Characterization of bovine ovarian surface epithelium and stromal cells: Identification of secreted proteins

Abstract

The majority of ovarian cancers are derived from a single layer of epithelial cells that covers the surface of the ovary termed the ovarian surface epithelium (OSE). Ovarian surface stromal cells underlie the OSE and have a morphology distinct from that of the interstitial stromal cells between follicles. Because of the similarities between bovine and human ovarian physiology, and to allow an adequate supply of tissue and cells bovine OSE and stromal cell cultures were established to investigate the cell biology of these cell types. Morphological analysis of bovine ovarian sections revealed that several layers of ovarian surface stromal cells can be identified and are structurally distinct from interstitial stromal cells. Both OSE and stromal cells can be isolated and cultured for weeks in the absence or presence of serum. The cell populations were found, through use of a keratin immunocytochemical stain for OSE, to be highly purified. To investigate the functional properties of the two cell types, radiolabeled secreted proteins were collected and electrophoretically analyzed. The radiolabeled secreted protein profiles of OSE and stromal cells were found to be distinct with a discrete number of secreted proteins. Major OSE secretory products were obtained from serum-free concentrated conditioned medium, electrophoretically separated, blotted, and sequenced. Two OSE secretory producers of 28 kDa and 40 kDa were sequenced and found to match a sequence in the computerized database. The 28-kDa OSE protein was identified as a tissue inhibitor of metalloproteinase, TIMP. The 40-kDa OSE protein was identified as the insulin-like growth factor (IGF) binding protein-2 (IGFBP2). The TIMP may play a role in regulating the local proteolytic activity of the OSE, and IGFBP2 in regulating IGF-1 actions on OSE. These proteins provide biochemical markers for OSE that can be used to further investigate the regulation of OSE function. The growth properties of cultured bovine OSE were also investigated. OSE proliferation was stimulated by epidermal growth factor (EGF) and was not influenced by keratinocyte or hepatocyte growth factors. Transforming growth factor-β was found to inhibit the growth stimulatory actions of EGF on OSE. Concentrated serum-free stromal cell-conditioned medium was also found to influence OSE growth. Therefore, the ovarian surface stromal cells appear to produce factors that can regulate the growth of the OSE. Combined results indicate that a culture system has been established to investigate the biology of OSE and ovarian surface stromal cells. Two OSE secretory products were identified as TIMP and IGFBP2. Preliminary data are presented that the ovarian surface stromal cells can regulate OSE growth. This stromal-epithelial cell interaction may have an important role in the normal cell biology of the OSE and be involved in the transformation of this cell to form an ovarian cancer.