Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium

Abstract

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Sperm interaction with the uterine innate immune system: toll-like receptor 2 (TLR2) is a main sensor in cattle

“…When sperm were incubated with luteal phase endometrial explants, it seemed that fewer sperm were gliding over the surface epithelium of luteal endometrium; however, sperm did not enter into the glands they encountered. These observations indicated that the uterine gland is a niche where sperm remain and interact with the uterus (Akthar et al 2020b;Elesh et al 2021). In cattle, this phenomenon was also reported in vivo, where glandular retention of sperm has been observed in histological sections made 24 h after AI (Koyama et al 1986).…”

“…1. We used an array of approaches to elucidate the inflammatory responses of the bovine endometrium to bull sperm, from a simple in vitro co-culture of bovine endometrial epithelial cells (BEECs) with frozen-thawed sperm (Elweza et al 2018;Ezz et al 2019), to an endometrial explant incubation model with fresh (Akthar et al 2020b) and frozen-thawed (Elesh et al 2021) sperm, to the in vivo experiments that were described above (Marey et al 2019). Initially, with our in vitro culture model, only live bull sperm stimulated an inflammatory cascade in BEECs, characterised by increased mRNA expression of the proinflammatory cytokines interleukin 8 (IL8), tumor necrosis factor-alpha (TNFA), interleukin 1 B (IL1B), and nuclear factor-kappa B2 (NFKB2), as well as complement factor 3 (C3) and prostaglandin E synthase (PGES).…”

The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

“…Other bacterial species, such as representatives of the Staphylococcus genus, may cause permanent sperm agglutination, through the secretion of agglutination or immobilization factors [ 29 ]. Furthermore, bacterial metabolites such as lipopolysaccharide, hemolysin, and peptidoglycan fragments may cause the flagellum to degenerate, tear off, or break, which has a negative effect on the ability of spermatozoa to reach the oocyte [ 30 , 31 , 32 ]. The exact mechanism by which such damage occurs is not yet fully understood, nevertheless previous reports indicated a significant role of membrane disintegration, promoted by ROS overproduction, either directly by the bacteria or indirectly by damaged spermatozoa [ 33 ].…”

Toll-like Receptor 2 is Involved in Calcium Influx and Acrosome Reaction to Facilitate Sperm Penetration to Oocytes During in vitro Fertilization in Cattle

“…Initially, preliminary dose-dependent and time-dependent experiments were conducted to identify cytotoxic or detrimental effects of TLR2 antagonist and agonist on sperm motility and viability as analyzed by CASA and flow cytometry ( Supplementary Table S1 and Supplementary Figure S1 ). Next, based on our previous investigations ( Ezz et al, 2019 ; Akthar et al, 2019 ; Morillo, et al, 2020 ; Elesh et al, 2021 ) and our initial trials, the pre-treatment of sperm by TLR2 antagonist (100 µM) or agonist (100 ng ml −1 ) for 30 min was designated as an effective model to test our hypothesis without generating any cytotoxic or detrimental effects on sperm motility and viability.…”