91 Treatment of bovine endometrial explants with interleukin-1 beta increases the relative abundance of transcripts for pro-inflammatory cytokines
M. Kuzniar A , R. L. White B C , J. J. Bromfield D and J. Block AA Department of Animal Science, University of Wyoming, Laramie, Wyoming, USA
B Teagasc, Animal and Grassland Research and Innovation Centre, Fermoy, Cork, Ireland
C School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland
D Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
Reproduction, Fertility and Development 35(2) 172-172 https://doi.org/10.1071/RDv35n2Ab91
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Previous work in cattle has demonstrated that exposure of endometrial cells or tissue to sperm or seminal plasma increases the abundance of the pro-inflammatory cytokine interleukin-1 β (IL-1β). Two experiments were conducted to determine the effect of treatment of bovine endometrial explants with IL-1β on subsequent changes in the transcript abundance of other pro-inflammatory cytokines. For Exp. 1, endometrial explants were collected from abattoir-derived uteri of mature beef cows at various stages of the oestrous cycle using an 8 mm punch biopsy. Harvested explants were randomly assigned to one of six treatments consisting of complete medium (RPMI 1640 plus 10% [v/v] fetal bovine serum, 1% [v/v] penicillin-streptomycin, and 1% [v/v] amphotericin B) supplemented with 0, 0.1, 1, 10, 100, or 1,000 ng/mL recombinant bovine IL-1β. Explants were cultured at 37°C in a humidified atmosphere with 5% CO2 for 24 h. After treatment, explants were harvested, placed in lysis buffer, and then stored at −20°C until subsequent analysis. The experiment included seven biological replicates. Exp. 2 was performed as described for Exp. 1, except that explants were collected from abattoir-derived uteri assessed to be between Day 0 and 5 of the oestrous cycle. Additionally, explants were randomly assigned to treatment with rbIL-1β for three timepoints: 2, 6, and 24 h. Explants were harvested from culture at the respective timepoints, placed into RNAlater, and stored at −20°C until subsequent analysis. The experiment included eight biological replicates. For both experiments, RNA was extracted and then submitted to qRT-PCR to assess the relative abundance of six gene transcripts: IL1R1, IL1B, IL6, IL8, CSF2, and TNFA. Relative expression of the genes of interest was calculated using the 2−DCt method with GAPDH as the reference. Data were analysed by least-squares analysis of variance. In Exp. 1, the relative abundance of IL1B was increased (P < 0.05) following treatment with 1, 10, and 100 ng/mL rbIL-1β. Moreover, treatment with 10 ng/mL rbIL-1β increased (P < 0.05) abundance of IL-6 and CSF2. There was no effect of rbIL-1β treatment on the relative abundance of IL1R1, IL8, or TNFA. In Exp. 2, abundance of IL1B was increased (P < 0.05) following treatment with 100 and 1,000 ng/mL rbIL-1β, irrespective of timepoint. Similar results were obtained for IL6, with 10, 100, and 1,000 ng/mL rbIL-1β increasing (P < 0.05) abundance, regardless of timepoint. Abundance of IL8 was increased (P < 0.05) following treatment with 1, 10, 100, and 1,000 ng/mL rbIL-1β, but the effect was only observed following 6 h of treatment. There was also a treatment × timepoint interaction for the abundance of TNFA. Treatment with 100 and 1,000 ng/mL rbIL-1β increased (P < 0.05) abundance of TNFA at 2 and 6 h, but not at 24 h. Treatment of endometrial explants with rbIL-1β altered the abundance of several pro-inflammatory cytokines, including IL1B, IL6, IL8, CSF2, and TNFA. Further work is necessary to elucidate the role of IL-1β during the early post-mating period.
