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Two New Male Contraceptives Exert Their Effects by Depleting Germ Cells Prematurely from the Testis¹

^a Population Council, The Rockefeller University, 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 three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3–7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4–14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

gametogenesis, Sertoli cells, sperm, spermatogenesis, testis

INTRODUCTION

During spermatogenesis, pre- and leptotene spermatocytes differentiated from type B spermatogonia residing near the basal lamina must translocate across the blood-testis barrier, which is formed by the inter-Sertoli tight junctions at the basal compartment of the seminiferous epithelium, entering the adluminal compartment for further development [1, 2]. This timely movement of developing germ cells across the epithelium coupled with the continual attachment of germ cells onto Sertoli cells throughout their development are conceivably essential for the completion of spermatogenesis. The cellular events of germ cell movement and germ cell attachment are also potential targets for fertility control. For instance, the disruption of the attachment of immature germ cells onto the epithelium prior to their maturation can induce infertility because spermatozoa released into the tubular lumen prematurely will lack the ability to fertilize the egg. Likewise, an increase or a decline in the pace of cell movement in the seminiferous epithelium will also lead to infertility because germ cells can either be detached from the epithelium prematurely or become aged and be removed by Sertoli cells via phagocytosis [1, 2].

Lonidamine (1-[2,4-dichlorobenzyl]-indazole-3-carboxylic acid, a derivative of 1-substituted 1H-indazole, Fig. 1) is an anticancer drug [3, 4]. During the course of examining its mechanism of action and toxicity, it was found that lonidamine did not target rapidly dividing cells; rather, it became associated with biological membranes causing conformational changes that result in the disruption of the respiratory process in cells that contained condensed mitochondria, such as tumor cells sensitized by X-irradiation and certain types of germ cells, such as spermatids and spermatocytes [3, 4]. Lonidamine was also shown to induce disruption of actin microfilaments and stress fibers in A431 (an epithelial squamous carcinoma cell line) and M14 cells (melanoma cells) in vitro [4]. It also caused vacuolation and retraction of the apical cytoplasm in Sertoli cells thereby inducing release of immature spermatids into the tubular lumen when administered in adult rats in vivo [5]. However, the antispermatogenic effects of lonidamine, if administered at high doses (at 400 mg/kg body weight [b.w.] and over), are irreversible and toxic. Needless to say, this compound, if properly modified to eliminate side-effects, could become a novel male contraceptive. Unfortunately, all efforts to develop new derivatives or analogues based on the core structure of lonidamine for male contraception ceased because of the high costs of screening by conventional methods [3] and the hesitation of the pharmaceutical industry to develop male contraceptives. In contrast to other medicines that are developed to curing life-threatening conditions, an optimal male contraceptive should be safe even when administered at high doses over a relatively long period of time.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Synthesis of new derivatives of 1-indazole-3-carboxylic acid

Based on a series of studies to understand the biology of Sertoli-germ cell interactions, our laboratory has shown that the expression of testin [6–8], a Sertoli cell glycoprotein of 37–40 kDa [6–8], was induced at the disruption of Sertoli-germ cell adherens junctions (AJ) [9–11]. For instance, a surge in testin expression is detected when germ cells are depleted from the testis in vivo following X-irradiation [12] and treatment with busulfan without impairing Leydig cell function [13]. Removal of germ cells from Sertoli-germ cell cocultures by hypotonic treatment that disrupts AJs between Sertoli and germ cells in vitro also induces a surge in Sertoli cell testin expression [10]. Germ cells, however, do not appear to release any soluble factors that can affect Sertoli cell testin expression in Sertoli-germ cell cocultures [11]. However, a disruption of the inter-Sertoli tight junctions by glycerol [11] or CdCl₂ [14] in vivo at the time when germ cells were not depleted from the epithelium had no apparent effect on the testicular testin steady-state mRNA level. These analyses reveal that testin is a sensitive marker to monitor the integrity of Sertoli-germ cell adhering or gap communicating junctions but not the inter-Sertoli tight junction. Based on these unusual features of testin, it was used to screen different newly synthesized analogues of lonidamine for their effects in disrupting Sertoli-germ cell junctions thereby inducing premature release of germ cells into the tubular lumen by monitoring their effects on the testicular testin expression. We have identified two new compounds having potent antispermatogenic effects without any noticeable toxicity as tested in preliminary laboratory studies with different formulations that appear to be promising candidate compounds for male contraception.

MATERIALS AND METHODS

Chemical Syntheses

A total of 27 new analogues of lonidamine (see Fig. 2) were synthesized by substituting different R₁ (Fig. 2B), R₂ (Fig. 2C), or R₃ (Fig. 2D) to the lonidamine core structure according to one of the two previously described methods [15–17]. In the first approach, direct alkylation of the corresponding 1H-indazole-3-carboxylic acids (Fig. 1a) with a benzyl halide was performed in aqueous NaOH (Fig. 1). When this method failed, benzylation of the ethyl ester (Fig. 1c) was performed in ethanol or dioxan solution followed by hydrolysis of the resulting ester (Fig. 1d) with aqueous ethanolic KOH (Fig. 1). The 2-substituted benzyl-2H-indazole-3-carboxylic acids were prepared by benzylation of the ester (Fig. 1c), and subsequent hydrolysis was performed as described [18]. While the reaction in ethanol in the presence of sodium ethoxide (C₂H₅ONa) is known to give very low yields of 2-benzyl-2H-indazole-3-carboxylate and very good yields of the isomeric 1-benzyl product (b), direct heating of the ester (c) with benzyl chloride without solvent at 120°C had been shown in our laboratory to give about equal quantities of the two isomers. The isomers were then separated by column chromatography or by simple crystallization.

View larger version (26K): [in this window] [in a new window]   FIG. 2. A–D) Synthesis of new analogues of 1H-indazole-3-carboxylic acid that were used to screen for their antispermatogenic effects in depleting germ cells from the seminiferous epithelium

Our earlier structure-antispermatogenic activity correlation studies [15, 16] have shown that in compounds having the general formula (Fig. 2A) where R₁ = CH₂-Me, the presence of a benzyl group is essential for antispermatogenic activity. The presence of one or more halogen or methyl in the benzyl ring is also necessary. If halogen is the only substitute, its position in the benzyl ring bears relatively little influence on activity. The ortho position, however, is essential in the case of a single methyl group. The highest antispermatogenic activity was observed with the two substitutes in the ortho and para positions, regardless of whether both are halogens or one is a halogen and the other is a methyl group. In addition to the acids, other compounds where R₂ is a residue of ethylene glycol or glycerol are active; several other esters were found to be ineffective. Mild activity was also found in unsubstituted amines and hydrazides, whereas the substitution of the carboxylic group with a tetrazole ring or a sulfonic group results in a loss of activity. An alcohol group in place of the carboxyl group would yield an inactive compound, while aldehyde had a similar effect as the acidic group; neither oxime nor thiosemicarbazone was active. The analogue (or R₁ = Me-2,4-Cl₂, R₂ = COOH or CO-NH-NH₂ and R₃ = H) of lonidamine was also active, while a higher homologue (R₁ = CH₂CHMe₂ or R₁ = CH₂CH(Me)-Me-2,4-Cl₂, R₂ = COOH, R₃ = H) was not.

Because two new analogues (out of the 27 chemicals that were synthesized and screened) selected by the in vivo bioassay were shown to have potent antispermatogenic effects (see below), their syntheses were described somewhat in detail herein.

Synthesis of AF-2785 (1-[2,4-dichlorobenzyl]-indazole-3-acrylic acid) Briefly, for the synthesis of AF-2785, a mixture of melted CH₃COOK (3 g, 0.03 mol), 1(2,4-dichlorobenzyl)-indazole-3-carboxaldehyde (14.2 g, 0.047 mol), and (CH₃CO)₂O (7.5 ml, 0.08 mol) was heated at 160°C for 6 h. The reaction mixture was then cooled at 50°C and partitioned between water and ethyl acetate. The residue of the organic phase was recrystallized first from acetic acid and then twice from isopropyl alcohol to give the title compound (8.5 g), melting point (m.p.) 198–199°C.

Synthesis of AF-2364 (1-[2,4-dichlorobenzyl]-indazole-3-carbohydrazide) For the synthesis of AF-2364, a solution of 85% hydrazine hydrate (100 ml) was added slowly to a solution of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid methyl ester (12.5 g) in acetic acid (25 ml), at 100°C while stirring. After 1 h at reflux temperature, the reaction mixture was cooled and filtered. The reaction product was washed with water (2 x 150 ml) and recrystallized from ethyl alcohol to give the title compound (11.5 g), m.p. 161–162°C.

Animals

Adult Sprague-Dawley rats weighing between 250 and 300 g were obtained from Charles Rivers Laboratories (Kingston, MA). Rats in groups of four to six at specified time points were killed by CO₂ asphyxiation. Rats were kept and cared for in the Laboratory Animal Research Center (LARC) at the Rockefeller University. Two adults rats were housed per cage. All animals had free access to standard rat chow and water ad libitum under controlled temperature (22°C) and constant light-dark cycles of 12 h:12 h. The Rockefeller University LARC has been fully accredited by the American Association for Accreditation of Laboratory Animal Care. These animals were maintained in accordance with the applicable portions 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 animals as described in this report had been approved by the Rockefeller University Animal Care and Use Committee (ACUC) with protocol numbers 91353-R2, 93140, 97101, and 00111.

In Vivo Assay to Screen New Analogues of Lonidamine

A bioassay was developed to screen the antispermatogenic activity of a target compound based on its ability to induce a transient but significant increase in testin expression in the testis; this in turn led to a massive depletion of germ cells from the seminiferous epithelium [9–11]. This bioassay was used to identify analogues of lonidamine that can selectively induce depletion of advanced postmeiotic germ cells such as round and elongated spermatids from the epithelium in vivo. The antispermatogenic effect of a target compound was subsequently verified by routine histological analysis. Adult rats (about 250 g b.w., n = 4 rats per time point) were fed with a single dose of an analogue of lonidamine (25 or 50 mg/kg b.w.) (see Fig. 2) suspended in 0.25% methylcellulose (w/v, in sterile water) to a concentration of 20 mg/ml. At 1-, 3-, 5-, 7-, and 14-days post-treatment, rats were killed, testes were removed for RNA extraction and histological analysis, and the steady-state testin mRNA level was quantified by Northern blotting using a 289-base pair (bp) testin cDNA probe as described [9–11]. A transient increase in testin expression within 24 h of treatment is an indication of germ cell depletion from the seminiferous epithelium that would become visible within 7 days post-treatment when the testis was examined by standard histology procedures [9, 19–22]. If the testin steady-state mRNA level remains elevated following treatment, rather than a transient induction of 1–6 days, this would indicate massive damage in the testis when examined histologically and the prognosis of recovery would be poor.

Immunohistochemistry

The antispermatogenic effects of different analogues of lonidamine were verified by immunohistochemistry techniques in addition to the bioassay using testin as a marker. Immunohistochemistry was performed essentially as previously described using frozen sections to preserve the testin antigenicity [19–20, 22]. Briefly, organs (testes, epididymides, kidney, liver, and brain) were removed and immediately frozen in liquid nitrogen. The unfixed tissues were sectioned to 5 µm thickness in a cryostat microtome at -22°C. Sections were then placed on poly-L-lysine-coated slides, air-dried, and fixed in modified Bouin solution containing 4% formaldehyde (v/v) in saturated picric acid for 3 min at room temperature. The endogenous peroxidase activity was blocked by treatment with 0.03% H₂O₂ (v/v). Nonspecific sites were blocked by treatment with 10% BSA in PBS (w/v) for 10 min, and the sections were dried at 4°C. Immunostaining with streptavidin-biotin complex was performed according to procedures specified by the manufacturer (Zymed Laboratories, Inc., South San Francisco, CA). Briefly, sections were incubated with the rabbit anti-testin antibody [6] at a dilution of 1:300 at 37°C for 1 h, washed three times in PBS (5 min each), and incubated with streptavidin-peroxidase complex for 10 min. Sections were washed thoroughly with PBS and developed in an AEC-H₂O staining system (Zymed) for 3–15 min. Slides were then washed in water for at least 15 min to stop the reaction, counterstained with Mayer hematoxylin, mounted, and examined microscopically. Controls were performed by substituting the primary antibody in adjacent sections with 1) antibody preabsorbed with purified testin, 2) substituting the primary antibody with preimmune serum, 3) substituting the primary antibody with normal rabbit serum, 4) substituting the second antibody with PBS, and 5) substituting the primary antibody with anti-testin antibody preabsorbed by protein A-Sepharose (antiserum:resin, 0.1 ml:10 ml). For each treatment group at a specified time point, about 100 cross-sections were examined microscopically using an Olympus BX40 microscope interfaced to an Olympus PM30 exposure control unit. All micrographs were digitally acquired and stored in a Compaq SP700 Workstation (Compaq Computer Corp., Houston, TX) and processed using Adobe Photoshop (Version 6.0) and Adobe PageMaker (Version 6.5) (Adobe Systems, Inc., San Jose, CA). Micrographs shown in this report are representations of these analyses.

Fertility Tests

For AF-2364, five different treatment regimens were used to assess the antifertility effects, efficacy, and reversibility as follows, that correspond to A–E in Figure 5. Adult Sprague-Dawley rats (250–280 g b.w., n = 4–6) were used for each treatment regimen. For the control, rats (n = 6) received vehicle only (0.25% methylcellulose, 0.75 ml by gavage, one dose). For regimen 1, rats (n = 4) received six doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every 2 wk. For regimen 2, rats (n = 4) received five doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 wk^-1. For regimen 3, rats (n = 4) received three doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every 2 wk. For regimen 4, rats (n = 5) received two doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 wk^-1. For regimen 5, rats (n = 5) received four doses of AF-2364 with 25 mg kg b.w.^-1 wk^-1. For AF-2785, four different regimens were used in adult rats (n = 4–6) as follows, that correspond to A–D in Figure 8. For the control, rats (n = 6) received vehicle only (0.25% methylcellulose, 0.75 ml by gavage, one dose). For regimen 1, rats (n = 4) received eight doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 wk. For regimen 2, rats (n = 6) received five doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 days. For regimen 3, rats (n = 5) received 10 doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 days. For regimen 4, rats (n = 4) received eight doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 day^-1. Following treatment of rats with the first dose of a either AF-2364 or AF-2785, each rat (4–6 rats per treatment group) was mated at specified time point (see Figs. 5 and 8) separately with a virgin female (about 270–300 g b.w.). All animals were kept and housed at the Rockefeller University Laboratory Animal Research Center. Mating was confirmed by the presence of sperm plugs. Thereafter, female rats were housed separately with free access to water and standard chow with a light:dark cycle of 12 h:12 h, to allow gestation to go to completion in 21 days. In control groups (n = 6), all female rats mated with the corresponding male and gave birth to pups, and the fertility efficacy was arbitrarily set at 100%. All newborn pups and the uterine horns of the mother rats at the end of the pregnancy were also examined for gross anatomical changes. No changes were detected with both compounds when the different treatment regimens outlined above were used.

View larger version (44K): [in this window] [in a new window]   FIG. 5. A–E) A study to assess the efficacy and antifertility effect of AF-2364 using five different treatment regimens. Each data point is a mean of four to six rats per treatment regimen

View larger version (39K): [in this window] [in a new window]   FIG. 8. A–D) A study to assess the efficacy and antifertility effect of AF-2785 using four different treatment regimens as described in Materials and Methods. Each data point is the mean of four to six rats for each treatment regimen

Radioimmunoassays for FSH, LH, and Testosterone

The concentrations of rat FSH and rat LH in serum samples were quantified by RIA using corresponding RIA kits obtained from Dr. A.F. Parlow at the National Hormone and Pituitary Program, National Institutes of Health (NIH). These assays have been validated as previously described from this laboratory [23, 24]. Iodinations of both rat FSH and rat LH were performed using Iodogen (1,3,4,6-tetrachloro-3,6-diphenylglycoluril) (Pierce, Rockford, IL) [25] with [¹²⁵I]Na from Amersham-Pharmacia Biotech (Arlington Heights, IL). Standard curves for both RIAs were calibrated using the corresponding purified FSH and LH from the NIH kits. These curves were parallel to curves generated by crude rat serum. Each sample was run in triplicate, and all samples within a given experiment were assayed simultaneously to eliminate interassay variations. All RIA data were computer analyzed by fitting standard curves to a four-parameter logistic function and interpolating unknowns from the resultant curve as previously described [26]. The minimal detectable dose for FSH and LH was 0.01 ng and 0.02 ng/assay tube, respectively; and 50% displacement was at 1.1 ng/assay for FSH and 0.9 ng/assay tube for LH. The inter- and intraassay coefficient of variations for FSH and LH RIAs were 12, 8 and 14, 9, respectively. [1,2,6,7-³H]Testosterone (specific activity 70–105 Ci/mmol) was obtained from Amersham-Pharmacia Biotech, and the serum testosterone level was quantified by using a kit from Sigma (St. Louis, MO). The minimal detectable dose for testosterone was 5 ng/assay tube, and 50% displacement was at 90 ng/assay tube.

Serum Microchemistry

Serum microchemistry to assess the kidney and liver function and to quantify other serum parameters was performed by Anatech Diagnostics (Farmingdale, NY). These include the concentration and/or activity of the following parameters: glucose, BUN (blood urea nitrogen), creatinine, total protein, albumin, globulin, total bilirubin, alkaline phosphatase, liver enzymes (SGOT, serum glutamic-oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase), cholesterol, sodium, and potassium.

General Methods

Routine histology was performed by licensed pathologists at the Rockefeller University LARC using standard histology techniques. Briefly, testes were removed and the seminiferous tubules were fixed in 10% formalin. Fixed samples were embedded in paraffin, and 6-µm-thick sections were stained with hematoxylin and eosin (Zymed Laboratories, South San Francisco, CA) by standard histology techniques. Statistical analyses were performed by ANOVA with Tukey HSD (honestly significant difference) test using the GB-STAT Statistical Analysis Software Package (Version 7.0) from Dynamic Microsystems, Inc. (Silver Spring, MD). Using Tukey test for ANOVA, results of individual samples were compared to controls and to samples subjected to the same treatment within the same group; thus, this test makes use of a single value against which all differences are compared.

RESULTS

Synthesis of New Analogues of Lonidamine

Three different groups of analogues, based on the core structure of lonidamine (Fig. 2A) with different substitution groups in R₁ (Fig. 2B), R₂ (Fig. 2C), and R₃ (Fig. 2D), were synthesized (a total of 27 chemical entities) as described in Materials and Methods. Prior to their use in the in vivo bioassay screening for antispermatogenic activity, each chemical entity was subjected to the following analytical tests to ensure their purity and confirm their structural formulae. These include melting point (m.p.) determination, elemental analysis, infrared spectra (KBr), NMR spectra (¹H and ¹³C), ultraviolet spectra, mass spectra, and HPLC as described [15].

Elemental Analysis of AF-2364 and AF-2785

The HPLC-purified AF-2364 (Table 1) and AF-2785 (Table 2) were subjected to elemental analysis and their results are shown in Tables 1 and 2. These results illustrate that both chemical entities were devoid of impurities because the percentage of C, H, and N found in each compound was virtually identical to the calculated percentage of C, H, and N (Tables 1 and 2).

Effects of the Lonidamine Analogues on Testicular Testin Induction—In Vivo Bioassay

For the in vivo assay, each compound was suspended in 0.25% methylcellulose in sterile water at a concentration of about 20 mg/ml and was administered to adult rats by gavage at doses ranging between 25 and 50 mg/kg b.w. Figure 3 shows the results of a typical bioassay where groups (20 rats including controls) of adult rats (about 300 g b.w., n = 4 for each time point) were fed with a single dose of the analogue (Fig. 3) at 50 mg/kg b.w., testes were removed at 1, 2, 3, and 14 days thereafter for RNA extraction, and the testin steady-state mRNA level quantified by northerns. Among four of the compounds that were screened and shown in Figure 3, A and B, lonidamine (positive control, Fig. 3C) induced a transient but drastic increase in testin expression. Likewise, AF-2364 and AF-2785 (Fig. 3C) that were analogues of lonidamine shown in Figure 2C where the R₂ was -CO-NH-NH₂ and -CH = CH-COOH, respectively, also induced a transient surge in testin expression that became clearly visible by as early as 1–2 days post-treatment versus control rats (Fig. 3, A and B) prior to the detachment of germ cells from the epithelium. Drugs D and J are also analogues of lonidamine where the R₂ group was replaced with -O-CH₂COOH and -O-CH(COOH)₂, respectively (see Fig. 2C); however, these two analogues (Fig. 3, A and B) along with 23 other analogues (data not shown) described in Figure 2, B through D had no apparent effects in inducing testin expression following a single dose of 50 mg/kg b.w.

View larger version (53K): [in this window] [in a new window]   FIG. 3. A–C) Bioassay using testin as a marker to screen new analogues of lonidamine. Northern blots showing the testin steady-state mRNA level in the rat testis after rats were fed (at Time 0) with a single dose of lonidamine or its analogues (AF-2364, AF-2785, drugs D and J) at 1-, 2-, 3-, and 14-day versus control rats at Day 0. Drugs D and J are analogues of lonidamine where the R2 group was replaced with -O-CH2COOH and -O-CH(COOH)2, respectively (see Fig. 2C). Control rats were killed at 1, 3, and 14 days after rats were fed with vehicle alone (0.25% methylcellulose). A) Lonidamine induced a more potent increase in testin expression than AF-2364 and AF-2785. Both new analogues induced only a mere transient increase in testin expression. Each lane had 10 µg of total RNA. B) This is the same blot of A but stained with ethidium bromide. Other analogues of lonidamine as shown in Figure 2 that had no apparent effects are not shown. C) Structural formulae of 1H-indazole-3-carboxylic acid, AF-2364, AF-2785, and lonidamine

Antispermatogenic Effects Assessed by Immunohistochemical Analysis

Immunohistochemical and morphological analyses of the testes were performed on these rats as described in Materials and Methods. Cross sections of the testis from control rats are shown in Figure 4, A through C. Figure 4, D and E are cross sections of typical seminiferous tubules from rats treated with a single oral dose of lonidamine (positive control) (50 mg/kg b.w.) by Day 14. Rats treated with AF-2364 (Fig. 4, H and I) and AF-2785 (Fig. 4, F and G) by Day 14 were found to possess seminiferous tubules that were virtually devoid of elongated and round spermatids, and a reduced number of spermatocytes when compared to untreated rats (Fig. 4, F–I, versus Fig. 4, A–C) and were similar to rats treated with lonidamine (Fig. 4, D and E). Also, an accumulation of immunoreactive testin protein was detected in the tubular lumen in rats treated with these compounds versus controls (Fig. 4, D–I, versus Fig. 4, A–C).

View larger version (132K): [in this window] [in a new window]   FIG. 4. A–I) A study to assess the antispermatogenic effects of AF-2364 and AF-2785 compared to lonidamine after a single oral dose of 50 mg/kg b.w. Cross sections of testes of control rats (A–C) received vehicle only. A, B) Control testes at stage V of the spermatogenic cycle stained with preimmune and anti-testin antibody, respectively. Immunoreactive testin appeared as a reddish-brown precipitate (arrowheads). Some immunoreactive testin was also found between the heads of migrating elongated spermatids and Sertoli cells (arrows). C) This is the cross section of a seminiferous tubule from control rat testes at stage VII of the cycle. Testin was found at the basal compartment at the site of Sertoli-germ cell junctions. D, E) Representations of tubules from rats 14 days after receiving a single oral dose of lonidamine at 50 mg/kg b.w. It is noted that there is an accumulation of testin in the tubular lumen (arrows) [10, 11]. F, G) Stage V tubules from rats 14 days post-treatment with AF-2785. Elongated spermatids were found in the tubular lumen in these stage V tubules. Arrowhead with an asterisk indicates the association of testin with the head of elongated spermatids. H, I) Tubules from rats 14 days after receiving an oral dose of AF-2364 at 50 mg/kg b.w. Virtually all elongated and round spermatids were depleted from the tubules with an accumulation of testin in the tubular lumen (arrows). P, Pachytene spermatocyte; rs, round spermatid; es, elongated spermatid. Bar = 25 µm

Effects of AF-2364 on Fertility, Its Efficacy, Morphology, Liver and Kidney Function

Antifertility effects of AF-2364 To assess the efficacy, antifertility effects, and reversibility of these compounds, adult rats (about 250–280 g b.w.) (n = 4–5) received five different formulations of AF-2364 (suspended in 0.25% methylcellulose and administered by gavage) as shown in (Fig. 5). Control rats (n = 6) receiving vehicle only (0.25% methylcellulose, w/v) displayed a fertility rate of 100% with an average litter size of 14.8 ± 2.3. Fertility tests were performed as described in Materials and Methods in which each male at a specified time point was caged separately with a virgin female rat for 1–3 days. Mating was confirmed by the presence of four to eight sperm plugs. AF-2364 could not suppress fertility by 100% during the first 2–3 wk after the first dose was administered (Fig. 5, A–E), possibly as a result of the epididymal sperm reserve because this compound neither induced any apparent changes in the epididymis when examined microscopically nor killed the epididymal sperm when assessed by erythrosine red-dye exclusion test (results not shown). Results of the treatment regimens are as follows: For regimen 1 (six doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every 2 wk), all rats became infertile between Days 56 and 140; fertility resumed by Day 154, but only 25% of the treated animals resumed fertility. For regimen 2 (five doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every week), rats became infertile between Days 29 and 90, and about 75% of the rat became fertile with normal litter size by Day 157. For regimen 3 (three doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every 2 wk), rats became infertile between Days 35 and 49, and about 75% of the rats became fertile with normal litter size by Day 84. For regimen 4 (two doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every week), rats became infertile on Day 35 for about 3 wk, and 100% of the treated animals became fertile with normal litter size by Day 76. For regimen 5 (four doses of AF-2364 with 25 mg kg b.w.^-1 dose^-1 every week), only 20% of the rats were fertile between Days 36 and 63, and 100% of the treated animals became fertile with normal litter size by Day 84.

The results shown in Figure 5 illustrate that AF-2364 is a potent male contraceptive. Its efficacy, however, depends on the dosing and the frequency of its administration. Based on the above studies, it is apparent that at least two consecutive doses that must be administered at least 1 wk apart were needed to induce complete infertility in the rat. Interestingly, the length of infertility could be manipulated by changing the dosing of AF-2364.

Effects of AF-2364 on testicular morphology When the testes of the treated animals were examined histologically using the formulation shown in Figure 5B (treatment regimen: five consecutive doses of AF-2364 with 50 mg kg b.w.^-1 dose^-1 every week), it was found that germ cells began to deplete from the seminiferous epithelium as early as 6 days after the first dose of AF-2364 (Fig. 6, B versus A, where Fig. 6A is the control rat testis). Greater than 80% of the tubules were devoid of germ cells by 28 days after the first oral dose of AF-2364 (Fig. 6C). The tubules were virtually devoid of germ cells by 40 days after treatment (Fig. 6D), and this morphological change persisted until 79 days (Fig. 6E), coinciding with the loss of fertility (Fig. 5B). When the testes were examined on 128 days, germ cells began to repopulate the seminiferous epithelium (Fig. 6F), and by Days 211–254, virtually 100% of the tubules appeared indistinguishable from control rats (Fig. 6, G and H versus A), which is consistent with the fertility data shown in Figure 5B illustrating the reversibility of this treatment.

View larger version (164K): [in this window] [in a new window]   FIG. 6. A–H) Changes in the morphology of the testes in rats treated with five doses of AF-2364 at 50 mg kg b.w.-1 dose-1 wk-1. Testes were removed from control rats (250–300 g b.w.) (A), and from rats at 6 (B), 28 (C), 40 (D), 79 (E), 128 (F), 211 (G), and 254 days (H) after the first dose of AF-2364. The disappearance of germ cells from the tubules and their reappearance are consistent with results of the fertility test shown in Figure 5B. Bar = 50 µm for A, E, and G; bar = 100 µm for B–D, F, and H

Effects of AF-2364 on serum FSH, LH, testosterone, and other serum parameters When the serum testosterone (Fig. 7B), FSH (Fig. 7E), and LH (Fig. 7H) levels from the AF-2364-treated rats using the treatment regimen shown in Figures 5B and 6 were quantified and compared to control rats (Fig. 7, A, D, and G), no significant changes were detected throughout the entire treatment period versus controls (Fig. 7). These data thus demonstrate that the hypothalamus-pituitary-testicular axis was not impaired. Moreover, serum microchemistry analysis revealed that the serum levels of SGOT, SGPT, alkaline phosphatase, BUN, creatinine, albumin, -globulins, sodium, and potassium were not altered when control rats were compared to treated animals using the treatment regimen outlined in Figure 5B, illustrating that liver and kidney function was not affected by AF-2364 (Table 3).

View larger version (42K): [in this window] [in a new window]   FIG. 7. A–I) Effects of AF-2364 and AF-2785 on the serum concentrations of testosterone (A–C), FSH (D–F), and LH (G–I) in rats receiving the treatment regimen shown in Figures 5B and 8D, respectively, and compared to control rats treated with vehicle (0.25% methylcellulose). For AF-2364, rats (250–300 g b.w., n = 6) received five consecutive doses of drug at 50 mg kg b.w.-1 dose-1 weekly). For AF-2785, rats (n = 6) received eight consecutive doses of drug at 50 mg kg b.w.-1 dose-1 daily

Effects of AF-2785 on Fertility of Rats, Its Efficacy, Serum FSH, LH, and Testosterone Levels, and Other Parameters

Antifertility effects and efficacy of AF-2785 Using adult rats, we have tested four different treatment regimens of AF-2785 (n = 4–6 for each treatment regimen) and their results (Fig. 8) are as follows: For regimen 1 (eight doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 wk), only 25% of the treated rats became infertile between Days 56 and 98, but all rats recovered from the treatment and became fertile by Day 112. For regimen 2 (five doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 days), only 16% of the treated rats were fertile by Day 42, and all rats recovered from the treatment and became fertile by Day 84. For regimen 3 (10 doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every 2 days), only 20% of the treated rats were fertile by Day 55, and all rats became fertile by Day 110. For regimen 4 (eight doses of AF-2785 with 50 mg kg b.w.^-1 dose^-1 every day), all rats became infertile by Day 20 and the infertility persisted until Day 57. By Day 73, 75% of the treated rats became fertile, and all rats became fertile by Day 97.

These results clearly demonstrate that AF-2785 is also a potent antispermatogenic compound. However, this chemical entity needs to be administered more frequently than AF-2364, preferably on a daily (or every 2-day) basis, in order to unleash its efficacy. More interestingly, treated rats recovered more rapidly after treatment with AF-2785 versus AF-2364 (Fig. 8 versus Fig. 5). As such, its antifertility effects can be manipulated by changing the dosing and frequency of administration.

Effects on testis morphology, serum hormone levels, and other serum parameters Similar to AF-2364 as shown in Figure 6, AF-2785 induced germ cell depletion, virtually all elongated and round spermatids, from the seminiferous epithelium beginning on Day 15 when a treatment regimen shown in Figure 8D was used when testes were morphologically examined by routine histology (data not shown). Testes were indistinguishable from control rats by Day 100 (data not shown) when fertility was restored in the AF-2785-treated rats (see Fig. 8D), indicating that germ cells repopulated more rapidly in AF-2785-treated rats than AF-2364-treated rats, consistent with data shown in Figure 8 (Fig. 8 versus Fig. 5). Moreover, AF-2785 did not alter the serum levels of testosterone, FSH, and LH in rats receiving the treatment regimen shown in Figure 8D when compared to control rats (Fig. 7, C, F, and I, versus Fig. 7, A, D, and G), illustrating that this compound, similar to AF-2364, also did not interfere with the hypothalamus-pituitary-testicular hormonal axis. Also, serum microchemistry analysis revealed that neither liver nor kidney function was affected by AF-2785 (Table 3).

Effects of AF-2364 and AF-2785 on Testicular Weights

The effects of both analogues of lonidamine on testicular weight were also examined using the treatment regimens such as the one shown in Figures 5B and 8D for AF-2364 (n = 2) and AF-2785 (n = 2), respectively, and compared to control rats (n = 2) (Fig. 9). Only two rats were killed at each time point to obtain testes for weighing. It was noted that AF-2364 induced a more rapid decline in testicular weight versus AF-2785 (Fig. 9), consistent with results of fertility tests shown in Figures 5 and 8 using another group of adult rats. Moreover, the testicular weight returned to normal more rapidly in rats treated with AF-2785 versus AF-2364 (Fig. 9).

View larger version (19K): [in this window] [in a new window]   FIG. 9. Effects of AF-2364 and AF-2785 on testicular weight. Adult rats (n = 2 for each time point) were treated with AF-2364 or AF-2785 using the treatment regimens shown in Figure 5B (five weekly doses of 50 mg/kg b.w.) or Figure 8D (eight daily doses of 50 mg/kg b.w.), respectively, and compared to control rats receiving vehicle only

DISCUSSION

Since the 1960s, a variety of effective and relatively safe contraceptives have become available to women, in part due to the extensive and collaborative efforts among scientists targeted to understand the hormonal regulation of female reproductive physiology [27]. However, studies in the male lag far behind that of the female, except in the area of morphological studies because the biochemistry and molecular biology of spermatogenesis are still poorly understood. As such, contraceptive choices for the male are still limited to the use of condoms that first appeared in 1564 and became widely used in Europe in the early 1700s [28]. While the use of the condom is relatively safe and convenient, its efficacy is poor. Other nonreversible sterilization procedures, such as vasectomy, have been available for some time and it is estimated that more than 500 000 men every year worldwide received vasectomy. This procedure, however, is associated with surgical complications, unwanted immunological consequences, and other pathological conditions [29]. More recent studies using androgens and their analogues have also promised a hormonal approach for male contraception that is safe, efficient, and reversible by disrupting the hypothalamus-pituitary-testicular hormonal axis [27, 30]. In this report, we present data demonstrating yet another approach for male contraception by disrupting germ cell attachment onto Sertoli cells. This approach does not have the drawback of the hormonal approaches by either suppressing sperm production or disrupting the hypothalamus-pituitary-testicular axis.

The mechanism by which these drugs exert their effects to induce premature release of germ cells from the seminiferous epithelium is not completely known. It is possible that they disrupt cell adhesion molecules such as N-cadherin:E-cadherin:ß-catenin complexes that are Ca²⁺-dependent cell adhesion molecules found between Sertoli and germ cells in the seminiferous epithelium [2, 31] that anchor germ cells onto Sertoli cells in the epithelium. Alternatively, they may induce disruption of the microfilament bundles that dissociates the intercellular junctions similar to the action of lonidamine in Sertoli cells [4]. Still, the precise cascade of events leading to disruption of cell adhesion is not known. Preliminary studies using inhibitors of different signal transducers investigating the mechanism of lonidamine-induced testin expression have shown that these analogues may transduce their signals via the cAMP/protein kinase A signal transduction pathway [14]. Work is in progress to determine whether an induction of testin expression causes the disruption of germ cell attachment onto the Sertoli cell or the observed testin induction is one of the many changes associated with junction disruption. Results presented in this study, however, have shown that a transient induction in testin expression correlates with the subsequent disappearance of more advanced stages of germ cells, such as round and elongated spermatids, from the epithelium. For instance, lonidamine that induced a much longer increase in testin expression [10, 32] compared to AF-2364 and AF-2785 when all compounds were administered to adult rats with the same dose (50 mg/kg b.w.), 1–14 days versus 1–3 days, illustrates that the expression of testin is a good indicator that can be used to select chemical entities exerting their effects specifically to deplete advanced stages of germ cells from the epithelium.

Recent studies on the assembly of tight and adherens junctions, such as those found in the blood-brain barrier created by opposing endothelial cells, have shown that they are regulated, at least in part, by the state of protein phosphorylation [33]. For instance, the assembly of tight junctions in MDCK cells requires the recruitment of highly phosphorylated occludin, a tight junction integral protein, at the site of tight junction; whereas non- or less phosphorylated occludin is distributed on the basolateral membranes illustrating the role of phosphorylation in tight junction formation [34]. Increased tyrosine phosphorylation also causes redistribution of tight and anchoring junction-associated proteins, which in turn perturbs the junction permeability barrier in several cell lines in vitro [35, 36]. Using Sertoli cells cultured in vitro and inhibitors of protein phosphatases and protein kinases, it was recently shown that the state of phosphoprotein content regulated by the interplay of protein kinases and phosphatases (e.g., myotubularin) indeed regulated the assembly and maintenance of the inter-Sertoli tight junction permeability barrier [37, 38]. It is therefore possible that these new chemical entities exert their effects to alter the phosphoprotein content in Sertoli and/or germ cells, which in turn destablizes the anchoring junctions in the testis and triggers testin expression. In addition, other studies have shown that the assembly and possibly maintenance of junctions between Sertoli and germ cells are associated with the timely induction or inhibition of multiple genes such as tryptase, ₂-macroglobulin, urokinase-type plasminogen activator, cathepsin L, N-cadherin, and sertolin [39–42].

Results of the present study also illustrate that both analogues of lonidamine induce a progressive loss of germ cells from the epithelium that begins with the latest stages, such as elongated spermatids, to earlier stages, such as round spermatids and some spermatocytes. This drug-induced stage-dependent germ cell loss can be explained as follows: we postulate that the two analogues of lonidamine exert their effects by activating the mechanism(s) that causes the cleavage of AJs between late spermatids and Sertoli cells through a yet-to-be defined cascade of events. For instance, lonidamine is known to induce rearrangement and disruption of cytoskeleton network in Sertoli cells, A431 epithelial squamous carcinoma cells, and M14 melanoma cells [4, 5, 43], thereby causing premature release of developing germ cells from the epithelium. Given the fact that these two new analogues share structural homology with lonidamine, they may indeed induce changes in the cytoskeletal network in Sertoli cells. However, the above AJ-disruptive activity between Sertoli cells and late spermatids is more efficient than that between early spermatids/spermatocytes and Sertoli cells. It is possible that the specialized AJs, such as ectoplasmic specializations and tubulobulbar junctions, between Sertoli cells and late spermatids, that are constituted by some yet-to-be identified AJ-associated proteins, are more susceptible to these analogues. Also, these specialized AJ proteins may also be part of the signaling pathway involving testin because a disruption of Sertoli-germ cell AJs is associated with a surge in testin expression both in vivo and in vitro [10, 11]. The relative long lag of response of germ cell loss, 6 days, to the analogue treatments seemingly signifies that the drug-induced AJ disruptive action consists of a cascade of events, that triggers an induction of testin expression along the pathway before physical depletion of germ cells from the epithelium can be visualized.

In this connection, it is noteworthy to mention that gossypol (a yellow pigment found in cotton seed oil) is another chemical entity that is known to induce germ cell depletion from the epithelium [44, 45]. However, the efficacy dose range and the safety margin of gossypol, similar to lonidamine, are very narrow; also, gossypol disrupts kidney function [44, 45]. More importantly, the antifertility effect of gossypol is irreversible in a large proportion of men after prolonged exposure to gossypol [44, 45]. While it remains to be determined if the two newly synthesized chemical entities are indeed chemical entities suitable for contraceptive use for human males without the side-effects of lonidamine and gossypol, some of the recently completed toxicity studies performed by licensed toxicologists according to Food and Drug Administration guidelines are promising. For instance, the recently completed acute toxicity study using the Irwin dose range (100–1000 mg/kg b.w.) by i.p. in mice revealed that AF-2364 does not influence any of the neurological or autonomic parameters (unpublished results). Moreover, both AF-2785 and AF-2364 do not induce reverse mutation in Salmonella typhimurium or Escherichia coli by standard mutagenicity tests (unpublished results).

In summary, we herein report the chemical synthesis of two potential male contraceptives. Preliminary laboratory studies illustrate their efficacy and reversibility in fertility control in the rat. Also, these chemical entities do not appear to affect the hypothalamus-pituitary-testicular hormonal axis or liver and kidney function.

ACKNOWLEDGMENTS

We thank the National Hormone and Pituitary Program, NIDDK, NIH, for the gifts of ovine FSH and RIA kits for both rat LH and rat FSH.

FOOTNOTES

First decision: 15 December 2000.

¹ Supported in part by grants from the CONRAD Program (CIG-96-05, CIG-96-05A), the Rockefeller Foundation, 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

³ These authors have contributed equally to the completion of this work.

Accepted: March 20, 2001.

Received: November 8, 2000.

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