Toll-like receptors (TLRs), particularly TLR2 and TLR4, are critical components of the innate immune system that play significant roles in reproductive biology beyond their well-established functions in immune defense. These receptors recognize distinct pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering signaling cascades that impact diverse reproductive processes. TLR2-induced physiological inflammation, characterized by transient and precisely controlled inflammatory responses, is essential for facilitating sperm-triggered uterine clearance and promoting embryo implantation, thus maintaining reproductive homeostasis. This homeostasis represents a dynamic equilibrium of immune, endocrine, and cellular interactions essential for successful reproduction. Conversely, pathological inflammation, often driven by TLR4, can result in severe tissue damage, impairing fertility and pregnancy outcomes. This review highlights the shared and distinct signaling pathways of TLR2 and TLR4, their interplay mediated by co-receptors (TLR1 and TLR6) and regulatory molecules such as SOCS-1 and A20, and the implications of their dimerization. Understanding how physiological and pathological inflammation overlap and influence reproductive processes is critical for advancing fertility treatments. Targeting these pathways presents a promising therapeutic approach to address inflammation-related infertility and improve reproductive health.

We previously reported that sperm binding to cultured monolayers of bovine uterine epithelial cells induces an acute inflammatory response involving the Toll-like receptor (TLR2) signaling pathway. This response serves to clear the uterus of sperm and thereby prepares the endometrium for implantation. The endometrium is lined by surface epithelial cells; however, epithelial cells also line uterine glands. To investigate the source of the immune response, we used an explant model. Explants of bovine endometrium were incubated with bull sperm illuminated by JC1 fluorescent labeling in their mitochondria. The sperm glided over the surface epithelium until they encountered and entered uterine glands, where they remained. Scanning electron microscopy of explants revealed polymorphonuclear neutrophils (PMNs) in uterine glands along with the sperm. In the absence of sperm, PMNs were not seen in glands. Incubation of sperm with explants resulted in an acute inflammatory response, seen as upregulation of mRNA expression of IL8, TNFA, IL1B, PGES, and TLR2 in whole explants, as well as increased TNFA protein expression in uterine glands. TLR1/2 antagonist reduced sperm numbers in the glands and inhibited the increase of TNFA. Our observations suggest that uterine glands serve as a site where sperm interact with uterine epithelium to trigger the innate immune response to clear excess sperm from the uterus.

During the passage through the female reproductive tract, sperm interact with various compartments and their immune systems. The immune system that protects the female against pathogens also could destroy sperm or prevent them from reaching the site of fertilisation. In particular, the uterine innate immune response is crucial from the perspectives of both the sperm and the uterus. Following insemination, sperm immediately start to trigger inflammation in the uterus by entering uterine glands and activating an innate immune response. In cattle, the activation occurs mainly via TLR2 signalling, if not the only one, between sperm and the uterine epithelium lining the glands. This acute immune response is manifested as the upregulation of mRNA expression of IL8, TNFA, IL1B, and PGES. As a consequence, many sperm are trapped by polymorphonuclear neutrophils, the first and major component of innate immunity. The sperm-induced uterine innate immune responses apparently serve to clear the uterus of excess sperm and, importantly, prepare the endometrium for implantation. Pathophysiological conditions in the uterus seriously disrupt this phenomenon, and thus could directly decrease fertility.

Both the uterus and oviduct are functionally adapted to facilitate sperm transport, oocyte fertilization, embryonic/fetal development, and deliver a calf. However, the mucosa of the uterus and oviduct is exposed to ascending infections at coitus, during pregnancy, and after parturition. Therefore, the mucosal immune system of the uterus and oviduct should exhibit a large degree of flexibility to potentially regulate maternal immune reactions to allogeneic spermatozoa and semi-allogeneic embryos without hindering the effective immune defense responses against infectious agents ( Ellington, 1991; Yaniz et al., 2000; Bauersachs and Wolf, 2013). Immunologically, sperm are allogeneic to the maternal immune system ( Gaunt, 1983; Lander et al., 1990). Therefore, once sperm is deposited into the uterus during artificial insemination (AI), a sequence of cellular dynamics and immunological inflammatory reactions toward sperm is anticipated. These inflammatory responses are regarded as the rapid and transient leukocytic infiltration, mostly polymorphonuclear neutrophils (PMNs), into the uterine lumen which remove the excess or dead sperm ( Katila, 2012). Very few sperm can escape from PMNs’ phagocytosis in the uterus and reach the oviduct where sperm reservoirs are formed to induce capacitation and to support sperm survival until fertilization ( Hunter, 2012).

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.

Toll-like receptor 2 (TLR2) signaling pathway is involved in the sperm-triggered uterine inflammatory response at insemination, but its precise mechanism at molecular-level remains unknown. According to the ligand specificity, TLR2 forms a heterodimer with TLR1 or TLR6 as an initial step to mediate intracellular signaling, leading to a specific type of immune response. Hence, the present study aimed to identify the active TLR2 heterodimer (TLR2/1 or TLR2/6) that is involved in sperm-uterine immune crosstalk in bovine using various models. First, in-vitro (bovine endometrial epithelial cells, BEECs) and ex-vivo (bovine uterine explant) models were employed to test different TLR2 dimerization pathways in endometrial epithelia after exposure to sperm or TLR2 agonists; PAM3 (TLR2/1 agonist), and PAM2 (TLR2/6 agonist). Additionally, in-silico approaches were performed to confirm the dimer stability using de novo protein structure prediction model for bovine TLRs. The in-vitro approach revealed that sperm triggered the mRNA and protein expression of TLR1 and TLR2 but not TLR6 in BEECs. Moreover, this model disclosed that activation of TLR2/6 heterodimer, triggers a much stronger inflammatory response than TLR2/1 and sperm in bovine uterine epithelia. In the ex-vivo model that mimics the intact uterine tissue at insemination, sperm also induced the protein expression of both TLR1 and TLR2, but not TLR6, in bovine endometrium, particularly in uterine glands. Importantly, PAM3 and sperm induced similar and low mRNA expression of pro-inflammatory cytokines and TNFA protein to a lesser extent than PAM2 in endometrial epithelia. This implied that sperm might trigger a weak inflammatory response via TLR2/TLR1 activation which is similar to that of PAM3. Additionally, the in-silico analyses showed that the existence of bridging ligands is essential for heterodimer stability in bovine TLR2 with either TLR1 or TLR6. Altogether, the present findings revealed that sperm utilize TLR2/1, but not TLR2/6, heterodimerization to trigger a weak physiological inflammatory response in the bovine uterus. This might be the way to remove excess dead sperm remaining in the uterine lumen without tissue damage for providing an ideal uterine environment for early embryo reception and implantation.