The macroscopic and microscopic parameters of semen were analysed and evaluated before any downstream experiments. The ejaculates that were either milky or creamy in colour, 2–4 mL in volume, free from coagulation, and homogenous in their consistency were considered for processing. The pH range of the ejaculates varied from 6.3-7.0. The samples were subsequently observed microscopically for normal morphology. The mean motility and viability of the used sample ejaculates after processing was 82.49 ± 1.33% and 84.76 ± 1.34%, respectively. The ejaculates with a mean sperm concentration of more than 600 x 106/mL were considered for the swim-up procedure.
Sperm Functional Parameters:
Membrane integrity assessment
The CFDA-PI dual staining distinguished three spermatozoa populations viz. live, dead and moribund (dying sperm, source of ROS) spermatozoa based on their fluorescence patterns indicative of the functional or compromised membrane integrity. The integrity of the buffalo spermatozoa plasma membrane appeared to be compromised upon protein extraction by elevated NaCl, PI-PLC treatment and in vitro capacitation (Fig. 1A and Fig. 2). Expectedly, the treatment controls indicated negligible effect of PI-PLC and capacitation on the membrane integrity since the physiologic medium, Sp-TALP was used in these extraction methods. Nonetheless, the presence of elevated salt (2X-DPBS) in the immediate milieu significantly reduced the percentage of live sperm (P < 0.05) and led to a rise in the moribund sperm population (P < 0.01) (Fig. 2). Interestingly, the effects of incubation media used for protein extraction (Sp-TALP and DPBS) on sperm plasma membrane integrity and other parameters employed in this study were also deemed statistically significant (P < 0.05-P < 0.00001) (Fig. 1 and Fig. 2).
Capacitation And Acrosome Integrity Status
Capacitation is a crucial process that allows the spermatozoa to mature and attain the fertilizing ability in the FRT. We used Chlortetracycline (CTC), to detect the redistribution of Ca2+ in the sperm head after the period of induced capacitation (6h). The fluorescence microscopy determined three fluorescent patterns classified as non-capacitated (NC), capacitated (C) and acrosome reacted (AR) spermatozoa (Fig. 1B and Fig. 2). The extraction treatments induced precocious capacitation in buffalo spermatozoa (Fig. 2). As expected, a time-dependent, considerable rise the occurrence of spontaneous acrosome reaction was observed in the treated sperm (Fig. 1C and Fig. 2).
Lipid Peroxidation
Lipid peroxidation (LPO) has been implicated in male sub- and in-fertility, in multiple mammalian species. The fluorescent dye BODIPY can incorporate into the spermatozoa and thereafter undergoes a spectral shift in emission upon interaction with the reactive oxygen species, thereby signifying oxidative stress. The incubation of buffalo spermatozoa in media with elevated salt, PI-PLC or capacitating factors significantly induced time-dependent LPO (Fig. 1D and Fig. 2). However, fluorescence was less evident in the sperm head or the rest of the sperm tail vis-à-vis the mid-piece indicating preferential localization of sperm mitochondria (Fig. 2).
Mitochondria Membrane Potential
The mitochondria membrane potential (MMP) is a potent marker of sperm health The JC-1 dye was used to assess the MMP of buffalo sperm after protein extraction from the surface. This dye provides bright green fluorescence to the spermatozoa with high MMP as whilst the spermatozoa with low MMP produced a dim green-ish fluorescence after labelling with JC-1 exhibiting the expected, preferential localization to sperm mid-piece (Fig. 1D). The mitochondria membrane potential (MMP) of buffalo spermatozoa significantly diminished after removal of surface proteins. Thus, a reduction in the population of sperm mitochondria bearing high MMP was observed (Fig. 1E and Fig. 2).
It appears that the ROS homeostasis (MMP & LPO) is considerably affected, not only by the media used (Sp-TALP and DPBS but by the time of incubation (0h, 30min, 120min, 6h) in the same medium.
Protein Tyrosine Phosphorylation
The monoclonal anti-phosphotyrosine antibody produced three fluorescent patterns (Fig. 1C). upon binding to buffalo spermatozoa viz. non-phosphorylated, NP (negligible or no fluorescence), phosphorylated sperm with fluorescence on the mid-piece and the equatorial region (EM) and acrosome (AEM). The NP spermatozoa population was the most abundant vis-à-vis the EM and AEM sperm across all the samples, as observed by immunocytochemistry (Fig. 2). Notably, the anti-phosphotyrosine antibody produced fluorescence simultaneously in the mid-piece and the equatorial region. The extraction treatments induced significant phosphorylation of tyrosine residues of sperm proteins, a conserved feature of mammalian sperm capacitation [39](Fig. 1F).
Buffalo Sperm Surface Has Thousands Of Distinct Proteins (And Their Isoforms)
A total of 64,290, 46,526 and 33,902 PSMs corresponding to 4881, 1781 and 2782 unique peptides, respectively were identified from buffalo sperm surface after salt-extraction, PI-PLC treatment and in vitro induced capacitated samples. These PSMs and peptides were mapped to 1342, 678 and 982 proteins and their isoforms (proteoforms) in the three samples, respectively as illustrated (Fig. 3A) by the distribution of common and exclusive proteins among the three groups. The β-defensin 129 (BuBD-129) was selected to validate the identified proteins because we have previously reported its presence on the entire buffalo sperm periphery [13]. The presence of BuBD-129 was detected by the appearance of a sharp band between ~ 35 kDa to ~ 40 kDa (Supplementary Fig. 1 and Fig. 5D). The initial screening of the buffalo sperm-surface proteins extracted by the three treatments exhibited the highly diversified nature of these proteins (Fig. 3B-G). The complete list of identified proteins and peptides identified by the three extraction treatments is provided in the Supplementary sheet-Results. The frequency distributions of molecular weight, peptide length, and amino acid composition of peptides belonging to three different groups have been shown in (Fig. 3B-G). The identified proteins and their isoforms exhibited a wide linear dynamic range in abundance, identity, the dynamic range of molecular mass, number of amino acids and pI indicating the differential effect of the used extraction methods. For example, the isoelectric point for XP_025127145.1 was predicted to be 4.06 whereas the highest pI in the sample was predicted for XP_006071250.1 as 11.87, a difference of seven orders of magnitude! Likewise, the lowest and highest molecular weight was predicted to be 7.8 and 781.4kDa for XP_025148992.1and XP_025129505.1 a difference of nearly 10 times!
The Perseus software platform (v. 2.0.1.0) was used for interpreting protein identification and quantification, normalization cross-replicate comparisons (and establishing the correlation between replicates from similar or distinct samples) and multi-hypothesis testing for the multi-dimensional proteomics data generated in this study (Fig. 4, Fig. 5). The PCA plots were constructed from the variance in abundance values of each protein wherein the proteins with similar abundance values tend to cluster together. The analysis separated the data into two sub-groups demonstrating that a major source of statistical variation can be attributed to the extraction treatment. (Fig. 4B, C). The cross-sample and replicate comparison and fold change dynamics of the DAPs (vis-à-vis respective controls) were tested for statistical significance by the t-test (assuming equal variance within the groups of replicates) with Benjamini and Hochberg false discovery rate (FDR)-correction at < 0.01 significance level and were used for functional annotation of the identified proteins (Fig. 4D-F and Fig. 5A-C). Only the entries with P-values < 0.05 and FDR ≤ 0.01 were considered to be statistically significant.
Chromosome Mapping Of Buffalo Sperm-surface Proteins
Next, we sought to determine the individual chromosomal localization of the identified peptides by creating Circos plots (chord plots) based on the NCBI data (Fig. 6A-C). We found the contribution from all the chromosomes (autosomes and allosomes), however, the distribution of protein expression was uneven. For example, in the first group (Salt-2X-DPBS), a maximum number of proteins (617) were mapped to chromosome 5 while chromosome 13 contributed minimally to the surface proteome [19] (Fig. 6A). The disruption of non-covalent electrostatic interaction by 2X-NaCl didn’t appear to change the observations. The incubation of spermatozoa in Sp-TALP also yielded similar results albeit no peptides could be mapped to either chromosome 13 or 19. Notably, the removal of the GPI-linked proteins by PI-PLC significantly changes the mapping dynamics with maximum peptides mapped on chromosome 2. Likewise, induction of in vitro capacitation led to the identification of hitherto unidentified proteins from chromosomes 13, and 19, for instance (Fig. 6B, C).
Molecular Characteristics Of Buffalo Sperm Surface Proteins
Enrichment and pathway analysis
The gene ontology (GO) and pathway analyses were carried out to hint at the biological processes and molecular functions associated with the non-covalently linked, GPI-APs and capacitation-related proteins. The BLAST2GO 5.0 uses the pair-wise similarity search algorithm developed by Altschul and colleagues [40] for finding similar/identical sequences in the NCBI. This analysis allowed a deduction of specific functions played by the novel, hitherto uncharacterized proteins/proteoforms dataset generated in this study. The maximum number of hits were returned from Bubalus bubalis (buffalo) followed by Bos taurus, cross-bred cattle and other ruminants (Fig. 6D-F). The overall frequency distribution of the GO-level functional annotation of the biological process, molecular function and cellular component terms of the gene ontology analysis is depicted in (Fig. 6G-I) indicating the quality of annotation. For instance, the non-covalently linked proteins were predicted to be primarily involved in the biological regulation of cellular and metabolic processes (FDR < 0.01) akin to the GPI-APs which, however, were also implicated in reproductive processes like capacitation and gamete interaction (Fig. 7A-C). The majority (two third) of the identified electrostatically bound protein were predicted to possess catalytic activity and were predicted to be implicated in protein binding (protein-2 interactions, PPIs. Only a fraction (one-tenth) of the GPI-APs were predicted to possess catalytic activity, nevertheless, they did have a DNA binding domain (DBD) whereas the capacitation-related proteins mainly had a hydrolase activity and functioned in metal-ion/protein binding (Fig. 7D-I). A small fraction of intracellular proteins was also observed among the surface protein population.
Interestingly, the pathway analysis of the identified proteins was done using KOBAS 3.0. It is a web server for annotating and identifying the enriched pathways in novel sequences (sequence-based) based on mapping to genes with known annotations. The data-set analysis revealed significant enrichment (P-valued < 0.05, FDR < 0.01) of metabolic, homeostasis and response to the extracellular signal, and signaling and immune-related pathways in the non-covalently bound (2X-DPBS), GPI-anchored and protein released upon induction of in vitro capacitation (Fig. 8).
We wanted to assess the quantum of proteoforms associated with the reproductive processes across all the samples. Surprisingly, doing an extensive literature search for each of the identified proteins and isoforms (1695) revealed that more than 50% of the proteins (873) were implicated in male reproductive physiology. Subsequently, functionally unannotated, uncharacterized, and unreported proteoforms were removed for generating an exclusive sperm-surface functional profile of reproduction-related proteins based on a thorough literature search. The analysis revealed that most of the protein families were implicated in male fertility, spermatogenesis and motility followed by protection, maturation, capacitation and immunomodulation (Fig. 9).