Recombinant buffalo (Bubalus bubalis ) cysteine-rich secretory protein 1 mature peptide acts as a potent sperm-quiescent and decapcitation factor by modulating nitric oxide/bicarbonate/adenylyl cyclase/Ca+2 signalling pathway

Recombinant buffalo (Bubalus bubalis ) cysteine-rich secretory protein 1 mature peptide acts as a potent sperm-quiescent and decapcitation factor by modulating nitric oxide/bicarbonate/adenylyl cyclase/Ca+2 signalling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Recombinant buffalo (Bubalus bubalis ) cysteine-rich secretory protein 1 mature peptide acts as a potent sperm-quiescent and decapcitation factor by modulating nitric oxide/bicarbonate/adenylyl cyclase/Ca+2 signalling pathway Nikki Kumari, Om Prakash, Rajani Kr. Paul This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6680127/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cysteine-rich secretory protein 1 (CRISP-1), an acidic glycoprotein of epididymis, acts as sperm decapacitation factor. Adding CRISP-1 protein to semen may extend the fertile life of preserved sperm by inhibiting premature sperm capacitation during preservation. To this end, production of bioactive recombinant buffalo CRISP-1 mature peptide in E. coli followed by it’s functional and mechanistic characterization on sperm motility and capacitation were described. A 687 bp cDNA fragment corresponding to C 15 -C 242 buffalo CRISP-1 mature peptide was cloned in pET22b expression vector and expressed in BL21(DE3)-codon plus E. coli strain. The recombinant peptide was expressed as insoluble form which was purified by Ni-NTA affinity chromatography and refolded by dialysis. Tissue expression analysis revealed that buffalo CRISP-1 protein was expressed mainly in cauda epididymis and vas deferens, but not in testis. In sperm, it localized on acrosome and principal piece of tail. The CRISP-1 peptide (20µg/mL) caused significant reduction in sperm progressive motility (61 vs. 33%) and capacitation. Further, it reduced tyrosine phosphorylation of two sperm proteins (p47, p72) under capacitating condition. The peptide was found active between pH 6 and 9, and optimal pH was pH 8. The activity of the peptide was reduced above 60°C. The peptide inhibited both bicarbonate and L-arginine mediated capacitation of buffalo sperm by modulating nitric oxide (NO)/adenylyl cyclase (AC)/cAMP/Ca + 2 signalling pathway. It was concluded that, the recombinant buffalo CRISP-1 mature peptide produced in E. coli was bioactive and it inhibited sperm motility and capacitation by interrupting NO/AC/cAMP/Ca + 2 signalling pathway. Hence, the recombinant CRISP-1 peptide could be utilized to prevent sperm capacitation during semen preservation for improving post-thaw quality of preserved buffalo semen. Recombinant CRISP-1 peptide production biological function mechanism of action tissue expression Buffalo Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction Conception rates in buffalo with cryopreserved semen are very low (30%) [ 1 , 2 ]. The reduced fertility of cryopreserved buffalo semen is considered due to sub-lethal damages in sperm, including oxidative changes in membrane lipids and capacitation-like changes in sperm such as increase in intracellular calcium ([Ca + 2 ] i ), cAMP, protein tyrosine phosphorylation, and cholesterol efflux. Several proteins of cauda epididymis are known to cause reversible inhibition of sperm motility and capacitation and are considered responsible for sperm-quiescence and prolonged survival of sperm in cauda epididymis [ 3 , 4 , 5 , 6 ]. Cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymis known to inhibit sperm capacitation and motility [ 7 ]. Studies conducted in vitro have demonstrated that the recombinant rat CRISP-1 peptide reversibly inhibits sperm capacitation and protein tyrosine phosphorylation [ 3 ], by modulating CatSper1 channel [ 8 ]. Therefore, addition of recombinant buffalo CRISP-1 peptide to diluted semen prior cryopreservation might enhance the post-thaw quality and fertility of buffalo spermatozoa by reducing sperm capacitation induced by freezing-thawing process. CRISP proteins belong to a sub-group of CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The CRISP proteins are characterised by the presence of N-terminal CAP domain (∼21 kDa) that contains six conserved cysteine residues, and the smaller C-terminal cysteine-rich CRISP domain (∼6 kDa) that contains 10 conserved cysteine residues [ 9 ]. The cysteine residues are engaged in intra-domain disulphide bonds [ 10 , 11 ]. Presence of this eight disulfide bonds render CRISP proteins structurally complex. In rat two forms of CRISP-1 protein (D and E) were identified. The D form is loosely associated to sperm and is released during capacitation and has been proposed as a decapacitation factor [ 3 ]. On ejaculation, the CRISP-1 protein is washed out by the secretions of accessory glands, thus allowing the pH i to increase and initiate sperm motility. In vitro study has shown that disassociation of CRISP-1 from the sperm membrane was required for capacitation to proceed in rat sperm [ 3 ].The E form of CRISP-1 protein binds tightly to sperm and is present even after capacitation. It is involved in both the interaction between sperm and the zona pellucida (ZP) as well as in the fusion of gametes by attaching to specific sites on the egg in mice [ 12 ]. In the male reproductive tract, expression of CRISP-1 is primarily detected in the epididymis both at the mRNA and protein levels [ 12 ]. The protein is secreted into seminal plasma and, to some degree, attaches to the surface of spermatozoa. CRISP-1 appears to be involved in post-testicular sperm maturation and inhibition of premature sperm capacitation, both essential for eventual fusion with the oocyte membrane [ 12 ]. The investigation into the structure-function relationship of both natural and bacterially-generated recombinant CRISP-1 (DE) unveiled that the protein's functionality does not engage with carbohydrates but rather resides within the polypeptide chain of the molecule [ 13 ]. Hence, ovine CRISP-1 protein produced in bacteria was found to be bioactive [ 7 ]. Recombinant CRISP-1 protein was shown to hinder sperm capacitation and impede protein tyrosine phosphorylation [ 3 ]. Further, it was demonstrated that CRISP-1 protein inhibits sperm capacitation by modulating Ca 2+ -transporting CatSper channels in mouse sperm [ 8 ]. However, the detailed mechanism of action and involvement of different signalling molecules associated with sperm capacitation for the activity of CRISP-1 protein are largely unknown. In view of the above, the current study was designed to produce recombinant buffalo CRISP-1 mature peptide in E. coli and characterize it’s biophysical and functional properties, tissue expression pattern in male reproductive tract and mechanism of action. 2. Materials and methods 2.1 Materials All the reagents used in cloning and protein expression studies were of molecular biology grade and obtained from Thermo Scientific (USA) and Sigma Aldrich (USA). The E. coli strains DH5α, BL21 (DE3) and BL21 (DE3)-codon plus and bacterial expression vector pET22b (+) plasmid were received from Animal Biotechnology Division, ICAR-NDRI as a gift. Other chemicals used were recombinant DNAse I (Takara, Japan), DNA ligase (Invitrogen, USA), AEBSF (SRL, India), mouse monoclonal anti-phosphotyrosine antibody (sc-81465, Santacruz, USA), PrecisionPlus® prestained protein marker (Bio-Rad, USA), N ω -nitro-L-arginine methyl ester HCL (L-NAME, Sigma), Forskolin (TCI, Japan) and FURA-2AM (Sigma). 2.2 Cloning of buffalo CRISP-1 cDNA in bacterial expression vector PCR primers against a 687bp buffalo CRISP-1 mRNA (NCBI acc. no. XM_006050714.4) corresponding to 228 amino acids length CRISP-1 mature peptide (C 15 -C 242 ) was designed by Primer select tool of NCBI. The forward and reverse primers 5’ - AA GGATCC TAGCTGCCTGCCTGTTTTGATT-3’ and 5’-TT AAGCTT ACACATACAACTAGCTTTGCAGA-3’ were flanked with Bam HI and Hind III sequences, respectively. Thermal cycle was optimized by doing gradient PCR: 94°C for 5 min, followed by 30 cycles each of 94°C for 30 s, 58–62°C for 30 s and 72°C for 1 min with a final extension at 72°C for 10 min. Amplified product was analysed on 1% agarose gel and purified by a kit. Then both PCR product and pET22b(+) plasmids were subjected to restriction endonuclease digestion and gel purified using a kit (Takara). Ligation reaction was carried out overnight at 4°C using 1:4 molar ratio of plasmid and PCR amplicon, respectively. For bacterial transformation, 10 µL of ligation mix was mixed with chemically competent E. coli DH5α cells and heat shock was given for 60 s at 42°C. Transformed colonies were initially screened by colony PCR and PCR-positive transformants were confirmed by carrying out RE digestion analysis of the isolated plasmids. 2.3 Optimization of protein expression in E. coli To optimize protein production in bacterial cells we tested four IPTG concentrations (0.1, 0.25, 0.5, and 0.75 mM), two incubation temperature (23 and 27°C), and two bacterial host strains BL21(DE3) and BL21(DE3)-codon plus and two induction times (6 and 14 h). The recombinant plasmid was used to transform chemically-competent E. coli strains and resultant colony was grown in multiple vials of 10 mL Luria-Bertani (LB) broth having antibiotics till OD 600 reached 0.6. Then different conc. of IPTG was added to the culture and shaking was continued either at 27°C for 6 h or 23°C for 14 h. Bacterial pellet was mixed with 2X Laemmli’s buffer, mixed by vortexing and lysed by 10 min incubation at 95°C in water bath. Protein expression was analysed by SDS-PAGE. Further, to check the solubility of the expressed peptide, bacterial pellet was initially lysed with native lysis buffer (20 mM Tris, pH 8.0 having 0.5M NaCl, 1 mg/mL lysozyme, 1 U/ml DNAse I, 1 mM AEBSF) by sonication (20 pulses of 10 s at 70% amplitude, 30 kHz) on ice. The supernatant was collected after centrifugation at 10,000 × g for 10 min at 4°C and the pellet was lysed with denaturing lysis buffer (8 M urea in 20 mM Tris, pH 8.0 having 0.5M NaCl, 20 mM 2-mercaptoethanol and 0.25% Triton X100) by vortexing followed by 1 h shaking at 25°C. Clear supernatant was collected after spinning at 10,000 × g for 30 min at 4°C. Both the lysates were analysed by SDS-PAGE to find the presence of the recombinant peptide. 2.4 Bulk production and purification of recombinant CRISP-1 peptide For bulk production of recombinant peptide freshly transformed colonies of BL21(DE3)-codon plus cell were prepared on a LB agar plate; and then one colony was transferred in to 10 mL LB broth having ampicillin and chloramphenicol and shaken for 4 h at 37°C. Then this culture was transferred in to a flask containing 1000 mL fresh LB with ampicillin and the flask was shaken for 1 h at 37°C or till the OD 600 reached 0.6. Then after bringing the culture at room temperature IPTG was added at 0.25 mM concentration and shaking was continued at 180 rpm for another 14 h at 23°C. The bacteria was pelleted by spinning at 7000 rpm and stored at -20°C. 2.5 Bacterial cell lysis and purification of recombinant peptide Bacterial pellet was thawed and lysed initially by native lysis buffer followed by denaturing lysis buffer as described before. For each gram of wet pellet 4 ml of native lysis buffer was added, mixed by vortexing, incubated at 25°C for 15 min and then sonicated on ice. After centrifugation and separation of the clear lysate, the pellet was washed twice with native lysis buffer (20 mM Tris, pH 8.0 having 0.5M NaCl) and finally lysed with denaturing lysis buffer. The clear lysate obtained from denaturing lysis was further clarified by passing through a 0.45 µm syringe filter. For purification of the recombinant peptide, Ni-NTA affinity chromatography was used. In brief, 5 mL of mixed gel slurry (70%; Takara) was washed twice each with distilled water and binding/column regeneration buffer (8 M urea in 20 mM Tris, 0.5M NaCl, pH 8.0) by spinning at 800 × g for 5 min. The gel slurry was mixed with denaturing protein lysate and placed on a shaker at 25°C for 1 h to allow protein binding to the resin. The unbound proteins were removed by washing the column with 10 bed volumes of wash buffer I (binding buffer having 10 mM imidazole) followed by 5 bed volumes of wash buffer II (binding buffer having 20 mM imidazole). The recombinant peptide was eluted sequentially with 4 bed volumes of elution buffer I and II having 40 and 80 mM imidazole in binding buffer, respectively. The fractions were analyzed by SDS-PAGE to check the purity of the peptide. 2.6 Dialysis and refolding of the peptide Both the 40 and 80 mM imidazole elutes were pooled and concentrated by using a 10 kDa cut-off centrifugal device (Amicon Ultra 4, USA) and dialyzed against 10 mM potassium phosphate buffer, pH 7.5 having urea at gradual decreasing concentrations starting from 4 M to 0.5 M in a 1000 mL flask at 4°C. The dialysis was carried out slowly over a period of 48 h in a 10 kDa cut-off dialysis bag by replacing buffer every 6 h. To assist in protein folding both reduced and oxidized glutathione (1:0.1 mM) and 0.25 M proline was added to the dialysis buffer. Finally, the dialysis was continued only in 10 mM potassium phosphate buffer, pH 7.5 for 6–8 h and peptide sample was concentrated by 10 kDa-cut-off centrifugal device and stored at -30°C in presence of 10% glycerol. 2.7 Functional characterization of recombinant buffalo CRISP-1 peptide 2.7.1 Processing semen sample Semen samples were collected from three buffalo bull by using an artificial vagina at Animal Breeding Research Centre of the institute. Following subjective analysis samples having mass motility score > 3 was only considered and diluted in Tris-Fructose-citric acid buffer having 15% egg yolk. Diluted sample vials were placed in a beaker having 30°C prewarmed water and the beaker was transferred in to a refrigerator maintained at 3–5°C. Live motile sperm were separated by 50% Percoll gradient centrifugation (600 × g for 10 min) and washed with spTALP media [ 14 ] by spinning at 150 ×g for 5 min and maintained in spTALP having 0.3% heat-inactivated bovine serum albumin (BSA). 2.7.2 Effect on sperm motility Sperm concentration in the Percoll-washed semen sample was adjusted to 20 × 10 6 sperm/mL with spTALP and 50 µL aliquots were taken in 0.5 mL eppendorf tubes. Recombinant buffalo CRISP-1 peptide was added to this sample at 20µg/mL and incubated at 37°C for 5 min. Sperm motility was analyzed from five random fields by using a CASA system (SCA-Vet, Microoptic, Spain). Sperm having curvilinear velocity > 25 µm/s and 80% straightness were considered progressively motile. 2.7.3 Effect on sperm capacitation Percoll-washed semen samples were adjusted to a concentration of 100 × 10 6 sperm/mL with spTALP. Capacitation buffer consisted of spTALP having 10 mM L-arginine and 25 mM sodium bicarbonate. To assess the effect of recombinant buffalo CRISP-1 peptide on sperm capacitation, 100 µL semen sample was added to 400 µL capacitation media (20 × 10 6 sperm/mL, final) and supplemented with either 20µg/mL CRISP-1 peptide or heat-inactivated CRISP-1 peptide. Sperm only in capacitation media was kept as a control. Samples were incubated at 37°C for 5 h in presence of 5% CO 2 and 95% humidity. Following incubation sperm were washed twice with spTALP and CTC (chlortetracycline fluorescent) staining was carried out for analysis of sperm capacitation. In brief, 100 µL sperm sample was mixed with 100 µL of CTC solution (600 µM CTC in 5 mM L-cysteine buffer, pH 7.8) and incubated in dark at 37°C and finally fixed with 10µL glutaraldehyde in 1 M Tris buffer, pH 7.0. Sperm were examined at 400 X magnification by using blue filter under a fluorescent microscope (BX51, Olympus) and 100 sperm were examined and three distinct patterns were recorded as described previously [ 15 ]. 2.7.4 Effect on sperm viability First, Percoll-washed buffalo sperm (20 × 10 6 sperm/mL) was treated with or without 20µg/mL recombinant CRISP-1 peptide for 10 min at 37°C. To assess sperm viability Eosin-Nigrosin staining was performed as described previously [ 16 ]. In brief, 10 µL of semen sample was mixed with 30 µL of pre-warmed (37°C) stain solution on a clean glass slide placed on a stage warmer (37°C) and a thin smear was drawn after 1 min incubation. Slides were examined at 400X magnification under brightfield objective of a microscope (Panthera2, Motic, Japan) and 200 sperm were counted randomly. 2.8 Biophysical characterization of recombinant buffalo CRISP-1 peptide 2.8.1 Effect of pH on the activity of CRISP-1 peptide Percoll-washed sperm was resuspended in spTALP media at 100 × 10 6 sperm/mL. Recombinant CRISP-1 peptide (20µg/mL) was pre-treated with spTALP having different pH starting from pH 5 to 9 for 10 min. Then sperm was added to this pre-treated peptide samples (20 × 10 6 sperm/mL, final) and incubated at 37°C for 5 min before sperm motility was analyzed by CASA. 2.8.2 Effect of temperature on the activity of CRISP-1 peptide First, recombinant CRISP-1 peptide (20µg/mL) was taken in 50 µL volume of spTALP media and incubated at different temperatures (40, 50, 60, 70, 80 and 90°C) for 10 min. After bringing back the samples to room temperature, 10 µL of Percoll-washed sperm was added (20 × 10 6 sperm/mL, final), incubated at 37°C for 5 min and sperm motility was analysed by CASA. 2.9 Mechanistic characterization of buffalo CRISP-1 peptide 2.9.1 Effect on protein tyrosine phosphorylation Sperm capacitation reaction was carried out in 1 mL spTALP media having both 10 mM L-arginine and 25 mM HCO3 (−) and either in presence or absence of 20 µg/mL recombinant CRISP-1 peptide, as described before. Protein extraction for phosphoprotein analysis was carried out as described previously [ 17 ]. In brief, sperm were washed twice with phosphate buffered saline (PBS) having 1mM Na-orthovanadate by centrifugation at 600 ×g for 5 min. To sperm pellet, 25µL 2mM Na-orthovanadate, 1µL of 50mM AEBSF and 25µL of 2X Laemmli’s buffer was added, mixed by repeated pipetting and vortexing for 2 min. Samples were boiled at 100°C for 5 min, cooled to 25°C and centrifuged at 10,000×g for 10 min to separate clear supernatant. Twenty microlitre of these protein samples were run on a 10% SDS-PAGE gel and proteins were electrotransferred on a 0.45 µM nitrocellulose membrane. After blocking with 3% BSA for 1 h, the membrane was incubated with mouse monoclonal anti-phosphotyrosine antibody (Santacruz, CAT# sc-81465) at 1:2000 dilution in tris-buffered saline (TBS; 25 mM Tris, pH 7.4, 150 mM NaCl, 2mM KCl) for overnight at 4˚C. Following three washes with TBS-0.1%Tween 20 (TBS-T) for 5 min each, the membrane was incubated with goat anti-mouse IgG-HRP conjugate (1:1000; Merck) for 1 h at 25°C. Following three washes with TBS-T as described before a final wash was given with TBS only. Membrane was incubated with DAB substrate solution, prepared by dissolving a DAB tablet (Amresco, USA) in 10 mL distilled water and adding 15 µL H 2 O 2 to it, at 25°C in dark until optimal colour was developed. Reaction was stopped by washing the membrane with distilled water. Membrane was scanned and relative intensity of the phosphoprotein bands was measured by Quantity 1 software (Bio-Rad, USA). 2.9.2 Effect of CRISP-1 peptide on NO and HCO 3 (−) signalling pathway of sperm capacitation To analyse which signalling pathway was involved in CRISP-1-mediated inhibition of sperm capacitation, the sperm capacitation reaction was induced either by 25 mM NaHCO 3 or 10 mM L-arginine or both in spTALP media. In addition, 0.5 mM L-NAME, a specific inhibitor of nitric oxide synthase (NOS), was used as a negative control. Recombinant peptide (20µg/mL) was added to these sperm capacitation reactions to examine it’s effect on capacitation. In brief, Percoll-washed buffalo sperm was resuspended in spTALP media at 100 × 10 6 sperm/mL. Triplicate samples (20 × 10 6 sperm/mL) were taken each for Control (spTALP only), L-arginine, HCO 3 (−) , L-arginine & HCO 3 (−) , L-arginine & L-NAME, HCO 3 (−) &L-NAME, L-arginine & CRISP-1, HCO 3 (−) & CRISP-1, L-arginine, HCO 3 (−) & CRISP-1 in spTALP media and incubated at 37°C in presence of 5% CO 2 and 95% humidity for 5 h. Then, acrosome reaction was induced with 30µM progesterone and the incubation was continued for 30 min. Sperm were pelleted by centrifugation at 300×g for 5 min and the pellet was re-suspended in 100 µL spTALP. Smear was drawn on clean glass slides in duplicates and air dried. Giemsa staining was performed to analyse acrosome-reacted (AR) sperm as an indicator of sperm capacitation [ 14 ]. 2.9.3 Effect of CRISP-1 peptide on adenylyl cyclase/cAMP pathway To assess if CRISP-1 peptide could modulate adenylyl cyclase (AC)/cAMP pathway during sperm capacitation, the capacitation reaction was carried out either in presence or absence of recombinant buffalo CRISP-1 peptide (20µg/mL). Sperm were pretreated with CRISP-1 peptide for 15 min at 37°C and then incubated in capacitation media (spTALP having 25 mM HCO 3 (−) and 10 mM L-arginine) at 50 × 10 6 sperm/mL. Forskolin (10 µM), a direct activator of adenylyl cyclase, was used either alone, as a positive control, or with CRISP-1 peptide to examine latter’s effect on AC/cAMP signalling pathway. Following 5 h incubation in capacitation media as before acrosome reaction was induced by adding 30µM progesterone to the samples and incubation was continued for another 30 min. Giemsa staining was performed to analyse acrosome reacted sperm. 2.9.4 Effect of CRISP-1 peptide on intracellular [Ca + 2 ] during sperm capacitation To examine if CRISP-1 peptide could affect influx of calcium increasing intracellular concentration of Ca + 2 during sperm capacitation, FURA-2AM ratiometric staining was performed. Free FURA-2 dye gives intense fluorescence at 380 nm excitation wavelength, while it’s fluorescence diminishes when it is bound to Ca + 2 . The Ca + 2 -bound FURA-2 gives fluorescence at 340 nm excitation wavelength. In brief, sperm capacitation reaction was carried out in presence or absence of CRISP-1 peptide (20µg/mL) as before. Forskolin (10 µM), an activator of both AC and Ca 2+ influx, was used either alone, as a positive control, or with the CRISP-1 peptide to examine latter’s inhibitory effect on Ca + 2 influx. After 3 h of incubation at 37°C in presence of 5% CO 2 and 95% humidity in respective buffers sperm were washed with spTALP by spinning at 300 x g for 5 min. Sperm were incubated with 2µM FURA-2AM in spTALP having 0.05% pluronic acid F-127 at 37°C for 30 min. After incubation, sperm were washed twice with spTALP as before to remove extra dye. Sperm were then mounted on a glass slide after mixing with DABCO anti-fading reagent (Fluoromount-G, Invitrogen). Sperm images were captured at 1000X magnification by using a confocal laser beam microscope (Olympus, Japan) at excitation and emission wavelengths of 380 and 510 nm, respectively. Sperm showing high tail fluorescence were due to free dye and considered having less Ca + 2 , while those showing low tail fluorescence were considered having more Ca + 2 bound to the dye. 2.10 Tissue expression analysis and sperm localization of CRISP-1 protein 2.10.1 Raising polyclonal antibody against buffalo CRISP-1 peptide Primary immunization was carried out in a female rabbit by sub-cutaneous injection of 100 µg of recombinant CRISP-1 peptide mixed with 250µL Freund’s complete adjuvant (Sigma, USA) at the flank region. Three booster injections of 50 µg CRISP-1 peptide mixed with Freund’s incomplete adjuvant (Sigma, USA) were given on 7th, 14th and 28th days. Blood was collected from the femoral vein after 7 days of last injection and serum was separated and stored at -20°C. 2.10.2 RT-PCR analysis of tissue samples cDNA was synthesized from the RNA samples isolated from the tissue samples of different parts of testes, epididymis and vas deferens. PCR was carried out for CRISP-1 gene by using cDNA, synthesized from the RNA samples, as template and analyzed by agarose gel electrophoresis. 2.10.3 Western blot analysis Tissue samples from testes, caput-, corpus- and cauda epididymis and vas deferens were homogenized in RIPA cell lysis buffer containing 1 µM AEBSF and clarified by centrifugation at 10,000 × g for 10 min at 4°C. The tissue lysates (30µg total peptide) were subjected to SDS-PAGE followed by electrotransfer on nitrocellulose membrane. Then the membrane was blocked with 3% BSA for 1 h at 25°C and incubated with anti-CRISP-1 antisera (1:1000) at 25°C for 2 h. Following three washes with TBS-T (TBS containing 0.1% Tween 20) the membrane was incubated with HRP-conjugated anti-rabbit IgG (1:2000) for 1 h at 25°C. Finally, the membrane was washed three times with TBS-T and incubated with DAB substrate solution at 25°C in dark. Once optimal color was developed the reaction was stopped by washing the membrane with distilled water. 2.10.4 Immunocytochemistry analysis Percoll-washed buffalo sperm was smeared on glass slides in duplicates and air dried. The smear was fixed with chilled methanol for 10 min and washed with PBS. Following blocking the non-specific sites with 5% BSA for 1 h, the slides were incubated with anti-CRISP-1 antisera (1:1000) for 2 h at 25°C. Following three washes with TBS-T the slides were incubated with anti-rabbit IgG-FITC-conjugate (1:2000) for 1 h at 25°C in dark. Following three washes with TBS-T, a drop of DABCO fixative (Invitrogen, USA) was put on slide and cover-slipped. Slides were examined at 1000X magnification under a confocal microscope (Olympus). 2.11 Statistical analysis The sperm motility and other data in percent were converted to arc sin square root transformation before statistical analysis. Mean ± SEM of different treatment groups was calculated by using GraphPad Prism 7.0. Statistical significance of differences between the means was evaluated by One-way ANOVA followed by Tukey’s post hoc test and was considered significant when P < 0.05. 3. Results 3.1 Cloning of buffalo CRISP-1 cDNA in bacterial expression vector The 687 bp buffalo CRISP-1 cDNA was successfully amplified at the annealing temperatures of 59, 60 and 61°C (Fig. 1 ). The PCR amplicon was cloned in to pET22b(+) bacterial expression vector by sequential steps of restriction endonuclease digestion, ligation and transformation in to E. coli DH5α cells. The transformed colonies were screened by carrying out colony-PCR which showed successful amplification of the 687 bp CRISP-1 product (Fig. 2 A). The restriction endonuclease digestion of the recombinant pET22b-CRISP1 plasmids isolated from the overnight cultures of the colony-PCR positive bacterial colonies showed release of the 687 bp CRISP-1 insert on agarose gel electrophoresis (Fig. 2 B). 3.2 Expression and purification of recombinant buffalo CRISP-1 peptide The recombinant buffalo CRISP-1 peptide was successfully expressed both in BL21(DE3) and BL21(DE3)-codon plus E. coli strains following induction with 0.1 to 1 mM IPTG both at 27°C for 6 h as well as at 23°C for 14 h. However, the expression was higher in BL21 (DE3)-codon plus cells as compared with BL21 (DE3); and after 14 h of induction at 23°C compared with 6 h induction at 27°C (Fig. 3 ). No significant effect of IPTG concentration on protein expression was observed, hence 0.25 mM IPTG level was randomly selected. The analysis of solubility of the expressed peptide revealed expression of the recombinant CRISP-1 peptide exclusively in insoluble form (Fig. 4 ). The purification of recombinant buffalo CRISP-1 peptide was carried out by Ni-NTA affinity chromatography and the peptide was successfully eluted both with 40 and 80 mM imidazole in almost pure form (Fig. 5 ). The affinity purified CRISP-1 peptide was subjected to dialysis to remove urea and assist in refolding. After the dialysis the recombinant peptide was successfully refolded in to soluble form with almost 100% recovery. The yield of the recombinant peptide was about 1 mg/ litre of E. coli culture. 3.3 Functional characterization of recombinant buffalo CRISP-1 peptide The recombinant CRISP-1 peptide at 20 µg /mL caused a significant inhibition of sperm progressive motility (about 46%) within 5 min of incubation. All sperm kinetic parameters (VSL, VAP and VCL) were significantly reduced in presence of the protein (Table 1 ). Table 1 Effect of recombinant buffalo CRISP-1 peptide on motility of buffalo sperm Sperm attributes Control CRISP-1 peptide PM (%) 61.27 ± 3.09 a 33.40 ± 2.09 b TM (%) 92.1 ± 0.96 a 88.65 ± 0.39 b VCL (µm/s) 75.48 ± 5.31 a 48.43 ± 1.87 b VAP (µm/s) 45.45 ± 2.34 a 25.77 ± 1.75 b VSL (µm/s) 33.80 ± 1.66 a 16.77 ± 1.39 b STR (%) 70.51 ± 1.44 a 59.66 ± 0.20 b WOB (%) 59.76 ± 1.16 a 51.34 ± 1.42 b BCF (Hz) 14.97 ± 0.42 a 8.47 ± 0.48 b Control: Percoll-washed buffalo sperm in spTALP media, CRISP-1 peptide: Percoll-washed sperm was incubated with 20µg /mL recombinant CRISP-1 peptide for 5 min at 37°C; PM: Progressive motility, TM: Total motility, VCL: Curvilinear velocity, VAP: Avg. path velocity, VSL: straight-line velocity, STR: Straightness index, LIN: Linearity index, WOB: Oscillation index, ALH: Amplitude lateral head, BCF: Beat crossfrequency; Data represents mean ± SEM. Mean values having different superscripts (a, b) differ significantly between columns (n = 3; p < 0.05) The effect of recombinant buffalo CRISP-1 peptide (20µg/mL) on sperm capacitation was also evaluated which showed a significant (P < 0.05) reduction in sperm capacitation in presence of the peptide (Fig. 6 ). The effect of CRISP-1 peptide on sperm viability was also assessed to rule out any toxic effect of the peptide on sperm. No significant change in sperm viability (98.0 ± 1.0 vs. 97.0 ± 1.2%) was observed following incubation of buffalo sperm with 20 µg/mL recombinant CRISP-1 peptide. 3.4 Biophysical characterization of recombinant buffalo CRISP-1 peptide 3.4.1 Effect of pH on the activity of CRISP-1 peptide In the control samples, sperm progressive motility was absent at pH 5.0, while no significant effect of pH between pH 6 and 9 was observed on sperm motility (Fig. 7 ). However, in presence of recombinant CRISP-1 peptide, progressive motility was significantly reduced between pH 6 and 9, and the maximum reduction was observed at pH 8.0. 3.4.2 Effect of temperature on the activity of CRISP-1 peptide Analysis of the effect of temperature on the activity of CRISP-1 peptide revealed that heat treatment of the protein up to 60°C did not affect it’s activity; however, heat treatment above this temperature caused a decrease in the activity of the peptide (Fig. 8 ). 3.5 Mechanistic characterization of recombinant buffalo CRISP-1 peptide 3.5.1 Effect on sperm protein tyrosine phosphorylation Phosphorylation at tyrosine residues of some target proteins following cAMP- mediated activation of protein kinase A (PKA) is a known molecular event during sperm capacitation. Results of this study clearly showed that presence of recombinant CRISP-1 peptide in capacitating media significantly reduced the tyrosine phosphorylation of both 72 (p72) and 47 (p47) kDa proteins in buffalo sperm (Fig. 9 ). 3.5.2 Effect of buffalo CRISP-1 peptide on NO and HCO 3 (−) signalling pathway After the incubation in capacitation media for 5 h the semen samples were further incubated with progesterone for 30 min to induce acrosome reaction in the capacitated sperm only as described previously [ 14 ] and assessed by Giemsa staining. Capacitation was found to be significantly higher both in L-arginine and -HCO 3 (−) groups as compared with control (Fig. 10 ); however, the capacitation was higher in presence of L-arginine than -HCO 3 (−) . Similarly, when both L-arginine and -HCO 3 (−) were present the capacitation was higher than either of these alone. L-NAME (specific inhibitor of NOS) reduced (P < 0.05) L-arginine-mediated sperm capacitation. Recombinant buffalo CRISP-1 peptide reduced (P < 0.05) sperm capacitation induced by either L-arginine or -HCO 3 (−) or both. 3.5.3. Effect of buffalo CRISP-1 peptide on adenylyl cyclase/cAMP pathway The acrosome reacted sperm (%) increased significantly both in capacitation media and in presence of 10µM forskolin as compared with the control (spTALP); while the presence of peptide decreased the acrosome reacted sperm (%) both in capacitation media and forskolin-treated samples (Fig. 11 ). 3.5.4. Effect of CRISP-1 peptide on intracellular Ca + 2 concentration during sperm capacitation The tail fluorescence was lower both in the capacitation and forskolin-treated groups as compared with the control. Presence of CRISP-1 peptide increased the tail fluorescence both in capacitation as well as forskolin groups as compared with these alone (Fig. 12 ). Based on the results obtained in this study, the possible mechanism of action of recombinant buffalo CRISP-1 peptide for inhibition of sperm motility and capacitation is outlined in the Fig. 13 . 3.6. Tissue expression analysis and sperm localization of CRISP-1 protein RT-PCR analysis of CRISP-1 gene expression in different tissues of male reproductive tract revealed CRISP-1 was expressed mainly in cauda epididymis, while a very scanty expression was found in caput epididymis of buffalo (Fig. 14 A). Western blot analysis of the proteins extracted from different regions of epididymis, testis and vas deferens revealed that buffalo CRISP-1 protein was expressed both in cauda epididymis and vas deferens; however, expression was not detected in other parts of epididymis or testis (Fig. 14 B). Immunocytochemistry analysis showed that CRISP-1 protein was located mainly on acrosome; however, reaction was also seen in principal piece of sperm tail (Fig. 14 C). 4. Discussion In the current study, prokaryotic expression yielded a modest amount (about 1 mg/ litre culture) of the histidine-tagged recombinant buffalo CRISP-1 peptide by using pET22b(+) expression vector and BL-21(DE3)-codon plus E. coli strain following induction with 0.25 mM IPTG at 23°C for 14 h. Since the CRISP-1 protein contains 16 cysteine residues that are engaged in intra-molecular disulfide bonds, earlier attempts to produce recombinant CRISP-1 protein by using bacterial expression system resulted in misfolded and insoluble protein [ 7 , 18 ]. Therefore, in the present study several strategies were adopted to obtain soluble expression of the recombinant peptide in E. coli. Firstly, the pET22b(+) expression vector was used to achieve better folding of the expressed protein. The pelB sequence present in the vector adds about 2.1 kDa pelB leader sequence at the N-terminus of the recombinant peptide which directs the peptide to bacterial periplasm. With disulfide oxidoreductase and isomerase located in the periplasm of E. coli, disulfide bonds can form, facilitating the accumulation of properly folded proteins [ 19 ]. Secondly, to facilitate proper folding of the recombinant peptide, the expression was regulated by keeping IPTG at a lower level (0.25 mM) and growing the cells at low temperature (23°C). However, despite all these measures the recombinant CRISP-1 peptide was expressed exclusively in insoluble form. This could be due to considerably higher number of disulfide bonds present in the peptide, those might be crucial for proper folding of the peptide. In a previous study, despite implementing various strategies for enhancing solubility of the expressed protein, the histidine-tagged rat CRISP-1 protein was expressed exclusively in insoluble form [ 18 ]. In a previous study in our lab the expression of thioredoxin-tagged ovine CRISP-1 peptide by using pET32b(+) vector and E. coli BL21(DE3) strain resulted in expression of the protein exclusively in insoluble form [ 7 ]. In the current study, the results suggested that the recombinant buffalo CRISP-1 peptide had successfully undergone proteolytic cleavage and the pelB leader sequence was removed from the mature CRISP-1 peptide. The above was evidenced from the molecular size of the peptide i.e. about 26.0 kDa instead of 28.5 kDa, if the pelB sequence were intact. Further, in the present study the protein refolding protocol consisting of slow dialysis in presence of proline and GSH/GS-SG, could successfully and completely solubilise the recombinant peptide. This might be due to periplasmic translocation of the peptide which might helped in formation of some of the disulfide bonds of the peptide in the presence of more oxidative environment of the periplasm facilitating refolding of the peptide during dialysis. The complete solubilization of the misfolded peptide after the dialysis, even though it contained a large number of disulfide bonds, achieved in the present study could also be due to the fact that the solubilization of CRISP proteins does not require all of it’s cysteine residues to be engaged in disulfide bonds [ 20 ]. In the current study, the recombinant buffalo CRISP-1 peptide (20µg/ml) demonstrated significant sperm-motility inhibitory effects, resulting in approximately 50% reduction in sperm progressive motility. The sperm-quiescent activity of CRISP-1 protein was not reported before and we were the first to report in sheep (Jorasia et al., 2021) [ 7 ]. Hence, the present study further confirmed the motility-inhibiting activity of CRISP-1 protein in buffalo sperm. Besides, several other sperm motility-inhibiting proteins were isolated from the reproductive tract of sheep, goat, and pig [ 4 , 5 , 6 , 21 ]. Therefore, it is quite pertinent that CRISP-1 protein, among others, plays an important role in sperm quiescence inside cauda epididymis. The results also suggested that the recombinant CRISP-1 peptide was active between pH 6 and 9 and the activity was optimum at pH 8. Further, the thermal stability study suggested that the peptide was thermolabile above 60°C temperature. The results also suggested that the recombinant CRISP-1 peptide was not toxic to the sperm, since sperm viability was not affected by the peptide. The recombinant buffalo CRISP-1 peptide also demonstrated a significant inhibition of sperm capacitation induced by either L-arginine or HCO 3 (−) which suggested that the peptide was a potent decapacitation factor. The decapacitating activity of CRISP-1 protein was also reported in earlier studies [ 3 , 8 , 22 ]. However, the detailed mechanism of action is yet to be understood. Therefore, in the present study the role of CRISP-1 peptide in modulating the signalling pathways associated with sperm capacitation was also investigated. The results suggested that both nitric oxide (NO) and HCO 3 (−) are involved in the capacitation of buffalo sperm, and the recombinant buffalo CRISP-1 peptide could interrupt the NO/HCO 3 (−) signalling pathway leading to reduction in sperm capacitation. Further, the peptide caused a significant reduction in sperm capacitation induced by forskolin, a potent activator of adenylyl cyclase/cAMP pathway, which suggested an interruption of the signalling pathway caused by the peptide. In addition, the present study also investigated the effect of CRISP-1 peptide on intracellular calcium concentration during sperm capacitation. The results clearly suggested that, presence of CRISP-1 peptide prevented increase of intracellular [Ca + 2 ] in buffalo sperm either during sperm capacitation or after treatment with forskolin. The decrease in intracellular [Ca + 2 ] in presence of CRISP-1 peptide might be due to blockage of CatSper1 ion channels caused by the peptide [ 8 ]. Further, the decrease in sperm motility observed in the presence of CRISP-1 peptide could be due to the decrease in intracellular [Ca + 2 ] caused by the peptide, since intracellular Ca + 2 is required for sperm motility [ 23 ]. Tyrosine phosphorylation of certain sperm proteins is a known molecular event during sperm capacitation. Hence, the present study also investigated the effect of CRISP-1 peptide on tyrosine phosphorylation of sperm proteins. The results suggested that the recombinant CRISP-1 peptide prevented tyrosine phosphorylation of several sperm proteins (p72, p47 and p28) under capacitating condition. Previous study also reported that the addition of exogenous CRISP-1 peptide resulted in reversible inhibition of protein tyrosine phosphorylation and modulation of the calcium-transporting CatSper1 ion channel [ 3 , 8 ]. However, the involvement of CatSper1 ion channel in the activity of recombinant buffalo CRISP-1 peptide has not been examined in this study; hence is an area for future research. Further, in the present study tissue expression pattern of CRISP-1 protein in male reproductive tract of buffalo was investigated. The results of RT-PCR and western blot analysis together suggested that the CRISP-1 protein was expressed predominantly in cauda epididymis and vas deferens, and very less expression was detected both in caput and corpus epididymis; however the expression was absent in testis. In mice, CRISP-1 was reported to express in all regions of epididymis, but was preferentially expressed in cauda [ 18 ]. In the present study, CRISP-1 was localized mainly on acrosome of buffalo sperm, but also found in principal piece of sperm tail. Similar to present study, CRISP-1 was localized on dorsal region of acrosome in non-capacitated rat sperm [ 24 ]. Therefore, the results of buffalo CRISP-1 tissue expression and sperm localization in the present study corroborated with those of previous studies. 5. Conclusion Recombinant buffalo CRISP-1 protein, a potent sperm-quiescent and decapacitation factor of epididymis was produced in E. coli in bioactive form by using pET22b(+) vector and E. coli BL21(DE3)-codon plus cells. The peptide was found non-toxic and thermolabile (above 60°C); and the optimal pH for it’s activity was pH 8. It inhibited sperm capacitation by modulating the NO/HCO 3 − /AC/cAMP/Ca + 2 signalling pathway and reduced protein tyrosine phosphorylation. However, the effect of CRISP-1 peptide on preservation of buffalo semen is still unknown and is an active area of future research. Declarations Competing Interest The authors have no relevant financial or non-financial interests to disclose. Ethics Approval This study was performed in line with the principles of the Declaration of Helsinki. An ethical clearance was obtained from the Institute Animal Ethics Committee before conducting experiments on animals (Approval No. 49/IAEC/2023/04). Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Rajani Kr. Paul, Nikki Kumari and Om Prakash. The first draft of the manuscript was written by Rajani Kr. Paul and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by SERB/DST, New Delhi, India (Grant No. SRG/2022/002038). Dr Rajani Kr. Paul has received research support from SERB/DST. Acknowledgement The authors were thankful to the Director, ICAR-NDRI, Karnal (India) for providing the necessary facilities to carry out the research work. Data availability statement The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. The nucleotide sequence of buffalo CRISP-1 CDS can be accessed through NCBI accession no. GenBank: PV545921.1 References Thirunavukkarasu, M. and Kathiravan, G. (2009). Factors affecting conception rates in artificially inseminated bovines. Indian Journal of Animal Sciences, 79(9), p.871. Singh, I. and Balhara, A. K. (2016). New approaches in buffalo artificial insemination programs with special reference to India. Theriogenology 86:194-199. Roberts, K.P., Wamstad, J.A., Ensrud, K.M. and Hamilton, D.W. (2003). Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1. Biology of Reproduction, 69(2), pp.572-581. Das, S., Saha, S., Majumder, G. C. and Dungdung, S. R. (2010). Purification and characterization of a sperm motility inhibiting factor from caprine epididymal plasma. PLoS One, 5(8): e12039. Ghosh, P., Mukherjee, S., Bhoumik, A. and Dungdung, S.R. (2018). A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO‐cGMP pathway. Journal of Cellular Physiology, 233(5), pp.4345-4359. Lal, P., Jorasia, K., Rathore, N.S., Kumar, V., Singh, R., Moolchandrani, A. and Paul, R.K. (2024). Purification and partial characterization of a sperm motility‐inhibitory protein of ram cauda epididymal plasma. Cell Biochemistry and Function, 42(1), p.e3930. Jorasia, K., Paul, R.K., Rathore, N.S., Lal, P., Singh, R. and Sareen, M. (2021). Production of bioactive recombinant ovine cysteine-rich secretory protein 1 in Escherichia coli. Systems Biology in Reproductive Medicine, 67(6), pp.471-481. Ernesto, J.I., Weigel Muñoz, M., Battistone, M.A., Vasen, G., Martínez-López, P., Orta, G., Figueiras-Fierro, D., De la Vega-Beltran, J.L., Moreno, I.A., Guidobaldi, H.A. and Giojalas, L. (2015). CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization. Journal of Cell Biology, 210 (7), pp.1213-1224. Gibbs, G.M., Roelants, K. and O'bryan, M. K. (2008). The CAP superfamily: cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins—roles in reproduction, cancer, and immune defense. Endocrine Reviews, 29(7), pp.865-897. Guo, M., Teng, M., Niu, L., Liu, Q., Huang, Q. and Hao, Q. (2005). Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold. Journal of Biological Chemistry, 280(13), pp.12405-12412. Roberts, K.P., Johnston, D.S., Nolan, M.A., Wooters, J.L., Waxmonsky, N.C., Piehl, L.B., Ensrud‐Bowlin, K.M. and Hamilton, D.W. (2007). Structure and function of epididymal protein cysteine‐rich secretory protein‐1. Asian Journal of Andrology, 9(4), pp. 508-514. Da Ros, V.G., Maldera, J.A., Willis, W.D., Cohen, D.J., Goulding, E.H., Gelman, D.M., Rubinstein, M., Eddy, E.M. and Cuasnicu, P.S. (2008). Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1). Developmental Biology, 320(1), pp.12-18. Ellerman, D.A., Da Ros, V.G., Cohen, D.J., Busso, D., Morgenfeld, M.M. and Cuasnicú, P.S. (2002). Expression and structure-function analysis of de, a sperm cysteine-rich secretory protein that mediates gamete fusion. Biology of Reproduction, 67(4), pp.1225-1231. Roy, S.C. and Atreja, S.K. (2008). Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway. International Journal of Andrology, 31(1), pp.12-24. Paul, R. K., Balaganur, K., Kumar, D. & Naqvi, S.M.K. (2018). Modulation of seminal plasma content in extended semen improves the quality attributes of ram spermatozoa following liquid preservation at 3 – 5 °C. Reproduction in Domestic Animals, 53 , 1200-1210. Bjorndahl, L., Soderlund, I., Kvist, U. (2003). Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Human Reproduction, 18 , 813–816. Galantino-Homer, H.L., Visconti, P.E. and Kopf, G.S. (1997). Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3', 5'-monophosphate-dependent pathway. Biology of Reproduction, 56(3), pp.707-719. Reddy, T., Gibbs, G.M., Merriner, D.J., Kerr, J.B. and O'Bryan, M.K. (2008). Cysteine‐rich secretory proteins are not exclusively expressed in the male reproductive tract. Developmental Dynamics: An official publication of the American Association of Anatomists, 237(11), pp.3313-3323. Kashani, H.H., and Moniri, R. (2015). Expression of recombinant pET22b-LysK-cysteine/histidine-dependent amidohydrolase/peptidase bacteriophage therapeutic protein in Escherichia coli BL21 (DE3). Osong Public Health and Research Perspectives, 6(4), 256-260. Sevier, C. S. and Kaiser, C.A. (2002). Formation and transfer of disulphide bonds in living cells. Nature Reviews Molecular Cell Biology, 3(11), pp.836-847. Jeng, H., Liu, K.M. and Chang, W.C. (1993). Purification and characterization of reversible sperm motility inhibitors from porcine seminal plasma. Biochemical and Biophysical Research Communications, 191(2), pp.435-440. Nixon, B., MacIntyre, D.A., Mitchell, L.A., Gibbs, G.M., O’Bryan, M. and Aitken, R.J. (2006). The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors. Biology of Reproduction, 74(2), pp.275-287. Chakraborty, S., Saha, S. (2022). Understanding sperm motility mechanisms and the implication of sperm surface molecules in promoting motility. Middle East Fertil Soc J, 27 , p.4. https://doi.org/10.1186/s43043-022-00094-7 Cohen D.J., Maldera J.A., Muñoz M.W., Ernesto J.I., Vasen G. and Cuasnicu P.S. (2011). Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization. Biol. Res.,44 (2),135-138 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6680127","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466248677,"identity":"7339c24f-0c46-4ac9-b9aa-192e82367869","order_by":0,"name":"Nikki Kumari","email":"","orcid":"","institution":"NDRI: National Dairy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Nikki","middleName":"","lastName":"Kumari","suffix":""},{"id":466248678,"identity":"fd31603f-04c2-4dff-a581-6d6a21d7fc52","order_by":1,"name":"Om Prakash","email":"","orcid":"","institution":"NDRI: National Dairy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Om","middleName":"","lastName":"Prakash","suffix":""},{"id":466248679,"identity":"1e7546c5-47a7-438a-b5ee-20f64d293afe","order_by":2,"name":"Rajani Kr. Paul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYDCCA0AswcDAw8befODAByCHjZ1YLXw8xxIfzgBpYSZGCwjISeQYG/OAWIS08N0++/CDRc0dGTaJtDRpm1/b5PmYGRg/fMzBrUXyXLqxhMSxZzxsPI+PSef23TZsY2Zglpy5DbcWgzNsDBISbIeB3gfakttzmxGohY2ZF78W5h8S/4BaGHLMpC17btsTo4VNQrINqIUD6H2GH7cTCWqRBGqxkOwDagEFcm/D7eQ2ZsZmvH7hAzrstsS3w/by7cCo/PHntu389uaDHz7i0QICzBIwFmMbmGzArx6k5AOc+Yeg4lEwCkbBKBiBAAC9Lk1Oy0hDDAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4794-0529","institution":"NDRI: National Dairy Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Rajani","middleName":"Kr.","lastName":"Paul","suffix":""}],"badges":[],"createdAt":"2025-05-16 11:03:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6680127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6680127/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84323368,"identity":"28d9f2f6-0770-4d61-a4e3-d94d7e270dbe","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39468,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis analysis of buffalo CRISP-1 PCR amplicon\u003c/p\u003e\n\u003cp\u003eL1: 1 Kb DNA ladder; Lane-2-4: CRISP-1 product at 59, 60 and 61° C T\u003csub\u003eA, \u003c/sub\u003erespectively\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/37a11dd7a1b6dc9acb58d616.png"},{"id":84325052,"identity":"2b72f995-88a9-4816-be5e-b707f034675c","added_by":"auto","created_at":"2025-06-10 14:59:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70820,"visible":true,"origin":"","legend":"\u003cp\u003eConfirmation of DNA cloning\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Colony PCR; \u003cstrong\u003eB\u003c/strong\u003e Restriction endonuclease digestion of recombinant pET22b-CRISP1 plasmid, M: 1 Kb DNA marker; Lane-1-5: CRISP-1 PCR amplicon from 5 colonies; DD: double RE digestion, SD: Single RE digestion\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/9347a7e6aad9578fe68ce46c.png"},{"id":84323373,"identity":"3c48caaa-6e86-4646-9def-21a218bd8e7a","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100456,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of recombinant buffalo CRISP-1 peptide expression in E. coli\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003eBL21(DE3) codon plus strain; \u003cstrong\u003eB\u003c/strong\u003eBL21(DE3) strain; M: protein marker, U: un-induced cell lysates; 0.1, 0.25, 0.5 and 1.0: protein expression induced with 0.1, 0.25, 0.5 and 1.0 mM IPTG\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/c4f23b6cbb031846ca1dc124.png"},{"id":84324133,"identity":"66c10578-672c-4f9e-af40-9f57378876f3","added_by":"auto","created_at":"2025-06-10 14:51:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90610,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of bacterial lysates to examine type of protein expression\u003c/p\u003e\n\u003cp\u003eUI: un-induced, N: native lysates, U: urea lysates, NB: native lysate of BL21(DE3); UB: urea lysates of BL21(DE3); NC+: native lysate of BL21(DE3) codon plus; UC+: urea lysate of BL21(DE3) codon plus\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/1bdf744a0217af7d83591ceb.png"},{"id":84323372,"identity":"9b1aa914-fbc8-499f-8fb6-ea876101db09","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95815,"visible":true,"origin":"","legend":"\u003cp\u003eNi-NTA affinity purification of recombinant CRISP-1 peptide\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Protein fractions after NI-NTA affinity chromatography, M: Protein marker, L1: crude lysates, L2: wash with 10mM imidazole in 8 M urea, L3-5: elutes with 20, 40 and 80 mM imidazole in 8 M urea buffer, respectively; \u003cstrong\u003eB\u003c/strong\u003e CRISP-1 peptide after dialysis, 80 mM: 80 mM imidazole fraction after dialysis\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/60d3ddd2f8646ed6a1d0f0a4.png"},{"id":84323378,"identity":"6e7f3e8d-4e0f-467f-8fc1-c6269b7b1988","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43292,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on sperm capacitation\u003c/p\u003e\n\u003cp\u003eControl: Percoll-washed sperm in spTALP, Capacitation: Washed sperm in capacitation media, Capacitation + Protein: Washed sperm in capacitation media having 20µg/ml CRISP-1 peptide. Data represented are mean ± SEM (n=3, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/4fd60f57590fbf64eaef7a2f.png"},{"id":84324134,"identity":"1c608d34-eb4a-4c5e-8438-131ab0b9fcb0","added_by":"auto","created_at":"2025-06-10 14:51:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":58159,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH on the activity of recombinant buffalo CRISP-1 peptide\u003c/p\u003e\n\u003cp\u003ePercoll-washed buffalo sperm was incubated in spTALP media having different pH for 10 min in presence or absence of CRISP-1 peptide (20µg/mL) and sperm progressive motility (PM) was analyzed by CASA. Data represented are mean ± SEM (n=3, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/ae91c5c0840343339a3fa977.png"},{"id":84324135,"identity":"e4da61b7-383e-43c0-86f7-42ac2b992b87","added_by":"auto","created_at":"2025-06-10 14:51:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":59109,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on sperm capacitation\u003c/p\u003e\n\u003cp\u003eBuffalo recombinant CRISP-1 peptide was pre-treated at different temperatures for 10 min, cooled to room temperature and added to Percoll-washed buffalo sperm at 20μg/ml and incubated for 5 min at 37 \u003cstrong\u003e˚\u003c/strong\u003eC. Sperm motility was analysed by CASA. Data represented are mean ± SEM (n=3, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/c08a285553659d862b6a60c5.png"},{"id":84325675,"identity":"70cca7f5-1e2d-4e18-84a1-1f8f0e16350e","added_by":"auto","created_at":"2025-06-10 15:07:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":80875,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CRISP-1 peptide on protein tyrosine phosphorylation\u003c/p\u003e\n\u003cp\u003eCon./Control: Percoll-washed buffalo sperm in spTALP, Cap./Capacitation: Washed sperm in capacitation media, Cap+Protein/Protein: Washed sperm in capacitation media having 20μg/mL recombinant buffalo CRISP-1 peptide. Data represented are mean ± SEM (n=3, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/3b338ffd6c5c29ec7a91f473.png"},{"id":84323385,"identity":"c1119d43-65f1-4247-bb0a-65a000729a1e","added_by":"auto","created_at":"2025-06-10 14:43:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":120260,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on L-arginine or bicarbonate mediated sperm capacitation\u003c/p\u003e\n\u003cp\u003eSperm capacitation was induced either with 10mM L-arginine or 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e or both in presence or absence of 20µg/mL recombinant buffalo CRISP-1 peptide. Following capacitation, AR was induced by 30µM progesterone. Sperm were stained with Giemsa and at least 200 sperm per sample were examined by a microscope at 400 X magnification. Data represented are mean ± SEM (n=5). Asterisk indicate significant difference between control and treatment groups (*p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/3ca0f1a285a4d83e34c567a3.png"},{"id":84323382,"identity":"dcf9d1ab-3d37-4b41-8144-519809d0532b","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":48035,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on AC/cAMP signalling during sperm capacitation, Control: spTALP, Cap.: capacitation buffer (25 mM HCO3 \u0026amp; 10 mM L-arginine in spTALP); Cap. + Protein: 20µg/mL CRISP-1 peptide in capacitation buffer, Forskolin: 10 µM Forskolin in spTALP; Forskolin + protein: 20µg/mL CRISP-1 peptide and 10 µM Forskolin in spTALP. Data represented are mean ± SEM (n=3, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/d588889f8d7473e998992880.png"},{"id":84323381,"identity":"1532e58e-026a-44ff-85d5-4c4e22a76a76","added_by":"auto","created_at":"2025-06-10 14:43:44","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":49001,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on intracellular Ca\u003csup\u003e+2\u003c/sup\u003e concentration during sperm capacitation\u003c/p\u003e\n\u003cp\u003eSperm tail fluorescence was captured by using a confocal microscope (Olympus, Japan) at 380 nm excitation and 510 nm emission wavelengths to analyse free FURA-2 dye. The free dye gives high fluorescence, while Ca\u003csup\u003e+2\u003c/sup\u003e-bound dye gives very less fluorescence at 380 nm excitation wavelength. Control: spTALP, Capacitation: capacitation buffer (25 mM HCO3 \u0026amp; 10 mM L-arginine in spTALP); Capacitation + Protein: 20µg/mL CRISP-1 peptide in capacitation buffer, Forskolin: 10 µM Forskolin in spTALP; Forskolin + protein:\u0026nbsp; 20µg/mL CRISP-1 peptide and 10 µM Forskolin in spTALP. Data represented are mean ± SEM (n=5, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/3adc4c2ee03a12328d9d1269.png"},{"id":84325056,"identity":"ae0211f0-27ef-4ca5-ac5a-68aaaed04231","added_by":"auto","created_at":"2025-06-10 14:59:45","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":204455,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram of possible mechanism of action of recombinant buffalo CRISP-1 peptide on buffalo sperm\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/e68553034755d6634a736a4d.png"},{"id":84325054,"identity":"cd1129ab-a23e-4fb0-bcc7-762ce6c8fded","added_by":"auto","created_at":"2025-06-10 14:59:45","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":218883,"visible":true,"origin":"","legend":"\u003cp\u003eTissue expression and sperm localization of buffalo CRISP-1 protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eRT-PCR analysis of CRISP-1 gene expression, Tes: testicular cortex, Cap: caput epididymis, Cor: corpus epididymis, Caud: cauda epididymis; \u003cstrong\u003eB \u003c/strong\u003eWestern blot analysis of CRISP-1 protein (in box) expression in different tissues of male reproductive tract, Testis: testis, Caput: caput epididymis, Corpus: corpus epididymis, Cauda: cauda epididymis, Vas: vas deferens; \u003cstrong\u003eC\u003c/strong\u003eLocalization of CRISP-1 protein on buffalo sperm by immuno-cytochemistry and confocal microscopy (1000 X magnification)\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/0e996a1e9b428f3ba83ec767.png"},{"id":84326724,"identity":"95938848-1f32-4766-be43-60972935dbaa","added_by":"auto","created_at":"2025-06-10 15:15:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2918305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6680127/v1/c89bbb95-27f0-4460-9f4d-927f525ed663.pdf"}],"financialInterests":"","formattedTitle":"Recombinant buffalo (Bubalus bubalis ) cysteine-rich secretory protein 1 mature peptide acts as a potent sperm-quiescent and decapcitation factor by modulating nitric oxide/bicarbonate/adenylyl cyclase/Ca+2 signalling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eConception rates in buffalo with cryopreserved semen are very low (30%) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The reduced fertility of cryopreserved buffalo semen is considered due to sub-lethal damages in sperm, including oxidative changes in membrane lipids and capacitation-like changes in sperm such as increase in intracellular calcium ([Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e), cAMP, protein tyrosine phosphorylation, and cholesterol efflux. Several proteins of cauda epididymis are known to cause reversible inhibition of sperm motility and capacitation and are considered responsible for sperm-quiescence and prolonged survival of sperm in cauda epididymis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymis known to inhibit sperm capacitation and motility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies conducted in vitro have demonstrated that the recombinant rat CRISP-1 peptide reversibly inhibits sperm capacitation and protein tyrosine phosphorylation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], by modulating CatSper1 channel [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, addition of recombinant buffalo CRISP-1 peptide to diluted semen prior cryopreservation might enhance the post-thaw quality and fertility of buffalo spermatozoa by reducing sperm capacitation induced by freezing-thawing process.\u003c/p\u003e \u003cp\u003eCRISP proteins belong to a sub-group of CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The CRISP proteins are characterised by the presence of N-terminal CAP domain (\u0026sim;21 kDa) that contains six conserved cysteine residues, and the smaller C-terminal cysteine-rich CRISP domain (\u0026sim;6 kDa) that contains 10 conserved cysteine residues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The cysteine residues are engaged in intra-domain disulphide bonds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Presence of this eight disulfide bonds render CRISP proteins structurally complex. In rat two forms of CRISP-1 protein (D and E) were identified. The D form is loosely associated to sperm and is released during capacitation and has been proposed as a decapacitation factor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. On ejaculation, the CRISP-1 protein is washed out by the secretions of accessory glands, thus allowing the pH\u003csub\u003ei\u003c/sub\u003e to increase and initiate sperm motility. In vitro study has shown that disassociation of CRISP-1 from the sperm membrane was required for capacitation to proceed in rat sperm [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].The E form of CRISP-1 protein binds tightly to sperm and is present even after capacitation. It is involved in both the interaction between sperm and the zona pellucida (ZP) as well as in the fusion of gametes by attaching to specific sites on the egg in mice [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the male reproductive tract, expression of CRISP-1 is primarily detected in the epididymis both at the mRNA and protein levels [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The protein is secreted into seminal plasma and, to some degree, attaches to the surface of spermatozoa. CRISP-1 appears to be involved in post-testicular sperm maturation and inhibition of premature sperm capacitation, both essential for eventual fusion with the oocyte membrane [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe investigation into the structure-function relationship of both natural and bacterially-generated recombinant CRISP-1 (DE) unveiled that the protein's functionality does not engage with carbohydrates but rather resides within the polypeptide chain of the molecule [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Hence, ovine CRISP-1 protein produced in bacteria was found to be bioactive [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recombinant CRISP-1 protein was shown to hinder sperm capacitation and impede protein tyrosine phosphorylation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Further, it was demonstrated that CRISP-1 protein inhibits sperm capacitation by modulating Ca\u003csup\u003e2+\u003c/sup\u003e-transporting CatSper channels in mouse sperm [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the detailed mechanism of action and involvement of different signalling molecules associated with sperm capacitation for the activity of CRISP-1 protein are largely unknown.\u003c/p\u003e \u003cp\u003eIn view of the above, the current study was designed to produce recombinant buffalo CRISP-1 mature peptide in E. coli and characterize it\u0026rsquo;s biophysical and functional properties, tissue expression pattern in male reproductive tract and mechanism of action.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eAll the reagents used in cloning and protein expression studies were of molecular biology grade and obtained from Thermo Scientific (USA) and Sigma Aldrich (USA). The E. coli strains DH5α, BL21 (DE3) and BL21 (DE3)-codon plus and bacterial expression vector pET22b (+) plasmid were received from Animal Biotechnology Division, ICAR-NDRI as a gift. Other chemicals used were recombinant DNAse I (Takara, Japan), DNA ligase (Invitrogen, USA), AEBSF (SRL, India), mouse monoclonal anti-phosphotyrosine antibody (sc-81465, Santacruz, USA), PrecisionPlus\u0026reg; prestained protein marker (Bio-Rad, USA), N\u003csub\u003eω\u003c/sub\u003e-nitro-L-arginine methyl ester HCL (L-NAME, Sigma), Forskolin (TCI, Japan) and FURA-2AM (Sigma).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cloning of buffalo CRISP-1 cDNA in bacterial expression vector\u003c/h2\u003e \u003cp\u003ePCR primers against a 687bp buffalo CRISP-1 mRNA (NCBI acc. no. \u003cb\u003eXM_006050714.4)\u003c/b\u003e corresponding to 228 amino acids length CRISP-1 mature peptide \u003cb\u003e(C\u003c/b\u003e\u003csup\u003e\u003cb\u003e15\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e-C\u003c/b\u003e\u003csup\u003e\u003cb\u003e242\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e was designed by \u003cb\u003ePrimer select\u003c/b\u003e tool of NCBI. The forward and reverse primers 5\u0026rsquo;\u003cb\u003e-\u003c/b\u003eAA\u003cb\u003eGGATCC\u003c/b\u003eTAGCTGCCTGCCTGTTTTGATT-3\u0026rsquo; and 5\u0026rsquo;-TT\u003cb\u003eAAGCTT\u003c/b\u003eACACATACAACTAGCTTTGCAGA-3\u0026rsquo; were flanked with Bam HI and Hind III sequences, respectively. Thermal cycle was optimized by doing gradient PCR: 94\u0026deg;C for 5 min, followed by 30 cycles each of 94\u0026deg;C for 30 s, 58\u0026ndash;62\u0026deg;C for 30 s and 72\u0026deg;C for 1 min with a final extension at 72\u0026deg;C for 10 min. Amplified product was analysed on 1% agarose gel and purified by a kit. Then both PCR product and pET22b(+) plasmids were subjected to restriction endonuclease digestion and gel purified using a kit (Takara). Ligation reaction was carried out overnight at 4\u0026deg;C using 1:4 molar ratio of plasmid and PCR amplicon, respectively. For bacterial transformation, 10 \u0026micro;L of ligation mix was mixed with chemically competent E. coli DH5α cells and heat shock was given for 60 s at 42\u0026deg;C. Transformed colonies were initially screened by colony PCR and PCR-positive transformants were confirmed by carrying out RE digestion analysis of the isolated plasmids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Optimization of protein expression in E. coli\u003c/h2\u003e \u003cp\u003eTo optimize protein production in bacterial cells we tested four IPTG concentrations (0.1, 0.25, 0.5, and 0.75 mM), two incubation temperature (23 and 27\u0026deg;C), and two bacterial host strains BL21(DE3) and BL21(DE3)-codon plus and two induction times (6 and 14 h). The recombinant plasmid was used to transform chemically-competent E. coli strains and resultant colony was grown in multiple vials of 10 mL Luria-Bertani (LB) broth having antibiotics till OD\u003csub\u003e600\u003c/sub\u003e reached 0.6. Then different conc. of IPTG was added to the culture and shaking was continued either at 27\u0026deg;C for 6 h or 23\u0026deg;C for 14 h. Bacterial pellet was mixed with 2X Laemmli\u0026rsquo;s buffer, mixed by vortexing and lysed by 10 min incubation at 95\u0026deg;C in water bath. Protein expression was analysed by SDS-PAGE.\u003c/p\u003e \u003cp\u003eFurther, to check the solubility of the expressed peptide, bacterial pellet was initially lysed with native lysis buffer (20 mM Tris, pH 8.0 having 0.5M NaCl, 1 mg/mL lysozyme, 1 U/ml DNAse I, 1 mM AEBSF) by sonication (20 pulses of 10 s at 70% amplitude, 30 kHz) on ice. The supernatant was collected after centrifugation at 10,000 \u0026times; g for 10 min at 4\u0026deg;C and the pellet was lysed with denaturing lysis buffer (8 M urea in 20 mM Tris, pH 8.0 having 0.5M NaCl, 20 mM 2-mercaptoethanol and 0.25% Triton X100) by vortexing followed by 1 h shaking at 25\u0026deg;C. Clear supernatant was collected after spinning at 10,000 \u0026times; g for 30 min at 4\u0026deg;C. Both the lysates were analysed by SDS-PAGE to find the presence of the recombinant peptide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Bulk production and purification of recombinant CRISP-1 peptide\u003c/h2\u003e \u003cp\u003eFor bulk production of recombinant peptide freshly transformed colonies of BL21(DE3)-codon plus cell were prepared on a LB agar plate; and then one colony was transferred in to 10 mL LB broth having ampicillin and chloramphenicol and shaken for 4 h at 37\u0026deg;C. Then this culture was transferred in to a flask containing 1000 mL fresh LB with ampicillin and the flask was shaken for 1 h at 37\u0026deg;C or till the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6. Then after bringing the culture at room temperature IPTG was added at 0.25 mM concentration and shaking was continued at 180 rpm for another 14 h at 23\u0026deg;C. The bacteria was pelleted by spinning at 7000 rpm and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Bacterial cell lysis and purification of recombinant peptide\u003c/h2\u003e \u003cp\u003e Bacterial pellet was thawed and lysed initially by native lysis buffer followed by denaturing lysis buffer as described before. For each gram of wet pellet 4 ml of native lysis buffer was added, mixed by vortexing, incubated at 25\u0026deg;C for 15 min and then sonicated on ice. After centrifugation and separation of the clear lysate, the pellet was washed twice with native lysis buffer (20 mM Tris, pH 8.0 having 0.5M NaCl) and finally lysed with denaturing lysis buffer. The clear lysate obtained from denaturing lysis was further clarified by passing through a 0.45 \u0026micro;m syringe filter.\u003c/p\u003e \u003cp\u003eFor purification of the recombinant peptide, Ni-NTA affinity chromatography was used. In brief, 5 mL of mixed gel slurry (70%; Takara) was washed twice each with distilled water and binding/column regeneration buffer (8 M urea in 20 mM Tris, 0.5M NaCl, pH 8.0) by spinning at 800 \u0026times; g for 5 min. The gel slurry was mixed with denaturing protein lysate and placed on a shaker at 25\u0026deg;C for 1 h to allow protein binding to the resin. The unbound proteins were removed by washing the column with 10 bed volumes of wash buffer I (binding buffer having 10 mM imidazole) followed by 5 bed volumes of wash buffer II (binding buffer having 20 mM imidazole). The recombinant peptide was eluted sequentially with 4 bed volumes of elution buffer I and II having 40 and 80 mM imidazole in binding buffer, respectively. The fractions were analyzed by SDS-PAGE to check the purity of the peptide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Dialysis and refolding of the peptide\u003c/h2\u003e \u003cp\u003eBoth the 40 and 80 mM imidazole elutes were pooled and concentrated by using a 10 kDa cut-off centrifugal device (Amicon Ultra 4, USA) and dialyzed against 10 mM potassium phosphate buffer, pH 7.5 having urea at gradual decreasing concentrations starting from 4 M to 0.5 M in a 1000 mL flask at 4\u0026deg;C. The dialysis was carried out slowly over a period of 48 h in a 10 kDa cut-off dialysis bag by replacing buffer every 6 h. To assist in protein folding both reduced and oxidized glutathione (1:0.1 mM) and 0.25 M proline was added to the dialysis buffer. Finally, the dialysis was continued only in 10 mM potassium phosphate buffer, pH 7.5 for 6\u0026ndash;8 h and peptide sample was concentrated by 10 kDa-cut-off centrifugal device and stored at -30\u0026deg;C in presence of 10% glycerol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Functional characterization of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.7.1 Processing semen sample\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eSemen samples were collected from three buffalo bull by using an artificial vagina at Animal Breeding Research Centre of the institute. Following subjective analysis samples having mass motility score\u0026thinsp;\u0026gt;\u0026thinsp;3 was only considered and diluted in Tris-Fructose-citric acid buffer having 15% egg yolk. Diluted sample vials were placed in a beaker having 30\u0026deg;C prewarmed water and the beaker was transferred in to a refrigerator maintained at 3\u0026ndash;5\u0026deg;C. Live motile sperm were separated by 50% Percoll gradient centrifugation (600 \u0026times; g for 10 min) and washed with spTALP media [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] by spinning at 150 \u0026times;g for 5 min and maintained in spTALP having 0.3% heat-inactivated bovine serum albumin (BSA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Effect on sperm motility\u003c/h2\u003e \u003cp\u003eSperm concentration in the Percoll-washed semen sample was adjusted to 20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL with spTALP and 50 \u0026micro;L aliquots were taken in 0.5 mL eppendorf tubes. Recombinant buffalo CRISP-1 peptide was added to this sample at 20\u0026micro;g/mL and incubated at 37\u0026deg;C for 5 min. Sperm motility was analyzed from five random fields by using a CASA system (SCA-Vet, Microoptic, Spain). Sperm having curvilinear velocity\u0026thinsp;\u0026gt;\u0026thinsp;25 \u0026micro;m/s and 80% straightness were considered progressively motile.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Effect on sperm capacitation\u003c/h2\u003e \u003cp\u003ePercoll-washed semen samples were adjusted to a concentration of 100 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL with spTALP. Capacitation buffer consisted of spTALP having 10 mM L-arginine and 25 mM sodium bicarbonate. To assess the effect of recombinant buffalo CRISP-1 peptide on sperm capacitation, 100 \u0026micro;L semen sample was added to 400 \u0026micro;L capacitation media (20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL, final) and supplemented with either 20\u0026micro;g/mL CRISP-1 peptide or heat-inactivated CRISP-1 peptide. Sperm only in capacitation media was kept as a control. Samples were incubated at 37\u0026deg;C for 5 h in presence of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity. Following incubation sperm were washed twice with spTALP and CTC (chlortetracycline fluorescent) staining was carried out for analysis of sperm capacitation. In brief, 100 \u0026micro;L sperm sample was mixed with 100 \u0026micro;L of CTC solution (600 \u0026micro;M CTC in 5 mM L-cysteine buffer, pH 7.8) and incubated in dark at 37\u0026deg;C and finally fixed with 10\u0026micro;L glutaraldehyde in 1 M Tris buffer, pH 7.0. Sperm were examined at 400 X magnification by using blue filter under a fluorescent microscope (BX51, Olympus) and 100 sperm were examined and three distinct patterns were recorded as described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4 Effect on sperm viability\u003c/h2\u003e \u003cp\u003eFirst, Percoll-washed buffalo sperm (20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL) was treated with or without 20\u0026micro;g/mL recombinant CRISP-1 peptide for 10 min at 37\u0026deg;C. To assess sperm viability Eosin-Nigrosin staining was performed as described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In brief, 10 \u0026micro;L of semen sample was mixed with 30 \u0026micro;L of pre-warmed (37\u0026deg;C) stain solution on a clean glass slide placed on a stage warmer (37\u0026deg;C) and a thin smear was drawn after 1 min incubation. Slides were examined at 400X magnification under brightfield objective of a microscope (Panthera2, Motic, Japan) and 200 sperm were counted randomly.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Biophysical characterization of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.8.1 Effect of pH on the activity of CRISP-1 peptide\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePercoll-washed sperm was resuspended in spTALP media at 100 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL. Recombinant CRISP-1 peptide (20\u0026micro;g/mL) was pre-treated with spTALP having different pH starting from pH 5 to 9 for 10 min. Then sperm was added to this pre-treated peptide samples (20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL, final) and incubated at 37\u0026deg;C for 5 min before sperm motility was analyzed by CASA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.8.2 Effect of temperature on the activity of CRISP-1 peptide\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFirst, recombinant CRISP-1 peptide (20\u0026micro;g/mL) was taken in 50 \u0026micro;L volume of spTALP media and incubated at different temperatures (40, 50, 60, 70, 80 and 90\u0026deg;C) for 10 min. After bringing back the samples to room temperature, 10 \u0026micro;L of Percoll-washed sperm was added (20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL, final), incubated at 37\u0026deg;C for 5 min and sperm motility was analysed by CASA.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Mechanistic characterization of buffalo CRISP-1 peptide\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Effect on protein tyrosine phosphorylation\u003c/h2\u003e \u003cp\u003eSperm capacitation reaction was carried out in 1 mL spTALP media having both 10 mM L-arginine and 25 mM HCO3\u003csup\u003e(\u0026minus;)\u003c/sup\u003e and either in presence or absence of 20 \u0026micro;g/mL recombinant CRISP-1 peptide, as described before. Protein extraction for phosphoprotein analysis was carried out as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In brief, sperm were washed twice with phosphate buffered saline (PBS) having 1mM Na-orthovanadate by centrifugation at 600 \u0026times;g for 5 min. To sperm pellet, 25\u0026micro;L 2mM Na-orthovanadate, 1\u0026micro;L of 50mM AEBSF and 25\u0026micro;L of 2X Laemmli\u0026rsquo;s buffer was added, mixed by repeated pipetting and vortexing for 2 min. Samples were boiled at 100\u0026deg;C for 5 min, cooled to 25\u0026deg;C and centrifuged at 10,000\u0026times;g for 10 min to separate clear supernatant. Twenty microlitre of these protein samples were run on a 10% SDS-PAGE gel and proteins were electrotransferred on a 0.45 \u0026micro;M nitrocellulose membrane. After blocking with 3% BSA for 1 h, the membrane was incubated with mouse monoclonal anti-phosphotyrosine antibody (Santacruz, CAT# sc-81465) at 1:2000 dilution in tris-buffered saline (TBS; 25 mM Tris, pH 7.4, 150 mM NaCl, 2mM KCl) for overnight at 4˚C. Following three washes with TBS-0.1%Tween 20 (TBS-T) for 5 min each, the membrane was incubated with goat anti-mouse IgG-HRP conjugate (1:1000; Merck) for 1 h at 25\u0026deg;C. Following three washes with TBS-T as described before a final wash was given with TBS only. Membrane was incubated with DAB substrate solution, prepared by dissolving a DAB tablet (Amresco, USA) in 10 mL distilled water and adding 15 \u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to it, at 25\u0026deg;C in dark until optimal colour was developed. Reaction was stopped by washing the membrane with distilled water. Membrane was scanned and relative intensity of the phosphoprotein bands was measured by Quantity 1 software (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.9.2 Effect of CRISP-1 peptide on NO and HCO\u003c/b\u003e\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e \u003cb\u003esignalling pathway of sperm capacitation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo analyse which signalling pathway was involved in CRISP-1-mediated inhibition of sperm capacitation, the sperm capacitation reaction was induced either by 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e or 10 mM L-arginine or both in spTALP media. In addition, 0.5 mM L-NAME, a specific inhibitor of nitric oxide synthase (NOS), was used as a negative control. Recombinant peptide (20\u0026micro;g/mL) was added to these sperm capacitation reactions to examine it\u0026rsquo;s effect on capacitation. In brief, Percoll-washed buffalo sperm was resuspended in spTALP media at 100 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL. Triplicate samples (20 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL) were taken each for Control (spTALP only), L-arginine, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e, L-arginine \u0026amp; HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e, L-arginine \u0026amp; L-NAME, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e \u0026amp;L-NAME, L-arginine \u0026amp; CRISP-1, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e \u0026amp; CRISP-1, L-arginine, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e \u0026amp; CRISP-1 in spTALP media and incubated at 37\u0026deg;C in presence of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity for 5 h. Then, acrosome reaction was induced with 30\u0026micro;M progesterone and the incubation was continued for 30 min. Sperm were pelleted by centrifugation at 300\u0026times;g for 5 min and the pellet was re-suspended in 100 \u0026micro;L spTALP. Smear was drawn on clean glass slides in duplicates and air dried. Giemsa staining was performed to analyse acrosome-reacted (AR) sperm as an indicator of sperm capacitation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3 Effect of CRISP-1 peptide on adenylyl cyclase/cAMP pathway\u003c/h2\u003e \u003cp\u003eTo assess if CRISP-1 peptide could modulate adenylyl cyclase (AC)/cAMP pathway during sperm capacitation, the capacitation reaction was carried out either in presence or absence of recombinant buffalo CRISP-1 peptide (20\u0026micro;g/mL). Sperm were pretreated with CRISP-1 peptide for 15 min at 37\u0026deg;C and then incubated in capacitation media (spTALP having 25 mM HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e and 10 mM L-arginine) at 50 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm/mL. Forskolin (10 \u0026micro;M), a direct activator of adenylyl cyclase, was used either alone, as a positive control, or with CRISP-1 peptide to examine latter\u0026rsquo;s effect on AC/cAMP signalling pathway. Following 5 h incubation in capacitation media as before acrosome reaction was induced by adding 30\u0026micro;M progesterone to the samples and incubation was continued for another 30 min. Giemsa staining was performed to analyse acrosome reacted sperm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.9.4 Effect of CRISP-1 peptide on intracellular [Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e] during sperm capacitation\u003c/h2\u003e \u003cp\u003eTo examine if CRISP-1 peptide could affect influx of calcium increasing intracellular concentration of Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e during sperm capacitation, FURA-2AM ratiometric staining was performed. Free FURA-2 dye gives intense fluorescence at 380 nm excitation wavelength, while it\u0026rsquo;s fluorescence diminishes when it is bound to Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e. The Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e-bound FURA-2 gives fluorescence at 340 nm excitation wavelength. In brief, sperm capacitation reaction was carried out in presence or absence of CRISP-1 peptide (20\u0026micro;g/mL) as before. Forskolin (10 \u0026micro;M), an activator of both AC and Ca\u003csup\u003e2+\u003c/sup\u003e influx, was used either alone, as a positive control, or with the CRISP-1 peptide to examine latter\u0026rsquo;s inhibitory effect on Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e influx. After 3 h of incubation at 37\u0026deg;C in presence of 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity in respective buffers sperm were washed with spTALP by spinning at 300 x g for 5 min. Sperm were incubated with 2\u0026micro;M FURA-2AM in spTALP having 0.05% pluronic acid F-127 at 37\u0026deg;C for 30 min. After incubation, sperm were washed twice with spTALP as before to remove extra dye. Sperm were then mounted on a glass slide after mixing with DABCO anti-fading reagent (Fluoromount-G, Invitrogen). Sperm images were captured at 1000X magnification by using a confocal laser beam microscope (Olympus, Japan) at excitation and emission wavelengths of 380 and 510 nm, respectively. Sperm showing high tail fluorescence were due to free dye and considered having less Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e, while those showing low tail fluorescence were considered having more Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e bound to the dye.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Tissue expression analysis and sperm localization of CRISP-1 protein\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1 Raising polyclonal antibody against buffalo CRISP-1 peptide\u003c/h2\u003e \u003cp\u003ePrimary immunization was carried out in a female rabbit by sub-cutaneous injection of 100 \u0026micro;g of recombinant CRISP-1 peptide mixed with 250\u0026micro;L Freund\u0026rsquo;s complete adjuvant (Sigma, USA) at the flank region. Three booster injections of 50 \u0026micro;g CRISP-1 peptide mixed with Freund\u0026rsquo;s incomplete adjuvant (Sigma, USA) were given on 7th, 14th and 28th days. Blood was collected from the femoral vein after 7 days of last injection and serum was separated and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2 RT-PCR analysis of tissue samples\u003c/h2\u003e \u003cp\u003ecDNA was synthesized from the RNA samples isolated from the tissue samples of different parts of testes, epididymis and vas deferens. PCR was carried out for CRISP-1 gene by using cDNA, synthesized from the RNA samples, as template and analyzed by agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3 Western blot analysis\u003c/h2\u003e \u003cp\u003eTissue samples from testes, caput-, corpus- and cauda epididymis and vas deferens were homogenized in RIPA cell lysis buffer containing 1 \u0026micro;M AEBSF and clarified by centrifugation at 10,000 \u0026times; g for 10 min at 4\u0026deg;C. The tissue lysates (30\u0026micro;g total peptide) were subjected to SDS-PAGE followed by electrotransfer on nitrocellulose membrane. Then the membrane was blocked with 3% BSA for 1 h at 25\u0026deg;C and incubated with anti-CRISP-1 antisera (1:1000) at 25\u0026deg;C for 2 h. Following three washes with TBS-T (TBS containing 0.1% Tween 20) the membrane was incubated with HRP-conjugated anti-rabbit IgG (1:2000) for 1 h at 25\u0026deg;C. Finally, the membrane was washed three times with TBS-T and incubated with DAB substrate solution at 25\u0026deg;C in dark. Once optimal color was developed the reaction was stopped by washing the membrane with distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e2.10.4 Immunocytochemistry analysis\u003c/h2\u003e \u003cp\u003ePercoll-washed buffalo sperm was smeared on glass slides in duplicates and air dried. The smear was fixed with chilled methanol for 10 min and washed with PBS. Following blocking the non-specific sites with 5% BSA for 1 h, the slides were incubated with anti-CRISP-1 antisera (1:1000) for 2 h at 25\u0026deg;C. Following three washes with TBS-T the slides were incubated with anti-rabbit IgG-FITC-conjugate (1:2000) for 1 h at 25\u0026deg;C in dark. Following three washes with TBS-T, a drop of DABCO fixative (Invitrogen, USA) was put on slide and cover-slipped. Slides were examined at 1000X magnification under a confocal microscope (Olympus).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe sperm motility and other data in percent were converted to arc sin square root transformation before statistical analysis. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of different treatment groups was calculated by using GraphPad Prism 7.0. Statistical significance of differences between the means was evaluated by One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test and was considered significant when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cloning of buffalo CRISP-1 cDNA in bacterial expression vector\u003c/h2\u003e \u003cp\u003eThe 687 bp buffalo CRISP-1 cDNA was successfully amplified at the annealing temperatures of 59, 60 and 61\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The PCR amplicon was cloned in to pET22b(+) bacterial expression vector by sequential steps of restriction endonuclease digestion, ligation and transformation in to E. coli DH5α cells. The transformed colonies were screened by carrying out colony-PCR which showed successful amplification of the 687 bp CRISP-1 product (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The restriction endonuclease digestion of the recombinant pET22b-CRISP1 plasmids isolated from the overnight cultures of the colony-PCR positive bacterial colonies showed release of the 687 bp CRISP-1 insert on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Expression and purification of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cp\u003eThe recombinant buffalo CRISP-1 peptide was successfully expressed both in BL21(DE3) and BL21(DE3)-codon plus E. coli strains following induction with 0.1 to 1 mM IPTG both at 27\u0026deg;C for 6 h as well as at 23\u0026deg;C for 14 h. However, the expression was higher in BL21 (DE3)-codon plus cells as compared with BL21 (DE3); and after 14 h of induction at 23\u0026deg;C compared with 6 h induction at 27\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No significant effect of IPTG concentration on protein expression was observed, hence 0.25 mM IPTG level was randomly selected. The analysis of solubility of the expressed peptide revealed expression of the recombinant CRISP-1 peptide exclusively in insoluble form (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe purification of recombinant buffalo CRISP-1 peptide was carried out by Ni-NTA affinity chromatography and the peptide was successfully eluted both with 40 and 80 mM imidazole in almost pure form (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The affinity purified CRISP-1 peptide was subjected to dialysis to remove urea and assist in refolding. After the dialysis the recombinant peptide was successfully refolded in to soluble form with almost 100% recovery. The yield of the recombinant peptide was about 1 mg/ litre of E. coli culture.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Functional characterization of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cp\u003eThe recombinant CRISP-1 peptide at 20 \u0026micro;g /mL caused a significant inhibition of sperm progressive motility (about 46%) within 5 min of incubation. All sperm kinetic parameters (VSL, VAP and VCL) were significantly reduced in presence of the protein (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of recombinant buffalo CRISP-1 peptide on motility of buffalo sperm\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm attributes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRISP-1 peptide\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVCL (\u0026micro;m/s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.48\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVAP (\u0026micro;m/s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVSL (\u0026micro;m/s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWOB (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCF (Hz)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eControl: Percoll-washed buffalo sperm in spTALP media, CRISP-1 peptide: Percoll-washed sperm was incubated with 20\u0026micro;g /mL recombinant CRISP-1 peptide for 5 min at 37\u0026deg;C; PM: Progressive motility, TM: Total motility, VCL: Curvilinear velocity, VAP: Avg. path velocity, VSL: straight-line velocity, STR: Straightness index, LIN: Linearity index, WOB: Oscillation index, ALH: Amplitude lateral head, BCF: Beat crossfrequency; Data represents mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Mean values having different superscripts (a, b) differ significantly between columns (n\u0026thinsp;=\u0026thinsp;3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003cp\u003eThe effect of recombinant buffalo CRISP-1 peptide (20\u0026micro;g/mL) on sperm capacitation was also evaluated which showed a significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in sperm capacitation in presence of the peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The effect of CRISP-1 peptide on sperm viability was also assessed to rule out any toxic effect of the peptide on sperm. No significant change in sperm viability (98.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 vs. 97.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%) was observed following incubation of buffalo sperm with 20 \u0026micro;g/mL recombinant CRISP-1 peptide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Biophysical characterization of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Effect of pH on the activity of CRISP-1 peptide\u003c/h2\u003e \u003cp\u003eIn the control samples, sperm progressive motility was absent at pH 5.0, while no significant effect of pH between pH 6 and 9 was observed on sperm motility (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, in presence of recombinant CRISP-1 peptide, progressive motility was significantly reduced between pH 6 and 9, and the maximum reduction was observed at pH 8.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Effect of temperature on the activity of CRISP-1 peptide\u003c/h2\u003e \u003cp\u003eAnalysis of the effect of temperature on the activity of CRISP-1 peptide revealed that heat treatment of the protein up to 60\u0026deg;C did not affect it\u0026rsquo;s activity; however, heat treatment above this temperature caused a decrease in the activity of the peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Mechanistic characterization of recombinant buffalo CRISP-1 peptide\u003c/h2\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Effect on sperm protein tyrosine phosphorylation\u003c/h2\u003e \u003cp\u003ePhosphorylation at tyrosine residues of some target proteins following cAMP- mediated activation of protein kinase A (PKA) is a known molecular event during sperm capacitation. Results of this study clearly showed that presence of recombinant CRISP-1 peptide in capacitating media significantly reduced the tyrosine phosphorylation of both 72 (p72) and 47 (p47) kDa proteins in buffalo sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.5.2 Effect of buffalo CRISP-1 peptide on NO and HCO\u003c/b\u003e\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e \u003cb\u003esignalling pathway\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAfter the incubation in capacitation media for 5 h the semen samples were further incubated with progesterone for 30 min to induce acrosome reaction in the capacitated sperm only as described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and assessed by Giemsa staining. Capacitation was found to be significantly higher both in L-arginine and -HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e groups as compared with control (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e); however, the capacitation was higher in presence of L-arginine than -HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e. Similarly, when both L-arginine and -HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e were present the capacitation was higher than either of these alone. L-NAME (specific inhibitor of NOS) reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) L-arginine-mediated sperm capacitation. Recombinant buffalo CRISP-1 peptide reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) sperm capacitation induced by either L-arginine or -HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e or both.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.5.3. Effect of buffalo CRISP-1 peptide on adenylyl cyclase/cAMP pathway\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe acrosome reacted sperm (%) increased significantly both in capacitation media and in presence of 10\u0026micro;M forskolin as compared with the control (spTALP); while the presence of peptide decreased the acrosome reacted sperm (%) both in capacitation media and forskolin-treated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.5.4. Effect of CRISP-1 peptide on intracellular Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u0026thinsp;2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003econcentration during sperm capacitation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe tail fluorescence was lower both in the capacitation and forskolin-treated groups as compared with the control. Presence of CRISP-1 peptide increased the tail fluorescence both in capacitation as well as forskolin groups as compared with these alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the results obtained in this study, the possible mechanism of action of recombinant buffalo CRISP-1 peptide for inhibition of sperm motility and capacitation is outlined in the Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.6. Tissue expression analysis and sperm localization of CRISP-1 protein\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eRT-PCR analysis of CRISP-1 gene expression in different tissues of male reproductive tract revealed CRISP-1 was expressed mainly in cauda epididymis, while a very scanty expression was found in caput epididymis of buffalo (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA). Western blot analysis of the proteins extracted from different regions of epididymis, testis and vas deferens revealed that buffalo CRISP-1 protein was expressed both in cauda epididymis and vas deferens; however, expression was not detected in other parts of epididymis or testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eB). Immunocytochemistry analysis showed that CRISP-1 protein was located mainly on acrosome; however, reaction was also seen in principal piece of sperm tail (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the current study, prokaryotic expression yielded a modest amount (about 1 mg/ litre culture) of the histidine-tagged recombinant buffalo CRISP-1 peptide by using pET22b(+) expression vector and BL-21(DE3)-codon plus E. coli strain following induction with 0.25 mM IPTG at 23\u0026deg;C for 14 h. Since the CRISP-1 protein contains 16 cysteine residues that are engaged in intra-molecular disulfide bonds, earlier attempts to produce recombinant CRISP-1 protein by using bacterial expression system resulted in misfolded and insoluble protein [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, in the present study several strategies were adopted to obtain soluble expression of the recombinant peptide in E. coli. Firstly, the pET22b(+) expression vector was used to achieve better folding of the expressed protein. The pelB sequence present in the vector adds about 2.1 kDa pelB leader sequence at the N-terminus of the recombinant peptide which directs the peptide to bacterial periplasm. With disulfide oxidoreductase and isomerase located in the periplasm of E. coli, disulfide bonds can form, facilitating the accumulation of properly folded proteins [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Secondly, to facilitate proper folding of the recombinant peptide, the expression was regulated by keeping IPTG at a lower level (0.25 mM) and growing the cells at low temperature (23\u0026deg;C). However, despite all these measures the recombinant CRISP-1 peptide was expressed exclusively in insoluble form. This could be due to considerably higher number of disulfide bonds present in the peptide, those might be crucial for proper folding of the peptide. In a previous study, despite implementing various strategies for enhancing solubility of the expressed protein, the histidine-tagged rat CRISP-1 protein was expressed exclusively in insoluble form [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In a previous study in our lab the expression of thioredoxin-tagged ovine CRISP-1 peptide by using pET32b(+) vector and E. coli BL21(DE3) strain resulted in expression of the protein exclusively in insoluble form [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, the results suggested that the recombinant buffalo CRISP-1 peptide had successfully undergone proteolytic cleavage and the pelB leader sequence was removed from the mature CRISP-1 peptide. The above was evidenced from the molecular size of the peptide i.e. about 26.0 kDa instead of 28.5 kDa, if the pelB sequence were intact. Further, in the present study the protein refolding protocol consisting of slow dialysis in presence of proline and GSH/GS-SG, could successfully and completely solubilise the recombinant peptide. This might be due to periplasmic translocation of the peptide which might helped in formation of some of the disulfide bonds of the peptide in the presence of more oxidative environment of the periplasm facilitating refolding of the peptide during dialysis. The complete solubilization of the misfolded peptide after the dialysis, even though it contained a large number of disulfide bonds, achieved in the present study could also be due to the fact that the solubilization of CRISP proteins does not require all of it\u0026rsquo;s cysteine residues to be engaged in disulfide bonds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, the recombinant buffalo CRISP-1 peptide (20\u0026micro;g/ml) demonstrated significant sperm-motility inhibitory effects, resulting in approximately 50% reduction in sperm progressive motility. The sperm-quiescent activity of CRISP-1 protein was not reported before and we were the first to report in sheep (Jorasia et al., 2021) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Hence, the present study further confirmed the motility-inhibiting activity of CRISP-1 protein in buffalo sperm. Besides, several other sperm motility-inhibiting proteins were isolated from the reproductive tract of sheep, goat, and pig [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, it is quite pertinent that CRISP-1 protein, among others, plays an important role in sperm quiescence inside cauda epididymis.\u003c/p\u003e \u003cp\u003eThe results also suggested that the recombinant CRISP-1 peptide was active between pH 6 and 9 and the activity was optimum at pH 8. Further, the thermal stability study suggested that the peptide was thermolabile above 60\u0026deg;C temperature. The results also suggested that the recombinant CRISP-1 peptide was not toxic to the sperm, since sperm viability was not affected by the peptide.\u003c/p\u003e \u003cp\u003eThe recombinant buffalo CRISP-1 peptide also demonstrated a significant inhibition of sperm capacitation induced by either L-arginine or HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e which suggested that the peptide was a potent decapacitation factor. The decapacitating activity of CRISP-1 protein was also reported in earlier studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the detailed mechanism of action is yet to be understood. Therefore, in the present study the role of CRISP-1 peptide in modulating the signalling pathways associated with sperm capacitation was also investigated. The results suggested that both nitric oxide (NO) and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e are involved in the capacitation of buffalo sperm, and the recombinant buffalo CRISP-1 peptide could interrupt the NO/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e(\u0026minus;)\u003c/sup\u003e signalling pathway leading to reduction in sperm capacitation. Further, the peptide caused a significant reduction in sperm capacitation induced by forskolin, a potent activator of adenylyl cyclase/cAMP pathway, which suggested an interruption of the signalling pathway caused by the peptide. In addition, the present study also investigated the effect of CRISP-1 peptide on intracellular calcium concentration during sperm capacitation. The results clearly suggested that, presence of CRISP-1 peptide prevented increase of intracellular [Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e] in buffalo sperm either during sperm capacitation or after treatment with forskolin. The decrease in intracellular [Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e] in presence of CRISP-1 peptide might be due to blockage of CatSper1 ion channels caused by the peptide [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Further, the decrease in sperm motility observed in the presence of CRISP-1 peptide could be due to the decrease in intracellular [Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e] caused by the peptide, since intracellular Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e is required for sperm motility [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTyrosine phosphorylation of certain sperm proteins is a known molecular event during sperm capacitation. Hence, the present study also investigated the effect of CRISP-1 peptide on tyrosine phosphorylation of sperm proteins. The results suggested that the recombinant CRISP-1 peptide prevented tyrosine phosphorylation of several sperm proteins (p72, p47 and p28) under capacitating condition. Previous study also reported that the addition of exogenous CRISP-1 peptide resulted in reversible inhibition of protein tyrosine phosphorylation and modulation of the calcium-transporting CatSper1 ion channel [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the involvement of CatSper1 ion channel in the activity of recombinant buffalo CRISP-1 peptide has not been examined in this study; hence is an area for future research.\u003c/p\u003e \u003cp\u003eFurther, in the present study tissue expression pattern of CRISP-1 protein in male reproductive tract of buffalo was investigated. The results of RT-PCR and western blot analysis together suggested that the CRISP-1 protein was expressed predominantly in cauda epididymis and vas deferens, and very less expression was detected both in caput and corpus epididymis; however the expression was absent in testis. In mice, CRISP-1 was reported to express in all regions of epididymis, but was preferentially expressed in cauda [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the present study, CRISP-1 was localized mainly on acrosome of buffalo sperm, but also found in principal piece of sperm tail. Similar to present study, CRISP-1 was localized on dorsal region of acrosome in non-capacitated rat sperm [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, the results of buffalo CRISP-1 tissue expression and sperm localization in the present study corroborated with those of previous studies.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eRecombinant buffalo CRISP-1 protein, a potent sperm-quiescent and decapacitation factor of epididymis was produced in E. coli in bioactive form by using pET22b(+) vector and E. coli BL21(DE3)-codon plus cells. The peptide was found non-toxic and thermolabile (above 60\u0026deg;C); and the optimal pH for it\u0026rsquo;s activity was pH 8. It inhibited sperm capacitation by modulating the NO/HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e/AC/cAMP/Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e signalling pathway and reduced protein tyrosine phosphorylation. However, the effect of CRISP-1 peptide on preservation of buffalo semen is still unknown and is an active area of future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. An ethical clearance was obtained from the Institute Animal Ethics Committee before conducting experiments on animals (Approval No. 49/IAEC/2023/04).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Rajani Kr. Paul, Nikki Kumari and Om Prakash. The first draft of the manuscript was written by Rajani Kr. Paul and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by SERB/DST, New Delhi, India (Grant No. SRG/2022/002038). Dr Rajani Kr. Paul has received research support from SERB/DST.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors were thankful to the Director, ICAR-NDRI, Karnal (India) for providing the necessary facilities to carry out the research work.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. The nucleotide sequence of buffalo CRISP-1 CDS can be accessed through NCBI accession no. \u003cb\u003eGenBank: PV545921.1\u003c/b\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThirunavukkarasu, M. and Kathiravan, G. (2009). Factors affecting conception rates in artificially inseminated bovines. Indian Journal of Animal Sciences, 79(9), p.871.\u003c/li\u003e\n\u003cli\u003eSingh, I. and Balhara, A. K. (2016). 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Res.,44 (2),135-138 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Recombinant CRISP-1 peptide, production, biological function, mechanism of action, tissue expression, Buffalo","lastPublishedDoi":"10.21203/rs.3.rs-6680127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6680127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCysteine-rich secretory protein 1 (CRISP-1), an acidic glycoprotein of epididymis, acts as sperm decapacitation factor. Adding CRISP-1 protein to semen may extend the fertile life of preserved sperm by inhibiting premature sperm capacitation during preservation. To this end, production of bioactive recombinant buffalo CRISP-1 mature peptide in E. coli followed by it\u0026rsquo;s functional and mechanistic characterization on sperm motility and capacitation were described. A 687 bp cDNA fragment corresponding to \u003cb\u003eC\u003c/b\u003e\u003csup\u003e\u003cb\u003e15\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e-C\u003c/b\u003e\u003csup\u003e\u003cb\u003e242\u003c/b\u003e\u003c/sup\u003e buffalo CRISP-1 mature peptide was cloned in pET22b expression vector and expressed in BL21(DE3)-codon plus E. coli strain. The recombinant peptide was expressed as insoluble form which was purified by Ni-NTA affinity chromatography and refolded by dialysis. Tissue expression analysis revealed that buffalo CRISP-1 protein was expressed mainly in cauda epididymis and vas deferens, but not in testis. In sperm, it localized on acrosome and principal piece of tail. The CRISP-1 peptide (20\u0026micro;g/mL) caused significant reduction in sperm progressive motility (61 vs. 33%) and capacitation. Further, it reduced tyrosine phosphorylation of two sperm proteins (p47, p72) under capacitating condition. The peptide was found active between pH 6 and 9, and optimal pH was pH 8. The activity of the peptide was reduced above 60\u0026deg;C. The peptide inhibited both bicarbonate and L-arginine mediated capacitation of buffalo sperm by modulating nitric oxide (NO)/adenylyl cyclase (AC)/cAMP/Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e signalling pathway. It was concluded that, the recombinant buffalo CRISP-1 mature peptide produced in E. coli was bioactive and it inhibited sperm motility and capacitation by interrupting NO/AC/cAMP/Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e signalling pathway. Hence, the recombinant CRISP-1 peptide could be utilized to prevent sperm capacitation during semen preservation for improving post-thaw quality of preserved buffalo semen.\u003c/p\u003e","manuscriptTitle":"Recombinant buffalo (Bubalus bubalis ) cysteine-rich secretory protein 1 mature peptide acts as a potent sperm-quiescent and decapcitation factor by modulating nitric oxide/bicarbonate/adenylyl cyclase/Ca+2 signalling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 14:43:39","doi":"10.21203/rs.3.rs-6680127/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93246f86-c21b-4eac-8103-d8b57c8dcbc9","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-10T14:43:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 14:43:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6680127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6680127","identity":"rs-6680127","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}