Patterns of Hormonally Regulated Endometrial Secretory Proteins as Detected by a Protein Chip Microarray

Abstract

Cytokines contribute to preimplantation endometrial development. Here we study steroid control of cytokines in a mouse endometrial model. Immature mouse endometrial cells were cultured with different hormonal media. Protein expression patterns were studied with a protein microarray. Endometrial cells were cultured from immature 3-week old brown BDF1 mice (Charles River Co., MA) The enzymatically digested glands and stroma (0.2x106 cell density) were seeded in four groups and incubated for 10 days with different hormonal media: Control, Estradiol (E2, 1x10-8 Molar), Progesterone (P, 1x10-8M) and E2+P. Protein array membranes for the detection of multiple cytokines at concentrations of 10 to 10,000 pg/ml (RayBio® Mouse Cytokine Antibody Array III) were incubated in serum free medium obtained from each group on day 10. The membranes were prepared per protocol and exposed to x-ray film. Primary analysis of the membranes was performed using Discovery Series Quantity One software (BioRad, CA). Expression was measured as the array spot area (mm2) x the optical density (OD). Mouse β-actin was amplified by RT-PCR as an internal control. Cell characteristics revealed hormonal differences in growth. E2 treated cells appeared longer and after 5 days proliferated in an overlapping, less orderly manner. P cells appeared fat and globular with large nuclei and abundant cytoplasm. E2+P cells grew abundantly and developed several glandular structures by day 8. RT-PCR was performed to confirm decidualization of cells by detection of decidual/trophoblastic prolactin-related protein (d/tPRP) mRNA in PCR products of P and E2+P treated cells (Kimura F et al., Gynecol Endocrinol 2001;15: 426-432). Of 62 cytokines, 41 were consistently detected by the array: L-selectin, Macrophage Inflammatory Protein-1α (MIP-1α), Vascular Cell Adhesion Molecule-1 (VCAM-1), Monocyte Chemo-attractant Protein-1 (MCP1), RANTES, and lymphotactin were expressed moderately strongly (OD>.04), and P-selectin and Tissue inhibitor of Metalloproteinase-1 (TIMP-1) were expressed most strongly (OD > .06). Cytokine array values were compared to control cell values for influence of hormonal treatment: E2 treatment increased secretion of 3 proteins, inhibited 22, and did not affect 13; P treatment increased secretion of 4, inhibited 19, and did not affect 8; E2+P treatment increased secretion of 9, decreased 26 and did not affect 6. Fractalkinine secretion was increased by both E2 and P alone but inhibited by E2+P. Secretion of eotaxin, eotaxin-2, and Interleukin-2 (IL-2) was not affected by E2 or P alone but was stimulated by E2+P. IGFBP-3, Macrophage Colony Stimulating Factor, MIP-3β, Soluble Tumor Necrosis Factor Receptor II, VEGF, and TIMP-1 were inhibited equally by E2, P and E2+P. MCP-1, MIP-2, Regulated upon Activation Normal T cell Expressed and Secreted (RANTES), and VCAM-1 were inhibited by E2 and P alone and further by E2+P. The simultaneous detection of multiple cytokines in a cultured mouse endometrial model suggests a steroid-based coordination of cytokines involved in implantation. Specific proteins demonstrated similar patterns of hormonal secretion, suggesting a common synthetic pathway or biologic function. These findings may be applied to create a more favorable milieu for embryo co-culture and to further study implantation.