NOS1 mediates LPS-induced oxidative stress and impaired endometrial receptivity in sheep during early pregnancy

In sheep production, bacterial infections often cause embryo implantation failure and early pregnancy loss, resulting in substantial economic losses. These detrimental effects are closely associated with bacterial lipopolysaccharide (LPS)-induced disruption of uterine function, mediated via inflammation and oxidative stress. Although nitric oxide synthase 1 (NOS1) has been established to play a regulatory role in the induction of oxidative stress, its precise role during the peri‑implantation period in sheep remains unclear. In this study, pregnant ewes during three critical periods of embryo implantation in sheep (days 12, 16, and 20) were intrauterinely perfused with LPS (50 μg/ewe) or PBS via laparoscopy and endometrial tissues were collected at the maternal-fetal interface at 24 h post-perfusion to evaluate dynamic changes in receptivity and redox status. A two-way ANOVA revealed that LPS had significant inhibitory effects on the expression of the implantation-related genes ITGB1 and ITGB5, while treatment-by-day interactions were detected for HOXA10, HOXA11, ITGB3, and RSAD2, indicating that LPS disrupts the dynamic transcriptional programming of implantation both gene- and stage-specifically. Furthermore, LPS significantly decreased superoxide dismutase (SOD) and catalase (CAT) activities, increased malondialdehyde (MDA) levels, and upregulated NOS1 expression in vivo. To clarify the role of NOS1, we performed siRNA-mediated knockdown in a sheep endometrial epithelial cell line primed to a receptive state sequentially with progesterone, 17β-estradiol, and recombinant ovine interferon-tau. RNA-seq of NOS1-knockdown cells revealed significant enrichment of oxidative stress pathways, particularly glutathione metabolism. Functionally, NOS1 knockdown attenuated LPS-induced reactive oxygen species (ROS) accumulation, restored SOD and CAT activities, alleviated MDA damage, and partially rescued the expression of key implantation-related genes. Collectively, our findings revealed that LPS impairs endometrial receptivity by inducing oxidative stress and upregulating NOS1. Targeted inhibition of NOS1 in vitro effectively mitigates these deleterious effects, highlighting NOS1 as a critical mediator of LPS-associated implantation failure and a potential therapeutic target for improving reproductive efficiency in sheep.