Health Branch (CRHB), National Institute of Child Health and Human Development (NICHD), Rockville, Maryland 20852
ABSTRACT
The goals of this study were to determine the CDB-4022 dose-response relationship for induction of acute decreases in testicular weight and germ cell depopulation in rats; establish the threshold dose of CDB-4022 required to induce infertility; and investigate whether CDB-4022-induced testicular damage could be prevented by a GnRH agonist (Lupron Depot). Reduction of testis weight and germ cell depopulation were observed 7 days after a single oral dose of 1 mg CDB-4022/kg, whereas 0.5 mg/kg had no observable effect. These effects were maximal at 12.5 or 25 mg CDB-4022/kg. After a single oral dose of either 2.5 or 5 mg/kg, CDB-4022 induced infertility in five of five treated rats by Week 5, whereas only one of five males was rendered infertile at a dose of 1 mg/kg. Proven fertile male rats (6/group) were treated with vehicle, CDB-4022 alone (2.5 mg/kg on Day 0), CDB-4022 plus Lupron Depot (on Weeks -1, 2, 5, and 8), or Lupron Depot alone. Control males demonstrated normal fertility throughout a 32-wk cohabitation period. Five of six rats were rendered transiently infertile with Lupron Depot alone, but all recovered fertility. CDB-4022 treatment resulted in infertility in all six rats, and only one of six regained fertility. Combined treatment also caused infertility in all six rats, but four of six recovered fertility (P = 0.08 compared to CDB-4022 alone). Testicular weight was decreased in the three treatment groups compared to vehicle controls; testicular weights were ranked from highest to lowest as follows: vehicle > Lupron Depot > Lupron Depot + CDB-4022 > CDB-4022. The tubule differentiation index of Lupron Depot-treated rats (96 ± 4%) was not different from vehicle-treated rats (100%). CDB-4022 treatment decreased the number of differentiating tubules (15 ± 8%). Lupron Depot plus CDB-4022 treatment resulted in a greater number of differentiating tubules (53 ± 12%) than CDB-4022 alone, but this was still lower than vehicle- or Lupron Depot-treated rats. These data indicate that 2.5 mg/kg of CDB-4022 was the oral threshold dose that caused testicular damage rendering the majority of adult male rats permanently infertile within the study interval; 12.5 mg/kg of CDB-4022 induced maximal testicular damage. Suppression of gonadotropins and/or testosterone production by treatment with Lupron Depot before and after CDB-4022 prevented the CDB-4022-induced irreversible testicular damage.
male reproductive tract, Sertoli cells, sperm, spermatogenesis, testis
INTRODUCTION
A series of compounds being considered as new antihistamines was inadvertently found to possess antispermatogenic activity during preliminary toxicology testing [1, 2]. Oral treatment of male dogs with an indenopyridine (Sandoz 20-438, [4aRS,5RS,-9bRS]-2-ethyl-2,3,4,4a,5,9b-hexahydro-7-methyl-5-p-tolyl-1H-indeno[1,2-c]pyridine) caused damage to the seminiferous tubules of the testes [1]. The investigators undertook an additional study to determine whether this phenomenon was reversible in rats and dogs. The indenopyridine resulted in germ cell depletion of the seminiferous epithelium in both dogs and rats. Serum levels of FSH and LH were elevated, indicating that the compound was not inhibiting spermatogenesis via suppression of gonadotropins. Recovery of normal spermatogenesis occurred 12 wk after cessation of drug treatment in dogs and after 10 wk in rats [2]. Treatment did not affect erection capability and ejaculation in dogs. These data suggested that this indenopyridine might have potential as a reversible male antifertility agent because the compound did not demonstrate genetic or overt toxicity or have other adverse effects [1–3].
These serendipitous findings [1, 2] prompted Cook and colleagues [4] to synthesize and test related indenopyridine analogues for antispermatogenic activity in mice. The antispermatogenic/antifertility activity of the new series of indenopyridines was diasteroselective, enantioselective, chemoselective, and dependent on the para substituent on the 5-aryl group [4]. Fertility of male mice was reduced following 35 days of treatment with 7.5 mg/kg/day of (4aRS,5RS,9bRS)-2-ethyl-7-methyl-2,3,4,4a,5,9b-hexahydro-5-(4-carbomethoxyphenyl)-1H-indeno[1,2-c]pyridine hydrochloride (designated as RTI 4587-056 by Research Triangle Institute and CDB-3632 by CRHB, NICHD), whereas a daily dose of 15 mg/kg rendered all male mice infertile. No adverse effect on libido was apparent because the mice continued to exhibit mating behavior as determined by the presence of vaginal copulatory plugs [4]. The results of a recent 28-day toxicological evaluation of this hexahydroindenopyridine analogue of Sandoz 20-438 (RTI 4587-056, CDB-3632) confirmed the relative safety of this compound [5].
In an initial long-term male antifertility study, a single oral dose of 100 mg/kg of the hexahydroindenopyridine, CDB-3632, resulted in infertility in five of five male rats by 4 wk after treatment (unpublished data). Fertility did not return by 26 wk as assessed by mating trials; the males were necropsied 28 wk after treatment. Histological examination of the testes revealed depletion of differentiated germinal elements in the seminiferous tubules with no signs of recovery in four of five treated rats; however, one rat demonstrated a tubule differentiation index of 44% but had not regained fertility. Although the remainder showed no differentiated cells, the nonrepopulating tubules did contain some A spermatogonia. This preliminary study suggested that infertility induced by highly potent indenopyridines might not be reversible in adult male rats. Because the damage to the seminiferous tubules in the CDB-3632-treated rats was similar to that observed in testes of irradiated rats or rats treated with procarbazine or cyclophosphamide [6–9], we hypothesized that the mechanism of cellular depletion of the seminiferous tubules might be the same or similar. Hormonal treatments such as GnRH antagonist and flutamide or testosterone and estradiol, prevented the decline in spermatogenesis resulting from the damaging effects of irradiation or chemotherapy drugs [7–9]. Therefore, we anticipated that similar treatments might facilitate recovery from infertility induced by indenopyridines.
A more potent analogue, [4aRS,5SR,9bRS]-2-ethyl-2,3,4,4a,5,9b-hexahydro-8-iodo-7-methyl-5-[4-carbomethoxyphenyl]-1H-indeno[1,2-c]pyridine-hydrochloride (RTI-4587-073), was recently synthesized by halogenation of the C-8 position of the hexahydroindenopyridine system [10]. This 8-iodo-hexahydroindenopyridine exhibited potent oral antispermatogenic activity in mice [10]. This lead indenopyridine has been designated as CDB-4022 by CRHB, NICHD, and we have conducted several studies on this promising compound. The goals of the present study were to determine the CDB-4022 dose-response relationship for induction of acute decreases in testicular weight and germ cell depopulation in male rats; establish the threshold dose of CDB-4022 required to induce infertility in treated adult male rats; and investigate whether CDB-4022-induced testicular damage could be prevented by a GnRH agonist (Lupron Depot).
MATERIALS AND METHODS
Animals
Sprague-Dawley CD rats (Crl:CD[SD]IGS BR Stock) were purchased from Charles River Laboratories (Kingston, NY). Males were singly housed except for mating trials, whereas females were group housed. For experiments that used proven fertile male rats, male fertility was assessed by in-house mating trials prior to study assignment. Male rats were cohabited with virgin females (one male per two females) over a period of three estrous cycles (approximately 17 days). Females were killed at the end of the cohabitation period and the number and condition of implantation sites determined. Males were considered fertile and assigned to study if they successfully impregnated at least one female rat with 10 conceptuses. All rats were housed in polycarbonate solid floor cages with Beta-Chip bedding (Andersons Industrial Products Group, Maumee, OH) and received Purina laboratory rodent diet (#5001) and tap water ad libitum. The photoperiod was 14L:10D. The environmental conditions of the animal rooms were maintained as recommended in the National Research Council, Guide for the Care and Use of Laboratory Animals to the maximum extent possible [11]. All study protocols were approved by BIOQUAL's institutional animal care and use committee.
Materials
CDB-4022, [4aRS,5SR,9bRS]-2-ethyl-2,3,4,4a,5,9b-hexahydro-8-iodo-7-methyl-5-[4-carbomethoxyphenyl]-1H-indeno[1,2-c]pyridine-hydrochloride, was provided by C.E. Cook and his colleagues, Research Triangle Institute, Research Triangle Park, NC. All assays used the same batch of CDB-4022. The structure of CDB-4022 is presented in Figure 1. Lupron Depot and its aqueous diluent were gifts from TAP Pharmaceuticals (Deerfield, IL). Food-grade sesame oil (Hain) was purchased from a local grocery store.
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Acute Testicular Effects of CDB-4022
In the first experiment, adult male rats weighing 240–280 g at the start of the study (5/group) received a single oral dose of either vehicle (10% ethanol/sesame oil) or CDB-4022 at 5.0, 12.5, or 25 mg/kg. Because fertility was not addressed in this initial experiment, proven fertile male rats were not used. A gross necropsy of the rats was performed 7 days after oral dosing, and paired testes weights were obtained. The testes were preserved for histological evaluation. Because testicular weight was suppressed by 55% at the lowest dose in this experiment, a second study was performed at lower CDB-4022 doses. In the second experiment, proven fertile male rats (490–560 g, 5/group) received a single oral dose of either vehicle (10% ethanol/sesame oil) or CDB-4022 at 0.5, 1.0, or 2.5 mg/kg. Necropsy was performed 7 days later, testis weights were obtained, and the testes preserved for morphological evaluation. The percentage of normal seminiferous tubules was determined for each testicular cross section. A tubule was considered abnormal if it showed germ cell depopulation (qualitative assessment), the presence of multinucleated giant cells, cells with pyknotic nuclei, vacuolization, or derangement of the seminiferous epithelium. Testes from vehicle-treated rats were assigned a value of 100% and data from CDB-4022-treated rats were expressed as a percentage of the control.
Dose-Dependent Effects of CDB-4022 on Male Fertility
Proven fertile male rats (400–555 g, 5/group) received a single oral dose of either vehicle (10% ethanol/sesame oil) or CDB-4022 at 1, 2.5, or 5 mg/kg (combination of two experiments). Male rats were cohabited with untreated virgin females (one male per two females) during Weeks 1, 2, 3, 4, 5, and 6 after dosing (Week 0). Vaginal washings were obtained daily from the female rats during the cohabitation period, and the presence of sperm, a copulatory plug, or vaginal cell types was recorded. These data were used to confirm mating and/or to establish that female rats were demonstrating normal estrous cycles. No males were cohabited with two acyclic females. Females were separated from the male rats at the end of 7 days of cohabitation and necropsied approximately 10 days later. At necropsy, the uteri of the female rats were examined for the presence and condition of implantation sites. The number of normal and resorbing implantation sites for each female rat was recorded. The males were killed after completion of the mating trials (Weeks 9 or 7 for experiments one and two, respectively). Testes were excised, weighed, and preserved for morphological evaluation.
Lupron Depot Prevention of CDB-4022-Induced Testicular Damage and Irreversible Infertility
Proven fertile male rats (495–615 g) were assigned to one of four treatment groups (6/group): 1) Vehicle control. 2) CDB-4022-treated. 3) Lupron Depot plus CDB-4022 treatment. 4) Lupron Depot treatment only. The experimental design is presented in Figure 2. Male rats were treated s.c. (Week -1) with 1.8 mg per rat of Lupron Depot or vehicle (aqueous diluent). One week later (Day 0, Week 0), the male rats were treated with a single oral dose of 2.5 mg/kg of CDB-4022 or vehicle (10% ethanol in sesame oil). Additional s.c. injections of Lupron Depot (1.8 mg per rat) or vehicle were administered on Weeks 2, 5, and 8 after the CDB-4022 treatment. The pre- and posttreatments of rats with Lupron Depot were designed to maximize the chance of observing a positive result [7, 8]. Individual male rats were weighed every 3 wk after oral dosing and at necropsy (Week 35). Mating trials were performed weekly during Weeks 1–8 and every 2 wk from Weeks 10 through 32 and the females examined for pregnancy status. At Week 35, the male rats were killed, and the testes and epididymides were excised, weighed, and preserved for morphological assessment. Tubule differentiation indices (TDIs) were determined by evaluating 200 seminiferous tubules per cross-section [12]. A differentiating seminiferous tubule was defined as having at least three or more spermatocytes or more advanced spermatogenic germ cells present in the tubule.
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Testicular Histology
The tunica albuginea (outer capsule) of the left and right testis was nicked, and the entire testes and epididymides were immersion fixed in 10% neutral buffered formalin for 48 h and postfixed in Bouin solution for 18 h. Tissues were washed with several changes of 50% ethanol. Following dehydration, both testes were embedded in paraffin for studies on acute and dose-dependent effects on fertility. For the Lupron Depot study the left testis was embedded in paraffin and the right testis was embedded in glycol methacrylate medium (GMA). Epididymides from all studies were embedded in paraffin. The histology of hematoxylin and Lee-stained GMA cross sections (2 µm) from the right testis and hematoxylin and eosin or periodic acid-Schiff (PAS)-stained paraffin cross sections (5 µm) of the left testis were evaluated at the light microscopic level. Hematoxylin and eosin-stained cross sections (5 µm, paraffin) of the head, body, and tail of the epididymides were also evaluated.
Statistical Analysis
Statistical tests were performed as follows: Male rat body weights were compared using a parametric two-way ANOVA for repeated measures; testicular weights and TDIs were compared using a parametric one-way ANOVA; the percentage of normal seminiferous tubules and number of normal implantation sites in pregnant females at Week 24 were compared using a nonparametric one-way Kruskal-Wallis ANOVA on ranks; and the proportion of male rats rendered infertile/treated and the proportion of male rats recovering fertility/rats rendered infertile were compared using a z-test of proportions (SigmaStat for Windows version 2.01; Jandel Scientific, San Rafael, CA). A Student-Newman-Keuls multiple range test was performed for an ANOVA with a significant F value (P < 0.05). Graphs were drawn using SigmaPlot for Windows version 4.0 (Jandel Scientific).
RESULTS
Acute Testicular Effects of CDB-4022
Acute dose-dependent effects of CDB-4022 on testicular weight and germ cell depopulation of the seminiferous epithelium were evaluated in order to determine effective doses of CDB-4022 for use in assessing effects on fertility. In the first experiment, CDB-4022 at 5, 12.5, or 25 mg/kg resulted in significant decreases (P < 0.05) in testicular weight at 7 days following treatment as compared to vehicle controls (Table 1). At 5 mg/kg, testis weight was decreased to 55% of control. Suppression of testicular weight to 51% and 47% of control was observed at 12.5 and 25 mg/kg of CDB-4022, respectively. Although testicular weight was not significantly different (P > 0.05) between the two higher dose groups, testicular weight was further suppressed (P < 0.05) at doses of 25 mg/kg as compared to 5 mg/kg. The testis from these rats demonstrated shrunken seminiferous tubules. In addition, the majority of these tubules were devoid of differentiating germinal elements with a maximal effect observed at the 12.5-mg/kg dose. A second experiment was performed at lower dose levels in order to determine the threshold dose of CDB-4022 resulting in significant effects on testis weight and histology (Table 1). No significant effect on either testis weight (P > 0.05) or testicular morphology (P > 0.05) was observed in rats treated with 0.5 mg CDB-4022/kg as compared to controls. A significant reduction (P < 0.05) in testis weight to 66% of control was observed in rats treated with a single oral dose of 1 mg CDB-4022/kg. This decrease in testis weight corresponded to a significant decrease (P < 0.05) in the percentage of normal seminiferous tubules. A dose of 2.5 mg CDB-4022/kg also significantly decreased (P < 0.05) testis weight compared to controls, but this effect was not significantly different (P > 0.05) from the 1-mg/kg dose. Although there was a tendency for a further decrease in the percentage of normal seminiferous tubules at the 2.5-mg/kg dose of CDB-4022, this also was not significantly different (P > 0.05) from the 1-mg/kg dose.
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Dose-Dependent Effects of CDB-4022 on Male Fertility
A dose-response assay was performed in order to determine the minimal dose of CDB-4022 that would consistently induce infertility in adult males. Male rats treated with vehicle alone demonstrated normal fertility throughout the study interval of both experiments (Fig. 3). At the lowest dose, 1 mg CDB-4022/kg, one of five rats became infertile by Week 3, and no other males in this group became infertile by Week 6 (Fig. 3). The testis weight at 9 wk after dosing with 1 mg CDB-4022/kg was 87% of vehicle control-treated rats and was not significantly different from controls (P > 0.05, first experiment). The testicular morphology appeared normal in the four fertile rats in this group. The one rat in this group that was infertile had many seminiferous tubules that were devoid of germ cells. In the two higher dose groups, CDB-4022 induced infertility in five of five male rats by 5 wk after a single oral dose of 2.5 or 5 mg/kg (second experiment, Fig. 3). At 7 wk following treatment with 2.5 or 5 mg CDB-4022/kg, CDB-4022 caused significant decreases (P < 0.05) in testis weight as compared to vehicle-treated rats (54% and 51% of control, respectively). Many of the seminiferous tubules were depleted of germ cells in rats treated with 2.5 or 5 mg CDB-4022/kg.
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Lupron Depot Prevention of CDB-4022-Induced Testicular Damage and Irreversible Infertility
Fertility In order to assess the ability of a GnRH agonist to prevent CDB-4022-induced irreversible testicular damage resulting in sterility, male rats were treated as indicated (Fig. 2). The threshold dose of CDB-4022 that induced infertility (2.5 mg/kg) was used for this study. All six male rats treated with the control vehicle demonstrated normal fertility throughout the 32-wk cohabitation period as indicated by a normal complement of uterine implantation sites in their female partners (Table 2). The treatment of male rats with Lupron Depot alone resulted in transient infertility in five of six rats by Week 3; all five infertile male rats recovered fertility by Week 18. One male rat treated with Lupron Depot alone remained fertile for the entire 32-wk cohabitation period. Treatment with CDB-4022 alone resulted in infertility by Week 5 in all six male rats. Fertility returned in one CDB-4022-treated male rat at Week 12, but the other five male rats remained infertile over the 32-wk cohabitation period. The combination of Lupron Depot and CDB-4022 treatment resulted in infertility in all six male rats by Week 2. Fertility was restored in two male rats by Week 12, and two additional male rats subsequently regained fertility, one at Week 20 and the other at Week 24. Hence, four of six males treated with Lupron Depot and CDB-4022 recovered fertility by 24 wk. More male rats treated with Lupron Depot plus CDB-4022 recovered fertility than rats treated with CDB-4022 alone; however, the proportions were not statistically different (Table 2, P = 0.08)
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Body and testicular weights The male rats gained weight over time as reflected by a significant time effect (P < 0.001); however, the treatments had no significant effect on body weight (P = 0.63, data not shown). At necropsy (Week 35), the paired testes weights were significantly different among all four treatment groups (P < 0.05, Fig. 4A). Treatment with CDB-4022 alone, Lupron Depot plus CDB-4022, and Lupron Depot alone significantly decreased (P < 0.05) the weight of the testes compared to controls. In addition, the testicular weight of Lupron Depot plus CDB-4022-treated rats was significantly higher (P < 0.05) than CDB-4022 alone-treated rats but not as high as those treated with Lupron Depot alone.
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Testicular histology Representative photographs of the testes from rats in each treatment group that recovered fertility or remained infertile for 32 wk are presented in Figures 5 and 6, respectively. The seminiferous tubules and Leydig cells of the testes from control animals appeared normal (Fig. 5A). Spermatogenesis was apparent in the seminiferous tubules (TDI = 100%, Fig. 4B). In addition, mature sperm were present in the epididymides (data not shown). These findings correlated with the normal fertility observed in these rats (Table 2).
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In the rats treated with Lupron Depot alone, all six rats had normal-appearing testes, except that some tubules were depleted of germ cells (Fig. 5D). These few depleted tubules typically appeared as groups or patches of tubules within the cross section of the testes. All six rats had mature sperm in their epididymides (data not shown), which was in accord with recovered fertility (Table 2).
The rats treated with CDB-4022 alone had normal-appearing Leydig cells. One of six rats that regained fertility had a tubule differentiation index of 53% (Fig. 5B). The remaining tubules in this rat's testes demonstrated signs of damage including germ cell depopulation and a shrunken, deranged appearance of the seminiferous tubules. Sperm were detected in the epididymides of this rat (data not shown). These findings corresponded with the recovered fertility exhibited by this animal. The testes of the remaining five rats were all similar in appearance: The majority of the seminiferous tubules appeared to be depleted of the germinal elements and contained only Sertoli cells (Fig. 6A). It was noted that 16% of the tubules that lacked differentiated germinal cells did contain undifferentiated type A spermatogonia. No sperm were present in the epididymides of these five rats (data not shown). These observations corresponded to the lack of fertility in these five rats.
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The histological appearance of the testes of the combined Lupron Depot/CDB-4022-treated rats varied. The interstitial tissue appeared normal in all six rats, but the percentage of damaged seminiferous tubules differed among the six rats. Nearly all of the seminiferous tubules in two of the rats were damaged as indicated by a lack of germinal elements, shrunken seminiferous tubules, derangement of the seminiferous tubules, and seminiferous tubules containing only Sertoli cells (Fig. 6B). These two rats did not have sperm present in their epididymides (data not shown), did not regain fertility, and had the smallest testes as indicated by testicular weight. The other four rats in this group demonstrated active spermatogenesis in 
36% of the seminiferous tubules (Fig. 5C). These four rats had sperm present in their epididymides (data not shown) and recovered fertility. The percentage of seminiferous tubules undergoing spermatogenesis correlated with testis weight. In these rats it was also noted that 18% of the tubules that lacked differentiated germ cells did have A spermatogonia present.
Overall, the group tubule differentiation indices corresponded with group testicular weights and fertility (Fig. 4B). All of the seminiferous tubules in vehicle-treated rats demonstrated active spermatogenesis. The tubule differentiation index for rats treated with Lupron Depot alone was not different (P > 0.05) from vehicle-treated rats and was in keeping with the reversible nature of GnRH agonist-induced infertility. CDB-4022 treatment significantly decreased (P < 0.05) the number of differentiating seminiferous tubules, whereas the combination of Lupron Depot and CDB-4022 resulted in a significantly greater (P < 0.05) number of repopulating seminiferous tubules than CDB-4022 treatment alone. However, the tubule differentiation index for combined Lupron Depot/CDB-4022-treated rat testis was still significantly lower (P < 0.05) than Lupron Depot-treated rats.
DISCUSSION
The major findings of this study were that CDB-4022 effects on the testis were dose dependent; the antispermatogenic activity of CDB-4022 was irreversible, rendering the majority of male rats in this study infertile; and Lupron Depot, a GnRH agonist, partially prevented the irreversible damage and infertility induced by CDB-4022. A single oral dose of 2.5 mg CDB-4022/kg was the minimal oral threshold dose that resulted in significant inhibition of both testis weight and fertility. At this dose, less than 50% of the seminiferous epithelium was undergoing normal spermatogenesis at 7 days after treatment. The presence of normal seminiferous tubules along with atrophied tubules could be the result of unequal distribution of the compound, differential sensitivity of various stages of the spermatogenic cycle, or random factors affecting the sensitivity of the different tubules to the drug. However, at a maximal dose (12.5 mg/kg), CDB-4022 induced testicular damage throughout the seminiferous epithelium implying that at high enough doses all seminiferous tubules are susceptible to its antispermatogenic effects. At doses of CDB-4022 that induced irreversible infertility, the seminiferous tubules consisted primarily of Sertoli cells only or Sertoli cells and type A spermatogonia. The surviving type A spermatogonia were incapable of repopulating the seminiferous tubules. However, treatment with a GnRH agonist, Lupron Depot, before and after CDB-4022 treatment permitted the surviving A spermatogonia to repopulate some of the seminiferous tubules resulting in recovery of fertility in four of six rats. Repopulation of approximately 50% of the seminiferous tubules was sufficient to confer normal fertility in these male rats, as expected, because the adult rat testis produces more sperm than necessary for fertility.
In contrast to previous studies testing the antispermatogenic/antifertility effects of indenopyridine analogues in dogs and mice [2, 13], a single oral dose of the 8-iodo-hexahydroindenopyridine, CDB-4022, induced irreversible infertility in the majority of adult male rats within the study interval. Differences in fertility outcome, reversible versus irreversible infertility, may be related to differences in the potency of indenopyridine analogues, the dosing regimen utilized, and/or the species used for testing. CDB-4022 has been reported to be a more potent antispermatogenic compound than either Sandoz 20-438 or RTI 4587-056 (CDB-3632) [4, 10]. The increased potency of this indenopyridine may explain, in part, the discrepancies observed in reversibility of infertility. The earlier indenopyridine analog, CDB-3632 (RTI 4587-056), induced irreversible infertility in adult male rats at a single oral dose of 100 mg/kg for the 26-wk study period (unpublished data), whereas infertility was reversible when adult male mice were treated with this same compound (designated RTI-056) at 15 mg/kg/day for 35 days [13]. Infertility was also reversible in male rats treated with a single oral dose of 10 mg/kg Sandoz 20-438; however, higher doses of 30 or 90 mg/kg resulted in incomplete recovery of fertility [2]. Hence, indenopyridine analogues may induce reversible infertility at low doses and permanent testicular damage and sterility at high doses. Lastly, reversibility of infertility may also be species specific. Irreversible infertility has been observed in adult male rats treated with both CDB-4022 and CDB-3632, whereas reversible infertility was observed in adult male mice treated with CDB-3632 (RTI-056) [13]. Rats are more susceptible to the induction of arrest of surviving spermatogonial stem cells from radiation or chemotherapy than are mice [14–16]. This arrest is characterized by the presence of cycling undifferentiated A spermatogonia that fail to differentiate but instead undergo apoptosis [17]. In addition, different rat strains demonstrate differential sensitivities to the form of radiation induced injury [6, 18, 19]. Therefore, radiation or chemical induction of reversible or irreversible suppression of spermatogenesis may be observed across strains or species. Because spermatogenesis in humans is particularly sensitive to injury from radiation and chemotherapy [20], more sensitive animal models, such as the rat, should be used for testing of potential contraceptive agents. Additional studies in other species, including nonhuman primates, would be necessary to elucidate the requirements for reversible infertility in adult human males following treatment with indenopyridines.
Results from the present study indicate that a GnRH agonist, Lupron Depot, prevented irreversible infertility in 67% of the CDB-4022-treated male rats. The presence of type A spermatogonia in some of the depopulated tubules of CDB-4022-treated rats suggested that the stem cells survived but were unable to repopulate the seminiferous epithelium within the study interval. These data are in agreement with those in a recent study that suggested that hormonal treatments prior to radiation do not necessarily protect the stem spermatogonia from death but rather enhance recovery by preserving the stem cell's subsequent ability to differentiate [21]. GnRH agonists/antagonists have been shown to prevent or reverse irreversible testicular damage induced by radiation [17, 22], chemotherapeutic agents such as procarbazine and cyclophosphamide [7, 9] and 2,5-hexanedione, a Sertoli cell toxicant [23]. However, the mechanism by which GnRH analogues prevent irreversible testicular damage is not well understood. Suppression of circulating gonadotropins, testosterone, and spermatogenesis induced by GnRH agonists/antagonists [24, 25] are all possible factors involved in prevention of irreversible testicular damage. A recent study suggests that suppression of intratesticular testosterone is the key GnRH antagonist-induced event that allows for repopulation of the rat seminiferous epithelium following irradiation [22]. Testosterone treatment blocked GnRH antagonist-induced recovery of spermatogenesis and coadministration of flutamide, an androgen antagonist, reversed the inhibitory effect of testosterone treatment on repopulation of the seminiferous tubules. The authors concluded that testosterone is capable of inhibiting repopulation via an androgen receptor-mediated pathway. Additional studies are required to determine whether suppression of intratesticular testosterone is a common critical event for recovery of spermatogenesis following treatment with radiation, chemotherapeutic drugs, hexanedione, or indenopyridines.
In conclusion, the 8-iodo-hexahydroindenopyridine, CDB-4022, induced testicular damage and irreversible infertility within the study interval in adult male rats with a threshold oral dose of 2.5 mg/kg; maximal damage was induced at a dose of 12.5 mg/kg. Lupron Depot, a GnRH agonist, partially prevented the development of testicular damage and irreversible infertility induced by CDB-4022. Other studies from our laboratory indicate that CDB-4022 induces infertility via direct action on the Sertoli cell resulting in apoptosis of spermatogonia/spermatocytes [26]. The 3- to 5-wk delay in the onset of CDB-4022-induced infertility suggests either that the antispermatogenic effect occurs at an early stage in the spermatogenic cycle or the damage to the Sertoli cell develops gradually. Additional studies are required to define the Sertoli cell factor(s) involved in CDB-4022-induced germ cell depopulation of the seminiferous epithelium and to determine the role of intratesticular testosterone in CDB-4022-induced irreversible infertility in adult male rats.
ACKNOWLEDGMENTS
The authors express thanks for the technical expertise of the following Bioqual technicians: Ms. Eileen Curreri, Mr. David Gropp, Ms. Brenda Hembrey, Ms. Sandra Menzies, and Mr. Bruce Till. Care of the animals was provided by Mr. Stuart Gormley and Mr. Osseh Saine. We also thank Mr. Gene Wilson for determining the tubule differentiation indices, Mr. Keith Rogers of Experimental Pathology Laboratories, Inc., Herndon, VA, for morphological processing of the testis, and Dr. Barbara Attardi for editorial assistance.
FOOTNOTES
First decision: 6 December 2000.
1 This work was supported by National Institute of Child Health and Human Development (NICHD) contract N01-HD-6-3259 awarded to BIOQUAL, Inc. A portion of this work was presented at the XIVth Testis Workshop: Germ Cell Development, Division, Disruption and Death (1997) Abstract I-32. 
2 Correspondence: Sheri Hild, BIOQUAL, Inc., 9600 Medical Center Dr., Rockville, MD 20850. FAX: 301 251 1260; sahild{at}compuserve.com
Accepted: February 22, 2001.
Received: November 21, 2000.
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RS,5SR,9ßRS]-2-ethyl-2,3,4,4,5,9ß-hexahydro-8-iodo-7-methyl-5-[4-carbomethoxyphenyl]-1H-indeno[1,2-c]pyridine-hydrochloride). In: 31st Annual Meeting of the Society for the Study of Reproduction; 1998; College Station, TX. Abstract 312 This article has been cited by other articles:
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  S. Koduri, S. A. Hild, L. Pessaint, J. R. Reel, and B. J. Attardi Mechanism of Action of l-CDB-4022, a Potential Nonhormonal Male Contraceptive, in the Seminiferous Epithelium of the Rat Testis Endocrinology, April 1, 2008; 149(4): 1850 - 1860. [Abstract] [Full Text] [PDF]
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 Y.-C. Chen, R. K Cochrum, M. T Tseng, D. T Ghooray, J. P Moore, S. J Winters, and B. J Clark Effects of CDB-4022 on Leydig Cell Function in Adult Male Rats Biol Reprod, December 1, 2007; 77(6): 1017 - 1026. [Abstract] [Full Text] [PDF]
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 S. A. Hild, J. R. Reel, M. J. Dykstra, P. C. Mann, and G. R. Marshall Acute Adverse Effects of the Indenopyridine CDB-4022 on the Ultrastructure of Sertoli Cells, Spermatocytes, and Spermatids in Rat Testes: Comparison to the Known Sertoli Cell Toxicant Di-n-pentylphthalate (DPP) J Androl, July 1, 2007; 28(4): 621 - 629. [Abstract] [Full Text] [PDF]
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S. A. Hild, G. R. Marshall, B. J. Attardi, R. A. Hess, S. Schlatt, D. R. Simorangkir, S. Ramaswamy, S. Koduri, J. R. Reel, and T. M. Plant Development of l-CDB-4022 as a Nonsteroidal Male Oral Contraceptive: Induction and Recovery from Severe Oligospermia in the Adult Male Cynomolgus Monkey (Macaca fascicularis) Endocrinology, April 1, 2007; 148(4): 1784 - 1796. [Abstract] [Full Text] [PDF]
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S. A. Hild, B. J. Attardi, and J. R. Reel The Ability of a Gonadotropin-Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone Biol Reprod, July 1, 2004; 71(1): 348 - 358. [Abstract] [Full Text] [PDF]
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 M. L. Meistrich, G. Wilson, K. L. Porter, I. Huhtaniemi, G. Shetty, and G. A. Shuttlesworth Restoration of Spermatogenesis in Dibromochloropropane (DBCP)-Treated Rats by Hormone Suppression Toxicol. Sci., December 1, 2003; 76(2): 418 - 426. [Abstract] [Full Text] [PDF]
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M. L. Meistrich and G. Shetty Inhibition of Spermatogonial Differentiation by Testosterone J Androl, March 1, 2003; 24(2): 135 - 148. [Full Text] [PDF]
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