A Higher Abundance of O-Linked Glycans Confers a Selective Advantage to High Fertile Buffalo Spermatozoa for Immune-Evasion From Neutrophils.

The glycans on the plasma membrane of cells manifest as the glycocalyx, which serves as an information-rich frontier that is directly in contact with its immediate milieu. The glycoconjugates (GCs) that adorn most of the mammalian cells are also abundant in gametes, especially the spermatozoa where they perform unique reproduction-specific functions e.g., inter-cellular recognition and communication. This study aimed to implicate the sperm glycosylation pattern as one of the factors responsible for low conception rates observed in buffalo bulls. We hypothesized that a differential abundance of glycans exists on the spermatozoa from bulls of contrasting fertilizing abilities endowing them with differential immune evasion abilities. Therefore, we investigated the role of glycan abundance in the phagocytosis and NETosis rates exhibited by female neutrophils (PMNs) upon exposure to such spermatozoa. Our results indicated that the spermatozoa from high fertile (HF) bulls possessed a higher abundance of O-linked glycans e.g., galactosyl (ß-1,3)N-acetylgalactosamine and N-linked glycans like [GlcNAc]1-3, N-acetylglucosamine than the low fertile (LF) bull spermatozoa. This differential glycomic endowment appeared to affect the spermiophagy and NETosis rates exhibited by the female neutrophil cells (PMNs). The mean percentage of phagocytizing PMNs was significantly different (P < 0.0001) for HF and LF bulls, 28.44 and 59.59%, respectively. Furthermore, any introduced perturbations in the inherent sperm glycan arrangements promoted phagocytosis by PMNs. For example, after in vitro capacitation the mean phagocytosis rate (MPR) rate in spermatozoa from HF bulls significantly increased to 66.49% (P < 0.01). Likewise, the MPR increased to 70.63% (p < 0.01) after O-glycosidase & α2-3,6,8,9 Neuraminidase A treatment of spermatozoa from HF bulls. Moreover, the percentage of PMNs forming neutrophil extracellular traps (NETs) was significantly higher, 41.47% when exposed to spermatozoa from LF bulls vis-à-vis the spermatozoa from HF bulls, 15.46% (P < 0.0001). This is a pioneer report specifically demonstrating the role of O-linked glycans in the immune responses mounted against spermatozoa. Nevertheless, further studies are warranted to provide the measures to diagnose the sub-fertile phenotype thus preventing the losses incurred by incorrect selection of morphologically normal sperm in the AI/IVF reproduction techniques.

Unusual interplay of contrasting selective pressures on ß-defensin genes implicated in male fertility of the Buffalo (Bubalus bubalis).

BACKGROUND: The buffalo, despite its superior milk-producing ability, suffers from reproductive limitations that constrain its lifetime productivity. Male sub-fertility, manifested as low conception rates (CRs), is a major concern in buffaloes. The epididymal sperm surface-binding proteins which participate in the sperm surface remodelling (SSR) events affect the survival and performance of the spermatozoa in the female reproductive tract (FRT). A mutation in an epididymal secreted protein, beta-defensin 126 (DEFB-126/BD-126), a class-A beta-defensin (CA-BD), resulted in decreased CRs in human cohorts across the globe. To better understand the role of CA-BDs in buffalo reproduction, this study aimed to identify the BD genes for characterization of the selection pressure(s) acting on them, and to identify the most abundant CA-BD transcript in the buffalo male reproductive tract (MRT) for predicting its reproductive functional significance. RESULTS: Despite the low protein sequence homology with their orthologs, the CA-BDs have maintained the molecular framework and the structural core vital to their biological functions. Their coding-sequences in ruminants revealed evidence of pervasive purifying and episodic diversifying selection pressures. The buffalo CA-BD genes were expressed in the major reproductive and non-reproductive tissues exhibiting spatial variations. The Buffalo BD-129 (BuBD-129) was the most abundant and the longest CA-BD in the distal-MRT segments and was predicted to be heavily O-glycosylated. CONCLUSIONS: The maintenance of the structural core, despite the sequence divergence, indicated the conservation of the molecular functions of the CA-BDs. The expression of the buffalo CA-BDs in both the distal-MRT segments and non-reproductive tissues indicate the retention the primordial microbicidal activity, which was also predicted by in silico sequence analyses. However, the observed spatial variations in their expression across the MRT hint at their region-specific roles. Their comparison across mammalian species revealed a pattern in which the various CA-BDs appeared to follow dissimilar evolutionary paths. This pattern appears to maintain only the highly efficacious CA-BD alleles and diversify their functional repertoire in the ruminants. Our preliminary results and analyses indicated that BuBD-129 could be the functional ortholog of the primate DEFB-126. Further studies are warranted to assess its molecular functions to elucidate its role in immunity, reproduction and fertility.