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.

Toll-like receptor 2 (TLR2) is a major membrane-bound receptor with ligand and species specificity that activates the host immune response. Heterodimerization of TLR2 with TLR1 (TLR2/1) or TLR6 (TLR2/6), triggered by ligand binding, is essential to initiating the signaling pathway. Bovine TLR2 (bTLR2) heterodimerization has not been defined yet compared with human and mouse TLR2s (hTLR2 and mTLR2). The aim of the present study was to model bovine TLRs (TLRs 1, 2 and 6) and create the heterodimeric forms of the bovine TLR2 using molecular dynamics (MD) simulations. We compared the intermolecular interactions in bTLR2/1-PAM3 and bTLR2/6-PAM2 with the hTLR2 and mTLR2 complexes through docking simulations and subsequent MD analyses. The present computational findings showed that bTLR2 dimerization could have a biological function and activate the immune response, similar to hTLR2 and mTLR2. Agonists and antagonists that are designed for hTLR2 and mTLR2 can target bTLR2. However, the experimental approaches to comparing the functional immune response of TLR2 across species were missing in the present study. This computational study provides a structural analysis of the bTLR2 interaction with bTLR1 and bTLR6 in the presence of an agonist/antagonist and reveals the three-dimensional structure of bTLR2 dimerization. The present findings could guide future experimental studies targeting bTLR2 with different ligands and lipopeptides.

In cattle, artificial insemination (AI) is a technique that allows breeding by depositing frozen-thawed and extended semen into the female reproductive tract. The semen contains sperm with various motility patterns including dead, progressive and hyperactivated. Sperm hyperactivation is high amplitude, asymmetrical beating of sperm tail which usually occurs in the oviduct as part of the capacitation process, but it can also be induced by cryopreservation. After insemination, sperm enter the uterine glands and trigger a pro-inflammatory response in the uterus. Hyperactivated sperm, stimulated by sperm-Toll-like receptor 2 (TLR2), penetrates the mucus and uterine glands more efficiently and enhances the immune response. This facilitates the clearance of excess and dead sperm from the uterus. Some sperm escape the immune response and reach the oviduct either before or after the immune response is initiated. In the oviduct, sperm bind to the epithelium and form a reservoir. This triggers an anti-inflammatory response and preserves the fertilization potential of sperm. Hyperactivation facilitates sperm detaching from the epithelium, swimming through the viscous mucus and cumulus cells, and penetrating the egg's zona pellucida. Sperm-TLR2 activation enhances Ca²⁺-influx and acrosome reaction, which enables sperm to penetrate and fertilize oocytes during in vitro fertilization. Altogether, post-AI in cattle, sperm and maternal immunity interact differentially depending upon the site of sperm hyperactivation – whether it occurs within the uterus or oviduct. Specifically, hyperactivated sperm that enter the uterus after AI or are triggered via sperm-TLR2 activation or other stimuli contribute to sperm-induced uterine inflammation. Such hyperactivated sperm may impede their capacity to ascend to the oviduct. Conversely, sperm that become hyperactivated within the oviduct modulate their interactions with the oviduct and oocytes, which is pivotal during fertilization process. Indeed, the location and timing of sperm hyperactivation partially via TLR2 activation are critical determinants of their different influence on fertility.

In vitro fertilization (IVF) and in vitro embryo production (IVP) are crucial technologies for accelerating genetic gain in cattle. However, IVP efficiency remains suboptimal, as in vitro -derived embryos frequently exhibit reduced developmental competence compared to their in vivo counterparts. Previous studies indicate that activation of the sperm Toll-like receptor 2/1 (TLR2/1) complex via Pam3CSK4 (PAM3) enhances sperm-oocyte interactions and improves cleavage and blastocyst rates; however, the underlying molecular mechanisms remain unexplored. In this study, frozen-thawed active sperm from three high-fertility Holstein bulls were allocated to control or PAM3-treated groups before IVF. Both sperm and the resulting day 7 blastocysts were subjected to RNA sequencing (RNA-seq) to investigate whether TLR2/1 activation modulates the paternal RNA cargo and if these alterations persist in the embryonic transcriptome. PAM3 treatment significantly improved developmental outcomes, specifically cleavage and blastocyst rates, although the magnitude of the effect varied among bulls. Transcriptomic analysis revealed distinct bull-specific RNA profiles and demonstrated that PAM3 treatment induced selective remodeling of sperm transcript abundance, involving both coding and non-coding RNA fractions. Conversely, day 7 embryos derived from PAM3-treated sperm did not exhibit global transcriptomic divergence from controls. This suggests that the molecular consequences of sperm TLR2/1 activation may be restricted to earlier developmental stages or regulated through specific pathways rather than broad transcriptomic remodeling at the blastocyst stage. In conclusion, these findings suggest that TLR2/1 signaling modulates paternal RNA cargo, potentially contributing to improved fertilization and bovine IVP efficiency by facilitating sperm penetration of the oocyte.