TLR2/4 signaling pathway mediates sperm-induced inflammation in bovine endometrial epithelial cells in vitro

Abstract

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro , however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes ( TNFA , IL-1B , IL-8 , and PGES ) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.

Mating to Intact, but Not Vasectomized, Males Elicits Changes in the Endometrial Transcriptome: Insights From the Bovine Model

“…Thus, it is possible that in intravaginal ejaculators, sperm can act as a vehicle of SP proteins that interact with the reproductive epithelium to induce changes in the maternal environment. Indeed, bull sperm has been shown to interact with endometrial cells and induce a proinflammatory response in vitro (Elweza et al, 2018;Ezz et al, 2019).…”

Persistent Breeding-Induced Endometritis in Mares—A Multifaceted Challenge: From Clinical Aspects to Immunopathogenesis and Pathobiology

“…Additionally, work in other species indicates that human and murine sperm express TLR2 and TLR4, although this has not been confirmed in the horse [151]. In bovine, in vitro studies suggested that sperm can stimulate transcription of pro-inflammatory cytokines, including chemokines (TNFα, IL1β, CXCL8) and prostaglandin E and activate the local complement system (C3) mediated via TLR2/4 [152][153][154].…”

Effect of Exposure to Seminal Plasma Through Natural Mating in Cattle on Conceptus Length and Gene Expression

“…In fact, some SP proteins that bind to sperm, such as Binder of Sperm Protein (Suarez and Pacey, 2006) and osteopontin (Souza et al, 2008), have previously been described to influence embryo development in vitro in pigs (Hao et al, 2008) and cattle (Gonçalves et al, 2008;Monaco et al, 2009;Rodríguez-Villamil et al, 2016). However, although bovine sperm can bind to both endometrial and oviductal cells in vitro and stimulate mRNA expression of different cytokines (Yousef et al, 2016;Elweza et al, 2018;Ezz et al, 2019), incubation of endometrial explants with cauda epididymis sperm (which are mature but have had no contact with SP) or Percollwashed ejaculated sperm did not induce differences in mRNA expression of some of those cytokines (Fernandez-Fuertes et al, 2019). Thus, it is not clear if SP-derived proteins, rather than intrinsic sperm proteins, are responsible for eliciting changes in the endometrium of cattle.…”