Results and Discussion
DDI-6 Discovered by Screening of a Chemical LibraryWe screened a chemical library to obtain compounds capable of activating spermiogenesis in spermatids from C. elegans him-8(tm611) males (for details, see “Experimental”). Eventually five compounds were found to trigger spermiogenesis in approx. 20–80% of total spermatids with no records about their activities on any biological phenomena. One of such compounds, named DDI-6, was a quinolinol analog and chosen for further analyses.
The Quinolinol Analog DDI-6 Synthesized Using a Four-Step ProcedureWe produced DDI-6 chemically to use it for C. elegans and mouse sperm activation assays. The synthesis started by dechlorination of 5-chloro-8-quinolinol (compound 1a) and its derivatives (compounds 1b–1d) with magnesium metal,7) but these reactions failed to yield 8-quinolinol derivatives. Therefore, we screened for reaction conditions using a palladium catalyst (Table 1). When compound 1a was treated with 10% palladium on carbon in various solvents, such as ethanol (EtOH) alone, tetrahydrofuran (THF) alone, and a mixture of THF, methanol (MeOH) and acetic acid (AcOH) (2 : 3 : 3), dechlorination did not occur (Table 1, entries 1–3). However, we found that a reaction performed in a mixture of THF and MeOH (1 : 2) could produce 1,2,3,4-tetrahydro-8-quinolinol (compound 3) without formation of 8-quinolinol derivatives such as compounds 2a–2d8) (Fig. 2 and Table 1, entry 4). The reaction was slightly improved in a mixture of THF and MeOH (1 : 1) to give compound 3 with a 72% yield (Table 1, entry 5). As shown in Fig. 2, N-sulfonylation of compound 3 was then conducted with pyridine and 4-acetamidobenzensulfonyl chloride to form compound 4 with a 56% yield.9,10) Deacetylation of the acetamido group in compound 4 under aqueous acidic conditions produced N-(4-aminobenzensulfonyl)-1,2,3,4-tetrahydro-8-quinolinol11) (compound 5; Fig. 2), and subsequent treatment of this with methyl chloroformate afforded the final product, DDI-6, with a 39% yield (Fig. 2).
Table 1. Screening of Reaction Conditions for Hydrogenolysis of Compound
1a |
|---|
| Entry | Solvent | Time (h) | Yield (%)a) |
|---|
| 1 | EtOH (0.18 M) | 97.5 | 0 |
| 2 | THF : MeOH : AcOH = 2 : 3 : 3 (0.19 M) | 52.5 | 0 |
| 3 | THF (0.50 M) | 13.5 | 0 |
| 4 | THF : MeOH = 1 : 2 (0.50 M) | 32.0 | 70 |
| 5 | THF : MeOH = 1 : 1 (0.50 M) | 24.5 | 72 |
We determined the ratios of cells completing spermiogenesis (spermatozoa formed with MO fusion) after stimulation of spermatids with several activators including the newly synthesized DDI-6 (Fig. 3). In the absence of activators (control), approx. 97% of total cells exhibited rounded morphology, and the PMs of the same cells were fluorescently stained with FM1-43 (Figs. 3A, B). Thus, neither pseudopod extension nor MO fusion were triggered substantially in spermatids in the absence of activators.
When spermatids were stimulated with 50 µM DDI-6, pseudopods were extended from approx. 28% of the cells. Moreover, punctate signals were observed by FM1-43-staining in nearly all of the pseudopod-bearing cells (Figs. 3A, B). We also tested the bacterial protease mixture Pronase (Pron)12) and the bacterial serine protease Proteinase K (ProK)6) as positive control activators. Indeed, 200 µg/mL Pron and ProK activated approx. 88 and approx. 99% of spermatids, respectively, to initiate spermiogenesis (Figs. 3A, B). Patterns of pseudopod extension and MO fusion with DDI-6 were indistinguishable from those induced by Pron and ProK (Fig. 3A), indicating that DDI-6 partly but significantly produced pseudopod-extended sperm with MO fusion.
Because Pron and ProK are water-soluble proteases, perhaps these two enzymes extracellularly degrade spermatid proteins, leading to initiation of spermiogenesis. Indeed, the previous genetic study implied that the C. elegans serine protease TRY-5 might be a physiological activator for spermiogenesis.13) Contrarily, DDI-6 indicated a low solubility in water, while it was highly soluble in organic solvents such as acetone, chloroform, dimethyl sulfoxide (DMSO), and EtOH (data not shown). This property presumably provides the PM-permeability to DDI-6, because of the PM’s hydrophobicity, so that the compound might act within cells. So far several agents have been reported to induce C. elegans spermiogenesis in vitro, such as monensin (cationic ionophore),14) triethanolamine (TEA, weak base),15) trifluoperazine (TFP, calmodulin inhibitor),12) chlorpromazine (CPZ, calmodulin inhibitor),12) N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7, calmodulin inhibitor),12) 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, chloride channel blocker),16) wortmannin (phosphatidylinositol-3 kinase inhibitor),17) labile zinc,18) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, mitogen-activated protein kinase activator).19) In particular, predicted targets of TFP, CPZ, W7, wortmannin, and AEBSF are intracellular proteins, thereby DDI-6 possibly interacts with intracellular molecules to induce spermiogenesis in C. elegans spermatids.
DDI-6 Also Triggers the Acrosome Reaction in Mouse SpermatozoaBecause DDI-6 seemed to be a moderate activator of C. elegans spermiogenesis (Fig. 3), we examined its capability to induce the acrosome reaction in mature spermatozoa from the cauda epididymis by Coomassie Brilliant Blue (CBB)-staining20) (Fig. 4). When mouse spermatozoa were mock stimulated, approx. 12% became acrosome-reacted (Figs. 4A, B). Therefore, it was likely that such levels of acrosome-reacted sperm would arise spontaneously in control treatments.
After being treated with 100 µM DDI-6, approx. 20% of all spermatozoa were acrosome-reacted. Because it is well known that the calcium ionophore A23187 can induce the acrosome reaction,21) this drug was also examined as a positive control activator. As expected, approx. 82% of sperm were acrosome-reacted in the presence of 10 µM A23187. Moreover, DDI-6-induced acrosome-reacted sperm were similar to A23187-induced acrosome-reacted sperm in cytology. These results demonstrate that DDI-6 is also a moderate activator for the acrosome reaction in mature mouse spermatozoa. It is worthwhile to note that A23187 does not affect C. elegans sperm activation.14,15)
DDI-6 Can Be Utilized to Investigate a Mechanism for Sperm Activation Shared by C. elegans and MiceThere is an evolutionary distance of approximately one billion years between C. elegans and rodents,22) and genes controlling reproductive traits generally evolve faster than somatic genes.23–25) However, it is not totally unlikely that these two species have a common mechanism for sperm activation. We have previously reported that C. elegans SPE-45 acts during gamete fusion as a functional equivalent to mouse IZUMO1, despite only approx. 9% identity of the entire amino acid sequences between these two proteins.26,27) Our present findings also imply that C. elegans and the mouse might have a common target of DDI-6 in signaling pathways for MO fusion and the acrosome reaction. Because there are no known effects for DDI-6 in any biological phenomena, we are currently seeking molecules that might interact directly or indirectly with DDI-6 in C. elegans and mouse spermatozoa by a combination of biochemical and genetic approaches. Identification of such DDI-6-related factors would provide important information regarding a common mechanism for sperm activation that are shared by C. elegans and mice.
From a human clinical aspect, the discovery of DDI-6 suggests a possible strategy in screening for compounds that can activate C. elegans spermatozoa. To develop therapeutic approaches or testing drugs for treating human male infertility, seed compound screening using mammals might be replaced by C. elegans-based screening, at least in part. Given that it is preferable to minimize the use of mammals such as mice for drug development, the availability of C. elegans for such screening is worthy of consideration. Moreover, it is also important to identify an elemental structure(s) of DDI-6 that is required for sperm activation. Studies on the structure–activity relationship of DDI-6 would lead to elucidating how it acts on sperm activation and possibly to developing it as clinically useful drugs.
Experimental
MaterialsThe Core Library used for screening of compounds was provided from the Drug Discover Initiative (DDI, at the University of Tokyo, Japan).28) Compounds contained in the library were stored as 10 mM solutions in DMSO at −80 °C until use. Pron and A23187 were purchased from Sigma-Aldrich (St. Louis, U.S.A.), ProK from Nacalai Tesque (Kyoto, Japan), and FM1-43 from Invitrogen (Carlsbad, U.S.A.). Other chemicals that we used were of the highest quality available.
AnimalsThe C. elegans him-8(tm611) IV strain (CA257) was provided from the Caenorhabditis Genetic Center (CGC, at the University of Minnesota, U.S.A.). This strain was used in this study as wild-type worms. ICR mice (approx. 10-week-old males) were purchased from Japan SLC (Shizuoka, Japan). This study was approved by the Institutional Animal Care and Use Committee (permission number: K19–34) and carried out according to the Setsunan University Animal Experimentation Regulations.
MicroscopyMost worm handling, such as picking and dissecting, were carried out under SZ61 or SZX10 dissecting microscopes (Olympus, Tokyo, Japan). To capture digital differential interference contrast (DIC) or fluorescent images, we used a BX53 microscope carrying a DP72 CCD camera with cellSens software (Olympus).
Screening for Compounds Triggering C. elegans SpermiogenesisThe Core Library from the DDI contained 9600 entries that are structurally divergent to each other. To obtain in vitro activators for C. elegans spermiogenesis, we primarily screened 480 entries of the Core Library as described below (see “C. elegans Spermiogenesis Assay”). Since 1% DMSO (solvent control) triggered spermiogenesis in approx. 3% of total C. elegans spermatids, compounds capable of activating more than 20% of total cells were subjected to a secondary screening. The selected compounds were tested to induce spermiogenesis in vitro at concentrations of 50 and 100 µM. If those compounds exhibited significant activities in a concentration-dependent manner, we judged that they are positive as C. elegans spermiogenesis activators.
C. elegans Spermiogenesis AssayThis assay was based on the previous report by Tajima et al.,6) and all steps were performed at room temperature (approx. 22 °C). Round spermatids were released by dissection of two to five him-8(tm611) males in 10 µL of 1 × Sperm Medium (1 × SM; 50 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes)–NaOH, pH 7.4, containing 45 mM NaCl, 5 mM KCl, 1 mM MgSO4, 5 mM CaCl2 and 10 mM glucose) containing 1% DMSO. After 5-min incubation, 10 µL of Activator Solution (1 × SM containing either of 1% DMSO (solvent control as a mock), 100 µM DDI-6, 400 µg/mL Pron or 400 µg/mL ProK) was added to the cells, and they were incubated for additional 35 min to induce spermiogenesis. To screen 480 entries that were contained in the Core Library, 1 × SM containing 100 µM either of the compounds was used as Activator Solution and then incubated with spermatids for 20 min. To stain cellular membranes, 5 µL of FM1–43 Solution (1 × SM containing 25 µg/mL FM1–43) was added to the 20-µL preparation. Following a 5-min incubation, digital DIC and fluorescent images were acquired as described in “Microscopy.”
Mouse Acrosome Reaction AssayThis assay was based on the previous report by Larson and Miller.20) Sperm were released from the cauda epididymis of ICR males into 200-µL drops of Human Tubal Fluid (HTF) medium29) omitting bovine serum albumin (HTF(-BSA) medium) and allowed to stand at 37 °C for 30 min in 5% CO2/95% air. Then, 2.0 × 107/mL epididymal sperm were incubated with either of 1% DMSO (solvent control as a mock), 100 µM DDI-6 or 10 µM A23187 in 50 µL of HTF(-BSA) medium at 37 °C for 30 min in 5% CO2/95% air. To quench the acrosome reaction by fixing sperm, 100 µL of 1 × phosphate-buffered saline (1 × PBS; 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl) containing 4% paraformaldehyde was added to the sperm suspension. After 20-min incubation at room temperature, cells were washed three times by centrifugation at 2200 × g for 5 min at room temperature in 200 µL of 100 mM ammonium acetate, pH 9.0, and then re-suspended in 100 µL of the same buffer. A 10-µL aliquot from the sperm suspension was put onto a slide glass and air-dried at room temperature. To stain the acrosomes, 50 µL of Staining Solution (0.22% CBB G-250, 50% MeOH and 10% AcOH) was added to the air-dried cells. After 10-min incubation at room temperature, cells were washed three times with ultrapure water in a chamber to remove excess CBB G-250. The stained cells were mounted with 10 µL of 1 × PBS, and digital DIC images were acquired as described in “Microscopy.”
Synthesis of DDI-6Chemicals required to synthesize DDI-6 were used without any purification unless otherwise stated. Below are the procedures for synthesis of intermediate and the final products. Each product name is the same as those shown in Fig. 2.
EquipmentsSpectra of 1H-NMR (400 MHz) and 13C-NMR (100 MHz) were measured on an ECZ 400S spectrometer (JEOL, Tokyo, Japan). Chemical shifts for 1H-NMR were determined as δ values by comparing with that of tetramethylsilane as an internal standard, and coupling constants are shown in Hz. The following abbreviations are used to express spin multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad. Chemical shifts for 13C-NMR were determined in ppm by comparing with that of the center line of a triplet for deuteriochloroform, on the basis of the assumtion that it appears at 77.16 ppm. Data on 1H-NMR and 13C-NMR of each compound are shown in “Supplementary Materials.” High-resolution mass spectroscopy (HR-MS) was carried out with electron ionization (EI) on a JEOL JMS-700T spectrometer. Analytical TLC was performed using Silica Gel 60 F254, precoated analytical plates (Merck, Darmstadt, Germany). For flash chromatography, SiliaFlash P60 (Silicycle, Quebec, Canada) was used unless otherwise noted.
1,2,3,4-Tetrahydro-8-quinolinol (Compound 3)8)Compound 1a (5-chloro-8-quinolinol: 1.26 g, 7.00 mmol) was dissolved in 14 mL of a mixture containing THF and MeOH (1 : 1), added to 10% Pd/C (744.9 mg, 0.70 mmol), and hydrogenated at 1 atm in H2 gas at room temperature. After being stirred for 24.5 h, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by recrystallization with CHCl3 to afford compound 3 (724.0 mg, 72%) as a pale orange crystal. Analytical data on compound 3 was as follows: 1H-NMR (CD3OD, 400 MHz) δ: 2.05–2.15 (m, 2H), 2.89 (t, J = 6.1 Hz, 2H), 3.41–3.49 (m, 2H), 6.78 (d, J = 7.5 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 7.20 (dd, J = 8.1, 7.5 Hz, 1H); 13C-NMR (CDCl3 + MeOH-d4 5 drop, 100 MHz) δ: 19.5, 24.9, 42.5, 113.8, 118.1, 120.5, 129.3, 132.2, 150.7; HR-MS (EI) Calcd for C9H11NO ([M]+): 149.0841. Found: 149.0836.
N-(4-Acetamidobenzensulfonyl)-1,2,3,4-tetrahydro-8-quinolinol (Compound 4)9,10)Compound 3 (300 mg, 2.01 mmol) was dissolved in 4 mL of CH3CN, and 4-acetamidobenzenesulfonyl chloride (516.4 mg, 2.21 mmol) and pyridine (240 µL, 3.02 mmol) were added to the solution at 0 °C. After stirring the reaction mixture for 2 h at room temperature, the reaction was quenched by addition of saturated, aqueous NH4Cl at 0 °C, followed by extraction with ethyl acetate (AcOEt). The organic phase was washed with brine and then dried over anhydrous Na2SO4. After filtration to remove solid Na2SO4, the resulting filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography in a mixture of hexane and AcOEt (4 : 6) to afford compound 4 (390.6 mg, 56%) as a pale orange solid. Analytical data on compound 4 was as follows: 1H-NMR (CDCl3, 400 MHz) δ: 1.76 (tt, J = 6.8, 6.8 Hz, 2H), 1.86 (t, J = 6.8 Hz, 2H), 3.74 (t, J = 6.8 Hz, 2H), 6.54 (d, J = 7.4 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 7.09 (dd, J = 8.0, 7.4 Hz, 1H), 7.32 (br s, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H); 13C-NMR (CDCl3 + DMSO-d6 1 drop, 100 MHz) δ: 22.9, 24.7, 25.1, 46.7, 116.5, 119.0, 120.6, 125.0, 128.4, 128.6, 131.7, 137.4, 143.5, 152.6, 169.4; HR-MS (EI) Calcd for C17H18N2O4S ([M]+): 346.0987. Found: 346.0992.
N-(4-Aminobenzensulfonyl)-1,2,3,4-tetrahydro-8-quinolinol (Compound 5)11)Compound 4 (200 mg, 0.58 mmol) was dissolved in 1.5 mL of MeOH, followed by addition of concentrated, aqueous HCl at 0 °C. Then, the reaction mixture was stirred at 80 °C until completion of the reaction, judged by TLC for 3 h. The mixture was concentrated under reduced pressure and neutralized with saturated, aqueous Na2CO3 at 0 °C. The resulting precipitate was filtered and then washed with cold water to afford compound 5 (94.6 mg, 54%) as an orange solid. Analytical data on compound 5 was as follows: 1H-NMR (DMSO-d6, 400 MHz) δ: 1.74 (tt, J = 6.6, 6.6 Hz, 2H), 2.04 (t, J = 6.8 Hz, 2H), 3.46 (t, J = 6.6 Hz, 2H), 6.08 (br s, 2H), 6.54 (d, J = 8.6 Hz, 2H), 6.54 (d, J = 7.7 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.98 (dd, J = 8.1, 7.7 Hz, 1H), 7.31 (d, J = 8.6 Hz, 2H), 8.90 (br s, 1H); 13C-NMR (DMSO-d6, 100 MHz) δ: 23.1, 24.7, 45.0, 112.6, 114.8, 118.9, 123.9, 125.2, 127.2, 129.3, 137.8, 153.2, 153.4; HR-MS (EI) Calcd for C15H16N2O3S ([M]+): 304.0882. Found: 304.0887.
N-(4-Methoxycarbonylaminobenzensulfonyl)-1,2,3,4-tetrahydro-8-quinolinol (DDI-6)Compound 5 (152.9 mg, 0.5 mmol) was dissolved in 1.7 mL of CH3CN, and pyridine (44 µL, 0.55 mmol) and methyl chloroformate (44 µL, 0.55 mmol) were added to the solution at 0 °C. After stirring the reaction mixture for 23.5 h at room temperature, the reaction was quenched by addition of saturated, aqueous NH4Cl at 0 °C. The mixture was extracted with CHCl3, and the organic phase was washed with brine and dried over anhydrous Na2SO4. After filtration to remove solid Na2SO4, the resulting filtrate was concentrated under reduced pressure. The crude product was subjected to flash chromatography and then eluted in a mixture of hexane and AcOEt with a linear gradient from 3 : 1 to 7 : 3 ratios to afford DDI-6 (70.3 mg, 39%) as a yellow solid. Analytical data on the final product, DDI-6, was as follows: 1H-NMR (CDCl3, 400 MHz) δ: 1.76 (t, J = 6.5, 6.5 Hz, 2H), 1.87 (t, J = 6.5 Hz, 2H), 3.74 (t, J = 6.5 Hz, 2H), 3.80 (s, 3H), 6.54 (d, J = 7.4 Hz, 1H), 6.85 (br s, 1H), 6.95 (d, J = 8.1 Hz, 1H), 7.08 (dd, J = 8.1, 7.7 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.71 (br s, 1H); 13C-NMR (CDCl3, 100 MHz) δ: 23.1, 25.2, 46.9, 53.0, 116.8, 117.9, 120.6, 125.1, 128.5, 129.0, 131.8, 137.4, 142.7, 152.8, 153.4; HR-MS (EI) Calcd for C17H18N2O5S ([M]+): 362.0936. Found: 362.0935.
