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.

Acute Exposure to Organophosphosphorus Pesticide Metabolites compromises mammalian sperm survival, functional and fertility

“…Furthermore, most couples often share lifestyle habits, and diet choices and occupy the same niche and together transmit the molecular memory of their past environmental experience to their offspring (123).…”

Probiotics and gut microbiota: mechanistic insights into gut immune homeostasis through TLR pathway regulation

“…β-Defensins are synthesised by various epithelial cells such as in the skin, respiratory tract, reproductive tract and gastrointestinal tract. 111,112 At the same time, the expression of human β-defensin-1 may be constitutive, while β-defensin-2 is induced by bacterial infection. 113 Several studies have shown the antagonistic effect of probiotics against various pathogenic microorganisms by releasing AMPs as a defensive response from the epithelial surface.…”

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

“…3A) by the distribution of common and exclusive proteins among the three groups. The β-defensin 129 (BuBD-129) was selected to validate the identi ed 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.…”

“…The sperm surface harbors 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 modi ed during its journey until it reaches the oviduct [11,14].…”