The Extracellular Matrix Protein, Spondin 1, Regulates Granulosa Cell Function and May Regulate Vascularization of the Corpus Luteum.

Abstract

Dramatic changes in the composition of the ovarian extracellular matrix (ECM) and cell-cell adhesion proteins occur throughout folliculogenesis. The ECM protein, Spondin 1 (SPON1), is a secreted adhesion protein best known for its ability to regulate patterning of the developing nervous system by promoting outgrowth and adhesion of neural cells. SPON1 has been previously identified in bovine follicular fluid, and was found to promote the proliferation of rat vascular smooth muscle cells. Although SPON1 is highly expressed in the human ovary, and dramatically overexpressed in ovarian cancer, a function for SPON1 in the ovary has never been studied. The objectives of our study were to: a) characterize the expression and localization of SPON1 in the mouse ovary, and b) identify what effect SPON1 may have on granulosa cell behaviour and function. Using qPCR, we found that SPON1 mRNA levels increased 5-fold in granulosa cells isolated from mice treated with PMSG for 48h compared to vehicle-treated controls, indicating that FSH increases SPON1 expression. Immunofluorescence studies localized SPON1 to regions between and surrounding both theca and granulosa cells of follicles of various sizes, from small secondary to preovulatory follicles. In the corpus luteum (CL), the pattern of SPON1 immunoreactivity was strikingly similar to that of platelet endothelial cell adhesion molecule (PECAM-1), which localizes to endothelial cells of the vasculature, suggesting that SPON1 may regulate vascularization during formation of the CL. Finally, we found that SPON1 has diverse effects when added as a recombinant protein to both granulosa cell lines and mouse primary granulosa cells. SPON1 enhanced the proliferation of KGN human granulosa cells, and in primary mouse granulosa cells, SPON1 enhanced granulosa cell viability by up to 50% compared to vehicle-treated controls, suggesting that SPON1 may regulate granulosa cell viability during folliculogenesis. Lastly, SPON1 dramatically induced aggregation of primary mouse granulosa cells, suggesting a role for SPON1 in granulosa cell adhesion, as has been reported in SPON1-treated neural cells. In summary, we have shown that the ECM protein, SPON1, regulates granulosa cell proliferation, survival, and cell-cell adhesion, and may also regulate vascularization of the corpus luteum. Given the diversity of its expression and effects on granulosa cells, we hypothesize that SPON1 plays a previously-unidentified important role in the regulation of various stages of folliculogenesis.