The maintenance and regression of corpus luteum are regulated by prostaglandin F_2α, but the role of prostaglandin D₂ (PGD₂) has not been thoroughly investigated. Its synthesis involves the key enzyme, hematopoietic prostaglandin D synthase (HPGDS) that mediates PGD₂ production in different cell types. The aim of this study was to investigate the biological role of endogenous PGD₂ on luteal cells in Hu sheep via HPGDS overexpression. For this purpose, luteal cells obtained from luteal tissue of 5 healthy adult Hu sheep at the mid-luteal phase immediately after slaughter were isolated via mechanical dissociation, enzymatic digestion, and Percoll density gradient centrifugation, cryopreserved in liquid nitrogen, and retrieved for subsequent experiments. Subsequently, quantitative real-time PCR (qRT-PCR) was used to confirm the efficiency of HPGDS overexpression. ELISA, qRT-PCR and Western blot (WB) were utilized to detect key indicators of luteal function and structural regression. Immunofluorescence was used to localize the PGD₂ receptors. Analytical results of lentiviral construction showed that HPGDS lentiviral plasmids were constructed, packaged in 293T cells, with MOI of 160 determined as optimal for luteal cell infection. HPGDS overexpression reduced P₄ concentration and StAR expression (P<0.01), showing impaired luteal endocrine function. Meanwhile, it increased BAX mRNA and protein and BAX/BCL-2 protein ratio (P<0.01), indicating enhanced luteal cell apoptosis. Moreover, HPGDS overexpression raised ROS concentration (P<0.01), upregulated mRNA levels of ATP6V1B1, SLCO2A1, PRKCE, and DP1 (P<0.01), while downregulating mRNA level of CREB (P<0.01), with enhanced DP1 fluorescence intensity. These findings indicate that HPGDS overexpression impairs progesterone secretion and promotes luteal cell apoptosis, suggesting that PGD₂ negatively regulates CL lifespan in small ruminants through HPGDS-related pathways.

To Cite This Article: Yang H, Lin R, Wu J, Long L, Li L, Xu H, Wei Z, Gan L, Wang G, Fu L and Dong X, 2026. Lentivirus-driven hematopoietic prostaglandin d synthase overexpression exerts a negative regulatory effect on the corpus luteum in sheep. Pak Vet J. http://dx.doi.org/10.29261/pakvetj/2026.018