Only the ejaculates that were milky or creamy in colour, homogenous in consistency i.e. free from flakes/clumps with a minimum sperm concentration of 600 x 106/ml were considered for swim-up and further downstream experiments. The average motility and viability of seven representative sample ejaculates after processing was 81.84 ± 1.20 and 85.85 ± 1.16 respectively. The samples were diluted according to the experiments, as mentioned, wherever required.
Hundreds of proteins are bound on buffalo sperm surface either through electrostatic interactions or by a GPI anchor
The extracted sperm-surface proteins indicated enough diversity among the types of protein removed using the seven treatment groups viz. 2X-30, 2X-60, 4X-30, 4X-60, and 1U/mL, 1.5U/mL and 2U/mL representing the elevated salt extractions (2X/4X-DPBS for 30/60 min) and PI-PLC extractions, respectively (Supplementary Figs. 1 and 2). All the elevated salt and PI-PLC treatments’ extracted sperm-surface proteins produced > 20000 PEP-XML spectra. The iprophet tool correctly identified more than 300 proteins in all of the treatments at p > = 0.99 where p indicates the probability that the spectra have been correctly matched to its analogous peptide (Table 1). A total of 317, 391, 395 and 432 proteins were identified in 2X-30, 4X-30, 2X-60 and 4X-60 (DPBS) treatments respectively. On the other hand, 385, 353 and 364 proteins were identified in the 1U, 1.5U and 2U/mL PI-PLC treatments, respectively. At p ≥ 0.99 zero proteins were found to be incorrectly identified. Many proteins were found to be unique to each sub-group of either treatment (elevated salt or PI-PLC) demonstrating that the individual combinations of incubation time and salt/enzyme concentration exerted disparate effects on disrupting the non-covalent/GPI interactions among the proteins of sperm surface. Moreover, nearly 30% of the proteins were observed as common between any two treatment subgroups (Supplementary Figs. 1 and 2).
Table 1
Treatments for sperm-surface protein extraction and corresponding TPP results indicating total spectra, correctly and incorrectly identified proteins at p ≥ 0.6 and 0.99
Sample
|
PEP-XML Total spectra
|
Ipro.pep.xml Total spectra
|
Prot.xml Total proteins
|
Total proteins with p ≥ 0.6
|
Incorrectly identified with iprophet p ≥ 0.6
|
Total proteins with p ≥ 0.99
|
Incorrectly identified with iprophet p ≥ 0.99
|
2X-DPBS-30 min
|
26535
|
7252
|
1733
|
875
|
76
|
317
|
0
|
4X-DPBS-30 min
|
26470
|
8399
|
1725
|
956
|
76
|
391
|
0
|
2X-DPBS-60 min
|
25947
|
7742
|
1898
|
1013
|
90
|
395
|
0
|
4X-DPBS-60 min
|
25951
|
8210
|
2162
|
1244
|
99
|
432
|
0
|
1 U/ml
|
24829
|
6557
|
1788
|
992
|
99
|
385
|
0
|
1.5 U/ml
|
24587
|
6343
|
1932
|
1005
|
76
|
353
|
0
|
2 U/ml
|
24881
|
5978
|
1662
|
793
|
63
|
364
|
0
|
Overall, we report a total of 352 buffalo sperm-surface proteins that were identified in the protein fractions extracted from sperm incubation with elevated salt (2X/4XDPBS) or enzyme (PI-PLC) concentration. The LC-MS/MS data analysis identified several BDs including the two Class-A beta-defensins (CA-BDs) viz. BD-129 and 126 amongst the other -surface proteins Notwithstanding, only 119 proteins were common among the elevated salt and PI-PLC treatments which were predicted to be extracellular (Supplementary Fig. 2 and Supplementary sheet-Results). A remarkable diversity in the range of M.W and pI was observed among the selected sperm-surface proteins (Supplementary sheet-Results). The BDs were found to be among the proteins with lowest molecular weight while the angiotensin-converting enzyme (ACE) and the two uncharacterized proteins (UniProt ID: F1MD73 and F1MQ37) were the on the other end of the scale (Mr=141.24, 190.10 and 227.10 respectively). The BDs Spag-11D and BD-129 had the highest pI while the Acrosin inhibitor 1 had the lowest pI (4.25). Only three (5%) proteins viz. Sperm acrosome membrane-associated protein1, Angiotensin-converting enzyme and an uncharacterized protein (F1MD73) were predicted to contain a transmembrane segment. A high level of PTMs, especially glycosylation appears to modify the analyzed proteins. More than 80% of the analyzed proteins were predicted to possess at least either one N-glycosylation site or one O-glycosylation site. The uncharacterized protein F1MQ37 and Keratin contained a maximum of 47 and 52 O-glycosylation sites. The BD-126 and 134 were predicted to contain one N-glycosylation site while the BD-129 was predicted to contain three N-glycosylation sites. BD-126 was predicted to contain two O-glycosylation sites whereas the BD-129 predicted to contain eight such sites (Supplementary sheet-Results).
Proteins involved in the immune response and reproductive processes adorn the buffalo sperm surface
GO analysis was performed on the identified 119 extracellular (EC) sperm-surface proteins (Supplementary Fig. 2) wherein the annotation terms for Biological Process, Molecular Function and Cellular Component were determined. The 119 buffalo sperm-surface proteins were successfully mapped to 63 entries in the background dataset. The singular enrichment analysis (SEA) for Biological Process terms’ identified reproductive processes, sexual reproduction, immune response and response to biotic/abiotic stimulus terms as the major GO annotations (Table 2) in the input list vis-à-vis the background reference dataset, the Bovine genome locus (Bovine Genome Database): GLEAN_03528. The scatter plot analysis (SPA) using SimRel for Biological Process similarly indicated semantic similarities between reproductive process functions, immune response and response to biotic/abiotic stimulus terms (Fig. 1) as observed by their closeness in the displayed two-dimensional space. The SEA for Molecular Function indicated that the majority of proteins were (Fig. 1) involved in catalytic and binding (carbohydrate or protein) functions. The SPA for Molecular Function (Fig. 1) also identified protein binding and catalytic activity as the major GO terms with the highest uniqueness index values and the least dispensability scores. Most of the proteins were found to be extracellular, vesicular or part of the plasma membrane as indicated by the SEA and SPA for the Cellular Component terms (Table 2 and Supplementary sheet-Results). The low p-values from the Fisher's test and the results of Yekutieli test (low FDRs) are indicative of high confidence in the determined annotation terms for the input list in the SEA (Table 2). Similarly, the lower log10 p-values and dispensability score with high uniqueness index indicate the reliability of the GO annotation terms for the input list in the SPA (Supplementary sheet-Results). Overall, the results suggest that the buffalo sperm surface is adorned with vesicular or extracellular proteins which are involved in reproduction specific activities, immune responses, responses to biotic/abiotic stimuli and perform catalytic or carbohydrate/protein binding functions.
Cytometry reveals removal of glycosylated proteins from buffalo sperm surface after elevated salt/PI-PLC treatment
The Flow cytometry analyses were performed on the control sample (NCM), spermatozoa incubated in elevated salt for 30 min (2X-DPBS) and spermatozoa incubated with 2U/mL PI-PLC to assess the corresponding changes in glycosylation after either treatment. The analyses revealed a reduction in O-linked, as well as N-linked glycans after elevated salt and PI-PLC treatments as illustrated by the decline in the MFI, produced upon binding of FITC-bound lectins on the buffalo sperm-surface (Fig. 2). A panel of five O-linked glycans specific lectins viz. ABL, JAC, MAL-II, LCA and PNA and one N-linked glycan specific lectin, LEL was used. The Brown-Forsythe test for all the lectins was found to produce a non-significant p-value indicating no differences in standard deviations of the MFIs produced in these groups (p < 0.05). The unstained spermatozoa were excluded from the analysis by gating and the singlets were chosen and the analyses were performed on single, stained spermatozoa. The O-linked glycan-binding lectin ABL has specificity towards Thomsen-Friedenreich antigen, galactosyl (β-1, 3) N-acetylgalactosamine (39). It produced a mean fluorescence intensity (MFI) of 1, 56,610.0 in the control sample which differed significantly (p < 0.001) from the MFI produced in the spermatozoa incubated in 2X-DPBS (1, 25,032.0) or treated with PI-PLC (1, 29,399.0) as assessed by one-way ANOVA (Fig. 2 and Supplementary Fig. 3). The lectin JAC which has a sugar specificity towards galactose of O-linked glycans preferring the structure galactosyl (β-1, 3) N-acetylgalactosamine also produced higher MFI in the control sample (2,47,848.0) in comparison to either the 2X-30 sample (1,71,757.0) or the PI-PLC treated sample (1,27,951.0). The post-hoc analysis indicated that the reduction in MFI for JAC after either the salt treatment (p < 0.001) or the PI-PLC treatment (p < 0.0001) was not only significantly different from the control sample but also each other (p < 0.01) (Fig. 2 and Supplementary Fig. 3). The N-linked glycan-binding lectin LEL which is specific for [GlcNAc] 1–3, N-acetylglucosamine is also removed from the sperm surface on exposing the spermatozoa to elevated salt milieu producing a diminished MFI of 48,715.0 which didn’t differ significantly from the MFI produced in control samples (92,968.0). (Fig. 2 and Supplementary Fig. 3). The exposure of PI-PLC, nonetheless, reduced the MFI fluorescence significantly to 32,161.0(p < 0.05). The LCA lectin specific for mannose and glucose produced significantly (p < 0.001) reduced MFI of 26,979.0 in the elevated salt-treated spermatozoa when compared to the control sample (36559.0) (Fig. 2 and Supplementary Fig. 3). Conversely, the MFI increased minutely to 38,451.0 after exposure to PI-PLC. The α-2, 3 linked sialic acid-binding lectin MAL-II (p < 0.001) similarly produced a significantly higher MFI in the control sample (6015.0) in comparison to the 2X-30 sample (4820.0) (Fig. 2 and Supplementary Fig. 3). Nevertheless, as observed for LCA binding, an MFI increased albeit significant (p < 0.001) after PI-PLC treatment (7435.0). The MFI produced upon MAL-II binding differed significantly (p < 0.0001) from each other (Fig. 2 and Supplementary Fig. 3). The acrosomal intactness indicator lectin PNA, which binds the asialylated galactosyl (β-1, 3) N-acetylgalactosamine produced higher MFI in both the salt-treated (28,334.0) and the PI-PLC exposed spermatozoa (23,075.0) when compared to the control spermatozoa (18,759.0). However, the rise was statistically insignificant for both the treatments (Fig. 2 and Supplementary Fig. 3).
Overall, both the treatments reduced the availability of respective cognate glycans for most lectins except the PNA after salt treatment. Contrarily, the PI-PLC treatment led to increased exposure of α-2, 3 linked sialic acid and asialylated galactosyl (β-1, 3) N-acetylgalactosamine. Furthermore, both the treatments were significantly different from each other vis-à-vis the MFI produced upon lectin binding on the surface of the buffalo spermatozoa.
Table 2
The major GO terms, Fisher’s p-values and Yekutieli result for Multi-test alignments (FDR under dependency) for SINGULAR ENRICHMENT ANALYSIS SEA of Biological Process, Molecular Function and Cellular Component for the sperm-surface proteins in the input list.
Biological Process
|
Molecular Function
|
Cellular Component
|
Term
|
p-value
|
FDR
|
Term
|
p-value
|
FDR
|
Term
|
p-value
|
FDR
|
Multicellular organismal process
|
2.00E-27
|
1.40E-24
|
Enzyme binding
|
2.90E-24
|
2.90E-22
|
Membrane-bounded vesicle
|
5.70E-85
|
6.10E-83
|
Sexual reproduction
|
1.90E-25
|
6.60E-23
|
Ubiquitin protein ligase binding
|
8.20E-16
|
4.10E-14
|
Vesicle
|
5.70E-85
|
6.10E-83
|
Reproduction
|
1.30E-24
|
3.00E-22
|
Protein binding
|
6.00E-11
|
2.00E-09
|
Extracellular region part
|
3.80E-70
|
2.70E-68
|
Reproductive process
|
1.20E-22
|
2.10E-20
|
Unfolded protein binding
|
3.60E-09
|
9.00E-08
|
Extracellular region
|
3.60E-62
|
1.90E-60
|
Positive regulation of biological process
|
1.90E-21
|
2.70E-19
|
Protein domain specific binding
|
1.80E-08
|
3.50E-07
|
Cytoplasm
|
3.90E-32
|
1.60E-30
|
Anatomical structure development
|
4.70E-21
|
5.50E-19
|
Carbohydrate binding
|
8.40E-06
|
0.00014
|
Membrane-bounded organelle
|
1.60E-29
|
5.70E-28
|
Regulation of biological quality
|
4.20E-18
|
4.20E-16
|
Binding
|
0.00037
|
0.0037
|
Extracellular space
|
9.30E-25
|
2.20E-23
|
Positive regulation of cellular process
|
5.30E-18
|
4.70E-16
|
Nucleotide binding
|
0.00041
|
0.0037
|
Organelle
|
1.60E-23
|
3.50E-22
|
System development
|
1.10E-17
|
8.90E-16
|
Enzyme regulator activity
|
0.00041
|
0.0037
|
Intracellular part
|
8.60E-20
|
1.70E-18
|
Response to stimulus
|
5.10E-17
|
3.60E-15
|
Catalytic activity
|
0.0022
|
0.012
|
Plasma membrane
|
7.80E-18
|
1.30E-16
|
Cellular developmental process
|
1.10E-16
|
7.00E-15
|
Receptor binding
|
0.0024
|
0.012
|
Cytoplasmic membrane-bounded vesicle
|
4.80E-15
|
6.10E-14
|
Response to biotic stimulus
|
1.90E-13
|
5.60E-12
|
|
|
|
Plasma membrane part
|
5.00E-09
|
3.90E-08
|
|
|
|
|
|
|
Cell part
|
4.80E-06
|
2.60E-05
|
|
|
|
|
|
|
Cell
|
4.80E-06
|
2.60E-05
|
Differential spatial distribution of BuBD-129 and 126
The peptides GRCKEYCNMDEKELDK for BuBD-129 and NKTGNCRSTCRNGEK for BuBD-126 were predicted to be highly antigenic and were thus adjudged as the best B-cell epitopes. This is because they were predicted to be preferentially present in turns and loops and had a comparatively higher probability for being found on the surface (Supplementary Fig. 4). Initially, the crude concentration of the isolated IgGs assayed by measuring the A280 was 155049 ng/µl and 168722 ng/µl for the CA-BDs, BuBD-129 and 126 respectively (Supplementary Fig. 5). Subsequently, Bradford’s assay was used to ascertain the more accurately and a concentration of 0.5ug/ml was fixed for further experiments like the IF, IVF studies.
The immunocytochemistry (ICC) studies, anti-BuBD-129 and 126 antibodies revealed that the two CA-BDs, BuBD-129 and 126 localized differentially on the surface of the buffalo bull spermatozoa (Fig. 3). Thus, a variation in the spatial distribution pattern was observed in the two class-A BuBDs (CA-BDs) wherein the BuBD-129 was present along the periphery of the spermatozoa, like the primate DEFB-126. The BuBD-126, however, was found to be present preferentially on the acrosomal and post-acrosomal and in the tail region while being absent in the mid-piece region (Fig. 3). The fluorescence produced by the BuBD-129 and 126 diminished when the spermatozoa were incubated in an elevated salt environment. The spermatozoa incubated in 2X-DPBS for 30 min appear to lose the sperm-surface bound BuBD-126 uniformly from the sperm surface, nonetheless, the sperm-surface bound BuBD-129 retained on the post acrosomal region although it was lost from the mid-piece and the tail region of the buffalo bull spermatozoa (Fig. 3). A thin band at the acrosomal-region fluoresced despite the loss of fluorescence signal from the remaining regions of the spermatozoa. The effect for the PI-PLC treatment, however, was markedly different for the sperm surface-bound BuBD-129. The spermatozoa exposed to 2U of PI-PLC lost the fluorescence signal for BuBD-129 from the entire spermatozoa. Nevertheless, the fluorescence pattern for BuBD-126 was similar to what was observed in 2X-DPBS treatment albeit weaker in intensity indicating partial loss of the bound BuBD-126 (Fig. 3).
Blocking BuBD-129 on sperm surface hinders cleavage, Morula and Blastocyst formation rates
The addition of anti-BuBD-129 antibody in the fertilization medium appeared to hamper the fertilization in a dose-dependent manner (Fig. 4). The percentage of cleaved oocytes decreased in the 1:15000 dilution group compared to the control group which further dropped significantly (P < 0.05) in the 1:10000 and 1:5000 (P < 0.00001) dilution. Both the 1:10000 and 1:15000 differed significantly (p < 0.001) from the 1:5000 dilution and the control group for the number of cleaved oocytes. The subsequent stages of embryo development e.g. the morula formation also exhibited a similar trend. The percentage of morula formed decreased in the 1:15000 dilution but declined significantly (P < 0.05) in 1:10000 dilution which further reduced (P < 0.00001) in the 1:5000 dilution group. As expected, the blastocyst formation rate was highest in control which declined (p < 0.01) on the addition of anti-BuBD-129 in 1:15000 and 1:10000 dilution groups (Fig. 4). No blastocyst was formed in the 1:5000 dilution group.