CD9 is expressed on the cell surface of human granulosa cells and associated with integrin α6β1

Abstract

The CD9 molecule is a 24-27 kDa cell surface glycoprotein which is reported to be involved in cell adhesion and migration. Recently, CD9 was shown to be associated with β1-related integrins. We have previously found that integrin α6β1 is expressed on human granulosa cells (GC) and regulates luteinization of GC in concert with its ligand laminin. In this study, we examined the expression of CD9 in human ovary and the relationship between CD9 and integrin α6β1 in GC. By immunohistochemistry, CD9 was detected on GC in a small antral follicle of <1 mm in diameter. In growing follicles, CD9 was moderately expressed on both GC and theca interna cells (TI). The expression intensity of CD9 on GC increased in preovulatory follicles. In the early luteal phase, CD9 was expressed in both luteinizing GC and TI. The expression intensity on large luteal cells decreased in the mid-luteal phase. In the corpus luteum (CL) of pregnancy, CD9 continued to be expressed on large luteal cells, but not on small luteal cells. By flow cytometry, CD9 was detected on the cell surface in ~90% of the isolated GC from patients undergoing in vitro fertilization. The molecular weight of CD9 in the isolated GC was shown to be 26.5 kDa by Western blotting. CD9 mRNA was also detected in the isolated GC and CL by reverse transcription-polymerase chain reaction (RT-PCR). The proteins purified from GC by immunoaffinity chromatography using anti-integrin α6 monoclonal antibodies were shown by Western blotting to include CD9 as well as integrin β1. These findings suggest that CD9 is a differentiation-related molecule of GC and TI and that it is associated with integrin α6β1 on the cell surface of GC, suggesting that CD9 is implicated in the function of human GC in cooperation with integrin α6β1.