Shotgun Proteomics-based distinctive fingerprint of sperm-surface revealed over- representation of proteins driving spermatogenesis, motility, and fertility

doi:10.21203/rs.3.rs-2345148/v1

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Shotgun Proteomics-based distinctive fingerprint of sperm-surface revealed over- representation of proteins driving spermatogenesis, motility, and fertility

https://doi.org/10.21203/rs.3.rs-2345148/v1

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Background

Numerous distinct secretagogues such as (glyco) proteins including the GPI-anchored proteins are added to the sperm surface during their transit through the male reproductive tract (MRT). This remodelling of the sperm surface is critical for sperm maturation, survival and function in the female reproductive tract (FRT). This study aimed to characterize the proteins present on the buffalo sperm surface.

Results

A buffalo sperm surface-specific proteomic fingerprint was generated using shotgun proteomics (LC-MS/MS). The protein informatics platform, Proteome Discoverer (v2.2) identified 1342, 678, and 982 distinct proteins and isoforms (P < 0.05, FDR < 0.01) in the salt-extracted, PI-PLC treated and capacitated samples, respectively. Overall, 1695 unique proteins (minimum 2 peptides) with ≥ 1 high-quality PSM/s and their isoforms (proteoforms) were identified. Descriptive statistical analysis indicated that these buffalo-specific proteoforms exhibit remarkable heterogeneity in their molecular weight, pI, distribution of expression from the genome and their functional roles in the MRT and the FRT. Subsequent analysis and a thorough literature search revealed that the fertility-related, reproduction-specific proteoforms constituted more than 50% (873) of the identified sperm-surface proteome (1695).

Discussion

These identified proteoforms are unique to buffalo since a buffalo-specific database, NCBI reference proteome (translated from the latest chromosome level genome assembly, (UOA_WB_1) was used as the search space. These proteoforms were mapped to 252 buffalo-specific proteins implicated in the regulation of various aspects of male reproductive physiology across multiple species. Besides, more than 200 orphan, buffalo-specific proteins and their isoforms (undefined locus, uncharacterized, P < 0.05, FDR < 0.01) were also identified using our computational strategy. This allowed us to consider these novel proteins for considered for mapping their ontology terms. This led to the elucidation of the biological functions of these hitherto unreported, buffalo-specific proteins by extrapolation of function from their sequence orthologs in more several ruminant and non-ruminant (e.g. Primates and Rodents) mammalian. These uncharacterized proteins constitute an extensive, yet unexplored, reproduction-specific sperm-surface proteome repertoire.

Conclusions

The proteomic signature driving the buffalo sperm production, maturation, survival and function discovered in this study is unparalleled vis-à-vis the depth identification of fertility-related and reproduction-specific cell-surface proteins. These results would facilitate advances in understanding the functional roles of proteins implicated in mammalian sperm function.

Buffalo

Male fertility

Sperm

Proteomics

The development of a male gamete entails three distinct physiological processes viz. spermatogenesis, spermiogenesis and sperm maturation (including capacitation in the female reproductive tract) each of which is controlled by proteins and their associated post-translational modifications. The concluding stages of sperm differentiation, including the tailoring of its surface, occur outside the gonads and do not appear to be regulated or mediated by the germline genome. This is because, as the differentiation progresses in the testes, the DNA starts condensing and consequently, the transcription in the germline DNA eventually diminishes and then ultimately halts [1–4]. The proteomic composition of the testicular spermatozoa surface changes continuously and progressively not only in the MRT but also during the secondary maturation (capacitation) in the FRT that endows the spermatozoa with motility and competence for fertilization [5, 6]. The transit of the spermatozoa in the FRT is arduous and tactical where they have to bind reversibly with numerous epithelial surfaces simultaneously circumventing the phagocytes, antibodies and complement proteins [6, 7–9]. The fertilizing spermatozoa, accordingly, must acquire surface properties primarily customized for this arduous voyage in the FRT. The proteins on the immature testicular gametes, for example, are modified, adsorbed, and inserted while new proteins in either covalently or non-covalently (transiently or permanently) on the surface during their transit in the MRT [10, 11]. It is now an established paradigm that the transit through the epididymis, secretions of the seminal plasma along the MRT and capacitation in the FRT regulate the maturational changes in sperm surface in a species-specific manner [6, 12].

The sperm surface harbours a hallmark proteome implicated in a multitude of immunological and reproductive processes [13]. It serves as the primary interface between the sperm and the various immunologic milieus, and immune surveillance mechanisms in the FRT and is radically modified during its journey until it reaches the oviduct [11, 14]. The sperm-surface proteins determine the functional competence of spermatozoa and eventually the success of sequential events of fertilization in the FRT. Many such proteins assist the spermatozoa during their stay in the male reproductive tract (MRT) and their voyage throughout the FRT. For example, the highly glycosylated and negatively charged, cysteine-rich antimicrobial peptides, β-defensins (BDs), that are added to the sperm surface during their transit through the epididymis are implicated in immune evasion and fertilization-related processes [15–18]. Besides, the re-distribution of sperm surface (glyco)proteins during capacitation are crucial to the subsequent fertilization process [19]. For determining the identity of such proteins, it has been posited that the proteomic analysis has a better potential to advance our understanding of male reproductive physiology as compared to genomic and transcriptomic analyses. This is because the gene expression varies vis-à-vis the cellular contexts and the transcribed mRNAs are not always translated. Moreover, the proteins reflect the events downstream of gene expression and are considered to more accurately predict the phenotype of the cell [20, 21]. Moreover, while the genome is relatively constant, the proteome is temporally dynamic and varies between cell types, particularly under different physiological states [20]. The sperm protein surface thus holds a potential repertoire of heterogeneous proteins, which are not only responsible for the phenotype (morphology, glyco-topography etc) but also for their protection, survival (Immune-evasion, ion binding/transport) and function in the FRT [22, 23]. Proteomic analysis can not only detect the proteins and measure their quantity but also assesses their associated PTMs [24, 25]. In addition, proteomics can yield accurate fertility biomarkers e.g. SPACA which is an exemplar of novel innovations in proteomics [26–30].

Buffalo is the major milk-producing animal in many countries, however, the poor reproductive efficiency of bulls (low conception rate with artificial insemination) constrains the realization of its full production potential [31, 32]. However, regardless of its economic significance in agriculture-based economies, the proteomic profile of buffalo sperm surface has not been investigated so far. Moreover, since the fertility of thousands of buffaloes is at stake by a single bull, it is therefore imperative to elucidate the buffalo sperm surface profile and their molecular functions that can help to understand their role vis-à-vis male fertility. Although a handful of studies are available on buffalo sperm proteome, these contemporary studies exhibit an inclination towards either seminal plasma proteins or biomarker discovery [33–37]. The proteomics analysis of semen is considered complicated since it contains secretions from various accessory glands and its proteome varies between and within individuals and exhibits seasonal and temporal fluctuations [20, 38].

Given the importance of sperm surface proteins in inter-cellular recognition, communication, and fertilization, an in-depth quantitative proteomic profile of buffalo sperm surface is needed for elucidation of molecular processes and pathways associated with spermatogenesis, fertilization and embryonic development. The role of the buffalo sperm-surface proteome has largely been ignored. The lack of studies on buffalo sperm proteome specifically makes it difficult for researchers to explore and investigate the role of sperm proteins in the regulation of mechanisms associated with sperm function. A comprehensive sperm-surface proteome will not only indicate the nature of the interaction of these proteins, and their reproduction-specific functions but also could be referred for the discovery of fertility-specific proteomic signatures. In this direction, the foremost thrust in the field of sperm proteomics is the profiling of various populations of sperm-surface proteins that would provide the most accurate functional characterization of buffalo sperm. We have previously reported a partial proteome of buffalo spermatozoa wherein we demonstrated the existence of proteins implicated in reproduction and immune-related processes on buffalo spermatozoa [13]. However, the study lacked the criteria for evaluating repeatability in the LC-MS/MS and appropriate controls. Consequently, to improve the depth of identification, we undertook this study to i) understand the proteome of buffalo sperm surface, in general, and ii) to functionally annotate the sperm surface proteins involved in sperm maturation, survival and performance in the FRT. This study can provide a framework for further studies on the functional aspects of buffalo spermatozoa.

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 (Light Microscopy) and viability (Eosin-Nigrosin) 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)

At the time of writing this manuscript, the Buffalo proteome was not available in the UniProt protein database; therefore, we used the NCBI buffalo database reference proteome database translated from the latest chromosome level genome assembly (UOA_WB_1) for peptide identification. 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. Besides, 563, 196 and 164 proteins were found to be treatment-specific and appeared only in the salt-extraction, PI-PLC treatment and in vitro-induced capacitated samples, respectively, indication differential effects of the three distinct sperm-surface protein extraction strategies used in this study (Fig. 3A). 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_025148992.1 and 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 activities and were predicted to be implicated in protein binding (protein-2 or protein-DNA interactions, PPIs and PDIs, respectively). 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 signalling 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).

Reproduction is a complex trait and the complexities of its associated processes indicate that there is more to predict male fertility than the handful of criteria such as motility and morphology, currently in use. Omics technologies offer great promise for improving the reproductive efficiency of males in a cost-effective and non-invasive manner e.g. through predictive measures and identifying the subfertile phenotypes. We presented a straightforward methodology for discovering the various populations of proteins that embellish the buffalo sperm surface and play crucial roles in sperm function (Supplementary Fig. 2). The in-depth functional proteome was deciphered through label-free shotgun proteomics (LC-MS/MS) which has not been explored earlier. The identification of 1342, 678, and 982 distinct proteoforms (FDR < 0.01) in the salt-extracted, PI-PLC treated and capacitated samples, respectively not only reflects the disparity in the interaction of these proteins with the buffalo sperm surface but also the varied influence of the extraction treatments in the removal of non-covalently attached, GPI-anchored and capacitation associated proteins (Supplementary sheet-Results.xlsx). Our study provides a repertoire of sperm surface proteins that will be valuable to decipher the maturational and functional aspects of sperm biology. The overall GO and pathway functional annotation analysis revealed a rich proteomic background covering a myriad of functions. As expected, there was an enrichment of proteins involved in the regulation of fertility, spermatogenesis and zona-binding or penetration, Most of the identified proteins were relevant in the context of male fertility and provide a platform for further studies on sperm biology.

We have previously reported the removal of sperm surface protein by these treatments [13]. However, we included capacitation as a natural method of protein shedding in this study and assessed five distinct sperm function parameters. Several of these parameters have reportedly been associated with male fertility and sperm health [41–44]. The effect of time and medium on function and protein extraction is more pronounced in the spermatozoa because of their elevated susceptible to environmental insults vis-à-vis the female gamete and other somatic cells. This is ascribed to the high unsaturated fatty acids (PUFAs) content in the plasma membrane, exceptionally high surface area/volume ratio (> 50:1) and near absence of repair, proofreading and protective enzymes [45–47]. Moreover, despite using N = 12 biological replicates, stringent search criteria and statistical rigour, our previous study had limitations in terms of limited technical replicates and a short MS run of 60 min [13]. Therefore, to increase the identification depth and confidence, we used this N = 12 biological replicates (minimum 4 ejaculates each, pooled) and additionally n = 3 technical replicates for each sample group apart from increasing the length of MS/MS run to 100 min. and incorporated the proteins released at the time of capacitation, in this study. A wide range of diversity was observed in the more than 1600 proteins and isoforms predicted to be present on the buffalo spermatozoa vis-à-vis their molecular weight, pI, distribution of expression across chromosomes and functional roles (Fig. 3), as reported in other species [48–54]. Several proteins were found to be unique to each of the treatment groups demonstrating indicating their disparate effects on disrupting the non-covalent (2X-DPBS) or covalent interactions (PI-PLC) among the proteins of sperm surface. This remarkable diversity in the proteome is reminiscent of any mammalian cell and sperm is no exception [2, 33, and 37]. Our computation strategy involved the buffalo NCBI RefSeq database which was rich in data on gene isoforms. The translated database used as search space thus included data on proteoforms as well. The interplay between various isoforms of the same protein has been implicated in the regulation of many crucial cellular processes. For instance, a wide spectrum of protamine proteoforms has been discovered in human sperm chromatin that is implicated production of infertile phenotypes. These include full-fledged proteins and truncated and post-translationally modified proteoforms [56]. Thus, the buffalo sperm surface proteome carries information about spermatogenesis, sperm function and paternal contribution to post-fertilization events in the FRT. The factors responsible for high expression from chromosome 5 and the minimal contribution of chromosome 13 (Fig. 6) to the buffalo sperm-surface protein are not yet known. The chromosome length appeared to dictate the gene density in buffalo since the size of chromosome 13 is 30% shorter than the chromosome (Bubalus bubalis, reference genome-UOA_WB_1) explaining lesser expression. Besides, we incorporated peptides rather than proteins in mapping analysis given that various subunits of a single protein complex may be expressed from distinctive chromosomes. For example, the two subunits of the human RBC metabolic enzyme, glucose-6-phosphate dehydrogenase (G6PD) are encoded by two distinct structural genes present on chromosome X and chromosome 6 to give rise to a fully functional enzyme, either by cross-translation of two mRNAs, or transpeptidation [56]. Nevertheless, further studies on whole sperm proteomic analysis of spermatozoa would shed light on this aspect.

Various studies in multiple mammalian species have indicated that the sperm proteins are reflective of the quality of semen or spermatozoa, can indicate the outcome of fertilization and embryogenesis and even identify the subfertile phenotypes [2, 33, 49–51]. Most of these studies have, however, compared seminal plasma, total or sub-populations of spermatozoa or their extracts or have only used regression models to identify stable fertility biomarkers [56–59]. The shotgun proteomics (LC-MS/MS) approach used in this allowed us to determine the effect of extraction treatments and helped to attain an unparalleled depth of 1695 buffalo-specific proteoforms. This increased depth contrasts with prior previous proteomic studies on buffalo sperm that have used less powerful methods e.g. 2D gel electrophoresis coupled to MS, which although provided valuable insights into protein patterns of fertility, however, addressed a limited number of proteins due to technical limitations [7, 28, 33 and 50]. The label-free quantification (LFQ) used in this study has been demonstrated to be better than the laborious gel-based techniques because of its better accuracy, less variation and experimental errors, and lower sample requirements [60]. The results obtained in this study reveal minimal variations across the sample loadings and among the technical replicates used for the three extraction treatments [Fig. 6C]. This is in concordance with other proteomic studies, wherein protein extraction and digestion have been demonstrated to be the major sources of technical variability in LC-MS proteomics [61].

One limitation of this study is that some intracellular proteins were also identified along with the surface proteins (Supplementary sheet-Results.xlsx). However, as expected, the integrity of the plasma membrane is likely compromised, with time, in a few sperm cells thus leading to the release of intracellular proteins in the incubation medium. The sub-cellular compartments of mammalian spermatozoa are known to be highly dynamic architectures which are sensitive to extraction treatments [19]. Nevertheless, it is worth mentioning that many proteins hitherto thought to be intracellular have now been identified on sperm surface e.g. HSP60, PGK2, AK1 [62, 63]. Furthermore, it has been reported that many core intracellular proteins e.g. nucleosomal histones (H1, H2A, H2B, H3 and H4) can directly translocate across the cellular membrane and act as carriers for the import of extracellular macromolecules into living mammalian cells [64].

We employed BLAST2GO 5.0 (OmicsBox) and KEGG Orthology Based Annotation System (KOBAS 3.0) for functional and pathway annotation of the identified proteins considering the lack of annotated bio-molecular data for buffalo [65, 66]. As mentioned earlier, buffalo is not listed in the commonly used gene/protein annotation software, gene ontology and functional annotation tools. The GO term cellular component indicated the enrichment of proteins localized to the plasma membrane, cell-periphery, and acrosome vesicle or secreted in the extracellular space. The identified proteins were predicted (by BLAST2GO) to be primarily involved in the regulation of metabolism, immune system (immunomodulation) and reproductive processes (e.g. gamete interaction) and a majority of them possessed one or other catalytic domains (Fig. 7). These findings indicated successful extraction of sperm-surface, plasma membrane proteins by the method used in this study. These observations are in agreement with the earlier studies that have also presented the evidence of expression of genes in spermatozoa that are crucial to sperm functional attributes, its maturation, survival, fertilization, and embryonic development (Fig. 7) [67–69]. Cross-species sequence-similarity mapping by KOBAS analysis corroborated the enrichment of various metabolic-regulatory pathways and those associated with immune-related and cellular homeostasis (Fig. 8). Overall, to the best of our knowledge, this is the first time that > 1500 non-covalently (electrostatically) linked, GPI-APs and capacitation-related proteoforms (mostly un-annotated) from buffalo sperm surface have been identified and functionally annotated by extrapolation of function from their respective annotated sequence orthologs (Figs. 9 and 10).

The results of this study again underpin the intricate relationship which entwines innate immunity, immune-evasion and and sexual reproduction (10, 13). During the transit of spermatozoa in the MRT, several surface proteins are either removed or modified and numerous novel antigens are either adsorbed or inserted in the spermatozoa plasma directly or associate with existing binding proteins e.g. clusterin, lipocalins and GPI-APs [70–73]. Such adhered proteins are thus present in a peripheral cellular environment bound to the sperm surface likely by the electrostatic/hydrophobic interactions. Several of these secretagogues, including BDs like SPAG 11, Bin1b and DEFB-126 are thus applied as a peripheral coat onto the surface of a mammalian spermatozoon during its passage through the MRT and are often reported to be overrepresented [73, 74–77]. The abundance of immune-related proteins has also been associated with the stabilization of the sperm membrane during the immune attack by immune cells and immunomodulation, particularly in the uterine lumen [59, 78 and 79]. For instance, we identified 11 unique DEFBs (β-defensins) in our study viz. DEFB-107A-like, 110, 112, 113, 114, 116, 119, 121, 123, 43-like and DEFB-129 (BuBD-129). Such a high abundance of host-defence peptides (HDPs) is in agreement with previous ‘omics studies that have also demonstrated a high expression of DEFB genes(in the epididymis) known to be implicated in evading the elicited immune responses(protection) and regulation of sperm function [80, 81]. Many of the spermatozoa transcripts of β-defensins such as DEFB-1, 7, 123,124,119 and DEFB-110 have been reported to be highly expressed in HF bulls and thus implicated in regulating fertility (Fig. 9). A pertinent question that arises from our study on buffalo is “Why so many”? The DEFBs (β-defensins) have been proposed to be crucial for the health of the MRT by providing a rapid, broad-spectrum response to disparate pathogens and are also required for interacting with immune cells in the FRT, functioning as chemokines or transduction of the information stored in their abundant O-linked glycans [21, 82]. It has been proposed that sperm competition, sexual conflict and sexual selection in combination or individually could provide the rapid adaptive evolution observed in reproduction-related genes [83–84]. An asymmetric evolution after tandem gene duplications appears to be the case for BuBDs, which have shaped the evolution of the β-defensin gene family in bovids including buffalo. This phenomenon is quite common in mammalian evolution contributing to varying gene numbers in different species [10, 79]. Alternatively, the high number of BD genes in buffalo as observed in cattle, especially on the sperm surface may be due to the domestication process that has subjected the animals to higher population densities vis-à-vis the wild bovids. This would have exposed them to more numbers of disparate microbes because herd structure and the polygynous mating system promote rapid transmission of the diseases [16–18, 85]. The seminal β-defensins also protect the sperm from LPS-mediated inflammation that is known to reduce motility [86]. Interestingly, BuBD-129 was the only β-defensin common to all the extraction methods. This novel defensin has been demonstrated to embellish the entire buffalo sperm and was predicted to be heavily O-glycosylated [10, 13]. This explains the anomaly between the observed band (~ 35-40kDa) and computed molecular weight (MW) which is ~ 19kDa. (Fig. 6D) Similar effects of O-glycosylation have been reported to cause a shift in the observed molecular weight [87–89]. Preliminary data such as ours indicate that the novel β-defensin, BuBD-129 is among the prominent buffalo sperm coat proteins, which could be a potential fertility biomarker [73, 87 and 89]. Future studies on elucidating its exact physiological function would unravel its pinpoint role in the modulation of fertility.

A shotgun proteomics-based deep proteomic profile of the buffalo sperm surface was generated in this study and > 1600 distinct proteoforms were reported on the sperm surface. The buffalo sperm-surface proteome fingerprint generated in this study would help to decipher the functional role of these proteins in the acquisition of fertilizing ability by sperm. The samples used in our study were taken from fertile bulls of proven fertility, however, it would be interesting to compare the pattern of abundance of sperm surface proteins with low fertile males. Different protein combinations are responsible for sperm maturation and preservation and the process appears to be species-specific, therefore this study is a welcome step to obtaining a greater understanding of this aspect of male reproduction which is fundamental for the survival of all species. These protein inventory lists, such as one generated in this work will shed light on peripheral and transmembrane proteins hitherto unknown to exist on the buffalo sperm surface.

Chemicals and plasticware

All chemicals, media and reagents were procured from Sigma Aldrich Chemical Co. Ltd, (USA) unless stated otherwise. All plasticware was procured from Nunc Inc. (Thermo Scientific, USA). Fetal bovine serum (FBS) was obtained from Hyclone, Canada.

Semen Sample Collection And Separation Of Seminal Plasma

Neat semen samples (mass motility ≥ 3) were collected from Murrah buffalo bulls of proven fertility (N = 12, 4 ejaculates each, aged between 3-5years) using an artificial vagina at the Artificial Breeding Research Centre (NDRI, Karnal, India). The ejaculates were collected in 15mL Falcon centrifuge tubes containing Sp-TALP i.e. HEPES buffered Tyrode’s medium (pH 7.4, 38°C) referred to as non-capacitating medium (NCM). The semen was transported to the laboratory at 38°C and was washed thrice by centrifugation at 600 x g for 10 min to separate the seminal plasma components. The motile spermatozoa were subsequently selected by subjecting the final pellet to the swim-up technique and thereafter the motile sperm fraction was resuspended in variable volume (200–800µL) of the NCM according to the downstream experiments. Subsequently, all the samples were subject to motility and viability analysis after processing by light microscopy and Eosin-Nigrosin staining. Only the samples with > 80% motility and viability were considered for further experiments

The Removal Of Buffalo Sperm-surface Proteins

We used three different strategies for extracting surface proteins from the motile spermatozoa fraction. The motile spermatozoa were subjected either to elevated salt extraction (2X-DPBS) or 2U/mL Phosphatidylinositol-specific phospholipase-C (PI-PLC) treatment or in vitro capacitation for releasing the non-covalently (electrostatic interactions) bound, GPI-anchored, capacitation induced proteins, respectively. The elevated salt extraction and PI-PLC extractions of buffalo sperm-surface proteins were done as per the procedure described by Batra et al [13]. Briefly, the post-swim-up spermatozoa were incubated in 2X-DPBS (elevated NaCl) for 30 min in siliconized micro-centrifuge tubes at 38°C with gentle shaking and subsequently pelletized by centrifugation at 280 x g for 10 min. The supernatants from 5 ejaculated were pooled and filtered through a 0.22µm filter. For PI-PLC extraction, 100 x 10⁶ spermatozoa were incubated with 2U/mL PI-PLC from Bacillus cereus (Sigma) for 2h with gentle shaking at RT. Subsequently, the samples were centrifuged at 1000 x g for 10 min and the supernatants from all the ejaculates from all the bulls were pooled and filtered. The extraction of proteins released during capacitation was done by inducing in vitro capacitation of buffalo spermatozoa as described by Batra et al. [81]. Briefly, 400 x 10⁶ spermatozoa were suspended in the capacitating medium (NCM supplemented with 6 mg/mL bovine serum albumin (BSA), 2 mM CaCl₂.2H₂O and 10 mM NaHCO₃) for 6h in a 5% CO₂ incubator at 38°C after washing twice in the capacitating medium (CM) containing 1 and 3 mg/mL BSA, sequentially. After the incubation period, the samples were centrifuged at 600 x g for 10 min and the supernatants from 4 ejaculates were pooled and filtered. The sperm capacitation was confirmed by chlortetracycline (CTC) staining, as mentioned below. Only the samples containing more than 75% of capacitated spermatozoa were used for further experimentation. The proteins in the filtrate obtained from the three treatments were precipitated using the acetone-precipitation method (1:9, supernatant/acetone ratio), concentrated on a speed-vac vacuum concentrator and subjected to SDS-PAGE after quantification by Quick Start™ Bradford protein assay.

Sperm Function Parameters (Cytomics)

After protein removal, the control and protein-extracted buffalo spermatozoa were evaluated for intactness of structural and functional integrity. The assessment of sperm-membrane integrity, acrosome reaction and lipid peroxidation were done by using carboxyfluoresceindiacetate-propidium iodide (CFDA-PI), Fluorescein isothiocyanate conjugated Arachis hypogaea (Peanut) Lectin (PNA) (FITC-PNA ) and 4, 4-Difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) dyes, respectively, as per the method described by Singh and colleagues [90]. However, the assessment of mitochondrial membrane potential (MMP) by JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) and capacitation status by Chlortetracycline (CTC), a fluorescent cheated probe of Ca²⁺ was done by the methods described by Saraf and co-workers [91]. After staining, the excess stains were removed by washing the stain-incubated spermatozoa with 200 µL of sperm-TALP by centrifugation at 800 x g for 3 min. The pelleted spermatozoa were used to make a thin smear onto which a few drops of mounting medium, Dabco® 33-LV were placed and was observed at 1000X magnification under an Olympus BX-51 fluorescence microscope using appropriate filters. A minimum of n = 250 spermatozoa (in triplicates) were evaluated, in a minimum of 10 fields for observing fluorescent patterns. The images of the two filters were merged to obtain the final image, wherever required.

2.4 Protein Tyrosine Phosphorylation (Ptp) Status

The protein tyrosine phosphorylation (PTP) status of the buffalo spermatozoa was assessed using an indirect immunofluorescence assay as described by Saraf et al [92]. The control and the protein-extracted spermatozoa were washed with PBS at 300 x g for 5min and a 20µL sperm suspension was then smeared onto a clean glass slide and air dried. Thereafter, the spermatozoa were fixed in 4% paraformaldehyde for 1h at 4˚C and washed with PBS (3X). The spermatozoa were permeabilized using methanol and the slides were blocked using 5% BSA in PBS for 2h at room temperature. Subsequently, the smears were incubated with monoclonal anti-phosphotyrosine antibody (P1869; Sigma, 1:100) in 1% BSA for 3h at 38˚C and then washed with PBS (3X). Afterwards, the smears were incubated with FITC-conjugated anti-mouse IgG antibody produced in goat (F4018; Sigma, 1:100) and then washed thoroughly with PBS. After the final washing step, the coverslip was mounted onto a dried glass slide. A drop of mounting medium, Dabco® 33-LVwas placed on the slide and the cells were then observed under a BX-51 Olympus fluorescence microscope at 1000X magnification using a FITC filter. The spermatozoa were assessed for the percentage of different phosphorylation patterns and a minimum of 250 spermatozoa were counted (in three technical replicates) across the slide. The three patterns of tyrosine phosphorylation (pattern NP – no fluorescence; pattern EM- fluorescence over the equatorial region and mid-piece and pattern AEM – fluorescence over the acrosomal area, equatorial region and mid-piece) were counted and expressed as percentages.

Electrospray Ionization, Tandem Mass Spectrometry (Lc-ms/ms) Analysis

A snapshot of the proteomics workflow followed in this study is depicted in Fig. 11. The protein pellets from the three extraction treatments were initially dissolved in 6M guanidium hydrochloride. Subsequently, 25µL of the dissolved samples were reduced with 5mM TCEP and then alkylated with 50mM iodoacetamide (in the dark). Next, the samples were digested with trypsin (1:50, trypsin/lysate ratio) for 16 h at 37°C (in-solution digestion). Afterwards, the digests were cleaned using a C18 silica cartridge and dried using a speed vac vacuum concentrator. The dried pellet was resuspended in Buffer A (5% acetonitrile, 0.1% formic acid). All the experiments were performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to a Thermo Fisher-QExactive mass spectrometer equipped with a nano-electrospray ion source. The peptide mixture (1µg) was resolved using a 25 cm PicoFrit column (360µm outer diameter, 75µm inner diameter, 10µm tip) filled with 1.8 µm of C18-resin (DrMaeisch, Germany). The peptides were loaded with Buffer A and eluted with a 0–40% gradient of Buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 300nL/min for 100min. The electron spray ionization (ESI) was the source of ions and the MS scan mode was Orbitrap. The collision-induced dissociation (CID) was the mode of fragmentation (y and b ions) and the linear ion trap was the MS/MS scan mode (400–2000 m/z). The MS1 resolution was set to 70000 while the MS2 resolution was 17500. The MS data were acquired using a data-dependent top10 (most intense peaks) method that dynamically selects the most abundant precursor ions from the survey scan (Supplementary Fig. 2). To check the performance of the instrument, a 20 fM standard BSA digest was parallel analyzed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [93] partner repository with the dataset identifier PXD028026.

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Data Processing And Analysis

All samples were processed and the generated RAW files were analyzed with Proteome Discoverer (ThermoFusion v2.2) against the NCBI buffalo database reference proteome database translated from the latest chromosome level genome assembly (UOA_WB_1). For the Sequest search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. The protease used to generate peptides, i.e. enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R: unless followed by “P”) along with a maximum missed cleavage value of two. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation were considered as variable modifications for database search. Both, the peptide spectrum match and protein false discovery rate (estimated by searching the data against a decoy database) were set to 1% to ensure only high-confidence protein identifications for the peptide-spectrum match (PSM) and site decoy fraction. The proteins identified with a minimum of one unique peptide (≥ 1 high-quality PSM per peptide) were used for analysis. Some of these peptides are likely legit and could be missed if the ‘minimum two peptides’ criterion was used. Therefore, the MS/MS spectra of these proteins were manually verified.

The downstream statistical analysis was performed in the Perseus environment [94] wherein the LFQ-intensity (abundance values from PD v.2.0) values were log₂ transformed. The data was imputed for missing values by assuming normal distribution using the implicit algorithm. The quantitative differences in the LFQ-intensity values of extracted proteins after treatments were analyzed by the two-tailed t-test (assuming equal variance within the groups of replicates) with Benjamini-Hochberg false discovery rate (FDR)-correction of the P-value (correction of the alpha error) at < 0.01 significance level. The volcano plots were used to analyze two-tailed significance values identified by determining fold change vs. –log₁₀ fold change. A P-value < 0.05 and FDR ≤ 0.01 were considered to be statistically significant.

Validation Of Identified Protein (β-defensin-129) In The Mass Spectrometric (Lc-ms/ms) Analysis

We have previously reported that the BuBD-129 and 126 were the most highly expressed β-defensins expressed in the male reproductive tract (10). The BuBD-129 reportedly ensnares the entire buffalo spermatozoa and was selected for western blot (WB) assays to validate its presence amongst the extracted proteins. The PVDF membranes (blots) were incubated with custom synthesized polyclonal antibody specific for BuBD-129 (1:25000) at 4°C overnight with slight agitation [13]. After probing with a primary antibody, the blots were washed thrice in TBST for 10min. Later, the blot was probed with the optimized dilution of HRP conjugated secondary antibody for 2 hours at RT: HRP-labeled anti-rat IgG (1:75,000 dilution; AP136P, Sigma Aldrich). Membranes were washed 4 times in TBST with each washing for 15 min. before detecting the bound antibodies. The immunoreactivity was detected by using chemiluminescence (Luminata forte substrate, Merck), as per the manufacturer’s instructions. The immunoreaction signals were captured using CL-XPosure Film (Thermo Scientific).

Enrichment And Pathway Analysis

The proteome for water buffalo (Bubalus bubalis) was not available at the time of proteomic data analysis for this manuscript. Besides, most of the gene/protein annotation software, gene ontology and functional annotation tools do not list buffalo as a reference organism. Therefore, we used the NCBI buffalo database reference proteome database (UOA_WB_1) and the translated coding sequence file, translated CDS (.faa) was used as the search database (decoy). BLAST2GO 5.0, a platform for high-quality functional annotation and analysis of high-throughput omics datasets was employed for the functional annotation of identified proteins. It transfers the existing functional annotation from existing annotated sequences to the uncharacterized novel sequences (e.g. Buffalo). The sequence-derived functional information was generated through BLAST2GO. It uses functional attributes of the gene ontology analysis to assess the biological meaning of the mined data, which was subsequently visualized using MS-excel. For the annotation and identification of enriched pathways, the functional enrichment analysis web tool, KOBAS 3.0 was used. It is a KEGG orthology-based annotation system (KOBAS), used for automated annotation and pathway identification of novel and uncharacterized proteins. The pie-doughnut diagrams were generated using HighCharts and the chord plots were generated using Circos and Graph1Bio.

BD/ DEFB: Beta-defensin

BuBD-129: Buffalo beta-defensin-129

GPI: Glycosylphosphatidylinositol

FRT: Female reproductive tract

IF: Immunofluorescence

KOBAS: KEGG orthology-based annotation system

LC-MS/MS: Tandem Mass Spectrometry

PI-PLC: Phosphoinositide-phospholipase C

Acknowledgements

The assistance of Balkar Singh and Sonu Kumar in logistics and catering support is gratefully acknowledged.

Author Contributions

The study was conceptualized and designed by V.B., R.K., and T.K.D. Sample collection, microscopy and ICC were done by V.B., K.D. & A.K. Proteomics experiments and data analysis were done by V.B., K.D. & M.P. K.D. & V.B. performed statistical analyses. R.K. and A.K. implemented and conducted Western Blotting. The manuscript was written by V.B. & M.P.D. Figures were designed by V.B., M.P. & A.K. All the authors contributed to the article and approved the submitted version.

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. The LC-MS/MS data are available via ProteomeXchange with the identifier PXD028026.

Consent for publication: Not Applicable

Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics approval and consent to participate

All the experiments were monitored and approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-National Dairy Research Institute and the methods were carried out per IAEC and ARRIVE guidelines.

Funding

This work was supported by the Bill & Melinda Gates Foundation (grant number OPP1154401).

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Shotgun Proteomics-based distinctive fingerprint of sperm-surface revealed over- representation of proteins driving spermatogenesis, motility, and fertility

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Abstract

Background

Results

Discussion

Conclusions

Figures

Background

Results

Sperm Functional Parameters:

Membrane integrity assessment

Capacitation And Acrosome Integrity Status

Lipid Peroxidation

Mitochondria Membrane Potential

Protein Tyrosine Phosphorylation

Buffalo Sperm Surface Has Thousands Of Distinct Proteins (And Their Isoforms)

Chromosome Mapping Of Buffalo Sperm-surface Proteins

Molecular Characteristics Of Buffalo Sperm Surface Proteins

Enrichment and pathway analysis

Discussion

Conclusions

Methods

Chemicals and plasticware

Semen Sample Collection And Separation Of Seminal Plasma

The Removal Of Buffalo Sperm-surface Proteins

Sperm Function Parameters (Cytomics)

2.4 Protein Tyrosine Phosphorylation (Ptp) Status

Electrospray Ionization, Tandem Mass Spectrometry (Lc-ms/ms) Analysis

Reviewer Account Details:

Data Processing And Analysis

Validation Of Identified Protein (β-defensin-129) In The Mass Spectrometric (Lc-ms/ms) Analysis

Enrichment And Pathway Analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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