Sperm Proteins

To extract proteins from the spermatozoa, 50 × 10⁶ spermatozoa were incubated in rehydration buffer containing 7 M urea (Sigma, St Louis, MO, USA), 2 M thiourea (Sigma, St Louis, MO, USA), 4% (w/v) CHAPS (USB, Cleveland, OH, USA), 0.05% (v/v) Triton X-100 (Sigma, St Louis, MO, USA), 1% (w/v) octyl β-D-glucopyranoside, 24 μM PMSF (Sigma, St Louis, MO, USA), 1% (w/v) DTT (Sigma, St Louis, MO, USA), 0.5% (v/v) IPG Buffer, and 0.002% (w/v) bromophenol blue at 4°C for 1 h. Then, 250 μg of solubilized protein from the sperm cells in 450 μL of rehydration buffer was placed in a rehydration tray with 24 cm-long NL Immobiline DryStrips (pH 3–11; Amersham, Piscataway, NJ, USA) for 12 h at 4°C. First dimension electrophoresis was performed using an IPGphor IEF device and then the strips were focused at 100 V for 1 h, 200 V for 1 h, 500 V for 1 h, 1,000 V for 1 h, 5,000 V for 1.5 h, 10 8,000 V for 1.5 h, and 8,000-90,000 Vhr. After iso-electrofocusing, the strips were equilibrated a second After iso-electrofocusing, the strips were equilibrated with equilibration buffer A containing 6 M urea, 75 mM Tris–HCl (pH 8.8), 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (w/v) bromophenol blue, and 2% (w/v) DTT for 15 min at RT. The strips were equilibrated for a second time with equilibration buffer B (equilibration buffer A with 2.5% [w/v] iodoacetamide [Sigma] but without DTT for 15 min at RT. Next, 2-DE was carried out with 12.5% (w/v) SDS-PAGE gels with the strips at 100 V for 1 h and 500 V until the bromophenol blue front began to migrate off the gels. The gels were silver-stained for image analysis according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). The gels were then scanned using a high-resolution GS-800 calibrated scanner (Bio-Rad, Hercules, CA, USA). Detected spots were matched and analyzed by comparing the gels from spermatozoa before- and after-capacitation using PDQuest 8.0 software (Bio-Rad, Hercules, CA, USA). The gel from before-capacitation spermatozoa was used as a control. Finally, the density of the spots was calculated and normalized as the ratio of the spot on the after-capacitation gel to that on the before-capacitation gel.

Because we sought to identify soluble, extracellular male Sfps, proteins were prepared in such a way so as to select specifically for soluble proteins. We also sought to reduce cell lysis and thus protein content from male sperm cells and female reproductive tract epithelial cells, since releasing intracellular proteins from these cells would dilute the concentration of transferred Sfps and render their identification more difficult. Female reproductive tracts were homogenized in the ammonium bicarbonate dissection buffer, which lacks any type of detergent and thus minimized cell lysis. The mixture was then centrifuged for 5 min at 18,000g. This process was repeated once, and the supernatant was retained. Protein concentration was estimated using a BCA assay (Pierce). Proteins were prepared for tandem mass spectrometry and digested with trypsin as previously described [57 (link)].

To perform ERC analysis, we first calculated the amount of amino acid divergence for each branch in the species tree for each of the 11,100 orthologous protein alignments produced above; this was done using ‘aaml’ of the PAML package [106] (link). Next, raw branch lengths were transformed into rates of evolution relative to the expected branch length. This projection operation, introduced by Sato et al. [58] (link), removes the inherent correlation of all proteins due to the underlying species tree and improves the power of ERC to resolve functionally related protein pairs from unrelated pairs [55] (link), [58] (link). Finally, we used these corrected branch-specific rates to calculate the correlations for all pairs of proteins, resulting in a proteome-by-proteome matrix of correlation coefficients, termed the ERC matrix. To limit the effect of outlier points, we limited all rates to 2 standard deviations from the mean.
In spite of our efforts (above) to improve species coverage, most alignments were missing at least one species. We set a minimum species threshold at 5, so species representation ranged from 5 to 12. This heterogeneity required us to create a flexible system to compare ERC results between different sets of species. A table of relative rates (projection operation, above) was produced for each unique set of species shared between protein pairs, resulting in 1,815 projections. Importantly, the distribution of ERC values varied depending on the particular set of species employed. For example, the variance of ERC values is consistently larger for smaller numbers of species (Figure S6). To correct for these effects we converted every observed ERC value in to an empirical p-value based on the observed distribution of ERC values for that particular set of species. The comparison of p-values allowed us to compare ERC results across all protein pairs. Hence, we report all ERC results as p-values ranging from 0 to 1, where a lower value indicates stronger evidence for rate correlation.
Significance testing for elevated ERC values in a set of proteins was performed using a proteome-wide permutation test (Figure S1A). The mean ERC value observed between all pairs in the tested set, such as the SP network, was compared to the mean ERC values of 10,000 sets of the same number of proteins randomly chosen from the entire proteome. A p-value for the tested set was computed as the proportion of random sets that had a mean ERC value equal to or greater than the tested set of proteins. Randomly chosen ERC values were taken from the same species-matched projections as in the observed set, which controlled for variation in ERC distributions due to different sets of species present in those genes.
The “reproductive protein only” analysis (Figure S1B–C) was performed as above, except that analysis was limited to the 756 Sfps, female proteins, and sperm proteins described above. We further limited this set to the 664 proteins that had detectable orthologs in at least 5 species. Significance testing for single pairs and for sets of proteins was performed as above, through empirical p-values. Calculations of pairwise correlations between pairs of known network proteins and between known network proteins and members of the sets of Sfps and female proteins were performed using this reproductive protein set.

Sperm proteins isolated from high and low fertile semen straw were subjected to western blotting. The protocol followed has been described earlier (Selvam et al., 2019 (link)) with slight changes. In brief, 15 µg of protein from each group was electrophoresed in 12% SDS denaturing gel. The gel was equilibrated for 20 min in a transfer buffer before semi-dry transfer and the proteins were transferred to PVDF membrane (0.2 microns) 25 V, 2.5 A at room temperature for 20 min using power blotter station (Invitrogen). After transfer, the membrane was washed with Tris buffer saline containing Tween-20 (TBS-T) for 2 min and then incubated in a blocking buffer containing 5% bovine serum albumin (BSA) overnight at 4°C. The membrane was washed with TBS-T and incubated with primary antibodies namely, mouse anti-β-actin (Thermo Fisher Scientific, catalogue no. AM4302, 1:20,000 dilution), rabbit anti-Sp17 (Invitrogen, catalogue no. PA5-69981, 1:15,000 dilution), goat anti-AKAP3 (biorbyt, catalogue no. orb 18,844, 1:25,000 dilution) and rabbit anti-DLD (Invitrogen, catalogue no. PA5-27367, 1:15,000 dilution) at room temperature with constant rocking for 3 h. After incubation, the membrane was washed with TBS-T thrice for 10 min. Then, the membrane was incubated in HRP conjugated secondary antibodies such as goat anti-mouse for β-actin (Sigma, 1:70,000 dilution, catalogue no. 665739), goat anti-rabbit for Sp17 (Sigma, catalogue no. 632131, 1:90,000 dilution), rabbit anti-goat for AKAP3 (Abcam, catalogue no. ab6741, 1:150,000 dilution) and goat anti-rabbit for DLD (Sigma, catalogue no. 632131, 1:50,000 dilution) at room temperature for 1 h, followed by three washes (5 min each) in TBS-T. The membrane was then washed with TBS-T and then the immunocomplexes were detected using the ECL-HRP substrate (Sigma, WBULS0100). The membrane was incubated with substrate for 5 min in the dark and the signal was captured by the system-ray film (Fuji). The bands were analysed using ImageJ software (https://imagej.nih.gov/ij/). The β-actin (42 kDa) was used as normalizing control to obtain the relative abundance of Sp17 (22 kDa), AKAP3 (95 kDa), and DLD (54 kDa) proteins in sperm. From the list of DAPs, AKAP3, Sp17, and DLD were shortlisted based on high fold change in mass spectrometry data and presence of the protein in all replicates along with peptide matches. More importantly, these proteins were selected based on their involvement in the major pathways (Gene Ontology) and vital roles in regulating the sperm fertilizing capacity. Availability of the antibodies was also a criteria for selecting the three DAPs.

To understand the functional role of the proteins in spermatozoa, DAPs and unique proteins of both fertility groups were subjected to GO analysis and KEGG (Kyoto Encyclopedia of Gene and Genomes) pathway analysis. To identify the functions of unique proteins in HF and LF, the proteins present in at least two technical replicates were selected. The functional profiling of DAPs and unique proteins in sperms was carried out using Gene Ontology (GO) of the sperm proteome. GO was carried out using Uniprot and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene enrichment tool v6.8. The proteins were then categrorized based on GO terms like molecular function (MF), biological function (BP), cellular component (CC) along with KEGG pathway enrichment. Top 10 BPs, MFs, and CCs and KEGG pathways were plotted in pie chart.

A western blot experiment was carried out to identify the SPAG7 protein expression level in sperm samples. Semen samples were obtained from an independent cohort of oligoasthenozoospermic men (n = 4) who attended the IVF center for infertility treatment and normozoospermic men served as controls (n = 4). Samples were thawed on ice and washed three times with PBS. The samples were then centrifuged at 14,000 × g at 4 °C for 15 min. The pellet was suspended in 100 µl RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor (Sigma–Aldrich). Then, the samples were sonicated at 20 J for 2 s × 10 at intervals of 10 s and incubated on ice overnight inside the fridge at 4 °C. The next day, samples were centrifuged at 4 °C for 10 min at 14,000 × g, supernatant from each sample was transferred into a new tube, and protein concentrations were quantified using the BCA protein assay kit (Pierce). Thirty micrograms of total protein from each sample were denatured with Laemmli buffer mixed with b-mercaptoethanol (1:4; Bio-Rad Laboratories) and heated at 95 °C for 5 min. Anti-SPAG7 antibody [EPR13391, Abcam] diluted in TBS Blotto A (1:1000) and GAPDH (14C10) rabbit mAb antibody (1:1000, 2118S, Cell Signaling Technology) were used to detect the protein expression level. Each primary antibody was incubated individually and precision plus protein standard (Bio-Rad Laboratories) was used for the size determination. Following incubation with an anti-rabbit secondary antibody (1:3,000, A0545, Sigma-Aldrich), the gray value of each protein band was analyzed on a ChemiDoc™ Touch system (Bio-Rad Laboratories) and normalized to that of GAPDH.