High Number of Neutrophils Exists in the Developing Corpus Luteum and Promotes Angiogenesis and Progesterone Secretion in the Cow.

Abstract

After ovulation, the bovine corpus luteum (CL) rapidly develops within a few days together with angiogenesis and progesterone (P) production. Ovulation and CL formation closely resemble an inflammatory response due to the influx of immune cells. It is well known that polymorphonuclear neutrophils (PMNs) play a role in host defense, inflammation, tissue remodeling and wound healing, and they secrete chemoattractants stimulating angiogenesis. We therefore hypothesized that PMNs and its chemoattractant interleukin-8 (IL-8) exist in the early CL just after ovulation and may play a physiological role in the CL development. PMNs were detected in CL tissue by Pas-staining, and IL-8 was measured in supernatant of the CL tissue culture by ELISA: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). Extremely low levels of PMNs and IL-8 were observed in the mid and late luteal phase, but increased during luteal regression. In immunohistochemical analysis, IL-8 protein was stained mainly in endothelial cells and immune cells such as PMNs and mononuclear cells in the CL. In transmigration assay of PMNs isolated from days 2, 5 and 10 of the estrous cycle, the different phase of estrous cycle did not affect the levels of PMN migration. On the other hand, the PMN migration was higher by the supernatant derived from the early CL as well as the recombinant IL-8 compared with that of the mid CL, and the level of PMN migration was inhibited by neutralizing with an anti-IL-8 antibody. These findings indicated that IL-8 is one of major chemoattractants to migrate PMNs within the early CL. We further investigated the role of PMNs and IL-8 in luteal function such as angiogenesis in luteal endothelial cells and P secretion from the early luteal cells. To assess the ability to stimulate angiogenesis, the peripheral PMNs from the early luteal phase were activated in culture with N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10 and 100 nM) or without fMLP as control at 37°C for 1 h. Supernatant of activated PMNs was harvested and used for the assessment of capillary tube formation. In in vitro matrigel assay, the supernatant of activated-PMNs as well as IL-8 (1-100 ng/ml) stimulated sprouting of capillary-like structures of luteal endothelial cells. Also, IL-8 (10 and 100 ng/ml) increased the proliferation of luteal endothelial cells. In co-culture model with early luteal cells (2x105 cells/well) and PMNs (1x105 cells/well), PMNs stimulated P secretion either with or without direct contact, but this was not accompanied with the increase of mRNA for steroidogenic acute regulatory protein (StAR) in luteal cells. On the other hand, IL-8 (1-100 ng/ml) did not affect P secretion and StAR mRNA. In conclusion, neutrophils are novel regulators of CL development after ovulation, and IL-8 secreted from the early CL especially endothelial and immune cells is a major chemoattractant for the migration of PMNs into the CL in the cow. Supported grants by Global COE and JSPS programs. (poster)