Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

Abstract

Background: Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins.

Results: The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments.

Conclusions: The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

Keywords: Beta-defensins; Buffalo; Epididymis; Glycosylation; Sperm.

Fig. 1

The semantic-similarity based scatter plots depicting the summarized lists of GO terms for a Biological Process, b Molecular Function and c Cellular Component domains for the buffalo sperm surface proteins

Fig. 2

Histogram plots of the observed mean fluorescent intensity (MFI) values produced upon binding of six different FITC-labelled lectins viz. a ABL, b JAC, c LEL, d LCA, e MAL-II, and f PNA on buffalo bull spermatozoa in NCM (control), 2x-DPBS (2x-30) or spermatozoa exposed to 2 U/mL PI-PLC. The differences being assessed by one way ANOVA followed by Tukey’s multiple comparison test

Fig. 3

Relative expression profiles of the two CA-BD genes, BuBD-126 a and BuBD-129 b in the peripheral blood and ejaculated spermatozoa obtained from Murrah buffalo bulls. Expression values were normalized to GAPDH & eEF-2. Horizontal bars represent the mean(s) and error bars represent the standard error of the mean (SEM)

Fig. 4

Immuno-localization pattern of the two CA-BDs viz. BuBD-126 and 129 using the in house generated anti-BuBD-129 and 126 antibodies, respectively in rabbit against selected B-epitopes. The decrease of the fluorescent signal intensity pertaining to the removal of the CA-BDs, BuBD-126 and 129 from the buffalo bull sperm surface is observable after both, the 2x-DPBS and PI-PLC treatments

Fig. 5

Bright-field images of a Grade A and B oocytes aspirated from buffalo ovaries, b Matured cumulus-oocyte complexes after IVM, c 2-celled stage, d 4-celled stage, e 8-celled stage, f 16-celled stage, g Morula and h Blastocyst stage of buffalo embryo observed during IVF studies

Fig. 6

Scatter plots showing the Mean ± SD for cleavage rate a, morula b and blastocyst formation rates c in the control group and samples treated with three different concentrations of anti-BuBD-129

Fig. 7

The overall research methodology followed for BuBD identification on the buffalo sperm surface by LC-MS/MS.