The proteomic landscape of sperm surface deciphers its maturational and functional aspects in buffalo

Abstract

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

Keywords: buffalo; male fertility; reproduction; shotgun-proteomics; sperm.

FIGURE 1

Sperm Functional Parameters (SFPs) The buffalo spermatozoa were subject to three surface protein extraction treatments viz. Elevated salt extraction (2X-DPBS) or 2 U/mL Phosphatidylinositol-specific phospholipase-C (PI-PLC) treatment or in vitro capacitation and the functional parameters were assessed along with the respective controls (see text). CFDA with PI (see text) was used to assess the membrane integrity of the spermatozoa that were categorized as live, dead or moribund (A, D) CTC (see text) was used to categorize the spermatozoa as non-capacitated (NC), capacitated (C), or acrosome-reacted (AR) (B, E) JC-1 (see text) dye was used to assess the mitochondrial membrane potential (MMP) and the spermatozoa were distinguished as with high or low MMP (C, F). Different letters denote statistical difference (p < 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

The sperm surface proteins, data pre-processing, statistical analysis, and cross-sample and replicate comparisons. Proteome Discoverer (v2.2) identified 1,342, 678, and 982 distinct proteins and isoforms (p < 0.05, FDR<0.01) in the salt-extracted, PI-PLC treated and capacitated samples, respectively (A). The Perseus software platform (v2.0.2.0) was used for normalizing the sample protein abundances and interpreting their quantification. Representative histograms depict the normalized abundances before transformation (B) and after transformation (C) with significantly abundant proteins marked in yellow. The computation of sample coefficient of variation to understand the sample variability (D) and dimensionality reduction using principal component analysis, PCA (E) were also performed in Perseus v2.0.2.0. The volcano plots were used to assess the statistical significance (-log10 p-value) vis-à-vis the magnitude of change (log₂ Fold change) for the differential abundance proteins across the three samples and their controls, e.g., elevated salt and its control (F).

FIGURE 3

Chromosome mapping of buffalo sperm-surface proteins Circos plots (chord plots) rendered as ratio layouts indicating the individual chromosomal localization of the identified peptides which revealed an uneven distribution of chromosomal protein expression, e.g., in before (A) and after (B) PI-PLC treatment of buffalo spermatozoa. Ribbons in the ratio layouts represent the relationship between the number of peptide-spectrum matches (PSMs) and the number identified peptides coded from each of the depicted chromosomes. Thickness of the ribbon indicates the number of PSMs/identified peptides (proteins) from that chromosome.

FIGURE 4

Overrepresented proteins The violin plots for overrepresented proteins beta-defensins (DEFBs) were computed for the assessment of the log2 normalized abundance values across the sample and technical replicates for all three treatment groups, e.g., capacitation (A). Blot images showing relative abundance of beta-tubulin and BuBD-129 in non-capacitated (NCM), control and capacitated (CM) spermatozoa (B). The in-house generated polyclonal anti-BuBD-129 produced a prominent band at ∼35–40 kDa in both control and CM sperm (B, right). The mouse monoclonal anti-β-tubulin antibody (T8328, Sigma-Aldrich) produced a band at 50 kDa (B, left) and served as the reference control. Densitometry analysis was done in ImageJ and the adjusted density values indicated the removal of BuBD-129 after induction of in vitro capacitation (C). **p < 0.01.

FIGURE 5

The functional annotation of the LC-MS/MS identified proteins Pie-donut charts indicating the results of BLAST2GO analyses illustrating biological process, molecular function and cellular component ontology terms across the elevated salt (A), PI-PLC treated (B) and capacitated spermatozoa (C). The GO IDs from every significant alignment for each sequence were directly annotated to the sequence if the alignments similarity passed the desired minimum and validated to remove the intermediate GO terms.

FIGURE 6

Exploratory visualization of the enriched terms from pathway analysis by KEGG orthology-based annotation system (KOBAS) KOBAS 3.0, was employed for automated annotation and pathway identification of novel and uncharacterized proteins which revealed a 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 elevated salt (A), PI-PLC treated (B) and capacitated spermatozoa (C). Bubbles represent p-values for pathway terms in different clusters consisting of colour mega-cluster (from C1 to C7) and top clusters not matched to any mega-cluster (light grey bubbles). Interrelated pathways are clustered and rendered in the same color.

FIGURE 7

Sperm surface-specific functional proteomic repertoire The literature-based functional characterization of buffalo sperm surface-specific N = 873 unique protein isoforms identified across the samples indicated their involvement in the regulation of various aspects of male reproductive physiology mainly sperm production, survival and function. Most of the identified proteins are implicated in spermatogenesis, sperm protection and regulation of male fertility.

FIGURE 8

Origin and Localization. Pie charts indicating the source or the origin (A) and sub-cellular location (B) of the buffalo sperm surface-specific 873 unique proteoforms identified across the samples, as inferred from the literature search. Almost a third of the proteins originate in testis and another third are added in the epididymis (and epididymosomes). Most of the proteins were predicted to be on the sperm exterior. SPP, Seminal Plasma Protein, PM, Plasma Membrane.