68 Lipopolysaccharide in follicular fluid of mares: effects on progesterone levels, cytokine expression, and oocyte developmental competence

Lipopolysaccharide (LPS), a glycolipid produced by the external cell membrane of gram-negative bacteria, plays a major role in bacterial pathogenicity and initiates the host immune response. Accumulation of LPS in follicular fluid (FF) impairs steroid production and oocyte developmental competence in cows and mice. We recently detected LPS in equine FF and found that it was associated with decreased follicular progesterone concentrations. This study aimed to (1) check the association between LPS concentrations in FF and the expression of IL-6 and TNF-α, and (2) investigate if the exposure to LPS during IVM of equine oocytes affects their developmental competence. Ovaries were collected, and a cross-section from the wall of the largest viable follicle (>30 mm in diameter; n = 16 slaughterhouse mares), where LPS was previously detected, was sampled. Immunohistochemical staining was followed by a semiquantitative scoring (H-score) of the expression of IL-6 and TNF-α in granulosa and theca cells of the follicle wall. Cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, held overnight, in vitro matured with LPS (LPS group, n = 80 COCs; 1 ng of LPS per 1 mL of maturation medium) and without LPS (control group, n = 77 COCs), and mature oocytes were fertilized by using ICSI. On Day 3 and from Day 7 to Day 11 post-ICSI, respectively, cleavage and blastocyst development were monitored. Spearman correlation coefficients were calculated. Independent samples t-test and Mann-Whitney U-test were used to compare means of maturation, cleavage, and blastocyst rates between control and LPS groups. Immunohistochemically, IL-6 and TNF-α were detected in the cytoplasm of granulosa cells (H-score = 127.17 and 146.17, respectively) and theca cells (H-score = 85.43 and 93.76, respectively). LPS concentrations in FF were not significantly (P > 0.05) associated with the H-score of IL-6 and TNF-α, in either granulosa cells or theca cells. There were no significant differences between maturation (59.44% and 59.82%), cleavage (72.23% and 64.31%), and blastocyst (21.53% and 14.58%) rates in control and LPS groups, respectively. Mature oocytes from the LPS group showed morphological abnormalities such as a large perivitelline space and dysmorphic or vacuolated cytoplasm. In conclusion, this report is the first to immunohistochemically investigate the expression of IL-6 and TNF-α in equine follicle cells. LPS concentrations in FF had no association with the expression of IL-6 and TNF-α in the follicle cells. Exposure to LPS during IVM was accompanied by morphological abnormalities in mature oocytes and a nonsignificant reduction in blastocyst rate. Further studies are ongoing to investigate the influence of LPS on the biological functions of oocytes.