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Regular Article |
a Population Council, Center for Biomedical Research, New York, New York 10021
b Department of Pharmacology of Natural Substances and General Physiology, University of Rome "La Sapienza," 00185 Rome, Italy
ABSTRACT
The oral male contraceptive agent 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF2364) is a new analogue of indazole-carboxylic acid. AF2364 was orally administered to rats at 50 mg/kg body weight once weekly for five consecutive weeks. The effects on fertility efficacy, hormonal profile, organ weights, tissue morphology, and serum microchemistry were examined. Complete infertility was noted in rats 29 days after the initial dose of AF2364 and continued until 90 days. Fertility resumed in 25% of the group after 104 days and had resumed in 75% of the rats by the last mating at 197 days. Morphological examination of the testis showed rapid exfoliation of elongated spermatids and the generation of large multinucleated cells 6 days after the first treatment, with depletion of most germ cells after 40 days. Normal spermatogenesis was noted in 95% of the tubules in the animals that were fertile at 210 days. Morphological analysis of the epididymal compartments revealed reduced lumen size, whereas the prostate exhibited an increase in the glandular lumen with a reduction in epithelium height. No morphological changes were detected in the kidney, liver, and cerebrum by light microscopy. Kidney and liver function, as evaluated by serum chemistry, were not affected by the drug treatment. AF2364 did not alter the levels of FSH, and only minimal changes were noted for LH and testosterone, suggesting that the hypothalamic-pituitary-testicular axis was not affected. These results illustrate the potential of AF2364 as a male contraceptive.
Sertoli cells, spermatogenesis, testis
INTRODUCTION
Development of new, safe, effective, and reversible male contraceptives has long been delayed in part because of the complexity of spermatogenesis. Although the morphological events throughout different phases of spermatogenesis are known, the participating molecules and the biochemical and molecular mechanisms regulating the process are not known. Thus, spermatogenesis per se offers many potential but unexplored targets of intervention. Prospective products are now based on the hormonal disruption of the hypothalamic-pituitary-testicular axis and may have long-term detrimental effects on the cardiovascular and prostatic health of men. These methods include elevating levels of androgens to inhibit spermatogenesis and inducing an immune response to GnRH, but they may be irreversible or ineffective [1]. We are currently focusing on other alternatives for the inhibition of spermatogenesis, i.e., the disruption of junctional complex sites between Sertoli cells and germ cells in the testis as a possible new target for antispermatogenic agents. Indazole-carboxylic compounds, which have profound inhibitory effects on spermatogenesis, are thought to disrupt the Sertoli-germ cell junctions by inducing premature release of germ cells into the lumen [2].
Lonidamine (1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) belongs to a group of indazole-carboxylic acids that were originally synthesized as anticancer drugs and were also found to be potent inhibitors of spermatogenesis [3]. Unlike other cancer drugs, lonidamine does not target rapidly dividing cells [4]. Rather, it becomes intimately associated with biological membranes causing conformational changes that result in the disruption of the respiratory process in cells that contain condensed mitochondria, such as cancer cells and certain germ cells [3, 5, 6]. In addition, the Sertoli cell in the testis is a major target of this drug; however, the respiratory process of this cell is unaffected. Studies indicate that lonidamine can induce vacuolation and retraction of the apical cytoplasm of the Sertoli cell [2, 7], presumably disrupting the Sertoli-germ cell junctions and inducing release of the immature spermatids into the lumen and resulting in infertility.
Lonidamine stimulates the expression of testin, a Sertoli cell secretory protein, approximately 100-fold in 24 h [8, 9]. Testin binds to the Sertoli cell membrane [10] and is localized at junctional complexes between Sertoli cells and germ cells in the testis [11, 12]. Testin was predominately localized on the concave side of elongating spermatids at the beginning of stage VIII tubules and became virtually undetectable in late stage VIII tubules after the release of the spermatids into the lumen [11]. These findings indicate that testin expression may be involved with the process of spermiation. Moreover, testin induction using many different methodologies, such as x-irradiation, glycerol, and busulfan [8, 10, 13, 14], has been correlated with depletion of germ cells from the testis.
Although lonidamine is currently used as an anticancer drug in Italy, its potential utilization as a male contraceptive had been abandoned because of two obstacles. First, the ratio in rats between the effective dose and its LD50 (
1:20–1:35) [15] advised caution for its use in healthy men as a contraceptive. Second, the cost of screening new analogues was very high because of the lack of a simple assay for their efficacy and to correlate structural modification, activity, and toxicity among the new analogues. However, indazole-carboxylic acids offer great hope for the development of new, safe, effective, and reversible male contraceptives because an increase in the antispermatogenic effect of these compounds is not correlated with an increase in toxicity [3]. Therefore, the only obstacle to the development of this compound as a male contraceptive is the screening of new analogues of lonidamine. By using testin expression as a measure of germ cell depletion, an in vivo assay was developed and used to screen several new analogues of lonidamine [9]. A transient increase in testin expression within 24 h of treatment is an indication of germ cell depletion, which would become visible within 7 days posttreatment when the testis is morphologically examined [8]. Analogues that cause only a brief induction of testin expression after a single dose deplete only the more advanced germ cells from the seminiferous epithelium, such as elongated spermatids, without affecting spermatocytes and spermatogonia. Therefore, these compounds are probably less toxic and their antifertility effects are more likely to be reversible (unpublished observations). Several analogues were screened, and 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF2364) was chosen over other derivatives as a possible candidate for a fertility trial because it mildly stimulates testin expression for only a brief period (72 h) after a single dose, and testin expression rapidly returns to normal [9]. The results of the comprehensive fertility study on AF2364, which included evaluation of the efficacy, reversibility, hormonal profile, effects on liver and kidney function, and morphological analysis of several organs, are presented here.
MATERIALS AND METHODS
Preparation of AF2364
Synthesis of AF2364 has been reported previously ([9] and Silvestrini B, Cheng CY, inventors; Aziende Chimiche Riunite Angelini Francesco [ACRAF], Rome, Italy, assignee. 3-Substituted 1-benzyl-1H-indazole derivatives as antifertility agents. US patent 6 001 865. December 14, 1999). The compound was suspended in 0.25% methylcellulose (w/v) and used at a concentration of 25 mg/ml.
Fertility and Efficacy Tests
A group of four male Sprague-Dawley rats were orally administered 50 mg/kg body weight of AF2364 once weekly for five consecutive weeks. Each of the four rats was separately housed with a virgin female for 1–4 days at the specified time points (29, 34, 48, 62, 76, 90, 104, 118, 126, 140, 157, and 197 days after initial dose of AF2364). Matings were confirmed by the presence of four to seven sperm plugs. Thereafter, the female was housed separately with free access to water and standard chow and a light:dark cycle of 12:12 h. The female rats were allowed to bring the pregnancy to term. All newborn pups and the uterine horns of the mother rats were examined for gross anatomical changes. In the control group for the fertility study (n = 6), male rats received only 0.25% methylcellulose (w/v) as a vehicle. All matings from this group gave birth to live healthy pups, and the fertility efficacy was set at 100%. All animals were housed at the Rockefeller University Laboratory Animal Research Center, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Rats were maintained in accordance with the applicable portion of the Animal Welfare Act and the guidelines in the Department of Health and Human Services publication Guide for the Care and Use of Laboratory Animals. The use of rats as described in this report has been approved by the Rockefeller University Animal Care and Use Committee with protocol numbers 97117 and 00111.
Morphological Analysis of Tissues
An additional 56 male Sprague-Dawley rats were orally administered the same AF2364 treatment regimen as stated above and were used for morphological and serum chemistry analysis. At 2, 6, 12, 20, 40, 50, 61, 69, 78, 100, 130, 163, 210, and 254 days after the initial dose of AF2364, groups of three or four rats were killed by excess inhalation of Metofane (Schering-Plough Animal Health Corporation, Union, NJ). Animals received a postmortem gross pathological evaluation. Tissues were carefully dissected to remove all excess fat. The organ weight for the brain comprised the cerebrum, cerebellum, and the medulla. Caput, corpus, and cauda were sectioned according to the method of Reid and Cleland [16] under a dissecting microscope. The caput and cauda were dissociated from the initial segment and vas deferens, respectively. Tissues were immediately removed, weighed, and placed in liquid nitrogen for storage at -80°C or fixed with 10% formalin. Fixed samples were embedded in paraffin, and 6-µm-thick sections were stained with hematoxylin and eosin by standard histology techniques and photographed using a BX40 microscope (Olympus, Tokyo, Japan) interfaced to a PM-30 Exposure Control Unit (Olympus). Blood was collected, and serum was prepared and stored at -20°C until further use. A second group of 25 animals was used as the control group, receiving only the vehicle (0.25% methylcellulose). At 0, 20, 40, 60, 90, 140, and 190 days after the initial dose of the vehicle, groups of three or four animals were killed and examined.
Hormone Measurements
Kits for rat FSH and rat LH RIAs were obtained from the National Hormone and Pituitary Program (Dr. A.F. Parlow, Harbor-UCLA Medical Center, Torrance, CA). Both FSH and LH were iodinated with 125I-Na using Iodogen (Pierce, Rockford, IL) with 5 µg of purified protein for each radioiodination [17]. Each standard or sample was run in duplicate or triplicate. All assays were analyzed by computer by fitting standard curves to a four-parameter logistic function and interpolating unknowns from the resultant curve using a program adapted from that of Robard et al. [18]. The minimal detectable dose for FSH and LH was 0.01 and 0.03 ng/assay tube, respectively. The 50% displacement was at 1.1 ng/assay tube for FSH and 0.85 ng/assay tube for LH. The intraassay and interassay coefficients of variation were 8.2% and 12%, respectively, for FSH and approximately 7.9% and 11.5%, respectively, for LH. Displacement curves generated by using crude rat serum samples were parallel to those obtained by purified rat FSH or rat LH provided in the corresponding RIA kits. [1,2,6,7-3H]Testosterone with a specific activity of 70–105 Ci/mmol was obtained from Amersham-Pharmacia Biotech (Arlington Heights, IL). Serum testosterone concentrations were then assayed in duplicate using a kit obtained from Sigma (St. Louis, MO). The minimal detectable dose for testosterone was 0.75 ng/assay tube, and the 50% displacement was approximately 94.5 ng/assay tube. The intra- and interassay coefficients of variation were approximately 7% and 12%, respectively.
Serum Chemistry
Serum microchemistry determinations were performed by Anatech Diagnostics (Farmingdale, NY). The following parameters were analyzed: glucose, blood urea nitrogen (BUN), creatinine, BUN:creatinine ratio, total protein, albumin, globulin, albumin:globulin (A:G) ratio, total bilirubin, alkaline phosphatase, liver enzymes serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT), calcium, phosphorus, cholesterol, sodium, potassium, and chloride. For each time point, only two of the control animals and three of the treated animals were subjected to serum chemistry analysis.
RESULTS
Effects of AF2364 on Fertility Efficacy and Litter Size
To assess the efficacy and reversibility of this new contraceptive drug, a group of four adult Sprague-Dawley male rats were treated with AF2364 (50 mg/kg body weight) once weekly for five consecutive weeks by gavage. At the first mating period, 29 days after the initial dose of the drug, the fertility efficacy dropped to zero (Fig. 1A). Thereafter, the fertility rate remained at 0% for up to 90 days. Recommencement of fertility was found at 104 days posttreatment, with 25% of the rats fostering a litter of normal size (Fig. 1, A and B). The fertility efficacy rose to 75% after 157 days, but at the last mating period at 197 days, one of the four rats remained infertile. Four to seven sperm plugs resulting from successful copulations were noted in the controls and throughout each mating session with treated animals, indicating that the number of copulations remained constant. Litter sizes of all fertile animals remained unchanged (Fig. 1B), indicating that when fertility is restored, litter size returns to normal. In addition, all newborn pups were examined for gross anatomical changes, and none were detected. The uterine horns of the mother rats were examined for increased fetal resorptions, and none were detected.
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Morphological Analysis of the Testis Following Treatment with AF2364
Morphological examination of the testis at specified intervals throughout the treatment period was performed by light microscopy. Figure 2A shows the morphology of a control animal, with tubules at several different stages of the spermatogenic cycle. Six days after the initial dose of AF2364, dramatic morphological changes had already occurred (Fig. 2B). Massive exfoliation of elongated spermatids was apparent in some tubules (small arrowheads). Large multinucleated cells were also present in a number of tubules (large arrow). In addition, prominent spaces detected within the germinal epithelium were consistent with structural disorganization (small arrow). The effectiveness of this drug was evident at 40 days after initial treatment (Fig. 2C). Ninety-eight percent of the tubules were completely devoid of the their germinal epithelium leaving behind only Sertoli cells and spermatogonia, and tubular diameter was decreased approximately 30% from the control (Fig. 2C). No apparent changes were seen within the interstitium. At 100 days, the tubules began regeneration of their germinal epithelium, and a few tubules showed signs of restructuring (Fig. 2D, large arrows). However, some tubules still exhibited a complete loss of the germinal epithelium (small arrow), and others were in the intermediate phase of reorganization (arrowhead). At 130 days posttreatment (Fig. 2E), signs of normal spermatogenic activity were observed, and other tubules were in the process of reorganizing. By 210 days (Fig. 2F), 95% of the tubules showed normal morphology, with different spermatogenic cycles.
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Morphological Analysis of the Caput, Corpus, and Cauda Epididymis Following Treatment with AF2364
Morphological examination of the epididymal compartments was performed on control animals during maximum infertility at 40 days and 210 days posttreatment (Fig. 3). Within all areas of the epididymis (caput, corpus, and cauda) at 40 days posttreatment, there was a slight reduction in the diameter of the lumen (Fig. 3, B, E, and H) in contrast to the controls (Fig. 3, A, D, and G). In addition, the tubule epithelium was slightly thicker and devoid of spermatozoa. A dramatic convolution of the epithelial lining was noted within the caudal segment of the epididymis (Fig. 3H) at 40 days posttreatment. However, by 210 days posttreatment, the tubules of the caput, corpus, and cauda were normal in size, exhibiting typical morphology and containing spermatozoa again (Fig. 3, C, F, and I) virtually indistinguishable from control rats.
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Morphological Analysis of the Prostate, Liver, Kidney, and Cerebrum Following Treatment with AF2364
Upon examination of the ventral prostate at 40 days and 210 days after treatment, there was some evidence of morphological changes within the columnar epithelial cells in this gland in comparison to the control. At 40 days after treatment, the epithelial cells surrounding the lumen were decreased in height and were less convoluted than the control (Fig. 4, B vs. A). The lumen of the prostate was enlarged and filled with fluid, indicating possible changes in the secretory processes of these cells. However, at the last morphological examination (210 days, Fig. 4C), the prostate had returned to its normal morphological state, with the epithelial lining becoming thicker and again more convoluted. Within the liver, kidney, and cerebrum, no morphological changes were detected by light microscopy at any time points (Fig. 4, D–L).
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Effect of AF2364 on Body and Organ Weights
Using an AF2364 treatment regimen of 50 mg/kg body weight once weekly for five consecutive weeks, body weight for the rats treated with the drug did not differ from that of the control rats (Fig. 5A). In contrast, testicular weight was reduced approximately 70% from Day 6 and until 210 days posttreatment (Fig. 5B). Although fertility increased to 25% by 104 days (Fig. 1A), the testicular weight did not show signs of recovery until 254 days posttreatment (Fig. 5B). Slight variations in the ventral prostate weight were observed in the treated group versus the control group at only a few time points (Fig. 5C). Caput, corpus, and cauda weights for the rats treated with AF2364 all dropped considerably compared with the control group (Fig. 6, A–C). Similar to the testicular weight, epididymal weights returned to the control level between 200 and 254 days posttreatment. Seminal vesicles, kidney, and brain (cerebrum, cerebellum, and medulla) were also examined for weight fluctuations. Only slight variations in organ weight were detected at various time points in these organs between the treated and control animals (Fig. 7, A–C).
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Effect of AF2364 on Serum FSH, LH, and Testosterone
Because of the dramatic decline in fertility and testicular and epididymal weight, the changes in the serum FSH, LH, and testosterone levels of the treated animals were evaluated by corresponding RIAs. Serum samples were taken on only the day animals were killed. No alterations of serum FSH were detected between the treated and control rats at most time points (Fig. 8A). However, at 40 and 163 days posttreatment there was a decline of serum LH (Fig. 8B). Serum testosterone concentration showed no notable changes compared with that of control rats until 163 days after the initial treatment of AF2364; a decline was noted at 163, 210, and 254 days posttreatment (Fig. 8C).
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Effect of AF2364 on Serum Chemistry
Serum chemistry was analyzed to monitor possible adverse effects of this drug on liver and kidney function. No changes were noted in alkaline phosphatase, SGOT, and SGPT (Table 1), indicating that liver function remained normal. There were no increases in glucose, BUN, creatinine, albumin, bilirubin, cholesterol, calcium, inorganic phosphorus, sodium, potassium, chloride, A:G ratio, and BUN:creatinine ratio, indicating that kidney function also remained unaltered (Table 1). The same parameters were analyzed in samples for all other time points, and no deviations from the control group were noted (data not shown).
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DISCUSSION
AF2364, a new analogue of lonidamine, is a potent antifertility agent with little or no apparent effects on organs other than those involved in the reproductive process. Rapid and dramatic depletion of the germinal epithelium and the eventual return of spermatogenesis are indicative of the potential of this drug as a male contraceptive. In addition, there were minimal changes in serum FSH, LH, and testosterone in rats treated with AF2364, indicating that the hypothalamic-pituitary-testicular axis was unaffected. Earlier studies using lonidamine and other analogues also indicate that the actions of this group of compounds are not mediated via an endocrine mechanism [19, 20].
AF2364 given at the treatment regimen of 50 mg/kg body weight once weekly for five consecutive weeks had overwhelming effects on the germinal epithelium by rapidly depleting elongated spermatids and round spermatids, with eventual depletion of most spermatocytes. Therefore, optimization of the treatment protocol and formulations will be necessary to achieve the desired outcome of premature release of elongated spermatids from the epithelium with little or no effect on other germ cell populations.
The decline of fertility to 0% after 29 days of the initial treatment with AF2364 does not necessarily indicate that the drug had adverse effects on the epididymal sperm reservoir. The epididymal capacity for sperm storage is approximately 30 days or beyond [21]; therefore, the sperm pool may have been resorbed instead of damaged. Sperm morphology was not examined during the treatment period; hence, we did not determine whether sperm became oligospermic or azoospermic during the 29 days posttreatment. However, utilizing a different treatment regimen of AF2364 (50 mg/kg body weight at 2-wk intervals for a total of six doses) resulted in a 50% drop in the fertility efficacy at 42 days after the initial dose, providing further evidence that AF2364 does not damage the epididymal sperm [9]. More importantly, the pups derived from matings of treated rats at 42 days showed no abnormalities, and there was no increased incidence of fetal resorptions [9]. Fertility efficacy using the current protocol was limited to 75% of the four animals at the last mating period, 197 days after the initial dose of AF2364; therefore, total recovery of fertility was not achieved. In some other dosage protocols in which two consecutive doses of 50 mg/kg body weight were administered weekly, animals were able to regain 100% of their fertility by 76 days after the initial dose with a brief infertile period of 2 wk by 35 days [9], illustrating that the inhibitory effects on spermatogenesis are completely reversible in some instances, depending on the dosage and frequency of administration.
Pronounced alterations in testicular histology with massive atrophy of 98% of the germinal epithelium at 40 days after the initial treatment was indicative of the infertility induced by AF2364. Testicular weight was dramatically reduced 6 days after treatment because of a massive loss of germ cells during the early stages of treatment and was indicative of the morphological changes that occurred. Spermatogenesis resumed by 100 days posttreatment, indicating that spermatogonia were obviously not affected. Similarly, a decrease in the epididymal weight reflected a loss of spermatozoa from the testis. Changes in the histology of the epididymis were possibly the consequence of the loss of spermatozoa from the tubular lumen, which can disrupt the absorption and secretory processes of the epithelial lining. The decrease in the size of the epididymal tubules may also be governed in part by a reduction of the volume and pressure normally exerted upon the tubules by luminal fluids and spermatozoa derived from the testis [22]. Morphological examination of the prostate at 40 days posttreatment indicated a decrease in the height of the epithelial lining and an increase in the size of the lumen. The epithelial lining had also become less convoluted, possibly because of an increase in the prostatic secretions. However, further analyses are needed to confirm this possibility and to determine the overall effect of AF2364 on the prostate. Kidney, liver, and cerebrum morphology at the light microscopic level were unchanged during the experimental period, indicating that although AF2364 can be incorporated into the plasma membrane of certain cell types, these tissue types are unaffected. In addition, serum chemistry analysis indicated that liver and kidney functions were not notably different from those of the control group, further emphasizing the value of AF2364 as a male contraceptive. In light of the specific action of AF2364 on the testis, this compound and other indazole-carboxylic acids are worthy of continued investigation as male contraceptives.
AF2364 is a potentially reversible antispermatogenic compound if given at the correct dosage and frequency. It can be delivered orally without any detrimental effects to liver or kidney function, does not alter the levels of FSH, and causes only minimal changes in LH and testosterone. In view of the potential clinical application of this compound, toxicity and mutagenicity studies are currently being investigated.
NOTE ADDED IN PROOF
A recently completed acute toxicity study with Irwin dose range (100 and 1000 mg/kg body weight, i.p.) in mice has shown that AF2364 does not affect any of the neurologic or autonomic parameters. In addition, bacterial mutation assays performed in Salmonella typhimurium (strains TA98 and TA100) and Escherichia coli (strain WP2 uvrA) have shown that AF2364 does not induce reverse mutation. Both sets of studies were performed by licensed toxicologists according to the U.S. FDA guidelines. These results further illustrate the potential of this new chemical entity for male contraception for human use. Work is in progress to complete the toxicity studies in anticipation of Phase 1 Clinical Trials.
ACKNOWLEDGMENTS
We thank Dr. Meng Yun Mo for his help in performing the fertility studies and RIAs. We also thank Dr. A.F. Parlow from the National Hormone and Pituitary Program for the RIA kits for rat FSH and rat LH and ovine FSH (biological grade).
FOOTNOTES
First decision: 11 September 2000.
1 Supported in part by grants from the CONRAD Program (CIG-96-05-A) and the Noopolis Foundation. 
2 Correspondence: C. Yan Cheng, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733; yan{at}popcbr.rockefeller.edu
Accepted: January 3, 2001.
Received: August 8, 2000.
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 C. Y. Cheng and D. D. Mruk Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development Physiol Rev, October 1, 2002; 82(4): 825 - 874. [Abstract] [Full Text] [PDF]
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S. Chung, S. P. Wang, L. Pan, G. Mitchell, J. Trasler, and L. Hermo Infertility and Testicular Defects in Hormone-Sensitive Lipase-Deficient Mice Endocrinology, October 1, 2001; 142(10): 4272 - 4281. [Abstract] [Full Text] [PDF]
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