The seminal acrosin-inhibitor ClTI1/SPINK2 is a fertility-associated marker in the chicken.

The seminal plasma is a very complex fluid, which surrounds sperm in semen. It contains numerous proteins including proteases and protease inhibitors that regulate proteolytic processes associated with protein activation and degradation. We previously identified a seminal protein, chicken liver trypsin inhibitor 1 (ClTI-1) over expressed in semen of roosters with high fertility, suggesting a role in male fertility. In the present study, we showed that ClTI-1 gene is actually SPINK2. Using normal healthy adult roosters, we showed that SPINK2 amount in seminal plasma was positively correlated with male fertility in chicken lines with highly contrasted genetic backgrounds (broiler and layer lines). Using affinity chromatography combined to mass spectrometry analysis and kinetic assays, we demonstrated for the first time that two chicken acrosin isoforms (acrosin and acrosin-like proteins) are the physiological serine protease targets of SPINK2 inhibitor. SPINK2 transcript was overexpressed all along the male tract, and the protein was present in the lumen as expected for secreted proteins. Altogether, these data emphasize the role of seminal SPINK2 Kazal-type inhibitor as an important actor of fertility in birds through its inhibitory action on acrosin isoforms proteins.

Design and synthesis of phenylisoxazole derivatives as novel human acrosin inhibitors.

Human acrosin is an attractive target for the discovery of novel male contraceptives. Isoxazole derivative ISO-1, a small-molecule weak human acrosin inhibitor, was used as the starting point for lead optimization. After two rounds of structure-based inhibitor design, a highly potent inhibitor B6 (IC50=1.44 µM) was successfully identified, which showed good selectivity over trypsin and represents one of the most active human acrosin inhibitors up to date.

Synthesis and acrosin inhibitory activities of 5-phenyl-1H-pyrazole-3-carboxylic acid amide derivatives.

A series of novel 5-phenyl-1H-pyrazole-3-carboxylic acid amide derivatives were designed, synthesized, and their acrosin inhibitory activities in vitro were evaluated. The results of the acrosin inhibitory activity showed that all target compounds were more potent than control TLCK. Compounds AQ-A1, AQ-D3, AQ-D4, AQ-E4 and AQ-E5 exhibited stronger acrosin inhibitory activities than control ISO-1. Especially, compound AQ-E5 displayed the most potent acrosin inhibitory activity in all the compounds, with an IC50 of 0.01µmol/mL. This study provided a new structural class for the development of novel acrosin inhibitory agents.

Synthesis and acrosin inhibitory activity of methyl 5-substituted-1H-benzo[d]imidazol-2-yl carbamate derivatives.

A series of novel methyl 5-substituted 1H-benzo[d]imidazol-2-ylcarbamates were designed, synthesized, and their acrosin inhibitory activities evaluated in vitro. The results of acrosin inhibitory activity showed that all title compounds were more potent than the control TLCK. Compound 4w displayed the most potent acrosin inhibitory activity among all the compounds, with an IC(50) of 6.3×10(-5)M. The studies provide a new structural class for the development of novel acrosin inhibitory agents.

Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica).

An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45  kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45  kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.

Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives.

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives.

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.

Discovery of novel human acrosin inhibitors by virtual screening.

Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC(50) = 14 µM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.

[Nandeshi: a powerful inhibitor of human acrosin activity].

OBJECTIVE: To evaluate the inhibitory effect of Nandeshi, an acrosin inhibitor, on human acrosin activity. METHODS: We collected sperm samples from 10 healthy fertile men and cultured them with Nandeshi at 30 degrees C for 5 minutes at the concentrations of 0. 100, 0.120, 0.144, 0.173, 0.207, 0.249, 0.299, 0.358 and 0.430 mmol/L, with the controls treated with a well-known acrosin inhibitor N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK) at 150.0, 189.8, 213.6, 240.3, 270.3, 304.1 and 342.1 mmol/L. Then we determined the residual activity of human acrosin by improved Kennedy assay. RESULTS: The residual activity of acrosin was negatively correlated with the Nandeshi concentration, and Nandeshi exhibited an inhibition rate about 800 times that of TLCK. CONCLUSION: Nandeshi has a powerful inhibitory effect on human acrosin, and improved Kennedy assay is a simple, practical and highly sensitive technique for the detection of human acrosin activity.

Acrosin inhibitor detection along the boar epididymis.

Epididymal sperm maturation represents a key step in the reproduction process. Spermatozoa are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in sperm membrane proteins are influenced by proteolytic, glycosylation and deglycosylation enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors of these enzymes in the epididymis is very important for the regulation of sperm membrane protein processing. In the present study, we monitored acrosin inhibitor distribution in boar epididymal fluid and in spermatozoa from different segments of the organ. Using specific polyclonal antibody we registered increasing signal of the acrosin inhibitor (AI) from caput to cauda epididymis. Mass spectroscopy examination of the immunoprecipitated acrosin inhibitor (12 kDa) unequivocally identified sperm-associated acrosin inhibitor (SAAI) in the epididymal tissue. Lectin staining showed N-glycosylation in AI from boar epididymis. Protein detection of AI was supported by the results of semi-quantitative RT-PCR showing the presence of mRNA specifically coding for SAAI and similarly increasing throughout the epididymal duct, from its proximal to distal part. Additionally, the immunofluorescence technique showed the AI localization in the secretory tissue of caput, corpus and cauda epididymis, and in the acrosome region and midpiece of the sperm.

Organization and sequence of the gene encoding the human acrosin-trypsin inhibitor (HUSI-II).

A complete cDNA encoding the acrosin-trypsin inhibitor, HUSI-II, was used as a probe to isolate genomic clones from a human placenta library. Three clones which cover the entire HUSI-II gene were isolated and characterized. The exon-intron organization of the gene was determined and found to be identical to other known Kazal-type inhibitor-encoding genes. The striking similarity in the amino acid sequences which was found previously in HUSI-II and glycoprotein hormone beta-subunits, is neither reflected in codon usage nor in the exon-intron arrangement of the genes. A 1.8-kb segment 5' of the gene was sequenced. The analysis of this sequence showed that HUSI-II contains a G + C-rich region upstream from the transcription start point (tsp) which fulfills the criteria for a CpG island. Furthermore, in the first intron, a potential glucocorticoid-responsive element was found as a half-palindrome flanked by two CACCC elements. Determination of the tsp by S1 mapping revealed that HUSI-II has multiple tsp. Genomic Southern hybridization was used to show that HUSI-II is a single-copy gene. The localization of the gene to chromosome 4 was determined by hybridization of a 5' genomic fragment to the DNA of a panel of somatic hybrids between human and rodent cells.

Amino acid sequence elucidation of human acrosin-trypsin inhibitor (HUSI-II) reveals that Kazal-type proteinase inhibitors are structurally related to beta-subunits of glycoprotein hormones.

The amino acid sequence of the acrosin-trypsin inhibitor HUSI-II from human seminal plasma is presented which unequivocally identifies HUSI-II as being of Kazal-type. In addition, the HUSI-II sequence shows a striking similarity to the middle part of glycoprotein hormone beta-subunits thus revealing a hitherto unknown structural and evolutionary relationship between Kazal-type inhibitors and glycoprotein hormones.

Boar spermadhesins AQN-1 and AWN are sperm-associated acrosin inhibitor acceptor proteins.

Trypsin-like inhibitors secreted by the male accessory sex glands have been identified in the seminal plasma of every mammalian species so far investigated. They bind to acceptor molecules on the anterior part of ejaculated sperm, and are thought to play a role in the capacitation of spermatozoa stabilizing zona pellucida binding sites during sperm uterine passage, and then dissociating to allow sperm-egg's zona pellucida interaction. Here we report the identification of acrosin inhibitor acceptor molecules isolated from boar seminal plasma. These proteins, termed AQN-1 and AWN, belong to the recently described spermadhesin protein family, whose members have been implicated in sperm-zona pellucida recognition events. Thus, members of the spermadhesin family, although not possessing detectable enzymatic activity, show features of serine proteinases, and may be involved in both sperm capacitation and sperm-egg recognition and binding events.

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor.

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm-associated and the seminal plasma inhibitors may confer to them different physico-chemical properties which are postulated to be of functional importance.

Matrix degrading properties of sperm serine proteinase, acrosin.

The serine proteinase acrosin plays an important role in sperm penetration of the zona pellucida. In the present study we investigated the effect of the enzyme on various matrix proteins. Acrosin degraded proteolytically fibronectin, type IV collagen and heat denatured type I collagen, whereas neither native type I collagen nor laminin were cleaved by the enzyme. The specific activity of acrosin with type IV collagen as substrate (66.6 g/h/g) was 125-fold higher than that of known type IV collagenase or stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase.

Structure-activity relationships for the inhibition of acrosin by benzamidine derivatives.

A series, consisting of 52 benzamidine derivatives, was evaluated for inhibitory activity against homogeneous boar sperm acrosin. All of the compounds in the series proved to be more potent than benzamidine (Ki = 4.0 x 10(-6) M), with one of the derivatives, alpha-(4-amidino-2,6-diiodophenoxy)-3-nitrotoluene (compound 16), showing outstanding potency with a Ki value of 4.5 X 10(-8) M. Although all of the derivatives were effective acrosin inhibitors, structural specificity was observed within homologous groups of compounds. The information gained from this preliminary study should prove extremely beneficial in the design and synthesis of future acrosin inhibitors.

Synthesis and inhibition of human acrosin and trypsin and acute toxicity of aryl 4-guanidinobenzoates.

The aryl 4-guanidinobenzoate, 4'-nitrophenyl 4-guanidinobenzoate (NPGB), is a potent inhibitor of sperm acrosin, an enzyme with an essential function in the fertilization process. NPGB prevents fertilization in a number of animal species and is a good lead compound for the development of contraceptive agents. In order to assess the efficacy of other aryl 4-guanidinobenzoates as acrosin inhibitors, 24 of these compounds were synthesized. Their inhibitory activity toward human acrosin was determined and compared with their activity toward human pancreatic trypsin in order to assess whether inhibitor sensitivity differed between these similar enzymes. Nine of the inhibitors were synthesized from phenols approved by the FDA for therapeutic use. The acute toxicity of these inhibitors in mice was determined and compared to that of nonoxynol-9, the most commonly used active ingredient in today's vaginal contraceptive preparations. All of the compounds proved to be potent inhibitors of human acrosin although 3 orders of magnitude difference were observed between the most and least effective inhibitors. Little specificity was present in regard to their inhibition of acrosin and trypsin. All the aryl 4-guanidinobenzoates synthesized from FDA-approved phenols were less toxic than nonoxynol-9, and it is concluded that these 4-guanidinobenzoates are of interest for further development and testing as nonhormonal contraceptive agents.

Inactivation of trypsin-like proteases by depsipeptides of p-guanidinobenzoic acid.

A number of esters of p-guanidinobenzoic acid have been synthesized which contain a glycolyl peptide as the departing group. In the case of several enzymes such as trypsin and plasma kallikrein, depsipeptides were obtained which were considerably more reactive than the ethyl ester in inactivation of the protease by acyl-enzyme formation; the depsipeptide processing -CH2CO-Phe-NH2 as a leaving group displayed the highest reactivity. They were less effective in the case of urokinase, plasmin, and urinary kallikrein. Boar acrosin was very susceptible to inactivation by both ethyl and peptidyl esters. Depsipeptides possessing a longer peptide chain and a secondary carbon as a leaving group showed lower activities. The results demonstrate the productive use of the departing group region of protease active centers to obtain selectivity.

Dansylalanyllysylchloromethyl ketone as a fluorescent probe for localization of acrosin activity in boar and human spermatozoa.

The localization of acrosin (EC 3.4.21.10) activity in mammalian spermatozoa was investigated by use of the fluorescent site-directed acrosin inhibitor, dansylalanyllysylchloromethyl ketone (DALCK). Fluorescence microscope preparations revealed, after the spermatozoa were subjected to a specific treatment, that acrosin activity is confined specifically to the inner acrosomal membrane (IAM). Spectrofluorometric and fluorescence polarization investigations verified that the fluorescent probe, once it is specifically bound to the treated spermatozoa, lies in a very hydrophobic environment and shows a remarkable reduction of rotational freedom. These results are compatible with the hypothesis that, under the experimental conditions used, active acrosin is tightly bound to the IAM and that the "specificity site" of the acrosin-active center is probably of a highly hydrophobic nature.

Molecular cloning and sequence analysis of the cDNA encoding porcine acrosin inhibitor.

A full-length cDNA encoding the porcine acrosin inhibitor has been isolated from a boar seminal vesicle cDNA library. Nucleotide sequence analysis of the 667-bp cDNA predicts a precursor protein of 97 amino acid residues, which includes a 26-residue signal peptide and a 71-residue secreted protein. The predicted amino acid sequence of the mature protein agrees completely with that of the sperm-associated acrosin inhibitor determined by conventional amino acid sequence analysis. However, the asparagine/aspartic acid and glutamine/glutamic acid substitutions, as reported in the seminal plasma counterpart, have not been observed. Southern blot analysis shows only a single hybridizing band with three different restriction endonucleases, suggesting the presence of a single copy of the acrosin inhibitor gene in the porcine genome.