Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium-regulating multifunctional protein

2.1 Sample collection

Fresh semen ejaculates of healthy breeding Murrah buffalo bulls (n = 3) maintained at Germ plasm Centre (Animal Reproduction Division, Indian Veterinary Research Institute, Izatnagar, India) were collected using the artificial vagina method and directly processed. Caudal semen samples were collected from mature buffalo testis procured from slaughter house (Bareilly, Uttar Pradesh, India) immediately after slaughter and transported to laboratory (n = 5). The epididymis was separated from the testis after thoroughly washing with PBS, and epididymal ducts were exposed by trimming the superficial tissue. Using a 16-gauge hypodermic needle, the caudal region was punctured at many points and subsequently squeezed to collect the spermatozoa-rich fluid in PBS. Samples containing at least 75% of motile spermatozoa and less than 20% morphological abnormality were used for the study. For studying testicular sperms, fresh testis (n = 5) collected from the slaughter house were used (four impression smears from each animal).

2.2 Primers

The forward (5′-GATCCGTTTGGATCCTGAGA-3′) and reverse primers (5′-TTGAAAATTCCACCAGCCTC-3′) for qPCR assay of buffalo RGN were designed using Gene Tool BioLite software from the published cDNA sequence of Bos taurus (Accession No: NM 173957.2). The theoretical length of the amplified product was ~197 bp.

2.3 Total RNA isolation and cDNA synthesis from spermatozoa

Three fresh ejaculates per buffalo were collected (n = 3), and 1 ml of each ejaculate was centrifuged at 700 g for 10 min at room temperature (28°C). The resulting pellet was reconstituted in 1 ml of PBS and purified using discontinuous 45/90% Percoll (Sigma, USA) in PBS (pH 7.4) to remove contaminating somatic cells, germ cells and leucocytes. Two millilitres of 45% Percoll was layered over 3 ml of 90% Percoll, and 1 ml of reconstituted sperm pellet was carefully layered on the top of this and centrifuged at 360 g for 30 min. The spermatozoa pellet was further used for total RNA and protein isolation.

The whole RNA isolation procedure was carried out in an area dedicated to RNA work after thoroughly cleaning with RNase Zap (Sigma, USA), using RNase/DNase-free water and RNase-free glass/plastic wares. The total RNA from sperm samples was isolated using the conventional TRIzol (Invitrogen, USA) method (Parthipan et al., 2015) with slight modification. TRIzol (Invitrogen, USA) pre-heated to 60°C was added to the sperm pellet and mixed well using a micropipette. Using a 26-G needle fixed to a syringe, the sperm cells were lysed by passing the contents through the needle several times, followed by incubation at 65°C for 30 min with intermittent vortexing at every 10 min. To facilitate complete lysis of spermatozoa and separation nucleoprotein complex, the contents were passed again through the needle for several times. Following the addition of 200 μl of chloroform for each ml of TRIzol used, the contents were mixed vigorously and incubated for 5 min at room temperature. The upper layer formed after centrifugation at 12,000 g for 15 min at 4°C was transferred into fresh tubes and incubated for 15 min at room temperature after adding 500 μl of nuclease-free isopropanol. The centrifugation step was repeated, and the RNA pellet formed was washed with 75% ethanol and air-dried in RNase-free environment. The RNA pellet was then reconstituted in nuclease-free water and treated with DNase I (Thermo Fisher Scientific, USA) as per manufacturer's instruction to remove any trace of DNA. The DNase was later inactivated by incubating at 65°C for 10 min. The quality and concentration of the isolated total RNA were assessed using the NanoDrop (Thermo Scientific, USA). The total RNA samples were then stored at −80°C for further analysis. Synthesis of cDNA was carried out from 2 μg of total RNA using Revert Aid™ H-Minus First-Strand cDNA Synthesis kit (Fermentas, USA) as per the protocol described by the manufacturer.

2.4 Real-time PCR

Following the manufacturer's instructions, the qualitative real-time PCR was performed using Maxima SYBR Green/ROX qPCR Mix (Fermentas, USA) in Mx3000P PCR cycler (Stratagene, the Netherlands). Reactions were set in triplicates for each batch of cDNA synthesized along with a non-template control (NTC). The qPCR was performed using 2 μl of cDNA (100 ng/μl), 12.5 μl of 2 ×  PCR master mix and 5 pmol of each primer in a final volume of 25 μl. Thermal cycling conditions were as follows: 10 min at 95°C followed by 40 repeats of 15 s at 95°C and 1 min at 60°C followed by 5 min at 72°C.

2.5 Western blot

Pure sperm pellet of three different buffalo bulls (three ejaculates per animal) obtained after Percoll purification as explained previously was treated with T^PER solution (Thermo Fisher, USA) to lyse spermatozoa. After adding 100 μl of T^PER solution to the pellet from 200 μl of semen, homogenized for 1–2 min over ice and centrifuged at 10,000 g for 15 min at 4°C. Total amount of particulate and supernatant fraction of sperm cell lysate were loaded and analysed by Western blot analysis.

Western blot of the particulate and supernatant fraction of sperm cell lysate was performed with primary anti-RGN monoclonal antibody (ab81721, Abcam, UK). The samples were resolved by a 12% SDS-PAGE after mixing with sample buffer containing 5% β-mercaptoethanol and denaturing for 10 min at 100°C. Resolved proteins were blotted onto a PVDF (polyvinylidene difluoride) membrane (Hybond-P, UK) by the semidry transfer method. Blotted membranes were later blocked overnight at 4°C with Tris-buffered saline containing 0.05% (v/v) Tween-20 and 5% (w/v) dry skimmed milk powder (Hi Media, India) and then incubated for 1 hr at 37°C with anti-RGN monoclonal antibody (1:2000). After washing out the unbound primary antibody, membrane was incubated with secondary antibody conjugated with horse radish peroxidase (1:5000; anti-mouse, Santa Cruz, USA) and developed for 5 min with DAB substrate (Sigma, USA).

To rule out the possibility of oligomer formation of RGN in the supernatant fraction, Western blotting was performed with a slightly modified procedure under strong reducing conditions (Berthold & Siedow, 1993). Supernatant fraction sample having 100 μg total protein was denatured for 10 min at room temperature in sample buffer containing 8.0 M urea. Later, β-mercaptoethanol was added to a final concentration of 5% and boiled for 15 min followed by the addition of 200 mM DTT just before loading on a 12% SDS-PAGE containing 2.5 M urea. The resolved proteins were further transferred and developed as explained before. The same experiment was performed using anti-RGN monoclonal antibody (1:2000) and anti-RGN polyclonal antibody (dilution 1:100) available in the laboratory for comparison (Harikrishna, Shende, Reena, et al., 2016).

2.6 Immunocytochemistry

Immunocytochemistry of buffalo testicular (n = 5), caudal (n = 5) and ejaculated spermatozoa (n = 3; three ejaculates per animal) was performed using anti-RGN polyclonal antibodies available in the laboratory. Three smears per spermatozoa type along with one negative control were set for each experiment. A 100 μl of ejaculated semen as well as caudal semen was centrifuged at 300 g for 10 min at 25°C followed by washing twice with 1 ml of PBS at 300 g for 10 min. Pellets were reconstituted in PBS, and the cell count was adjusted roughly to 1 × 10⁷ cells/ml using hemocytometer. Cell smears were prepared on clean grease-free cover slips and allowed to air-dry. For testicular spermatozoa, impression smears of the testes were made by cutting open the testis cross-sectionally and pressing the interior portion against clean grease-free cover slips. Cells were simultaneously fixed and permeabilized with acetone at −20°C for 20 min. Cover slips were washed three times for 5 min each with PBS. Non-specific sites were blocked with 10% normal goat serum in PBS by incubating for 30 min at room temperature. The cover slips were further washed thrice with 0.05% Triton X-100 in PBS of 5 min each. Later, cover slips were incubated with anti-RGN polyclonal antibodies (1:50 dilution) for 45 min at 37°C. Negative control was set for each sperm type by incubating with pre-immunized sera (1:50) instead of primary antibody. Washing step was repeated, followed by incubation with FITC-labelled rabbit anti-mouse IgGs (ab6724, Abcam, 1:5000) in PBS for 15 min, and unbound conjugate was washed thoroughly. The cover slips were mounted with Fluoroshield containing DAPI (F6057, Sigma, USA) and inverted on clean dry glass slides. Images were captured using a fluorescent microscope (IX 71, Olympus, Japan).

3.1 Regucalcin mRNA expression in spermatozoa

The mRNA expression of RGN in buffalo spermatozoa was confirmed by qPCR of the spermatozoa cDNA. Concentrations of total RNA isolated were in the range of 50–100 ng/μl with A_260/280 values between 1.8 and 1.9. The RGN gene amplification plot in spermatozoa is observed (Figure 1a). A single peak in dissociation curve as well as no amplification in NTC (non-template control) showed the specificity of the amplified product (Figure 1b). Further, the amplified PCR product showed the expected 197 bp size on 2% agarose gel electrophoresis (Figure 1c).

3.2 Regucalcin protein expression in spermatozoa

The presence of ~34-kDa isoform of regucalcin in the particulate fraction of a sperm cell lysate was confirmed by Western blot probed with anti-RGN monoclonal antibodies, whereas it was missing in the supernatant (Figure 2). The supernatant of the buffalo spermatozoa lysate showed the presence of other isoforms of regucalcin by Western blot analysis probed using both anti-RGN monoclonal and polyclonal antibodies. Three bands of 44 kDa, 28 kDa and 24 kDa sizes were obtained with monoclonal antibodies, whereas an additional band of 48 kDa along with the other three bands was visualized with polyclonal antibody. Further, the 44 kDa band appeared to be very prominent on the blot probed with polyclonal antibody (Figure 3).

3.3 Immunocytochemistry of spermatozoa

The immunofluorescence studies of spermatozoa from testicular, epididymal and ejaculate showed localization of RGN predominantly in the acrosomal region (Figures 4-6). However, the cytosol and acrosomal regions of the testicular spermatozoa showed immunofluorescence (Figure 4) whereas the immunofluorescence had been restricted to only acrosomal region in case of spermatozoa of cauda epididymis (Figure 5) and ejaculated spermatozoa (Figure 6). Slight fluorescence of the tail axonal region was also observed in few spermatozoa of all samples.